Environmental Toxicology and Pharmacology 40 (2015) 156–163
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Caffeic acid phenethyl ester attenuates ionize radiation-induced intestinal injury through modulation of oxidative stress, apoptosis and p38MAPK in rats Liu-gen Jin a , Jian-Jun Chu b , Qing-feng Pang c , Fu-zheng Zhang b , Gang Wu b , Le-Yuan Zhou b , Xiao-jun Zhang b , Chun-gen Xing a,∗ a
Department of Surgery, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, 215004 Suzhou, China Department of Oncology, The Affiliated Hospital of Jiangnan University, 200 Huihe Road, 214122 Wuxi, China c Wuxi Medical School, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, China b
a r t i c l e
i n f o
Article history: Received 1 March 2015 Received in revised form 26 May 2015 Accepted 26 May 2015 Available online 31 May 2015 Keywords: Caffeic acid phenethyl ester Radiation Intestine Apoptosis p38MAPK
a b s t r a c t Caffeic acid phenyl ester (CAPE) is a potent anti-inflammatory agent and it can eliminate the free radicals. This study aimed to investigate the radioprotective effects of CAPE on X-ray irradiation induced intestinal injury in rats. Rats were intragastrically administered with 10 mol/kg/d CAPE for 7 consecutive days before exposing them to a single dose of X-ray irradiation (9 Gy) to abdomen. Rats were sacrificed 72 h after exposure to radiation. We found that pretreatment with CAPE effectively attenuated intestinal pathology changes, apoptosis, oxidative stress, bacterial translocation, the content of nitric oxide and myeloperoxidase as well as the concentration of plasma tumor necrosis factor-␣. Pretreatment with CAPE also reversed the activation of p38MAPK and the increased expression of intercellular cell adhesion molecule-1 induced by radiation in intestinal mucosa. Taken together, these results suggest that pretreatment with CAPE could be a promising candidate for treating radiation-induced intestinal injury. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Radiation therapy has become one of the most important treatments for human malignancy. However, this therapy affects not only malignant tumors, but also surrounding normal tissues. Radiation injury to the intestinal tissue is among the most significant complications encountered in patients receiving ionizing radiation directed at the abdominal or pelvic cavity (Hakansson and Molin, 2011). It is reported that radiotherapy induces intestinal crypt cell death, disruption of the epithelial barrier, and mucosal inflammation (Hamama et al., 2012, 2014; Hauer-Jensen et al., 2007; Toomey et al., 2006). Therefore, it is of great practical significance to research safe and efficient drugs to protect the body against radiation-induced intestinal damage. Reactive oxygen species (ROS) are produced through electron transport mechanisms during physiological functions such as mitochondrial substrate oxidation and phagocytic activity. They are also produced in response to differently environmental factors and
∗ Corresponding author. Tel.: +86 13812291973. E-mail address: [email protected] (C.-g. Xing). http://dx.doi.org/10.1016/j.etap.2015.05.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
metabolic diseases including ionized and non-ionized electromagnetic radiation (Kayan et al., 2009, 2010). Since ROS play a major role in the initiation and progression of radiation-induced toxicity, antioxidants might offer protection against radiation-induced damage. Many synthetic compounds or natural antioxidants have been investigated as potential radioprotective agents (Nazıro˘glu et al., 2013). However, the inherent toxicity of these agents at the radioprotective doses warrant further search for safer and more effective radioprotectors (Nair and Nair, 2013). Recently, several researches have focused on the potential use of caffeic acid phenethyl ester (CAPE) as free radical scavengers to prevent oxidative damage (Tolba et al., 2014). CAPE is a biologically active ingredient of honeybee propolis. CAPE has properties of anti-virus, anti-inflammation and antioxidation (Banskota et al., 2001; Murtaza et al., 2014). Several reports have showed CAPE had a protective effect on multi-organs damage induced by radiation, such as heart, lung, liver and kidney (Chu et al., 2015; Mansour and Tawfik, 2012; Yildiz et al., 2008). However, it is still unknown that whether CAPE attenuates radiation-induced intestinal injury in rats. Therefore, the aim of this study was to investigate the therapeutic effect of CAPE on radiationinduced intestinal injury and the potential molecular mechanism.
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2. Materials and methods 2.1. Antibodies and chemicals The following reagents were purchased from the indicated sources: CAPE (cat# C8221-1G) and N-acetylcysteine (NAC) was purchased from Sigma Chemical (USA). Rabbit polyclonal antibodies specific for total p38MAPK, p-p38MAPK and intercellular cell adhesion molecule-1 (ICAM-1) were purchased from Boao Sen Biotechnology (Beijing, China). Tumor necrosis factor-␣ (TNF␣) kits and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) detection kit was provided by KeyGen Biotechnology Co., Ltd. (Nanjing, China). Superoxide dismutase (SOD) and nitric oxide (NO) detection kit were from Jiancheng Biological Engineering Institute (Nanjing, China). All other reagents were of analytical grade and obtained commercially. 2.2. Animals and drug administration Male Sprague-Dawley rats (5–6-week-old) rats were supplied by the experiment animal center of Jiangnan University. All animal experiments were started after 1 week of acclimation and performed according to protocols in accordance with institutional guidelines. The rats were individually housed with free access to food and water. The animals were randomly divided into four groups (n = 10). Control group: rats were gavaged with 0.2 ml normal saline once daily for 7 days; IR (9 Gy) group: rats were exposed to a single dose of 9 Gy abdominal irradiation (Morel et al., 2003; Wang et al., 2009); IR + CAPE group: rats were gavaged for 7 consecutive days of 10 mol/kg/d CAPE and a single abdominal irradiation (Cikman et al., 2014); IR + N-acetylcysteine (NAC) group (positive group): rats were gavaged for 7 consecutive days of NAC 200 mg/kg/d and a single abdominal irradiation.
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3 days. After complete polymerization, samples were ready for ultra-microtomy. Semi-thin sections (1 m) were cut using an ultra-microtome (Leica Microsystems, Wetzlar, Germany) and initially stained with toluidine blue for light microscope examination. Selected ultra-thin sections were double-stained with uranylacetate/lead citrate and examined under a transmission electron microscope (JEOL Ltd., Tokyo, Japan).
2.6. Oxidative stress assays The activities of superoxide dismutase (SOD), the levels of nitric oxide (NO), myeloperoxidase (MPO) and methane dicarboxylic aldehyde (MDA) were determined by using the Beckman Coulter DU80 (Beckman, USA) and kits (Nanjin Jiancheng Biotechnology, China). Briefly, intestine tissues were homogenized in RIPA lysis buffer. The mixture was centrifuged at 12,000 × g for the supernatant. The subsequent assay was performed according to the manufacturers’ instructions. The determination of SOD activities was measured at 550 nm wavelengths using the Beckman Coulter DU80. The concentration of NO, MPO and MDA were measured by the absorbance at 550 nm, 460 nm and 532 nm, respectively.
