Summary Studying Of The Function And Countermeasures Of Activin, Integrin And Endothelin In Fibrosis Of The Chronic Pancreatitis

prefacePancreatic fibrosis is a special histopathological changes of the pancreas accompanying with chronic pancreatitis,which often leads to functional losing of organization and deposition of the cell-rich connective tissue of extracellular matrix. However,to date,the precise regulatory mechanism of pancreatic fibrosis remains unclear.Pancreatic stellate cell(PSC) is a key cell in pancreatic fibrosis,which eventually led to the formation of chronic pancreatitis.A variety of cytokines involved in this process,and different cytokines have different origins as well as mechanism of interaction.Activin was first found in the gonadal glycoprotein hormone,but it has a variety of biological functions,it belongs to TGF-βsuperfamily members,and its three types of receptor belong to serine / threonine protein kinase.Activin organism involved in inflammatory response and tissue repair process,A certain level of activin not only promote the PSC proliferation in a dose-dependent manner to expressing a smooth muscle actin(a-SMA),but also enhance its secretion of collagen.Endothelin (endothelin ET) as the strongest known vasoconstrictor substances,its mechanism associates with increasing synthesis and secretion of PSC collagen as well as improving its ability of uptake and synthesis.Integrins are glycoprotein receptor family molecules located on cell surface,as mediating membrane signaling molecules and mediating cell -cell and cell-extracellular matrix molecules they play an important role in many pathophysiological processes:such as cell proliferation,differentiation,spreading and migration,apoptosis,inflammatory response,tissue repair and tumor invasion and metastasis.Cell factors control PSC through the cell signal-transduction pathways in the process of promoting activation,proliferation,migration of PSC.c-Jun N-terminal kinase(JNK) are signal transduction pathway members of the mitogen-activated protein kinase(MAPK) family.MAPK widely involved in variety of life processes of cells,including growth,differentiation,cell cycle regulation,tumorigenesis,apoptosis and so on.JNK mainly involved in the synthesis of ECM.Han-Dan-Gan-Le is consisted by salvia,astragalus,Chinese Tetrandrine,gingko, flavors Chishao and other sides of the Chinese Medicines Board on the basis of the clinical differentiation.Research has confirmed that it can exert anti-liver fibrosis effect through many mechanisms,such as reducing lipid peroxidation in liver cells, protecting liver cells,inhibiting HSC proliferation and collagen synthesis,enhancing collagenase activity,promoting collagen degradation and so on.This issue detected dynamic change of activin,integration and endothelin in the process of pancreatic tissue repair and fibrosis,and applied drug intervention of pancreatic fibrosis,by using cell biology and molecular biology techniques(including immunohistochemistry,RT-PCR,western blot,etc.).The purpose of this issue is to discuss the role of activin,integration and endothelin in the process of the occurrence and development of chronic pancreatic fibrosis,discuss the intracellular transduction pathway as well as the effect of drug intervention in this process.This topic is divided into three parts:(1) setting up rat model of chronic pancreatitis;(2),Expression activin, integration and endothelin in chronic pancreatitis tissues;(3) studying of the intervention of Khan-Dan-Gan-Le-Grand in rats with chronic pancreatitis.methods1,The establishment of experimental animal modelsInjecting 10%ethanol solution PBS containing 2%trinitrobenzene sulfonic acid (TNBS) into pancreatic duct of Wistar rats in order to replicate animal model of chronic pancreatitis,and injecting equivalent 10%ethanol solution PBS as the control group, Three days later we give oral Han-Dan-Gan-Le to the intervention group(model group) and observe after the 1,2,3,4,5,6,7-week.2,Preparation of experimental sampleswe determinated serum amylase and C-peptide of serum samples by using rate method and electrochemiluminescence immunoassay respectively.Then,some of this pancreatic tissue was taken immediately to observe ultrastructure by the electron microscope lens,some was prepared to form histology specimens,and some was preserved in -80℃.3,ImmunohistochemistryDewaxing,from benzene,hydration Detecting FN collagen,ACT-A,ET-1, Integrin a5β1,JNK,a-SMA,after dewaxing,from benzene,and hydration of histology specimens through immunohistochemistry,we took this result as positive staining:the pancreas appeared interstitial yellow and brown under light microscopy.Expression degree of Immunohistochemical semi-quantitative analysis:By using color image analysis system,we selected 10 sections vision randomly to measure the pancreatic samples of rats in each group,calculated the area of positive staining area then the total pancreatic vision,and gave quantitative analysis expressed as stained area in percentage of specimens4,Masson staining of collagenThe histological specimens of pancreatic tissue was stained by using of Masson staining of collagen method after dewaxing,froming benzene and hydration,resulting in judgments:acinar red,collagen fibers were blue or green.Semi-quantitative analysis of collagen area:selected 10 different visions of each specimen under the same size region,measured area of the collagen percentage in the region,and choose the average as the value of the relative content of collagen. 