The Expression Of A20 And Its Function In Liver Fibrosis

PartⅠ Hepatic expression of A20 in cirrhosis livers is upregulatedObjective: To investigate whether A20 protein level in cirrhosis livers has changed. Methods: Liver tissues from patients with hepatic cirrhosis(n=7) and healthy individuals(n=4) were included and studied for A20 protein level by immunohistochemistry. Results: A20 was highly expressed in human cirrhosis livers, mainly expressed in hepatic stellate cells and hepatocytes. Conclusions: Hepatic expression of A20 in cirrhosis livers is upregulated.PartⅡ Hepatic expression of A20 in fibrosis livers of mouse model is upregulatedObjective: To investigate the A20 expression level in fibrosis livers of mouse model. Methods: To build the model of liver fibrosis with feeding C57/B6 male mice containing DDC or MCD food or BDL operation, and to investigate the expression of A20 through western bloting, PCR. Results: The expression of A20 was significantly higher in BDL operation 、DDC or MCD feeding mice than the normal controls, A20 m RNA also increased compared with the control group. Conclusions: Upregulation of A20 in liver tissue around play a certain role in the development of liver fibrosis.Part Ⅲ A20 expression in LPS-induced liver fibrosis model in vitroObjective: To investigate the A20 expression level in LPS-induced fibrosis cell model and the mechanisms by which it employed. Methods: LX-2 cells were exposed to LPS at the concentration of 0.1 μg/m L, then expression of A20 were detected by western blot and PCR. The level of α-SMA、col-1、col-3、TGF-β、TNF-α、MCP-1、TLR4 cytokines were also examined by PCR. Results: A20 could be induced by LPS in LX-2 cells. A20 protein expression had increased at 6 h, and then atenuated to the basal level at 24 h. LX-2 Cells treated by LPS at 0, 0.001, 0.01, 0.1 and 1 μg/m L for 6 h showed dose-dependent increase of A20 protein expression. The expression of α-SMA、col-1、col-3、TGF-β、TNF-α、MCP-1、TLR4 cytokines were increased after LPS treatment in a time-dependent manner. Conclusions: LPS-mediated liver fibrosis model in vitro could induce the expression of A20 and production of inflammatory cytokines.Part IV Overexpression of A20 in vitro could inhibit LX-2 cells activation and reduce the expression of inflammation cytokinesObjective: To explore whether overexpression of A20 could inhibit liver fibrosis in vitro and its possible mechanisms. Methods: LX-2 cells were infected with control adenovirus or A20-expressing adenovirus for 24 h and then replaced with standard culture medium, when reached 80%–90% confluency, then washed with PBS and incubated with serum-free DMEM/F12. After subjected to LPS at 0.1 μg/m L concentration for 24 h, cells were harvested. The m RNA level of α-SMA、col-1、col-3、TGF-β、TNF-α、MCP-1、TLR4 were examined by PCR. And western blot was used to detect the expression of A20, α-SMA. Results: Overexpression of A20 in LX-2 could inhibit α-SMA deposition and col-1、col-3 secretion. TGF-β、TNF-α、MCP-1、TLR4 m RNA was surprisingly reduced in A20-overexpression LX-2 cell lines. Conclusions: Overexpression of A20 could inhibit the activation of LX-2 cell and reduced the expression of inflammation cytokines.