CX3CR1 silencing-mediated macrophage polarization attenuates CCl4 -induced liver fibrosis in mice

Abstract

Objective To investigate the role of CX3CR1 in liver fibrosis induced by CCl4 in mice. Methods The mice were divided into normal group (n=24), CCl4 group (n=36), CCl4+sh-NC group (n=36), and CCl4+sh-CX3CR1 group (n=36), and all the groups were induced liver fibrosis by intraperitoneal injection of CCl4 except the normal group. From the 4th week of CCl4 injection, the mice of CCl4+sh-NC group and CCl4+sh-CX3CR1 group were respectively injected with shRNA or shRNA-CX3CR1 by tail vein weekly, for 2 weeks. The expression of CX3CR1 in the liver tissues was determined by immunohistochemistry, and the number of M1 macrophages (F4/80+iNOS+) or M2 macrophages (F4/80+CD206+) in the liver tissues was measured by immunofluorescence staining. ELISA was used to detect the serum levels of IL-1β, IL-6 and TGF-β1 in mice, and flow cytometry was adopted to analyze the phenotypic changes of M1 and M2 macrophages after co-culture of primary liver macrophages with activated hepatic stellate cells (HSC). Results CX3CR1 was hardly detected in healthy non-fibrotic control livers, but strongly up-regulated 4~6 weeks after CCl4 treatment. By silencing CX3CR1 expression in CCl4-induced liver fibrosis mice, the severity of inflammatory cell recruitment and fibrous tissue deposition in liver tissues was significantly reduced, and the protein levels of α-SMA, N-cadherin and Vimentin were decreased, while the expression of E-cadherin increased. As compared with the CCl4+sh-NC group, the serum levels of IL-1β, IL-6 and TGF-β1 were down-regulated by 4.6-8.1 times in the CCl4+sh-CX3CR1 group (P < 0.01), and the percentage of M2-macrophages as well as the M2/M1 ratio were remarkably elevated (P < 0.01). In the co-cultures of HSC and macrophages extracted from CX3CR1 silencing mice, collagen production was inhibited due to the high M2/M1 ratio. Conclusion CX3CR1 silencing plays an anti-hepatic fibrosis role by promoting polarization of M1 to M2 macrophage.

Keywords

• cx3cr1
• macrophage polarization
• mice
• liver fibrosis