2.7. TUNEL assay Apoptosis in the intestinal mucosa cells was determined by the TUNEL method using an in situ cell detection kit (KeyGen Biotechnology Co., Ltd. Nanjing, China). The fluorescence microscope (Olympus IX51, ×200) was used to observe and photograph specimens. The cells with green fluorescent particles or fragments were deemed to apoptosis cells. Five microscope fields of each section were picked to calculate apoptosis index (AI) by using the formula: AI = account of positive cells/account of total cells × 100%.
2.3. Radiation exposure 2.8. Measurement of serum tumor necrosis factor-˛ (TNF-˛) The rats were anesthetized with 6% chloral hydrate, fixed on a special plexi glass board, and irradiated a single 9 Gy X-ray on abdominal region by 6 MV medical linear accelerator (Varian 2300 CD23EX, USA), at 4.5 Gy/min for 2 min, from the pubic symphysis to the xiphoid, 8.5 cm × 5.5 cm area, and 100 cm source skin distance. Rats were sacrificed 72 h after exposure to radiation. Blood samples were collected for serum separation; segments of small intestine were excised from the proximal part of the jejunum for histological examination. The remaining intestinal tissue were washed with ice-cold isotonic saline and were homogenized in RIPA lysis buffer using homogenizer (Glas-Col, Terre Haute, IN, USA). 2.4. Histological examination The section from the proximal jejunum was fixed in 10% formalin, embedded in paraffin wax, sectioned serially into 4 m thick sections, and stained with hematoxylin and eosin. Tissues were examined under light microscopy by a blinded pathologist and were scored using a system described by Chiu and coworkers (Chiu et al., 1970). 2.5. Transmission electron microscopy The jejunum tissue was cut into small pieces and immersed in Karnowsky solution (4% paraformaldehyde + 1% glutaraldehyde in 0.1 M sodium cacodylate buffer) for 2 h. The specimens were fixed in buffered 1% osmium tetroxide (pH 7.4) and dehydrated in a graded ethanol series. Each specimen was embedded in a mold filled with epoxy resin and kept in an oven at 60 ◦ C for
The full blood specimens were centrifuged at 3000 rpm for 5 min. The suctions of upper layer serum were obtained and the content of TNF-␣ was determined by ELISA using the manufacturer’s protocol (KeyGen Biotechnology Co., Ltd. Nanjing, China). Briefly, a monoclonal antibody specific for TNF-␣ had been precoated onto a microplate. Standards and samples were pipetted into the wells and any TNF-␣ present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TNF-␣ was added to the wells. Following a wash to remove any unbound antibodyenzyme reagent, a substrate solution was added to the wells and color develops in proportion to the amount of TNF-␣ bound in the initial step. The color development was stopped and the intensity of the color is measured.
2.9. Bacterial translocation Spleen and 3 mesenteric lymph nodes were removed and placed in sterile glass bottles containing sterile brain-heart infusion medium. The bottles were re-weighed and tissue homogenates were prepared in 2 ml brain-heart infusion medium. 0.1 ml portion of each homogenate was cultured on blood agar, chocolate agar, eosin methylene blue agar, or Sabouraud-dextrose agar. All agar plates were analyzed after 48 h of incubation at 37 ◦ C. The incidence of bacterial translocation was calculated by determining the number of rats with positive bacterial culture divided by the total number of rats.
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Fig. 1. Histopathological changes in intestine tissues (n = 10). Intestine in the control group showed normal histology (A). In the IR (9 Gy) group, the intestinal villus, mucous membrane, gland and recessus were obviously damaged (B). Both CAPE and NAC administration attenuated intestine damages (C and D). Radiation increased the injury score of intestine tissue while CAPE and NAC decreased the injury score (E). H&E staining, original magnification ×400.
2.10. Immunohistochemistry The intestinal samples had been routinely fixed in 4% neutral formalin and embedded in paraffin blocks. 4 m-thick sections mounted on poly-l-lysine-coated slides were deparaffinized, rehydrated, immersed in 10 mmol/L citrate buffer, pH 6.0, and submitted to heat-induced epitope retrieval using a vapor lock for 45 min. After heating, the slides were cooled to room temperature and were briefly washed with Tris-buffered saline solution. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 5 min and nonspecific sites were blocked with bovine serum albumin for 30 min at room temperature. Sections were then incubated overnight at 4 ◦ C with primary ICAM-1 antibody (diluted 1:100). After washing with PBS, sections were subsequently incubated with biotin-labeled secondary antibodies at 37 ◦ C for 1 h. The immunoreaction was detected using horseradish peroxidase- labeled antibodies at 37 ◦ C for 1 h and visualized with the diaminobenzidine tetrachloride system (brown
color). The images were observed using a microscope (Olympus, Tokyo, Japan). 2.11. Western blotting Total proteins were extracted from samples using protein extracted kid (KG Nanjing Ltd., Nanjing, China) according to the manufacturer’s instructions. The extract was mixed with sample buffer (3:1, v/v) containing 200 mM Tris–HCl (pH 7.6), 8% SDS, 40% glycerol, 40 mM dithiothreitol, boiled them for 10 minutes, and then the mixture were stored at −80 ◦ C. The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in phosphate buffered saline containing 0.1% Tween-20 for 1 h at room temperature, washed in PBST 4 times, following by incubation in a 1:1000 dilution of primary antibody in PBST, overnight at 4 ◦ C. After three washes in PBST, membranes were incubated for 1 h at room temperature in
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Fig. 2. Electron micrographs of rat jejunum. (A) Normal non-irradiated rats showing normal nuclei, mitochondria, rough endoplasmic reticulum and regular arrangement of microvilli with almost uniform diameter. (B) Irradiated rats showing lysis of cytoplasm matrix, degenerated nuclei, swollen mitochondria with rupture of its cristae, dilated rough endoplasmic reticulum and active lysosomes. Microvilli are disorganized and relatively short. (C) Rats pretreated CAPE (10 mol/kg/d) before irradiation with 9 Gy showing normal nuclei, mitochondria, lysosomes, rough endoplasmic reticulum and regular arrangement of microvilli. (D) Rats pretreated NAC showing normal nuclei, mitochondria, lysosomes, rough endoplasmic reticulum and regular arrangement of microvilli with almost uniform diameter.
a 1:5000 dilution of HRP-conjugated secondary antibody. The blot was detected by the enhanced chemiluminescence system according to the recommended procedure. 2.12. Statistical analysis Data were analyzed with SPSS 13.0 software and expressed as mean ± SEM. Differences between groups were calculated by using one-way analysis of variance. Differences were considered to be significant for P values less than 0.05.
