LPS-TLR4/MD-2–TNF-α signaling mediates alcohol-induced liver fibrosis in rats

• Published in: Journal of Toxicologic Pathology 2022, Vol.35(2), pp.193-203
• Main Authors: Mou, Wen-Ling, Chen, Shi-Ru, Wu, Zhen-Ting, Hu, Li-Hua, Zhang, Ji-Ye, Chang, Hong-Jie, Zhou, Hang, Liu, Ying
• Format: Article
• Language: English
• Subjects: Alcohol; Alcohols; Animal models; Animal tissues; Antibodies; Cirrhosis; Fibrosis; hepatic fibrosis; hepatic stellate cells; Kinases; Kupffer cells; Lipopolysaccharides; Liver cancer; Liver cirrhosis; Liver diseases; Original; Signal transduction; Signaling; Staining; Stellate cells; TLR4 protein; TLR4/MD-2; Toll-like receptors; Tumor necrosis factor-TNF; Tumor necrosis factor-α; Western blotting
• ISSN: 0914-9198; 1881-915X; 1347-7404
• DOI: 10.1293/tox.2021-0018

Liver fibrosis results from liver inflammation and progresses to liver cirrhosis or liver cancer. It is known that nonalcoholic liver disease is mediated by the Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2)–tumor necrosis factor-alpha (TNF-α) signaling pathway. This study aimed to investigate whether alcoholic liver disease is also mediated by this pathway. To this end, we first established rat models of liver fibrosis by administering alcohol. Next, the rats were injected with anti-TLR4 and anti-MD-2 antibodies. Real Time Quantitative PCR (RT-qPCR) and Western blotting were used to detect the activation of the TLR4/MD-2–TNF-α signaling pathway and hepatic stellate cells (HSCs). Moreover, the expression of molecules related to liver fibrosis was estimated. The morphology of rat liver tissue was observed through hematoxylin–eosin staining and Masson staining. For in vitro studies, Kupffer cells (KCs) isolated from the liver were transfected with si-TLR4 and si-MD-2 and co-cultured with HSCs to determine the activity of HSCs. It was found that alcohol treatment activated the TLR4/MD-2–TNF-α signaling pathway and upregulated the molecules associated with liver fibrosis. However, inhibition of TLR4 and MD-2 partially reversed this trend. Notably, in vitro studies indicated that knockdown of TLR4 and MD-2 in KCs partially inhibited LPS-induced activation of KCs and HSCs. Overall, this study showed that alcohol induces liver fibrosis via the LPS-TLR4/MD-2–TNF-α signaling pathway.