Effects Of Telmisartan Ameliorating Chronic Pancreatitis In Rats And The Study Of Its Mechanism

Chronic pancreatitis(CP) is a fibroinflammatory process of the pancreatic tissue that multiple factors are involved in. Histological changes include periductal, intra-acinar and inter-acinar fibrosis with destruction of exocrine parenchyma and acinar and ductular distention. Activated pancreatic stellate cells (PSCs) is a characteristic feature of CP, lead-ing to the development of pancreatic fibrosis. The activation and cell functions in PSCs are regulated by the coordinated activation of intracellular signaling pathways and signaling molecules, such as peroxisome proliferator-activated receptor-γ(PPAR-γ), extracellular signal-regulated kinase (ERK) and Smads proteins pathways, whose stimulants are ligands of PPAR-γ, angiotensinⅡ(AngⅡ) and transforming growth factor-β1(TGF-β1). TGF-β1 is a multifunctional cytokine that also plays an important role in progressive pancreatic fibr-osis, efficiently promoting extracellular matrix (ECM) synthesis and stimulating PSCs activation.PPARs are members of subfamily of nuclear hormone receptors and ligand-activated transcription factors, including PPAR-a,PPAR-β,PPAR-d and PPAR-γ. PPAR-γis the most extensively studied of the 3 PPAR subtypes to date, involving in inflammatory res-ponse, lipid synthesis and glycometabolism, as well as differentiation, proliferation and apoptosis of many kinds of cells. PPAR-y exert antiinflammatory effects both at the cellul-ar level and the level of the organism by suppressing gene expression of inflammatory response. In the model of female C57BL/6 mice with chronic pancreatitis induced by repeated intraperitoneal injections of the cholecystokinin analogue cerulein, PPAR-y agonists troglitazone suppressed the activation of PSCs induced by TGF-β1 and prevented the development of pancreatic fibrosis both in earlier and advanced stage.The renin-angiotensin system (RAS) is classically known as a circulating or hormo-nal system regulating blood pressure, electrolyte and fluid homeostasis. These functional local RASs have been found in such diverse organ systems. Such local RASs operate in an autocrine, paracrine and/or intracrine manner and exhibit multiple physiological effects at the cellular level which adds to, and/or differs from, the circulating RAS. Local RASs exist in major cell types of the endocrine and exocrine pancreas, the activities of which are sub-jected to regulation by physiological and pathophysiological stimuli. The biological actions of the RAS are mediated by the angiotensinⅡtype 1 and type 2 receptors(AT1R and AT-2R); most of the functions are, however, mediated by the AT1R. These functional RASs have important roles in the regulation of islet, acinar and duct cell secretion and their activation is involved in AT1R-mediated oxidative stress-induced cell inflammation, apoptosis and fibrosis. Interference with such a local RAS may be promising in the prevention and treatment of pancreatic inflammation and disease. The fundamental effect of blocking the RAS is anti-inflammatory in nature. Candesartan alleviates chronic pan-creatitis and fibrosis by suppressing the overexpression of TGF-β1, resulting in prevention of activation of pancreatic stellate cells in male WBN/Kob rats.Telmisartan(Tel) is a unique AT1R blocker and partial agonist of PPAR-γ. Theoreti-cally speaking, it may preferably prevent chronic inflammation and fibrotic disease throgh its dual actions. Privous study reported that Tel inhibited chemokineinduced migration of CD4-positive lymphocytes by reducing PI3K activity with subsequent inhibition of F-actin formation and ICAM3 translocation. These effects of Tel on cell migration are mediated by PPAR-γand independent of the AT1R. Meanwhile,in vitro, Tel inhibited AT1R gene expression through PPARy activation.. The dual inhibition of angiotensinⅡfunction by Tel-AT1R blockade and downregulation-would contribute to more complete inhibition of the renin-angiotensin system.The aim of the present study is to identify the anti-inflammatory and anti-fibrotic effects of Tel in experimental CP. Furthermore, relative mechanisms of these effects were studied, providing the rationale to clinic treatment of CP.1,To establish the model of CP in Wistar ratsObjective To establish the model of CP in Wistar rats, treatment with Tel on CP is based on this model and observe expressions of a-SMA and PPAR-γpotein during the course of pancreatic fibrosis in Wistar rats. Methods Dibutyltin Dichloride (DBTC) was dissolved first in 100% ehanol (two parts) and then mixed with glycerol (three parts). DBTC (8 mg/kg body weight) in a volume of 200 mL solvent was injected into the tail vein. Wistar rats were randomly divided into seven groups according to weights. In the control group, animals were treated only with the solvent. At days 1,3,5,7,14,21 and 28 after application of DBTC or solvent, several indexes were observed. According to pancreatic tissue samples stained with hematoxylin and eosin(HE), pathologic changes were assessed in a blinded manner. Collagen accumulation in pancreatic sections was determined by staining for Sirius Red, quantitated with image analysis. Activity of amylase, alanine aminotransferase and alkaline phosphatase in blood was assayed using an full automatic biochemical analysor. Pancreatic myeloperoxidase activity was determined using the spectrophotometer. Expressions of a-SMA and PPAR-γprotein were assessed in the Envision immunohistochemical method. Result Light microscopy showed signs of a acute pancreatitis with interstitial edema and infiltration of neutrophilic granulocytes lweek after DBTC injection. Extensive infiltration with neutronphilic granulocytes, mononuclear cells, and an elevated number of fibroblasts could be observed starting at day 7. The acini seem-ed dilated, and scattered acinar cell necrosis was found. Beginning deposition of connec-tive tissue was observed at day 14 with a predominance of periductal areas spreading dur-ing the following time into the interstitium. At the end of our observation period 6 weeks after DBTC