Arch. Pharm. Res. DOI 10.1007/s12272-014-0465-7
RESEARCH ARTICLE
Therapeutic effect of human clonal bone marrow-derived mesenchymal stem cells in severe acute pancreatitis Kyung Hee Jung • TacGhee Yi • Mi Kwon Son Sun U. Song • Soon-Sun Hong
•
Received: 23 June 2014 / Accepted: 30 July 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Severe acute pancreatitis (SAP), a common necroinflammatory disease initiated by the premature activation of digestive enzymes within the pancreatic acinar cells, is associated with significant morbidity and mortality. In this study, we investigated whether human bone marrowderived clonal mesenchymal stem cells (hcMSCs), isolated from human bone marrow aspirate according to our newly established isolation protocol, have potential therapeutic effects in SAP. SAP was induced by three intraperitoneal (i.p.) injections of cerulein (100 lg/kg) and sequential LPS (10 mg/kg) in Sprague-Dawley (SD) rats. hcMSCs (1 9 106/head) were infused on 24 h after LPS injection via the tail vein. The rats were sacrificed 3 days after infusion of hcMSCs. We observed that infused hcMSCs reduced the levels of serum amylase and lipase. Infused hcMSCs ameliorated acinar cell necrosis, pancreatic edema, and inflammatory infiltration. Also, infused hcMSCs decreased the level of malondialdehyde, and increased the levels of glutathione peroxidase and superoxide dismutase. The number of TUNEL positive acinar cells was reduced after hcMSCs infusion. In addition, hcMSCs reduced the expression levels Kyung Hee Jung and TacGhee Yi contributed equally to this work. K. H. Jung T. Yi M. K. Son S.-S. Hong Department of Drug Development, Inha University School of Medicine, 7-241, 3-ga, Sinheung-dong, Jung-Gu, Incheon 400-712, Korea K. H. Jung T. Yi M. K. Son S. U. Song (&) S.-S. Hong (&) Translational Research Center and Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon, Republic of Korea e-mail: [email protected] S.-S. Hong e-mail: [email protected]
of pro-inflammation mediators and cytokines, and increased the expression of SOX9 in SAP. Taken together, hcMSCs could effectively relieve injury of pancreatitis as a promising therapeutics for SAP. Keywords Human clonal bone marrow-derived mesenchymal stem cell (hcMSCs) Severe acute pancreatitis (SAP) Lipase Amylase
Introduction Acute pancreatitis (AP) is clinically a common disease, seriously affecting the human health (Frossard et al. 2008). About 10–20 % of AP patients develop severe acute pancreatitis (SAP), which is associated with poor prognosis and mortality rates as high as 15–25 % (Berezina et al. 2005). SAP is initiated by the premature activation of digestive enzymes within the pancreatic acinar cells, leading to self-digestion of the pancreatic tissue (Shah et al. 2009). During the early phase of SAP, inflammatory responses occur in the ductal cells and lead to systemic inflammatory response. These inflammatory response would be mediated by pro-stimulate the inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukine (IL)-4, and interferon gamma (IFN-c) from the inflammatory cells (Pandol et al. 2007). Long-term inflammation response in pancreatitis finally leads to long distance organ damages and multiple organ dysfunction syndromes (MODS) (Bhatia 2009). The overall mortality of SAP patients with MODS and infectious pancreatic necrosis is as high as 47 % (Chan and Leung 2007). Current conservative therapies, including inhibition of pancreatic enzyme synthesis and secretion, use of antibiotics, and nutritional support, are frequently utilized as clinical
123
K. H. Jung et al.
treatment of SAP (Gong et al. 2014). However, there are no effective strategies for the treatment of SAP, thus far. Therefore, it is needed to develop a novel therapeutic strategy to reduce complications and mortality of SAP. Stem cell based therapies for the repair and regeneration of tissue and organs offer promising therapeutic solutions for various diseases. Mesenchymal stem cells (MSCs) belong to adult stem cells with self-renewal and multilineage differentiation potentials (Friedenstein et al. 1987). They have been used to treat a lot of diseases, including pulmonary injury, acute kidney failure, and myocardial infarction (Humphreys and Bonventre 2008; Williams and Hare 2011). Also, MSCs has been reported to act as immune regulators, which can inhibit inflammatory injury (Najar et al. 2009). All these advantages are beneficial to the cell replacement therapy for the treatment of various inflammatory diseases. Meanwhile, MSCs have been isolated from various sources, such as bone marrow, adult adipose tissue, umbilical cord blood, and neonatal tissues. Among those, the most clinical applications are isolated from the bone marrow. Especially, the capacity of MSCs isolated from various tissues is different according to the method of mononuclear cell fractionation. Indeed, preclinical and clinical studies have reported that cultured, adherent MSCs are heterogeneous and showed various differentiation potential and clinical outcomes (Uccelli et al. 2008; Rosenzweig 2006). A solution to the mixed outcomes from the heterogeneous cell populations is to produce highly homogeneous and well-characterized MSCs. Recently, we have developed a new protocol for the isolation of a homogeneous population of MSCs called the subfraction culturing method (SCM), and established a library of human clonal MSC (hcMSC) lines (Song et al. 2008). In this study, we investigated whether hcMSCs, obtained via SCM, can improve impaired pancreas and have anti-inflammatory effects in SAP.
Materials and methods Animal experiments Sprague-Dawley rats weighing 180–200 g were used for this study. Animal care and all experimental procedures were conducted in accordance to the Guide for Animal Experiments, published by the Korea Academy of Medical Science. SAP was induced by three intraperitoneal injections of cerulein (Sigma-Aldrich, St. Louis, MO, USA), given a total dose of 100 lg/kg body weight at 2 h intervals, with each injection containing 50 % of the dose. The rats then received an intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS) (Sigma), immediately after the third cerulein injection, as previously described (Wang et al. 2012). Afterwards, CM1,10 -dioctadecyl-3,3,30 -tetramethylindo-carbocyanine
123
perchloride (CM-DiI)-labeled hcMSCs (1 9 106) were infused 24 h after the last injection of LPS by the tail vein. Thirty-two rats were randomly divided into four groups; Con (n = 8), Con ? hcMSCs (n = 8), SAP (n = 8), and SAP ? hcMSCs (n = 8). The rats were sacrificed by decapitation 3 days after hcMSCs infusion, and the blood was collected and centrifuged (5009g, 25 min, 4 °C). Pathology Pancreas samples were fixed in 10 % buffered formaldehyde embedded in paraffin and sectioned. The 8 lm-thick sections were stained with hematoxylin and eosin (H&E) for routine histology. For H&E staining, the sections were stained with hematoxylin for 3 min, washed, and stained with 0.5 % eosin for an additional 3 min. After washing with water, the slides were dehydrated in 70, 96, and 100 % ethanol, and then in xylene. To quantify acinar cell injury, 20 randomly chosen microscopic fields were scored as previously described (Van Laethem et al. 1995). In brief, edema was graded from 0 to 3 (0: absent; 1: focally increased between lobules; 2: diffusely increased between lobules; 3: acini disrupted and separated), inflammatory cell infiltration was graded from 0 to 3 (0: absent; 1: in ducts (around ductal margins); 2: in parenchyma, \50 % of the lobules; 3: in parenchyma, [50 % of the lobules), and acinar necrosis was graded from 0 to 3 (0: absent; 1: periductal necrosis, \5 %; 2: focal necrosis, 5–20 %; 3: diffuse parenchymal necrosis, 20–50 %). Determination of activities of amylase, lipase and myeloperoxidase (MPO) Amylase activity was assessed with a commercial kit (Bioassay, Hayward, CA, USA) using cibachron blue-amylose as a chromogenic substrate. The soluble chromogen in 0.1 mL of serum was measured spectrophotometrically at 580 nm. The absorbance was linear to the enzyme activity. Plasma lipase activity was also determined using a commercial kit (Bioassay), following the manufacturer’s instructions. The titrimetric method is based on the degradation of triolein by lipase and the consequent release of diacetylglycerol, which leads to formation of hydrogen peroxide (H2O2). The latter reacts with a leuco dye, which results in the formation of a chromophore that can be measured colorimetrically at 412 nm. Next, sequestration of neutrophils within the pancreas was evaluated by measuring the tissue MPO activity (Lau et al. 2005). Tissue samples were homogenized with 0.5 % hexadecyltrimethyl-ammonium bromide in 50 mM phosphate buffer (pH 6.0). The suspension underwent a freezing-thawing cycle four times, which was further disrupted by sonication (40 s). Then, the sample was centrifuged (10,0009g, 5 min, 4 °C) and MPO activity in the
Human clonal bone marrow-derived mesenchymal stem cells
