| BackgroundLiver fibrosis refers to the liver extracellular matrix(Extracellular matrix,ECM)excessive deposition of a variety of pathological processes common to chronic liver disease and must pass through.Among them,the accumulation of collagen is the major pathological changes of liver fibrosis.Studies have shown that activation of hepatic stellate cells(HSC)is the main source ECM of the liver,plays a key role in the process of liver fibrosis.Recent studies found that liver cells can be transformed by phenotype by epithelial cells into mesenchymal cells(epithelial mesenchymal transition,EMT)and the synthesis and secretion of extracellular matrix proteins such as collagen,participate in tissue repair and promote the development of fibrosis,become liver fibrosis research focus,revealing in the process of liver fibrosis an important mechanism.Epithelial mesenchymal transition(EMT),refers to the loss of its original features of epithelial cells and acquire characteristics of mesenchymal cells in the process.Mainly in the epithelial cell marker protein E-cadherin(E-cadherin)were down-regulated,mesenchymal cell marker proteins such as vimentin(vimentin)upregulation,the main component of the extracellular matrix,such as collagen type I(collagen I)expression enhancement.Now that the EMT in embryonic development,tumor invasion and metastasis,tissue healing and organ fibrosis and other physiological and pathological processes play an important role.EMT can be divided into three types,type ? EMT occurs in wound healing,tissue remodeling and fibrosis,closely related to the formation of liver fibrosis,also discussed in this paper type.Oxidative stress,as another important mechanism involved in liver fibrosis,namely the body or intracellular reactive oxygen species(ROS)overproduction of endogenous antioxidant defenses weakened,resulting in an imbalance between tissue or cause cell damage state.Studies have shown,ROS can activate HSC proliferation and activation,generating a large number of ECM,promote liver fibrosis.Meanwhile,ROS itself can directly or indirectly act on the liver cells,liver cell damage caused by necrosis and apoptosis,and inflammation of liver tissue.Among them,the non-phagocytic cells oxidase(NOX)family play in chronic liver injury in a key role,the main biological function of NOX is to produce ROS,under abnormal conditions such as the body by growth factor,platelet-derived factors,cytokines,inflammation after the media and other stimuli,NOX can produce large amounts of ROS,resulting in the generation of oxidative stress,thus contributing to liver fibrosis.Bile duct ligation-induced liver fibrosis in rats is the classic model of the principle mechanisms that occur in the human body model with secondary biliary cirrhosis consistent simulated by bile duct blockage,biliary congenital malformations,parasites and other factors biliary cirrhosis caused by siltation caused.This method of modeling takes short and bile duct ligation can form only.On the basis of the bile duct ligation,we invented a new model,the bile duct fixed and ligation on the hose,resulting in short-term state of the bile duct ligation,and after a certain time to form bile duct ligation lifted recanalization,remove the obstruction as a way to achieve the established mode of treatment to alleviate the purpose of liver fibrosis,in order to observe the intrahepatic bile duct epithelial cells,affecting the quality between phenotype after recanalization,while observing oxidative stress-related protein expression of NOX4.ObjectiveSet up rats model of liver fibrosis and treatment by bile duct ligation and recanalization surgery.By detecting NOX4 protein expression and liver epithelial,mesenchymal markers change,to explore the effects and related mechanisms of bile duct ligation recanalization for bile duct ligation-induced rat liver fibrosis epithelial,interstitial phenotype.Methods1.An animal model:four 12-week-old male Wistar rats,weighing as 200-250g,were randomly divided into four groups,labeled sham group(SHAM),bile duct ligation two weeks group(BDL2w),bile duct ligation four weeks group(BDL4w),BDL2 weeks + 2 weeks recanalization surgery group(BDL recanalization group).The rats in each group were treated for surgery,after 24h fasting,the corresponding processing is complete slaughter rat liver tissue specimens from paraffin and frozen specimen preparation for further study.2.Histopathological evaluation:After the prepared rat liver tissue paraffin sectioning,dewaxing,hydration,respectively HE staining and Masson staining,using an upright microscope according to staining for pathological specimens in each group after taking assessment,using Ishak score for each group of rat liver fibrosis score.3.Immunohistochemistry:the prepared paraffin sections of rat liver tissue and dewaxing,hydration,antigen retrieval by microwave repair method,followed by inactivation of endogenous peroxidase,the closure of non-specific antigen,and incubated The first antibody and a second antibody,and finally to DAB chromogenic dye hematoxylin and nuclei were stained.After completion of staining were mounted upright