阿尔泰金莲花有效成分抗炎作用的研究

Anti-Inflammatory Effects of Active Components in Trollius altaicus

目的:研究阿尔泰金莲花总黄酮及其成分荭草素2″-O-β-L-半乳糖苷(OGA)的抗炎作用。方法:以环氧合酶1/2(COX 1/2)及5-脂氧酶(5-LO)抑制剂筛选试剂盒检测阿尔泰金莲花总黄酮和荭草素2″-O-β-L-半乳糖苷对COX 1/2和5-LO的抑制作用;脂多糖(LPS)刺激小鼠巨噬细胞RAW 264.7细胞建立炎症细胞模型,采用MTS法检测阿尔泰金莲花总黄酮和荭草素2″-O-β-L-半乳糖苷对RAW264.7细胞活力的影响,Griess试剂检测一氧化氮(NO)的分泌量,用2’,7’-氯荧光黄双乙酸酯(DCFH-DA)荧光探针法检测细胞内活性氧(ROS)释放情况,ELISA法测定肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)和前列腺素E;(PGE;)的分泌量,RT-qPCR法测定炎症因子Tnf-α、Il-1β和Il-6的mRNA表达。结果:阿尔泰金莲花总黄酮对COX-1、COX-2酶的半数抑制浓度分别106.66μg/mL,6.94μg/mL;荭草素2″-O-β-L-半乳糖苷对COX-1、COX-2的半数抑制浓度分别为140.34μg/mL,5.01μg/mL,阿尔泰金莲花总黄酮和荭草素2″-O-β-L-半乳糖苷对5-LO无明显影响;阿尔泰金莲花总黄酮和荭草素2″-O-β-L-半乳糖苷在1.95μg/mL~125μg/mL条件下对RAW264.7细胞不产生毒性作用。与正常对照组比较,模型对照组细胞中NO、ROS、TNF-α、IL-6、MCP-1和PGE;含量显著升高(P<0.01),Tnf-α、Il-1β和Il-6 mRNA表达显著上调(P<0.01);与模型对照组相比,阿尔泰金莲花总黄酮和荭草素2″-O-β-L-半乳糖苷呈剂量依赖性地降低细胞中NO、ROS、TNF-α、IL-6、MCP-1和PGE;的分泌(P<0.05或P<0.01),下调Tnf-α、Il-1β和Il-6的mRNA表达(P<0.05或P<0.01)。结论:阿尔泰金莲花总黄酮具有良好的抗炎作用,荭草素2″-O-β-L-半乳糖苷是其发挥抗炎作用的物质基础之一,其作用机制可能是通过抑制COX-2活性,减少RAW 264.7细胞NO、PGE;、MCP-1及炎性因子的分泌,下调Tnf-α、Il-6、Il-1βmRNA的表达有关。

Objective:To investigate the anti-inflammatory effects of the total flavonoids and orientin-2″-O-β-L-galactopyranosylide(OGA)from Trollius altaicus.Methods:Cyclooxygenase 1/2(COX 1/2)and 5-lipoxygenase inhibitor(5-LO)screening kits were employed to evaluate the inhibitory effects of the total flavonoids and OGA against COX 1/2 and 5-LO.The mouse macrophage RAW 264.7 cells were stimulated with lipopolysaccharide(LPS)for inducing inflammation,followed by the detection of the effects of total flavonoids and OGA on RAW264.7 cell viability using MTS method.Griess assay kit was employed to detect the secretion of nitric oxide(NO).The level of intracellular reactive oxygen species(ROS)was detected by fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA).The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1),and prostaglandin E;(PGE;)were detected by ELISA,and the mRNA expression levels of inflammatory cytokines TNF-α,IL-1β,and IL-6 by RT-qPCR.Results:The IC;values of the total flavonoids against COX-1 and COX-2 were 106.66μg/mL and 6.94μg/mL,respectively.The IC;values of OGA against COX-1 and COX-2 were 140.34μg/mL and 5.01μg/mL,respectively.The total flavonoids and OGA did not obviously affect 5-LO,and they were not toxic to RAW264.7 cells within the range of 1.95μg/mL~125μg/mL.Compared with the normal control group,the model group exhibited significantly increased NO,ROS,TNF-α,IL-6,MCP-1,and PGE;levels(P<0.01)and up-regulated mRNA expression of TNF-α,IL-1β,and IL-6(P<0.01).Compared with the model group,the total flavonoids and OGA of T.altaicus decreased NO,ROS,TNF-α,IL-6,MCP-1,and PGE;in a dose-dependent manner(P<0.01 or P<0.05)and down-regulated the mRNA expression of TNF-α,IL-1β,and IL-6(P<0.01 or P<0.05).Conclusion:The total flavonoids of T.altaicus have good anti-inflammatory effects,with OGA being an active component.The action mechanism may lie in the inhibition of COX-2 activity,the reduction of the release of NO,PGE;,and MCP-1 in RAW264.7 cells,and the down-regulation of TNF-α,IL-6,and IL-1βmRNA expression.