Melatonin Alleviates Liver Fibrosis Through PI3K/AKT Signaling Pathway In Rats

Backgrounds:Liver fibrosis is a process of repeated injury and repair of chronic liver diseases caused by various factors.If it is not effectively treated,it will eventually progress to liver cirrhosis,liver cancer and so on.Studies have demonstrated that liver fibrosis is reversible.Activation of hepatic stellate cells?HSCs?is an important process leading to liver fibrosis.Studies have shown that the inhibition of PI3K/AKT signaling pathway could alleviate liver fibrosis.Melatonin?MEL?has anti-inflammatory and anti-fibrotic effects,but its mechanism of anti-fibrosis is unclear.In this study,the relationship between melatonin antifibrosis and PI3K/AKT signaling pathway was further explored in rats with liver fibrosis induced by carbon tetrachloride?CCl₄?and HSC-T6 cells induced by transforming growth factor-?1?TGF-?1?.Methods:Animal experiment:Male SD rats were randomly divided into 5 groups:normal group,model group,melatonin low dose group?2.5mg/kg,MEL-1 group?,melatonin medium dose group?5.0mg/kg,MEL-2 group?,and melatonin high dose group?10.0mg/kg,MEL-3 group?,15 in each group.The rat model of liver fibrosis was established by the subcutaneous injection of CCl₄ for 6 weeks.The expression of PI3K/AKT signaling pathway in liver tissue was detected by immunohistochemistry and Western Blotting.Cell experiment:HSC-T6 cells were divided into 5 groups:control group,model group,melatonin low concentration group?1nmol/L,MEL-1 group?,melatonin medium concentration group?1?mol/L,MEL-2 group?,and melatonin high concentration group?0.1mmol/L,MEL-3 group?.After being cultured for 24h,the cells were cultured in FBS-free medium.Except the control group,the other groups were all added with TGF-?1?10 ng/ml?,and different concentrations of melatonin groups were added with the corresponding concentrations of melatonin.After drugs incubation for48h,the effect of melatonin on the proliferation of HSC-T6 cells was detected by MTT;the expression of PI3K/AKT signaling pathway in HSC-T6 cells was detected by immunohistochemistry and Western Blotting;the expressions of?-SMA and Collagen I in HSC-T6 cells were detected by qPCR.Results:In animal experiments,the results of immunohistochemistry showed that the expression of P-AKT was significantly increased in the model group compared with the normal group?P<0.01?;Compared with the model group,the expression of P-AKT was significantly reduced in each dose of melatonin group?P<0.01?.Western Blotting results showed that the expressions of PI3K and P-AKT were significantly increased in the model group compared with the normal group?P<0.01?;Compared with the model group,the expressions of PI3K and P-AKT were significantly reduced in each dose of melatonin group?P<0.01?.In cell experiments,MTT results showed that TGF-?1 could significantly induce the proliferation of HSC-T6 cells compared with the control group?P<0.01?;compared with the model group,melatonin high concentration group could significantly inhibit the proliferation of HSC-T6 cells?P<0.01?.The results of immunohistochemistry showed that the expression of P-AKT was significantly increased in the model group compared with the control group?P<0.01?;Compared with the model group,the expression of P-AKT was significantly reduced in each concentration of melatonin group?P<0.01?.Western Blotting results showed that the expressions of PI3K and P-AKT were significantly increased in the model group compared with the control group?P<0.01?;Compared with the model group,the expressions of PI3K and P-AKT were significantly reduced in each concentration of melatonin group.qPCR results showed that the expressions of?-SMA and Collagen I were significantly increased in the model group compared with the control group?P<0.01?;Compared with the model group,the expressions of?-SMA and Collagen I were significantly reduced in each concentration of melatonin group?P<0.01?.Conclusions:1)Melatonin can inhibit the expressions of?-SMA and Collagen I,possibly by inhibiting the activation of HSCs and reducing the synthesis of extracellular matrix to alleviate liver fibrosis;2)Melatonin can inhibit the expressions of PI3K and P-AKT,possibly by inhibiting PI3K/AKT signaling pathway to alleviate liver fibrosis.