International Journal of

Molecular Sciences Article

Rutin-Enriched Extract from Coriandrum sativum L. Ameliorates Ionizing Radiation-Induced Hematopoietic Injury Xiaodan Han, Xiaolei Xue, Yu Zhao, Yuan Li, Weili Liu, Junling Zhang * and Saijun Fan * Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Peking Union Medical College and Chinese Academy of Medical Science, Tianjin 300192, China; [email protected] (X.H.); [email protected] (X.X.); [email protected] (Y.Z.); [email protected] (Y.L.); [email protected] (W.L.) * Correspondence: [email protected] (J.Z.); [email protected] (S.F.); Tel.: +86-22-8568-2315 (J.Z.); +86-22-8568-5301 (S.F.) Academic Editors: David Arráez-Román and Ana Maria Gómez Caravaca Received: 16 December 2016; Accepted: 24 April 2017; Published: 29 April 2017

Abstract: Hematopoietic injury is a major cause of mortality in radiation accidents and a primary side effect in patients undergoing radiotherapy. Ionizing radiation (IR)-induced myelosuppression is largely attributed to the injury of hematopoietic stem and progenitor cells (HSPCs). Coriander is a culinary herb with multiple pharmacological effects and has been widely used in traditional medicine. In this study, flavonoids were identified as the main component of coriander extract with rutin being the leading compound (rutin-enriched coriander extract; RE-CE). We evaluated the radioprotective effect of RE-CE against IR-induced HSPCs injury. Results showed that RE-CE treatment markedly improved survival, ameliorated organ injuries and myelosuppression, elevated HSPCs frequency, and promoted differentiation and proliferation of HSPCs in irradiated mice. The protective role of RE-CE in hematopoietic injury is probably attributed to its anti-apoptotic and anti-DNA damage effect in irradiated HSPCs. Moreover, these changes were associated with reduced reactive oxygen species (ROS) and enhanced antioxidant enzymatic activities in irradiated HSPCs. Collectively, these findings demonstrate that RE-CE is able to ameliorate IR-induced hematopoietic injury partly by reducing IR-induced oxidative stress. Keywords: ionizing radiation; coriander extract; total body irradiation; hematopoietic stem and progenitor cells; reactive oxygen species

1. Introduction Exposure to ionizing radiation (IR) may induce injury in various tissues and organs, among which bone marrow (BM) is the most radiosensitive tissue [1]. Acute myelosuppression is the primary cause of death after accidental or intentional exposure to a high dose of total body irradiation (TBI) [2,3]. Several studies have demonstrated that IR-induced myelosuppression is mainly due to impaired proliferation and differentiation ability and increased apoptosis and senescence of hematopoietic stem and progenitor cells (HSPCs) [2–5]. Therefore, it should be a primary goal to protect HSPCs in the development of novel medical countermeasures against IR, and there is a critical need to develop effective radioprotective agents that can ameliorate IR-induced HSPCs injury. It has been well established that reactive oxygen species (ROS) play a critical role in IR-induced hematopoietic injury [6,7]. IR induces the excessive production of ROS including superoxide, hydroxyl radicals, and hydrogen peroxide derived from the radiolysis of water. Oxidative stress from ROS may induce DNA damage, cell apoptosis, and senescence [5,7,8]. Moreover, ROS have been illustrated

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in several studies to be responsible for the loss of self-renewing ability and premature exhaustion of hematopoietic stem cells [9,10]. Fortunately, ROS can be eliminated by exogenous administration of antioxidants or by enhancing endogenous antioxidant enzyme activities, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT). Antioxidants have been extensively studied as ROS scavengers, which may mitigate the oxidative stress induced by IR [11–16]. Coriander (Coriandrum sativum L.) is an annual herb belonging to the Apiaceae family that has been used as a flavoring agent and traditional remedy. Essential oil, avonoids, phenolic acids, and polyphenols are important constituents of the aerial parts of coriander, and essential oil and fatty oil are the major components of coriander seeds [17,18]. Different parts of coriander have been reported for multiple health functions and biological activities, including antioxidant, antimicrobial, anti-diabetic, antidyslipidemic, anticonvulsant, anxiolytic, diuretic, antihypertensive, anti-inflammatory, and antimutagenic activities [17–20]. Hwang and his colleagues found that coriander possessed the potential to prevent ultraviolet radiation-induced skin photoaging [21]. More importantly, coriander extracts have been used to scavenge ROS as well as up-regulate endogenous cellular antioxidant systems [22,23]. These findings suggest that coriander may act as a radioprotective agent to mitigate IR-induced hematopoietic injury due to its antioxidant activity. In this study, we assessed the protective effects of the aqueous and ethanol extract mixture from the aerial parts of coriander on IR-induced hematopoietic injury in a well-established TBI mouse model [12]. Our data showed that rutin-enriched coriander extract (RE-CE) ameliorated myelosuppression, elevated HSPCs frequency, and improved the proliferation and differentiation ability of HSPCs, probably by inhibiting apoptosis and DNA damage in irradiated HSPCs. These protective effects of RE-CE may be attributed to scavenging ROS and activating antioxidant enzymes in irradiated HSPCs. All these findings suggest that CE treatment is able to protect the hematopoietic system from IR-induced injury. 2. Results 2.1. RE-CE Ameliorates IR-Induced Organ Injury It has been found that IR can cause damage to multiple organs, leading to changes of organ indexes, including a decline in the spleen index and thymus index, but a rise in lung index [24,25]. To determine whether RE-CE treatment protected mice from IR-induced organ index changes, mice were exposed to 4 Gy TBI and treated with the vehicle or RE-CE as described in the Materials and Methods. As shown in Figure 1A,B,D,E, 50 mg/kg RE-CE treatment significantly attenuated the reduction in the spleen index and thymus index of irradiated mice, while the 25 mg/kg RE-CE treatment showed a slight effect. Interestingly, the lung index of irradiated mice was markedly decreased by consumption of 50 mg/kg CE as well as 25 mg/kg RE-CE (Figure 1C,F). The thymus and spleen of irradiated mice atrophied and exhibited decreased lymphocytes compared to the controls. Congestion and inflammatory cell infiltration could be observed in the lungs of irradiated mice. CE treatment alleviated these pathological changes (Figure A1). In addition, 50 mg/kg RE-CE treatment attenuated the declines in total splenocyte and thymocyte counts in 4 Gy irradiated mice (Figure A1C,D). These findings suggest that RE-CE plays a protective role in IR-induced organ injury in mice, and the 50 mg/kg RE-CE treatment exhibits higher efficiency than the 25 mg/kg RE-CE treatment.

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Figure 1. RE-CE rescues organ index in irradiated mice. Mice were daily treated with vehicle or Figure 1. RE-CE rescues organ index in irradiated mice. Mice were daily treated with vehicle or Figure 1. RE-CE rescues organ index in irradiated mice. Mice were daily treated vehicle different different concentrations of rutin-enriched coriander extract (RE-CE) 30 minwith before 4 Gyortotal body different concentrations of rutin-enriched coriander extract (RE-CE) 30 min before 4 Gy total body concentrations of rutin-enriched coriander extract (RE-CE) min sham-irradiated. before 4 Gy total body irradiation (TBI) and up to 7 days after TBI. Control mice30were Spleenirradiation index (A), irradiation (TBI) and up to 7 days after TBI. Control mice were sham-irradiated. Spleen index (A), (TBI) and up to 7(B), daysand after TBI.index Control werewere sham-irradiated. index thymus index thymus index lung (C)mice of mice calculated onSpleen the 14th day(A), after exposure to(B), TBI. thymus index (B), and lung index (C) of mice were calculated on the 14th day after exposure to TBI. and lung index (C) of mice were calculated on the 14th day after exposure to TBI. 50 mg/kg RE-CE 50 mg/kg RE-CE treatment significantly increased the spleen index and thymus index, as well as 50 mg/kg RE-CE treatment significantly increased the spleen index and thymus index, as well as treatment increased the spleen index and thymus images index, as as (D), decreased the(E) lung decreasedsignificantly the lung index of irradiated mice. Representative of well spleen thymus and decreased the lung index of irradiated mice. Representative images of spleen (D), thymus (E) and index mice. Representative images ofare spleen (D), thymus (E) andare lung (F) on theas14th day ± lung of (F)irradiated on the 14th day after exposure to TBI shown. Organ indices presented means lung (F) on the 14th day after exposure to TBI are shown. Organ indices are presented as means ± after SEMexposure (n = 5). to TBI are shown. Organ indices are presented as means ± SEM (n = 5). SEM (n = 5).

