The Influence And Mechanism Of G6PD On Autophagy Induced By Benzoquinone

Benzene is a common industrial raw material and environmental pollutant.Chronic benzene exposure can cause damage to the hematopoietic system,including the decrease of leukocytes,granulocytopenia and thrombocytopenia and severe aplastic anemia,myelodysplastic syndrome and even leukemia.1,4-benzoquinone is one of the ultimate metabolites of benzene,and the toxicity mechanism of benzene is mainly related to oxidative damage,gene mutation and chromosomal aberration caused by its oxidative metabolites.Glucose-6-phosphate dehydrogenase(G6PD)deficiency is a common haemolytic disease,commonly known as "broad bean disease",affecting nearly 400 million people worldwide.G6 PD is the initial enzyme of the pentose phosphate pathway,which can protect cells from reactive oxygen species(ROS)damage by catalyzing the synthesis of NADPH and maintaining the reductive nature of glutathione.Autophagy,also known as "self-phagocytosis," is a process of cell degradation and recycling of intracellular components and organelles.Autophagy can provide cells energy and maintain the homeostasis through the breakdown of cellular components,usually activated by starvation,hypoxia,oxidative stress,and inflammatory response.Our previously study found that G6 PD deficiency could increase the oxidative damage and cytotoxicity induced by benzoquinone exposure in K562 cells.This study will further investigate the effect of G6 PD deficiency on benzoquinone-induced autophagy and its mechanism.Firstly,K562 cells were treated with 1,4-BQ to study the effect and mechanism of 1,4-BQ on autophagy in K562 cells;Secondly,the autophagy level of G6 PD defective K562 cells induced by 1,4-BQ was detected,through constructing K562 cells line with G6 PD gene silencing,to study the role of G6 PD in the regulation of autophagy induced by 1,4-BQ.Then,glutathione(GSH)was added to detect the changes of autophagic activity in G6 PD defective K562 cells induced by 1,4-BQ,and to explore the possible mechanism of G6 PD regulation of autophagy induced by 1,4-BQ.一.Effect and mechanism of 1,4-benzoquinone on autophagy in K562 cellsK562 cells were treated with with 0,10,20μmol/L of benzoquinone.The proliferation of K562 cells was dected with MTT assay,qualitative and quantitative autophagy level by fluorescent staining and flow cytometry assay using CYTO-ID fluorescence probe,lysosome pH value by acridine orange staining method,and the gene and protein expression of LC3 and P62 by real-time quantitive PCR and Western blot,respectively.The results showed that the CYTO-ID autophagic activity in K562 cells increased,lysosome pH value decreased,and gene and protein expression of LC3 and P62 were significantly up-regulated after exposed to 1,4-BQ.The results suggest that 1,4-BQ may induce autophagy activity in K562 cells or inhibit autophagy flow.In order to study the effect of 1,4-BQ on autophagy flow in K562 cells,the autophagy inhibitor LY294002 was added to detect the change of autophagy level induced by 1,4-BQ in K562 cells.The results showed that CYTO-ID autophagy level and LC3-II protein expression level were remarkably declined in 1,4-BQ+LY294002 group than that of 1,4-BQ group(P<0.05).Then the autophagy blocker CQ was added to detect the effect of autophagy blocking on autophagy induced by 1,4-BQ in K562 cells.The results showed that the CYTO-ID autophagy level and the LC3-II protein expression induced by 1,4-BQ were not significantly changed after the treatment of CQ(P>0.05).In summary,this study found that 1,4-BQ could increase the autophagy level of K562 cells,while blocking autophagy flow.The expression of key proteins in the PI3K/AKT/mTOR signaling pathway were detected in 1,4-BQ-infected K562 cells.The results showed that the expression of PI3K(P85)protein in K562 cells decreased after 1,4-BQ exposure,and the phosphorylation level of AKT and mTOR was inhibited(P<0.05).It indicated that 1,4-BQ activated K562 cells autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway.To study the role of autophagy in the toxicity of 1,4-BQ-induced K562 cells,the proliferation was dected with MTT assay,ROS level and apoptosis rate by flow cytometry,and lipid peroxidation by elisa assay when autophagy was inhibited by treating with LY294002.The results showed that the proliferation inhibition rate,ROS,apoptosis and lipid peroxidation in K562 cells caused by 1,4-BQ were decreased in the presence of LY294002(P<0.05).The study suggested that inhibition of autophagy by LY294002 could reduce the oxidative damage in K562 cells caused by 1,4-BQ.二.Effect of G6 PD deficiency on autophagy in K562 cells induced by 1,4 benzoquinone.G6PD plays an important role in protecting cells from damage by oxidants,especially peroxides,and G6 PD deficiency was reported that enhanced the oxidative damage of benzoquinone to K562 cells.Firstly,stable K562 cells line with G6 PD gene silencing was constructed.The morphological characteristics of autophagic structures in G6 PD low activity K562 cells and normal K562 cells were detected by transmission electron microscopy,the gene and protein expression of LC3 and P62 by real-time quantitive PCR and Western blot,respectively.The results showed that compared with G6 PD normal cells,autophagy level in G6 PD low-expression cells was significantly decreased,which showed that the number of autophagosomes and autophagosomes decreased,the expression level of LC3-II protein decreased and the mRNA expression level of LC3 decreased correspondingly(P<0.05).This study indicated that the low expression of G6 PD inhibited the autophagy activity of K562 cells.Secondly,G6 PD low activity K562 cells and normal K562 cells were treated with 0,10 and 20μmol/L 1,4-BQ.And autophagy level was detected.The results showed that after 1,4-BQ treatment,the number of autophagosomes and autolysosomes in G6 PD low-expression cells were significantly increased,the mRNA expression level of LC3 and P62 increased and the protein expression of LC3-II and P62 were also up-regulated(P<0.05).The results indicate that G6 PD deficiency increased the autophagy level of K562 cells induced by 1,4-BQ.Finally,we treated G6 PD low activity K562 cells and normal K562 cells with 0,20μmol /L 1,4-BQ and intervened with 1.5 mmol/L GSH respectively.The results showed that after GSH intervention,the protein expression of LC3-II and P62 were significantly decreased both in G6 PD low expression cells and normal K562 cells,and there was no diffence in two cells(P>0.05).In addition,the expression of PI3K(P85)protein increased after GSH intervention,and the expression of AKT,p-AKT,mTOR and p-mTOR proteins increased correspondingly.The above results showed that GSH could reduce the autophagy induced by 1,4-BQ in G6 PD low activity K562 cells by activating PI3K/AKT/mTOR signaling pathway.ConclutionThe study found that 1,4-BQ activated K562 cell autophagy by inhibiting PI3K/AKT/mTOR pathway,and affected the degradation process of autophagy,which block the autophagy flow to a certain extent.Then,inhibition of autophagy by LY294002 could reduce the oxidative damage and cytotoxicity in K562 cells caused by 1,4-BQ.Furthermore,we successfully established the G6 PD low activity of K562 cell model and found that G6 PD deficiency increased the autophagy level of K562 cells induced by 1,4-BQ,and GSH could reduce the autophagy induced by 1,4-BQ by activating PI3K/AKT/mTOR signaling pathway.These studies provide a theoretical basis for the mechanism of G6 PD deficiency in increasing the susceptibility to benzoquinone toxicity.