The Role Of Autophagy In Liver Fibrosis And The Effect Of Melatonin On It

Backgrounds:Liver fibrosis represent a common pathological procedure caused by chronic liver injury,containing many signing pathways,and finally leads to liver cirrhosis.Recently,studys have reported that liver fibrosis or even early liver cirrhosis could be reversed under effective therapy.Researches indicate that hepatic stellate cells play crucial role in liver fibrosis.Autophagy in wildly found in eukaryotes cells.In normal cells,aged organelles and structures would be engulfed by autophagosome and delivered to mitochondrion for beita-oxidation to provide energy for cells.In hepatic stellate cells,lipid droplets stored in the cells could be engulfed by autophagosome and delivered to mitochondrion for beita-oxidation to provide energy for activation of HSCs.Studys have reported that liver fibrosis could be alleviated byinhibiting autophagy in HSCs.Melatonin has powerful anti-inflammatory effects.In our previous study,we found that melatonin could alleviate fibrosis by reducing oxidative stress and inhibiting TGF-β/Smad signing pathways.However,it is still mysterious how melatonin affects autophagy in liver fibrosis.In our present study,liver fibrosis models were established by CCl₄ and HSC-T6 cells were stimulated by PDGF-BB to explore the effect of melatonin on liver fibrosis and autophagy.Methods:In vivo,Seventy-five Male SD rats（180g²20 g）were divided into five groups randomly:the normal group,the model group and the melatonin-treated groups（2.5 mg/kg,5.0 mg/kg,10.0 mg/kg;n=15）.In model group and melatonin-treated group,CCl₄（3 ml/kg）diluted with peanut oil（40%CCl₄ in peanut oil,vol/vol）was administered by hypodermic injection twice a week for 6 weeks to establish liver fibrosis models.HE staining and VG staning were performed to evaluate pathology.Immunohistochemical staining,western blotting and qRT-PCR were performed to evaluate autophagy level.In vitro,The HSCs were divided into the followinggroups:controlgroup,modelgroupandmelatonintreated groups（concentration of 1nmol/L,1μmol/L and 0.1mmol/L）.After being cultured for24h,they were replaced with FBS-free medium,and all groups were given with PDGF-BB（10ng/ml）excepted the control group.Besides,melatonin ofdifferent concentrations（1nmol/L,1μmol/L and 0.1mmol/L）were added immediately in three treated groups.After incubated for forty-eight hours,the proliferation of HSCs was detected by MTT assay,and the levels of autophagy were detected to assess by western blotting and qRT-PCR.Results:In vivo,the pathology results showed that liver injury were reduced compared with model group（P<0.01）.Immunohistochemical staining showed that the expression ofα-SMA and Beclin1 were up-regulated in model group compared with normal group,and that ofα-SMA and Beclin1 were downregulated in treatment groups compared with model group（P<0.01）.The results of Western blotting showed that in treatment group,the expression ofα-SMA,Beclin1,LC3 were reduced in melatonin treated groups compared with model group（P<0.05）.The result of qRT-PCT showed that Beclin1 and LC3B related genes were up-regulated in model group compared with the normal group,and the genes could be down-regulated in melatonin treated groups compared with the model group（P<0.05）.In vitro,MTT assay showed that melatonin could inhibit the proliferation of hepatic stellate cells（P<0.01）.Western blotting showed that the expression ofα-SMA,COLⅠ,Beclin1 and LC3Ⅱwere up-regulated in model group compared with the control group（P<0.05）,and that ofα-SMA,COLⅠ,Beclin1 and LC3Ⅱwere reduced in melatonin treated groups compared with model group（P<0.05）.qRT-PCR showed that the expression of Beclin1 and LC3B related genes were up-regulated in model group compared with control group（P<0.01）,and the expression of the genes were reduced in melatonin treated groups compared with model group（P<0.05）.Conclusion:1)Autophagy is associated with the development of liver fibrosis and may play a role in the activation of hepatic stellate cells;2)Melatonin can inhibit liver fibrosis and inhibit autophagy of hepatic stellate cells may be one of its effects.