Endogenous production of n−3 polyunsaturated fatty acids protects mice from carbon tetrachloride-induced liver fibrosis by regulating mTOR and Bcl-2/Bax signalling pathways

Abstract

New Findings: What is the central question of this study? What is the protective benefit of n−3 polyunsaturated fatty acids (PUFAs) on liver fibrosis and what are the relevant signalling pathways in a transgenic mouse model overexpressing the mfat-1 enzyme? What is the main finding and its importance? n−3 PUFA elevation strongly prevented carbon tetrachloride (CCl4)-induced hepatic damage and inhibited the activation of hepatic stellate cells. n−3 PUFAs suppressed CCl4-induced activation of mTOR, elevated Bcl-2 expression, and reduced Bax level, suggesting that n−3 PUFAs can render strong protective effects against liver fibrosis and point to the potential of mfat-1 gene therapy as a treatment modality. Abstract: Liver fibrosis is a reversible wound healing response with excessive accumulation of extracellular matrix proteins. It is a globally prevalent disease with ultimately severe pathological consequences. However, very few current clinical therapeutic options are available. Nutritional addition of n−3 polyunsaturated fatty acids (PUFAs) can delay and lessen the development of liver fibrosis. Herein, this study examined the protective benefit of n−3 PUFAs on liver fibrosis and the relevant signalling pathways using a transgenic mouse model overexpressing the mfat-1 enzyme that converts n−6 to n−3 PUFAs. Male C57BL/6 wild-type and mfat-1 transgenic mice were administered carbon tetrachloride (CCl4) or control corn oil by intraperitoneal injection. Blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were subsequently measured. CCl4-induced hepatic damage and fibrosis were assessed using haematoxylin–eosin and Masson's trichrome staining. Western blot assays were used to detect and quantify fibrosis-related proteins and mechanistic target of rapamycin (mTOR) and B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) signalling components. The direct effect of docosahexaenoic acid (DHA) on primary hepatic stellate cells (HSCs) was also investigated in a co-culture experiment. n−3 PUFAs, as a result of mfat-1 activity, had a strong protective effect on liver fibrosis. The elevation of ALT and AST induced by CCl4 was significantly lessened in the mfat-1 mice. Histological determination revealed the protective effects of n−3 PUFAs on liver inflammation and collagen deposition. Co-incubation with DHA reduced the expression of profibrogenic factors in the primary HSCs. Moreover, mfat-1 transgenic mice showed significant reduction of proteins that are involved in mTOR and Bcl-2/Bax signalling pathways. Collectively, these results suggest that n−3 PUFA elevation strongly prevents CCl4-induced hepatic damage by directly inhibiting the activation of HSCs and regulating the basal activity of the mTOR and Bcl-2/Bax signalling pathways. Gene therapy applying mfat-1 and elevating n−3 PUFAs represents a promising treatment strategy to prevent liver fibrosis.