2.2. 2.2.RE-CE RE-CEAlleviates AlleviatesIR-Induced IR-InducedMyelosuppression Myelosuppressionand andPromotes PromotesMyeloid MyeloidSkewing SkewingRecovery Recovery 2.2. RE-CE Alleviates IR-Induced Myelosuppression and Promotes Myeloid Skewing Recovery ItIthas hasbeen beenwell wellestablished establishedthat thatIRIRcan caninduce inducemyelosupression myelosupressionwhich whichisischaracterized characterizedby by It has been well established that IR can induce myelosupression which is characterized by cytopenia accompanied with myeloid skewing ininperipheral blood [26,27]. To determine whether cytopenia accompanied with myeloid skewing peripheral blood [26,27]. To determine whether cytopenia accompanied with myeloid skewing in peripheral blood [26,27]. To determine whether RE-CE treatment promoted recovery in irradiated irradiated mice, mice, we weanalyzed analyzedthe thenumber numberof RE-CE promoted hematopoietic hematopoietic recovery RE-CE treatment treatment promoted hematopoietic in irradiated mice, we analyzed the number of ofdifferent different types of peripheral blood using a hematology analyzer, andpercentage the percentage of peripheral blood cellscells using hematology analyzer, andthe the ofBBcells, cells, different types types of of peripheral blood cells using aa hematology analyzer, and percentage of BTcells, T cells,myeloid and myeloid cells using flow cytometry. As illustrated in Figures and A1, irradiated cells using using flow cytometry. cytometry. As illustrated illustrated in Figures Figures and2A1, A1,irradiated irradiated mice T cells, cells, and and myeloid cells flow As in 22 and mice mice exhibited a significant decline in white blood cells (WBCs), lymphocyte percentage (LY%), exhibited decline in in white white blood blood cells cells (WBCs), (WBCs), lymphocyte lymphocyte percentage percentage (LY%), (LY%), and and exhibited aa significant significant decline and percentage of B cells and T cells, but an increase in neutrophil percentage (NE%) and percentage percentage of B cells and T cells, but an increase in neutrophil percentage (NE%) and percentage of percentage of B cells and T cells, but an increase in neutrophil percentage (NE%) and percentage of ofmyeloid myeloid cells in peripheral blood at day 14 after 4 Gy TBI, when compared to unirradiated cells in peripheral blood at day 14 after 4 Gy TBI, when compared to unirradiated controls. myeloid cells in peripheral blood at day 14 after 4 Gy TBI, when compared to unirradiated controls. controls. Treatment of 50RE-CE mg/kg RE-CE and 25 mg/kg RE-CEWBC elevated WBC counts, LY%, B cell Treatment of mg/kg RE-CE and 25 mg/kg mg/kg RE-CE elevated WBC counts, LY%, cell percentage Treatment of 50 50 mg/kg and 25 RE-CE elevated counts, LY%, BBcell percentage percentage T cell percentage (Figuresand 2A,B,D,E andreduced A1), butNE% reduced NE% and myeloid cell and percentage (Figures 2A,B,D,E 2A,B,D,E and A1), but but reduced NE% and myeloid myeloid cell percentage and T T cell cell and percentage (Figures A1), and cell percentage percentage (Fiugres 2C,F and A1) in the peripheral blood of irradiated mice. The decline in WBC (Figures 2C,F and A1) in the peripheral blood of irradiated mice. The decline in WBC count was still (Figures 2C,F and A1) in the peripheral blood of irradiated mice. The decline in WBC count was still count still found 2 months afterand 6 Gy TBI, the 50 mg/kg RE-CE treatment attenuated such found 22 months after Gy TBI, TBI, and the 50and mg/kg RE-CE treatment attenuated such aa decline decline foundwas months after 66 Gy the 50 mg/kg RE-CE treatment attenuated such a (Figure decline (Figure A1B). These results suggest that RE-CE treatment promotes recovery from IR-induced A1B). These results suggest that RE-CE treatment promotes recovery from IR-induced (Figure A1B). These results suggest that RE-CE treatment promotes recovery from IR-induced myelosuppression myelosuppression andmyeloid myeloidskewing skewingtoto tomaintain maintainhematopoietic hematopoietichomeostasis. homeostasis. myelosuppressionand and myeloid skewing maintain hematopoietic homeostasis. (A) (A)

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Figure Figure2.2.RE-CE RE-CEalleviates alleviatesionizing ionizingradiation radiation(IR)-induced (IR)-inducedmyelosuppression myelosuppressionand andpromotes promotesmyeloid myeloid Figure 2. RE-CE alleviates ionizing radiation (IR)-induced myelosuppression and promotes myeloid skewing recovery in irradiated mice. Mice were sham-irradiated as the control or irradiatedwith with skewing recovery in irradiated mice. Mice were sham-irradiated as the control or irradiated skewing recovery in irradiated mice. Mice were sham-irradiated as the control or irradiated with 4 4 4Gy GyTBI TBIafter afterreceiving receivingthe the vehicle vehicle or or RE-CE RE-CE treatment treatment as as described described in in the Materials and Methods. Materials Methods. Gy TBI after receiving the vehicle or RE-CE treatment as described in thethe Materials andand Methods. The number of white blood cells (WBCs) (A), lymphocyte percentage (LY%) (B), and neutrophil The (WBCs) (A), (A), lymphocyte lymphocytepercentage percentage (LY%) neutrophil Thenumber numberof of white white blood blood cells cells (WBCs) (LY%) (B),(B), andand neutrophil percentage (NE%) (C) (C) in peripheral peripheralblood bloodwere werequantified quantified on the 14th day after exposure to TBI. percentage the 14th day after exposure to TBI. percentage(NE%) (NE%) (C) in in peripheral blood were quantified ononthe 14th day after exposure to TBI. TheThe The percentage of B cells (D), T cells (E), and myeloid cells (F) in peripheral blood were analyzed by percentage (E), and and myeloid myeloidcells cells(F) (F)ininperipheral peripheral blood were analyzed percentageofofBB cells cells (D), (D), T T cells cells (E), blood were analyzed by by Fluorescence-Activated Cell Sorting (FACS) on the 14th day after exposure to TBI. RE-CE treatment Fluorescence-Activated (FACS)on onthe the14th 14thday dayafter afterexposure exposure TBI. RE-CE treatment Fluorescence-Activated Cell Cell Sorting Sorting (FACS) to to TBI. RE-CE treatment significantly elevated WBC counts, LY%, B cell percentage, T cell percentage, and reduced NE% and significantly LY%, BBcell cellpercentage, percentage,T Tcell cellpercentage, percentage, and reduced NE% significantlyelevated elevated WBC WBC counts, LY%, and reduced NE% andand myeloid cell percentage. All data are presented as means means ± SEM(n(n= 5). = 5). myeloidcell cellpercentage. percentage. All All data are myeloid are presented presentedas as means± ±SEM SEM (n = 5).

2.3. RE-CE Dysfunction Cells and Erythrocytes 2.3. RE-CEMitigates MitigatesIR-Induced IR-InducedDifferentiation-Related Differentiation-Related Dysfunction BBB Cells and Erythrocytes in in BM 2.3. RE-CE Mitigates IR-Induced Differentiation-Related Dysfunctionofofof Cells and Erythrocytes inBM BM There in BM after Gy TBI, but RE-CE treatment Therewas wasaaanotable notabledecline decline in in cell percentage TBI, but RE-CE treatment There was notable decline in BBB cell cell percentage percentagein inBM BMafter after444Gy Gy TBI, but RE-CE treatment + Ter119 + immature + + increased the B cell percentage (Figure 3A). Additionally, the percentage of CD71 increasedthe theBBcell cellpercentage percentage (Figure (Figure 3A). of of CD71 Ter119 immature +Ter119 + immature increased 3A).Additionally, Additionally,the thepercentage percentage CD71 erythrocytes was elevated after 4 Gy but treatment alleviated this effect of IR on erythrocytes was elevated after 44 TBI, Gy but treatment alleviated this effect ofimmature IR IR on on erythrocytes was elevated after Gy TBI, TBI,RE-CE but RE-CE RE-CE treatment alleviated this effect of immature erythrocytes (Figure 3B). erythrocytes (Figure 3B). immature erythrocytes (Figure 3B). (A)

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Figure RE-CEmitigates mitigatesIR-induced IR-induced differentiation-related differentiation-related dysfunction ofof B cells and erythrocytes. Figure 3. 3. RE-CE dysfunction B cells and erythrocytes. Mice were sham-irradiated as control or irradiated with 4 Gy TBI after receiving the vehicle or or RE-CE Mice were sham-irradiated as control ordifferentiation-related irradiated with 4 Gy TBI after receiving the vehicle RE-CE Figure 3. RE-CE mitigates IR-induced dysfunction of B cells and erythrocytes. +Ter119+ treatment as described in the Materials and Methods. B cells percentage (A) and CD71 + Ter119+ treatment as described in the Materials and Methods. B cells percentage (A) and CD71 Mice were sham-irradiated as control or irradiated with 4 Gy TBI after receiving the vehicle or RE-CE immature erythrocyte percentage (B) in bone marrow (BM) were analyzed by FACS on the 14th day immature erythrocyte (B) in bone marrow (BM) were analyzed (A) by FACS on the 14th+ +Ter119 treatment as describedpercentage in the Materials and Methods. B cells percentage and CD71 after exposure to TBI. A representative FACS analysis shows the B cells percentage (C) and day after exposure to TBI. A representative FACS analysis shows the B cells percentage (C) and immature erythrocyte percentage (B) percentage in bone marrow analyzed byincreased FACS on the the B14th +Ter119 + immature erythrocyte (D) in(BM) BM. were RE-CE treatment cellday CD71 + Ter119 + immature CD71 erythrocyte percentage (D) in BM. RE-CE treatment increased the B cell after exposure TBI. A representative FACS analysis erythrocyte shows the percentage. B cells percentage +Ter119 + immature The data(C) areand percentage andto decreased the CD71 + + percentage and decreased the CD71 Ter119 immature percentage. Theincreased data are presented +Ter119 + immature erythrocyte percentage (D) erythrocyte in BM. RE-CE treatment the B cell CD71 presented as means ± SEM (n = 5). as means ± SEM (n = 5). + + percentage and decreased the CD71 Ter119 immature erythrocyte percentage. The data are

presented as means ± SEM (n = 5).

