Keratin Participated In Primary Biliary Cirrhosis, Bile Duct Epithelial Cell Apoptosis, Mitochondrial Damage And Its Signal Transduction Mechanism

IntroductionPrimary biliary cirrhosis(PBC) is an autoimmune liver disease characterized by the progressive destruction of intrahepatic bile ducts,resulting in chronic cholestasis. It is considered as a representative autoimmune disease particularly due to the high specificity of the antimitochondrial antibodies(AMA) found in 90–95% of patients. The role of immune tolerance broken of mitochondria during PBC pathogenesis is not clear.Membrance damage,curls and valgus may induce cellular contents leak,abnormal apoptosis which enforce mitochonria component degrading in trouble. Furthermore, immunogenic fragments may induce PBC onset.Cytokeratins(CK) are the largest subgroup of intermediate filament proteins,which is an important constituent of the cellular cytoskeleton. CK’s functions included maintenance of cell integrity and protection from oxidative injury,apoptosis and also maintain normal mitochondrial structures. Furthermore, CK’s mutations are associated with several diseases,for example, transgenic mice that overexpress of CK8 develop mild chronic pancreatitis;CK8-null mice may develop spontaneous colitis;In breast cancer, CK8 expression significantly increased.In recent years,absence or mutation of CK8 were reported to be associated with liver diseases,CK8/CK18 m RNA and protein levels increase 3-fold in response to liver injury as noted in mice. Similarly, transgenic mice that knockdowning CK8 may develop mild chronic hepatitis, fragile hepatocytes and marked susceptibility to drug-induced liver injury. CK8-/-(FVB/N) mice manifest 94% embryo lethality with extensive liver hemorrhage and susceptibility to liver injury. CK8-/-(C57/B1) mice 55% have a normal life span but harbor a marked susceptibility to liver injury. Several observations support that CK8 variants may predispose to PBC. Anti-cytokeratin8 antibodies have been found in serum of PBC patients. The patients whose developed with primary biliary cirrhosis that liver CK8/CK18 protein increases 2-4 folds. Furthermore,CK8 variants in PBC was significantly higher than healthy people. In addition,serum anti-mitochondrial antibodies(AMA) are found in CK8-null mice accomplied with alterations in mitochondrial size,organization,and function.These observations lead us to hypothesis that CK8 variants may predispose people to PBC,but the role of variants CK8 in PBC is unclear.The research aim to investigate whether CK8 variants participate in PBC by regulation of mitochondrial organization/function which promote apoptosis.Objective1. To investigate the CK8 expression in human bile duct cell, RBE and its effect on apoptosis of human bile duct cell;2. To study morphology and function of mitochondria in overexpression or knockdown CK8 of human bile duct cell;3. To explore the signal transfuction pathway through which overexpression or knockdown CK8 regulate RBE cell in apoptosis.Methods1.To build lentiviral vector encoding CK8,transfect into human bile duct cell RBE to establish gain-of-function models,using RT-PCR and Western Blot detect expression of CK8 in human bile duct cell RBE;2.To investigate cell apoptosis and percentage of apoptosis through immunohistochemistry and flow cytometry analysis;3.To verify mitochondrial morphology and function changes through immunohistochemistry and flow cytometry analysis;4.To test the key protein changes in cell apoptosis by western blot;5.To investigate the effect of GCDC on apoptosis and percentage of apoptosis changes in human bile duct cell RBE through immunohistochemistry and flow cytometry analysis;6.To verify the effect of GCDC on mitochondrial morphology and function changes in human duct cell RBE through immunohistochemistry and flow cytometry analysis;7.To test the key protein changes in cell apoptosis in human bile duct cell RBE after the stimulation of GCDC by western blot.Results1.Abnormal expression of CK8 promote apoptosis of human bile duct cell RBE.We found that nucleus condensation and distribution of keratin changed in overexpression or knockdown CK8 of human bile duct cell RBE when compared with the control group by immunohistochemistry. We discovered that the cell apoptosis rate in overexpression or knockdown of CK8 of bile duct cancer cells rised significantly by flow cytometry technology.2.Abnormal expression of CK8 induce mitochondria morphological and functional changes.We found that the destruction of mitochondrial morphology and changed distribution of mitochondria in overexpression or knockdown CK8 when compared with the control group by immunocytochemistry. We discovered the significantly increased cells percentage of reactive oxygen species in overexpression or knockdown CK8 of human bile duct cell RBE by flow cytometry.3.Abnormal expression of CK8 may cause cell apoptosis through AKT signaling pathwayWe found that phosphorylation of AKT protein levels was significantly declined and the amount of total protein levels did not change. Furthermore, we found that AKT downstream of apoptosis-related protein such as Bcl-2 and cleaved Caspase-3 had changed subsequently.4.GCDC can promote cytokeratin destruction, mitochondrial damage and cell apoptosisWe found that GCDC can promote the distribution change of keratin in overexpression or knockdown CK8 of human bile duct cell RBE by immunohistochemistry. We discovered that GCDC can increase cell apoptosis rate and the percentage of reactive oxygen species in overexpression or knockdown CK8 of human bile duct cell RBE significantly by flow cytometry technology. In 1m M, apoptosis is most obviously.ConclusionAbnormal expression of CK8 may promote cell apoptosis, morphological and functional changes of mitochondria. And abnormal expression of CK8 induced apoptosis of human bile duct cell RBE may possibly through Akt signal pathway. GCDC can promote cytokeratin destruction, mitochondrial damage and cell apoptosis in human bile duct cell RBE of abnormal expression of CK8 and the effect of GCDC present with concentration-dependent.