异丙酚通过TRPV1离子通道抑制LPS诱导的小胶质细胞炎症反应

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

目的:探讨异丙酚(propofol,P)对脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞炎症反应的影响及其机制。方法:LPS(100μg/L)处理建立小鼠小胶质细胞BV2神经炎症损伤模型,将细胞分为对照(C)组、模型(L)组、L+P组和LPS+AMG517(L+A)组。ELISA法检测各组细胞培养液中肿瘤坏死因子α(TNF-α)的含量;real-timePCR法检测各组细胞瞬时受体电位阳离子通道V亚族成员1(TRPV1) mRNA的表达;Western blot法检测各组细胞TRPV1、TNF-α、白细胞介素1β(IL-1β)、IL-6和磷酸化钙/钙调蛋白依赖性蛋白激酶Ⅱ(p-CaMKⅡ)的蛋白表达;荧光探针Fluo-3 AM标记法检测细胞内游离的Ca2+含量。结果:与C组相比,L组细胞培养液中TNF-α水平显著升高(P<0.01);单独propofol处理TNF-α水平无显著差异;与L组相比,L+P组细胞培养液中在4 h内TNF-α水平显著降低(P<0.01)。与C组相比,L组TRPV1 mRNA表达显著上调(P<0.01);与L组相比,L+P组TRPV1 mRNA表达显著下调(P<0.01)。相比L组,L+P和L+A组TNF-α、IL-1β、IL-6和p-CaMKⅡ蛋白表达显著下调(P<0.01)。相比L组,L+P和L+A组细胞内Ca2+浓度显著降低(P<0.01)。结论:Propofol通过下调TRPV1的表达,下调p-CaMKⅡ的表达,降低细胞内Ca2+浓度,从而下调TNF-α、IL-1β和IL-6的表达,抑制小胶质细胞的炎症反应。

AIM: To investigate the effects of propofol(P) on the inflammatory response of microglia induced by lipopolysaccharide(LPS) and the mechanisms. METHODS: Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups: control group(C group), model group(L group), L+P group and LPS+AMG517 group(L+A group). The level of tumor necrosis factor-α(TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1(TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β(IL-1β), interleukin-6(IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ(p-CaMKⅡ) were determined by Western blot. The content of free Ca2+ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS: Compared with C group, the level of TNF-α was significantly increased in L group(P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h(P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group(P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group(P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca2+ concentration were significantly lower than those in L+P group and L+A group(P<0.01). CONCLUSION: Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca2+ concentration.