小胶质细胞参与视网膜神经节细胞死亡机制的实验研究

目的探讨小胶质细胞活化在急性高眼压引起的视网膜缺血再灌注损伤视网膜神经节细胞(RGC)死亡中的作用及其机制。方法实验研究。取C57BL/6小鼠原代RGC与小鼠小胶质细胞BV2细胞共培养或单独培养,建立体外氧糖剥夺/再灌注(OGD/R)模型模拟体内视网膜缺血再灌注损伤(复氧时间分别设为6 h、24 h、36 h、48 h),使用小胶质细胞特异性离子钙接头蛋白(iba1)免疫荧光染色评估BV2细胞活化程度;采用活细胞计数试剂盒检测RGC的细胞活性;应用细胞凋亡检测试剂盒检测RGC凋亡率;通过半定量逆转录PCR、Western印迹、细胞免疫荧光检测BV2细胞中Toll样受体-4(TLR4)与Nod样受体家族含pyrin结构域蛋白3(NLRP3)的mRNA及蛋白水平;应用半胱氨酸天冬氨酸蛋白酶-8(caspase-8)染色试剂盒检测BV2细胞中caspase-8活性;酶联免疫吸附试验检测BV2细胞上清液中白细胞介素1β(IL-1β)含量;应用TLR4小干扰RNA(siRNA)转染和caspase-8抑制剂阻断相应通路,对比TLR4、NLRP3的表达、caspase-8的活性变化、IL-1β含量的差异以及共培养RGC的细胞活性变化。采用方差分析进行统计学分析。结果RGC与BV2细胞共培养下,细胞免疫荧光检测显示OGD/R模型中BV2细胞iba1表达增多,BV2细胞显著激活。RGC与BV2细胞共培养下,OGD/R模型中RGC的凋亡率(71.1%±3.2%)高于RGC单独培养下OGD/R模型中RGC的凋亡率(35.1%±1.8%),差异有统计学意义(t=10.10,P<0.01)。细胞免疫荧光检测显示BV2细胞单独培养下,OGD/R模型中BV2细胞TLR4、NLRP3表达显著增加,其mRNA水平均随复氧时间延长显著上升,差异均有统计学意义(F=64.45,72.74;均P<0.01),且在OGD/R复氧24 h时达到峰值(TLR4 mRNA与对照的相对比值为2.83±0.23,NLRP3 mRNA与对照的相对比值为3.12±0.27);caspase-8的活性也随复氧时间延长显著增加,差异有统计学意义(F=93.57,P<0.01),且在OGD/R复氧24 h时达到峰值(与对照的相对比值为2.92±0.31)。应用TLR4 siRNA转染BV2细胞后其caspase-8的活性明显受到抑制,应用caspase-8抑制剂并不影响BV2细胞中TLR4的表达上调,而OGD/R作用下BV2细胞分泌的成熟IL-1β在caspase-8抑制剂干预下显著减少(由3.52±0.55降低为1.39±0.37,t=7.19,P<0.01),同时NLRP3的表达量在caspase-8抑制剂干预下显著降低(由2.79±0.23降低至1.37±0.19,t=9.37,P<0.01)。OGD/R模型中,与TLR4 siRNA转染BV2细胞共培养的RGC细胞活性为74.5%±1.2%,与经caspase-8抑制剂处理BV2细胞共培养的RGC细胞活性为62.8%±1.5%,均高于与对照BV2细胞共培养的RGC细胞活性(36.7%±0.3%),差异均有统计学意义(t=11.60,6.83;均P<0.01)。结论视网膜缺血再灌注损伤促使小胶质细胞活化,激活TLR4-caspase-8-NLRP3炎性反应小体信号通路,介导RGC死亡。

Mechanism of microglia promoting retinal ganglion cell death in vitro

Objective To investigate the role and mechanism of microglial activation in the process of retinal ganglion cell(RGC)death in the oxygen-glucose deprivation/reperfusion(OGD/R)model which mimicked retinal ischemia/reperfusion injury in vitro.Methods Experimental study.Primary RGCs from C57BL/6 mice and BV2 microglia were co-cultured or cultured alone.The OGD/R model was established in vitro(reoxygenation time was set to 6 h,24 h,36 h,48 h).BV2 microglial activation was assessed by immunofluorescence staining of ionized calcium binding adapter molecule 1(iba1),and the survival rate of RGCs was detected by the Cell Counting Kit-8.The apoptosis rate of RGC was detected by using apoptosis detection kit.The levels of Toll-like receptor-4(TLR4)and Nod-like receptor family pyrin domain containing protein 3(NLRP3)in BV2 cells were detected by PCR,Western-blot and immunofluorescence staining.The activity of caspase-8 in BV2 cells was detected by the CaspGLOW Kit,and the content of interleukine-1β(IL-1β)in the supernatant was detected by enzyme linked immunosorbent assay.After the corresponding pathways were blocked by TLR4 small interfering RNA(siRNA)transfection or caspase-8 inhibitor,the expression changes of TLR4 and NLRP3,the activity of caspase-8,and the difference of IL-1βcontent could be observed as well as the activity of RGCs co-cultured with BV2.Statistical analysis was performed using analysis of variance.Results Under co-culture of RGC and BV2 cells,cellular immunofluorescence detection showed that the expression of iba1 in BV2 cells increased,which indicated BV2 cells were activated significantly in the OGD/R model.In the OGD/R model,the apoptosis rate of RGC co-cultured with BV2 cells(71.1%±3.2%)was significantly higher than that of RGC cultured alone(35.1%±1.8%)(t=10.10,P<0.01).Cellular immunofluorescence detection showed that the expression of TLR4 and NLRP3 in BV2 cells in the OGD/R model increased significantly when BV2 cells were cultured alone,and their mRNA levels increased significantly with prolongation of reoxygenation time(F=64.45,72.74;P<0.01),and peaked at OGD/R 24 h(TLR4 mRNA,relative ratio to control was 2.83±0.23;NLRP3 mRNA,relative ratio to control was 3.12±0.27).Caspase-8 activity also increased with prolonged reoxygenation time,the difference was statistically significant(F=93.57,P<0.01),and peaked at OGD/R 24 h(relative ratio to control was 2.92±0.31).After transfection of BV2 cells with TLR4 siRNA,its caspase-8 activity was significantly inhibited,but using caspase-8 inhibitor did not affect the up-regulation of TLR4 expression in BV2 cells.However,the mature IL-1βsecreted by BV2 cells exposed to OGD/R was significantly reduced by using caspase-8 inhibitor(from 3.52±0.55 to 1.39±0.37,t=7.19,P<0.01),meanwhile,the expression of NLRP3 was also significantly decreased after caspase-8 inhibitor pretreatment(from 2.79±0.23 to 1.37±0.19,t=9.37,P<0.01).In the OGD/R model,the activity of RGC cells co-cultured with TLR4 siRNA-transfected BV2 cells was 74.5%±1.2%,and the activity of RGC cells co-cultured with BV2 cells treated with caspase-8 inhibitor was 62.8%±1.5%,those were both higher than that of RGC cells co-cultured with untreated BV2 cells(36.7%±0.3%),and the difference was statistically significant(t=11.60,6.83;both P<0.01).Conclusion TLR4-caspase-8-NLRP3 inflammasome pathway is activated in microglia exposed to OGD/R,resulting in the production of IL-1β,thereby contributing to the death of RGCs.