application, the pancreatic tissue was characterized by an extended interstitial fibrosis with infiltrating mononuclear cells. The islets were not involved in the fibrotic pro-cess. In control rats that were administered only the solvent, no histological changes were observed during the whole observation period. Contents of collagen fibers stained for Sirius Red in the pancreatic tissue reached the peak value 1 week after DBTC injection, maintaining a high-er level 2 weeks after DBTC injection. Collagen fibers accumulated from periductal to intra-acinar and/or inter-acinar areas. Activity of amylase in blood reached the peak value 3 to 5 days after DBTC injection, gradually decreasing 1 week after DBTC injection. Acti-vity of alanine aminotransferase in blood reached the peak value respectively 1 and 14 days after DBTC injection, gradually decreasing 2 weeks after DBTC injection. Activity of alkaline phosphatase in blood reached the peak value respectively 3 and 14 days after DBTC injection, gradually decreasing 2 weeks after DBTC injection. Pancreatic myeloper-oxidase activity reached the peak value 1 and 5 days after DBTC injection, maintaining a slightly high level 1 week after DBTC injection. In control rats, contents of collagen fibers, serum parameters and pancreatic myeloperoxidase activity were in the normal range. During the course of pancreatic fibrosis in rats, the expression of a-SMA protein initially located in periductal areas, gradually involving intra-acinar and/or interacinar areas. The expression of a-SMA increased significantly in 1 week, maintaining higher levels 2 weeks after DBTC injection. The kind of change was consistent with that of collagen content. In control rats, the expression of a-SMA only located in vessel wall. The expression of PPAR-y protein was positive 3 days after DBTC injection and gradually strong during the course of pancreatic fibrosis in rats. In control rats, the expression of PPAR-y protein was negative. Conclusion The model of CP in Wistar rat was established according to pathologic changes, contents of collagen fibers and myeloperoxidase activity in pancreatic tissue. The expres-sion of a-SMA protein may show when PSCs were activated, how many PSCs were active-ated and why treating CP in rats with Tel was at two time points. During the course of pancreatic fibrosis in rats, the expression of PPAR-y protein is gradually strong, associated with pathogenetic condition.2,To observe effects of Tel ameliorating CP in rats and approach its mechanismObjective Fourty-two animals were randomly divided into the control group, Tel control group, CP group, CP with Tel (earlier stage/large dose) group, CP with Tel (earlier stage/small dose) group, CP with Tel (advanced stage/large dose) group and CP with Tel (advanced stage/small dose) group. Large dose of Tel means 8.33mg/kg. Small dose of Tel means 4.17mg/kg. Earlier stage means that application of Tel and DBTC was at the same time. Advanced stage means that application of Tel was at 2 weeks after DBTC injection. Effects of Tel ameliorating CP in rats were observed. To observe effects of Tel ameliorating CP in rats and approach its mechanism. Methods According to pancreatic tissue samples stained with hematoxylin and eosin(HE), pathologic changes were assessed in a blinded manner. Collagen accumulation in pancreatic sections was determined by staining for Sirius Red, quantitated with image analysis. Pancreatic myeloperoxidase activity was determined using the spectrophotometer. The level of insulin in blood was assayed using enzyme linked immunosorbent assay(ELISA). Expressions of a-SMA, PPAR-γ, AT-1R and AT-2R protein were assessed in Envision immunohistochemical method. Expressions of TGF-β1 and PPAR-γmRNA were assessed in the method of real-time quantitative polymerase chain reaction(RT-PCR). Result Expressions of a-SMA, PPAR-γ, AT-1R and AT-2R protein in CP with Tel (earlier stage/large dose) group and CP with Tel (advanced stage/large dose) group decreased, in comparison with CP group (P<0.05), but CP with Tel (earlier stage/small dose) group and CP with Tel (advanced stage/small dose) group were not(P>0.05). Expressions of TGF-β1 and PPAR-γmRNA in CP with Tel (earlier stage/large dose) group and CP with Tel (advanced stage/large dose) group decreased, in comparison with CP group(P<0.05),but CP with Tel (earlier stage/small dose) group and CP with Tel (advanced stage/small dose) group were not(P >0.05). Expressions of PPARy protein and mRNA were no significant difference in the control group and Tel control group (P>0.05). Result Results of HE stain, collagen content and pancreatic myeloperoxidase activity in CP with Tel (earlier stage/large dose) group and CP with Tel (advanced stage/large dose) group were all improved, in compar-ison with CP group(P<0.05), but CP with Tel (earlier stage/small dose) group and CP with Tel (advanced stage/small dose) group were not(P>0.05). The level of INS in blood was no significant difference between all groups(P>0.05). Expressions of a-SMA, PPAR- γ, AT-1R and AT-2R protein in CP with Tel (earlier stage/large dose) group and CP with Tel (advanced stage/large dose) group decreased, in comparison with CP group(P<0.05), but CP with Tel (earlier stage/small dose) group and CP with Tel (advanced stage/small dose) group were not(P>0.05). Expressions of TGF-β1 and PPAR-γmRNA in CP with Tel (earlier stage/large dose) group and CP with Tel (advanced stage/large dose) group decreased, in comparison with CP group(P<0.05),but CP with Tel (earlier stage/small dose) group and CP with Tel (advanced stage/small dose) group were not(P>0.05). Expressions of PPARy protein and mRNA were no significant difference in the control group and Tel control group (P>0.05). Conclusion At two time points, Tel with large dose ameliorated the degree of inflammation and fibrosis in CP rats. Tel down-regulated AT-1R, decreased the production of TGF-β1 and activation of PSCs. Meanwhile Tel may cause the conformational change of PPAR-γ, promting the combination between PPAR-γand its ligands. These changes may contribute to the antiinflammatory effect of PPAR-γ.