The Role And Mechanism Of ATM In Hydroquinone-induced Autophagy In Hepatocytes

Objective:Autophagy is the predominant intracellular degradation system that delivers cytoplasmic contents to the lysosome and degrades them.However,the purpose of autophagy is not simply to remove substances,but to act as a dynamic recycling system that provides power and energy for cell renewal and homeostasis.Previous studies in our group elucidated that hydroquinone(HQ)can induce autophagy in hepatocytes and found its effect on DNA damage in hepatocytes.In recent years,the role of ataxia-telangiectasia mutated kinase(ATM)in autophagy has been gradually discovered.ATM is an important member of the phosphatidylinositol-3 kinase-related kinase family and has been identified due to its key role in the DNA damage response.Currently,the role of ATM in autophagy and its regulatory mechanism have been extensively studied under the effects of hypoxia,starvation,reactive oxygen species,and ionizing radiation,but the role of ATM in HQ-induced autophagy and its regulatory mechanism have not been clarified.This study aims to elucidate the role of ATM in HQ-induced autophagy and its regulatory mechanism,in order to provide a scientific and theoretical basis for the prevention and treatment of HQ poisoning,as well as to explore new ways of ATM regulation of autophagy and further understand its role in cellular dynamics and homeostasis.Methods:(1)The role of ATM activity inhibition in HQ-induced autophagy in hepatocytes and mechanism study: firstly,the optimal action concentration of ATM inhibitor(KU-60019)to inhibit ATM protein activity without affecting cell viability was determined by CCK-8 and Western blot;secondly,after the action of HQ combined with ATM inhibitor on L02 hepatocytes,the effect of HQ was investigated by Inverted microscopy to observe cell morphology,CCK-8 method to detect cell viability,flow cytometry to detect apoptosis,transmission electron microscopy to observe autophagic morphology,m Cherry-GFP-LC3 B tandem fluorescent protein to detect autophagic flow,MDC method to detect autophagic vesicle aggregation status,Western blot to detect the effect of autophagy-related protein and its autophagic signaling pathway protein expression,and Immunofluorescence assay to detect subcellular localization and expression of autophagy-associated proteins.(2)Study on the role and mechanism of ATM gene silencing in HQ-induced hepatocyte autophagy: firstly,the defective cell line was constructed using sh RNA gene technology,and the interference effect of the target gene was identified by Western blot to confirm the stability of ATM-deficient cells;secondly,the cell morphology was observed by inverted microscopy,the expression of autophagy-related proteins was detected by Western blot,and the subcellular localization and expression of autophagy-related proteins were detected by immunofluorescence assay under the effect of HQ.Results:1.Effect of different concentrations of HQ on the expression of ATM phosphorylated protein in hepatocytesThe results of Western blot showed that the relative grayness values of p-ATM in the 40 μmol/L and 80 μmol/L concentration groups were significantly higher than those in the 0 μmol/L concentration group(P<0.05);the relative grayness values of p-ATM in the 160 μmol/L concentration group were significantly lower than those in the 0 μmol/L concentration group(P<0.05).Considering the previous research results of our group,the optimum concentration of HQ in this study was determined to be 80μmol/L.2.Effect of ATM inhibitor on hepatocyte viability and ATM phosphorylated proteinsThe results of CCK-8 experiments showed that only the 1‰ DMSO group significantly inhibited cell survival compared with the control group(P<0.05).western blot experiments showed that 0.5,1,5 and 10 μmol/L KU-60019 significantly reduced the expression of ATM phosphorylated protein compared with the control group(P<0.01).Combining the results of the above two experiments,subsequent experiments will use a low dose of 0.5 μmol/L KU-60019 and the corresponding 0.05‰ DMSO as the experimental dose.3.ATM activity inhibition does not affect the morphology,viability and apoptosis of HQ-acting hepatocytesThe