Autophagy Induced By Enterovirus 71 Regulates Apoptosis And Pyroptosis Through NLRP3 Inflammasome

Objective: Hand-foot-mouth Disease(HFMD)is a seasonal epidemic disease caused by enterovirus infections.They are mostly caused by human enterovirus 71(EV71)and coxsackievirus A16(Cox A16).It threatens children under six years of age prevailingly.In recent years,the fatality rate of HFMD ranks the first in class c infectious diseases in China.Among the confirmed cases of severe HFMD,EV71 infection is the main reason,accompanied by neurological complications.At present,the inductions of programmed cell death induced by EV71,such as autophagy,apoptosis and pyroptosis have become a research focus,but the mutual regulations among them have not been clear.Therefore,starting from the definite result that EV71 can induce autophagy,apoptosis and pyroptosis,this study aimed to explore the regulatory mechanism of autophagy induced by EV71 infection on apoptosis and pyroptosis,unveil the mysterious relationship between the regulation of programmed cell death induced by EV71 and provide a new reference for the treatment of HFMD.Methods: 1.Cell culture Human normal gastric epithelial cells(GES-1)and rhabdomyosarcoma cells(RD)were cultured in Dulbecco's modified Eagle's medium(DMEM)medium containing 10% fetal bovine serum(FBS),100 ?g/ml penicillin,and 100 U/ml streptomycin.Cells were incubated with 5% CO2 at 37?.GES-1 cells and RD cells were digested by trypsin containing EDTA at 90% confluence.2.Virus propagation and infection EV71 virus was kindly provided by Professor Zhendong Zhao.RD cells were infected with EV71 for 24 h and then centrifuged at 5000 g for 10 min at 4?.The supernatant was collected and frozen at-80 ?.The titer of EV71 was measured by 50% tissue culture infective dose(TCID50).Virus infection was carried out as follows.Briefly,GES-1 cells were infected by EV71 at the indicated multiplicity of infection(MOI),and then cultured in fresh culture medium for certain time periods.At last,culture supernatant and cells were harvested.3.Western blotting Cells were collected after EV71 infection at the indicated time points.Cells were then lysed with RIPA buffer,and total protein was quantified using the BCA protein quantification assay kit.Samples were separated by 12% SDS-PAGE and transferred to a nitrocellulose(NC)membrane.The NC membrane was blocked with 5% skimmed milk for 2 h at room temperature and then incubated with the corresponding antibodies at 4? overnight.After three washes,the NC membrane was incubated with HRP-labeled goat anti-rabbit Ig G(H+L)antibody at room temperature for 2 h.Immunocomplexes were detected using the Western blotting detection kit reagent.4.DAPI nuclear staining experiment Lay in the logarithmic growth phase cells into 24 orifice,12×104 cells/ well and then cells were incubated with 5% CO2 at 37?.When the cell growth density reached about 80%,the old medium was removed and the new medium was replaced.GES-1 cells were infected with EV71(MOI = 5)for 24 h.Cells growing on orifice were rinsed with PBS three times for 5 min each and then fixed with 4% paraformaldehyde for 20 min.After three washes,the cells were incubated with DAPI for 10 min.A high content imaging analysis system was used to observe the morphology of nuclear.5.Correlation detection of pyroptosis(1)Dil staining detection Lay in the logarithmic growth phase cells into 24 orifice,12×104 cells/ well and then cells were incubated with 5% CO2 at 37?.When the cell growth density reached about 80%,the old medium was removed and the new medium was replaced.GES-1 cells were infected with EV71(MOI = 5)for 24 h.Cells growing on orifice were rinsed with PBS three times for 3 min each and then fixed with 4% paraformaldehyde for 20 min.After three washes,the cells were incubated with Dil solution at 37? for 30 min.Then the cells were incubated with DAPI for 10 min after three washes for 5 min each.A fluorescence microscope was used to observe the changes of cell membrane.(2)Enzyme-linked immunosorbent assay(ELISA)Supernatants of GES-1 cells infected with EV71 were collected and IL-1? levels were determined using a commercial ELISA kit according to the manufacturer's instructions.Experiments were performed at least in triplicate.(3)lactate dehydrogenase(LDH)activity detection Lay in the logarithmic growth phase cells into 96 orifice,1×104 cells/ well and then cells were incubated with 5% CO2 at 37?.10% LDH releasing reagent was added 1 h before EV71 intervention.Then the cells were cultured continuously.After a certain time of virus intervention,the supernatant was collected in a new 1.5 ml tube by centrifugation.For each well,120 ?l supernatant was added to a new 96-well plate,and 60 ?l prepared LDH working solution was added to the 96-well plate.The solution was rocked at room temperature for 30 min,and OD value was measured at 490 nm with a microplate reader.LDH activity =(OD value of samples in the experimental group-OD value of samples in the control group)/(OD value of maximum cell enzyme activity-OD value of samples in the control group)× 100.6.Real-time Quantitative PCR(q PCR)Total RNA was extracted using Trizol reagent,then reverse transcription was performed using Beyo RT II First Strand c DNA Synthesis Kit according to the manufacturer's instructions.c DNA was synthesized at 42 ? for 60 min,and then treated at 80 ? for 10 min.Real-time quantitative PCR was performed using Beyo Fast SYBR Green q PCR Mix.Amplification of c DNA was performed for 40 cycles of 95 ? for 2 min,95 ? for 15 s,and 60 ? for 15~30 s.The primers for IL-1? and glyceraldehyde 3-phosphate dehydrogenase(GAPDH)were as follows,IL-1?,sense primer 5'- ACAGATGAAGTGCTCCTTCCA-3',antisense primer 5'-GTCGGAGATTCGTAGCTGGAT-3';GAPDH,sense primer 5'-ACAACTTTGGCATTGTGGAA-3',antisense primer 5'-GATGCAGGGATGATGTTCTG-3'.7.Transfection experiment(1)transfection of GFP-LC3 plasmid Lay in the logarithmic growth phase cells into 24 orifice,12×104 cells/ well and then cells were incubated with 5% CO2 at 37?.When the cell growth density reached about 80%,the old medium was removed and the new medium containing serum not antibiotics was replaced.GES-1 cells were transfected with GFP-LC3 plasmid and transfection reagent lipo6000 TM at a certain ratio for 6 h and then the medium was transformed into a complete culture medium.After GES-1 cells were transfected for 24 h,EV71 virus at MOI of 5 was added for 24 h,and the expressions of autophagosomes were observed under a high content imaging analysis system or a fluorescence microscope.