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目的观察瓜子金皂苷己(polygalasaponin F,PGSF)对脂多糖(LPS)诱导的BV-2小胶质瘤细胞激活的作用及其作用机制。方法以0.1μg·mL~(-1)LPS刺激BV-2细胞24 h构建神经炎症模型,以酶联免疫吸附分析法(ELISA)检测细胞培养基上清中的白介素1β(IL~(-1)β)、肿瘤坏死因子(TNF-α)浓度,以Griess法检测一氧化氮(NO)的浓度;以逆转录-聚合酶链式反应(RT-PCR)法或免疫印迹法(Western Blot)检测细胞炎性蛋白酶诱导型一氧化氮合酶(iNOS)和环氧合酶-2(COX-2)的蛋白、mRNA表达,以及Toll样受体4(TLR4)mRNA表达。结果PGSF(10,1μmol·L~(-1))可明显降低LPS诱导的BV-2细胞培养基中TNF-α(P<0.01)和IL~(-1)β(P<0.05)浓度,同时下调细胞内iNOS蛋白(P<0.05)和mRNA(P<0.01,P<0.05)表达;PGSF(10μmol·L~(-1))可降低NO的浓度(P<0.05)、下调COX-2的蛋白和mRNA(P<0.05)表达,并逆转TLR4 mRNA表达的上调(P<0.05)。结论PGSF能够负调控LPS诱导的BV-2小胶质细胞炎性介质的释放和炎性蛋白酶的表达,抑制小胶质细胞激活,发挥对抗神经炎症的作用,此抗炎作用可能由TLR4受体介导。
Objective To observe the effect of PGSF on activation of lipopolysaccharide (LPS) -induced BV-2 microglial cells and its mechanism. Methods The model of neuroinflammation was induced by stimulating BV-2 cells with 0.1μg · mL -1 LPS for 24 hours. The level of interleukin-1β (IL-1β) in the supernatant of the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA) ) β, TNF-α, and the concentration of nitric oxide (NO) were determined by Griess method. The expression of nitric oxide was detected by reverse transcriptase-polymerase chain reaction (RT- The expressions of protein, mRNA and Toll-like receptor 4 (TLR4) mRNA of inflammatory proteinase inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) Results PGSF (10,1 μmol·L -1) significantly decreased the concentration of TNF-α (P <0.01) and IL-1β (P <0.05) in BV-2 cells induced by LPS, (P <0.01, P <0.05). PGSF (10μmol·L -1) decreased the concentration of NO (P <0.05) and decreased the expression of COX-2 (P <0.05), and reversed the up-regulation of TLR4 mRNA expression (P <0.05). Conclusion PGSF can negatively regulate LPS-induced release of BV-2 microglial inflammatory mediators and expression of inflammatory proteases, inhibit microglial activation and exert anti-neuroinflammatory effects. This anti-inflammatory effect may be mediated by TLR4 receptor mediate.