microscope and photographed,analyzed the experimental results.4.Protein electrophoresis and immunoblotting experiments:Fresh liver tissue immersed in liquid nitrogen added and quickly grinding mortar,prepare tissue,followed by centrifugation,concentration determination,adding loading buffer and temperature variability,etc.the good preparation of protein samples prepared in accordance with the proportion of added acrylamide gel electrophoresis for protein electrophoresis tank,after separation of the target protein,the proteins in the gel was transferred to a membrane by a wet transfer PVCD membrane,further nonspecific closed antigen antibody incubation and color.Results collected using image analysis software for semi-quantitative analysis of the results.ResultA histopathological evaluation1.HE stainingSHAM:liver cells arranged rules,complete and clear lobular structure of liver cell cords arranged radially change without liver cell degeneration and necrosis;BDL2w:liver cells arranged slightly irregular,partial destruction of the structural integrity of lobular,hepatic cord structural disorder,portal area near the inflammatory cells increased and infiltrate adjacent tissue,partial liver cell degeneration;BDL4w:liver cells arranged in a serious disorder,hepatic cord structure illegible,periportal lot stromal hyperplasia,showing formation of false lobules,a large number of liver cell degeneration and necrosis;BDL recanalization group:liver cells arranged in irregular,hyperplastic periportal relatively BDL4w rats reduced,still visible part pseudolobules,partial liver cell degeneration watery.Compared to SHAM group,BDL2w and BDL4w varying degrees of cell degeneration,necrosis,and BDL4w BDL2w between the degree of liver cell necrosis between BDL recanalization group.2.Masson stainingCompared to SHAM group,BDL2w and liver tissue Masson staining of collagen Aizen range BDL4w rats with varying degrees of expanding,especially BDL4w most obvious.BDL rats recanalization collagen Aizen range SHAM group was still increasing,but significantly less than the simple BDL4w rats.Compared with SHAM group,BDL2w and BDL4w rat hepatic collagen content was significantly increased(p<0.01),BDL recanalization group collagen content was significantly less than in rat liver though BDL4w group(p<0.01),but still higher than SHAM group,the difference was statistically significant.Description of each bile duct ligation group of liver fibrosis,collagen content increased significantly heavier with fibrosis,liver fibrosis in rats BDL recanalization group has been reduced by way of surgical intervention after bile duct patency and reduce collagen content.3.Ishak score resultsCompared with SHAM group,BDL2w group and BDL4w group ISHAK fibrosis score with prolonged bile duct ligation and have significantly increased(p<0.01),while BDL recanalization group scores were more simple BDL4w group decreased(p<0.01),the difference was statistically significant.Description of each bile duct ligation group with bile duct ligation extend the time,the degree of fibrosis gradually increased,BDL recanalization group due to join the surgical intervention,liver fibrosis improved scores compared BDL2w and BDL4w group were all reduced.The results of immunohistochemical stainingCompared to SHAM group,with the extension of the bile duct ligation between time,fibrosis aggravating,BDL2w and BDL4w group a-smooth muscle actin(?-SMA),collagen I,NOX4 and protein cells vimentin were significantly increased,especially in the most obvious BDL4w group.Compared to BDL4w group,BDL recanalization above each protein expression in rats in varying degrees of ease,but the expression of SHAM group still want more.The result of Western blotCompared to the a-SMA SHAM group,BDL2w and BDL4w groups,collagen ?,and vimentin NOX4 expression of bile duct ligation with the extension of time,obvious increasing trend(P<0.05)was,and epithelial cell marker protein E-cadherin expression of the bile duct ligation with the extension of time was significantly decreased,especially in the most obvious BDL4w group(P<0.05),the difference was statistically significant.Recanalization rats in BDL,protein ?-SMA,collagen ?,the expression of NOX4,vimentin than pure BDL4w rats was significantly lower(P<0.05),the difference was statistically significant,but still significantly SHAM group,and the expression of E-cadherin was significantly increased(P<0.05).Conclusion1.Fibrosis after joining hose on the basis of the bile duct ligation established after a certain time releasing bile duct ligation recanalization form,may reduce the development of liver fibrosis.2.In rat liver fibrosis induced by bile duct ligation,as vimentin,a-SMA,collagen I increased with the aggravation of fibrosis,and increased expression of NOX4,such as E-cadherin reduced with the aggravation of liver fibrosis.3.After the adoption of surgical recanalization with reduction of liver fibrosis in rats,such as vimentin,?-SMA,collagen I reduce as fibrosis mitigate,and reduce expression of NOX4,and E-cadherin increased with the mitigation of liver fibrosis. |
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