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目的:探讨复方毒素清对肺上皮细胞A549内核因子κB(NF-κB)和丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号通路的影响,从细胞分子水平揭示毒素清治疗细菌性肺炎的作用机制。方法:LPS刺激肺泡上皮细胞A549,将培养好的A549细胞分为空白组(10%空白药物血清)、模型组(10%空白药物血清和1μg/ml LPS)、毒素清(14g/kg)组(10%含药血清和1μg/ml LPS)、毒素清(28g/kg)组(10%含药血清和1μg/ml LPS)、毒素清(56g/kg)组(10%含药血清和1μg/ml LPS),然后ELISA检测IL-1、IL-6、IL-8;实时荧光定量PCR测定TLR4mRNA和NF-κB mRNA的表达;Western Blotting检测P38MAPK、JNK46/54、ERK42/44总蛋白和磷酸化蛋白。结果:与空白组相比,模型组细胞L-1β、IL-6、IL-8、TLR4mRNA和NF-κB mRNA的表达以及P38MAPK、JNK46/54、ERK42/44蛋白的磷酸化水平均明显升高;与模型组相比,毒素清(14g/kg、28g/kg、56g/kg)的含药血清组L-1β、IL-6、IL-8、TLR4mRNA和NF-κB mRNA的表达以及P-P38MAPK、P-ERK42/44蛋白的磷酸化水平均明显降低,P-JNK46/54无显著性差异。结论:LPS能够激活肺上皮细胞A549内NF-κB和MAPK信号通路,诱导细胞因子的分泌。毒素清可通过抑制该两条信号通路,减少细胞因子的释放,从而减轻炎症反应。
Objective: To investigate the effect of compound toxin on the expression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in lung epithelial cells A549, Pneumonia mechanism of action. Methods: Human lung adenocarcinoma A549 cells were induced by LPS. The cultured A549 cells were divided into blank group (10% blank serum), model group (10% blank serum and 1μg / ml LPS), and toxin Qing (14g / (10% drug-containing serum and 1μg / ml LPS), toxins (28g / kg) (10% serum and 1μg / ml LPS) / ml LPS). The levels of IL-1, IL-6 and IL-8 were detected by ELISA. The expressions of TLR4 mRNA and NF-κB mRNA were detected by real-time fluorescence quantitative PCR. The expressions of P38MAPK, JNK46 / 54 and ERK42 / The protein. Results: Compared with the blank group, the expression of L-1β, IL-6, IL-8, TLR4 mRNA and NF-κB mRNA and phosphorylation of P38MAPK, JNK46 / 54 and ERK42 / 44 in model group were significantly increased ; Compared with the model group, the expression of L-1β, IL-6, IL-8, TLR4 mRNA and NF-κB mRNA in the serum containing 14g / kg, 28g / kg and 56g / P38MAPK, P-ERK42 / 44 protein phosphorylation levels were significantly lower, P-JNK46 / 54 no significant difference. Conclusion: LPS can activate NF-κB and MAPK signal pathways in lung epithelial A549 cells and induce the secretion of cytokines. Toxin Qing can inhibit the two signal pathways, reducing the release of cytokines, thereby reducing the inflammatory response.