右美托咪啶通过HSP60-TLR4-NF-κB通路抑制热应激诱导的小胶质细胞活化

Dexmedetomidine inhibits the activation of microglia induced by heat stress through the HSP60-TLR4-NF-κB signaling pathway

目的热射病是一种死亡率极高的重症中暑,所导致的脑损伤是其防治的焦点。本研究拟探讨右美托咪啶(Dexmedetomidine,Dex)对热应激诱导小鼠小胶质细胞株BV2细胞活化的影响,以期为热射病脑损伤的防治研究提供实验依据。方法采用体外培养BV2细胞,分为空白对照组、热应激组(43℃)、Dex处理组、热应激(43℃)联合药物处理组。采用MTT[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide]法检测细胞活力的改变。ELISA(enzyme linked immunosorbent assay)方法检测热休克蛋白60(heat shock protein 60,HSP60)和相关炎性因子如肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)、白细胞介素-1β(interleukin-1 beta,IL-1β)、白细胞介素-6(interleukin-6,IL-6)的释放。Western-blot检测Dex对热应激活化的BV2细胞信号通路的作用。结果本实验条件下,Dex以及热应激不会对细胞活力产生影响(P>0.05)。与空白对照组相比,热应激将导致HSP60及炎性因子(TNF-α、IL-1β、IL-6)的释放增加,而Dex的预处理能够减轻HSP60及炎性因子的释放,差异均具有统计学意义(P<0.05)。与空白对照组相比,热应激将导致Toll样受体-4(Toll-like receptor-4,TLR4)和核转录因子(nuclear factor kappa B,NF-κB)的表达上调,而Dex预处理能够减弱这样的表达上调,差异均具有统计学意义(P<0.05)。结论 Dex可能通过抑制HSP60-TLR4-NF-κB信号通路阻止热应激导致的小胶质细胞活化。

Objective Heat stroke is a severe heat stroke with high mortality, and brain damage is the focus of prevention and treatment. The aim of our study is to investigate the effect of dexmedetomidine（Dex） on the activation of BV2 cells induced by heat stress.Methods Microglia（BV2 cell line） was cultured in vitro. The experiment was divided into 4 groups, such as control group, heat stress group（43 ℃）, Dex treatment group, heat stress combined Dex treatment group.The changes of cell vitality were detected by MTT [3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide] method. The release of heat shock protein 60（HSP60） and related inflammatory factors, such as tumor necrosis factor-α（TNF-α）, interleukin-1β（IL-1β）,and Interleukin-6（IL-6） was detected by enzyme linked immunosorbent assay（ELISA）.Western-blot detected the effect of Dex on the signaling pathway of BV2 cell activation induced by heat stress, and to provide experimental basis for the prevention and treatment of brain injury caused by heat stroke.Results Under our experiment conditions, Dex and heat stress did not have a significant effect on cell viability（P〉0.05）. Compared with the control group, heat stress would cause HSP60 and inflammatory factor（TNF-alpha, IL-1 beta, IL-6） increased release, while Dex pretreatment could reduce the release of HSP60 and inflammatory factors. The differences were statistically significant（P〈0.05）.Compared with the control group, heat stress would increase Toll like receptor-4（Toll-like receptor-4, TLR4） and nuclear transcription factor（nuclear factor kappa B, NF-κB） expression, while Dex pretreatment attenuated the up-regulated expression of these proteins. The differences were statistically significant（P〈0.05）. Conclusion Dex may inhibit the activation of microglia caused by heat stress by inhibiting the HSP60-TLR4-NF-κB signaling pathway.