Experimental Study On The Intervention Mechanism Of The Rat With Blood Stasis Type Of Liver Fibrosis

Objective:by carbon tetrachloride?CCl4?and bovine serum albumin?BSA?and norepinephrine?NE?multiple factors induced liver fibrosis in rat blood stasis and blood stasis animal model of hepatic fibrosis was established,plumbagin as research object,observe the plumbagin before and after the intervention of rat liver function index?AST,ALT,SOD,etc.?and liver fibrosis related factors?HA,LN and PC ??content change,using HE staining and Masson staining,to observe the changes of plumbagin intervention before and after hepatic fibrosis degree,the WB assay LSEC cell HSPG2 protein of the expression.To explore the intervention mechanism of the blood stasis type of liver fibrosis in the blood stasis type of liver fibrosis,and to lay a foundation for the prevention and treatment of liver fibrosis.Methods: 1 blood stasis type of hepatic fibrosis model preparation.2 To detect the content of ALT?AST?TBil?ALP?SOD and MDA in serum.3 Content changes of LN,? Col,? PC and HA in serum were measured by chemiluminescence method.4 ELISA method for the determination of CTGF in serum.5 HE staining and Masson collagen staining of liver tissue were observed in liver fibrosis degree and staging score.6 WB to detect the expression of HSPG2 protein in LSEC cells.Results: The first part: the effect of the intervention of the blood stasis type of liver fibrosis rats before and after the intervention of the liver function related indicators,liver fibrosis related factors and the degree of liver fibrosis?1?the content changes of serum ALT?AST?TBil?ALP?SOD and MDA compared with the blank control group,model control group,serum ALT?AST?ALP?TBil?SOD and MDA content were significantly increased,compared with extremely significant difference?P<0.01?,confirmed the model of hepatic fibrosis the rat has been formed;compared with the model group,plumbagin treatment group,colchicine group and salvianolic acid B group rats serum ALT?AST?ALP?TBil?SOD and MDA were significantly decreased?P<0.05?,the high dose group plumbagin is better than the low dose group;compared with colchicine group,high dose group plumbagin and TBil SOD decreased,compared with significant difference?P<0.05?;compared with salvianolic acid B of plumbagin group,high dose group and colchicine group,LN?HA ? PC ? and HA content decreased,compared with Fruit with significant difference?P<0.05?.?2?chemiluminescence method for the determination of content of serum LN?Col ??PC ? and HA: compared with the blank control group,LN?HA?PC increased ? and HA content in model group,compared with extremely significant difference?P<0.01?;compared with the model group,all LN?HA?PC ? and HA content of plumbagin in the treatment group,compared with significant difference?P<0.05?which plumbagin in high dose group is better than the low dose group;compared with colchicine group,high dose group plumbagin in LN?HA?PC ? and HA content drop comparison results have significant difference?P < 0.05?; compared with salvianolic acid B of plumbagin group,high dose group and colchicine group in LN?HA?PC ? and HA decreased,compared with significant difference?P<0.05?.?3?the changes of CTGF content in serum was determined by ELISA assay: compared with the control group,the serum levels of CTGF increased significantly in the model group,compared with extremely significant difference?P<0.01?;compared with the model group,CTGF treatment group plumbagin content in serum were decreased,compared with significant results the difference?P<0.05?,which decreased greatly compared with the low dose group plumbagin high dose group;compared with colchicine group,decreased the content of CTGF in serum of rats in high dose group plumbagin in comparison.Results there was significant difference?P<0.05?;compared with salvianolic acid B group,plumbagin serum quinone in high dose group and colchicine group rats were low,the comparison results had significant difference?P<0.05?.?4?HE staining and Masson collagen staining were used to observe the degree of liver fibrosis and the staging of the liver fibrosis.In the normal group,the liver tissue of the liver tissue in the A group was in the central vein,which was in a radial arrangement.There was no fatty degeneration in the liver cells.No hyperplasia was found in the fibrous tissue.In the B group,there were obvious fibrous tissue proliferation,formation of false leaflets,degeneration of hepatic steatosis,inflammatory infiltration of the liver cells,which confirmed that