Catechin Alleviates APAP-induced Liver Injury By Reducing Mitochondrial Oxidative Stress Through Inhibition Of JNK Signaling Pathway

Objectives: 1.To evaluate the effect of catechin antagonism on acetaminophen(APAP)liver injury at the whole animal and in vitro cellular levels,respectively.2.To reveal the mechanism of catechin antagonism on acetaminophen liver injury through the regulation of JNK signaling pathway at the cellular level.Research methods:1.To evaluate the effect of catechins on APAP-induced liver injury: mice were stained with 400 mg/kg APAP intraperitoneally to establish an animal injury model.Serum glutamic acid/glutamic oxalacetic transaminase(ALT/AST)levels were measured by Lai’s method,and the pathological changes of the liver were observed by H&E staining.2.Effect of catechins on APAP metabolizing enzyme activity: The expression of hepatic microsomal mixed function oxidase 450 was detected by in vitro enzyme activity assay.3.Effect of catechins on APAP-induced oxidative stress: The activity of Glutathione(GSH)and Superoxide dismutase(SOD)was detected by activity assay,and the level of Reactive oxygen species(ROS)was detected by fluorescence staining The levels of reactive oxygen species(ROS)were measured by fluorescent staining.4.The effects of catechins on APAP-induced mitochondrial dysfunction: the levels of Adenosine triphosphate(ATP)and mitochondrial respiratory chain complex I were detected by colorimetric assay,the expression of mitochondrial membrane potential was detected by fluorescence staining,the expression of Smac,AIF,Bax in hepatocytes was detected by Western-blot assay,and the protein expression of mitochondrial membrane potential was detected by Western-blot,The protein expression of Smac,AIF and Bax in hepatocytes and Mfn2,Drp1 and Bax in mitochondria were detected by Western-blot.5.Effect of catechins on APAP-induced JNK pathway: Protein expression of P-JNK in hepatocytes was detected by Western-blot assay.6.To verify the key role of JNK signaling pathway in the antagonism of APAP liver injury by catechins: the protein expression of P-JNK,Bax,AIF and Smac in hepatocytes was detected by Western-blot method in combination with the application of JNK activator anisomycin;the protein expression of Drp1,Mfn2 and Bax in mitochondria was detected;the protein expression of Adenosine triphosphate was detected by colorimetric method.Adenosine triphosphate(ATP)and mitochondrial respiratory chain complex I were detected by colorimetric assay,and the expression of mitochondrial membrane potential was detected by fluorescence staining;cell survival rate was detected by CCK8 assay.Results of the study:1.In vivo experiments revealed that after giving APAP to mice,serum ALT and AST levels increased,liver tissues showed a large number of hepatocyte necrosis,hepatic sinusoidal congestion and other manifestations,and the expression of antioxidant enzymes SOD and GSH decreased.After treatment with catechins,the levels of appeal indexes were reversed.2.The effects of catechin on CYP2E1,CYP1A2 and CYP3A4 enzyme activities were investigated in vitro.The results showed that 100 u M of catechin only inhibited CYP3A4 activity.These results suggest that inhibition of metabolic enzymes of APAP is a part of mechanisms why catechin alleviates the hepatotoxicity.3.In vitro,L02 cells were treated by CCK-8 and protein immunoblotting assay methods,and it was found that the survival rate of L02 cells was reduced and P-JNK protein expression was enhanced after APAP treatment,indicating that APAP could induce apoptosis and enhance JNK phosphorylation in hepatocytes.When catechin was given,the survival rate of L02 cells increased and the expression level of P-JNK was changed.This indicates that catechins can inhibit APAP-induced JNK protein phosphorylation in L02 cells and restore hepatocyte survival.4.The levels of mitochondrial functional indicators ATP,mitochondrial membrane potential and mitochondrial respiratory chain complex I were measured on L02 cells,and cytoplasmic and mitochondrial protein expression was detected by protein immunoblotting,and it was found that APAP reduced the levels of mitochondrial functional indicators and altered protein expression.After treatment with catechin administration,the levels of mitochondrial functional indicators were found to increase and reversed protein expression,indicating that catechin was able to reduce APAP-induced mitochondrial damage.Anisomycin,a JNK activator,was administered in the same way,and it was found that anisomycin attenuated the protective effect of catechin on hepatocytes,which fully demonstrated that catechin could attenuate the hepatotoxicity of APAP by inhibiting the JNK signaling pathway.Conclusion: Catechin attenuates APAP-induced liver injury and affects oxidative stress,increasing the expression of mitochondrial ATP,mitochondrial complex I,mitochondrial membrane potential and mitochondria-associated proteins,but these effects can be affected by the JNK activator anisomycin.Thus,we speculate that the mechanism of action of catechins antagonizing APAP-induced liver injury is related to their inhibition of JNK signaling pathway.