results of inverted microscopy showed that no significant changes in cell morphology occurred in all treatment groups compared with the control group.The results of the CCK-8 assay showed that the cell survival rate was significantly lower in the HQ and HQ+KU groups compared with the control group(P<0.05);the cell survival rate was not significantly affected in the HQ+KU group compared with the HQ group(P>0.05).The results of apoptosis assay showed that the apoptosis rate was significantly higher in both the HQ and HQ+KU groups compared with the control group(P<0.05);there was no significant change in the apoptosis rate in the HQ+KU group compared with the HQ group(P>0.05).It showed that inhibition of ATM activity did not affect the survival rate and apoptosis rate of hepatocytes by HQ.4.ATM activity inhibition reduces HQ-induced autophagy levels in hepatocytesThe results of transmission electron microscopy showed that autophagosomes were visible within the control,DMSO,and HQ+KU groups;a small number of autophagosomes were visible in the KU group;and an increase in autophagosomes and autophagolysosomes could be observed in the HQ group.The number of autophagosomes was reduced in the HQ+KU group compared with the HQ group.m Cherry-GFP-LC3 B tandem fluorescent protein results showed that the number of yellow fluorescent spots was increased in the HQ group compared with the control group.The number of yellow fluorescent spots was significantly reduced in the HQ+KU group compared with the HQ group(P<0.05),and the red lamellar spots also showed a trend of reduction.western blot experiments showed that inhibition of ATM activity by the effect of HQ could upregulate the expression of p62 protein and downregulate the expression of LC3Ⅱ/LC3Ⅰ protein and Beclin 1 protein;immunofluorescence experiments The results showed that inhibition of ATM activity by HQ enhanced the fluorescence intensity of p62 protein and decreased the fluorescence intensity of LC3 protein and Beclin 1 protein;increased the distribution of p62 protein in the cytoplasm and decreased the distribution of LC3 protein and Beclin 1 protein in the cytoplasm.The results of MDC positive cell rate by flow cytometry showed that the MDC positive cell rate was significantly higher in the HQ group compared with the control group(P<0.05),and there was no significant difference between the MDC positive cell rate in the DMSO,KU and HQ+KU groups(P>0.05).5.ATM activity inhibition reduces HQ-induced hepatocyte autophagy via AMPK/m TOR signaling pathwayThe results of Western blot assay showed that the expression of PI3 K,p-PI3 K,AKT,p-AKT,p-AMPK proteins was significantly lower in the HQ+KU group compared with the control group(P<0.05),and the expression of m TOR and p-m TOR proteins was significantly higher in the KU and HQ+KU groups(P<0.05).Compared with the HQ group,the expression of PI3 K,p-PI3 K,AKT,p-AKT,AMPK,p-AMPK proteins was significantly lower(P<0.05),and the expression of m TOR,p-m TOR proteins was significantly higher(P<0.05)in the HQ+KU group.6.ATM gene silencing reduces HQ-induced autophagy levels in hepatocytesThe results of inverted microscopy showed that the cell morphology and cell number of L02-ATM-sc and L02-ATM-sh cells under the effect of HQ were not significantly changed compared with normal L02 cells.western blot showed that the ratio of LC3Ⅱ/LC3Ⅰ protein was significantly lower in L02-ATM-sh cells compared with normal L02 cells under the effect of HQ(P<0.05);the expression of p62 protein was elevated and that of Beclin 1 protein was decreased,but none of the differences were statistically significant.Immunofluorescence results showed that the fluorescence intensities of LC3 and Beclin 1 proteins were decreased and that of p62 protein was increased in the L02-ATM-sh+HQ group compared with the L02+HQ group.Conclusion:(1)HQ can induce the increase of ATM phosphorylation level in hepatocytes;(2)ATM activity inhibition reduces the level of HQ-induced autophagy in hepatocytes;(3)Inhibition of ATM activity enhances the negative regulation of hepatocyte autophagy by m TOR and can regulate it through the AMPK/m TOR signaling pathway;(4)ATM gene silencing reduces the level of HQ-induced autophagy in hepatocytes.