羟基积雪草苷对LPS诱导小胶质细胞增殖的抑制作用及机制研究

Inhibitory effect of madecassoside on LPS-stimulated microglia

目的:研究羟基积雪草苷对LPS刺激的小胶质细胞增殖的抑制作用及机制。方法:取SD大鼠的新生小鼠,进行小胶质细胞的原代培养,分离纯化小胶质细胞;MTT法筛选LPS刺激小胶质细胞增殖的最佳浓度,观察不同浓度羟基积雪草苷对LPS刺激小胶质细胞后的作用。ELISA检测细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)的含量,流式细胞术检测细胞周期及细胞凋亡,Western blotting法检测Toll样受体4(TLR4)蛋白的表达,RT-PCR检测NF-κB mRNA的表达。结果:LPS可以明显诱导体外培养小胶质细胞的增殖和炎症因子释放。与LPS组比较,羟基积雪草苷对LPS诱导的小胶质细胞增殖具有显著抑制作用,且具有剂量依赖性,羟基积雪草苷处理小胶质细胞48 h的IC50为10.97 nmol/L。同时羟基积雪草苷使小胶质细胞TNF-α和IL-6的释放显著降低(P<0.05);羟基积雪草苷使小胶质细胞的G2期细胞与细胞凋亡率增加,并降低小胶质细胞TLR4和NF-κB的表达。结论:羟基积雪草苷对LPS刺激的小胶质细胞的增殖和炎症因子的生成具有抑制作用,其作用机制可能与抑制TLR-4和NF-κB表达、改变细胞周期并诱导细胞凋亡有关。

AIM：To observe the inhibitory effect of madecassoside on the LPS-stimulated microglia and to inves-tigate its possible mechanism.METHODS：Microglia cells of neonatal Sprague-Dawley （ SD） rats were cultured, isolated and purified.Microglia cells were activated with lipopolysaccharide （ LPS） .The inhibitory effect of madecassoside on micro-glia was measured by MTT assay.Tumor necrosis factor alpha （TNF-α）, interleukin 1β（IL-1β） were detected by ELISA. Cell cycle and apoptotic rate were evaluated by flow cytometry.The expression of TLR4 was detected by Western blotting. The expression of NF-κB was detected by RT-PCR.RESULTS： LPS induced the proliferation of microglia and release in-flammatory cytokines significantly.Compared with LPS group, madecassoside inhibited the proliferation of microglia induced by LPS in a dose dependent manner.The IC50 value of madecassoside was 10.97 nmol/L to microglia after incubation for 48 h.Madecassoside also decreased the levels of TNF-αand IL-6, increased the ratios of microglia at the G2 phase and the ap-optotic rate, decreased the expression of TLR4 and NF-κB significantly （P〈0.05）.CONCLUSION：Madecassoside has in-hibitory effects on the proliferation of LPS-stimulated microglia, by which the mechanism may be related to inhibition of the expression of TLR4 and NF-κB, change of cell cycle distribution and induction of microglia apoptosis.