supernatant was assessed spectrophotometrically at 630 nm, using tetramethylbenzidine as the substrate. The results were corrected in terms of the protein concentration, and expressed as the activity per protein of the tissue (U/mg). Determination of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) The pancreas tissues were washed with phosphate buffered saline (PBS) to remove the blood and homogenized in 50 mM Tris buffer (pH 7.4) for 2 min. Tissue homogenates were used for the measurement of MDA. For GPx assay, tissue homogenates were centrifuged at 7,0009g for 30 min, and then the supernatant was examined. For SOD assay, tissue homogenates were mixed with the same volume of ethanol/chloroform (5:3) and centrifuged at 3,5009g for 20 min; transparent upper layer was then analyzed. MDA assay was performed according to the thiobarbituric acid method (Niehaus and Samuelsson 1968). SOD and GPx levels were measured using a commercial assay kit (Cayman, Michigan, MI). Reverse transcription-polymerase chain reaction (RT-PCR) To test the effect of hcMSCs on inflammatory mediators and cytokines, the total RNA was extracted from the pancreas sample with Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. An aliquot of the total RNA was reverse-transcribed and amplified using reverse transcriptase and Taq DNA polymerase (Promega, Madison, WI, USA), respectively. The PCR product was electrophoresed on a 1.5 % agarose gel. The results were recorded by an imaging system (Kodak Molecular Imaging Systems, New Haven, CT, USA), and bands were quantified using a densitometry. Enzyme-linked immunosorbent assay (ELISA) For the analysis of IFN-c in AP serum, we used rat ELISA kits (R&D Systems). The plates were coated overnight with 2 or 4 lg/mL IFN-c capture monoclonal antibodies (in 0.1 M Na2HPO4 pH 9 buffer) and washed with PBS-Tween 20. A biotin-labeled 1 or 2 lg/mL, anti-IFN-c detecting antibodies were used. The plates were developed using streptavidin-horseradish peroxidase (Vector, Burlingame, CA, USA) and 2, 2-azino-bis substrate (Sigma). Immunohistochemistry Immunostaining was performed on 8 lm-thick sections after deparaffinization. Microwave antigen retrieval was performed in citrate buffer (pH 6.0) for 10 min prior to
peroxidase quenching with 3 % H2O2 in PBS for 10 min. The sections were then washed in water and preblocked with a normal goat or rabbit serum for 10 min. In the primary antibody reaction, slides were incubated for 1 h at room temperature in a 1:100 dilution of the antibodies. The sections were then incubated with biotinylated secondary antibodies (1:500) for 1 h. Following a washing step with PBS, streptavidin-HRP was applied. Finally, the sections were developed with diaminobenzidine tetrahydrochloride substrate for 10 min, and then counterstained with hematoxylin. At least five random fields of each section were examined at a magnification of 2009 and analyzed by a computer image analysis system (Media Cybernetics, Silver Spring, MD, USA). Immunofluorescence The 8 lm-thick pancreas sections were washed twice with PBS and were blocked in blocking solution 1 h at room temperature, and then incubated overnight at 4 °C with primary antibody in a humidified chamber. After washing twice with PBS, the tissues were incubated with rabbit tetramethylrhodamine isothiocyanate (TRITC)-labeled secondary antibody (1:100, Dianova, Germany) for 1 h at room temperature. The tissues were also stained with 4,6-diamidino-2phenylindole (DAPI) to visualize the nuclei. The slides were then washed twice with PBS, and covered with DABCO (Sigma-Aldrich, St. Louis, MO) before confocal laser scanning microscopy was performed (Olympus, Tokyo, Japan). Statistical analysis Data are expressed as the mean ± S.D. Statistical analysis was performed using ANOVA and an unpaired Student0 s t test. A P value of 0.05 or less was considered statistically significant. Statistical calculations were performed using SPSS software for Windows operating system (Version 10.0; SPSS, Chicago, IL).
Results Histological analysis after hcMSCs infusion Pancreatic tissue from the SAP group showed mass edema and inflammation with necrosis compared to the control group. After infusion with hcMSCs, edema formation was reduced about 30 % compared with the SAP group (Fig. 1). Also, inflammatory cell infiltration was marked in the SAP group, whereas the hcMSCs-infused group showed minimal inflammatory cell infiltration. The pathological scores were indicative of a significant difference between the SAP and hcMSCs-infused groups (P \ 0.05 or
123
K. H. Jung et al.
P \ 0.01). In addition, necrosis was prominent in the SAP group, and the increased necrosis was reduced 1.8-fold after hcMSCs infusion. Serum amylase, lipase, and MPO levels after infusion of hcMSCs In this study, serum amylase, lipase, and MPO were quantified to evaluate the severity of pancreatitis. As shown in Fig. 2, serum amylase after hcMSCs infusion decreased about 40 % in SAP. Lipase levels were highly elevated in the SAP group compared with the control group (2.5-fold); whereas, hcMSCs significantly reduced the lipase levels in SAP (1.5-fold; P \ 0.01). Also, MPO is located in the neutrophil azurophilic granules and in monocyte lysosomes; therefore it can be used to measure the extent of tissue infiltration in these cells. In our study MPO activities of the hcMSCs-infused group were decreased 2.0-fold in comparison to the SAP group.
infused group displayed fluorescence. The hcMSCs aloneinfused group without pancreas injury showed a much smaller number of CM-DiI labeled cells than the hcMSCsinfused group with SAP. However, we could not observe any CM-DiI labeled hcMSCs in other organs, such as the heart, kidneys, and lungs, except for the spleen (data not shown). MDA, SOD, and GPx after infusion of hcMSCs Oxidative stress was assayed through the levels of MDA, SOD, and GPx in the pancreas tissue homogenates. MDA, a product of lipid peroxidation, was increased in the SAP group. However, the level of MDA in the hcMSCs-infused group was decreased compared to the SAP group (Fig. 4). The SOD level of the SAP group was significantly lower than those of the control (P \ 0.05); whereas the SOD level of the hcMSCs-infused group was 1.9-fold higher than the SAP group (P \ 0.01). Furthermore, the GPx level in the hcMSCs-infused group showed a 1.5-fold increase compared to the SAP group (P \ 0.05).
In vivo tracking of hcMSCs Effect of hcMSCs on inflammatory response Using a confocal microscopy, we investigated whether hcMSCs migrate to the injured pancreas after infusion. CMDiI was used to label for in vivo cell tracking of hcMSCs. hcMSCs were labeled with CM-DiI with an optimal concentration of 1 lg/L (Fig. 3). Unlabeled cells did not display red fluorescence, while CM-DiI labeled cells in the hcMSCs-
Con
Con+hcMSCs
*
2.5
3.0
Edema
2.5
2.0
We next investigated the effects of hcMSCs infusion on the expression and production of pro-inflammatory mediators that are linked to SAP. The mRNA of TNF-a, IL-1b, iNOS and IL-6 were amplified by RT-PCR. As shown in Fig. 5a, hcMSCs significantly reduced the expression level of pro-
SAP
SAP+hcMSCs
**
3.5
Infiltration
3.0 2.5
1.0
Score
Score
Score
2.0 1.5
** Necrosis
1.5 1.0
2.0 1.5 1.0
0.5
0.5
0.0
0.0
Con Con+hcMSCs SAP SAP+hcMSCs
0.5 0.0
Con Con+hcMSCs SAP SAP+hcMSCs
Fig. 1 Pancreatic pathological findings in rats with SAP. Pancreatic edema, infiltration, and necrosis were highly observed in SAP group, whereas were decreased after fusion of hcMSC in SAP. *P \ 0.05 and **P \ 0.01, compared to SAP group. Con, control;
123
Con Con+hcMSCs SAP SAP+hcMSCs
Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group. Original magnification 9200
Human clonal bone marrow-derived mesenchymal stem cells
**
**
Con
Con+hcMSCs
SAP
Con
SAP+hcMSCs
Con+hcMSCs
SAP
SAP+hcMSCs
**
Con
Con+hcMSCs
Fig. 2 Serum amylase (U/L), lipase (U/L), and myeloperoxidase (U/ mL) after infusion of hcMSCs in SAP. Each value represents the mean ± SD of three separate experiments. **P \ 0.01, compared to
inflammatory cytokines and mediators, including TNF-a, IL-1b, iNOS, and IL-6 in SAP (P \ 0.01). In addition, the anti-inflammatory activity of hcMSCs in SAP was accompanied by suppression of the systemic inflammatory response, showing a decreased production of IFN-c by ELISA (Fig. 5b, P \ 0.01).
SAP
SAP+hcMSCs
SAP group. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group
Con+hcMSCs
SAP+hcMSCs
Effect of hcMSCs on acinar cell apoptosis and macrophage response TUNEL staining showed that there were almost no apoptotic cells in the control group. In the SAP group, massive apoptotic cells were observed in the pancreas. After infusion of hcMSCs, the numbers of apoptotic cells were decreased. Based on the anti-inflammatory effect of hcMSCs, we investigated whether hcMSC suppressed macrophage infiltration into the pancreas. Immunostaining of CD11b, a macrophage marker revealed a marked infiltration of macrophages in the SAP group. However, the number of cells positively stained for CD11b was much lower in the hcMSCs-infused group. Furthermore, we investigated the expression of SOX9, which play an important role in organ maintenance. As shown in Fig. 6, the expression of SOX9 in the SAP group was decreased, whereas that of hcMSCs-infused group was increased.
100 µm
Fig. 3 Tracking of infused hcMSCs. Pancreas sections of CM-DiIlabeled hcMSCs (1 9 106), injected into rats with or without SAP. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group. Original magnification 9200
Discussion SAP is a mortal digestive disease. However, no fully effective treatment is available to date, with the main therapy being only symptomatic treatment. Stem cell therapy shows the possibility of repairing acutely or chronically injured tissues and has the potential to regulate the immune function and reduce inflammatory changes.