(2)transfection of si RNA Atg5 si RNA and NLRP3 si RNA and their respective non-interfering control si RNAs were synthesized chemically.In order to reduce the expressions of Atg5 and NLRP3,GES-1 cells were transfected with control si RNA and Atg5 si RNA/NLRP3 si RNA and transfection reagent lipo6000 TM at a certain ratio for 6 h and then were infected with EV71 virus at MOI of 5 for 24 h.The cells and supernatant were collected for different experiments.8.Statistical analysis All data are presented as mean ± standard error of the mean(S.E.M.).The data were analyzed using Graph Pad Prism software.The mean values between groups were compared with Student's t test.A p value < 0.05 was considered statistically significant.Results:1.EV71 infection induced autophagy in GES-1 cells and RD cells.Western blotting results showed that with the increase of EV71 infection time points and dosages,autophagy related protein P62 gradually degraded,the conversion of LC3-? to LC3-? was induced;GES-1 cells infected with EV71 were transfected with GFP-LC3,and marked punctate aggregations were observed under fluorescence microscope.2.EV71 infection induced apoptosis in GES-1 cells and RD cells.Western blotting results showed that the apoptosis-related proteins PARP and caspase-3 were degraded gradually with an increase of EV71 infection time points and dosages.DAPI nuclear staining results under high content imaging analysis system showed that EV71 infection resulted in nuclear shrinkage,dense granular fluorescence and decreased cell number.3.EV71 infection induced pyroptosis in GES-1 cells and RD cells.Western blotting results showed that with the increase of EV71 infection time points and dosages,the pyroptosis related protein GSDMD,caspase-1 and IL-1? precursors were degraded gradually,and the expression of mature IL-1? protein was increased gradually.The results of Dil staining showed that the membrane of GES-1 cells was incomplete and its contour became smaller after EV71 infection.ELISA results showed that the secretion of IL-1? in the supernatant increased when GES-1 cells were infected with EV71 for 24 h.q PCR showed that EV71 infection could induce the expression of IL-1? at the transcriptional level.The LDH activity detection results showed that the LDH activity in the supernatant increased after GES-1 cells were infected with EV71 at MOI of 5 for 24 h.4.Autophagy induced by EV71 occurred before apoptosis and pyroptosis Previous results showed that EV71 infection could induce autophagy,apoptosis and pyroptosis,but the sequence of the three has not been clear at present.To investigate the sequence of the three,Western blotting was performed.The results showed that autophagy related proteins changed significantly at 12 h after EV71 infection,while apoptosis or pyroptosis related proteins began to change at 18 h after EV71 infection,and the changes were most significant at 24 h.GES-1 cells infected with EV71 were transfected with GFP-LC3,and the punctuated accumulation was observed under high content imaging analysis system at 12 h.5.Inhibition of autophagy induced by EV71 infection could inhibit apoptosis and pyroptosis The above results showed that after GES-1 cells were infected with EV71,autophagy occurred earlier than apoptosis and pyroptosis,so we speculated whether autophagy,as a protective mechanism of the body,could regulate the occurrence of apoptosis and pyroptosis.In order to prove our hypothesis,autophagy related drugs 3-methyladenine(3-MA)and chloroquine(CQ)were introduced to intervene in the process of autophagy,and the autophagy promoter molecule Atg5 was knocked down with RNAi technology to observe the effect of the inhibition of autophagy on apoptosis and pyroptosis.Western blotting results showed that after GES-1 cells were infected with EV71 for 24 h,the treatment of 3-MA and CQ inhibited apoptosis and pyroptosis induced by EV71 infection,at the same time,knocking down the autophagy promoter Atg5 inhibited the expressions of apoptosis and pyroptosis related proteins.The results of q PCR showed that the treatment of 3-MA and CQ reduced the expression of IL-1? at the transcription level.LDH activity detection results showed that the treatment of 3-MA,CQ or Atg5 si RNA could inhibit the release of LDH secreted by EV71 infection.6.Autophagy induced by EV71 infection regulated apoptosis and pyroptosis through NLRP3 inflammasome In order to explore the mechanism of autophagy regulating apoptosis and pyroptosis,Western blotting assay was performed.The results showed that the expressions of NLRP3 proteins were increased with the increase of EV71 infection time points and dosages in GES-1 cells and RD cells.Knockdown of NLRP3 was found to inhibit the expression of apoptosis and pyroptosis related proteins.Conclusion: EV71 infection induced autophagy,apoptosis and pyroptosis in GES-1 cells and RD cells.Moreover,autophagy induced by EV71 infection may regulate apoptosis and pyroptosis through NLRP3 inflammasome.These results may provide a theoretical basis for us to further explore the effects of this regulatory action on virus replication and antiviral activity in the body.In addition,revealing the relationship between autophagy,apoptosis and pyroptosis can provide a reference for the prevention and treatment of EV71 infection.