D). Radiation increased the injury score of intestine tissue while CAPE and NAC decreased the injury score (Fig. 1E, P < 0.05). The results of electron microscope showed that IR (9 Gy) group had extensive microstructure abnormality, such as shortened intestinal microvilli, swelled chondriosome, vacuoles and crista breaking (Fig. 2B); In the IR + CAPE and IR + NAC groups, theses intestinal structure had kept relatively complete; array had relatively in order and the chondriosome was slight swollen (Fig. 2C and D). 3.2. CAPE rescued radiation-induced intestinal epithelial cells apoptosis
3. Results 3.1. Pretreatment of CAPE attenuated radiation-induced intestinal pathological changes Clinical observations indicate that radiation therapy of abdominal and pelvic malignancies may lead to acute radiation enteropathy (Hauer-Jensen et al., 2014). To explore whether pretreatment of CAPE could attenuate radiation-induced intestinal injury. The sample tissues were examined under light microscopy. The control group had normal mucosal thickness, villus arranged in neat rows (Fig. 1A). However, in the IR (9 Gy) group, we observed that the intestinal villus, mucous membrane, gland and recessus were obviously damaged. Moreover, the submucous membrane had obvious vessel hyperemia expansion accompany with neutrophils accumulate (Fig. 1B). Pretreatment of CAPE or NAC could significantly reduce radiation-induced intestinal lesion (Fig. 1C and
Apoptosis in intestinal mucosa is also triggered by RT (Bonnaud et al., 2010). To determine the ability of CAPE to inhibit radiationinduced intestinal apoptosis, TUNEL assays were done on jejunum sections 3 days after radiation. High amount of apoptotic cells (green staining) was observed within the intestinal epithelial cells from irradiated rats compared with the rats in control group (Fig. 3B). Pretreatment CAPE or NAC inhibited radiation-induced damage by reducing the amount of apoptotic cells within intestinal mucosa cells (Fig. 3C and D). Radiation increased the apoptosis index of intestine tissue while CAPE and NAC decreased this parameter (Fig. 3E, P < 0.05). 3.3. CAPE lessened radiation-induced oxidative stress in intestine To examine whether CAPE has a protective effect against radiation-induced oxidative stress, the levels of MDA and
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Fig. 3. The effect of CAPE on apoptosis induced by radiation in the intestinal tissue. (A) Normal non-irradiated rats. (B) Rats subjected to 9 Gy(R). (C) Rats were gavaged for 7 consecutive days of CAPE 10 mol/kg/d and followed by 9 Gy radiation (CAPE). (D) Rats were gavaged for 7 consecutive days of NAC 200 mg/kg/d and followed by 9 Gy radiation (NAC). (E) Bar graphs show the number of apoptotic cells per crypt in the jejunum sections subjected to TUNEL staining. Values are mean ± SEM (n = 3). # P < 0.05 vs. Control group; * P < 0.05 vs. IR (9 Gy) group.
NO, and the activity of SOD in the intestinal mucosa were measured. Radiation significantly increased NO and MDA levels and decreased the activity of SOD compared with that in the control group (Table 1, P < 0.05). Pretreatment with CAPE or NAC attenuated the increased NO and MDA levels induced by radiation and increased the activities of SOD (P < 0.05).
3.4. Pretreatment of CAPE reversed radiation-induced upregulation of plasma TNF-˛ The levels of TNF-␣ in the IR (9 Gy) group increased almost three folds compared with that of the control group (P < 0.01). However, pretreatment of CAPE or NAC effectively reversed the elevation (Fig. 4).
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Fig. 4. The influence of CAPE on TNF-a level in irradiated-treated rats. Values are expressed as mean ± SEM (n = 10). # P < 0.05 vs. Control group, * P < 0.05 vs. IR (9 Gy) group.
3.5. Pretreatment of CAPE reduced the radiation-induced bacterial translocation The culture results, expressed as the number of rats with positive bacterial culture divided by the total number of rats, were presented in Table 2. No significant difference in peritoneal culture results was observed among IR + CAPE and IR + NAC groups. Comparison of the IR (9 Gy) group with the other groups revealed a significantly higher proportion of positive culture results in the spleen and mesenteric lymph node cultures (P < 0.05). CAPE treatment significantly decreased the proportion of positive mesenteric lymph node and spleen cultures (P < 0.05). 3.6. CAPE-mediated radioprotection might be mediated through the inactive of p38MAPK To further explore the mechanisms which CAPE ameliorated intestinal damage induced by radiation, we detected the levels of pp38MAPK by western blotting. As Fig. 5 shown, immune-reactivity of p-p38MAPK in the radiation group significantly increased, while pretreatment of CAPE or NAC significantly decreased the phosphorylated levels of p38MAPK. In addition, we also observed that ICAM-1 (the downstream effector p38MAPK) were significantly enhanced by radiation and were reduced with pretreatment of CAPE compared to irradiated rats (Fig. 6). 4. Discussion The aim of the present study was to assess the protective effect of CAPE on acute radiation-induced intestinal injury in rats. We found that pretreatment with CAPE attenuated intestinal histopathological changes, apoptosis, oxidative stress, bacterial translocation as
Fig. 5. CAPE inhibited p38 MAPK phosphorylation induced by radiation in the intestinal tissue. (A) Representative banding from triplicate experiments was offered. (B) Densitometric analysis for levels of p38 MAPK phosphorylation protein relative to -actin are presented as mean ± SEM (n = 3). *P < 0.05 vs. other groups.
