Study On The Effects And The Mechanism Of Epigallocatechin-3-Gallte On Experimental Pancreatic Fibrosis In Rats

BackgroundsFibrosis is the characteristic histopathological feature of chronic pancreatitis and the imbalance between synthesis and degradation of extracellular matrix (ECM) results in the formation of fibrosis. Recent studies display that oxidative stress, pancreatic stellate cells (PSCs) and cytokines are involved in the pancreatic fibrosis. Increasing evidences suggest that oxidative stress and lipid peroxidation have tight relation with pancreatic fibrosis. They not only induce pancreatic cell damage directly, but also can activate PSCs, so promote the fibrotic process. Activated PSCs have been identified as an important cellular source of ECM in pancreatic fibrosis. Transforming growth factor-β₁ (TGF-β₁) is the strongest pro-fibrotic cytokine. It regulates PSCs activation via both paracrine and autocrine manner, and moreover, it efficiently promotes PSCs to synthesize and secrete ECM. TGF-β₁ could impede secretion of matrix metalloproteinase and stimulate secretion of tissue inhibitors of metalloproteinase by PSCs, thus decrease of degradation of ECM. Furthermore, TGF-β₁ can modulate other cytokines. Connective tissue growth factor (CTGF) is a downstream effector of TGF-β₁. Platelet-derived growth factor (PDGF) is considered to be the most effective mitogen. It not only irritates PSCs activation and enhances the migratory capacity of PSCs but also promotes the product of collagen and fibronectin. In conclusion, oxidative stress, activation of PSCs and cytokines are the important mechanisms of pancreatic fibrosis and they interact. Lipid peroxidation activates PSCs, and meanwhile oxygen free radical endlessly attacks pancreatic cells to form chronic injury; activated PSCs secrete ECM and cytokines ( such as TGF-β₁ ) to further the fibrosis; cytokines (such as TGF-β₁) promote the product of ECM and irritate the activation of PSCs via paracrine or autocrine manner to facilitate the formation of fibrosis.Epigallocatechin-3-gallte (EGCG) is an extract from green tea and it has been shown to have the strong activity to eliminate free radical, inhibit lipid peroxidation and strengthen other antioxidants. EGCG has a wide range of pharmacologic action, including anti-tumour effect, hypolipidemic effect, anti-inflammatory and anti-fibrotic effects. Accordingly, we guess that EGCG has the protective effects on pancreatic fibrosis induced by oxidative stress.So far, there was no systemic research in treating pancreatic fibrosis by EGCG. Therefore, we use EGCG treating rat pancreatic fibrosis induced by diethyldithiocarbamate (DDC), and investigate the effects of EGCG on rat pancreatic fibrosis, and explore the mechanisms from oxidative stress, cellular level and molecular level.Objectives1. To investigate the protective effects of EGCG on oxidative stress injury in rat pancreatic tissue by observing the pancreatic histopathological changes and the effects of EGCG on malondialdehyde (MDA) content, copper/zinc superoxide dismutase (CuZn-SOD) and glutathion peroxidase (GSH-PX) activity in rat pancreas.2. To investigate the effects of EGCG on activation of PSCs and explore the mechanism of anti-fibrotic effect of EGCG from cellular level by observing the change of expression ofα-smooth muscle actin (α-SMA) in rat pancreatic tissue and the pancreatic collagen deposition. 3. To evaluate the effects of EGCG on TGF-β₁/Smads signaling pathway and explore the mechanism of anti-fibrotic effect of EGCG from molecular level by observing the changes of protein and mRNA expression of TGF-β₁, Smad3 and Smad7 in rat pancreatic tissue.Methods1. Experimental groups Forty male Wistar rats were randomly divided into five groups of eight rats each, labeled model group, low-dose EGCG group, medium-dose EGCG group, high-dose group and control group. Except control group, other four groups of rats were received intraperitoneal injection of lml/100g body weight of DDC (75mg/ml, diluted in distilled water) twice a week for 10 weeks. From the third week, the low-, medium- and high-dose EGCG groups were given intragastric administration of lml/100g body weight of EGCG (corresponding concentration: 5mg/ml, 10mg/ml and 20mg/ml, diluted in 0.5% sodium carboxymethyl cellulose) once a day for 8 weeks. The rats of control group were received intraperitoneal injections of isodose distilled water twice a week and from the third week rats were received intragastric administration of isodose 0.5% sodium carboxymethyl cellulose once a day. Then, the rats of five groups were sacrificed at the end of 10^th week, and pancreases were immediately excised. Some pancreas samples were immediately embedded in 10% formalin for histological assessment and immunohistochemical examination. The remainder was immediately frozen and stored at - 80°C for molecular analysis.2. Pancreatic tissue samples were used for histopathologic examination by H-E staining.3. Applying the tissue homogenate and spectrophotometric methods to determine the content of MDA and the activity of CuZn-SOD and GSH-PX in rat pancreatic tissue.4. Applying picrosirius red-polarization method to determine the content of collagen in pancreatic tissue, and applying the computer aided video systerm to analyze quantitatively. 