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2.4. RE-CE Attenuates IR-Induced Alterations in HSPCs Frequency 2.4. RE-CE Attenuates IR-Induced Alterations in HSPCs Frequency Since the exhaustion of HSPCs is a critical cause of myelosuppression, we examined whether Since the exhaustion of HSPCs is a critical cause myelosuppression, examined whether RE-CE treatment attenuated the IR-induced decline inof BM cells (BMCs) and we HSPCs frequency. The RE-CE treatment attenuated themice IR-induced decline in BM cells BMCs, (BMCs)but andRE-CE HSPCstreatment frequency.attenuated The 4 Gy 4 Gy and 6 Gy TBI irradiated both exhibited decreased and 6 Gy TBI irradiated miceasboth but RE-CE these declines in the 4 Gy wellexhibited as 6 Gy decreased irradiated BMCs, mice (Figure 4A).treatment As shownattenuated in Figure these 4B–F, declines in the Gy as as 6caused Gy irradiated mice (Figure 4A).(hematopoietic As shown in Figure 4B–F, 4cells, Gy 4 Gy and 6 4Gy TBIwell both a decrease in HPC progenitor −+Scal− c-kit+ ), − − + − + + + + − + and 6 Gy TBI both caused a decrease in HPC (hematopoietic progenitor cells, Lineage Lineage Scal c-kit ), LSK (Lineage Scal c-kit ), and CD34 LSK (CD34 Lineage Scal c-kit ) frequency, − Scal+ c-kit+−), and CD34−+ LSK (CD34 − Scal+ c-kit+ ) frequency, and an increase in LSK and (Lineage an increase in CD34 LSK (CD34 Lineage−Scal++Lineage c-kit+) frequency at day 14 after TBI. Furthermore, − − − + + CD34 (CD34a Lineage Scal 2c-kit ) frequency at day after TBI. Furthermore, HSPCsattenuated exhibited HSPCsLSK exhibited similar trend months after 6 Gy TBI 14 (Figure 4G–J). RE-CE treatment + −LSK athese similar trend 2 months after 6 Gy TBI (Figure 4G–J). RE-CE treatment attenuated these effects in HPC effects in HPC (Figure 4B,G), LSK (Figure 4C,H), CD34 LSK (Figure 4E,J), and CD34 + − (Figure 4B,G), LSK (Figure 4C,H), results CD34 LSK (Figure and treatment CD34 LSKpromotes frequencyrecovery (Figure 4D,I). frequency (Figure 4D,I). These suggest that4E,J), RE-CE from These resultsalternations suggest thatinRE-CE treatment promotes recoverygating from IR-induced in HSPCs IR-induced HSPCs frequency. The complete strategy foralternations HSPCs is presented frequency. The complete gating strategy for HSPCs is presented in Figure A2. in Figure A2. (A)

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Figure 4. RE-CE attenuates IR-induced alterations in hematopoietic stem and progenitor cells (HSPCs) frequency. Mice were daily treated with the vehicle or 50 mg/kg RE-CE by gavage for 30 min before 4 Gyattenuates or 6 Gy IR-induced TBIIR-induced and upalterations to 7alterations daysinafter radiation. Mice were sham-irradiated ascells the Figure 4.4.RE-CE hematopoietic stem and progenitor cells (HSPCs) Figure RE-CE attenuates in hematopoietic stem and progenitor control or CE group. (A) The number of BM cells (BMCs) per femur on the 14th day after exposure to frequency. Mice were daily treated with the vehicle or 50 mg/kg RE-CE by gavage for 30 min before (HSPCs) frequency. Mice were daily treated with the vehicle or 50 mg/kg RE-CE by gavage for 30 44min Gy 6 Gy TBI and up to 7 days after radiation. Mice were sham-irradiated as the control or CE Gyor or 6 Gy TBI; (B – D) The percentages of HSPCs were analyzed by FACS on the 14th day after before 4 Gy or 6 Gy TBI and up to 7 days after radiation. Mice were sham-irradiated as the group. The4group. number BM cells femur on the 14th day exposure to 4 Gy or 6cells, Gy exposure Gy or 6The Gynumber TBI; (BMCs) (B) percentage of (hematopoietic control(A) or to CE (A)of of The BMper cells (BMCs) perHPCs femur onafter the 14th day progenitor after exposure to − − + TBI; (B–D) percentages of HSPCs analyzed by were FACS on the day after 4 Gy or Lineage the were lineage-negative cells; (C)14th The percentage ofday LSKs 4 Gy or sca1 6 The Gyc-kit TBI;) (B–among D) The percentages of HSPCs analyzed by FACS onexposure the 14thto after − sca1− c-kit + ) among the +c-kit +) amongof 6(Lineage Gy TBI; −(B) percentage HPCs (hematopoietic progenitor cells, Lineageof sca1 lineage-negative cells; (D)ofThe percentage CD34−progenitor LSKs amongcells, the exposure toThe 4 Gy or 6 Gythe TBI; (B) The percentage HPCs (hematopoietic − + + +LSKs lineage-negative cells; (C) The percentage of LSKs (Lineage sca1 c-kit ) among the lineage-negative among the LSK cells; (F) Representative FACS analyses LSK cells; (E) The percentage of CD34 − − + Lineage sca1 c-kit ) among the lineage-negative cells; (C) The percentage of LSKs − LSKs +among the LSK cells; + LSKs −LSKs; cells; of the CD34 (E)(GThe of CD34 –J) percentage The percentages of HSPCs of the(D) percentage HPCs, LSKs, CD34 LSKs, and −The −LSKs sca1+percentage c-kit+of ) among lineage-negative cells;CD34 (D) The percentage of CD34 among the (Lineage + LSKs, among the by LSK cells; (F) Representative FACSamong analyses ofLSK the of HPCs, LSKs, CD34analyses +LSKs analyzed 2 months to 6 the Gy TBI;percentage (G) (F) TheRepresentative percentage ofFACS HPCs among cells; LSK cells; (E) FACS The percentage ofafter CD34exposure − LSKs; (G–J) The percentages of HSPCs analyzed by FACS 2 months after exposure to and CD34 −LSKs; (G–J) Thecells; lineage-negative cells; (H) The percentage of LSKs (I) The percentage andamong CD34lineage-negative percentages of HSPCs of the percentage of HPCs, LSKs, CD34+LSKs, +LSKscells; 6ofGy TBI;−LSKs (G) The percentage of(J) HPCs among lineage-negative (H) Thecells. percentage of LSKs CD34 among LSK cells; The percentage of CD34 among LSK treatment analyzed by FACS 2 months after exposure to 6 Gy TBI; (G) The percentage RE-CE of HPCs among − LSKs among LSK cells; (J) The percentage +LSK, among lineage-negative cells; (I) The percentage of CD34 of significantly increased the number of BMCsofper femur, the frequencies of HPC, LSK, and CD34 lineage-negative cells; (H) The percentage LSKs among lineage-negative cells; (I) The percentage + LSKs among LSK cells. RE-CE treatment significantly CD34 increased the number of BMCs per femur, − The +data presented as mean ± treatment SEM (n = and resulted decreased frequencies of CD34 LSK. −LSKsin of CD34 among LSK cells; (J) The +percentage of CD34 LSKsare among LSK cells. RE-CE the frequencies HPC, LSK, and CD34 LSK, and resulted in decreased frequencies of CD34−+LSK. # p < 0.05 of 5−15); vs. control; * p < 0.05 vs. 4 Gy. significantly increased the number of BMCs per femur, the frequencies of HPC, LSK, and CD34 LSK, # p < 0.05 vs. control; * p < 0.05 vs. 4 Gy. The data are presented as mean ± SEM (n = 5−15); and resulted in decreased frequencies of CD34−LSK. The data are presented as mean ± SEM (n = 2.5. RE-CE Colony Forming and Abilities of HSPCs in Irradiated Mice 5−15); #Improves p < 0.05 vs. control; * p < 0.05 vs.Engraftment 4 Gy.

2.5. RE-CE Improves Colony Forming and Engraftment Abilities of HSPCs in Irradiated Mice We conducted colony of granulocyte macrophage cells (CFU-GM) assays, spleen 2.5. RE-CE Improves Colony and and Engraftment Abilities of HSPCs in Irradiated Mice We conducted colony ofForming granulocyte macrophage cellsbone (CFU-GM) assays, spleen colony-forming colony-forming units (CFU-S) assays, competitive marrow transplantation to evaluate units (CFU-S) assays, and competitive bone marrow transplantation evaluate whether RE-CE whether RE-CE treatment improved the colony forming and engraftment abilities of HSPCs in We conducted colony of granulocyte macrophage cells to (CFU-GM) assays, spleen treatment improved the colony forming and engraftment abilities of HSPCs in irradiated mice. irradiated mice. The results revealed that the CFU-GM number remarkably decreased in irradiated colony-forming units (CFU-S) assays, and competitive bone marrow transplantation to evaluate The results revealed the improved CFU-GM number remarkably in irradiated mice compared to mice compared tothat control mice, but treatment attenuated this abilities decline (Figure 5A). whether RE-CE treatment theRE-CE colony formingdecreased and engraftment of HSPCs in control mice, but RE-CE treatment attenuated this decline (Figure 5A). In addition, 6 Gy TBI stimulated In addition, 6 Gy TBI stimulated the formation of spleen colonies and RE-CE treatment increased the irradiated mice. The results revealed that the CFU-GM number remarkably decreased in irradiated the formation of spleen coloniesmice, and RE-CE increased the4number CFU-S in irradiated mice number of CFU-S in irradiated mice (Figure 5B). At day 14 after Gy TBI,of hypoplastic bone marrow mice compared to control but treatment RE-CE treatment attenuated this decline (Figure 5A). (Figure 5B). At day 14 stimulated after 4 Gy TBI, hypoplastic marrow was observed in irradiated mice, but was observed in TBI irradiated mice, RE-CE-treated mice exhibited increased cellularity in bone In addition, 6 Gy thebut formation ofbone spleen colonies and RE-CE treatment increased the RE-CE-treated mice exhibited increased cellularity in bone marrow (Figure 5C). More importantly, 4 Gy marrow (Figure 5C). More importantly, 4 Gy and 6 Gy TBI both induced a reduction in engraftment number of CFU-S in irradiated mice (Figure 5B). At day 14 after 4 Gy TBI, hypoplastic bone marrow and Gy TBI both a reduction inRE-CE-treated engraftment but after competitive bone marrow transplantation, after competitive bone marrow transplantation, RE-CE treatment promoted donor cell was6 observed in induced irradiated mice, but mice exhibited increased cellularity in bone but RE-CE(Figure treatment promoted in4TBI 4 months Gyboth and induced 6 after Gy irradiated mice in 4bone months after engraftment in 5C). 4 Gy andimportantly, 6donor Gy cell irradiated mice competitive marrow marrow More 4engraftment Gy and 6 Gy a reduction engraftment competitive bone(Figures marrow transplantation (Figures 5Dbut andRE-CE A3). These data demonstrate that RE-CE transplantation and A3). These data demonstrate that RE-CE treatment the after competitive bone 5D marrow transplantation, treatment promotedimproves donor cell treatment improves the colony forming and engraftment abilities of irradiated HSPCs. colony forming engraftment abilities of irradiated engraftment in and 4 Gy and 6 Gy irradiated mice 4HSPCs. months after competitive bone marrow

transplantation (Figures 5D and A3). These data demonstrate that RE-CE treatment improves the (B) colony forming and engraftment(A) abilities of irradiated HSPCs. (A)

