GSDMD Serves As A Key Executioner Of Pyroptosis In Experimental Cerebral Ischemia And Reperfusion Model Both In Vivo And In Vitro

Part Ⅰ Pyroptosis was induced in brain tissues after I/RObjectiveTo investigate the expression of NLRP3 inflammasome proteins,including ASC,NLRP3,pro-Caspases-1,Caspases-1 p20,GSDMD and GSDMD-N in brain after cerebral ischemia/reperfusion in rats and to evaluate the interactions of ASC,NLRP3,and pro-Caspase-1 in brain injury after cerebral ischemia/reperfusion.Methods1.Animal groups:42 SD rats initially underwent surgery.Of these,36 survived and were randomly divided(via use of a randomization table)into six groups:Sham group(n=6);MCAO/R group(n=30)arranged by time 6,12,24,48,and 96 hr.(n=6 for each time point).2.The brain tissue samples were obtained separately from rats.At 6,12,24,48,and 96 hr.after reperfusion,the cerebrospinal fluid(CSF)was collected for enzyme-linked immunosorbent assay(ELISA),and the brain tissue samples were obtained for western blot analysis and immunoprecipitation(IP)analysis.Results1.The level of Caspase-1 p20(active form of Caspase-1)was progressively upregulatedafter I/R injury,and peaked at 24 hr after MCAO/R.2.The protein level of GSDMD and the N-terminal of GSDMD(GSDMD-N)was increased following the I/R injury,and peaked at 24 hr after MCAO/R.3.Compared with the Sham group,the expressions of NLRP3 inflammasome proteins,including ASC,NLRP3,and pro-Caspases-1 were remarkably increased in ischemic penumbra of brain tissue following MCAO/R.The interactions of ASC,NLRP3,and pro-Caspase-1 were increased in brain tissues 24 hr.after MCAO/R.4.The levels of IL-1β and IL-18 release were upregulated following MCAO/R and peaked at 24 hr.Conclusions1.The formation of NLRP3 inflammasomes was significantly increased in brain tissues after I/R injury.2.GSDMD induced pyroptosis was activated by I/R injury post-stroke.Part Ⅱ Effects of gene intervention and drug inhibition on pyroptosis induced by MCAO/RObjectiveTo investigate the incidence of pyroptosis and prognosis of model animals when treated with Caspase-1 specific inhibitor y-VAD and gene intervention after MCAO/RMethods1.Animal groups:95 rats underwent surgery.Of these,90 survived and were randomly divided(via use of a randomization table)into 9 groups:Sham group(n=12),MCAO/R group(n=12),MCAO/R+vector group(n=12);MCAO/R+GSDMD-over group(n==12),MCAO/R+Control siRNA group(n=12),MCAO/R+GSDMD siRNA group(n=12),MCAO/R+Vehicle group(n=6),MCAO/R+Z-YVAD-FMK group(n=6),and MCAO/R+GSDMD-FL+Z-YVAD-FMK group(n=6).2.Animal model making:Under an operating microscope,focal cerebral ischemia was achieved by right-sided endovascular MCAO.Rectal temperature was maintained with a heating pad between 36.5 and 37.5℃.After 2h of ischemia,the filament was withdrawn to allow reperfusion.3.Detection methods and indicators:brain tissues were collected for western blot.Z-yVAD-FMK,a selective inhibitor for Caspase-1,was used to create a Control group in this study,which would be used to indicate whether the role played by GSDMD was dependent on Caspase-1 activation.For the Sham group,MCAO/R group,MCAO/R+vector group,MCAO/R+GSDMD-over group,MCAO/R+Control siRNA group,and MCAO/R+GSDMD siRNA group,CSF was collected for ELISA and TTC staining was performed using an additional six rats in each group.Results1.With the treatment of yVAD,the levels of GSDMD-N and the Caspase-1 p20 were reduced,while Caspase-land GSDMD-FL was not.The IL-1β and IL-18 secretion in CSF was significantly downregulated.2.The protein level of GSDMD and GSDMD-N in the siRNA-GSDMD group was significantly reduced versus the non-target siRNA Control group,while Caspase-1 and Caspase-1 p20 was not.The IL-1β and IL-18 secretion and volume of infarction was significantly decreased after