Endogenous Production Of Omega-3 Polyunsaturated Fatty Acids Protects Hepatic Fibrosis By Regulating MTOR Pathway

Liver fibrosis is a reversible wound healing response that occurs due to chronic liver diseases,such as non-alcoholic fatty liver,hepatitis B virus infection,hepatitis C virus infection,autoimmune liver disease and alcoholic liver disease.Pathologically,its essence is the excessive accumulation of extracellular matrix（ECM）proteins after liver injury.Liver fibrosis will further lead to liver cirrhosis and liver failure,and greatly increase the risk of liver cancer without intervened in time.Due to its complex etiology and pathogenesis,liver fibrosis is a worldwide health issue with limited therapeutic options.Recent studies have demonstrated that the Omega-3 polyunsaturated fatty acids（PUFAs）are important regulators of cell proliferation,apoptosis,and anti-inflammation,and thus play an important role in the pathogenesis of many clinical diseases,including asthma,cancers,autoimmune diseases,and non-alcoholic fatty liver disease.It is impossible to convert Omega-6 into Omega-3 PUFAs with lack of Omega-3desaturase in mammals.Therefore,many studies related to liver fibrosis are mostly through oral,intravenous injection,or local treatment of Omega-3 PUFAs.Until 2004,Dr.Kang et al transferred the Omega-3 desaturase gene from Caenorhabditis elegans into C57BL/6 mice,leading to elevation of tissue content of Omega-3 PUFAs.However,the effect of endogenous Omega-3 PUFAs against liver fibrosis is not clear.As the serine/threonine protein kinase,the mammalian target of rapamycin（mTOR）signaling is widely involved in maintaining the normal physiological functions of cells,such as cell growth and proliferation,translation,differentiation,and metabolism.Studies have found that mTOR signal plays an important role in the occurrence and development of liver fibrosis.Inhibition of mTOR signal pathway is an important target for the treatment of liver fibrosis by inhibiting the ECM synthesis,HSCs activation and proliferation.In addition,it has been reported that Omega-3PUFAs play an important role in regulating the activity of mTOR signal pathway.Herein,this study assumes that endogenous Omega-3 PUFAs can inhibit the activation of HSCs and reduce the hepatocyte apoptosis,and finally to improve the development of liver fibrosis by regulating the mTOR signal pathway.Objective:To study the effect of endogenous Omega-3 PUFAs on the development of liver fibrosis and the potential molecular events underlying the phenotypes,to provide a potential therapeutic modality of anti-fibrosis strategy.Methods:（1）The mice were identified and divided into mfat-1 transgenic mice and WT mice（C57BL/6）according to the identification results of genotypes.At the same time,the proportion of Omega-3/Omega-6 PUFAs was further detected by gas chromatography in every group.（2）Establishment of hepatic fibrosis mouse model:WT and mfat-1 transgenic mice were randomly divided into 4 groups:WT-CCl₄,WT-Corn oil,mfat-1-CCl₄,mfat-1-Corn oil.（3）To collect the serum and liver tissue.1)Measurement of serum parameters:the levels of serum ALT and AST were detected by ELISA kits.2)Tissue sections:（1）the pathological changes of liver tissue were detected by HE staining,（2）the level of fibrosis was detected by Masson staining and Sirius red staining,（3）the activation level of HSCs was detected by immunohistochemicalα-SMA,（4）the degree of hepatocyte apoptosis was detected by TUNEL staining,（5）the relationship between mTOR signal and HSCs activation in liver tissue was detected by immunofluorescence double staining p-S6 andα-SMA.3)Molecular biology experiment:the expression of apoptosis-related proteins Bax,Bcl-2,Caspase-3,mTOR-related protein p-S6,α-SMA and Collagen-1 were detected by Western Blot.Results:（1）The mfat-1 gene was detected in mfat-1 transgenic mice by PCR.The results showed that mfat-1 gene was found in mfat-1 transgenic mice,while not in WT mice.At the same time,the polyunsaturated fatty acids in mice were detected by gas chromatography.The results showed that proportion of Omega-3/Omega-6 PUFAs was higher in mfat-1 mice than that in WT,indicating that mfat-1 mice could transform Omega-6 into Omega-3 PUFAs.（2）By detecting the levels of ALT and AST in serum,the results showed that the levels of ALT and AST were significantly higher in CCl₄ group than those in Corn oil group,indicating that the liver function was impaired during hepatic fibrosis;while compared to CCl₄ group,the levels of ALT and AST were significantly lower in mfat-1 mice,indicating that endogenous Omega-3 PUFAs rendered strong protective effects against CCl₄-induced chronic hepatic damage mice.（3）Endogenous Omega-3 PUFAs protect against CCl₄-induced liver injury.Compared to Corn oil group,the liver tissue structure was seriously damaged in CCl₄group,and with many hepatocyte necrosis and inflammatory cell infiltration,indicating that liver injury occurred in the CCl₄-induced hepatic fibrosis model mice;Compared to CCl₄ group,the liver tissue damage was significantly improved in mfat-1 mice.The results showed that the endogenous Omega-3 PUFAs rendered strong protective effects against CCl₄-induced chronic hepatic damage mice.（4）Endogenous Omega-3 PUFAs reduce CCl₄-induced HSCs activation.Compared to Corn oil group,the expression ofα-SMA was significantly up-regulated in CCl₄ group,indicating that HSCs was activated in CCl₄-induced hepatic fibrosis model mice.Compared to CCl₄ group,the expression ofα-SMA was significantly down-regulated in mfat-1 mice.At the same time,through Sirius red,Masson staining and Western Blot,it was found that in mfat-1 mice,the collagen protein was significantly lower than that in CCl₄ group.It is suggested that the endogenous Omega-3 PUFAs in the liver can reduce hepatic fibrosis through inhibiting the activation of HSCs.（5）Endogenous Omega-3 PUFAs inhibited the mTOR signaling pathway in CCl₄-induced liver fibrosis.Compared to Corn oil group,the expression of p-S6 was significantly up-regulated in CCl₄ group,indicating that mTOR pathway was activated in CCl₄-induced hepatic fibrosis model mice.Compared to CCl₄ group,the expression of p-S6 protein was lower in mfat-1 mice,indicating that endogenous Omega-3 PUFAs protected the liver against CCl₄-induced fibrosis by inhibiting the activation of mTOR signaling pathway.（6）Endogenous Omega-3 PUFAs decrease CCl₄-induced hepatocyte apoptosis.Compared to Corn oil group,the number of hepatocyte apoptosis was increased in CCl₄group,and the expression of hepatocyte apoptosis-related protein Caspase-3 and pro-apoptotic protein Bax were increased,while the expression of anti-apoptotic protein BCl-2 was decreased,indicating that hepatocyte apoptosis was increased in CCl₄-induced hepatic fibrosis model mice.Compared to CCl₄ group,the number of hepatocyte apoptosis was significantly lower in mfat-1 mice,and the expression of Caspase-3 and Bax were significantly decreased,while the expression of BCl-2 was increased.It is suggested that endogenous Omega-3 PUFAs could reduce the level of hepatocyte apoptosis.Conclusion:Endogenous Omega-3 PUFAs could inhibit the activation of HSCs,reducing the production of collagen fibers,reducing the hepatocyte apoptosis,and improving the level of liver function by regulating the mTOR signal pathway.Thus,these findings highlighted that endogenously synthesized n-3 PUFAs maybe a prospective therapeutic strategy for CCl₄-induced liver fibrosis.