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Figure Figure5.5.RE-CE RE-CEimproves improvescolony colonyforming formingand andengraftment engraftmentabilities abilitiesof ofHSPCs HSPCsininirradiated irradiatedmice. mice. Mice were treated with the vehicle or RE-CE as described in the Materials and Methods. MiceFigure weredaily daily treated with the vehicle or RE-CE as described in the Materials and Methods. 5. RE-CE improves colony forming and engraftment abilities of HSPCs in irradiated mice. (A) 5 bone marrow cells (A) The number ofcolonies colonies ofgranulocyte granulocyte macrophage cells (CFU-GM) per bone marrow The Mice number cells (CFU-GM) per 10 105and wereof daily treatedofwith the vehiclemacrophage or RE-CE as described in the Materials Methods. (A)cells (BMCs); The number of of spleen colony-forming units (CFU-S) on the day to TBI;to bone marrow cells The(B) number of colonies of granulocyte macrophage cells(CFU-S) (CFU-GM) per 14th 105 after (BMCs); (B) The number spleen colony-forming units on14th the day exposure after exposure (C) Representative stained femurs (×40)(×40) on the 14th dayday after TBI; (D) (BMCs); (B) TheH&E number of stained spleen colony-forming units (CFU-S) onafter theexposure 14th day to after exposure to TBI; (C) Representative H&E femurs on the 14th exposure to TBI; (D)Donor Donor cell engraftment in the peripheral blood of recipients 4 months after competitive bone marrow TBI; (C) Representative H&E stained femurs (×40) on the 14th day after exposure to TBI; (D) Donor cell engraftment in the peripheral blood of recipients 4 months after competitive bone marrow cell engraftment in the peripheral blood of recipients months afterofof competitive bone marrow transplantation. RE-CE treatment significantly increased number CFU-GM and transplantation. RE-CE treatment significantly increased4the the number CFU-GMand andCFU-S, CFU-S, and transplantation. RE-CE treatment significantly increased the number ofmice. CFU-GM and CFU-S, and promoted HSPCs colony forming and donor cell engraftment in irradiated The data are presented promoted HSPCs colony forming and donor cell engraftment in irradiated mice. The data are promoted HSPCs forming and donor cell engraftment aspresented means ± as SEM (n = ±5 colony in panel B, n A = 10 inB, panel C).in panelinC).irradiated mice. The data are means SEM (n =A5 and in panel and n = 10 presented as means ± SEM (n = 5 in panel A and B, n = 10 in panel C).

2.6. Inhibits IR-Induced Apoptosis and DNA Damage ininHSPCs 2.6.RE-CE RE-CE Inhibits IR-Induced Apoptosis HSPCs 2.6. RE-CE Inhibits IR-Induced Apoptosisand andDNA DNA Damage Damage in HSPCs IRIRmay induce DNA damage in HSPCs, namely double breaks (DSBs), leading totocell may induce DNA damage doublestrand strandbreaks breaks (DSBs), leading cell IR may induce DNA damageininHSPCs, HSPCs, namely namely double strand (DSBs), leading to cell apoptosis, dysfunctional cell growth, and ultimately hematopoietic disorders [4]. As shown in Figure 6, apoptosis, dysfunctional hematopoieticdisorders disorders shown apoptosis, dysfunctionalcell cellgrowth, growth, and and ultimately ultimately hematopoietic [4].[4]. As As shown in in there is a noticeable increase in both apoptosis (Annexin V+) and the mean flourescence intensity (MFI) Figure 6, there is ais noticeable (AnnexinV+) V+)and and mean flourescence Figure 6, there a noticeableincrease increasein in both both apoptosis apoptosis (Annexin thethe mean flourescence ofintensity phospho-histone H2AX (γH2AX) in irradiated LSKs, but RE-CE treatment markedly attenuated intensity (MFI) of phospho-histone H2AX (γH2AX) in irradiated LSKs, but RE-CE treatment (MFI) of phospho-histone H2AX (γH2AX) in irradiated LSKs, but RE-CE treatment these trends (Figure 6). Similar results were obtained in c-kit positive cells (Figure A4). These results markedly attenuated these trends (Figure 6). Similar results were obtained in c-kit positive cells markedly attenuated these trends (Figure 6). Similar results were obtained in c-kit positive cells suggest that RE-CE promotes hematopoietic recovery probably by inhibiting IR-induced apoptosis (Figure These resultssuggest suggest that that RE-CE RE-CE promotes probably by by (Figure A4).A4). These results promotes hematopoietic hematopoieticrecovery recovery probably inhibiting IR-induced apoptosisand andDNA DNAdamage damage in and DNA damage in HSPCs. inhibiting IR-induced apoptosis in HSPCs. HSPCs. (A)

(B)

(C)

(D)

(A)

(C)

(B)

(D)

Figure 6. RE-CE inhibits IR-induced apoptosis and DNA damage in LSKs. Mice were daily treated

Figure 6. RE-CE inhibits IR-induced apoptosis and DNA damage in LSKs. Mice were daily treated with vehicle or RE-CE as described in the Materials and Methods. (A) The percentage of apoptosis in with vehicle or RE-CE as described in the Materials and Methods. (A) The percentage of apoptosis LSKs; The mean fluorescence intensity (MFI)and of phospho-histone H2AX (γH2AX) in LSKs; (C)treated A Figure 6. (B) RE-CE inhibits IR-induced apoptosis DNA damage in LSKs. Mice were daily in LSKs; (B) The mean fluorescence intensity (MFI) of phospho-histone H2AX (γH2AX) in LSKs; representative FACS analysis of the percentage of cell apoptosis in LSKs; (D) A representative with vehicle or RE-CE as described in the Materials and Methods. (A) The percentage of apoptosis in (C) Aanalysis representative FACS analysis of thebypercentage of cellRE-CE apoptosis in LSKs; (D) A representative of γH2AX expression inintensity LSKs flow cytometry. treatment significantly LSKs; (B) The mean fluorescence (MFI) of phospho-histone H2AX (γH2AX)reduced in LSKs;the (C) A analysis of γH2AX expression in LSKs by flow cytometry. RE-CE treatment significantly reduced percentage of cell apoptosis, and decreased the MFI of γH2AX in irradiated LSKs. All data are representative FACS analysis of the percentage of cell apoptosis in LSKs; (D) A representative the percentage ofmeans cell apoptosis, presented as ± SEM (n =and 5). decreased the MFI of γH2AX in irradiated LSKs. All data are analysis of γH2AX expression in LSKs by flow cytometry. RE-CE treatment significantly reduced the presented as means ± SEM (n = 5). percentage of cell apoptosis, and decreased the MFI of γH2AX in irradiated LSKs. All data are presented as means ± SEM (n = 5).

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2.7. RE-CE Scavenges IR-Induced ROS in HSPCs 2.7.The RE-CE Scavenges IR-Induced in HSPCscaused by ROS, which contribute to IR-induced indirect effect of IR ROS is mainly hematopoietic injury. determine whether RE-CE treatment reduced IR-induced ROS, we The indirect effectTo of IR is mainly caused by ROS, which contribute to IR-induced hematopoietic measured MFI of 2,7-dichlorofluorescein (DCF), MitoSox, and dihydroethidium (DHE) in LSKs injury. Tothe determine whether RE-CE treatment reduced IR-induced ROS, we measured the MFI of and c-kit positive cells 14 days after 4 Gy TBI to evaluate the total cellular ROS, 2,7-dichlorofluorescein (DCF), MitoSox, and dihydroethidium (DHE) in LSKs and c-kit positive cells mitochondria-derived ROS (superoxide), andcellular superoxide radicals levels, respectively. The MFI 14 days after 4 Gy TBI to evaluate the total ROS, free mitochondria-derived ROS (superoxide), ofand 2,7-dichlorodihydrofluorescein diacetate (DCFDA) (Figure A5), MitoSox (Figure 7B), diacetate and DHE superoxide free radicals levels, respectively. The MFI of 2,7-dichlorodihydrofluorescein (Figure 7C)(Figure in c-kit positive cells was7B), significantly elevated radiation, but RE-CE treatment (DCFDA) A5), MitoSox (Figure and DHE (Figure 7C)after in c-kit positive cells was significantly attenuated the radiation, IR-inducedbutelevation in ROS levels. Similar results of total cellular ROSlevels. were elevated after RE-CE treatment attenuated the IR-induced elevation in ROS Similar results of total cellular ROS were observed in LSKs (Figure 7A). These results suggest that observed in LSKs (Figure 7A). These results suggest that RE-CE serves as a free radical scavenger in RE-CE serves irradiated mice.as a free radical scavenger in irradiated mice. (A)

(B)

(C)

Figure7.7.RE-CE RE-CEscavenges scavengesIR-induced IR-induced reactive reactive oxygen oxygen species species (ROS) Figure (ROS) in in HSPCs. HSPCs. Mice Mice were weredaily daily treated with the vehicle or RE-CE as described in the Materials and Methods. (A) The total ROS treated with the vehicle or RE-CE as described in the Materials and Methods. (A) The total ROS levels in LSKs were presented as the MFI of 2,7-dichlorodihydrofluorescein diacetate (DCFDA); levels in LSKs were presented as the MFI of 2,7-dichlorodihydrofluorescein diacetate (DCFDA); (B) (B) The mitochondria-derived ROS levels in c-kit positive cells were presented as MFI of MitoSox; The mitochondria-derived ROS levels in c-kit positive cells were presented as MFI of MitoSox; (C) (C) The levels of superoxide free radicals were presented as the MFI of dihydroethidium (DHE). RE-CE The levels of superoxide free radicals were presented as the MFI of dihydroethidium (DHE). RE-CE treatment significantly decreased the MFI of DCF, MitoSox, and DHE in irradiated HSPCs. The MFI of treatment significantly decreased the MFI of DCF, MitoSox, and DHE in irradiated HSPCs. The MFI DCF, MitoSox and DHE are presented as means ± SEM (n = 5). of DCF, MitoSox and DHE are presented as means ± SEM (n = 5).