well as the plasma TNF-␣. Meanwhile, pretreatment with CAPE reversed the activation of p38MAPK and the increased expression of intercellular cell adhesion molecule-1 induced by radiation in intestinal mucosa. Most importantly, the protection of CAPE at low dose (10 mol/kg/d i.e. 2.84 mg/kg/d) was comparable as that of the dose of NAC (a positive control) at the high dose of 100 mg/kg/d according to our present study. High-dose radiation often induced extensive damage to intestine (Langberg et al., 1996). In the radiation group, we observed that the pathological changes were characterized by epithelial barrier breakdown and mucosal inflammation. However, pretreatment of CAPE ameliorated the histological intestinal alterations induced by radiation. Generally, neutrophils accumulate in intestine damaged by radiation, and then release inflammatory cytokines, like TNF-␣, which also have toxic effects in radiation-induced intestinal injury (Moon et al., 2005; Veeraraghavan et al., 2011). In the current study, treatment of rats with X-ray radiation resulted in a 3-fold increased level in TNF-␣. However, pretreatment with CAPE significantly inhibited the increased content of TNF-␣. Meanwhile, CAPE reduced the radiation-induced bacterial translocation. These findings indicated that CAPE efficiently attenuated radiation-induced intestinal injury in rats. Excessive apoptosis is one of the main etiological factors contributing to intestinal injury after radiation exposure (Goo et al., 2013). Similar findings were observed in this study. The results
Table 1 The effect of CAPE on the SOD activity and the content of NO, and MDA in the intestine tissue (n = 10, mean ± SEM). Group
SOD (U/mg tissue)
MDA (nmol/mg tissue)
NO (mol/g tissue)
MPO (U/g tissue)
Control IR (9 Gy) IR + CAPE IR + NAC F value P value
90 ± 14 47 ± 6* 67 ± 9# 63 ± 8# 11.915 0.000
0.43 ± 0.06 0.90 ± 0.14* 0.68 ± 0.07# 0.70 ± 0.08# 15.131 0.000
0.23 ± 0.11 0.56 ± 0.10* 0.37 ± 0.08# 0.36 ± 0.11# 5.208 0.004
0.13 ± 0.06 1.27 ± 0.15* 0.33 ± 0.08# 0.30 ± 0.10# 10.036 0.000
* #
P < 0.01 vs. Control group. P < 0.01 vs. IR (9 Gy) group.
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Fig. 6. Representative immunohistochemical images of the ICAM in the intestinal tissue. (A) Control group; (B) IR (9 Gy) group; (C) IR + CAPE group; (D) IR + NAC group. Original magnification ×400.
of TUNEL demonstrated that radiation increased significantly the number of TUNEL positive cells. Radiation produces reactive oxygen species (ROS), which can lead to the damage of lipids, proteins, nucleic acids, and therefore destroy cancer cells in the treated area. Unfortunately, normal cells in the surrounded area also can be injured (Han et al., 2005). ROS is thought to play a key role in the promotion of apoptosis by affecting mitochondrial permeability, release of cytochrome c, activation of p53 and caspases (Kobashigawa et al., 2015; Yazlovitskaya et al., 2013). Low et al. (2006) demonstrated a dose-dependent intracellular generation of ROS at 1 h post-irradiation, which was thought to be an important triggering factor in the apoptotic process. The fact that ROSinduced oxidative stress was involved in the apoptotic process was evidenced by an increase in the tissue levels of MDA and a corresponding decrease in SOD, as well as an increase in NO. Our results showed that CAPE pretreatment could restore the activity of SOD and scavenge radiation-induced ROS. Radiation induces various cells apoptosis through the activation of various signaling pathways. Important signaling events that regulate the cell death of radiation-damaged cells include: p53dependent pathways (Bertout et al., 2009; Zheng et al., 2005), MAPK protein (Verheij et al., 1996). The p38MAPK pathway plays an
Table 2 Positive microbiological culture results in each group. Group
Control IR (9 Gy) IR + CAPE IR + NAC * #
Spleen culture
0/10 7/10* 4/10# 4/10#
P < 0.05 vs. IR + CAPE and IR + NAC group. P < 0.05 vs. IR (9 Gy) group.
indispensable role in promoting inflammatory responses elicited by DNA damaging stressors such as chemotherapeutics, oxidative stress, and radiation (Zhang et al., 2013; Zhou et al., 2006). In response to radiation, the stress kinase (p38MAPK) is activated by dual phosphorylation at Thr180 and Tyr182 to promote ICAM1 protein expression (Derijard et al., 1995; Hirama et al., 2003; Meineke et al., 2002; Raingeaud et al., 1995; Sun et al., 2001; Yan et al., 2002). The data from this study demonstrated that radiation challenge activated p38MAPK and increased the expression of the downstream effector protein of ICAM-1. However, CAPE pretreatment could reduce the change in signaling pathways induced by radiation.
5. Conclusions This study demonstrates that CAPE has potent radioprotective effects against X-ray radiation-induced intestinal damage in rats. The radio-protective effect of CAPE is probably due to multiple mechanisms involving modulation of oxidative stress, apoptosis, p38MAPK and ICAM-1 protein. Thus, administration of CAPE appears to be an effective and promising method for the prevention of radiation-induced intestinal diseases.
Conflict of interest Mesenteric lymph node culture 1/10 8/10* 3/10# 4/10#
The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
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Acknowledgements This work was supported by the following grant: the National Natural Science Foundation of China (81270126), the Fundamental Research Funds for the Central Universities (JUSRP51412B), the Wuxi hospital management key program (yGzxl 1314) and the Jiangnan university Students Innovation Training (2014317). References Banskota, A.H., Tezuka, Y., Kadota, S., 2001. Recent progress in pharmacological research of propolis. Phytother. Res. 15, 561–571. Bertout, J.A., Majmundar, A.J., Gordan, J.D., Lam, J.C., Ditsworth, D., Keith, B., Brown, E.J., Nathanson, K.L., Simon, M.C., 2009. HIF2alpha inhibition promotes p53 pathway activity, tumor cell death, and radiation responses. Proc. Natl. Acad. Sci. U. S. A. 106, 14391–14396. Bonnaud, S., Niaudet, C., Legoux, F., Corre, I., Delpon, G., Saulquin, X., Fuks, Z., Gaugler, M.H., Kolesnick, R., Paris, F., 2010. Sphingosine-1-phosphate activates the AKT pathway to protect small intestines from radiation-induced endothelial apoptosis. Cancer Res. 70, 9905–9915. Chiu, C.J., Scott, H.J., Gurd, F.N., 1970. Intestinal mucosal lesion in low-flow states. II. The protective effect of intraluminal glucose as energy substrate. Arch. Surg. 101, 484–488. Chu, J., Zhang, X., Jin, L., Chen, J., Du, B., Pang, Q., 2015. Protective effects of caffeic acid phenethyl ester against acute radiation-induced hepatic injury in rats. Environ. Toxicol. Pharmacol. 39, 683–689. Cikman, O., Taysi, S., Gulsen, M.T., Demir, E., Akan, M., Diril, H., Kiraz, H.A., Karaayvaz, M., Tarakcioglu, M., 2014. The radio-protective effects of caffeic acid phenethyl ester and thymoquinone in rats exposed to total head irradiation. Wien. Klin. Wochenschr. 