123
K. H. Jung et al.
Based on previous findings, we set to explore the role of hcMSCs, obtained from our new protocol in a model of SAP. In this study, we observed that administration of hcMSCs alleviated SAP, presenting pancreatic edema, infiltration of inflammation cells, and necrosis of acinar cells. After systemic infusion of CM-DiI labeled hcMSCs, they were detected more in the pancreas of the SAP group than the normal pancreas group. Also, hcMSCs reduced oxidative stress in SAP. In addition, hcMSCs improved pancreas function by decreasing the expression of inflammatory mediators/cytokines. In this study, we report that hcMSCs improved pancreatic injury, inhibiting oxidative stress and inflammatory responses in an animal with SAP. In the pathological examination and pancreatic enzyme activity, the main pathological findings were pancreatic edema, infiltration of massive inflammatory cells, pancreatic parenchymal bleeding, and acinar cell necrosis. However, hcMSCs inhibited inflammation and acinar cell degeneration in the SAP group (Fig. 1). Also, hcMSCs significantly decreased the activities of amylase, lipase, and MPO (Fig. 2). These findings indicate that hcMSCs infusion facilitates the recovery of pancreatic pathology and exocrinal function. However, the specific mechanisms are still poorly understood. Previous studies have shown that MSCs can migrate into the injured pancreas and repair it (Gong et al. 2014; Jung et al. 2011). Since it is crucial that systemically delivered MSCs reach the site of injury during stem cell therapy, we used CMDiI, a cell tracker, and identified whether CM-DiI-labeled hcMSCs can migrate into the injured pancreas. As expected, we observed that considerable numbers of CM-DiI-labeled hcMSCs migrated into the site of inflammation of the pancreas, compared to the normal pancreas (Fig. 3). These results are consistent with the previous findings showing that MSCs can home into the lung, liver, hind limb, and pancreas after injury (Moodley et al. 2009; Jung et al. 2009; Laurila et al. 2009; Gong et al. 2014). Pancreatic acinar cells are the functional unit for external secretion of the pancreas. SAP is caused by a functional disorder and impairment of the pancreatic acinar cells. During SAP, pro-inflammatory mediators and oxygenderived free radicals may reduce the antioxidative ability of the pancreatic cells, causing tissue hemorrhage, thereby resulting in necrosis of the pancreas (Bhatia et al. 2005; Liu et al. 2009). Accordingly, maintaining the function of pancreatic cells has an important significance in relieving the severity of SAP. Previous studies have reported that MSCs reduce oxygen-derived free radical levels and maintain the stability of membranes (Lanza et al. 2009; Cassatella et al. 2011). Given that MSCs affect oxidative stress, we explored whether hcMSC reduce MDA and increase antioxidant enzymes, protecting free-radical derived oxidative stress. In this study, the MDA levels in the SAP ? hcMSCs group were lower than in the SAP group (Fig. 4). Also, hcMSC
123
increased SOD and GPx levels in the SAP group, indicating that MSCs could impact the oxidative stress level of the injured pancreatic acinar cells, abrogate lipid peroxidation, and improve the scavenging ability of free radicals by increasing antioxidant enzymes. Inflammation plays a crucial role in the pathophysiology of SAP. Several studies have shown that multiple pro-inflammatory cytokines and mediators are involved in the pathogenesis of SAP (de Waal Malefyt et al. 1991; Zyromski and Murr 2003). Among those, TNF-a, IL-1b, and IL-6 as proinflammatory cytokines are mainly produced during SAP (Zyromski and Murr 2003; Norman et al., 1995). Also, IFN-c is reported to be involved in various kinds of inflammatory diseases (Dominguez Rojas 1950; Tagawa et al. 1997; Ishida et al. 2002). In addition, iNOS caused the pathogenesis of human and experimental pancreatitis (Genovese et al. 2006; Wildi et al. 2007; Al-Mufti et al. 1998). Thus, we investigated whether if hcMSCs decrease pro-inflammatory cytokines and mediators, including TNF-a, IL-1b, IL-6, IFN-c, and iNOS in the SAP group. We observed that SAP increased the expression of pro-inflammatory cytokines, such as TNF-a, IL-1b, IL-6, and other inflammatory cytokines and mediators, including IFN-c and iNOS. However, hcMSCs decreased the expression and production of TNF-a, IL-1b, IL-6, IFN-c, and iNOS (Fig. 5). These results are in line with the studies of Meng et al. and Tu et al, which demonstrated that human bone marrow and umbilical cord-derived MSCs attenuated the levels of TNF-a, IL-1b, IL-6, and IFN-c in SAP, respectively (Tu et al. 2012; Meng et al. 2013). Thus, our results suggest that hcMSCs have the potential for the treatment of SAP due to its anti-inflammatory effect. The apoptosis of pancreatic acinar cells is initiated as a response for inflammation related stimulation to the cells (Xiong et al. 2013). Also, the apoptosis and necrosis of pancreatic acinar cells have reciprocal transformation. Thus, we identified hcMSCs-mediated anti-apoptotic effect of acinar cells by TUNEL staining. In the present study, a lot of apoptotic acinar cells were observed in the pancreas of SAP rats. However, hcMSCs reduced significantly the apoptotic acinar cells in SAP (Fig. 6). This result showed that hcMSCs attenuated pancreatitis and inhibited the apoptotic initiation, which prevent further attacks on the acinar cells. Furthermore, hcMSCs increased the expression of SOX9 (Fig. 6), which play a role in organogenesis and the maintenance of adult pancreas as a pancreatic progenitor cell marker (Kawaguchi 2013). In addition to role of SOX9 in organ maintenance, recent studies have reported other functions of SOX9 such as cell survival, homeostasis, cancer development, and inflammation response (Perera et al. 2010; Daisuke et al.; 2011; Janel et al. 2013). Especially, Schaefer et al. and Murakami et al. have reported that expression of SOX9 were inhibited by treatment of pro-inflammatory cytokines such TNF-a and
Human clonal bone marrow-derived mesenchymal stem cells
** SOD (Unit/mg tissue)
MDA (nmol/mg tissue)
*
Con
Con+hcMSCs
SAP
SAP+hcMSCs
Con
Con+hcMSCs
SAP
SAP+hcMSCs
GPx (Unit/mg tissue)
*
Con
SAP+hcMSCs
experiments. *P \ 0.05 and **P \ 0.01, compared to SAP group. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group
Fig. 4 Anti-oxidant effect of hcMSCs on malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in SAP. Each value represents the mean ± SD of three separate
mRNA relative expressions
A 2.5
Con Con+hcMSCs SAP SAP+hcMSCs
** **
2.0
B
600 500
**
**
1.5 1.0 0.5 0.0
IL-1β
TNF-α
IFN-r (pg/ml)
Fig. 5 Inflammatory cytokines and mediators after infusion of hcMSCs in SAP. a mRNA expression levels of TNF-a, iNOS, IL-1b, and IL-6. b IFN-c serum levels after hcMSCs infusion by enzyme-linked immunosorbent assay. Data are the mean ± SD for at least three separate experiments. **P \ 0.01, compared to SAP group. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group
SAP
Con+hcMSCs
IL-6
iNOS
**
400 300 200 100
IL-1b in chondrosarcoma cells and chondrocytes (Schaefer et al. 2003; Murakami et al. 2000; Kolettas et al. 2001). Also, loss of SOX9 has been reported to be associated with
apoptosis or neural crest cells and chondrocytes (Cheung et al. 2005; Daisuke et al. 2011). Considering these previous findings, it is likely that the decrease of SOX9 in SAP
123
K. H. Jung et al.
Con
Con+hcMSCs
SAP
SAP+hcMSCs
TUNEL
CD11b
SOX9 30 µm
Fig. 6 Effect of hcMSCs on acinar cell apoptosis, macrophage response, and cell differentiation in SAP. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group. Original magnification 9200
is occurred by exposure of pro-inflammatory cytokines, leading to apoptosis of acinar cells. After treatment of hcMSCs in SAP, inflammation surroundings were improved and expression of SOX9 was increased, which may partly be involved in apoptotic inhibition of acinar cells. However, to understand relation between SOX9 and SAP is still insufficient and needs further study. In conclusion, our study showed that hcMSC may protect the structural integration of the acinar cells and improve pancreatic pathology. In addition, hcMSC regulated the inflammatory response and attenuated acinar cell apoptosis. Therefore, we suggest that hcMSCs are a promising tool to stem cell-based clinical therapies in various inflammatory diseases, including SAP. Acknowledgments This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Korea (A120266).
References Al-Mufti, R.A., R.C. Williamson, and R.T. Mathie. 1998. Increased nitric oxide activity in a rat model of acute pancreatitis. Gut 43: 564–570.
123
Berezina, T.L., S.B. Zaets, D.J. Mole, Z. Spolarics, E.A. Deitch, and G.W. Machiedo. 2005. Mesenteric lymph duct ligation decreases red blood cell alterations caused by acute pancreatitis. American Journal of Surgery 190: 800–804. Bhatia, M. 2009. Acute pancreatitis as a model of SIRS. Frontiers in Bioscience (Landmark Ed) 14: 2042–2050. Bhatia, M., F.L. Wong, Y. Cao, H.Y. Lau, J. Huang, P. Puneet, and L. Chevali. 2005. Pathophysiology of acute pancreatitis. Pancreatology 5: 132–144. Cassatella, M.A., F. Mosna, A. Micheletti, V. Lisi, N. Tamassia, C. Cont, F. Calzetti, M. Pelletier, G. Pizzolo, and M. Krampera. 2011. Toll-like receptor-3-activated human mesenchymal stromal cells significantly prolong the survival and function of neutrophils. Stem Cells 29: 1001–1011. Chan, Y.C., and P.S. Leung. 2007. Acute pancreatitis: animal models and recent advances in basic research. Pancreas 34: 1–14. Cheung, M., M.C. Chaboissier, A. Mynett, E. Hirst, A. Schedl, and J. Briscoe. 2005. The transcriptional control of trunk neural crest induction, survival, and delamination. Developmental Cell 8: 179–192. Daisuke, I., A. Haruhiko, S. Akira, N. Takashi, N. Toru, Y. Hideki, and T. Noriyuki. 2011. Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways. Development 138: 1507–1519. De Waal, Malefyt R., J. Abrams, B. Bennett, C.G. Figdor, and J.E. De Vries. 1991. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. Journal of Experimental Medicine 174: 1209–1220.