the rat model of hepatic fibrosis was already formed.Proliferation of fibrous tissue of group C of plumbagin in high dose group and D group white Dan quinone low dose group,the hepatocytes with fatty degeneration,pathological damage compared to group B in the model group were reduced,which the protective effect on the liver plumbagin high dose group greater than low dose group.Hyperplasia of fibrous tissue in the colchicine group in Group E and group F of salvianolic acid B group Dudu,hepatocytes with fatty degeneration,inflammatory cell infiltration,pathological damage compared to group B in the model group were reduced,which colchicine group on the liver protective effect of greater than low dose group.The second part the effect of the HSPG2 protein expression of LSEC cells before and after the intervention.This thesis chooses the LSEC cell plumbagin intervention mechanism of liver fibrosis as research object,specific reasons are the following several points: normal differentiation of LSEC can induce activation of HSC reversed to a state of calm,effectively preventing the development of liver fibrosis.The activation effect of HSC will be allowed when the LSEC is stopped or the hepatic sinus is stopped.When the liver fibrosis and liver injury,LSEC becomes an efficient and proinflammatory cytokine,and secrete a series of cytokines and chemokines,which can induce the acceleration of liver inflammatory response and the change of liver immune function.LSEC cells were divided into 7 groups: A group LSEC group;group B model control group?leptin activation and activation of Leptin culture medium added a final concentration of 100ug/L LSEC cells?;group C1 of plumbagin in high concentration group?the first cell activation by Leptin after adding a final concentration of plumbagin 15umol/L?;group C₂ concentrations of plumbagin in?the first cell activation by Leptin after adding plumbagin final concentration of 8umol/L?;group C₃?low dose group plumbagin to cell activation by Leptin after adding plumbagin final concentration of 2umol/L?;D group forcolchicine intervention the control group?the first cell activation by Leptin after the drug concentration was6.25ug/L?;group E,salvianolic acid B group(the first cell activation by Leptin after the drug concentration was 10^-6mol/L).Collected during the logarithmic growth phase of the seven groups of cells,with Ripa lysis buffer and cell protein extraction,BCA method for protein quantification and take 50ug/ hole protein 6%SDS-PAGE,electrophoretically separated proteins were transferred to PVDF membrane on,containing 5% skim milk tbst?closed liquid?soaking PVDF membrane,room temperature into closed 2 hours in shaker and add anti?HSPG2 and beta actin?,4? for the night,after the addition of second antibody?HRP?,incubation for 2 hours at room temperature and enhanced chemical ECL development using bandscan film analysis gray value.Expressed protein HSPG2 high doses of HSPG2 high doses of HSPG2 quinone HSEG2 results show that: with the liver sinusoidal endothelial cells group,leptin activates the model control group protein expression level increased significantly?P<0.01?,and leptin activation model control group comparison of Plumbago zeylanica treatment group,colchicine group and salvianolic acid B group in LSEC cell protein expression level decreased significantly?P<0.05?.The plumbago zeylanica quinone high dose group is superior to middle dose and low dose group and colchicine group,plumbago zeylanica quinone group in LSEC cell protein expression were significantly decreased?P<0.05?;and salvianolic acid B group compared with white flower Dan quinone group level is low,the comparison results have significant difference?P<0.05?.Conclusion: 1 the content of liver function related indexes AST?ALT?ALP and so on in liver fibrosis of blood stasis type of liver fibrosis can be significantly reduced,which is beneficial to protect the liver function and reduce the occurrence of liver inflammation.2 the content of LN?HA and PC ? in liver fibrosis related factors of blood stasis type of hepatic fibrosis can be significantly reduced,and the degree of liver fibrosis can be reduced.3 using HE staining and Masson staining,it was proved that the degree of hepatic fibrosis in the liver fibrosis model of blood stasis type and the pathological changes.4 The expression of HSPG2 protein in LSEC cells was determined by4.WB method.The expression of protein was confirmed by the regulation of HSPG2 protein,and the formation of hepatic fibrosis was induced by indirect intervention.