Low-dose LPS promotes progress of non-alcoholic steatohepatitis by up-regulating TLR4 in macrophages

Abstract

Objective To investigate the role and underlying mechanisms of lipopolysaccharide (LPS) in the promotion of non-alcoholic steatohepatitis. Methods Wild type B6 (B6.WT) mice aged 6~8 weeks were randomly divided into 3 groups and fed with normal diet (ND), high-fat diet (HFD) and HFD combined LPS injection (HFD+LPS), respectively. LPS (0111∶B4, 1.25 μg/g body weight) was injected intraperitoneally to the mice of the HFD+LPS group once a week, and normal saline of same amount was given to the mice of the other 2 groups. After 10 weeks' feeding, the levels of triglycerides (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate transaminase (AST) in the serum and liver tissue were detected by enzyme based assays. The lipid deposition in the liver was observed by oil red O staining. The levels of IL-1β, TNF-α, CCL5, CCL2 and CCL4 in the serum and liver tissue were measured by ELISA. The mRNA levels of IL-1β, TNF-α, CCL5, CCL2 and CCL4 in the liver tissue were detected by quantitative real time-PCR (RT-qPCR). The pathological changes of liver tissue and inflammatory cell infiltration were detected by HE staining. Infiltration of macrophages and neutrophils, and the expression of TLR4 in the liver tissue was analyzed by immunofluorescence (IF) assay. Proportion of macrophages and neutrophils in the liver was assessed by flow cytometry. Results The serum levels of TG and TC were significantly higher in the HFD group than the ND group (P < 0.05). Their contents in the liver were markedly increased in the HFD+LPS group than the HFD group (P < 0.01). The mice from the HFD+LPS group had more obvious vacuolar degeneration of liver cells, infiltration of inflammatory cells, higher NAS score (P < 0.05) and larger number of infiltrated mononuclear cells (P < 0.01) when compared with the HFD group. IF assay and flow cytometry proved that HFD+LPS treatment induced significantly obvious infiltration of hepatic macrophages and neutrophil accumulation (P < 0.05). ELISA proved that HFD+LPS treatment increased serum levels of IL-1β, TNF-α, CCL5 and CCL2 (P < 0.05), elevated mRNA levels of IL-1β, CCL5 and CCL2 in the liver tissue (P < 0.05). IF revealed that TLR4 showed obvious cell membrane translocation in liver tissue after LPS treatment, and the co localization of TLR4 and macrophages showed that the expression of TLR4 on the surface of macrophages in HFD + LPS group was significantly higher than that in HFD group(P < 0.01). Conclusion LPS mediates intrahepatic infiltration of macrophages and neutrophils and aggravates the process of NASH in mice fed with HFD through upregulating TLR4 expression on macrophages to induce chemokines and inflammatory cytokines.

Keywords

• non-alcoholic steatohepatitis
• high-fat diet
• lipopolysaccharide
• chemokine
• inflammatory cytokine
• toll-like receptor 4