Effect Of An Endogenous Ligand Of Toll Like Receptor4on Hepatic Stellate Cells Biology

Background and Aim:Hepatic stellate cells (HSC) are the primary source of extracellular matrix (ECM) in fibrotic liver. Toll-like receptors4(TLR4) is a member of the pattern-recognition receptor superfamily. It plays an important role in recognizing bacterial lipopolysaccharide (LPS) and mediating inflammatory responses and innate immunity. Some injury related endogenous ligands such as High mobility group box-1(HMGB1) also activate TLR4. It has been found that HSC have intact TLR4signaling and respond to LPS stimulation. The aim of the present study was to investigate the activation of TLR4signaling by HMGB1in HSC and its effect on the cells biology.Methods:1. Immortalized wild type(JS1), TLR4-/-(JS2) and MyD88-/-(JS3) mouse HSC lines were subcultured in plates or dishes and divided into the normal control, LPS and HMGB1treated groups. They were transfected with transcriptional factors NF-κB or AP-1responsive luciferase reporter plasmids, followed by stimulation with normal saline, LPS (100ng/ml) or HMGB1(100ng/ml), respectively. The activation of NF-κB or AP-1was detected by a Dual-luciferase reporter assay system. The induction of monocyte chemotactic protein-1(MCP-1) mRNA in these three cell lines after LPS or HMGB1stimuli was determined by real time quantitative PCR (RT-qPCR). The protein level of MCP-1in the culture supernatants was determined by ELISA.2. JS1, JS2and JS3HSC lines were subcultured and divided into the normal control, LPS(100ng/ml), HMGB1(100ng/ml), TGF-β1(10μg/ml), TGF-β1combined with HMGB1, or LPS combined with HMGB1groups. RT-qPCR and Western blot are used to evaluate the expression of a-SMA and Col I mRNA or protein in each group of cells with the relative treatment.Results:1. The activation of NF-κB and AP-1responsive reporters were significantly upregulated in JS1cells treated with HMGB1or LPS, which were coincident with a markedly upregulated expression and secretion of MCP-1. Less responsiveness of NF-κB and AP-1activity, and the expression and secretion of MCP-1were found in JS2or JS3cells after HMGB1or LPS treatment.2. TGF-β1induced a significant upregulation of fibrogenic proteins expression including Coll and a-SMA in all these three cell lines. Co-stimulation with HMGB1and TGF-β1on JS1cells further enhanced the TGF-β1induced Col I and a-SMA expression. This effect was not observed in JS2or JS3cells.Conclusion:As an endogeous ligand of TLR4, HMGB1activates TLR4signaling on HSC. HMGB1enhances the TLR4-mediated inflammatory phenotype of HSC. It also has a synergistic effect with TGF-β1to stimulate fibrogenic protein expression, which is likely TLR4dependent.