DJ-1 Participates In The Mechanism Of Protective Autophagy To Protect H9C2 Cells From Hypoxia Injury Through RACK1/AMPK/mTOR Pathway

Objective:Ischemic heart disease(IHD)is determined by an imbalance of the cross talk between myocardial energy state and coronary blood flow.It has occupied the leader in the adverse cardiovascular events.From an evolutionary perspective,autophagy is commonly regarded as highly conserved degradation mechanism.It is conductive to cell survival in response to stress and has been proved an adaptive response during myocardial ischemia/hypoxia.As a multifunctional protein,DJ-1 is involved in a variety of cellular life processes including the regulation of autophagy.This research aims to study DJ-1 participates in the mechanism of protective autophagy in hypoxic H9c2 cells.Methods:1.DJ-1 knockdown(shDJ-1),overexpression(LV-DJ-1)and overexpression mutant of C106 reduced state(DJ-1(C106S))stable H9c2 cell lines were constructed by infecting H9c2 cells with lentivirus vectors p LVX-shDJ-1,p LVX-DJ-1,and p LVX-DJ-1(C106S)for 48 h,and then positive clones were screened with puromycin.Western Blot was used to evaluate the expression of Flag or DJ-1 to identify stable cell lines.2.To determine the effects of different expression of DJ-1 on protective autophagy in hypoxic H9c2 cells,we cultured the H9c2,H9c2/shDJ-1 and H9c2/LV-DJ-1 cells pretreated with or without 5 m M autophagy inhibitor3-Methyladenine(3-MA)for 12 h.Cell injury was achieved by hypoxia for 3 h.The expression of autophagy markers LC3-II/LC3-I and P62 were assessed by Western Blot.CCK-8 and LDH kits were used to evaluate the cell viability and degree of cell damage.3.Cell hypoxia model was established in H9c2,H9c2/shDJ-1 and H9c2/LV-DJ-1 cells.To determine whether DJ-1 could activate AMPK/mTOR pathway in the process of the regulation protective autophagy in hypoxic H9c2 cells,the expression of AMPK,p-AMPK,mTOR and p-mTOR were detected by Western Blot.4.Cell hypoxia model was established in H9c2,H9c2/shDJ-1,H9c2/LV-DJ-1cells and H9c2/DJ-1(C106S)cells.To determine whether DJ-1 interacts with RACK1 in a C106-dependent manner to form complex in response to autophagy that is responsible for activating the AMPK/mTOR pathway,the change of co-localization and interaction of DJ-1 and RACK1 were evaluated by Immunofluorescence co-localization and Immunoprecipitation,and the expression of AMPK,p-AMPK,mTOR and p-mTOR were assessed by Western Blot.5.The H9c2 and H9c2/LV-DJ-1 cells were transfected with 50 n M RACK1 siRNA or negative control(NC)siRNA for 48 h.Cell injury was achieved by hypoxia for 3 h.To determine the effects of silencing RACK1 on AMPK/mTOR signaling pathway and protective autophagy induced by DJ-1 in hypoxic H9c2 cells,we measured the following indicators.(1)The expression of RACK1,AMPK,p-AMPK,mTOR,p-mTOR,LC3-II/LC3-I and P62 were assessed by Western Blot.(2)The number of autophagosomes was observed by Transmission Electron Microscopy.(3)CCK-8 and LDH kits were used to evaluate the cell viability and the degree of cell damage.6.The H9c2 and H9c2/LV-DJ-1 cells were incubated with or without 10 μM Compound C for 12 h prior to hypoxia.To determine the upstream and downstream relationship between DJ-1 and RACK1/AMPK/mTOR signaling pathway,and the effect of blocking AMPK/mTOR signaling pathway on protective autophagy by DJ-1induced in hypoxic H9c2 cells,we measured the following indicators.(1)The expression of AMPK,p-AMPK,mTOR,p-mTOR,LC3-II/LC3-I and P62 were assessed by Western Blot.(2)Autophagy flux changes were observed with the confocal scanning microscope.