Baicalin Activates The M-TOR Signaling Pathway To Promote The Repair Of Acetaminophen Liver Injury

Objective: In this study,we investigated the detoxifying and repairing effects of baicalin on acetaminophen（APAP）hepatotoxicity at the in vivo animal level,and further investigated its mechanism of promoting the repairing effect of APAP liver injury,so as to provide an experimental basis for further exploration of the hepatoprotective effect of baicalin.Methods:1.To evaluate the effect of baicalin on APAP-induced liver injury: mice were stained with 300 mg/kg APAP intraperitoneally to establish an animal injury model.The serum glutamic/glutamic transaminase（ALT/AST）levels were measured by Lai’s method,and the pathological changes of the liver were observed by H&E staining.2.Evaluation of the proliferative effect of baicalin on hepatocytes after APAP liver injury: m RNA expression of Hepatocyte growth factor（HGF）and Epidermal Growth Factor（EGF）in mouse liver was measured by real-time fluorescent quantitative PCR（RT-q PCR）.The expression of PCNA and Cyclin D1 in mouse liver was measured by Western-blot method.The expression of PCNA and Cyclin D1 in mouse liver was measured by Western-blot.3.To reveal the role of baicalin in activating the m-TOR signaling pathway: p62,LC-3B,p-m TOR and p-S6 protein expression were detected in mouse liver by Western-blot method.4.Verification of the key role of m-TOR signaling pathway in baicalin promoting the repair of APAP liver injury: detection of p62,LC-3B,p-m TOR,p-S6,PCNA and Cyclin D1 protein expression assay in mouse liver by Western-blot method in combination with the application of m-TOR inhibitor rapamycin;detection of serum glutathione/glutamate/AST levels by Lai’s method;pathological changes in the liver by H&E staining;cell proliferation by Beyo Click^TM Ed U method;and the number of PCNA-positive cells by immunohistochemical staining.Results:1.In the APAP-induced acute liver injury model of C57BL/6 mice,baicalin therapeutic administration had significant hepatoprotective effects: intraperitoneal injection of APAP（300 mg/kg）followed by gavage of baicalin（40,80 mg/kg）in mice for 1 hour significantly reduced APAP-induced elevated ALT and AST enzyme activity levels（p<0.05）;observation of liver H&E staining It was found that baicalin（40,80 mg/kg）significantly improved APAP-induced extensive hepatic hemorrhage and necrotic hepatocytes;after quantifying the area of injury,it was found that baicalin（40,80 mg/kg）significantly reduced the area of APAP-induced hepatic necrosis（p<0.01,p<0.05）.2.By observing the mitotic number of hepatocytes in liver histopathological sections,it was found that baicalin（40,80 mg/kg）significantly increased the mitotic number of hepatocytes（p<0.05）.edu cell proliferation results showed that baicalin（40,80 mg/kg）significantly enhanced edu fluorescence intensity（p<0.05）.Baicalin increased the expression of HGF and EGF m RNA reduced by APAP.Also PCNA immunohistochemical staining sections showed that baicalin（40,80 mg/kg）significantly increased the number of PCNA-positive cells（p<0.05）.Protein electrophoresis experiments revealed that baicalin significantly increased the expression of PCNA and Cyclin D1 proteins that were reduced by APAP.3.In the study of m-TOR signaling pathway,baicalein was found to decrease the expression of LC-3B protein increased by APAP and increase the expression of p62,p-m TOR and p-S6 protein decreased by APAP.4.The activation of m-TOR signaling pathway by baicalin was inhibited after the application of m-TOR inhibitor rapamycin,resulting in the abolition of the effect of baicalin in reducing ALT and AST enzyme activities and attenuating liver pathological damage（p<0.05）;The effect of baicalin in increasing the mitotic number of hepatocytes and the fluorescence intensity of liver sections EDU and the increased number of PCNA-positive cells（p<0.05）was also diminished after the application of the m-TOR inhibitor rapamycin.Protein electrophoresis experiments revealed that rapamycin significantly decreased the expression of PCNA and Cyclin D1 protein in the liver of mice increased by baicalein.Conclusion: Baicalin attenuated APAP-induced liver injury and promoted hepatocyte proliferation,increased the expression of EGF,HGF and PCNA,Cyclin D1 thereby accelerating liver repair,but these effects could be inhibited by rapamycin.Thus,we speculate that the mechanism of action of baicalin to promote liver repair is closely related to its activation of the m-TOR signaling pathway for pro-cell proliferation and inhibition of its mediated autophagy.