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目的观察丹皮酚对体外培养的星形胶质细胞炎性因子分泌的影响,并探讨其作用机制。方法采用神经胶质原纤维酸性蛋白(GFAP)免疫荧光染色法鉴定星形胶质细胞;实验分为对照组,模型组和2.5、5、10μmol·L-1丹皮酚组,0.5 mg·L-1脂多糖(LPS)诱导炎症反应。采用ELISA法测定培养液中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平;采用Western blot检测细胞IκBα蛋白表达和磷酸化水平及胞核NF-κB(p65)蛋白表达水平。结果与对照组相比,模型组星形胶质细胞上清液中IL-1β、IL-6和TNF-α水平显著增加(P<0.01),胞浆IκBα蛋白表达受抑(P<0.01),IκBα蛋白磷酸化和胞核NF-κB(p65)蛋白表达水平上调(P<0.01);5、10μmol·L-1丹皮酚能减少LPS活化的星形胶质细胞上清液中IL-1β、IL-6和TNF-α水平(P<0.05或P<0.01),增加胞浆IκBα蛋白表达(P<0.05或P<0.01),抑制LPS上调的IκBα蛋白磷酸化和胞核NF-κB(p65)蛋白表达水平(P<0.05或P<0.01)。结论丹皮酚能抑制LPS诱导的星形胶质细胞炎性因子IL-1β、IL-6和TNF-α的分泌,IκBα/NF-κB信号通路可能参与了丹皮酚对星形胶质细胞炎症反应的抑制作用。
Objective To observe the effect of paeonol on the secretion of inflammatory cytokines in cultured astrocytes and to explore its mechanism. Methods Astrocytes were identified by immunofluorescence staining with glial fibrillary acidic protein (GFAP). The experiment was divided into control group, model group and 2.5, 5, 10μmol·L -1 paeonol group, 0.5 mg · L -1 lipopolysaccharide (LPS) induces an inflammatory response. The levels of IL-1β, IL-6 and TNF-α in the culture medium were measured by ELISA. The expression of IκBα protein in the culture medium was detected by Western blotting Phosphorylation and nuclear NF-κB (p65) protein expression levels. Results Compared with the control group, the levels of IL-1β, IL-6 and TNF-α in astrocyte supernatant of model group were significantly increased (P <0.01), and the expression of IκBα protein in cytoplasm was inhibited (P <0.01) , Phosphorylation of IκBα protein and up-regulation of nuclear NF-κB (p65) protein expression (P <0.01). Paeonol at 5 and 10μmol·L -1 could reduce the level of IL- 1β, IL-6 and TNF-α (P <0.05 or P <0.01), increased IκBα protein expression (P <0.05 or P <0.01), inhibited IκBα phosphorylation and nuclear NF-κB (p65) protein expression level (P <0.05 or P <0.01). Conclusions Paeonol can inhibit the secretion of IL-1β, IL-6 and TNF-α induced by LPS, and the IκBα / NF-κB signaling pathway may be involved in the paracrine effect of paeonol on astrocytes Inflammatory response inhibition.