127, 103–108. Derijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J., Davis, R.J., 1995. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267, 682–685. Goo, M.J., Park, J.K., Hong, I.H., Kim, A.Y., Lee, E.M., Lee, E.J., Hwang, M., Jeong, K.S., 2013. Increased susceptibility of radiation-induced intestinal apoptosis in SMP30 KO mice. Int. J. Mol. Sci. 14, 11084–11095. Hakansson, A., Molin, G., 2011. Gut microbiota and inflammation. Nutrients 3, 637–682. Hamama, S., Gilbert-Sirieix, M., Vozenin, M.C., Delanian, S., 2012. Radiation-induced enteropathy: molecular basis of pentoxifylline-vitamin E anti-fibrotic effect involved TGF-beta1 cascade inhibition. Radiother. Oncol. 105, 305–312. Hamama, S., Noman, M.Z., Gervaz, P., Delanian, S., Vozenin, M.C., 2014. MiR-210: a potential therapeutic target against radiation-induced enteropathy. Radiother. Oncol. 111, 219–221. Han, Y., Platonov, A., Akhalaia, M., Yun, Y.S., Song, J.Y., 2005. Differential effect of gamma-radiation-induced heme oxygenase-1 activity in female and male C57BL/6 mice. J. Korean Med. Sci. 20, 535–541. Hauer-Jensen, M., Denham, J.W., Andreyev, H.J., 2014. Radiation enteropathy – pathogenesis, treatment and prevention. Nat. Rev. Gastroenterol. Hepatol. 11, 470–479. Hauer-Jensen, M., Wang, J., Boerma, M., Fu, Q., Denham, J.W., 2007. Radiation damage to the gastrointestinal tract: mechanisms, diagnosis, and management. Curr. Opin. Support. Palliat. Care 1, 23–29. Hirama, T., Tanosaki, S., Kandatsu, S., Kuroiwa, N., Kamada, T., Tsuji, H., Yamada, S., Katoh, H., Yamamoto, N., Tsujii, H., Suzuki, G., Akashi, M., 2003. Initial medical management of patients severely irradiated in the Tokai-mura criticality accident. Br. J. Radiol. 76, 246–253. Kayan, M., Naziro˘glu, M., Celik, O., Yalman, K., Köylü, H., 2009. Vitamin C and E combination modulates oxidative stress induced by X-ray in blood of smoker and nonsmoker radiology technicians. Cell Biochem. Funct. 27, 424–429. Kayan, M., Naziro˘glu, M., Barak, C., 2010. Effects of vitamins C and E combination on element levels in blood of smoker and nonsmoker radiology X-ray technicians. Biol. Trace Elem. Res. 136, 140–148. Kobashigawa, S., Kashino, G., Suzuki, K., Yamashita, S., Mori, H., 2015. Ionizing radiation-induced cell death is partly caused by increase of mitochondrial reactive oxygen species in normal human fibroblast cells. Radiat. Res. 183, 455–464.
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Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Caffeic acid phenethyl ester attenuates ionize radiation-induced intestinal injury through modulation of oxidative stress, apoptosis and p38MAPK in rats Liu-gen Jin a , Jian-Jun Chu b , Qing-feng Pang c , Fu-zheng Zhang b , Gang Wu b , Le-Yuan Zhou b , Xiao-jun Zhang b , Chun-gen Xing a,∗ a
Department of Surgery, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, 215004 Suzhou, China Department of Oncology, The Affiliated Hospital of Jiangnan University, 200 Huihe Road, 214122 Wuxi, China c Wuxi Medical School, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, China b
a r t i c l e
i n f o
Article history: Received 1 March 2015 Received in revised form 26 May 2015 Accepted 26 May 2015 Available online 31 May 2015 Keywords: Caffeic acid phenethyl ester Radiation Intestine Apoptosis p38MAPK
a b s t r a c t Caffeic acid phenyl ester (CAPE) is a potent anti-inflammatory agent and it can eliminate the free radicals. This study aimed to investigate the radioprotective effects of CAPE on X-ray irradiation induced intestinal injury in rats. Rats were intragastrically administered with 10 mol/kg/d CAPE for 7 consecutive days before exposing them to a single dose of X-ray irradiation (9 Gy) to abdomen. Rats were sacrificed 72 h after exposure to radiation. We found that pretreatment with CAPE effectively attenuated intestinal pathology changes, apoptosis, oxidative stress, bacterial translocation, the content of nitric oxide and myeloperoxidase as well as the concentration of plasma tumor necrosis factor-␣. Pretreatment with CAPE also reversed the activation of p38MAPK and the increased expression of intercellular cell adhesion molecule-1 induced by radiation in intestinal mucosa. Taken together, these results suggest that pretreatment with CAPE could be a promising candidate for treating radiation-induced intestinal injury. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Radiation therapy has become one of the most important treatments for human malignancy. However, this therapy affects not only malignant tumors, but also surrounding normal tissues. Radiation injury to the intestinal tissue is among the most significant complications encountered in patients receiving ionizing radiation directed at the abdominal or pelvic cavity (Hakansson and Molin, 2011). It is reported that radiotherapy induces intestinal crypt cell death, disruption of the epithelial barrier, and mucosal inflammation (Hamama et al., 2012, 2014; Hauer-Jensen et al., 2007; Toomey et al., 2006). Therefore, it is of great practical significance to research safe and efficient drugs to protect the body against radiation-induced intestinal damage. Reactive oxygen species (ROS) are produced through electron transport mechanisms during physiological functions such as mitochondrial substrate oxidation and phagocytic activity. They are also produced in response to differently environmental factors and
∗ Corresponding author. Tel.: +86 13812291973. E-mail address: [email protected] (C.-g. Xing). http://dx.doi.org/10.1016/j.etap.2015.05.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
metabolic diseases including ionized and non-ionized electromagnetic radiation (Kayan et al., 2009, 2010). Since ROS play a major role in the initiation and progression of radiation-induced toxicity, antioxidants might offer protection against radiation-induced damage. Many synthetic compounds or natural antioxidants have been investigated as potential radioprotective agents (Nazıro˘glu et al., 2013). However, the inherent toxicity of these agents at the radioprotective doses warrant further search for safer and more effective radioprotectors (Nair and Nair, 2013). Recently, several researches have focused on the potential use of caffeic acid phenethyl ester (CAPE) as free radical scavengers to prevent oxidative damage (Tolba et al., 2014). CAPE is a biologically active ingredient of honeybee propolis. CAPE has properties of anti-virus, anti-inflammation and antioxidation (Banskota et al., 2001; Murtaza et al., 2014). Several reports have showed CAPE had a protective effect on multi-organs damage induced by radiation, such as heart, lung, liver and kidney (Chu et al., 2015; Mansour and Tawfik, 2012; Yildiz et al., 2008). However, it is still unknown that whether CAPE attenuates radiation-induced intestinal injury in rats. Therefore, the aim of this study was to investigate the therapeutic effect of CAPE on radiationinduced intestinal injury and the potential molecular mechanism.
