SARS-COV-2 Spike Protein Promotes RPE Cell Senescence Via The ROS/P53/P21 Pathway In Vivo

BackgroundThe Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)has triggered a massive global epidemic since it was first reported in 2019,is highly infectious,and can cause multiple organ damage through the body.A growing number of studies have demonstrated the ability of SARS-CoV-2 to infect various tissues of the fundus.Numerous studies have also reported the development of fundus inflammation and microangiopathy in virus-infected and recovered individuals.These case reports suggest the existence of complex effects of SARS-CoV-2 on fundus tissues,but studies of SARS-CoV-2-associated fundus lesions are scarce and the mechanisms of occurrence are unclear.Current studies suggest that the disruption of the blood-retinal barrier is crucial to SARS-CoV-2-mediated fundus pathology,and RPE cells are the structural basis of the blood-retinal outer barrier,and that senescence of RPE cells is closely related to a variety of chronic fundus pathologies such as AMD.Stimulation by different factors can induce an increase in ROS levels in RPE cells and trigger cellular senescence through mechanisms such as P53/P21,then loss of original cellular functions.Current studies have shown that SARS-CoV-2 can induce different cellular senescence processes through upregulation of interferon and Toll-like receptors,triggering chronic pathological changes in the corresponding organs.As one of the target organs of the virus,viral receptors are expressed in the cornea,conjunctiva,iris,RPE and retinal capillaries,which suggests that SARS-CoV-2 can trigger chronic damage in the eye by inducing the aging process of ocular cells,but the specific mechanism is not yet studied.In this experiment,we will research the effect of SARS-CoV-2 spike protein on human retinal pigment epithelial senescence and investigate the mechanism of its occurrence.ObjectThe purpose of this experiment was to investigate the effect of SARS-CoV-2 S protein on human retinal pigment epithelial cells in vitro and to investigate the mechanism of S protein-mediated senescence of RPE cells.To provide ideas for the study of the mechanism of SARS-CoV-2-mediated chronic damage in the fundus,to explore the regulatory relationship between SARS-CoV-2 S proteins and chronic damage in the retina,and to provide a theoretical basis for the prevention and treatment of COVID-19 sequelae.Methods1.To detect the effects of SARS-CoV-2 S protein on the growth,cell cycle and senescence of RPE cells by using RTCA system,flow cytometry,β-Gal staining and RT-qPCR using administration of S protein or transfection of Flag-S plasmid.2.Validate the role of P21 in the senescence process of RPE cells induced by ectopic Flag-S or administration of S protein through P21 siRNA transfection3.Treat RPE cells with ROS inhibitor NAC and detect the role of ROS in the senescence process induced by administration of S protein or ectopic Flag-S in RPE cells using flow cytometry,β-Gal staining and immunoblotting.4.Cells were pretreated with NF-κB pathway inhibitor BAY for 30 min,and the role of NF-κB pathway in the senescence process of RPE cells induced by administration of S protein or ectopic Flag-S was detected using immunoblotting,RT-qPCR and other methods.5.The role of endoplasmic reticulum stress in the senescence process of RPE cells induced by administration of S protein or ectopic Flag-S was verified using immunofluorescence to detect the localization of S protein in cells and immunoblotting to detect endoplasmic reticulum stress-related proteins.Results1.Addition of S protein or transfection of Flag-S plasmid to RPE cells resulted in decreased cell growth level(P<0.05),cell cycle G1 phase arrest(P=0.0005,P=0.013)and increased proportion of senescent cells(P=0.0001,P=0.0001).RT-qPCR showed that cells in the experimental groups had decreased expression of P53,P21 P14ARF,IL-1,IL-6,TGF-β,VEGFA,ICAM,BCL-XL mRNA expression levels were increased,MMP-3 expression was decreased in the S protein treated group and MCP-1 expression was decreased in the overexpressed Flag-S protein group(P<0.01).Immunoblotting showed increased expression of P53,P21,BCL-2 and β-Gal proteins after S protein treatment,and Elisa showed increased levels of IL-1 and IL-6 in cell supernatants(P<0.05).2.S protein treatment or overexpression of Flag-S protein induced an increase in the proportion of senescent cells(P<0.0001,P=0.0005),and after pre-transfection with P21 siRNA,the RPE senescence induced by the addition of S protein or overexpression of Flag-S protein was eliminated and the proportion of senescent cells was reduced(P<0.0001,P=0.0003).3.the level of ROS was increased in RPE cells treated with S protein or transfected with Flag-S plasmid(P=0.0001,P=0.0001)and the proportion of senescent cells was increased(P=0.0003,P=0.0001),and the proportion of senescent cells was decreased after the addition of ROS inhibitor NAC in both cases(P=0.0003,P=0.0002).the S protein-induced P53,P21 protein expression increase(P=0.0035,P=0.0359)was also eliminated after the addition of NAC co-treatment,and P53,P21 protein expression was reduced(P=0.0067,P=0.0098).4.Immunofluorescence showed that S protein was localized to the endoplasmic reticulum after the addition of S protein or transfection of Flag-S plasmid.48h after S protein treatment,the expression levels of endoplasmic reticulum stress-related proteins ATF3,ATF6 and CHOP were increased,and the expression levels of ATF4 and Calnexin were decreased.After the addition of NAC and S protein co-treatment,the expression levels of ATF6 protein decreased and those of ATF3,ATF4,and Calnexin increased(P<0.05).5.Immunofluorescence showed that P65 in the control group accumulated in the cytoplasm,and most of P65 was transferred from the cytoplasm to the nucleus after adding S protein or transfecting Flag-S plasmid.The results of nucleoplasmic separation showed that the content of P65 in the nucleus increased after S protein treatment.phosphorylated iκB protein expression decreased in RPE cells after S protein treatment(P=0.0147),and phosphorylated iκB protein expression increased with the addition of NAC and S protein co-treatment(P=0.0383).S protein induced elevated P53 and P21 protein expression in RPE cells(P=0.0351,P=0.0416),and the addition of S protein after pretreatment with BAY for 30 min decreased cellular P53 protein expression levels(P=0.0104)and further increased P21 protein expression levels(P=0.0202),mRNA levels of senescence-related factors P53,P21,S protein-induced increase in RPE cell senescence model P14ARF,TGF-β and ICAM expression was reduced in the BAY pretreatment group(P<0.01).ConclusionsThe SARS-CoV-2 Spike protein has an inhibitory effect on the growth of human retinal pigment epithelium,blocks the cell cycle in the G1 phase,and induce the cellular senescence of APRE cells by activating endoplasmic reticulum stress,ROS and NF-κB.