5. Applying immunohistochemical staining to determine the protein expression ofα-SMA, TGF-β₁, Smad3 and Smad7, and the results were quantitatively analyzed by the computer aided video systerm.6. Applying the technique of reverse transcription polymerase chain reaction (RT-PCR) to examine the expression of CuZn-SOD,α-SMA, TGF-β₁, Smad3 and Smad7 mRNA in pancreatic tissue, and the results were quantitatively analyzed by the computer aided video systerm..7. Statistical analysis Data were expressed as the mean±S.D., the significance of these differences among five groups was analyzed by means of One-Way ANOVA using SPSS12.0 for windows. p-values less than 0.05 were considered statistically significant.Results1. Sections from rats of control group displayed normal structure and no pathologic changes were detected. In model group, DDC succeeded in inducing pancreatic fibrosis. H-E stained sections of the pancreas showed inflammatory cells infiltration, interstitial edema, and acinar cells atrophy. Microvesicles were observed in acinar and islet cells. In some local areas the acinar vacuolization was extensive. In addition, interlobular space was enlarged and the structure of some lobules was destroyed in pancreatic specimens. Interlobular and intralobular fibrosis was observed. Administration of EGCG resulted in conspicuous attenuation of these pathologic changes. Results showed the vacuolization in pancreas tissues of low-dose EGCG group was decreased compared with that of model group. More interestingly, the histopathology of pancreas tissue in high-dose EGCG group was close to that of control group.2. Compared with control group, the activity of CuZn-SOD and GSH-PX was degraded and the content of MDA was upgraded in rat pancreas of model group. The activity of CuZn-SOD and GSH-PX was notably heighten and the content of MDA was markedly degraded in rat pancreas of different dose EGCG groups, which had significant differences compared with model group (p<0.001), and there was also significant difference between high-dose and low-dose EGCG group (p<0.001).3. The expression of CuZn-SOD mRNA was significantly supressed in rat pancreas of model group compared with that of control group (p<0.001). EGCG enhanced the expression of CuZn-SOD mRNA, and there was also significant difference between every two groups of three EGCG groups (p<0.001).4. In rats of control group, no or only slight collagen was present in pancreas. In contrast, the pancreas of model rats exhibited abundant collagen accumulation. EGCG treated groups showed the content of collgen was remarkably degraded compared with that of model rats (p<0.001). Besides, there was also significant difference between every two groups of three EGCG groups (p<0.001).5.α-SMA-positive cells were found very rare in rat pancreatic tissue of control group, whereas they were observed easily in model group.α-SMA-positive cells were localized in the periacinar fibrotic areas, vascular and duct walls in rats of model group. Compared with model group, the number ofα-SMA-positive cells was decreased in rat pancreatic tissue of three EGCGgroups. The result of quantitative analysis displayed the expression ofα-SMA in three different doses EGCG groups was significantly decreased in comparison with that of model group (p<0.01). Besides, there was also significant difference between low-dose and high-dose EGCG group (p<0.01). The changes of expression ofα-SMA mRNA were similarly to the results of immunohistochemical staining.6. In rat pancreas of control group, the expression of Smad7 protein was abundant, it generally present in cytoplasm of ductal and acinar cells; TGF-β₁ and Smad3 protein was scattered present in a few ductal and acinar cells. In the model group, the protein expression of TGF-β₁ and Smad3 was up-regulated, but the expression of Smad7 was down-regulated in rat pancreatic tissue. Compared with model group, the overexpression of TGF-β₁ and Smad3 was suppressed and the expression of Smad7 was enhanced in rat pancreatic tissue of three EGCG groups. The changes of mRNA expression of TGF-β₁, Smad3 and Smad7 were similarly to the results of immunohistochemical staining.Conclusions1. EGCG effectually ameliorated rat pancreatic fibrosis induced by DDC.2. EGCG could block the oxidative process and suppress lipid peroxidation by inducing the expression of CuZn-SOD mRNA and elevating the activity of CuZn-SOD and GSH-PX, so to lessen the oxidative stress injury induced by DDC.3. EGCG could marked suppress the activation of PSCs, which may be one of mechanism of EGCG treating pancreatic fibrosis.4. TGF-β₁/Smads signaling pathway was activated in the development of pancreatic fibrosis and exerted important role in the pancreatic fibrosis. EGCG had a potent influence on expression of TGF-β₁/Smads: it could not only inhibit the expression of TGF-β₁ directly but also suppress the expression of regenerative Smad3 and enhance the expression of degenerative Smad7 to regulate the balance between them. Therefore EGCG could impede signal transduction of TGF-β₁ and thwart the pro-fibrotic effect of TGF-β₁ in the multilevel.SignificanceThe present study first time applied in vivo experiment to explore the effects and the mechanism of EGCG on rat pancreatic fibrosis in rats from oxidative stress, cellular level and molecular level. Our results demonstrate that EGCG can markedly ameliorate the pathological change of pancreatic tissue and display that EGCG has notable anti-fibrotic effect on rat pancreatic fibrosis induced by DDC. The mechanism may be that EGCG suppresses oxidative stress, inhibits the activation of PSCs, declines the production of TGF-β₁ and impedes TGF-β₁/ Smads signal transduction. The present study provides preliminary evidences that support to apply EGCG to clinical application of anti-fibrosis in chronic pancreatitis patients.