5,RT-PCRUsing RT-PCR technology we can detect the expression of.endothelin-mRNA, activin-mRNA and integrin-mRNA in the model group.After Rnase treatment of items used in the experiment,extraction of Organizational total RNA,reaction of reverse transcription(RT),polymerase chain reaction(PCR),as well as electrophoresis and photographed of gene amplificational products in 1.5%agarose gel we can give such analysis:entered the gel electrophoresis image,used automatic electrophoresis gel image analysis system to analysis the degree of expression,according to this formula to get the relative coefficient:relative coefficient = intensity of protein expression /β-actin expression.Each electrophoresis map repeated 3 times,and calculated the mean.6,western blotwestern blot detected the expression ofα-SMA,JNK.After extraction of organizational total protein,electrophoresis of gel polypropylene(SDS ONE PAGE), transferred membrane and blotting,the film image was entered to gel image analysis system for expression analysis,calculated the relative coefficient as this formula: relative coefficient = intensity of protein expression /β-actin expression.Each electrophoresis map repeated 3 times,and calculated the mean.results1,experimental animal general and morphology changeModel group rats body weight extremely decreased in the first week after operation,then increased later step by step,but still lighter than control group.Model group rats pathological change:fibrosis around the pancreatic foliole and the stoma,in the second week and reached pick at the forth weak,the changes of pancreatic tissue in the fifth,sixth,seventh week were similar with the forth week.2,observation under electron microscope model group appeared PBS cell in the pancreatic stroma in the first and second week and the Vitamin A lipid droplet disappeared in it,pancreatic cell mitochondrial swelling and multiple cyst formatted.Clearing activated PSC and fibroblasts increased markedly in pancreatic stroma at the third week,mitochondrial swelling and hyalinized apparently.In the forth week,activated PSC,fibroblasts and macrophages increased significantly in pancreatic stroma and generous fibers appeared in the stroma, pancreatic cell rough endoplasmic reticulum extended and cell volume decrease.The changes in the fifth,sixth,seventh week were the same with the forth week.3,serum detectmodel group every time point serum diastase was significantly higher than control group,C-peptide had no difference in the first and second week,buy model group became lower from the third week to the seventh week.4,immunohistochemisty detect(1) model group FN collagen expression increased steeply and reached pick in the forth week,significantly higher than intervention and control group.The changes of model group in the fifth,sixth,seventh week were the same with the forth week.(2) Control group:Activin expressed low level in alveolus cell,pancreatic duct wall cell and pancreatic stroma.Model group and intervention group:positive cell located in pancreatic stroma,fibrosis septation area and inflammatory cell invasion area. Mode group increased gradually form the first to the seventh week,significantly higher than control group especially in the post 4 weeks,compared with model group the intervention group went down-regulation.(3) Control group:endothelin slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive cell located in fibrosis septation area and inflammatory cell invasion area.Model group appeared tow pick in the first and the forth week and significantly higher than control group,compared with model group the intervention group went down-regulation markedly. (4) Control group:integrin slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive cell located in fibrosis area and inflammatory cell invasion area.Model group went up-regulation step by step and appeared pick in the third or the forth week then went down and significantly higher than control group,compared with model group the intervention group went down-regulation markedly.(5) Control group:JNK slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive stain located in pancreatic storma and fibrosis area cell plasma and nucleus.Both were significantly higher than control group,compared with model group the intervention group went down-regulation markedly.(6) Control group:a-SMA slightly expressed at blood vessel wall cell;model group and intervention group:a-SMA located in fibrosis area and around pancreatic duct.Both were significantly higher than control group,compared with model group the intervention group went down-regulation markedly.5,pancreatic tissue collagen distributioncontrol group appeared slightly collagen fiber stain in the pancreatic fibrosis septation area;model group showed plenty of blue stained collagen fiber deposition, especially between foliole and parapancreatic duct,scattered distributed in the foliole, compared with model group,intervention group interfoliole and parapancreatic duct collagen fiber deposition significantly decreased,there were only a little collagen fiber deposition in the foliole.6,RT-PCR(1) activin mRNA could not be detected from the first to the third week in model group,progressive increased from the forth week and reached pick in the sixth or seventh week.(2) Endothelin mRNA appeared tow pick in the first week and the forth week in model group. (3) Integrin mRNA expression progressively increased and reached pick in the forth week,then went down.7,western blot(1) a-SMA protein expression in pancreatic tissue compared with control group: model and intervention group significantly up-regulation;compared with model group the intervention group went down-regulation markedly.(2) JNK protein expression in pancreatic tissue compared with control group: model and intervention group significantly up-regulation;compared with model group the intervention group went down-regulation markedly.Conclusion1,Injecting 10%ethanol solution PBS containing 2%trinitrobenzene sulfonic acid (TNBS) into pancreatic duct of Wistar rats in order to replicate animal model of chronic pancreatitis witch was successful.2,this methods can image the process of acute pancreatitis transformed into chronic pancreatitis and consistent with human chronic pancreatitis succession.It can be satisfied with any study after 4 weeks.3,activin,Endothelin and Integrin can promote chronic pancreatitis fibrosis.4,activin promote chronic pancreatitis fibrosis in the late stage of chronic pancreatitis,Endothelin and Integrin work in the early and middle stage.5,Han-Dan-Gan-Le can inhibit rats chronic pancreatitis fibrosis.6,JNK signal transport pathway does important work in the process of chronic pancreatitis fibrosis.7,the effct of activin,Endothelin and Integrin witch can improve PSC activation and proliferation may have correlation with JNK signal transport pathway activity.8,the effect of Han-Dan-Gan-Le witch can inhibit pancertitis fibrosis may have correlation with decrease activin,Endothelin and Integrin expresson and inhibit JNK signal transport pathway activity.