ATP敏感性钾通道开放剂对氧糖剥夺/复氧小胶质细胞炎性反应的影响及其机制

Effects and mechanisms of ATP-sensitive potassium channel openers on microglial inflammatory response induced by oxygen-glucose deprivation/reoxygenation

目的探讨ATP敏感性钾通道(ATP-sensitive potassium channel,KATP)开放剂尼可地尔对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)所致BV2小胶质细胞焦亡和炎性反应的影响及作用机制。方法将BV2细胞分为对照组、OGD/R组和OGD/R+尼可地尔组。BV2细胞氧糖剥夺3 h后复氧24 h建立OGD/R细胞模型,OGD/R+尼可地尔组细胞在氧糖剥夺3 h后加入5μg/mL尼可地尔培养液孵育24 h。采用CCK8法检测细胞增殖活性;钙黄绿素/碘化丙啶(calcein/PI)试剂盒检测各组细胞膜成孔破裂率;Western blot检测核因子-κB(nuclear factor-κB,NF-κB)、磷酸化NF-κB(phosphorylated NF-κB,p-NF-κB)、NF-κB抑制蛋白α(inhibitor of nuclear factor-κBα,IκB-α)、磷酸化IκB-α(phosphorylated IκB-α,p-IκB-α)、黑色素瘤缺乏因子2(absent in melanoma 2,AIM2)、活化的半胱氨酸蛋白酶-1(cleaved-caspase-1)、消皮素D N-末端片段(gasdermin D-N,GSDMD-N)、白细胞介素18(interleukin-18,IL-18)和白细胞介素1β(interleukin-1β,IL-1β)的蛋白表达情况;细胞免疫荧光检测各组AIM2、GSDMD-N蛋白表达情况。使用SPSS 26.0软件进行统计分析,多组间比较采用单因素方差分析,进一步两两比较采用LSD检验。结果(1)3组细胞膜成孔破裂率差异有统计学意义(F=615.882,P<0.05),OGD/R+尼可地尔组细胞膜成孔破裂率低于OGD/R组[(41.50±3.04)%,(59.44±3.66)%,P<0.05]。(2)Western blot结果显示,3组细胞p-NF-κB、NF-κB、p-IκB-α和IκB-α的蛋白表达水平均差异有统计学意义(F=10.000,62.652,67.121,101.023,均P<0.05)。OGD/R+尼可地尔组细胞p-NF-κB、NF-κB、p-IκB-α蛋白水平[(0.60±0.13),(0.87±0.06),(0.55±0.06)]均低于OGD/R组[(1.02±0.09),(1.03±0.09),(0.86±0.04)](均P<0.05),OGD/R+尼可地尔组细胞IκB-α[(0.63±0.05),(0.46±0.06)]表达水平高于OGD/R组(P<0.05)。(3)3组细胞AIM2、cleaved-caspase-1、GSDMD-N、IL-18和IL-1β的蛋白表达水平均差异有统计学意义(F=65.926,12.428,66.447,44.831,52.960,均P<0.05)。OGD/R+尼可地尔组细胞AIM2、cleaved-caspase-1、GSDMD-N、IL-18和IL-1β蛋白水平[(0.78±0.04),(0.71±0.09),(0.54±0.04),(0.72±0.07),(0.50±0.08)]均低于OGD/R组[(0.94±0.09),(0.89±0.09),(0.85±0.04),(0.90±0.07),(0.99±0.03)](均P<0.05)。(4)免疫荧光结果显示,3组细胞焦亡特征性蛋白分子AIM2和GSDMD-N的荧光强度均差异有统计学意义(F=36.353,46.817,均P<0.05)。OGD/R+尼可地尔组AIM2[(124.36±7.91),(140.19±5.63)]和GSDMD-N[(134.16±5.18),(147.45±5.63)]的蛋白荧光强度均低于OGD/R组(均P<0.05)。结论尼可地尔可以减轻氧糖剥夺后BV2细胞损伤,抑制促炎因子释放,其机制可能与其下调NF-κB相关蛋白表达,抑制AIM2炎性小体介导的细胞焦亡有关。

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 5μg/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 analysis was performed to detect the protein expression levels of nuclear factor-κB(NF-κB),phosphorylated NF-κB(p-NF-κB),inhibitor of nuclear factor-κBα(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 interleukin-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-αwere 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-αin 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-αin 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-1βwere 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-1βin 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 results 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 intensities 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).Conclusion 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.