摘要
目的探讨雷帕霉素对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium iodide,MPP+)诱导的小胶质细胞中Nod样受体蛋白3(nod-like receptor protein 3,NLRP3)炎症小体激活的影响。方法将BV2小胶质细胞分为对照组、模型组和雷帕霉素组,模型组和雷帕霉素组以MPP+激活NLRP3炎症小体,雷帕霉素组予雷帕霉素预处理。实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)检测NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein,ASC)和caspase-1的mRNA水平,免疫荧光检测NLRP3和白细胞介素-1β(interleukin-1β,IL-1β)的表达情况,Western blot检测NLRP3、ASC、caspase-1、beclin1和微管相关蛋白l轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达情况。结果模型组的NLRP3、ASC和caspase-1 mRNA水平较对照组升高(t=4.825,3.015,5.853,均P<0.05),雷帕霉素组的NLRP3和caspase-1 mRNA水平较模型组降低(t=2.75,2.89,均P<0.05)。模型组NLRP3(1.54±0.22)、ASC(1.02±0.13)和caspase-1(1.42±0.30)蛋白表达较对照组NLRP3(0.66±0.15)、ASC(0.41±0.14)和caspase-1(0.70±0.10)显著增加(t=5.653,5.602,3.964,均P<0.01),而beclin1(0.28±0.09)蛋白表达和LC3II/LC3I(0.69±0.14)比值较对照组beclin1(0.60±0.11)和LC3II/LC3I(1.29±0.23)显著降低(t=4.010,3.982,均P<0.01)。与模型组比较,雷帕霉素组NLRP3(0.80±0.18)和ASC(0.68±0.14)蛋白表达下降(t=4.413,3.077,均P<0.05),而beclin1(0.65±0.20)蛋白表达和LC3II/LC3I(1.42±0.36)比值增加(t=2.965,3.278,均P<0.05)。结论MPP+可激活小胶质细胞中的NLRP3炎症小体,并损害细胞的自噬活动;雷帕霉素通过提高自噬活动抑制MPP+诱导的NLRP3炎症小体激活。
Objective To explore the effect of rapamycin on 1-methyl-4-phenylpyridinium iodide(MPP+)-induced activation of Nod-like receptor protein 3(NLRP3)inflammasome in microglia.Methods The BV2 microglia cells were divided into control group,model group and rapamycin group.The model group and rapamycin group were treated by MPP+to activate NLRP3 inflammasome,and rapamycin group was pretreated with rapamycin.Quantitative real-time PCR(RT-qPCR)was used to detect the mRNA levels of NLRP3,apoptosis-associated speck-like protein(ASC)and caspase-1.Immunofluorescence was used to detect the protein expression of NLRP3 and interleukin-1β(IL-1β).Western blot was carried out to assess the protein expression of NLRP3,ASC,caspase-1,beclin1 and microtubule-associated protein 1 light chain 3(LC3).Results The mRNA levels of NLRP3,ASC and caspase-1 in model group were higher than those in control group(t=4.825,3.015,5.853,all P<0.05).The mRNA levels of NLRP3 and caspase-1 in rapamycin group were lower than those in model group(t=2.75,2.89,both P<0.05).In model group,the protein expressions of NLRP3(1.54±0.22),ASC(1.02±0.13)and caspase-1(1.42±0.30)were higher than NLRP3(0.66±0.15),ASC(0.41±0.14)and caspase-1(0.70±0.10)in control group(t=5.653,5.602,3.964,all P<0.01),while the protein expression of beclin1(0.28±0.09)and LC3II/LC3I ratio(0.69±0.14)were lower than beclin1(0.60±0.11)and LC3II/LC3I(1.29±0.23)in control group(t=4.010,3.982,both P<0.01).The protein expressions of NLRP3(0.80±0.18)and ASC(0.68±0.14)in rapamycin group were lower than those in model group(t=4.413,3.077,both P<0.05),while the protein expression of beclin1(0.65±0.20)and LC3II/LC3I ratio(1.42±0.36)were higher than those in model group(t=2.965,3.278,both P<0.05).Conclusion MPP+activates NLRP3 inflammasome and impairs autophagic function in microglia.Rapamycin inhibits MPP+-induced activation of NLRP3 inflammasome by restoring autophagic impairment in microglia.
作者
黄树宣
卢欢欢
范秉林
陈志
姜炳坚
吴月娟
李晓峰
李燕华
Huang Shuxuan;Lu Huanhuan;Fan Binglin;Chen Zhi;Jiang Bingjian;Wu Yuejuan;Li Xiaofeng;Li Yanhua(Department of Neurology,the People’s Hospital of Guangxi Zhuang Autonomous Region,Nanning 530021,China)
出处
《中华行为医学与脑科学杂志》
CAS
CSCD
北大核心
2020年第9期774-779,共6页
Chinese Journal of Behavioral Medicine and Brain Science
基金
国家自然科学基金项目(81560210)
广西壮族自治区人民医院院内青年基金项目(QN2019-5)。
