The Oxidative Stress In Endoplasmic Reticulum Regulates Aging By Modulating Mitophagy

Objective:To study the role of oxidative stress in endoplasmic reticulum on mitophagy and aging by employing the natural aging models both in vivo and in vitro,and try to elucidate the possible molecular mechanism underlying.Methods:1.SD rats aged 3 months and 24 months were employed as the natural aging model.Human cell lines HUVEC and WI38 cells were passaged to 50 generations to establish replicative cell senescence model.2.Gene manipulation on the key target molecules:GPx8 overexpression cell line was established by infected lentivirus packaged with antioxidant enzyme GPx8.Parkin knockdown cell line was established by infected lentivirus packaged with Parkin sh RNA.3.Senescence detection:β-gal staining and activity of cells were detected by kits;the m RNA and protein levels of p21,p16 and p27,markers of cell senescence,were detected by Q-PCR and Western Blot;CCK8 kit was used to detect cell proliferation activity.4.GPx8 detection:m RNA and protein levels of antioxidant enzyme GPx8 in the endoplasmic reticulum of rats and cells,were detected by Q-PCR and Western Blot.5.H₂O₂ level in the endoplasmic reticulum detection:ER-hyper adenovirus infected cells were co-located with ER Tracker probe,and H₂O₂ level in the endoplasmic reticulum was detected by confocal fluorescence imaging.6.Sensitivity to endoplasmic reticulum stress（ERS）detection:Tunicamycin was used to treat the cells,and the sensitivity of cells to ERS was detected by cell viability kit.7.Autophagy detection:Western Blot was used to detect the levels of LC3 and p62.8.Mitophagy detection:GFP-LC3 adenovirus-infected cells were co-localized with Mitotracker,and mitophagy level was detected by confocal fluorescence imaging.DNA was extracted by genomic DNA kit,and the copy number of mitochondrial DNA in cells was detected by fluorescence quantitative PCR.The mitophagy-related protein levels of PIKN1,Parkin,Nix and FUNDC1 were detected by Western Blot.Result:1.During the process of aging,the level of key antioxidant enzyme GPx8 in endoplasmic reticulum decreased.The results of senescence markers showed that the rats were naturally aging.The results of Q-PCR and Western Blot showed that the m RNA and protein levels of GPx8 in endoplasmic reticulum were significantly decreased in the aged rats,suggesting that GPx8in endoplasmic reticulum may be involved during the aging process.2.During the process of cell senescence,the oxidative stress level of endoplasmic reticulum increased,the sensitivity of ERS decreased,and the autophagy was inhibited.The results of senescence markers showed that the replication senescence models of cells were established successfully.Both Q-PCR and Western Blot results showed that m RNA and protein levels of the antioxidant enzyme GPx8 in the endoplasmic reticulum were significantly decreased during cell senescence.At the same time,ER-Hyper result showed that the level of H₂O₂ in endoplasmic reticulum increased.These results indicated that the oxidative stress in endoplasmic reticulum occurred druing the process of cell senescence.The sensitivity test results of ERS cells showed that the sensitivity of ERS cells decreased during cell senescence.In addition,Western Blot results showed that during cell senescence,LC3-II/LC3-I decreased,p62 level increased,indicating the autophagy level was inhibited.All these data suggested that the increased oxidative stress endoplasmic reticulum may lead to cell senescence by inhibiting autophagy.3.Overexpression of GPx8 inhibited endoplasmic reticulum oxidative stress,enhanced autophagy and alleviated cell senescence.ER-Hyper results showed that after the overexpression of GPx8,the H₂O₂ level in endoplasmic reticulum was reduced,and the oxidative stress level was inhibited.Western Blot results showed that after overexpression of GPx8,LC3-II/LC3-I was increased and p62 level was decreased,indicating the autophagy level was increased.The results of senescence marker detection showed that after the overexpression of GPx8,the coloration and activity ofβ-gal decreased,the cell proliferation activity increased,the m RNA level of cell senescence marker p21 decreased,suggesting the cell senescence was alleviated.These results indicated that oxidative stress in endoplasmic reticulum inhibited autophagy and promoted cell senescence.4.Inhibition of UPR pathway activity by specific inhibitor of UPR pathway antagonized the improvement of autophagy level and the alleviating effect of GPx8overexpression on cell senescence.The sensitivity of the cells to ERS showed that the sensitivity of the cells to ERS increased after GPx8 overexpression.However,the UPR pathway inhibitor treatment significantly inhibited the improvement of sensitivity to ERS by GPx8 overexpression.The levels of mitophagy related proteins showed that the mitophagy level increased after the overexpression of GPx8.However,after the administration of inhibitor,the levels of mitophagy related proteins Parkin and PINK1 decreased,the level of mt DNA increased,the fusion part of GFP-LC3 with mitochondria decreased,demonstrating the level of mitophagy was inhibited.The results of senescence marker detection showed that the cell senescence was alleviated after the overexpression of GPx8.However,after inhibitor administration,the coloration and activity of senescence markerβ-gal were increased,cell proliferation activity was decreased,m RNA levels of cell senescence markers p21,p27 and p16 were increased,indicating cell senescence was accelerated.All these results suggested that improving endoplasmic reticulum oxidative stress alleviated cell senescence by activating UPR pathway and promoting autophagy.5.The knockdown of Parkin,a key protein of mitophagy,inhibited the level of mitophagy and blocked the increase of autophagy level and the alleviating effect of GPx8 overexpression on cell senescence.After overexpression of GPx8 and knockdown the key protein of mitophagy of Parkin,the protein levels of Parkin and PINK1 were significantly decreased,the mt DNA level was significantly increased,the fusion part of GFP-LC3 and mitochondria was reduced,suggesting the level of mitophagy was inhibited.The results of senescence markers showed that after the inhibition of mitophagy,the coloration and activity of senescence markerβ-gal were increased,cell proliferation activity was decreased,the degree of cell senescence was aggravated,and the anti-senescence effect caused by the GPx8overexpression disappeared.All these results suggested that improving endoplasmic reticulum oxidative stress alleviated senescence by increasing the mitophagy level.Conclusions:1.During the process of aging,the oxidative stress in endoplasmic reticulum increases,the activity of UPR decreases,the sensitivity of ERS decreases,and the level of mitophagy decreases.2.Improving the oxidative stress of endoplasmic reticulum can increase the activity of UPR,increase the sensitivity to ERS,promote mitophagy and alleviate cell senescence.