Role Of Toll-like Receptor4-mediated Downstream Inflammatory Factors In Carbon Tetrachloride-induced Acute Liver Injury And Repair

Objective Liver diseases have severely affected human health and acute liver injury can induce acute and chronic liver diseases, so clarifying the mechanisms of acute liver injury play an important role for protecting and treating acute and chronic liver diseases. Toll-like receptor (TLR)4-mediated innate immune responses play an important role for liver diseases, and TLR4-mediated downstream inflammatory factors have been involved in liver injury and repair. On the other hand, vitamin D(Vit D) can down-regulate the expression of TLR4-mediated signal and is involed in inflammation. The present study will clarify the role of TLR4-mediated downstream inflammatory factors in carbon tetrachloride (CCl4)-induced acute liver injury and repair in mice by investigation about (1) the role of TLR4in CCl4-induced acute liver injury and repair in mice and (2) the effects of vitamin D deficiency on hepatic inflammation during CCl4-induced acute liver injury in mice.Methods The present study includes two parts.(1)24healthy male TLR4-/-mice and24healthy male TLR4+/+mice were sacrificed at0,24,48and72hours after a single intraperitoneal injection of carbon tetrachloride (CCl4,0.3ml/kg), and serum and liver samples were collected immediately. Serum alanine aminotransferase (ALT) was determined and hepatic histopathological damage was analyzed with HE staining. Apoptosis was detected by TUNEL. PCNA was detected by western boltting and immunohistochemistry. Hepatic Cyclin D1, phagocytosis related genes(CD36, Gpnmb, Cd81and Cd51), pro-inflammatory cytokines(TNF-α, IL-1β and IL-6), chemokines(KC, MCP-1and MIP-1α), anti-inflammatory cytokines(IL-4和IL-10)and TGF-β1mRNAs were measured by real-time RT-PCR.(2)36healthy male ICR mice were randomly divided into LVD and STD groups. All mice in the LVD group were fed with vitamin D deficient diet for one week and all mice in the STD group were fed with standard diet for one week. All mice were sacrificed at0,24and72hours after a single intraperitoneal injection of carbon tetrachloride (CCl4,0.3ml/kg), and serum and liver specimens were collected immediately. Serum alanine aminotransferase (ALT) level was determined and hepatic histopathological damage was analyzed with HE staining. Hepatic KC, MCP-1, MIP-1α, TNF-α, TGF-β1and α-SMA mRNAs were measured by real-time RT-PCR.Results (1) Serum ALT level and necrosis area increased significantly at24h in TLR4+/+mice, and recovered obviously at72h. No significant difference on serum ALT level and apoptosis between TLR4-/-mice and TLR4+/+mice, but necrosis area were both more significant than that of TLR4+/+mice at the same time points. Compared to TLR4+/+mice, the cell proliferative response was delayed in TLR4-/-mice after CCl4adminstration. By Western Blotting and real-time RT-PCR technology, we found that PCNA level and Cyclin D1mRNA level in liver tissue nucleoprotein of TLR4+/+mice were significantly higher than that of TLR4-/-mice.Compared with the results of TLR4+/+mice at same time point after CCl4treatment, mRNA levels of hepatic chemokines, pro-inflammatory and anti-inflammatory cytokine were significantly decreased in TLR4-/-mice. Moreover, phagocytosis related genes were significantly down-regulated in TLR4-/-mice, but mRNA level of pro-fibrogenic factor TGF-β1was significantly higher than that of TLR4+/+.(2) No significant difference on serum ALT level and hepatic histopathological damage was observed between the LVD group and the STD group. Compared with the results of the STD group at same time point after CCl4treatment, mRNA levels of hepatic KC and MCP-1, two chemokines, and TNF-α, pro-inflammatory cytokine, were significantly increased in mice fed with LVD diet. Moreover, TGF-β1and α-SMA mRNAs were significantly up-regulated in LVD diet-fed mice.Conclusions (1) Toll-like receptor4can up-regulate pro-inflammatory cytokines, chemokines and pro-fibrogenic factor in CCl4-induced acute liver injury, and significantly up-regulate mRNA expression of proliferation and phagocytosis related genes. TLR4signaling may play an important role in liver repair after CCl4adminstration.(2) Vitamin D deficiency can aggravate inflammation and pro-fibrosis in CCl4-induced acute liver injury.