冬凌草甲素调节IL-6/STAT3信号通路对氧糖剥夺/复氧小胶质细胞增殖、自噬、凋亡及炎症反应的影响

The effect of oridonin regulating the IL-6/STAT3 signaling pathway on proliferation，autophagy， apoptosis，and inflammatory response in microglia subjected to oxygen-glucose deprivation/reoxygenation

关键词:  冬凌草甲素  白细胞介素-6/转录活化因子3信号通路  氧糖剥夺/复氧  小胶质细胞  增殖  自噬  凋亡

Key words:  oridonin  IL-6/STAT3 signaling pathway  oxygen-glucose deprivation/reoxygenation  microglia  proliferation  autophagy  apoptosis

摘要:

[目的] 探讨冬凌草甲素（ORI）调节白细胞介素（IL）-6/转录活化因子3（STAT3）信号通路对氧糖剥夺/复氧（OGD/R）小胶质细胞增殖、自噬、凋亡及炎症反应的影响。[方法] 将小胶质细胞BV2细胞分成：对照组、OGD/R组、ORI低、中、高浓度（ORI-L、ORI-M、ORI-H）组、ORI与重组IL-6联合处理组。各组给予相应干预并培养24 h。细胞计数试剂盒8（CCK8）法和5-乙炔基-2’-脱氧尿苷（EdU）染色法检测BV2细胞增殖；透射电子显微镜观察BV2细胞自噬；流式细胞仪检测BV2细胞凋亡；酶联免疫吸附法检测BV2细胞中炎症因子[肿瘤坏死因子-α（TNF-α）、IL-18、IL-1β]水平；蛋白印迹法检测BV2细胞中IL-6、STAT3蛋白表达。[结果] 与对照组比较，OGD/R组BV2细胞存活率、EdU阳性细胞率降低，自噬空泡数、凋亡率、TNF-α、IL-18、IL-1β、IL-6、STAT3蛋白表达升高（P<0.01）；与OGD/R组比较，ORI-L组、ORI-M组、ORI-H组BV2细胞存活率、EdU阳性细胞率呈浓度依赖性升高，自噬空泡数、凋亡率、TNF-α、IL-18、IL-1β、IL-6、STAT3蛋白表达呈浓度依赖性降低（P<0.05）；与ORI-H组比较，ORI与重组IL-6联合处理组BV2细胞存活率、EdU阳性细胞率降低，自噬空泡数、凋亡率、TNF-α、IL-18、IL-1β、IL-6、STAT3蛋白表达升高（P<0.01）。[结论] ORI能够促进OGD/R诱导的小胶质细胞增殖，抑制OGD/R诱导的小胶质细胞自噬、凋亡及炎症反应，其机制可能是通过抑制IL-6/STAT3信号通路实现的。

Abstract:

[Objective] To investigate the effect of oridonin（ORI） on proliferation，autophagy，apoptosis，and inflammatory response in microglia subjected to oxygen-glucose deprivation/reoxygenation（OGD/R） and its potential mechanism involving the interleukin-6（IL-6）/signal transducer and activator of transcription 3（STAT3） signaling pathway. [Methods] BV2 microglial cells were divided into the following groups：control，OGD/R，ORI（low，medium，and high concentration：ORI-L，ORI-M，ORI-H），and ORI combined with recombinant IL-6. After respective treatments for 24 h，cell proliferation was assessed by Cell Counting Kit-8（CCK-8） assay and 5-ethynyl-2'-deoxyuridine（EdU） staining. Autophagy was observed under a transmission electron microscope. Apoptosis was detected by flow cytometry. The levels of inflammatory factors [tumor necrosis factor-α（TNF-α），IL-18，and IL-1β] were measured using enzyme-linked immunosorbent assay（ELISA）. The protein expression levels of IL-6 and STAT3 were determined by Western blotting. [Results] Compared with the control group，the OGD/R group showed decreased cell viability and EdU-positive cell rate，along with increased number of autophagic vacuoles，apoptosis rate，levels of TNF-α，IL-18，IL-1β，and protein expression of IL-6 and STAT3（P<0.01）. Compared with the OGD/R group，the ORI-L，ORI-M，and ORI-H groups exhibited a concentration-dependent increase in cell viability and EdU-positive cell rate，and a concentration-dependent decrease in the number of autophagic vacuoles，apoptosis rate，levels of TNF-α，IL-18，IL-1β，and protein expression of IL-6 and STAT3（P<0.05）. Compared with the ORI-H group，the ORI combined with recombinant IL-6 group demonstrated decreased cell viability and EdU-positive cell rate，and increased number of autophagic vacuoles，apoptosis rate，levels of TNF-α，IL-18，IL-1β，and protein expression of IL-6 and STAT3（P<0.01）. [Conclusion] ORI can promote proliferation and inhibit autophagy，apoptosis，and inflammatory response in OGD/R-induced microglia. Its mechanism may be associated with the inhibition of the IL-6/STAT3 signaling pathway.