基于Notch通路探讨缺氧对小胶质细胞IL-1β表达的影响  

The effect of Notch pathway on IL-1β expression in hypoxic BV-2 microglia

作　　者：韩永丽 黎春常 丁洪光 黄林强 刘新强 陈胜龙 曾红科 Han Yongli;Li Chunchang;Ding Hongguang;Huang Linqiang;Liu Xinqiang;Chen Shenglong;Zeng Hongke(The Department of Emergency&Crital Care Medince,Guangdong Provincial People's Hospital,Guangdong Academy of Medical Sciences,Guangzhou,510080,China)

机构地区：[1]广东省人民医院广东省医学科学院急危重症医学部,广州510080

出　　处：《中华急诊医学杂志》2020年第12期1584-1589,共6页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金青年项目(81701875);广州市科技计划(20190410039);广东省医学科研基金(A2017284)。

摘　　要：目的探讨缺氧后小胶质细胞Notch信号通路是否激活,及其对IL-1β表达的影响。方法将BV-2小胶质细胞分为对照组、缺氧无糖培养组(oxygen glucose deprivation,OGD组)、及缺氧无糖培养+10μmol/L DAPT组(DAPT组);对照组细胞正常培养,OGD组、DAPT组缺氧培养4 h(含3%O2,5%CO2,92%N2培养箱中培养)后在正常培养箱中复氧24 h。先用CCK8分析10μmol/L DAPT对BV-2小胶质细胞活性的影响;Western blot检测各组BV-2小胶质细胞中活化的Notch信号通路下游蛋白NICD的表达,以及免疫荧光法、RT-PCR、Western blot检测各组BV-2小胶质细胞IL-1β的表达。数据采用单因素方差分析,用LSD-t法进行组间两两比较,P<0.05为差异有统计学意义。结果CCK8检测OGD组及DAPT组的OD值分别为:0.269±0.013、0.265±0.025(P>0.05);表明10μmol/L DAPT对BV-2小胶质细胞活性无影响。Western blot提示:与对照组比较,OGD组与DAPT组BV-2小胶质细胞NICD蛋白表达明显增加;与OGD组比较,DAPT抑制Notch信号通路后BV-2小胶质细胞NICD蛋白表达明显减少(对照组:0.632±0.065,OGD组:1.276±0.049,DAPT组:0.938±0.049,P<0.05)。免疫荧光提示:对照组BV-2小胶质细胞少量表达IL-1β;OGD组与DAPT组BV-2小胶质细胞IL-1β的表达明显增加;与OGD组比较,DAPT组BV-2小胶质细胞IL-1β的表达明显减少。RT-PCR提示:与对照组比较,OGD组与DAPT组BV-2小胶质细胞IL-1βmRNA表达明显增加;与OGD组比较,DAPT组BV-2小胶质细胞IL-1βmRNA表达明显减少(对照组:1.000±0.173,OGD组:2.741±0.207,DAPT组:1.762±0.177,P<0.05)。Western blot提示:与对照组比较,OGD组与DAPT组BV-2小胶质细胞IL-1β蛋白表达明显增加;与OGD组比较,DAPT组BV-2小胶质细胞IL-1β蛋白表达明显减少(对照组:0.422±0.030,OGD组:1.236±0.143,DAPT组:0.730±0.047,P<0.05)。结论缺氧可激活小胶质细胞Notch信号通路,抑制Notch信号通路可减少缺氧小胶质细胞IL-1β的表达。Objective To investigate whether the Notch pathway is activated in hypoxic microglia and its effect on IL-1βexpression.Methods The cells were divided into the control group,oxygen glucose deprivation group(OGD group),and oxygen glucose deprivation+10μmol/L DAPT group(DAPT group).Cells in the control group were cultured normally,and cells in the OGD and DAPT groups were incubated in an air-tight hypoxia chamber with 3%O2,5%CO2,92%N2 for 4 h and followed re-oxygenation for 24 h in a normoxia incubator.CCK8 was used to analyze the effect of 10μmol/L DAPT on BV-2 microglia cells viability.Western blot was performed to detect the expression of Notch receptor domain(NICD).Immunofluorescence double labeling,RT-PCR,and Western blot were performed to detect the expression of IL-1β.All data were analyzed by one-way analysis of variance(ANOVA).The intergroup comparisons were analyzed by the least-significant-difference(LSD)test.Differences were considered statistically significant if P<0.05.Results The OD values of the OGD group and DAPT group detected by CCK8 were 0.269±0.013 and 0.265±0.025,respectively(P>0.05);it showed that 10μmol/L DAPT had no effect on the activity of BV-2 microglia.Western blot showed that the protein expression of NICD were significantly increased in the OGD and DAPT groups compared to the control group;however,the protein expression of NICD were significantly reduced in the DAPT group compared to the OGD group(control group:0.632±0.065;OGD group:1.276±0.049;DAPT group:0.938±0.049;P<0.05).Immunofluorescence showed that the expressions of IL-1βwere significantly increased in the OGD and DAPT groups compared to the control group;however,the expression of IL-1βwas significantly reduced in the DAPT group compared to the OGD group.RT-PCR showed that the expression of IL-1βmRNA were significantly increased in the OGD and DAPT groups compared to the control group;however,the expression of IL-1βmRNA was significantly reduced in the DAPT group compared to the OGD group(control group:1.000±0.17

关 键 词：缺氧 小胶质细胞 Notch信号通路 OGD DAPT NICD IL-1Β 脑缺血-再灌注损伤 

分 类 号：R743.3[医药卫生—神经病学与精神病学]