Effect Of ?-3 Polyunsaturated Fatty Acids On Liver Fibrosis

Objective:Recently,the research about the roles of?-3 polyunsaturated fatty acids?PUFAs?in prevention and treatment of diseases has drawn greater attention.However,so far,few researchs demonstrate the effect and mecinisams of?-3 PUFAs in liver fibrosis.In this study,we intend to observe the effect of?-3 PUFAs on liver fibrosis,and study the mechanisms involved in these process,which may provide a new way for the research on the occurrence and development of liver fibrosis.Methods:1.After the mice models were established,liver tissues were taken and examined by means of macroscopic morphology,HE staining,Sirius red staining and immunohistochemistry.2.The expression of?-SMA and col1?1 in liver tissues was detected by qRT-PCR and western blot.3.Hydroxyproline content in liver tissues was measured.4.The proliferation effect of DHA and EPA on LX-2 and HSC-T6 cells was measured by MTT.5.The effect of DHA and EPA on the expression of pro-fibrosis genes in LX-2 and HSC-T6 cells was measured by qRT-PCR.6.The expression of pro-fibrosis genes in liver tissues was measured by qRT-PCR.7.The protein expression of YAP/TAZ in control group and CCl₄ group was detected by western blot.8.The expression of Yap/Taz mRNA in primary isolated quiescent HSCs?day 3? and in vitro activated HSCs?day 12?was detected by qRT-PCR.The expression of Yap//Taz mRNA in primary isolated quiescent HSCs?day 3?from healthy and fibrosis mice was detected by qRT-PCR.9.The expression of YAP/TAZ in liver tissues was measured by western blot and IHC.10.After the treatment of DHA,EPA in LX-2 and HSC-T6 cells,the expression of YAP/TAZ was measured by western blot.11.Knockdown of YAP with siRNA,then perform western blot to detect the effect of DHA or EPA on pro-fibrosis genes expression.12.After the treatment of CHX,CHX+DHA,CHX+EPA in LX-2 cells,the expression of YAP/TAZ was measured by western blot.13.After the treatment of ethoal,DHA,MG132,MG132+DHA in LX-2 cells,the expression of YAP/TAZ was measured by western blot.Results:1.There were fewer nodules on hepatic surfaces in the fish oil/CCl₄ group compared to CCl₄ group,indicating lower degree of fibrosis.HE staining shows for gross morphology and the Sirius red staining for the determination of specific fibrotic regions in the liver tissues.Immunohistochemical staining revealed that CCl₄-treated liver exhibited increased expression of?-SMA and collagen1.Only faint traces of?-SMA and collagen1-positive cells were detected in fish oil+CCl₄ group.2.The expression of?-SMA and col 1?1 mRNA in liver tissues were upregulated by CCl₄,while decreased by fish oil,which were conformed by western blot.3.Hydroxyproline content showed an approximate 30%reduction in fish oil+CCl₄ group compared with CCl₄ group.4.The inhibition of proliferation by DHA and EPA is dose-and time-dependency.5.After the treatment of DHA or EPA in LX-2 and HSC-T6 cells,the expression of pro-fibrosis genes and?-SMA was decreased.6.Liver fibrosis-related genes were increased in CCl₄ group.7.YAP and TAZ proteins were over-expressed in CCl₄–induced liver fibrosis.8.Yap and Taz mRNAs were over-expressed in activated HSCs.9.Yap and Taz mRNAs were increased in CCl₄ liver tissue,while did not change in CCl₄+fish oil group.10.After the treatment of DHA or EPA,the expression of YAP/TAZ was decreased,while the expression of Yap/Taz mRNA did not change.11.The inhibition on pro-fibrosis genes of DHA or EPA did not strengthen after YAP knockdown.12.YAP/TAZ protein did not been changed after the treatment of CHX+DHA or CHX+EPA,compared with CHX treatment.13.YAP and TAZ proteins were increased after the treatment of MG132+DHA or MG132+EPA.Conclusions:1.DHA and EPA inhibit the proliferation of LX-2 and HSC-T6 in vitro.2.?-3 PUFAs down-regulate pro-fibrogenic genes in LX-2 and HSC-T6 cell lines.3.Fish oil attenuates nodules and hydroxyproline content in CCl₄-induced liver fibrosis in vivo.4.?-3 PUFAs down-regulate pro-fibrogenic genes in liver fibrosis tissue.5.YAP and TAZ are over-expressed in liver fibrosis and fish oil decreases the protein level of YAP/TAZ without changing their mRNA level in liver tissue.6.DHA and EPA decrease the protein level but not the mRNA level of YAP/TAZ in liver fibrosis tissue and HSCs.7.DHA and EPA induce the degradation of YAP/TAZ through proteasome-dependent pathway,thus reducing the expression of pro-fibrogenic genes and attenuating liver fibrosis.