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为明确电压门控质子通道(voltage-gated proton channel,Hv1)对小胶质细胞的影响,研究了Hv1在正常、激活及抑制后对小胶质细胞活性氧、促炎因子产生的影响及机制,该研究使用原代培养的小胶质细胞,利用免疫荧光染色明确Hv1表达。用Hv1非特异性抑制剂Zn Cl2阻断该通道,用DCFH-DA染色、Real-time PCR观察糖氧剥夺(oxygen glucose deprivation,OGD)处理后小胶质细胞产生活性氧(reactive oxygen species,ROS)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)水平的变化。采用Western blot检测Hv1和Nox2蛋白质水平。实验结果显示,小鼠脑部小胶质细胞可检测到Hv1通道的表达;单纯ODG组缺氧后小胶质细胞ROS、TNF-α、IL-1β的产生明显增加;而OGD加Zn Cl2干预组,小胶质细胞ROS、TNF-α、IL-1β的产生较单纯OGD组显著降低;Western blot显示,OGD导致小胶质细胞中Hv1和Nox2蛋白质水平显著增加。以上结果表明,小鼠脑部小胶质细胞存在Hv1通道蛋白质,Zn Cl2阻断该通道可降低OGD诱导的小胶质细胞ROS和TNF-α、IL-1β的产生,该抑制作用可能与其抑制NADPH氧化酶的作用相关。
To clarify the effect of voltage-gated proton channel (Hv1) on microglia, the effect of Hv1 on the production of reactive oxygen species and proinflammatory cytokines in microglial cells after normal, activated and inhibited , Which uses primary cultured microglia and uses immunofluorescence staining to confirm Hv1 expression. The channel was blocked with ZnCl2, a non-specific inhibitor of Hv1. DCFH-DA staining and Real-time PCR were used to observe the effects of oxygen glucose deprivation (OGD) treatment on microglial cells producing reactive oxygen species (ROS) , Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels were measured. Western blot was used to detect Hv1 and Nox2 protein levels. The experimental results showed that the expression of Hv1 channel was detected in the mouse microglia cells. The production of ROS, TNF-α and IL-1β in microglia in ODG group was significantly increased after hypoxia in pure ODG group. However, The microglia ROS, TNF-α, IL-1β production was significantly lower than that of OGD alone group. Western blot showed that OGD resulted in a significant increase of Hv1 and Nox2 protein levels in microglia. The above results showed that the Hv1 channel protein was present in mouse microglia cells and the blockade of this channel by ZnCl2 could reduce the OGD-induced microglia ROS and TNF-α, IL-1β production, which may be related to its inhibition The role of NADPH oxidase is related.