Screening And Mechanism Of Natural Compounds For DNA Damage Repairing Of Host Cells Caused By EV-d68 Virus

Background: Human enterovirus 68(Enterovirus D68,EV-D68)belongs to the group of enterovirus D.In recent years,respiratory diseases caused by EV-D68 have broken out in various regions.Unfortunately,there is no definite effective vaccine and medicine.Our previous studies have shown that EV-D68 virus can induce cell cycle arrest in G0/G1 phase and apoptosis,this suggests us that EV-D68 virus infection of host cells may induce DNA damage.This research is dedicated to clarify this hypothesis,find the target of EV-D68 virus inducing host cell DNA damage,and screen out antiviral natural compounds on this basis.Methods: RD cells(Human rhabdomyosarcoma cells)were used as host cells.First,it is clear whether EV-D68 induces DNA damage in RD cells.Secondly,analyze Ataxia telangiectasia mutated(ATM),ATM and Rad3 related kinase(ATR)and DNA-dependent protein kinase(DNA-PK)inhibitors And the corresponding si RNA(ATM,ATR,DNA-PK)on the propagation of EV-D68 virus and the impact on host cells.Finally,the virus structural protein VP1 and γ-H2 AX protein were used as the detection indicators to screen the antiviral effects of natural compounds,and the DNA damage(γ-H2AX)was used as the target to analyze the treatment of each natural compounds after adding ATM,ATR and DNA-PK activators The expression of γ-H2 AX protein in the cells was further analyzed for positive drugs that inhibit virus production to activate the DNA damage response pathway.Results and Conclusions: 1.EV-D68 induces DNA damage in RD cells: EV-D68 virus infection of RD cells caused cell morphology changes,induced cell arrest in G0/G1 phase;γ-H2 AX protein expression increased(P<0.001).Therefore,it can be determined that the EV-D68 virus can induce DNA damage in host cells.2.EV-D68 infection activates ATM and DNA-PK pathways in RD cells:(1)ATM and DNA-PK inhibitors can inhibit virus production and produce damage repair effects:After EV-D68 virus infects RD cells for 24 hours,the expression of viral structural proteins VP1 and γ-H2 AX in the ATM inhibitor treatment group and the DNA-PK inhibitor treatment group decreased in a dose-dependent manner;while the ATR inhibitor treatment group the expression of virus structural proteins VP1 and γ-H2 AX increased in a dose-dependent manner.TCID results showed that the number of virus particles in the supernatant of the ATM inhibitor treatment group and the DNA-PK inhibitor treatment group decreased(P<0.05);while the number of virus particles in the supernatant of the ATR inhibitor treatment group increased(P<0.05).(2)Transfection of small interfering RNA to knock down ATM and DNA-PK can inhibit virus production and produce damage repair effects: The result trend is the same as(1).3.ATM inhibitor and DNA-PK inhibitor have a protective effect on EV-D68-induced cytopathic effect: ATM inhibitors and DNA-PK inhibitors can reduce the cytopathic effect induced by EV-D68 virus and increase the number of RD cells(P<0.001).4.The active oxygen scavenger NAC has no effect on the production of EV-D68 virus: Compared with Mock+Con,the results of flow cytometry showed that the ROS level in the Infected+Con group was not high;Compared with Infected+Con,the viral protein VP1,cell number,viral genome and supernatant virus particles of the Infected+NAC group had not been changed.5.Tanshinone II-A,resveratrol and silymarin can inhibit virus production and have a protective effect on host cells: The viral structural proteins VP1 and γ-H2 AX protein in the drug treatment group of tanshinone II-A,resveratrol and silibinin were down-regulated in a dose-dependent manner,and the number of virus particles was reduced(P<0.001);rutin and quercetin were compared with The expected therapeutic effect is opposite.The viral structural proteins VP1 and γ-H2 AX protein are both up-regulated;oleanolic acid and vanillin have no effect on the viral proteins VP1 and γ-H2 AX protein.6.Tanshinone II-A inhibit the expression of γ-H2 AX protein by inhibiting the ATM pathway,Resveratrol and Silymarin inhibits the expression of γ-H2 AX protein by inhibiting the ATM and DNA-PK pathway,Rutin and Quercetin Inhibiting the expression of γ-H2 AX protein by inhibiting the ATR pathway: Tanshinone II-A can inhibit the expression of γ-H2 AX protein up-regulated byATM activator;Resveratrol can inhibit the expression of γ-H2 AX protein up-regulated by ATM and DNA-PK activator;Silymarin can inhibit ATM and DNA-PK activation γ-H2 AX protein expression up-regulated by the agent.Innovation: 1.EV-D68 virus is positively correlated with the expression of γ-H2 AX protein.2.EV-D68 virus activates ATM and DNA-PK pathways to initiate DNA damage response.3.Tanshinone II-A,Resveratrol and Silymarin can inhibit the production of EVD68 virus and have a protective effect on host cells.4.Tanshinone II-A inhibit the expression of γ-H2 AX protein by inhibiting the ATM pathway;Resveratrol and Silymarin inhibits the expression of γ-H2 AX protein by inhibiting the ATM and DNA-PK pathway.