【摘要】 目的 探讨右美托咪定介导白细胞介素(IL)-17对脂多糖(LPS)诱导的人Ⅱ型肺泡上皮A549细胞(以下简称“A549细胞”)炎症、增殖、迁移、上皮间质转化及转化生长因子-β1(TGF-β1)/Smad蛋白3(Smad3)信号通路的调控作用。方法 体外培养A549细胞,将其分为对照组(不做干预)、LPS组(10.00 μg/ml LPS)和实验组(10.00 μg/ml LPS+1.25、2.50、5.00、10.00 μg/ml右美托咪定),分别采用CCK-8试剂盒和反转录-聚合酶链反应(RT-qPCR)法测定细胞活力和IL-17 mRNA表达水平以筛选右美托咪定最适实验浓度;随后将细胞分为对照组、LPS组、IL-17组、右美托咪定组和IL-17+右美托咪定组,干预24 h。酶联免疫吸附试验(ELISA)检测炎症因子IL-17、IL-8和IL-6的表达水平;5-乙炔基-2’脱氧尿嘧啶核苷(Ed U)试剂盒用来检测细胞增殖率;划痕实验用来检测细胞迁移率;蛋白免疫印迹(WB)法测定肺泡上皮间质转化(EMT)相关蛋白、TGF-β1/Smad3通路相关蛋白及IL-17蛋白表达水平。结果 右美托咪定逆转了LPS对A549细胞活力的抑制作用和IL-17 mRNA表达水平的促进作用,且10.00 μg/ml浓度的右美托咪定组的效果最好,因此选择10.00 μg/ml右美托咪定用于后续实验。LPS组细胞中IL-17、IL-8、IL-6表达水平、细胞迁移率、N-钙黏蛋白、波形蛋白、纤维粘连蛋白(FN)、Smad3的磷酸化(p-Smad3)/Smad3、TGF-β1和IL-17蛋白表达水平高于对照组,差异有统计学意义(P<0.05);LPS组细胞增殖率、E-钙黏蛋白和Smad7蛋白表达水平低于对照组,差异有统计学意义(P<0.05)。IL-17+右美托咪定组IL-17、IL-8、IL-6表达水平、细胞迁移率、N-钙黏蛋白、波形蛋白、FN、p-Smad3/Smad3、TGF-β1和IL-17蛋白表达水平低于LPS组,差异有统计学意义(P<0.05);IL-17+右美托咪定组细胞增殖率、E-钙黏蛋白和Smad7蛋白表达水平高于LPS组,差异有统计学意义(P<0.05)。结论 右美托咪定通过抑制IL-17的分泌恢复LPS诱导的人Ⅱ型肺泡上皮细胞损伤,促进LPS诱导的人Ⅱ型肺泡上皮细胞增殖并抑制其迁移和EMT进程,其作用机制与抑制TGF-β1/Smad3通路的信号转导有关。更多还原

【Abstract】 Objective To explore the regulatory effects of Dexmedetomidine mediating interleukin(IL)-17 on lipopolysaccharide(LPS) induced inflammation, proliferation, migration, epithelial interstitium transformation and transforming growth factor-β1(TGF-β1)/Smad protein 3(Smad3) signaling pathway in human type Ⅱ alveolar epithelial A549 cells(hereinafter referred to as "A549 cells"). Methods The A549 cells were cultured in vitro and they were divided into control group(no intervention), LPS group(10.00 μg/ml LPS) and experimental group(10.00 μg/ml LPS+1.25, 2.50, 5.00, 10.00 μg/ml Dexmedetomidine). CCK-8 kit and quantitative real-time PCR(RT-qPCR) were used to determine cell viability and IL-17 mRNA expression levels, respectively, to screen the optimal concentration of Dexmedetomidine. The cells were then divided into control group, LPS group, IL-17 group,Dexmedetomidine group and IL-17+Dexmedetomidine group, and they were intervened for 24 h. The expression levels of inflammatory cytokines IL-17, IL-8 and IL-6 were detected by enzyme-linked immunosorbent assay(ELISA). 5-acetylidene-2’ Deoxyuracil nucleoside(Ed U) kit was used to detect cell proliferation rate. Scratch assay was used to detect cell mobility. The expression levels of epithelial-mesenchymal transition(EMT) related protein, TGF-β1/Smad3 pathway related protein and IL-17 protein were determined by western blot(WB). Results Dexmedetomidine reversed the inhibition effect of LPS on A549 cell viability and the promotion effect of IL-17 mRNA expression level, and the 10.00 μg/ml Dexmedetomidine concentration group had the best effect, so 10.00 μg/ml Dexmedetomidine was selected for follow-up experiments. The expression levels of IL-17, IL-8, IL-6, cell mobility, N-cadherin, vimentin, fipronectin(FN), phosphorylation of Smad3(p-Smad3)/Smad3, TGF-β1 and IL-17 protein in LPS group were higher than those in control group, the differences were statistically significant(P<0.05). The cell proliferation rate, E-cadherin and Smad7 protein expression levels in LPS group were lower than those in control group, and the differences were statistically significant(P<0.05). The expression levels of IL-17, IL-8, IL-6, cell mobility, N-cadherin, Vimentin, FN, p-Smad3/Smad3, TGF-β1 and IL-17 in IL-17+ Dexmedetomidine group were lower than those in LPS group, with statistical significances(P<0.05). The cell proliferation rate, E-cadherin and Smad7 protein expression levels in IL-17+Dexmedetomidine group were higher than those in LPS group, and the differences were statistically significant(P<0.05). Conclusion Dexmedetomidine restored LPS-induced human type Ⅱ alveolar epithelial cell injury by inhibiting the secretion of IL-17, promoted LPS-induced proliferation of human type Ⅱ alveolar epithelial cell, and inhibited their migration and EMT process. The mechanism of action is related to the inhibition of TGF-β1/Smad3 pathway signal transduction.更多还原