2.8. RE-CE Ameliorates IR-Induced Repression of Antioxidant Enzymatic Activities in c-Kit Positive Cells 2.8. RE-CE Ameliorates IR-Induced Repression of Antioxidant Enzymatic Activities in c-Kit Positive Cells Antioxidant enzymes minimize the perturbations caused by ROS under normal conditions. Antioxidant enzymes minimize the perturbations caused by ROS under normal conditions. However, the significantly elevated production of ROS under pathological conditions overcomes However, the significantly elevated production of ROS under pathological conditions overcomes cellular levels of antioxidants, inducing oxidative stress. To assess the effects of RE-CE on antioxidant cellular levels of antioxidants, inducing oxidative stress. To assess the effects of RE-CE on enzymes, we measured the enzymatic activity of SOD, GSH-PX, and CAT in c-kit positive cells. antioxidant enzymes, we measured the enzymatic activity of SOD, GSH-PX, and CAT in c-kit Moreover, we also measured the glutathione (GSH) level which is a global cellular antioxidant. positive cells.showed Moreover, measured the glutathione (GSH) levelofwhich is a global cellular The results that 4we Gy also TBI down-regulated the enzymatic activities SOD, GSH-PX, and CAT, antioxidant. The results showed that 4 Gy TBI down-regulated the enzymatic activities of and decreased the GSH level in c-kit positive cells, but RE-CE treatment dramatically amelioratedSOD, the GSH-PX, and CAT, and decreased the GSH level in c-kit positive cells, but RE-CE treatment repression of these enzymatic activities and increased the GSH level. (Figure 8). We also confirmed the dramatically ameliorated the repression of these enzymatic activities c-kit and positive increased theafter GSH level. increase in transcripts of SOD1, SOD2, CAT, and GSH-PX in irradiated cells RE-CE (Figure 8). We also A6). confirmed the increase in transcripts of SOD1, SOD2, CAT, and GSH-PX in treatment (Figure These findings indicate that the effects of RE-CE treatment on IR-induced irradiated c-kit positive cells after RE-CE treatment (Figure A6). These findings indicate that the hematopoietic injury may be, at least in part, attributed to ROS scavenging and enhancing antioxidant effects of RE-CE treatment on IR-induced hematopoietic injury may be, at least in part, attributed to enzymatic activities in irradiated c-kit positive cells. ROS scavenging and enhancing antioxidant enzymatic activities in irradiated c-kit positive cells. (A)

(B)

(C)

GSH-PX, and CAT, and decreased the GSH level in c-kit positive cells, but RE-CE treatment dramatically ameliorated the repression of these enzymatic activities and increased the GSH level. (Figure 8). We also confirmed the increase in transcripts of SOD1, SOD2, CAT, and GSH-PX in irradiated c-kit positive cells after RE-CE treatment (Figure A6). These findings indicate that the Int. J. Mol. Sci. 2017, 18, 942 9 of 20 effects treatment on IR-induced hematopoietic injury may be, at least in part, attributed to Int. J. Mol.of Sci.RE-CE 2017, 18, 942 9 of 20 ROS scavenging and enhancing antioxidant enzymatic activities in irradiated c-kit positive cells. (A)

(D) (B)

(C)

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(D) of antioxidant enzymatic activities in c-kit Figure 8. RE-CE ameliorates IR-induced repression positive cells. Mice were daily treated with the vehicle or RE-CE as described in the Materials and Methods. (A) Enzymatic activity of SOD in c-kit positive cells; (B) Enzymatic activity of GSH-PX in c-kit positive cells; (C) Enzymatic activity of CAT in c-kit positive cells. RE-CE treatment significantly increased the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT) in irradiated c-kit positive cells. The enzymatic activities are presented as means ± SEM (n = 3).

2.9. RE-CE Improves Survival of Lethally Irradiated Mice Figure ameliorates IR-induced repression of antioxidant enzymatic activitiesactivities in c-kit positive Figure8.8.RE-CE RE-CE ameliorates IR-induced repression of antioxidant enzymatic in c-kit

To test whether RE-CE affected the survival of irradiated mice, we fed mice with 50 mg/kg cells. Mice were daily treated with the vehicle orvehicle RE-CE or as described in the Materials Methods. positive cells. Mice were daily treated with the RE-CE as described in the and Materials and RE-CE or vehicle 30 min before radiation and up to 7 days after radiation. As shown in Figure 9, all (A) Enzymatic of SOD in c-kit positive cells;positive (B) Enzymatic activity of GSH-PX in of c-kit positive Methods. (A) activity Enzymatic activity of SOD in c-kit cells; (B) Enzymatic activity GSH-PX in vehicle-treated mice died within 24 days following 7 Gy TBI. However, 25% of RE-CE-treated mice cells; (C) Enzymatic activity of CAT in c-kit positive cells. RE-CE treatment significantly increased the c-kit positive cells; (C) Enzymatic activity of CAT in c-kit positive cells. RE-CE treatment significantly were enzymatic alive 30 days afterofTBI. These findings suggest that RE-CE peroxidase treatment significantly increases activities superoxide dismutase (SOD), glutathione (GSH-PX), and catalase the increased the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), survival rate of irradiated (CAT) in irradiated c-kitmice. positive cells. The enzymatic activities are presented as means ± SEM (n = 3). and catalase (CAT) in irradiated c-kit positive cells. The enzymatic activities are presented as means ± SEM (n = 3).

2.10. Flavonoids are Identified as the Major Component of Coriander Extract 2.9. RE-CE Improves Survival of Lethally Irradiated Mice 2.9. To RE-CE Improves of Lethally Irradiated Mice as a radioprotector for the hematopoietic identify the Survival major component that functioned To test whether RE-CE affected the survival of irradiated mice, we fed mice with 50 mg/kg system in coriander extract, we performed liquid chromatography-mass spectrometry (LC/MS) Toortest whether RE-CE affected the survival we fed with mg/kg RE-CE vehicle 30 min before radiation and up toof7irradiated days aftermice, radiation. Asmice shown in 50 Figure 9, analyses using liquid chromatography. As shown in Figure 10 and Table 1, flavonoids, phenolic RE-CE or vehicle mice 30 min before radiation up to 77days afterHowever, radiation. Asof shown in Figuremice 9, all all vehicle-treated died within 24 daysand following Gy TBI. 25% RE-CE-treated acids, and coumarins were the main components in coriander extract, with rutin, quercetin vehicle-treated mice died within days following 7 GyRE-CE TBI. However, of RE-CE-treated were alive 30 days after TBI. These24 findings suggest that treatment25% significantly increasesmice the 3-glucuronide, and nicotiflorin as the major compounds in the flavonoids. were alive after TBI. These findings suggest that RE-CE treatment significantly increases the survival rate30ofdays irradiated mice. survival rate of irradiated mice. 2.10. Flavonoids are Identified as the Major Component of Coriander Extract To identify the major component that functioned as a radioprotector for the hematopoietic system in coriander extract, we performed liquid chromatography-mass spectrometry (LC/MS) analyses using liquid chromatography. As shown in Figure 10 and Table 1, flavonoids, phenolic acids, and coumarins were the main components in coriander extract, with rutin, quercetin 3-glucuronide, and nicotiflorin as the major compounds in the flavonoids.

Figure 9. RE-CE RE-CEimproved improvedsurvival survival lethally irradiated mice. with the Figure 9. of of lethally irradiated mice. MiceMice werewere daily daily treatedtreated with the vehicle vehicle or 50 RE-CE mg/kg by RE-CE by gavage for before 30 min7 before Gy TBI then 7 days TBI.show Curves or 50 mg/kg gavage for 30 min Gy TBI7and thenand 7 days after TBI.after Curves the show therate survival rateafter of exposure 30 days to after exposure a lethal of TBI. RE-CE treatment survival of 30 days a lethal dose oftoTBI. RE-CEdose treatment significantly increases significantly increases theirradiated survival rate irradiated mice (n = 20). the survival rate of 7 Gy miceof(n7=Gy 20).

2.10. Flavonoids Are Identified as the Major Component of Coriander Extract To identify the major component that functioned as a radioprotector for the hematopoietic system in coriander extract, we performed liquid chromatography-mass spectrometry (LC/MS) analyses using liquidFigure chromatography. As shown in Figure 10 andirradiated Table 1, flavonoids, andwith coumarins 9. RE-CE improved survival of lethally mice. Mice phenolic were dailyacids, treated the vehicle or 50 mg/kg RE-CE by gavage for 30 min before 7 Gy TBI and then 7 days after TBI. Curves show the survival rate of 30 days after exposure to a lethal dose of TBI. RE-CE treatment significantly increases the survival rate of 7 Gy irradiated mice (n = 20).

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were the main components in coriander extract, with rutin, quercetin 3-glucuronide, and nicotiflorin Int.the J. Mol. Sci. 2017, 18, 942 10 of 20 as major compounds in the flavonoids.

