豨莶草水洗脱部位对人肺纤维细胞的毒性与药效作用

Toxicity and Efficacy of Water-eluted Fraction from Siegesbeckiae Herba Against Human Embryonic Lung Fibroblasts

目的:检测不同浓度豨莶草水洗脱部位(SWEF)对MRC-5人肺纤维细胞的毒性作用及对MRC-5细胞α7烟碱型乙酰胆碱受体(α7nAChR)表达与炎症因子的影响,从而发现豨莶草毒效与药效作用物质基础。方法:以MRC-5细胞为研究对象,采用细胞增殖与活性检测(CCK-8)法结合流式细胞术、台盼蓝染色判定SWEF(1,6,10,20,50 g·L^(-1))对MRC-5细胞的毒效作用;通过测定沉默基因前后α7nAChR的表达及炎症因子水平的变化探讨SWEF对MRC-5细胞的药效作用。结果:SWEF(≥6 g·L^(-1))显著降低MRC-5细胞的存活率(P<0.01),促进MRC-5细胞凋亡与坏死(P<0.01),其半数抑制浓度(IC50)为6.03 g·L^(-1);MRC-5细胞α7nAChR表达与炎症因子检测显示SWEF含有α7nAChR激动剂样物质,该物质可增高α7nAChR基因和蛋白的表达(P<0.05,P<0.01),降低炎症因子水平(P<0.05,P<0.01),SWEF下调炎症因子的作用可能与其上调α7nAChR mRNA表达有关,二者呈负相关(P<0.01)。结论:SWEF(≥6 g·L^(-1))对MRC-5细胞具有显著的毒性作用,药效作用研究证实SWEF含有α7nAChR激动剂样物质,该物质可增高α7nAChR的表达,降低炎症因子水平,推测过量的α7nAChR激动剂样物质可能为豨莶草的毒性成分之一。

Objective: To detect the toxicity of water-eluted fraction from Siegesbeckiae Herba(SWEF)at different concentrations against MRC-5 human embryonic lung fibroblasts and its impacts on the expression of α7 nicotinic acetylcholine receptor(α7nAChR)and inflammatory factors,so as to figure out the active components responsible for toxicity and efficacy. Method: The toxicities of SWEF at 1,6,10,20,and 50 g·L^(-1) against MRC-5 cells were determined by cell counting kit-8(CCK-8)assay combined with flow cytometry and Trypan blue staining. The changes in α7nAChR expression and inflammatory factor levels before and after α7nAChR gene silencing were detected to reveal the pharmacodynamic effect of SWEF on MRC-5 cells. Result: SWEF(≥6 g·L^(-1))obviously inhibited the viability of MRC-5 cells(P<0.01)and promoted their apoptosis and necrosis(P<0.01),with the half-maximal inhibitory concentration(IC50)being 6.03 g·L^(-1). The determination of α7nAChR expression and inflammatory factor levels in MRC-5 cells showed that SWEF contained α7nAChR agonist-like substance,which enhanced α7nAChR mRNA and protein expression(P<0.05,P<0.01) and decreased the inflammatory factor levels(P<0.05, P<0.01). SWEF down-regulated the inflammatory factors possibly by re-regulating α7nAChR mRNA expression,exhibiting a negative correlation between them(P<0.01). Conclusion: SWEF( ≥6 g·L^(-1))is highly toxic to MRC-5 cells. Pharmacodynamic studies have confirmed that α7nAChR agonist-like substance contained in SWEF was responsible for the elevated α7nAChR expression and reduced inflammatory cytokines. It is inferred that excessive α7nAChR agonist-like substance may trigger the toxicity of Siegesbeckiae Herba.