低温对小胶质细胞氧糖剥夺/复糖复氧时细胞极化及TLR4/NF-κB信号通路的影响

Effect of hypothermia on polarization of microglia and TLR4/NF-κB signaling pathway during oxygen-glucose deprivation/restoration

目的评价低温对小胶质细胞氧糖剥夺/复糖复氧(OGD/R)时细胞极化及TLR4/NF-κB信号通路的影响。方法体外培养BV2小胶质细胞,采用随机数字表法分为3组(n=18):对照组(C组)、OGD/R组和OGD/R+低温组(OGD/R+HT组)。C组正常培养24 h,OGD/R组氧糖剥夺2 h后复糖复氧24 h,低温组在33℃低温培养箱中对细胞进行氧糖剥夺2 h复糖复氧24 h处理。采用CCK-8法检测细胞存活率,分别采用免疫荧光及RT-qPCR法检测M1型小胶质细胞标志物CD32、iNOS及其mRNA、M2型小胶质细胞标志物CD206、Arg-1及其mRNA的表达,分别采用Western blot法和RT-qPCR法检测TLR4、NF-κB及其mRNA的表达,采用ELISA法检测细胞上清液TNF-α、IL-1β及IL-10浓度。结果与C组比较,OGD/R组和OGD/R+HT组细胞存活率下降,CD32、iNOS、CD206、Arg-1、TLR4、NF-κB及其mRNA表达上调,上清液TNF-α、IL-1β及IL-10浓度升高(P<0.05);与OGD/R组比较,OGD/R+HT组细胞存活率升高,CD32、iNOS、TLR4和NF-κB及其mRNA表达下调,CD206、Arg-1及其mRNA表达上调,上清液TNF-α、IL-1β浓度降低,IL-10浓度升高(P<0.05)。结论低温可明显抑制小胶质细胞OGD/R时向M1型极化,并增加M2型水平,抑制炎症反应,其机制可能与抑制TLR4/NF-κB信号通路有关。

Objective To evaluate the effect of hypothermia on the polarization of microglia and TLR4/NF-κB signaling pathway during oxygen-glucose deprivation/restoration(OGD/R).Methods BV2 microglia were cultured in vitro and divided into 3 groups(n=18 each)using the random number table method:control group(group C),group OGD/R and OGD/R plus hypothermia group(group OGD/R+HT).Group C was cultured normally for 24 h.In group OGD/R,the cells were exposed to 5%CO2-1%O2 at 37℃for 2 h in a glucose-free medium,followed by restoration of glucose and oxygen for 24 h.In group OGD/R+HT,the high-glucose medium was replaced with a glucose-free medium,the cells were exposed to 5%CO2-1%O2 for 2 h in a 33℃cryostat,followed by restoration of glucose and oxygen for 24 h.The cell survival rate was measured by CCK-8 assay.The expression of M1 microglia markers CD32 and iNOS protein and mRNA and M2 microglia markers CD206 and Arg-1 and mRNA was detected by immunofluorescence and real-time quantitative polymerase chain reaction.The expression of TLR-4 and NF-κB in cells was detected by Western blot,and the expression of TLR4 and NF-κB mRNA in cells was detected by real-time quantitative polymerase chain reaction.The concentrations of tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β)and interleukin 10(IL-10)in the cell supernatant were detected by enzyme-linked immunosorbent assay.Results Compared with group C,the cell survival rate was significantly decreased,the expression of CD32,iNOS,CD206,Arg-1,TLR4 and NF-κB protein and mRNA was up-regulated,and the concentrations of TNF-α,IL-1βand IL-10 in supernatant were increased in OGD/R and OGD/R+HT groups(P<0.05).Compared with group OGD/R,the cell survival rate was significantly increased,the expression of CD32,iNOS,TLR4 and NF-κB protein and mRNA was down-regulated,the expression of CD206 and Arg-1 protein and mRNA was up-regulated,the concentrations of TNF-αand IL-1βin supernatant were decreased,and the concentration of IL-10 was increased in group OGD/R+HT(P<0.05).Conclusions Hypothermia can significantly inhibit microglia polarization toward M1 phenotype,increase microglia polarization toward M2 phenotype and inhibit the development of inflammatory responses during OGD/R,and the mechanism may be related to inhibition of TLR4/NF-κB signaling pathway.