Figure 10.10. ion chromatography (TIC) analysisextract. of liquid coriander extract. liquid Figure TotalTotal ion chromatography (TIC) analysis of coriander chromatography-mass chromatography-mass spectrometry (LC/MS) analyses were performed usingthe liquid spectrometry (LC/MS) analyses were performed using liquid chromatography to identify active ◦ chromatography to identify theReverse active phase component of coriander extract.atReverse phase component of coriander extract. separation was performed 40 C using an separation ACOUITY was BEH-C18 performed at (1.740µm). °C using phase an consisted ACOUITY UPLC acid BEH-C18 Column UPLC Column The mobile of 0.1% formic in water (solvent A) (1.7 HPLC μm). The mobile phase(solvent consisted 0.1% formicprogram acid in water (solvent A)B and grade and grade methanol B).of The gradient consisted of: 5% for 0HPLC min, 95% B methanol (solvent B). The gradient program consisted of: 5% B for 0 min, 95% B for 50 min, and 95% for 50 min, and 95% B for 60 min. The eluted peaks were. 1: Chlorogenic acid; 2: Esculin or B for 60 min. The eluted peaks were. 1: Chlorogenic acid; 2: Esculin or Daphnetin-8-O-glucoside; 3: Daphnetin-8-O-glucoside; 3: Hydroxybenozyl Tryptophan; 4: Methoxycinnamic acid glucoside; Hydroxybenozyl Tryptophan; 4: Methoxycinnamic glucoside; acid glucoside; 6: 5: Ferulic acid glucoside; 6: Citroside A, Citroside B,acid or Icariside B2;5:7:Ferulic Coriandrone E hexoside; Citroside A, 3-glucuronide; Citroside B, or9:Icariside B2;Nicotiflorin; 7: Coriandrone E hexoside; 8: Quercetin 3-glucuronide; 9: 8: Quercetin Rutin; 10: 11: Dihydrocoriandrin; 12: Coriandrone A or B; Rutin; 10: Nicotiflorin; 11: Dihydrocoriandrin; 12: Coriandrone A or B; 13: Coriandrin. 13: Coriandrin. Table 1. Identified constituents the coriander extract obtained from the aerial parts of Coriandum Table 1. Identified constituents of theofcoriander extract obtained from the aerial parts of Coriandum sativum L. sativum L. Peak Retention Molecular Ion Molecular Compound Ion Mode Compound Name Number Retention Time (min)Molecular Peak m/z Peak MolecularFormula Compound Type Ion Peak Ion Mode Compound Name Number Time (min) Formula Type 377.0836 [M + Na]+ Chlorogenic acid C16 H18 O9 8.452 m/z 1 Phenolic acid 353.0790 [M − H]− 377.0836 [M + Na]+ 1 8.452 C16H18O9 Phenolic acid Chlorogenic acidor Esculin 363.0687 [M + Na]+ C15 H16 O9 353.0790 [M − H]− 9.735 Coumarin 2 Daphnetin-8-O-glucoside 339.0614 [M − H]− 363.0687 [M + Na]+ Esculin or 2 9.735 C15H16O9 Coumarin Hydroxybenozyl 3 10.010 339.0614 371.1245 [M − [M Animo acidDaphnetin-8-O-glucoside H]−+ HCOO]− C18 H18 N2 O4 tryptophan Animo acid Hydroxybenozyl tryptophan 3 10.010 371.1245 [M + HCOO]− C18H18N2O4 Methoxycinnamic acid 365.1189 [M + Na]+ C18 H22 O8 [M + Na]+ 10.487 365.1189 4 Phenolic acid glucoside 341.1163 [M − H]− 4 10.487 C18H22O8 Phenolic acid Methoxycinnamic acid glucoside 341.1163 [M − H]− 379.0975 [M + Na]+ 379.0975 [M + Na]+ Ferulic acid glucoside C H O9 5 11.220 Phenolic acid 355.0943 [M − H]− acid Ferulic acid glucoside 5 11.220 C16H20O9 16 20Phenolic 355.0943 [M − H]− 409.1818 [M + Na]+ Citroside A or B or [M + Na]+ C H O8 6 11.917 409.1818 Norcarotenoid Citroside A or B 431.1830 [M + HCOO] −H30O8 19 30 Norcarotenoid 6 11.917 C19 Icariside B2 or Icariside B2 431.1830 [M + HCOO]− 433.1079 [M + Na]+ [M + Na]+ C H O10 14.080 433.1079 Coumarin 7 Coriandrone E hexoside 455.1075 [M + HCOO] Coriandrone E hexoside 7 14.080 C19−H22O1019 22 Coumarin 455.1075 [M + HCOO]− 479.0805 [M + H]+ [M + H]+ Quercetin 3-glucuronide C H O13 16.848 479.0805 Flavonoid 88 16.848 C21H18O1321 18 Flavonoid Quercetin 3-glucuronide 477.0564 [M − H]− 477.0564 [M − H]− 611.1590 [M + H]+ [M + H]+ C H O16 17.398 611.1590 Flavonoid RutinRutin 99 17.398 C27H30O1627 30 Flavonoid 609.1390 [M − H]− 609.1390 [M − H]− 617.1465 [M + Na]+ [M + Na]+ C H O15 19.800 617.1465 Flavonoid Nicotiflorin 1010 19.800 C27H30O1527 30 Flavonoid Nicotiflorin 593.1367 [M − H]− 593.1367 [M − H]− 22.220 255.0614 255.0614 [M + Na]+ [M + Na]+ H O4 Coumarin Dihydrocoriandrin 1111 22.220 C13H12OC 4 13 12 Coumarin Dihydrocoriandrin 24.200 293.1311 293.1311 [M + H]+ [M + H]+C16H20OC Coumarin Coriandrone Coriandrone 1212 24.200 5 16 H20 O Coumarin A orAB or B 5 1313 25.282 C13H10OC 4 Coumarin Coriandrin 25.282 253.0453 253.0453 [M + Na]+ [M + Na]+ H O Coumarin Coriandrin 13 10 4

3. Discussion Despite the wide use of coriander as a medicinal herb to treat various diseases, the therapeutic potential of coriander as a radioprotective agent is poorly understood. In this study, we explored whether RE-CE treatment could ameliorate IR-induced hematopoietic injury in a TBI mouse model. Our results indicated that RE-CE treatment improved the survival of irradiated mice, and rescued IR-induced injury in the spleen, thymus, and lung. It seems that there is no direct connection between IR-induced hematopoietic injury and lung injury; however, under our experimental

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3. Discussion Despite the wide use of coriander as a medicinal herb to treat various diseases, the therapeutic potential of coriander as a radioprotective agent is poorly understood. In this study, we explored whether RE-CE treatment could ameliorate IR-induced hematopoietic injury in a TBI mouse model. Our results indicated that RE-CE treatment improved the survival of irradiated mice, and rescued IR-induced injury in the spleen, thymus, and lung. It seems that there is no direct connection between IR-induced hematopoietic injury and lung injury; however, under our experimental conditions, radioprotective agents including RE-CE both protected the hematopoietic system from injury and alleviated IR-induced lung injury 14 days after 4 Gy TBI. A recent study reported that the lung is a site of platelet biogenesis and a reservoir for hematopoietic progenitors [28]. When hematopoietic stem cells decrease in the bone marrow, hematopoietic progenitors migrate out of the lung and reconstitute the bone marrow. In the light of these findings, one may speculate that RE-CE alleviates IR-induced HSPCs injury both in the bone marrow and lung, and HSPCs may play a role in IR-induced lung injury. Further work is required to establish the potential connection between IR-induced hematopoietic injury and lung injury. Myelosuppression is one of the common symptoms of hematopoietic injury, mainly caused by the exhaustion and dysfunction of HSPCs. The RE-CE treatment mitigated IR-induced myelosuppression and promoted the recovery of HSPCs populations to provide adequate reserves for hematopoietic injury. It has been demonstrated that lymphoid-biased HSCs are more sensitive to IR-induced differentiation than myeloid-biased HSCs, resulting in an imbalance in myeloid-lymphoid differentiation in irradiated mice [26]. The RE-CE treatment mitigated myeloid skewing in irradiated mice and promoted balanced differentiation of irradiated HSPCs. IR may also impair the self-renewing ability of HSPCs, causing long-term or permanent damage to the hematopoietic system [29,30]. The RE-CE treatment not only mitigated IR-induced myelosuppression, but also promoted HSPCs colony forming abilities and engraftment. Together, we demonstrate that the RE-CE treatment ameliorates IR-induced HSPCs injury in cell number, differentiation function, and colony forming abilities. To explore the underlying mechanisms, we measured cell apoptosis and DNA damage in LSKs and c-kit positive cells which are enriched with HSPCs. Results indicated that RE-CE treatment inhibited apoptosis and DNA damage in HSPCs, which may benefit the recovery of HSPCs populations and functions. Parsley, a similar herb to coriander in the Apiaceae family, has been reported to protect mouse fibroblasts from DNA damage induced by H2 O2 and induce apoptosis of cancer cells [31,32]. We confirm the protective effects of RE-CE on DNA damage in irradiated HSPCs, and demonstrate for the first time that the RE-CE treatment inhibits IR-induced apoptosis of HSPCs. However, the mechanisms by which RE-CE protects HSPCs from apoptosis are unknown and further exploration is warranted. Rutin, a bioactive flavonoid, was identified as a leading compound in coriander extract, consistent with previously reported findings [17]. Rutin has been reported to decrease oxidative stress by regulating oxidative stress related genes and proteins [33–35]. Quercetin, also identified as a component of RE-CE, together with rutin have antioxidant and radioprotective potential in mice exposed to γ-radiation [36–38]. These compounds may be largely responsible for the radioprotective effect of RE-CE on the hematopoietic system, observed in our studies. Our results indicate that RE-CE alleviates IR-induced HSPCs injury probably by reducing oxidative stress. As ROS may be the primary cause of DNA damage and apoptosis in HSPCs 14 days after radiation, they may also be partly responsible for the disturbance of proliferation and differentiation in irradiated HSPCs. Furthermore, Coriandrone A or B were also the major compounds in RE-CE, however, there are no reports about the biological and radioprotective effect of Coriandrone A or B, which is worthy of further exploration. Subsequent studies focusing on the effect of the individual or combined compounds identified in coriander extract are warranted.

