Effects and mechanisms of ATP-sensitive potassium channel openers on microglial inflammatory re-sponse induced by oxygen-glucose deprivation/reoxygenation
Objective To investigate the effects and mechanisms of Nicorandil,an ATP-sensitive potassium channel(KATP)opener,on pyroptosis and inflammatory responses in microglia(BV2)induced by oxygen-glucose deprivation/reoxygenation(OGD/R).Methods BV2 cells were divided into control group,OGD/R group,and OGD/R+Nicorandil group.And the cells were subjected to oxygen-glucose deprivation for 3 hours and then reoxygenated for 24 hours to establish an OGD/R cell model.OGD/R+Nicorandil group cells were incubated with 5 μg/mL Nicorandil culture medium for 24 hours after oxygen-glucose deprivation for 3 hours.The cell proliferation activity was detected by CCK8 assay.Calcein/propidium iodide(calcein/PI)assay kit was used to detect the membrane porosity rupture rate of cell in each group.Western blot analy-sis was performed to detect the protein expression levels of nuclear factor-κB(NF-κB),phosphorylated NF-κB(p-NF-κB),inhibitor of nuclear factor-κB α(IκB-α),phosphorylated IκB-α(p-IκB-α),absent in melanoma 2(AIM2),cleaved-caspase-1,gasdermin D-N(GSDMD-N),interleukin-18(IL-18),and inter-leukin-1β(IL-1β).Immunofluorescence was used to detect the protein expression levels of AIM2 and GSDMD-N in each group.Statistical analysis was performed by SPSS 26.0 software.One-way ANOVA was used for multiple group comparisons,and LSD test was used for pairwise comparisons.Results(1)There were statistically significant differences in the membrane porosity rupture rates among the three groups(F=615.882,P＜0.05).The membrane porosity rupture rate in the Nicorandil group was lower than that in the OGD/R group((41.50±3.04)％,(59.44±3.66)％,P＜0.05).(2)Western blot results showed that the protein expression levels of p-NF-κB,NF-κB,p-IκB-α,and IκB-α were significantly different among the three groups(F=10.000,62.652,67.121,101.023,all P＜0.05).The levels of p-NF-κB,NF-κB and p-IκB-α in the OGD/R+Nicorandil group((0.60±0.13),(0.87±0.06),(0.55±0.06),respectively)were lower than those in the OGD/R group((1.02±0.09),(1.03±0.09),(0.86±0.04),respectively)(all P＜0.05).The level of IκB-α in the OGD/R+Nicorandil group((0.63±0.05),(0.46±0.06))was higher than that in the OGD/R group(P＜0.05).(3)The protein expression levels of AIM2,cleaved-caspase-1,GSDMD-N,IL-18,and IL-1β were significantly different among the three groups(F=65.926,12.428,66.447,44.831,52.960,all P＜0.05).The levels of AIM2,cleaved-caspase-1,GSDMD-N,IL-18 and IL-1β in the OGD/R+Nicorandil group((0.78±0.04),(0.71±0.09),(0.54±0.04),(0.72±0.07),(0.50±0.08),respectively)were lower than those in the OGD/R group((0.94±0.09),(0.89±0.09),(0.85±0.04),(0.90±0.07),(0.99±0.03),respectively)(all P＜0.05).(4)Immunofluorescence re-sults showed statistically significant differences in the fluorescence intensity of pyroptosis marker proteins AIM2 and GSDMD-N among the three groups(F=36.353,46.817,both P＜0.05).The fluorescence inten-sities of AIM2((124.36±7.91),(140.19±5.63))and GSDMD-N((134.16±5.18),(147.45±5.63))in the OGD/R+Nicorandil group were lower than those in the OGD/R group(both P＜0.05).Conclusi-on Nicorandil can mitigate BV2 cell damage following oxygen-glucose deprivation,inhibiting the release of pro-inflammatory factors.The mechanism may be related to the downregulation of the expression of NF-κB related proteins and inhibition of AIM2 inflammasome-mediated pyroptosis after OGD/R.
ATP-sensitive potassium channelNicorandilOxygen-glucose deprivationPyroptosisAIM2 inflammasomeNF-κB signal pathway
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