The Expression Of MMP7 In Human Biliary Epithelial Cells Induced By Rotavirus And Bacterial Endotoxin

Objective:In vitro experiments were conducted to explore the inducible factors for the expression of matrix metalloproteinase 7(MMP7)in biliary epithelial cells(BECs),and to investigate the molecular mechanism by which rotavirus(RRV)and bacterial endotoxin(LPS)up-regulated MMP7 expression through TLR4.Methods:MA104 cells underwent cell passage for RRV amplification and titer determination.Collected enough RRV to freeze.Immortalized human intrahepatic biliary epithelial cells(BECs)were cultured,subcultured and cryopreserved to reach the number of cells required for the experiment.Part 1: BECs was infected with RRV at different titers(MOI=0.1,1,10),and the levels of supernatant factors MMP7、IL-1、IL-12 and IFN-γ were detected by enzyme-linked immunosorbent assay(ELISA)at different time points(6,12,24,48,72h).Immunofluorescence cell assay(IFC)observed the effect of RRV on the expression of TLR3 and TLR4.Cell death at each time point was detected by trypan blue staining method.The optimal titer and action time of RRV were selected for follow-up experiments.Part 2: cells were treated with different concentrations of LPS(0.1,1,5,10ug/ml),and the levels of supernatant factors MMP7,IL-6,and IL-8 were detected by ELISA at different times(6,12,24,48,72 h).IFC observed the effects of different concentrations of LPS on the expression of TLR3,TLR4 and MMP7.Cell death at each time point was detected by trypan blue staining method.Part 3:RRV and LPS acted synergistically on BECs.The RRV of confirmed concentration(experiment 1)was selected to act on BECs with the appropriate concentration of LPS respectively,and the corresponding concentration of LPS and RRV was used as the control,and no treatment was used as the blank control.The levels of supernatant factor MMP7,IL-6 and IL-8 were detected by ELISA.Cell death in each group was detected by trypan blue staining.IFC detected the expression of TLR3,TLR4 and MMP7 in each group.Results:Part 1: the secretion of IL-1,IL-12 and IFN-γ increased significantly after RRV infection with different titers compared with the control group(P <0.05),while there was no difference in the secretion of MMP7 between the two groups.Compared with the blank control group,the expression of TLR3 in the RRV infected BECs group was significantly increased(P <0.05),and the expression of TLR4 was also significantly up-regulated(P <0.01).The cell death showed an increasing effect of virus dose and time of action(P <0.05).The cytokine secretion was the most significant when MOI=0.1RRV acted BECs for 12 h.Part 2: IFC and ELISA showed that 0.1ug/ml LPS acted on BECs,with no significant difference compared with the blank control group(P >0.05).After BECs treatment with 1ug/ml LPS,the production of cytoplasmic MMP7 and the secretion of supernatant IL-6,IL-8 and MMP7 increased significantly(P <0.05).Cell death was significant after the action of 5ug/ml or 10ug/ml LPS for 12 h,and the death difference was observed after the action of 1ug/ml LPS for 24 h,while the cells treated with 0.1ug/ml LPS remained active throughout the observation period.Part 3: After BECs were infected with MOI= 0.1 RRV,BECs produced a significant response to 0.1ug/ml LPS and secreted MMP7(P <0.01),showing a dose-dependent effect.When treated with 5ug/ml LPS,cell death was significant and the expression level of MMP7 decreased(P <0.05).Conclusion:1.BECs infected with RRV significantly up-regulated the expression of TLR4 on the cell surface,but no MMP7 was produced.2.BECs did not respond to low concentration(0.1ug/ml)LPS stimulation,while MMP7 secretion was significant after high concentration(≥1ug/ml)LPS stimulation.3.After BECs were infected with RRV,they had an obvious response to low concentration of LPS,and the expression of MMP7 was significantly up-regulated;The expression level of MMP7 increased first and then decreased with the increase of LPS concentration.