Human clonal bone marrow-derived mesenchymal stem cells Dominguez Rojas, R. 1950. [Amebiasis of infants and children]. Revista Colombiana de Pediatria y Puericultura 10: 5–47. Friedenstein, A.J., R.K. Chailakhyan, and U.V. Gerasimov. 1987. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell and Tissue Kinetics 20: 263–272. Frossard, J.L., M.L. Steer, and C.M. Pastor. 2008. Acute pancreatitis. Lancet 371: 143–152. Genovese, T., E. Mazzon, R. Di Paola, C. Muia, C. Crisafulli, G. Malleo, E. Esposito, and S. Cuzzocrea. 2006. Role of peroxisome proliferator-activated receptor-alpha in acute pancreatitis induced by cerulein. Immunology 118: 559–570. Gong, J., H.B. Meng, J. Hua, Z.S. Song, Z.G. He, B. Zhou, and M.P. Qian. 2014. The SDF-1/CXCR4 axis regulates migration of transplanted bone marrow mesenchymal stem cells towards the pancreas in rats with acute pancreatitis. Molecular Medicine Reports 9(5): 1575–1582. Humphreys, B.D., and J.V. Bonventre. 2008. Mesenchymal stem cells in acute kidney injury. Annual Review of Medicine 59: 311–325. Ishida, Y., T. Kondo, T. Ohshima, H. Fujiwara, Y. Iwakura, and N. Mukaida. 2002. A pivotal involvement of IFN-gamma in the pathogenesis of acetaminophen-induced acute liver injury. FASEB Journal 16: 1227–1236. Jung, K.H., H.P. Shin, S. Lee, Y.J. Lim, S.H. Hwang, H. Han, H.K. Park, J.H. Chung, and S.V. Yim. 2009. Effect of human umbilical cord blood-derived mesenchymal stem cells in a cirrhotic rat model. Liver International 29: 898–909. Jung, K.H., S.U. Song, T. Yi, M.S. Jeon, S.W. Hong, H.M. Zheng, H.S. Lee, M.J. Choi, D.H. Lee, and S.S. Hong. 2011. Human bone marrow-derived clonal mesenchymal stem cells inhibit inflammation and reduce acute pancreatitis in rats. Gastroenterology 140: 998–1008. Kopp, J.L., G. von Figura, E. Mayes, F.F. Liu, C.L. Dubois, J.P. Morris 4th, F.C. Pan, H. Akiyama, C.E. Wright, K. Jensen, M. Hebrok, and M. Sander. 2013. Identification of Sox9-dependent acinar-toductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma. Cancer Cell 22: 1–14. Kawaguchi, Y. 2013. Sox9 and programming of liver and pancreatic progenitors. Journal of Clinical Investigation 123: 1881–1886. Kolettas, E., H.I. Muir, J.C. Barrett, and T.E. Hardingham. 2001. Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor. Rheumatology (Oxford) 40(10): 1146–1156. Lanza, C., S. Morando, A. Voci, L. Canesi, M.C. Principato, L.D. Serpero, G. Mancardi, A. Uccelli, and L. Vergani. 2009. Neuroprotective mesenchymal stem cells are endowed with a potent antioxidant effect in vivo. Journal of Neurochemistry 110: 1674–1684. Lau, H.Y., F.L. Wong, and M. Bhatia. 2005. A key role of neurokinin 1 receptors in acute pancreatitis and associated lung injury. Biochemical and Biophysical Research Communication 327: 509–515. Laurila, J.P., L. Laatikainen, M.D. Castellone, P. Trivedi, J. Heikkila, A. Hinkkanen, P. Hematti, and M.O. Laukkanen. 2009. Human embryonic stem cell-derived mesenchymal stromal cell transplantation in a rat hind limb injury model. Cytotherapy 11: 726–737. Liu, Z.H., J.S. Peng, C.J. Li, Z.L. Yang, J. Xiang, H. Song, X.B. Wu, J.R. Chen, and D.C. Diao. 2009. A simple taurocholate-induced model of severe acute pancreatitis in rats. World Journal of Gastroenterology 15: 5732–5739. Meng, H.B., J. Gong, B. Zhou, J. Hua, L. Yao, and Z.S. Song. 2013. Therapeutic effect of human umbilical cord-derived mesenchymal stem cells in rat severe acute pancreatitis. International Journal of Clinical and Experimental Pathology 6: 2703–2712.
Moodley, Y., D. Atienza, U. Manuelpillai, C.S. Samuel, J. Tchongue, S. Ilancheran, R. Boyd, and A. Trounson. 2009. Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin-induced lung injury. American Journal of Pathology 175: 303–313. Murakami, S., V. Lefebvre, and B. de Crombrugghe. 2000. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. Journal of Biological Chemistry 275(5): 3687–3692. Najar, M., R. Rouas, G. Raicevic, H.I. Boufker, P. Lewalle, N. Meuleman, D. Bron, M. Toungouz, P. Martiat, and L. Lagneaux. 2009. Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6. Cytotherapy 11: 570–583. Niehaus Jr, W.G., and B. Samuelsson. 1968. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. European Journal of Biochemistry 6: 126–130. Norman, J., M. Franz, J. Messina, A. Riker, P.J. Fabri, A.S. Rosemurgy, and W.R. Gower Jr. 1995. Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis. Surgery 117: 648–655. Pandol, S.J., A.K. Saluja, C.W. Imrie, and P.A. Banks. 2007. Acute pancreatitis: bench to the bedside. Gastroenterology 132: 1127–1151. Perera, P.M., E. Wypasek, S. Madhavan, B. Rath-Deschner, J. Liu, J. Nam, B. Rath, Y. Huang, J. Deschner, N. Piesco, C. Wu, and S. Agarwal. 2010. Mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation during inflammation via integrinlinked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes. Arthritis Research and Therapy 12(3): R106. Rosenzweig, A. 2006. Cardiac cell therapy–mixed results from mixed cells. New England Journal of Medicine 355: 1274–1277. Schaefer, J.F., M.L. Millham, B. de Crombrugghe, and L. Buckbinder. 2003. FGF signaling antagonizes cytokine-mediated repression of Sox9 in SW1353 chondrosarcoma cells. Osteoarthritis Cartilage 11(4): 233–241. Shah, A.U., A. Sarwar, A.I. Orabi, S. Gautam, W.M. Grant, A.J. Park, J. Liu, P.K. Mistry, D. Jain, and S.Z. Husain. 2009. Protease activation during in vivo pancreatitis is dependent on calcineurin activation. American Journal of Physiology. Gastrointestinal and Liver Physiology 297: G967–G973. Song, S.U., C.S. Kim, S.P. Yoon, S.K. Kim, M.H. Lee, J.S. Kang, G.S. Choi, S.H. Moon, M.S. Choi, Y.K. Cho, and B.K. Son. 2008. Variations of clonal marrow stem cell lines established from human bone marrow in surface epitopes, differentiation potential, gene expression, and cytokine secretion. Stem Cells and Development 17: 451–461. Tagawa, Y., K. Sekikawa, and Y. Iwakura. 1997. Suppression of concanavalin A-induced hepatitis in IFN-gamma(-/-) mice, but not in TNF-alpha(-/-) mice: role for IFN-gamma in activating apoptosis of hepatocytes. J of Immunology 159: 1418–1428. Tu, X.H., J.X. Song, X.J. Xue, X.W. Guo, Y.X. Ma, Z.Y. Chen, Z.D. Zou, and L. Wang. 2012. Role of bone marrow-derived mesenchymal stem cells in a rat model of severe acute pancreatitis. World Journal of Gastroenterology 18: 2270–2279. Uccelli, A., L. Moretta, and V. Pistoia. 2008. Mesenchymal stem cells in health and disease. Nature Reviews Immunology 8: 726–736. Van Laethem, J.L., A. Marchant, A. Delvaux, M. Goldman, P. Robberecht, T. Velu, and J. Deviere. 1995. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. Gastroenterology 108: 1917–1922. Wang, Y., J. Wang, R. Liu, H. Qi, Y. Wen, F. Sun, and C. Yin. 2012. Severe acute pancreatitis is associated with upregulation of the ACE2-angiotensin-(1-7)-Mas axis and promotes increased circulating angiotensin-(1-7). Pancreatology 12: 451–457.
123
K. H. Jung et al. Wildi, S., J. Kleeff, J. Mayerle, A. Zimmermann, E.P. Bottinger, L. Wakefield, M.W. Buchler, H. Friess, and M. Korc. 2007. Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations. Gut 56: 685–692. Williams, A.R., and J.M. Hare. 2011. Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic
123
implications for cardiac disease. Circulation Research 109: 923–940. Xiong, J., J. Ni, G. Hu, J. Shen, Y. Zhao, L. Yang, G. Yin, C. Chen, G. Yu, Y. Hu, M. Xing, R. Wan, and X. Wang. 2013. Shikonin ameliorates cerulein-induced acute pancreatitis in mice. Journal of Ethnopharmacology 145: 573–580. Zyromski, N., and M.M. Murr. 2003. Evolving concepts in the pathophysiology of acute pancreatitis. Surgery 133: 235–237.