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4. Materials and Methods 4.1. Reagents Anti-mouse CD34 FITC (clone RAM34), anti-mouse CD3 APC (clone145-2C11), anti-mouse CD117 (c-kit) APC (clone 2B8), and anti-mouse Ly-6 A/E (Sca1) CE/Cy7 (clone D7) were purchased from eBioscience (San Diego, CA, USA). Biotin anti-mouse CD4 (clone GK1.5), CE anti-mouse CD4 (clone GK1.5), CE anti-mouse/human CD45R/B220 (clone RA3-6B2), biotin anti-mouse/human CD45R/B220 (clone RA3-6B2), PerCP anti-mouse/human CD45R/B220 (clone RA3-6B2), PerCP anti-mouse/human CD11b (clone M1/70), FITC anti-mouse/human CD11b (clone M1/70), biotin anti-mouse/human CD11b (clone M1/70), PerCP anti-mouse Ly-6G/ Ly-6C(Gr1) (clone RB6-8C5), biotin anti-mouse Ly-6G/ Ly-6C(Gr1) (clone RB6-8C5), CE/Cy7 anti-mouse Ly-6G/Ly-6C(Gr1) (clone RB6-8C5), biotin anti-mouse Ter119 (clone TER119), FITC anti-mouse Ter119 (clone TER119), FITC anti-mouse CD8 (clone 53-6.7), biotin anti-mouse CD8 (clone 53-6.7), CE anti-mouse CD71 (clone RI7217), and PerCP streptavidin were obtained from Biolegend (San Diego, CA, USA). Anti-γH2AX rabbit monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). FITC goat anti-rabbit IgG was obtained from ZSGB-BIO Origene (Beijing, China). 4.2. Mice Male C57BL/6 (CD45.2) mice were purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). Male C57BL/6 (CD45.1) mice were purchased from the Institute of Hematology and Blood Disease, Chinese Academy of Medical Sciences and Peking Union Medical College (Tianjin, China). Male C57BL/6 (CD45.1/45.2) mice were bred in the experimental animal center of the Institute of Radiation Medicine. Mice were used at approximately 6 to 8 weeks of age. Animal experiments in our study were approved by the Animal Care and Ethics Committee of the Institute of Radiation Medicine (Permit number 1505, 1 Jan 2015). The study was performed in accordance with the principles of the Institutional Animal Care and Ethics Committee guidelines. 4.3. TBI and RE-CE Administration For evaluations of the organ index, histomorphology, peripheral blood cell counts, and HSPCs differentiation, mice were randomly divided into 4 groups, namely control, TBI, TBI + 25 mg/kg RE-CE, and TBI + 50 mg/kg RE-CE. For the other experiments, mice were also divided into 4 groups consisting of control, 50 mg/kg RE-CE, TBI, and TBI + 50 mg/kg RE-CE. Mice received 7 Gy TBI for the survival experiment, and 6 Gy or 4 Gy TBI in other experiments at a dosage rate of 0.99 Gy/min. Mice in control and RE-CE groups were sham-irradiated. CE was dissolved in distilled water (vehicle), and then administrated once a day by gavage 30 min before radiation and up to 7 days after radiation in RE-CE and TBI + RE-CE groups. Mice in the control and TBI groups received the same volume of vehicle for the same frequency and duration as those in CE or TBI + RE-CE groups. Mice were finally euthanized on the 14th or 60th day after TBI. 4.4. Weight, Organ Index, Counts of Splenocyte and Thymocyte, and HE Staining The body weight of individual mice was measured at the 14th day after exposure to 4 Gy TBI. The spleen, thymus, and lung were removed and weighed. The organ index was calculated according to the following formula: Organ index = [organ weight (g)/body weight (g)] × 10. Single cell suspensions of the spleen and thymus obtained by mechanical trituration were filtered and cells were counted. Specimens from spleen, thymus, and lung tissue were fixed with 4% formalin, embedded with paraffin, serially sectioned, and stained with hematoxylin and eosin. Specimens from the femur were decalcified with microwave and 10% ethylenediaminetertraacetic acid (EDTA) before being embedded.

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4.5. Peripheral Blood Cell Counts and Wright-Giemsa Staining Blood was obtained via the orbital sinus and was collected in EDTA tubes. The cell counts of peripheral blood including WBC counts, and LY% and NE% were analyzed with a hematology analyzer (Nihon Kohden, Japan). A Wright-Giemsa staining kit (Solarbio life sciences, Beijing, China) was used according to the manufacturer’s instructions. 4.6. Isolation of BM Cells (BMCs) and Flow Cytometry Analysis BM cells (BMCs) were isolated from tibias and femurs, suspended in phosphate-buffered saline (PBS), filtered, and counted prior to antibody staining. For B cell, T cell, and myeloid cell analysis in peripheral blood, 50 µL peripheral blood was harvested and incubated with premixed antibodies of B220, CD3, Gr1, and CD11b at room temperature for 30 min, and subsequently red blood cells were removed with BD FACSTM Lysing Solution. For B cell analysis in BM, a 1 × 106 BMC suspension was incubated with CD3 and B220 antibodies at 4 ◦ C for 30 min. For immature erythrocytes analysis in BM, a 1 × 106 BMC suspension was incubated with CD71 and Ter119 antibodies at 4 ◦ C for 30 min. For the analysis of HPSC frequency, 5 × 106 BMCs were incubated with biotin-conjugated antibodies specific for Gr1, Ter119, CD11b, B220, CD8, and CD4 and then stained with streptavidin, sca1, c-kit, and CD34 antibodies. Data acquisition was performed on a BD Accuri C6 and analyzed by BD Accuri C6 software (BD Bioscience, San Jose, CA, USA). 4.7. CFU-GM and CFU-S Assays For CFU-GM assay, 1 × 104 BMCs from unirradiated mice and 1 × 105 BMCs from irradiated mice were cultured in M3534 methylcellulose medium (Stem Cell Technologies, Vancouver, BC, Canada) for 5 days. The number of CFU-GM containing more than 30 cells was counted according to the manufacturer’s instructions, and the results are expressed as the number of CFU-GM per 105 BMCs. For the CFU-S assay, the spleen was soaked in trinitrophenol for 6 h and then the number of CFU-S was counted. 4.8. Competitive Bone Marrow Transplantation 1 × 106 BMCs from C57BL/6 (CD45.2) treated mice and 1 × 106 BMCs from C57BL/6 (CD45.1/45.2) mice were mixed and transplanted into lethally irradiated C57BL/6 mice (CD45.1). The percentage of donor-derived (CD45.2) cells in the peripheral blood of recipients was examined 4 months after transplantation. 4.9. Isolation of c-Kit Positive Cells BMCs were stained with c-kit APC antibody for 30 min on ice. Then the cells were washed with PBS, and incubated with anti-APC microbeads (Miltenyi Biotec, Teterow, Germany) for 15 min at room temperature. C-kit positive cells were sorted by MACS using a LS column in the QuadroMACS™ Separator (Miltenyi Biotec, Teterow, Germany). 4.10. Apoptosis Assay 1 × 106 c-kit positive cells or 5 × 106 BMCs stained with LSK (Gr1, Ter119, CD11b, B220, CD8, CD4, sca1 and c-kit) antibodies were prepared to perform the apoptosis assay with an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Samples were collected by a BD Accuri C6 and analyzed using the BD Accuri C6 software (BD Bioscience, San Jose, CA, USA). 4.11. Analysis of γH2AX Staining in c-Kit Positive Cells 1 × 106 c-kit positive cells or 5 × 106 BMCs stained with LSK antibodies were fixed and permeabilized with BD Cytofix/Cytoperm buffer for 30 min at room temperature. After washed

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with BD perm/Wash buffer twice, cells were incubated with anti-γH2AX (1:100) for 1 h and then stained with FITC goat anti-rabbit IgG for 30 min at room temperature. The MFI of γH2AX in c-kit positive cells was detected by flow cytometry. 4.12. Analysis of Intracellular ROS Levels 1 × 106 c-kit positive cells or 5 × 106 BMCs stained with LSK antibodies were stained with 2,7-dichlorodihydrofluorescein diacetate (DCFDA, Beyotime Biotechnology, Nanjing, China; 10 µM), MitoSox (Life Technologies, Grand Island, NY, USA; 10 µM), and dihydroethidium (DHE, Beyotime Biotechnology, Nanjing, China; 5 µM) for 20 min, 30 min, and 10 min, respectively, in a 37 ◦ C water bath. The intracellular ROS levels of c-kit positive cells were analyzed by measuring the MFI of DCF, Mitosox, and DHE using a flow cytometer. 4.13. Analysis of SOD, GSH-PX, GSH, and CAT Activities SOD, GSH-PX, GSH, and CAT enzymatic activities of c-kit positive cells were determined using the total superoxide dismutase assay kit with WST-8, total glutathione peroxidase assay kit, total glutathione assay kit, and catalase assay kit (Beyotime Institute of Biotechnology, Nanjing, China), respectively, according to the manufacturer’s instructions. Briefly, 1 × 106 c-kit positive cell lysate or homogenate was added into the detection buffer, and the maximum absorption wavelength was examined at 450, 340, 412, and 520 nm by the colorimetric method for the detection of enzymatic activities of SOD, GSH-PX, GSH, and CAT, respectively. 4.14. Quantitative Real-Time PCR The total RNA of c-kit positive cells was extracted with TRIzol reagent (Life Technologies, Grand Island, NY, USA). Reverse transcription was performed with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. All PCRs were conducted under an ABI 7500 Sequence Detection System and GAPDH (Thermo, Waltham, MA, USA) was used as control. Three pairs of primers were designed for SOD2, CAT, and GSH, according to the sequences shown in a previous study [39]. The forward primer sequence of SOD1 was AACCAGTTGTGTTGTCAGGAC, and its reverse primer sequence was CCACCATGTTTCTTAGAGTGAGG. 4.15. Preparation and Component Identification of CE RE-CE was purchased from ICTORY Biological Technology Co., Ltd. (Xi’an, China). The raw materials of coriander herbs were dried without access to direct sunlight after copious washing in running water. The dried materials were ground into a fine power and passed through a 40-mesh sieve. This powered product (500 g) was submitted to extraction with aquaous ethanol (30%, 5 L) in a shaker at 60 ◦ C for 2 h. The extract waste was filtered out and the solution was concentrated by a rotary evaporator and dried under vacuum. The extract was stored at 4 ◦ C for further use. LC/MS analyses were performed on a Shimadzu LC-30AD liquid chromatography interfaced with an IT-TOF mass spectrometer (Shimadzu Corp., Kyoto, Japan) with electrospray ionization. An ACOUITY UPLC BEH-C18 Column (1.7 µm) was used. The column temperature was 40 ◦ C and the elution velocity was 0.3 mL/min. The sample extracts were analyzed using a gradient program, and the mobile phase consisted of 0.1% formic acid in water (solvent A) and HPLC grade methanol (solvent B). The gradient program consisted of: 5% B for 0 min, 95% B for 50 min, and 95% B for 60 min. The nebulizer gas flow rate was 1.5 L/min. CDL and block heater temperatures were both 200 ◦ C. The spray and detector voltages were 4.5 and 1.58 kV, respectively. The scan time and mass range were 1.5 s and 50–1000 m/z, respectively. The injected volume was 2 µL.