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2. Materials and methods 2.1. Antibodies and chemicals The following reagents were purchased from the indicated sources: CAPE (cat# C8221-1G) and N-acetylcysteine (NAC) was purchased from Sigma Chemical (USA). Rabbit polyclonal antibodies specific for total p38MAPK, p-p38MAPK and intercellular cell adhesion molecule-1 (ICAM-1) were purchased from Boao Sen Biotechnology (Beijing, China). Tumor necrosis factor-␣ (TNF␣) kits and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) detection kit was provided by KeyGen Biotechnology Co., Ltd. (Nanjing, China). Superoxide dismutase (SOD) and nitric oxide (NO) detection kit were from Jiancheng Biological Engineering Institute (Nanjing, China). All other reagents were of analytical grade and obtained commercially. 2.2. Animals and drug administration Male Sprague-Dawley rats (5–6-week-old) rats were supplied by the experiment animal center of Jiangnan University. All animal experiments were started after 1 week of acclimation and performed according to protocols in accordance with institutional guidelines. The rats were individually housed with free access to food and water. The animals were randomly divided into four groups (n = 10). Control group: rats were gavaged with 0.2 ml normal saline once daily for 7 days; IR (9 Gy) group: rats were exposed to a single dose of 9 Gy abdominal irradiation (Morel et al., 2003; Wang et al., 2009); IR + CAPE group: rats were gavaged for 7 consecutive days of 10 mol/kg/d CAPE and a single abdominal irradiation (Cikman et al., 2014); IR + N-acetylcysteine (NAC) group (positive group): rats were gavaged for 7 consecutive days of NAC 200 mg/kg/d and a single abdominal irradiation.
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3 days. After complete polymerization, samples were ready for ultra-microtomy. Semi-thin sections (1 m) were cut using an ultra-microtome (Leica Microsystems, Wetzlar, Germany) and initially stained with toluidine blue for light microscope examination. Selected ultra-thin sections were double-stained with uranylacetate/lead citrate and examined under a transmission electron microscope (JEOL Ltd., Tokyo, Japan).
2.6. Oxidative stress assays The activities of superoxide dismutase (SOD), the levels of nitric oxide (NO), myeloperoxidase (MPO) and methane dicarboxylic aldehyde (MDA) were determined by using the Beckman Coulter DU80 (Beckman, USA) and kits (Nanjin Jiancheng Biotechnology, China). Briefly, intestine tissues were homogenized in RIPA lysis buffer. The mixture was centrifuged at 12,000 × g for the supernatant. The subsequent assay was performed according to the manufacturers’ instructions. The determination of SOD activities was measured at 550 nm wavelengths using the Beckman Coulter DU80. The concentration of NO, MPO and MDA were measured by the absorbance at 550 nm, 460 nm and 532 nm, respectively.
2.7. TUNEL assay Apoptosis in the intestinal mucosa cells was determined by the TUNEL method using an in situ cell detection kit (KeyGen Biotechnology Co., Ltd. Nanjing, China). The fluorescence microscope (Olympus IX51, ×200) was used to observe and photograph specimens. The cells with green fluorescent particles or fragments were deemed to apoptosis cells. Five microscope fields of each section were picked to calculate apoptosis index (AI) by using the formula: AI = account of positive cells/account of total cells × 100%.
2.3. Radiation exposure 2.8. Measurement of serum tumor necrosis factor-˛ (TNF-˛) The rats were anesthetized with 6% chloral hydrate, fixed on a special plexi glass board, and irradiated a single 9 Gy X-ray on abdominal region by 6 MV medical linear accelerator (Varian 2300 CD23EX, USA), at 4.5 Gy/min for 2 min, from the pubic symphysis to the xiphoid, 8.5 cm × 5.5 cm area, and 100 cm source skin distance. Rats were sacrificed 72 h after exposure to radiation. Blood samples were collected for serum separation; segments of small intestine were excised from the proximal part of the jejunum for histological examination. The remaining intestinal tissue were washed with ice-cold isotonic saline and were homogenized in RIPA lysis buffer using homogenizer (Glas-Col, Terre Haute, IN, USA). 2.4. Histological examination The section from the proximal jejunum was fixed in 10% formalin, embedded in paraffin wax, sectioned serially into 4 m thick sections, and stained with hematoxylin and eosin. Tissues were examined under light microscopy by a blinded pathologist and were scored using a system described by Chiu and coworkers (Chiu et al., 1970). 2.5. Transmission electron microscopy The jejunum tissue was cut into small pieces and immersed in Karnowsky solution (4% paraformaldehyde + 1% glutaraldehyde in 0.1 M sodium cacodylate buffer) for 2 h. The specimens were fixed in buffered 1% osmium tetroxide (pH 7.4) and dehydrated in a graded ethanol series. Each specimen was embedded in a mold filled with epoxy resin and kept in an oven at 60 ◦ C for
The full blood specimens were centrifuged at 3000 rpm for 5 min. The suctions of upper layer serum were obtained and the content of TNF-␣ was determined by ELISA using the manufacturer’s protocol (KeyGen Biotechnology Co., Ltd. Nanjing, China). Briefly, a monoclonal antibody specific for TNF-␣ had been precoated onto a microplate. Standards and samples were pipetted into the wells and any TNF-␣ present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TNF-␣ was added to the wells. Following a wash to remove any unbound antibodyenzyme reagent, a substrate solution was added to the wells and color develops in proportion to the amount of TNF-␣ bound in the initial step. The color development was stopped and the intensity of the color is measured.
2.9. Bacterial translocation Spleen and 3 mesenteric lymph nodes were removed and placed in sterile glass bottles containing sterile brain-heart infusion medium. The bottles were re-weighed and tissue homogenates were prepared in 2 ml brain-heart infusion medium. 0.1 ml portion of each homogenate was cultured on blood agar, chocolate agar, eosin methylene blue agar, or Sabouraud-dextrose agar. All agar plates were analyzed after 48 h of incubation at 37 ◦ C. The incidence of bacterial translocation was calculated by determining the number of rats with positive bacterial culture divided by the total number of rats.
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Fig. 1. Histopathological changes in intestine tissues (n = 10). Intestine in the control group showed normal histology (A). In the IR (9 Gy) group, the intestinal villus, mucous membrane, gland and recessus were obviously damaged (B). Both CAPE and NAC administration attenuated intestine damages (C and D). Radiation increased the injury score of intestine tissue while CAPE and NAC decreased the injury score (E). H&E staining, original magnification ×400.