RESEARCH ARTICLE
Therapeutic effect of human clonal bone marrow-derived mesenchymal stem cells in severe acute pancreatitis Kyung Hee Jung • TacGhee Yi • Mi Kwon Son Sun U. Song • Soon-Sun Hong
•
Received: 23 June 2014 / Accepted: 30 July 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Severe acute pancreatitis (SAP), a common necroinflammatory disease initiated by the premature activation of digestive enzymes within the pancreatic acinar cells, is associated with significant morbidity and mortality. In this study, we investigated whether human bone marrowderived clonal mesenchymal stem cells (hcMSCs), isolated from human bone marrow aspirate according to our newly established isolation protocol, have potential therapeutic effects in SAP. SAP was induced by three intraperitoneal (i.p.) injections of cerulein (100 lg/kg) and sequential LPS (10 mg/kg) in Sprague-Dawley (SD) rats. hcMSCs (1 9 106/head) were infused on 24 h after LPS injection via the tail vein. The rats were sacrificed 3 days after infusion of hcMSCs. We observed that infused hcMSCs reduced the levels of serum amylase and lipase. Infused hcMSCs ameliorated acinar cell necrosis, pancreatic edema, and inflammatory infiltration. Also, infused hcMSCs decreased the level of malondialdehyde, and increased the levels of glutathione peroxidase and superoxide dismutase. The number of TUNEL positive acinar cells was reduced after hcMSCs infusion. In addition, hcMSCs reduced the expression levels Kyung Hee Jung and TacGhee Yi contributed equally to this work. K. H. Jung T. Yi M. K. Son S.-S. Hong Department of Drug Development, Inha University School of Medicine, 7-241, 3-ga, Sinheung-dong, Jung-Gu, Incheon 400-712, Korea K. H. Jung T. Yi M. K. Son S. U. Song (&) S.-S. Hong (&) Translational Research Center and Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon, Republic of Korea e-mail: [email protected] S.-S. Hong e-mail: [email protected]
of pro-inflammation mediators and cytokines, and increased the expression of SOX9 in SAP. Taken together, hcMSCs could effectively relieve injury of pancreatitis as a promising therapeutics for SAP. Keywords Human clonal bone marrow-derived mesenchymal stem cell (hcMSCs) Severe acute pancreatitis (SAP) Lipase Amylase
Introduction Acute pancreatitis (AP) is clinically a common disease, seriously affecting the human health (Frossard et al. 2008). About 10–20 % of AP patients develop severe acute pancreatitis (SAP), which is associated with poor prognosis and mortality rates as high as 15–25 % (Berezina et al. 2005). SAP is initiated by the premature activation of digestive enzymes within the pancreatic acinar cells, leading to self-digestion of the pancreatic tissue (Shah et al. 2009). During the early phase of SAP, inflammatory responses occur in the ductal cells and lead to systemic inflammatory response. These inflammatory response would be mediated by pro-stimulate the inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukine (IL)-4, and interferon gamma (IFN-c) from the inflammatory cells (Pandol et al. 2007). Long-term inflammation response in pancreatitis finally leads to long distance organ damages and multiple organ dysfunction syndromes (MODS) (Bhatia 2009). The overall mortality of SAP patients with MODS and infectious pancreatic necrosis is as high as 47 % (Chan and Leung 2007). Current conservative therapies, including inhibition of pancreatic enzyme synthesis and secretion, use of antibiotics, and nutritional support, are frequently utilized as clinical
123
K. H. Jung et al.
treatment of SAP (Gong et al. 2014). However, there are no effective strategies for the treatment of SAP, thus far. Therefore, it is needed to develop a novel therapeutic strategy to reduce complications and mortality of SAP. Stem cell based therapies for the repair and regeneration of tissue and organs offer promising therapeutic solutions for various diseases. Mesenchymal stem cells (MSCs) belong to adult stem cells with self-renewal and multilineage differentiation potentials (Friedenstein et al. 1987). They have been used to treat a lot of diseases, including pulmonary injury, acute kidney failure, and myocardial infarction (Humphreys and Bonventre 2008; Williams and Hare 2011). Also, MSCs has been reported to act as immune regulators, which can inhibit inflammatory injury (Najar et al. 2009). All these advantages are beneficial to the cell replacement therapy for the treatment of various inflammatory diseases. Meanwhile, MSCs have been isolated from various sources, such as bone marrow, adult adipose tissue, umbilical cord blood, and neonatal tissues. Among those, the most clinical applications are isolated from the bone marrow. Especially, the capacity of MSCs isolated from various tissues is different according to the method of mononuclear cell fractionation. Indeed, preclinical and clinical studies have reported that cultured, adherent MSCs are heterogeneous and showed various differentiation potential and clinical outcomes (Uccelli et al. 2008; Rosenzweig 2006). A solution to the mixed outcomes from the heterogeneous cell populations is to produce highly homogeneous and well-characterized MSCs. Recently, we have developed a new protocol for the isolation of a homogeneous population of MSCs called the subfraction culturing method (SCM), and established a library of human clonal MSC (hcMSC) lines (Song et al. 2008). In this study, we investigated whether hcMSCs, obtained via SCM, can improve impaired pancreas and have anti-inflammatory effects in SAP.
Materials and methods Animal experiments Sprague-Dawley rats weighing 180–200 g were used for this study. Animal care and all experimental procedures were conducted in accordance to the Guide for Animal Experiments, published by the Korea Academy of Medical Science. SAP was induced by three intraperitoneal injections of cerulein (Sigma-Aldrich, St. Louis, MO, USA), given a total dose of 100 lg/kg body weight at 2 h intervals, with each injection containing 50 % of the dose. The rats then received an intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS) (Sigma), immediately after the third cerulein injection, as previously described (Wang et al. 2012). Afterwards, CM1,10 -dioctadecyl-3,3,30 -tetramethylindo-carbocyanine
123
perchloride (CM-DiI)-labeled hcMSCs (1 9 106) were infused 24 h after the last injection of LPS by the tail vein. Thirty-two rats were randomly divided into four groups; Con (n = 8), Con ? hcMSCs (n = 8), SAP (n = 8), and SAP ? hcMSCs (n = 8). The rats were sacrificed by decapitation 3 days after hcMSCs infusion, and the blood was collected and centrifuged (5009g, 25 min, 4 °C). Pathology Pancreas samples were fixed in 10 % buffered formaldehyde embedded in paraffin and sectioned. The 8 lm-thick sections were stained with hematoxylin and eosin (H&E) for routine histology. For H&E staining, the sections were stained with hematoxylin for 3 min, washed, and stained with 0.5 % eosin for an additional 3 min. After washing with water, the slides were dehydrated in 70, 96, and 100 % ethanol, and then in xylene. To quantify acinar cell injury, 20 randomly chosen microscopic fields were scored as previously described (Van Laethem et al. 1995). In brief, edema was graded from 0 to 3 (0: absent; 1: focally increased between lobules; 2: diffusely increased between lobules; 3: acini disrupted and separated), inflammatory cell infiltration was graded from 0 to 3 (0: absent; 1: in ducts (around ductal margins); 2: in parenchyma, \50 % of the lobules; 3: in parenchyma, [50 % of the lobules), and acinar necrosis was graded from 0 to 3 (0: absent; 1: periductal necrosis, \5 %; 2: focal necrosis, 5–20 %; 3: diffuse parenchymal necrosis, 20–50 %). Determination of activities of amylase, lipase and myeloperoxidase (MPO) Amylase activity was assessed with a commercial kit (Bioassay, Hayward, CA, USA) using cibachron blue-amylose as a chromogenic substrate. The soluble chromogen in 0.1 mL of serum was measured spectrophotometrically at 580 nm. The absorbance was linear to the enzyme activity. Plasma lipase activity was also determined using a commercial kit (Bioassay), following the manufacturer’s instructions. The titrimetric method is based on the degradation of triolein by lipase and the consequent release of diacetylglycerol, which leads to formation of hydrogen peroxide (H2O2). The latter reacts with a leuco dye, which results in the formation of a chromophore that can be measured colorimetrically at 412 nm. Next, sequestration of neutrophils within the pancreas was evaluated by measuring the tissue MPO activity (Lau et al. 2005). Tissue samples were homogenized with 0.5 % hexadecyltrimethyl-ammonium bromide in 50 mM phosphate buffer (pH 6.0). The suspension underwent a freezing-thawing cycle four times, which was further disrupted by sonication (40 s). Then, the sample was centrifuged (10,0009g, 5 min, 4 °C) and MPO activity in the
Human clonal bone marrow-derived mesenchymal stem cells
supernatant was assessed spectrophotometrically at 630 nm, using tetramethylbenzidine as the substrate. The results were corrected in terms of the protein concentration, and expressed as the activity per protein of the tissue (U/mg). Determination of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) The pancreas tissues were washed with phosphate buffered saline (PBS) to remove the blood and homogenized in 50 mM Tris buffer (pH 7.4) for 2 min. Tissue homogenates were used for the measurement of MDA. For GPx assay, tissue homogenates were centrifuged at 7,0009g for 30 min, and then the supernatant was examined. For SOD assay, tissue homogenates were mixed with the same volume of ethanol/chloroform (5:3) and centrifuged at 3,5009g for 20 min; transparent upper layer was then analyzed. MDA assay was performed according to the thiobarbituric acid method (Niehaus and Samuelsson 1968). SOD and GPx levels were measured using a commercial assay kit (Cayman, Michigan, MI). Reverse transcription-polymerase chain reaction (RT-PCR) To test the effect of hcMSCs on inflammatory mediators and cytokines, the total RNA was extracted from the pancreas sample with Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. An aliquot of the total RNA was reverse-transcribed and amplified using reverse transcriptase and Taq DNA polymerase (Promega, Madison, WI, USA), respectively. The PCR product was electrophoresed on a 1.5 % agarose gel. The results were recorded by an imaging system (Kodak Molecular Imaging Systems, New Haven, CT, USA), and bands were quantified using a densitometry. Enzyme-linked immunosorbent assay (ELISA) For the analysis of IFN-c in AP serum, we used rat ELISA kits (R&D Systems). The plates were coated overnight with 2 or 4 lg/mL IFN-c capture monoclonal antibodies (in 0.1 M Na2HPO4 pH 9 buffer) and washed with PBS-Tween 20. A biotin-labeled 1 or 2 lg/mL, anti-IFN-c detecting antibodies were used. The plates were developed using streptavidin-horseradish peroxidase (Vector, Burlingame, CA, USA) and 2, 2-azino-bis substrate (Sigma). Immunohistochemistry Immunostaining was performed on 8 lm-thick sections after deparaffinization. Microwave antigen retrieval was performed in citrate buffer (pH 6.0) for 10 min prior to
peroxidase quenching with 3 % H2O2 in PBS for 10 min. The sections were then washed in water and preblocked with a normal goat or rabbit serum for 10 min. In the primary antibody reaction, slides were incubated for 1 h at room temperature in a 1:100 dilution of the antibodies. The sections were then incubated with biotinylated secondary antibodies (1:500) for 1 h. Following a washing step with PBS, streptavidin-HRP was applied. Finally, the sections were developed with diaminobenzidine tetrahydrochloride substrate for 10 min, and then counterstained with hematoxylin. At least five random fields of each section were examined at a magnification of 2009 and analyzed by a computer image analysis system (Media Cybernetics, Silver Spring, MD, USA). Immunofluorescence The 8 lm-thick pancreas sections were washed twice with PBS and were blocked in blocking solution 1 h at room temperature, and then incubated overnight at 4 °C with primary antibody in a humidified chamber. After washing twice with PBS, the tissues were incubated with rabbit tetramethylrhodamine isothiocyanate (TRITC)-labeled secondary antibody (1:100, Dianova, Germany) for 1 h at room temperature. The tissues were also stained with 4,6-diamidino-2phenylindole (DAPI) to visualize the nuclei. The slides were then washed twice with PBS, and covered with DABCO (Sigma-Aldrich, St. Louis, MO) before confocal laser scanning microscopy was performed (Olympus, Tokyo, Japan). Statistical analysis Data are expressed as the mean ± S.D. Statistical analysis was performed using ANOVA and an unpaired Student0 s t test. A P value of 0.05 or less was considered statistically significant. Statistical calculations were performed using SPSS software for Windows operating system (Version 10.0; SPSS, Chicago, IL).
