当归芍药散含药血清对脂多糖诱导的RAW 264.7细胞炎症因子及信号通路的影响

Effects of drug-containing serum of Danggui Shaoyao powder on RAW 264.7 cell inflammatory factor and its signal pathway induced by lipopolysaccharide

目的探讨当归芍药散含药血清对脂多糖(lipopolysaccharide,LPS)诱导的RAW 264.7细胞炎症因子及炎症信号通路的影响。方法在体外培养的小鼠腹腔巨噬细胞脂多糖刺激,以建立炎症细胞模型。将细胞分为正常对照组、LPS诱导组、空白血清组、LPS+空白血清组、当归芍药散含药血清组和LPS+当归芍药散含药血清组。采用CCK-8法测定细胞生长活力,流式细胞术检测细胞因子白细胞介素-6(interleukin 6,IL-6)、单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)水平,Western Blot检测过氧化物酶体增殖剂激活受体(peroxisome proliferators-activated receptorγ,PPARγ)、核因子κB(nuclear factor kappa B,NF-κB)蛋白的表达,RT-PCR法检测PPARγ、NF-κB mRNA的表达。结果(1)RAW 264.7细胞在1μg/mL的LPS和10%、20%的空白血清、10%、20%的当归芍药散含药血清干预24小时后,各组细胞之间生长活力未见明显统计学差异(P>0.05)。(2)炎症因子水平,与正常对照组相比,LPS诱导组炎症因子IL-6和MCP-1水平均明显升高(P<0.05);与LPS+空白血清组相比,LPS+当归芍药散含药血清组炎症因子IL-6和MCP-1水平明显降低(P<0.05)。(3)与正常对照组相比,LPS诱导组NF-κB mRNA和蛋白表达水平明显升高(P<0.05),PPARγmRNA和蛋白表达水平降低(P<0.05);与LPS+空白血清组相比,LPS+当归芍药散含药血清组NF-κB mRNA和蛋白表达降低(P<0.05),PPARγmRNA和蛋白水平明显升高(P<0.05)。结论当归芍药散含药血清对NF-κB、PPARγ信号通路有显著的调控作用,并可降低其下游促炎因子IL-6、MCP-1的分泌水平,对代谢性炎症有明确的治疗作用,减缓了动脉粥样硬化的病理进程。

Objective To investigate effects of drug-containing serum of Danggui Shaoyao powder on RAW 264.7 cell inflammatory factor and inflammatory signaling pathway induced by lipopolysaccharide(LPS).Methods The inflammatory cell model was established by stimulating macrophages in mouse peritoneal cultured in vitro with LPS.The cells were divided into 6 groups,including normal control group,LPS induced group,blank serum group,LPS combining with blank serum group,drug-containing of Dangui Shaoyao powder group,and LPS combining with drug-containing of Dangui Shaoyao powder group.The cell growth activity was determined by CCK-8 determined.The cytokine levels of IL-6 and MCP-1 were detected by flow cytometry.The protein expression levels of PPARγand NF-κB were detected by Western Blot.The mRNA expression levels of PPARγand NF-κB were detected by RT-PCR method.Results After RAW 264.7 cells being intervened with 1ug·mL-1 LPS,10%and 20%blank serum,10%and 20%drug-containing serum of Danggui Shaoyao powder for 24 hours respectively,there was no statistically significant difference in the growth activity of the cells in each group(P>0.05).(2)Compared with normal control group,the cytokine levels of IL-6 and MCP-1 in LPS induced group both increased significantly(P<0.05);Compared with LPS combining with blank serum group,the cytokine levels of IL-6 and MCP-1 in LPS combining with drug-containing serum of Danggui Shaoyao powder group decreased significantly(P<0.05).(3)Compared with normal control group,the protein and mRNA expression levels of NF-κB in LPS induced group both increased significantly(P<0.05),while the protein and mRNA expression levels of PPARγboth decreased significantly(P<0.05);Compared with LPS combing with blank serum group,the protein and mRNA expression levels of NF-κB in LPS combining with drug-containing serum of Danggui Shaoyao powder group both decreased significantly(P<0.05),while the protein and mRNA expression levels of PPARγboth increased significantly(P<0.05).Conclusion Drug-containing serum of Danggui Shaoyao powder has a significant regulatory effect on signaling pathways of NF-κB and PPARγ,reducing the secretion levels of pro-inflammatory cytokines IL-6 and MCP-1 on the signaling pathway’s downstream,having definite therapeutic effect on metabolic inflammation,and slowing down the pathological process of atherosclerosis.