2.10. Immunohistochemistry The intestinal samples had been routinely fixed in 4% neutral formalin and embedded in paraffin blocks. 4 m-thick sections mounted on poly-l-lysine-coated slides were deparaffinized, rehydrated, immersed in 10 mmol/L citrate buffer, pH 6.0, and submitted to heat-induced epitope retrieval using a vapor lock for 45 min. After heating, the slides were cooled to room temperature and were briefly washed with Tris-buffered saline solution. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 5 min and nonspecific sites were blocked with bovine serum albumin for 30 min at room temperature. Sections were then incubated overnight at 4 ◦ C with primary ICAM-1 antibody (diluted 1:100). After washing with PBS, sections were subsequently incubated with biotin-labeled secondary antibodies at 37 ◦ C for 1 h. The immunoreaction was detected using horseradish peroxidase- labeled antibodies at 37 ◦ C for 1 h and visualized with the diaminobenzidine tetrachloride system (brown
color). The images were observed using a microscope (Olympus, Tokyo, Japan). 2.11. Western blotting Total proteins were extracted from samples using protein extracted kid (KG Nanjing Ltd., Nanjing, China) according to the manufacturer’s instructions. The extract was mixed with sample buffer (3:1, v/v) containing 200 mM Tris–HCl (pH 7.6), 8% SDS, 40% glycerol, 40 mM dithiothreitol, boiled them for 10 minutes, and then the mixture were stored at −80 ◦ C. The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in phosphate buffered saline containing 0.1% Tween-20 for 1 h at room temperature, washed in PBST 4 times, following by incubation in a 1:1000 dilution of primary antibody in PBST, overnight at 4 ◦ C. After three washes in PBST, membranes were incubated for 1 h at room temperature in
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Fig. 2. Electron micrographs of rat jejunum. (A) Normal non-irradiated rats showing normal nuclei, mitochondria, rough endoplasmic reticulum and regular arrangement of microvilli with almost uniform diameter. (B) Irradiated rats showing lysis of cytoplasm matrix, degenerated nuclei, swollen mitochondria with rupture of its cristae, dilated rough endoplasmic reticulum and active lysosomes. Microvilli are disorganized and relatively short. (C) Rats pretreated CAPE (10 mol/kg/d) before irradiation with 9 Gy showing normal nuclei, mitochondria, lysosomes, rough endoplasmic reticulum and regular arrangement of microvilli. (D) Rats pretreated NAC showing normal nuclei, mitochondria, lysosomes, rough endoplasmic reticulum and regular arrangement of microvilli with almost uniform diameter.
a 1:5000 dilution of HRP-conjugated secondary antibody. The blot was detected by the enhanced chemiluminescence system according to the recommended procedure. 2.12. Statistical analysis Data were analyzed with SPSS 13.0 software and expressed as mean ± SEM. Differences between groups were calculated by using one-way analysis of variance. Differences were considered to be significant for P values less than 0.05.
D). Radiation increased the injury score of intestine tissue while CAPE and NAC decreased the injury score (Fig. 1E, P < 0.05). The results of electron microscope showed that IR (9 Gy) group had extensive microstructure abnormality, such as shortened intestinal microvilli, swelled chondriosome, vacuoles and crista breaking (Fig. 2B); In the IR + CAPE and IR + NAC groups, theses intestinal structure had kept relatively complete; array had relatively in order and the chondriosome was slight swollen (Fig. 2C and D). 3.2. CAPE rescued radiation-induced intestinal epithelial cells apoptosis
3. Results 3.1. Pretreatment of CAPE attenuated radiation-induced intestinal pathological changes Clinical observations indicate that radiation therapy of abdominal and pelvic malignancies may lead to acute radiation enteropathy (Hauer-Jensen et al., 2014). To explore whether pretreatment of CAPE could attenuate radiation-induced intestinal injury. The sample tissues were examined under light microscopy. The control group had normal mucosal thickness, villus arranged in neat rows (Fig. 1A). However, in the IR (9 Gy) group, we observed that the intestinal villus, mucous membrane, gland and recessus were obviously damaged. Moreover, the submucous membrane had obvious vessel hyperemia expansion accompany with neutrophils accumulate (Fig. 1B). Pretreatment of CAPE or NAC could significantly reduce radiation-induced intestinal lesion (Fig. 1C and
Apoptosis in intestinal mucosa is also triggered by RT (Bonnaud et al., 2010). To determine the ability of CAPE to inhibit radiationinduced intestinal apoptosis, TUNEL assays were done on jejunum sections 3 days after radiation. High amount of apoptotic cells (green staining) was observed within the intestinal epithelial cells from irradiated rats compared with the rats in control group (Fig. 3B). Pretreatment CAPE or NAC inhibited radiation-induced damage by reducing the amount of apoptotic cells within intestinal mucosa cells (Fig. 3C and D). Radiation increased the apoptosis index of intestine tissue while CAPE and NAC decreased this parameter (Fig. 3E, P < 0.05). 3.3. CAPE lessened radiation-induced oxidative stress in intestine To examine whether CAPE has a protective effect against radiation-induced oxidative stress, the levels of MDA and
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Fig. 3. The effect of CAPE on apoptosis induced by radiation in the intestinal tissue. (A) Normal non-irradiated rats. (B) Rats subjected to 9 Gy(R). (C) Rats were gavaged for 7 consecutive days of CAPE 10 mol/kg/d and followed by 9 Gy radiation (CAPE). (D) Rats were gavaged for 7 consecutive days of NAC 200 mg/kg/d and followed by 9 Gy radiation (NAC). (E) Bar graphs show the number of apoptotic cells per crypt in the jejunum sections subjected to TUNEL staining. Values are mean ± SEM (n = 3). # P < 0.05 vs. Control group; * P < 0.05 vs. IR (9 Gy) group.
NO, and the activity of SOD in the intestinal mucosa were measured. Radiation significantly increased NO and MDA levels and decreased the activity of SOD compared with that in the control group (Table 1, P < 0.05). Pretreatment with CAPE or NAC attenuated the increased NO and MDA levels induced by radiation and increased the activities of SOD (P < 0.05).
3.4. Pretreatment of CAPE reversed radiation-induced upregulation of plasma TNF-˛ The levels of TNF-␣ in the IR (9 Gy) group increased almost three folds compared with that of the control group (P < 0.01). However, pretreatment of CAPE or NAC effectively reversed the elevation (Fig. 4).
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Fig. 4. The influence of CAPE on TNF-a level in irradiated-treated rats. Values are expressed as mean ± SEM (n = 10). # P < 0.05 vs. Control group, * P < 0.05 vs. IR (9 Gy) group.