Results Histological analysis after hcMSCs infusion Pancreatic tissue from the SAP group showed mass edema and inflammation with necrosis compared to the control group. After infusion with hcMSCs, edema formation was reduced about 30 % compared with the SAP group (Fig. 1). Also, inflammatory cell infiltration was marked in the SAP group, whereas the hcMSCs-infused group showed minimal inflammatory cell infiltration. The pathological scores were indicative of a significant difference between the SAP and hcMSCs-infused groups (P \ 0.05 or
123
K. H. Jung et al.
P \ 0.01). In addition, necrosis was prominent in the SAP group, and the increased necrosis was reduced 1.8-fold after hcMSCs infusion. Serum amylase, lipase, and MPO levels after infusion of hcMSCs In this study, serum amylase, lipase, and MPO were quantified to evaluate the severity of pancreatitis. As shown in Fig. 2, serum amylase after hcMSCs infusion decreased about 40 % in SAP. Lipase levels were highly elevated in the SAP group compared with the control group (2.5-fold); whereas, hcMSCs significantly reduced the lipase levels in SAP (1.5-fold; P \ 0.01). Also, MPO is located in the neutrophil azurophilic granules and in monocyte lysosomes; therefore it can be used to measure the extent of tissue infiltration in these cells. In our study MPO activities of the hcMSCs-infused group were decreased 2.0-fold in comparison to the SAP group.
infused group displayed fluorescence. The hcMSCs aloneinfused group without pancreas injury showed a much smaller number of CM-DiI labeled cells than the hcMSCsinfused group with SAP. However, we could not observe any CM-DiI labeled hcMSCs in other organs, such as the heart, kidneys, and lungs, except for the spleen (data not shown). MDA, SOD, and GPx after infusion of hcMSCs Oxidative stress was assayed through the levels of MDA, SOD, and GPx in the pancreas tissue homogenates. MDA, a product of lipid peroxidation, was increased in the SAP group. However, the level of MDA in the hcMSCs-infused group was decreased compared to the SAP group (Fig. 4). The SOD level of the SAP group was significantly lower than those of the control (P \ 0.05); whereas the SOD level of the hcMSCs-infused group was 1.9-fold higher than the SAP group (P \ 0.01). Furthermore, the GPx level in the hcMSCs-infused group showed a 1.5-fold increase compared to the SAP group (P \ 0.05).
In vivo tracking of hcMSCs Effect of hcMSCs on inflammatory response Using a confocal microscopy, we investigated whether hcMSCs migrate to the injured pancreas after infusion. CMDiI was used to label for in vivo cell tracking of hcMSCs. hcMSCs were labeled with CM-DiI with an optimal concentration of 1 lg/L (Fig. 3). Unlabeled cells did not display red fluorescence, while CM-DiI labeled cells in the hcMSCs-
Con
Con+hcMSCs
*
2.5
3.0
Edema
2.5
2.0
We next investigated the effects of hcMSCs infusion on the expression and production of pro-inflammatory mediators that are linked to SAP. The mRNA of TNF-a, IL-1b, iNOS and IL-6 were amplified by RT-PCR. As shown in Fig. 5a, hcMSCs significantly reduced the expression level of pro-
SAP
SAP+hcMSCs
**
3.5
Infiltration
3.0 2.5
1.0
Score
Score
Score
2.0 1.5
** Necrosis
1.5 1.0
2.0 1.5 1.0
0.5
0.5
0.0
0.0
Con Con+hcMSCs SAP SAP+hcMSCs
0.5 0.0
Con Con+hcMSCs SAP SAP+hcMSCs
Fig. 1 Pancreatic pathological findings in rats with SAP. Pancreatic edema, infiltration, and necrosis were highly observed in SAP group, whereas were decreased after fusion of hcMSC in SAP. *P \ 0.05 and **P \ 0.01, compared to SAP group. Con, control;
123
Con Con+hcMSCs SAP SAP+hcMSCs
Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group. Original magnification 9200
Human clonal bone marrow-derived mesenchymal stem cells
**
**
Con
Con+hcMSCs
SAP
Con
SAP+hcMSCs
Con+hcMSCs
SAP
SAP+hcMSCs
**
Con
Con+hcMSCs
Fig. 2 Serum amylase (U/L), lipase (U/L), and myeloperoxidase (U/ mL) after infusion of hcMSCs in SAP. Each value represents the mean ± SD of three separate experiments. **P \ 0.01, compared to
inflammatory cytokines and mediators, including TNF-a, IL-1b, iNOS, and IL-6 in SAP (P \ 0.01). In addition, the anti-inflammatory activity of hcMSCs in SAP was accompanied by suppression of the systemic inflammatory response, showing a decreased production of IFN-c by ELISA (Fig. 5b, P \ 0.01).
SAP
SAP+hcMSCs
SAP group. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group
Con+hcMSCs
SAP+hcMSCs
Effect of hcMSCs on acinar cell apoptosis and macrophage response TUNEL staining showed that there were almost no apoptotic cells in the control group. In the SAP group, massive apoptotic cells were observed in the pancreas. After infusion of hcMSCs, the numbers of apoptotic cells were decreased. Based on the anti-inflammatory effect of hcMSCs, we investigated whether hcMSC suppressed macrophage infiltration into the pancreas. Immunostaining of CD11b, a macrophage marker revealed a marked infiltration of macrophages in the SAP group. However, the number of cells positively stained for CD11b was much lower in the hcMSCs-infused group. Furthermore, we investigated the expression of SOX9, which play an important role in organ maintenance. As shown in Fig. 6, the expression of SOX9 in the SAP group was decreased, whereas that of hcMSCs-infused group was increased.
100 µm
Fig. 3 Tracking of infused hcMSCs. Pancreas sections of CM-DiIlabeled hcMSCs (1 9 106), injected into rats with or without SAP. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group. Original magnification 9200
Discussion SAP is a mortal digestive disease. However, no fully effective treatment is available to date, with the main therapy being only symptomatic treatment. Stem cell therapy shows the possibility of repairing acutely or chronically injured tissues and has the potential to regulate the immune function and reduce inflammatory changes.
