| Background:Primary biliary cholangitis(PBC)is a chronic autoimmune cholestatic liver disease.PBC is specially targeting the small bile duct and interlobular biliary duct.So intrahepatic biliary epithelial cells(BEC)play a vital role in the physiological and pathological of PBC.Mesenchymal stem cells(MSCs)have the potential to differentiate into hepatocyte and the capability of tissue repair.Recent studies have demonstrated that MSCs transplantation is safe and effective in PBC patients.However the mechanism remains unclear.Objective:To investigate the protective role and mechanism of MSCs on GCDC induced BEC injury and provide the evidence of MSCs transplantation on PBC patients.Methods:We cultured BEC in vitro with different concentration of GCDC first and detected the apoptosis rate of BEC with flow cytometry.Then BEC were cultured in the presence or absence of MSCs,and harvested them at different times.We detected the apoptosis,mitochondrial transmembrane potential and reactive oxygen species of BEC by flow cytometry.We used the cell counting kit 8 to detect the viability of BEC,and the expression of the E2 components of the pyruvate dehydrogenase complex(PDC-E2),caspase3/8/9,microtubule-associated protein 1 light chain 3 II(LC3II)was measured by Western blotting.CK-19 the specific marker of BEC was detected by mmunohistochemistry.The LC3B was observed by immunofluorescence.The serum levels of vascular endothelial growth factor(VEGF)in PBC patients,health controls and the supernatant level of the cultured cells were detected by enzyme linked immunosorbent assay(ELISA).Results:1.GCDC induced BEC injury.The apoptosis of BEC was significantly higher in the GCDC stimulated group in a dose and time dependent manner.With the simulation of 1,000 ?M for 2h,6h,24h,the apoptosis rate were much higher than the control group,(37.6 ± 5.5.74.2 ± 4.0.62.6 ± 7.7)%vs(6.0 ± 0.5?16.0 ± 4.3?16.4 ± 4.0)%,(p<0.001).2.MSCs reduced apoptosis of BEC caused by GCDC,(19.3 ± 4.8)%vs(38.9 ± 4.7)%,(p<0.05).MSCs inhibited the mitochondrial transmembrane potential damage of BEC.Western blotting showed that there was a trend of activation of caspase8,9 after GCDC stimulation and inhibition of actived caspase8,9 by MSCs.3.The expression of CK-19 in BEC was decreased in GCDC stimulated group while MSCs increased its expression by immunohistochemical staining;Western blotting showed that the PDC-E2 level was increased after GCDC stimulation compared with control group,(1.4 ± 0.2)vs(0.7 ± 0.1),(p<0.05)while MSCs decreased the expression of PDC-E2,(0.9 ± 0.1)vs(1.4 ± 0.2),(p<0.05).4.MSCs inhibited the autophagy of BEC.GCDC increased the expression of LC3II,while MSCs down regulated LC3II expression compared with GCDC stimulated group.The immunofluorescence also showed that GCDC activated autophagy while MSCs down regulated it.5.MSCs alleviated the vitality of BEC,GCDC inhibited the viability of BEC both in 2h and 6h.6.GCDC partly increased the level of ROS expression,and MSCs partly decreased ROS level of BEC,but showed no statistical differences.7.MSCs decreased the level of VEGF secreted by GCDC stimulated BEC,(296.5 ± 38.8)pg/ml vs(712.7 ± 90.2)pg/ml,(p<0.001).The serum levels of VEGF from PBC patients were much higher than health controls,(1128.0 ±650.4)pg/ml vs(92.6 ± 39.8)pg/ml,(p<0.05).Conclusions:GCDC caused the BEC injury,MSCs protected BEC through inhibition of apoptosis and autophagy,reduction of PDC-E2,improvement of CK-19 expression.The mechanism of MSCs treatment in PBC may relate to the down regulation of VEGF. |
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