GSDMD knockdown,whereas the neurological score was significantly increased.3.The protein level of GSDMD and GSDMD-N shows an obvious increase observed in the GSDMD overexpression group compared with vector group,while Caspase-1 and Caspase-1 p20 was not.The up regulation of IL-1β and IL-18 by MCAO/R was significantly aggravated by GSDMD overexpression.The volume of infarction was also significantly increased and resulted in a large decrease in the neurological score after MCAO/R.4.The protein level of GSDMD-N、Caspase-1 and Caspase-1 p20 was partly reduced by Z-YVAD-FMK+GSDMD-overexpression treatment after MCAO/R while the variation of GSDMD was no significance compared to the group that only received GSDMD overexpression.Meanwhile,the levels of IL-1β and IL-18 in GSDMD overexpression+Z-YVAD-FMK were decreased.Conclusions1.GSDMD-N should be considered an important factor for pyroptosis after MCAO/R.GSDMD played the role of substrates of activated Caspase-1,and GSDMD-N was cleaved to induce pyroptosis after I/R brain injury.Respectively,GSDMD knockdown and overexpression were able to ameliorate and exacerbate neurological behavior.2.GSDMD cleaved and induced pyroptosis partly dependent on Caspase-1 p20activation.Z-YVAD-FMK as Caspase-1 inhibition could reverse GSDMD induced pyroptosis after MCAO/R.Part Ⅲ To confirm the subcellular location of GSDMD in microglia after I/R injury in vitro and evaluating the effect to microglia by intervention of GSDMD amino acid sitesObjective:To investigate the mechanisms of GSDMD induced pyroptosis in I/R injury and explore the specific amino acid sites for GSDMD to induce pyroptosis after OGD/R in microgliaMethods:1.Primary cortical microglia cells group:primary cultured microglia were used and partitioned into nine groups:Control group;glucose deprivation/reinfusion(OGD/R)groups,including the medium group,the medium+GSDMD-FL group,the medium+GSDMD-N group and the medium+GSDMD-C group,the medium+GSDMD D280A group,the medium+GSDMD E15K/L156D group,the medium+GSDMD-FL+GSDMD-C group,the medium+ GSDMD-N+GSDMD-C group2.Detection methods and indicators:After required treatment,the cells were collected for immunofluorescence staining,and the supernatant of the cultures was collected for ELISA analysis.Results1.GSDMD-NeGFP could be recruited to the membranes after OGD/R in the GSDMD-NeGFP overexpression group,whereas GSDMD-CeRFP located in the cytoplasm was observed in the GSDMD-CeRFP transfection group.In the GSDMD-NeGFP+GSDMD-CeRFP co-transfection group,GSDMD-NeGFP was bound in the cytoplasm by GSDMD-CeRFP,which prevented it from moving to the membrane.2.GSDMD-CeRFP overexpression could inhibit IL-1β and IL-18 secretion after OGD/R.Moreover,the levels of IL-1β and IL-18 in the GSDMD-CeRFP+GSDMD-NeGFP co-transfection group were significantly decreased compared to the GSDMD-NeGFPoverexpression group,which exhibited a similar consequence in the GSDMD-CeRFP+GSDMD-FL group.3.GSDMD could not move to the membrane after E15K/L156D mutation.The secretion levels of IL-1β and IL-18 were significantly reduced after E15K/L156D,mutations when compared to the GSDMD-FL overexpression group.4.Similarly,GSDMD was also unable to move to the membrane following D280A mutation with the OGD/R in microglia,and the secretion levels of IL-1β and IL-18 were significantly reduced after D280A mutations compared with the GSDMD-FL overexpression group.Conclusions1.GSDMD-N was a key executioner of pyroptosis.GSDMD-C exerted an auto-inhibitory function by restricting GSDMD-N to the cytoplasm,rather than the membrane,resulting in the inhibition of pyroptosis after OGD/R in microglia.2.D280was the amino acid sites of GSDMD-FL for Caspase-1 cleavage.3.E15 and L156 were important sites for the membrane association of GSDMD-N.