123
K. H. Jung et al.
Based on previous findings, we set to explore the role of hcMSCs, obtained from our new protocol in a model of SAP. In this study, we observed that administration of hcMSCs alleviated SAP, presenting pancreatic edema, infiltration of inflammation cells, and necrosis of acinar cells. After systemic infusion of CM-DiI labeled hcMSCs, they were detected more in the pancreas of the SAP group than the normal pancreas group. Also, hcMSCs reduced oxidative stress in SAP. In addition, hcMSCs improved pancreas function by decreasing the expression of inflammatory mediators/cytokines. In this study, we report that hcMSCs improved pancreatic injury, inhibiting oxidative stress and inflammatory responses in an animal with SAP. In the pathological examination and pancreatic enzyme activity, the main pathological findings were pancreatic edema, infiltration of massive inflammatory cells, pancreatic parenchymal bleeding, and acinar cell necrosis. However, hcMSCs inhibited inflammation and acinar cell degeneration in the SAP group (Fig. 1). Also, hcMSCs significantly decreased the activities of amylase, lipase, and MPO (Fig. 2). These findings indicate that hcMSCs infusion facilitates the recovery of pancreatic pathology and exocrinal function. However, the specific mechanisms are still poorly understood. Previous studies have shown that MSCs can migrate into the injured pancreas and repair it (Gong et al. 2014; Jung et al. 2011). Since it is crucial that systemically delivered MSCs reach the site of injury during stem cell therapy, we used CMDiI, a cell tracker, and identified whether CM-DiI-labeled hcMSCs can migrate into the injured pancreas. As expected, we observed that considerable numbers of CM-DiI-labeled hcMSCs migrated into the site of inflammation of the pancreas, compared to the normal pancreas (Fig. 3). These results are consistent with the previous findings showing that MSCs can home into the lung, liver, hind limb, and pancreas after injury (Moodley et al. 2009; Jung et al. 2009; Laurila et al. 2009; Gong et al. 2014). Pancreatic acinar cells are the functional unit for external secretion of the pancreas. SAP is caused by a functional disorder and impairment of the pancreatic acinar cells. During SAP, pro-inflammatory mediators and oxygenderived free radicals may reduce the antioxidative ability of the pancreatic cells, causing tissue hemorrhage, thereby resulting in necrosis of the pancreas (Bhatia et al. 2005; Liu et al. 2009). Accordingly, maintaining the function of pancreatic cells has an important significance in relieving the severity of SAP. Previous studies have reported that MSCs reduce oxygen-derived free radical levels and maintain the stability of membranes (Lanza et al. 2009; Cassatella et al. 2011). Given that MSCs affect oxidative stress, we explored whether hcMSC reduce MDA and increase antioxidant enzymes, protecting free-radical derived oxidative stress. In this study, the MDA levels in the SAP ? hcMSCs group were lower than in the SAP group (Fig. 4). Also, hcMSC
123
increased SOD and GPx levels in the SAP group, indicating that MSCs could impact the oxidative stress level of the injured pancreatic acinar cells, abrogate lipid peroxidation, and improve the scavenging ability of free radicals by increasing antioxidant enzymes. Inflammation plays a crucial role in the pathophysiology of SAP. Several studies have shown that multiple pro-inflammatory cytokines and mediators are involved in the pathogenesis of SAP (de Waal Malefyt et al. 1991; Zyromski and Murr 2003). Among those, TNF-a, IL-1b, and IL-6 as proinflammatory cytokines are mainly produced during SAP (Zyromski and Murr 2003; Norman et al., 1995). Also, IFN-c is reported to be involved in various kinds of inflammatory diseases (Dominguez Rojas 1950; Tagawa et al. 1997; Ishida et al. 2002). In addition, iNOS caused the pathogenesis of human and experimental pancreatitis (Genovese et al. 2006; Wildi et al. 2007; Al-Mufti et al. 1998). Thus, we investigated whether if hcMSCs decrease pro-inflammatory cytokines and mediators, including TNF-a, IL-1b, IL-6, IFN-c, and iNOS in the SAP group. We observed that SAP increased the expression of pro-inflammatory cytokines, such as TNF-a, IL-1b, IL-6, and other inflammatory cytokines and mediators, including IFN-c and iNOS. However, hcMSCs decreased the expression and production of TNF-a, IL-1b, IL-6, IFN-c, and iNOS (Fig. 5). These results are in line with the studies of Meng et al. and Tu et al, which demonstrated that human bone marrow and umbilical cord-derived MSCs attenuated the levels of TNF-a, IL-1b, IL-6, and IFN-c in SAP, respectively (Tu et al. 2012; Meng et al. 2013). Thus, our results suggest that hcMSCs have the potential for the treatment of SAP due to its anti-inflammatory effect. The apoptosis of pancreatic acinar cells is initiated as a response for inflammation related stimulation to the cells (Xiong et al. 2013). Also, the apoptosis and necrosis of pancreatic acinar cells have reciprocal transformation. Thus, we identified hcMSCs-mediated anti-apoptotic effect of acinar cells by TUNEL staining. In the present study, a lot of apoptotic acinar cells were observed in the pancreas of SAP rats. However, hcMSCs reduced significantly the apoptotic acinar cells in SAP (Fig. 6). This result showed that hcMSCs attenuated pancreatitis and inhibited the apoptotic initiation, which prevent further attacks on the acinar cells. Furthermore, hcMSCs increased the expression of SOX9 (Fig. 6), which play a role in organogenesis and the maintenance of adult pancreas as a pancreatic progenitor cell marker (Kawaguchi 2013). In addition to role of SOX9 in organ maintenance, recent studies have reported other functions of SOX9 such as cell survival, homeostasis, cancer development, and inflammation response (Perera et al. 2010; Daisuke et al.; 2011; Janel et al. 2013). Especially, Schaefer et al. and Murakami et al. have reported that expression of SOX9 were inhibited by treatment of pro-inflammatory cytokines such TNF-a and
Human clonal bone marrow-derived mesenchymal stem cells
** SOD (Unit/mg tissue)
MDA (nmol/mg tissue)
*
Con
Con+hcMSCs
SAP
SAP+hcMSCs
Con
Con+hcMSCs
SAP
SAP+hcMSCs
GPx (Unit/mg tissue)
*
Con
SAP+hcMSCs
experiments. *P \ 0.05 and **P \ 0.01, compared to SAP group. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group
Fig. 4 Anti-oxidant effect of hcMSCs on malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in SAP. Each value represents the mean ± SD of three separate
mRNA relative expressions
A 2.5
Con Con+hcMSCs SAP SAP+hcMSCs
** **
2.0
B
600 500
**
**
1.5 1.0 0.5 0.0
IL-1β
TNF-α
IFN-r (pg/ml)
Fig. 5 Inflammatory cytokines and mediators after infusion of hcMSCs in SAP. a mRNA expression levels of TNF-a, iNOS, IL-1b, and IL-6. b IFN-c serum levels after hcMSCs infusion by enzyme-linked immunosorbent assay. Data are the mean ± SD for at least three separate experiments. **P \ 0.01, compared to SAP group. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group
SAP
Con+hcMSCs
IL-6
iNOS
**
400 300 200 100
IL-1b in chondrosarcoma cells and chondrocytes (Schaefer et al. 2003; Murakami et al. 2000; Kolettas et al. 2001). Also, loss of SOX9 has been reported to be associated with
apoptosis or neural crest cells and chondrocytes (Cheung et al. 2005; Daisuke et al. 2011). Considering these previous findings, it is likely that the decrease of SOX9 in SAP
123
K. H. Jung et al.
Con
Con+hcMSCs
SAP
SAP+hcMSCs
TUNEL
CD11b
SOX9 30 µm
Fig. 6 Effect of hcMSCs on acinar cell apoptosis, macrophage response, and cell differentiation in SAP. Con, control; Con ? hcMSCs, hcMSCs alone-infusion group; SAP, severe AP; SAP ? hcMSCs, hcMSCs infusion in SAP group. Original magnification 9200
is occurred by exposure of pro-inflammatory cytokines, leading to apoptosis of acinar cells. After treatment of hcMSCs in SAP, inflammation surroundings were improved and expression of SOX9 was increased, which may partly be involved in apoptotic inhibition of acinar cells. However, to understand relation between SOX9 and SAP is still insufficient and needs further study. In conclusion, our study showed that hcMSC may protect the structural integration of the acinar cells and improve pancreatic pathology. In addition, hcMSC regulated the inflammatory response and attenuated acinar cell apoptosis. Therefore, we suggest that hcMSCs are a promising tool to stem cell-based clinical therapies in various inflammatory diseases, including SAP. Acknowledgments This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Korea (A120266).
References Al-Mufti, R.A., R.C. Williamson, and R.T. Mathie. 1998. Increased nitric oxide activity in a rat model of acute pancreatitis. Gut 43: 564–570.
123
Berezina, T.L., S.B. Zaets, D.J. Mole, Z. Spolarics, E.A. Deitch, and G.W. Machiedo. 2005. Mesenteric lymph duct ligation decreases red blood cell alterations caused by acute pancreatitis. American Journal of Surgery 190: 800–804. Bhatia, M. 2009. Acute pancreatitis as a model of SIRS. Frontiers in Bioscience (Landmark Ed) 14: 2042–2050. Bhatia, M., F.L. Wong, Y. Cao, H.Y. Lau, J. Huang, P. Puneet, and L. Chevali. 2005. Pathophysiology of acute pancreatitis. Pancreatology 5: 132–144. Cassatella, M.A., F. Mosna, A. Micheletti, V. Lisi, N. Tamassia, C. Cont, F. Calzetti, M. Pelletier, G. Pizzolo, and M. Krampera. 2011. Toll-like receptor-3-activated human mesenchymal stromal cells significantly prolong the survival and function of neutrophils. Stem Cells 29: 1001–1011. Chan, Y.C., and P.S. Leung. 2007. Acute pancreatitis: animal models and recent advances in basic research. Pancreas 34: 1–14. Cheung, M., M.C. Chaboissier, A. Mynett, E. Hirst, A. Schedl, and J. Briscoe. 2005. The transcriptional control of trunk neural crest induction, survival, and delamination. Developmental Cell 8: 179–192. Daisuke, I., A. Haruhiko, S. Akira, N. Takashi, N. Toru, Y. Hideki, and T. Noriyuki. 2011. Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways. Development 138: 1507–1519. De Waal, Malefyt R., J. Abrams, B. Bennett, C.G. Figdor, and J.E. De Vries. 1991. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. Journal of Experimental Medicine 174: 1209–1220.