(3)The number of autophagosomes was detected by Transmission Electron Microscope.(4)CCK-8 and LDH kits were used to evaluate the cell viability and the degree of cell damage.Results:1.Compared with the H9c2 cells,DJ-1 was downregulation in H9c2/shDJ-1cells significantly.In addition,DJ-1 and Flag were expressed significantly in H9c2/LV-DJ-1 cells and H9c2/DJ-1(C106S)cells.The above results indicated that H9c2/shDJ-1,H9c2/LV-DJ-1 and H9c2/DJ-1(C106S)cell lines were successfully constructed.2.In contrast to hypoxia group,low DJ-1 expression significantly inhibited hypoxia-induced autophagy in H9c2 cells,that is,LC3-II/LC3-I ratio was significantly decreased,P62 expression was increased,and cell damage was further aggravated,including decreased cell viability and improved LDH release.The high expression of DJ-1 further induced autophagy in hypoxia H9c2 cells,that is,increased LC3-II/LC3-I ratio,decreased P62,and significantly alleviated hypoxia damage,including increased cell viability and decreased LDH release.Concerns were raised that the above effects were reversed in H9c2/LV-DJ-1 cells pretreatment with3-MA.3.Compared to the hypoxia group,DJ-1 overexpression further elevated the AMPK phosphorylation level and reduced mTOR phosphorylation.In contrast,downregulation of DJ-1 produced the opposite effect.The above results indicated that DJ-1 could induce cytoprotective autophagy against hypoxia-induced injury by activating the AMPK/mTOR pathway.4.The results of Immunofluorescence co-localization showed that DJ-1 and RACK1 were mainly distributed in cytoplasm.The low expression of DJ-1 presented an attenuated degree of co-localization and interaction between DJ-1 and RACK1,while high expression of DJ-1 presented a heightened degree of co-localization and interaction between DJ-1 and RACK1.It is noteworthy that,due to the mutation of C106 site of DJ-1,the above effects caused by the high expression of DJ-1 was reversed,along with decreased the expression of p-AMPK,increased the expression of p-mTOR.The above results indicated that DJ-1 interacted with RACK1 in a C106-dependent manner in H9c2 cells,thus activating AMPK/mTOR signaling pathway.5.The deletion of RACK1 in DJ-1 overexpression H9c2 cells compromised activation of the AMPK/mTOR pathway.Meanwhile,silencing of RACK1 resulted in cell viability decrease,LDH release increase,the number of autophagosomes decrease,a reduction in LC3-II/LC3-I and accumulation of the P62 compared to the NC group.The above results indicated that RACK1 plays a key role in activation of the AMPK/mTOR signaling pathway,and the silence of RACK1 inhibits DJ-1-induced protective autophagy in H9c2 cells.6.The expression of p-AMPK decreased and p-mTOR increased obviously compared with DMSO control group,while it has no effect on DJ-1 and RACK1 in H9c2/LV-DJ-1 cells pretreatment with the AMPK inhibitor Compound C.In addition,with Compound C,the effects of cytoprotective autophagy induced by DJ-1 high expression were weakened,namely,reduced LC3-II/LC3-I ratio,raised P62,decreased autophagosome number and autophagy flow,LDH release increase and cell viability decrease.The above results indicated that DJ-1 and RACK1 serves as the key upstream factor of activation the AMPK/mTOR pathway,namely,DJ-1 interacted with RACK1,and then activated AMPK/mTOR pathway,participated in the regulation of protective autophagy,and ultimately protected H9c2 cells from hypoxia injury.Conclusion:In this experiment,it is clear that DJ-1 interacts with RACK1 in a C106-dependent manner,and then activates AMPK/mTOR signaling pathway,participates in the regulation of protective autophagy in hypoxic H9c2 cell.