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4.16. Statistical Analysis Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Prism Software Inc., La Zolla, CA, USA) with an unpaired t test (two-tails) and Welch’s correction t-test for mean comparisons. Survival rates were analyzed with the Kaplan Meier method and Log rank test. Data are presented as means ± SEM, and differences were considered statistically significant at p < 0.05. 5. Conclusions Our study demonstrates for the first time that RE-CE treatment protects mice from IR-induced hematopoietic injury. RE-CE treatment improves the proliferation and differentiation function, and inhibits apoptosis and DNA damage in irradiated HSPCs. Our studies also suggest the potential of RE-CE to scavenge ROS and enhance antioxidant enzyme activities in irradiated HSPCs. Further studies are warranted to explore the effects of RE-CE as a radioprotective agent to ameliorate IR-induced injuries. Acknowledgments: We thank Yiliang Li and Qi Shan for the excellent work on composition identification and analysis of CE. This study was supported by a grant from the National Natural Science Foundation of China (81572969) ,the Technology and Development and Research Projects for Research Institutes, Ministry of Science and Technology (2014EG150134), the Tianjin Science & Technology Pillar Program (14ZCZDSY00001), and The CAMS Innovation Fund for Medical Sciences (CIFMS, No. 2016-I2M-1-017) to Saijun Fan; National Natural Science Foundation of China (81402633), and Natural Science Foundation of Tianjin (16JCQNJC13600) to Junling Zhang; Fundamental Research Funds for the Central Universities and PUMC Youth Fund (3332016099) to Weili Liu. Author Contributions: Xiaodan Han, Junling Zhang, and Saijun Fan conceived and designed the experiments; Xiaodan Han, Xiaolei Xue, Yu Zhao, Yuan Li, and Weili Liu performed the experiments; Xiaodan Han, Junling Zhang, and Saijun Fan analyzed the data; Xiaolei Xue and Yuan Li contributed reagents/materials/analysis tools; Xiaodan Han wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations IR HSPCs CE ROS BM TBI SOD GSH-PX CAT WBC LY NE BMCs HPCs CFU-S CFU-GM

ionizing radiation hematopoietic stem and progenitor cells coriander extract reactive oxygen species bone marrow total body irradiation superoxide dismutase glutathione peroxidase catalase white blood cell lymphocyte neutrophil bone marrow cells hematopoietic progenitor cells spleen colony-forming units colony of granulocyte macrophage cells

LY lymphocyte NE neutrophil BMCs bone marrow cells HPCs hematopoietic progenitor cells CFU-S Int. J. Mol. spleen colony-forming units Sci. 2017, 18, 942 CFU-GM colony of granulocyte macrophage cells

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Appendix A

Appendix A

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Figure organ and and peripheral peripheralblood. blood.Mice Micewere were FigureA1. A1.CE CEtreatment treatmentameliorates ameliorates IR-induced IR-induced injury injury in in organ

Figuredaily A1. CE treatment ameliorates IR-inducedininjury in organ and peripheral blood. Mice were the Materials Materials andMethods. Methods. (A)Representative Representative dailytreated treatedwith withthe thevehicle vehicleor orCE CE as as described described in the and (A) daily treated withspleen, the vehicle CE and as described in stained the Materials and Methods. (A)14th Representative H&E thymus, and wright-giemsa stained peripheral blood (×40)on the 14th H&Estained stained spleen, thymus,orlung, lung, wright-giemsa peripheral blood (×40) on the day day after exposure to TBI; (B) The number of WBCs in peripheral blood 2 months after 6 Gy TBI; (C) the 14th after exposure TBI; (B) The number WBCs in peripheral blood 2peripheral months afterblood 6 Gy TBI; (C) Total H&E stained spleen,tothymus, lung, andofwright-giemsa stained (×40)on Total splenocyte on theday 14th dayexposure after exposure to TBI; Total thymocyte on the TBI; (C) splenocyte counts on the 14th after to TBI; Total(D) thymocyte counts oncounts the 14th day after exposure tocounts TBI; (B) The number of WBCs in (D) peripheral blood 2 months after 6day Gy 14th day after exposure after exposure to TBI. to TBI. Total splenocyte counts on the 14th day after exposure to TBI; (D) Total thymocyte counts on the 14th day after exposure to TBI.

FigureA2. A2. Gating Gating strategy strategy for for HSPCs. Figure HSPCs.

Figure A2. Gating strategy for HSPCs.

Figure A3. CE treatment promotes donor cell engraftment in irradiated mice. Mice were daily treated with the vehicle or CE as described in the Materials and Methods. A representative analysis

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Figure A2. Gating strategy for HSPCs.

Figure A3. CE treatment promotes donor cell engraftment in irradiated mice. Mice were daily treated with the vehicle or CE as described in the Materials and Methods. A representative analysis Figure A3. CE CEtreatment treatmentpromotes promotes donor engraftment in irradiated Mice daily weretreated daily Figure A3. donor cellcell engraftment in irradiated mice. mice. Mice were of the percentage of donor cell engraftment in the peripheral blood of recipients by flow cytometry. treated with the vehicle or CE as described in the Materials and Methods. A representative analysis with the vehicle or CE as described in the Materials and Methods. A representative analysis of the of the percentage of donor cell engraftment the peripheral of recipients bycytometry. flow cytometry. percentage of donor cell engraftment in the in peripheral blood blood of recipients by flow (A)

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FigureA4. A4. treatment inhibits IR-induced apoptosis and DNA damage in HSPCs. Micedaily were Figure CECE treatment inhibits IR-induced apoptosis and DNA damage in HSPCs. Mice were daily treated the or vehicle CE as described in the Materials and Early Methods. Early(Annexin apoptosis treated with thewith vehicle CE asor described in the Materials and Methods. apoptosis − ) (A), late apoptosis+ (Annexin +PI−) (A), late V+ PIapoptosis V+MFI PI+ ) of (B)γH2AX and MFI(C) of in γH2AX (C) in c-kit V(Annexin (Annexin V PI+) (B) and c-kit positive cellspositive were cells wereon measured the after 14th day after exposure to TBI; (D) A representative analysis measured the 14thonday exposure to TBI; (D) A representative FACS FACS analysis of theof the percentage of apoptosis in c-kit positive A representative analysis of γH2AX expression percentage of apoptosis in c-kit positive cells;cells; (E) A(E) representative analysis of γH2AX expression in in c-kit positive by flow cytometry; Gating strategy analysis LSKapoptosis. apoptosis.CE CE c-kit positive cellscells by flow cytometry; (F) (F) Gating strategy forfor thethe analysis ofofLSK treatmentsignificantly significantlyreduced reducedthe thepercentage percentageof ofcell cell apoptosis, apoptosis, and decreased the treatment the MFI MFI of ofγH2AX γH2AXin inirradiated irradiatedc-kit c-kitpositive positivecells. cells.All Alldata dataare arepresented presentedasasmeans means±± SEM SEM (n (n == 5).

measured on the 14th day after exposure to TBI; (D) A representative FACS analysis of the treatment significantly reduced the percentage of cell apoptosis, and decreased the MFI of γH2AX percentage of apoptosis in c-kit positive cells; (E) A representative analysis of γH2AX expression in in irradiated c-kit positive cells. All data are presented as means ± SEM (n = 5). c-kit positive cells by flow cytometry; (F) Gating strategy for the analysis of LSK apoptosis. CE treatment significantly reduced the percentage of cell apoptosis, and decreased the MFI of γH2AX in irradiated c-kit positive cells. All data are presented as means ± SEM (n = 5). Int. J. Mol. Sci. 2017, 18, 942 18 of 20

Figure A5. CE scavenges IR-induced ROS in c-kit positive cells. Mice were daily treated with the vehicle or CE as described in the Materials and Methods. The total ROS levels in c-kit positive cells Figure A5. CEasscavenges IR-induced ROS in c-kit positive cells. Mice were daily with the c-kit were presented MFI of DCF. CE treatment significantly decreased the MFItreated in irradiated Figure A5. CE scavenges IR-induced ROS in c-kit positive cells. Mice were daily treated with the vehicle or CE as described in the Materials and Methods. The total ROS levels in c-kit positive cells positive cells. The MFI of DCF, are presented as means ± SEM (n = 5). vehicle CE as described in the and Methods. The total ROSinlevels in c-kit wereor presented as MFI of DCF. CE Materials treatment significantly decreased the MFI irradiated c-kit positive positive cells were presented MFI are of presented DCF. CE astreatment significantly cells. The MFI as of DCF, means ± SEM (n = 5). decreased the MFI in irradiated c-kit positive cells. The MFI of DCF, are presented as means ± SEM (n = 5). (A) (B) (A)

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Figure A6. CE ameliorates IR-induced repression of antioxidant enzyme transcripts in c-kit positive

Figure A6. CE ameliorates IR-induced repression of antioxidant enzyme transcripts in c-kit positive cells. Mice were daily treated with the vehicle or CE as described in the Materials and Methods. cells. Mice were daily treated with the vehicle or CE as described in the Materials and Methods. (A) (A) Relative mRNA expression of SOD1 in c-kit positive cells; (B) Relative mRNA expression of SOD2 Relative mRNA of SOD1 in c-kit positive (B) Relative expression of SOD2 in in c-kit positiveexpression cells; (C)Relative mRNA expression of cells; CAT in c-kit positivemRNA cells; (D) Relative mRNA c-kitexpression positive cells; (C)Relative mRNA expression of CAT in c-kit positive cells; (D) Relative mRNA of GSH-PX in c-kit positive cells. CE treatment significantly increased antioxidant enzyme expression of in GSH-PX in c-kit c-kitpositive positivecells. cells. CErelative treatment significantly increased antioxidant enzyme transcripts irradiated The mRNA expression of SOD2, CAT, and GSH-PX are presented as means ± SEMpositive (n = 3). cells. The relative mRNA expression of SOD2, CAT, and transcripts in irradiated c-kit GSH-PX are presented as means ± SEM (n = 3). References

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Rutin-Enriched Extract from Coriandrum sativum L. Ameliorates Ionizing Radiation-Induced Hematopoietic Injury.

Hematopoietic injury is a major cause of mortality in radiation accidents and a primary side effect in patients undergoing radiotherapy. Ionizing radi...
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