Human clonal bone marrow-derived mesenchymal stem cells Dominguez Rojas, R. 1950. [Amebiasis of infants and children]. Revista Colombiana de Pediatria y Puericultura 10: 5–47. Friedenstein, A.J., R.K. Chailakhyan, and U.V. Gerasimov. 1987. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell and Tissue Kinetics 20: 263–272. Frossard, J.L., M.L. Steer, and C.M. Pastor. 2008. Acute pancreatitis. Lancet 371: 143–152. Genovese, T., E. Mazzon, R. Di Paola, C. Muia, C. Crisafulli, G. Malleo, E. Esposito, and S. Cuzzocrea. 2006. Role of peroxisome proliferator-activated receptor-alpha in acute pancreatitis induced by cerulein. Immunology 118: 559–570. Gong, J., H.B. Meng, J. Hua, Z.S. Song, Z.G. He, B. Zhou, and M.P. Qian. 2014. The SDF-1/CXCR4 axis regulates migration of transplanted bone marrow mesenchymal stem cells towards the pancreas in rats with acute pancreatitis. Molecular Medicine Reports 9(5): 1575–1582. Humphreys, B.D., and J.V. Bonventre. 2008. Mesenchymal stem cells in acute kidney injury. Annual Review of Medicine 59: 311–325. Ishida, Y., T. Kondo, T. Ohshima, H. Fujiwara, Y. Iwakura, and N. Mukaida. 2002. A pivotal involvement of IFN-gamma in the pathogenesis of acetaminophen-induced acute liver injury. FASEB Journal 16: 1227–1236. Jung, K.H., H.P. Shin, S. Lee, Y.J. Lim, S.H. Hwang, H. Han, H.K. Park, J.H. Chung, and S.V. Yim. 2009. Effect of human umbilical cord blood-derived mesenchymal stem cells in a cirrhotic rat model. Liver International 29: 898–909. Jung, K.H., S.U. Song, T. Yi, M.S. Jeon, S.W. Hong, H.M. Zheng, H.S. Lee, M.J. Choi, D.H. Lee, and S.S. Hong. 2011. Human bone marrow-derived clonal mesenchymal stem cells inhibit inflammation and reduce acute pancreatitis in rats. Gastroenterology 140: 998–1008. Kopp, J.L., G. von Figura, E. Mayes, F.F. Liu, C.L. Dubois, J.P. Morris 4th, F.C. Pan, H. Akiyama, C.E. Wright, K. Jensen, M. Hebrok, and M. Sander. 2013. Identification of Sox9-dependent acinar-toductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma. Cancer Cell 22: 1–14. Kawaguchi, Y. 2013. Sox9 and programming of liver and pancreatic progenitors. Journal of Clinical Investigation 123: 1881–1886. Kolettas, E., H.I. Muir, J.C. Barrett, and T.E. Hardingham. 2001. Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor. Rheumatology (Oxford) 40(10): 1146–1156. Lanza, C., S. Morando, A. Voci, L. Canesi, M.C. Principato, L.D. Serpero, G. Mancardi, A. Uccelli, and L. Vergani. 2009. Neuroprotective mesenchymal stem cells are endowed with a potent antioxidant effect in vivo. Journal of Neurochemistry 110: 1674–1684. Lau, H.Y., F.L. Wong, and M. Bhatia. 2005. A key role of neurokinin 1 receptors in acute pancreatitis and associated lung injury. Biochemical and Biophysical Research Communication 327: 509–515. Laurila, J.P., L. Laatikainen, M.D. Castellone, P. Trivedi, J. Heikkila, A. Hinkkanen, P. Hematti, and M.O. Laukkanen. 2009. Human embryonic stem cell-derived mesenchymal stromal cell transplantation in a rat hind limb injury model. Cytotherapy 11: 726–737. Liu, Z.H., J.S. Peng, C.J. Li, Z.L. Yang, J. Xiang, H. Song, X.B. Wu, J.R. Chen, and D.C. Diao. 2009. A simple taurocholate-induced model of severe acute pancreatitis in rats. World Journal of Gastroenterology 15: 5732–5739. Meng, H.B., J. Gong, B. Zhou, J. Hua, L. Yao, and Z.S. Song. 2013. Therapeutic effect of human umbilical cord-derived mesenchymal stem cells in rat severe acute pancreatitis. International Journal of Clinical and Experimental Pathology 6: 2703–2712.
Moodley, Y., D. Atienza, U. Manuelpillai, C.S. Samuel, J. Tchongue, S. Ilancheran, R. Boyd, and A. Trounson. 2009. Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin-induced lung injury. American Journal of Pathology 175: 303–313. Murakami, S., V. Lefebvre, and B. de Crombrugghe. 2000. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. Journal of Biological Chemistry 275(5): 3687–3692. Najar, M., R. Rouas, G. Raicevic, H.I. Boufker, P. Lewalle, N. Meuleman, D. Bron, M. Toungouz, P. Martiat, and L. Lagneaux. 2009. Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6. Cytotherapy 11: 570–583. Niehaus Jr, W.G., and B. Samuelsson. 1968. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. European Journal of Biochemistry 6: 126–130. Norman, J., M. Franz, J. Messina, A. Riker, P.J. Fabri, A.S. Rosemurgy, and W.R. Gower Jr. 1995. Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis. Surgery 117: 648–655. Pandol, S.J., A.K. Saluja, C.W. Imrie, and P.A. Banks. 2007. Acute pancreatitis: bench to the bedside. Gastroenterology 132: 1127–1151. Perera, P.M., E. Wypasek, S. Madhavan, B. Rath-Deschner, J. Liu, J. Nam, B. Rath, Y. Huang, J. Deschner, N. Piesco, C. Wu, and S. Agarwal. 2010. Mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation during inflammation via integrinlinked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes. Arthritis Research and Therapy 12(3): R106. Rosenzweig, A. 2006. Cardiac cell therapy–mixed results from mixed cells. New England Journal of Medicine 355: 1274–1277. Schaefer, J.F., M.L. Millham, B. de Crombrugghe, and L. Buckbinder. 2003. FGF signaling antagonizes cytokine-mediated repression of Sox9 in SW1353 chondrosarcoma cells. Osteoarthritis Cartilage 11(4): 233–241. Shah, A.U., A. Sarwar, A.I. Orabi, S. Gautam, W.M. Grant, A.J. Park, J. Liu, P.K. Mistry, D. Jain, and S.Z. Husain. 2009. Protease activation during in vivo pancreatitis is dependent on calcineurin activation. American Journal of Physiology. Gastrointestinal and Liver Physiology 297: G967–G973. Song, S.U., C.S. Kim, S.P. Yoon, S.K. Kim, M.H. Lee, J.S. Kang, G.S. Choi, S.H. Moon, M.S. Choi, Y.K. Cho, and B.K. Son. 2008. Variations of clonal marrow stem cell lines established from human bone marrow in surface epitopes, differentiation potential, gene expression, and cytokine secretion. Stem Cells and Development 17: 451–461. Tagawa, Y., K. Sekikawa, and Y. Iwakura. 1997. Suppression of concanavalin A-induced hepatitis in IFN-gamma(-/-) mice, but not in TNF-alpha(-/-) mice: role for IFN-gamma in activating apoptosis of hepatocytes. J of Immunology 159: 1418–1428. Tu, X.H., J.X. Song, X.J. Xue, X.W. Guo, Y.X. Ma, Z.Y. Chen, Z.D. Zou, and L. Wang. 2012. Role of bone marrow-derived mesenchymal stem cells in a rat model of severe acute pancreatitis. World Journal of Gastroenterology 18: 2270–2279. Uccelli, A., L. Moretta, and V. Pistoia. 2008. Mesenchymal stem cells in health and disease. Nature Reviews Immunology 8: 726–736. Van Laethem, J.L., A. Marchant, A. Delvaux, M. Goldman, P. Robberecht, T. Velu, and J. Deviere. 1995. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. Gastroenterology 108: 1917–1922. Wang, Y., J. Wang, R. Liu, H. Qi, Y. Wen, F. Sun, and C. Yin. 2012. Severe acute pancreatitis is associated with upregulation of the ACE2-angiotensin-(1-7)-Mas axis and promotes increased circulating angiotensin-(1-7). Pancreatology 12: 451–457.
123
K. H. Jung et al. Wildi, S., J. Kleeff, J. Mayerle, A. Zimmermann, E.P. Bottinger, L. Wakefield, M.W. Buchler, H. Friess, and M. Korc. 2007. Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations. Gut 56: 685–692. Williams, A.R., and J.M. Hare. 2011. Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic
123
implications for cardiac disease. Circulation Research 109: 923–940. Xiong, J., J. Ni, G. Hu, J. Shen, Y. Zhao, L. Yang, G. Yin, C. Chen, G. Yu, Y. Hu, M. Xing, R. Wan, and X. Wang. 2013. Shikonin ameliorates cerulein-induced acute pancreatitis in mice. Journal of Ethnopharmacology 145: 573–580. Zyromski, N., and M.M. Murr. 2003. Evolving concepts in the pathophysiology of acute pancreatitis. Surgery 133: 235–237.