3.5. Pretreatment of CAPE reduced the radiation-induced bacterial translocation The culture results, expressed as the number of rats with positive bacterial culture divided by the total number of rats, were presented in Table 2. No significant difference in peritoneal culture results was observed among IR + CAPE and IR + NAC groups. Comparison of the IR (9 Gy) group with the other groups revealed a significantly higher proportion of positive culture results in the spleen and mesenteric lymph node cultures (P < 0.05). CAPE treatment significantly decreased the proportion of positive mesenteric lymph node and spleen cultures (P < 0.05). 3.6. CAPE-mediated radioprotection might be mediated through the inactive of p38MAPK To further explore the mechanisms which CAPE ameliorated intestinal damage induced by radiation, we detected the levels of pp38MAPK by western blotting. As Fig. 5 shown, immune-reactivity of p-p38MAPK in the radiation group significantly increased, while pretreatment of CAPE or NAC significantly decreased the phosphorylated levels of p38MAPK. In addition, we also observed that ICAM-1 (the downstream effector p38MAPK) were significantly enhanced by radiation and were reduced with pretreatment of CAPE compared to irradiated rats (Fig. 6). 4. Discussion The aim of the present study was to assess the protective effect of CAPE on acute radiation-induced intestinal injury in rats. We found that pretreatment with CAPE attenuated intestinal histopathological changes, apoptosis, oxidative stress, bacterial translocation as
Fig. 5. CAPE inhibited p38 MAPK phosphorylation induced by radiation in the intestinal tissue. (A) Representative banding from triplicate experiments was offered. (B) Densitometric analysis for levels of p38 MAPK phosphorylation protein relative to -actin are presented as mean ± SEM (n = 3). *P < 0.05 vs. other groups.
well as the plasma TNF-␣. Meanwhile, pretreatment with CAPE reversed the activation of p38MAPK and the increased expression of intercellular cell adhesion molecule-1 induced by radiation in intestinal mucosa. Most importantly, the protection of CAPE at low dose (10 mol/kg/d i.e. 2.84 mg/kg/d) was comparable as that of the dose of NAC (a positive control) at the high dose of 100 mg/kg/d according to our present study. High-dose radiation often induced extensive damage to intestine (Langberg et al., 1996). In the radiation group, we observed that the pathological changes were characterized by epithelial barrier breakdown and mucosal inflammation. However, pretreatment of CAPE ameliorated the histological intestinal alterations induced by radiation. Generally, neutrophils accumulate in intestine damaged by radiation, and then release inflammatory cytokines, like TNF-␣, which also have toxic effects in radiation-induced intestinal injury (Moon et al., 2005; Veeraraghavan et al., 2011). In the current study, treatment of rats with X-ray radiation resulted in a 3-fold increased level in TNF-␣. However, pretreatment with CAPE significantly inhibited the increased content of TNF-␣. Meanwhile, CAPE reduced the radiation-induced bacterial translocation. These findings indicated that CAPE efficiently attenuated radiation-induced intestinal injury in rats. Excessive apoptosis is one of the main etiological factors contributing to intestinal injury after radiation exposure (Goo et al., 2013). Similar findings were observed in this study. The results
Table 1 The effect of CAPE on the SOD activity and the content of NO, and MDA in the intestine tissue (n = 10, mean ± SEM). Group
SOD (U/mg tissue)
MDA (nmol/mg tissue)
NO (mol/g tissue)
MPO (U/g tissue)
Control IR (9 Gy) IR + CAPE IR + NAC F value P value
90 ± 14 47 ± 6* 67 ± 9# 63 ± 8# 11.915 0.000
0.43 ± 0.06 0.90 ± 0.14* 0.68 ± 0.07# 0.70 ± 0.08# 15.131 0.000
0.23 ± 0.11 0.56 ± 0.10* 0.37 ± 0.08# 0.36 ± 0.11# 5.208 0.004
0.13 ± 0.06 1.27 ± 0.15* 0.33 ± 0.08# 0.30 ± 0.10# 10.036 0.000
* #
P < 0.01 vs. Control group. P < 0.01 vs. IR (9 Gy) group.
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Fig. 6. Representative immunohistochemical images of the ICAM in the intestinal tissue. (A) Control group; (B) IR (9 Gy) group; (C) IR + CAPE group; (D) IR + NAC group. Original magnification ×400.
of TUNEL demonstrated that radiation increased significantly the number of TUNEL positive cells. Radiation produces reactive oxygen species (ROS), which can lead to the damage of lipids, proteins, nucleic acids, and therefore destroy cancer cells in the treated area. Unfortunately, normal cells in the surrounded area also can be injured (Han et al., 2005). ROS is thought to play a key role in the promotion of apoptosis by affecting mitochondrial permeability, release of cytochrome c, activation of p53 and caspases (Kobashigawa et al., 2015; Yazlovitskaya et al., 2013). Low et al. (2006) demonstrated a dose-dependent intracellular generation of ROS at 1 h post-irradiation, which was thought to be an important triggering factor in the apoptotic process. The fact that ROSinduced oxidative stress was involved in the apoptotic process was evidenced by an increase in the tissue levels of MDA and a corresponding decrease in SOD, as well as an increase in NO. Our results showed that CAPE pretreatment could restore the activity of SOD and scavenge radiation-induced ROS. Radiation induces various cells apoptosis through the activation of various signaling pathways. Important signaling events that regulate the cell death of radiation-damaged cells include: p53dependent pathways (Bertout et al., 2009; Zheng et al., 2005), MAPK protein (Verheij et al., 1996). The p38MAPK pathway plays an
Table 2 Positive microbiological culture results in each group. Group
Control IR (9 Gy) IR + CAPE IR + NAC * #
Spleen culture
0/10 7/10* 4/10# 4/10#
P < 0.05 vs. IR + CAPE and IR + NAC group. P < 0.05 vs. IR (9 Gy) group.
indispensable role in promoting inflammatory responses elicited by DNA damaging stressors such as chemotherapeutics, oxidative stress, and radiation (Zhang et al., 2013; Zhou et al., 2006). In response to radiation, the stress kinase (p38MAPK) is activated by dual phosphorylation at Thr180 and Tyr182 to promote ICAM1 protein expression (Derijard et al., 1995; Hirama et al., 2003; Meineke et al., 2002; Raingeaud et al., 1995; Sun et al., 2001; Yan et al., 2002). The data from this study demonstrated that radiation challenge activated p38MAPK and increased the expression of the downstream effector protein of ICAM-1. However, CAPE pretreatment could reduce the change in signaling pathways induced by radiation.
5. Conclusions This study demonstrates that CAPE has potent radioprotective effects against X-ray radiation-induced intestinal damage in rats. The radio-protective effect of CAPE is probably due to multiple mechanisms involving modulation of oxidative stress, apoptosis, p38MAPK and ICAM-1 protein. Thus, administration of CAPE appears to be an effective and promising method for the prevention of radiation-induced intestinal diseases.
Conflict of interest Mesenteric lymph node culture 1/10 8/10* 3/10# 4/10#
The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
L.-g. Jin et al. / Environmental Toxicology and Pharmacology 40 (2015) 156–163
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