益气养阴逐瘀方对LPS诱导的RAW 264.7细胞炎症模型体外抗炎活性的影响

Effect of Yiqi Yangyin Zhuyu Recipe in Anti-inflammation Activities in Vitro on LPS-induced RAW 264.7 Cells

目的:研究益气养阴逐瘀方对脂多糖(LPS)诱导的小鼠单核巨噬细胞系RAW264.7细胞炎症模型相关细胞因子表达及经典活化(M1)/选择性活化(M2)炎症表型转化的影响。方法:运用噻唑蓝(MTT)比色法检测不同给药浓度的益气养阴逐瘀方对RAW264.7细胞增殖情况的影响;利用LPS诱导RAW264.7细胞,构建体外炎症模型,采用格里斯试剂法(Griess)检测细胞上清液中一氧化氮(NO)的分泌量;酶联免疫吸附测定法(ELISA)检测细胞上清中M1/M2型相关炎症因子肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),白细胞介素-1β(IL^-1β),一氧化氮合酶(iNOS),白细胞介素-10(IL^-10),转化生长因子-β(TGF-β),精氨酸-1(Arg-1)的表达量;实时荧光定量PCR(Real-time PCR)检测M1型巨噬细胞标志基因TNF-α,IL-6,IL^-1β,iNOS和M2型巨噬细胞标志基因IL^-10,TGF-β,Arg-1mRNA的表达;蛋白免疫印迹法(Western blot)检测细胞内相关蛋白TNF-α,IL-6,IL^-1β,iNOS的表达。结果:MTT比色法显示,与空白组比较,益气养阴逐瘀方2.0g·L^-1及以下时对细胞增殖无明显影响。Griess法显示,与空白组相比,LPS组NO释放量显著上调(P<0.01);与LPS组相比,各给药组NO释放量均显著下调(P<0.01)。ELISA显示,与空白组相比,LPS组M1/M2型巨噬细胞相关炎症因子表达均显著上调(P<0.01);与LPS组相比,益气养阴逐瘀方各给药组可明显下调M1型巨噬细胞促炎症因子表达(P<0.01),对M2型巨噬细胞抗炎症因子无影响。Real-time PCR显示,与空白组相比,LPS组M1/M2型巨噬细胞相关mRNA表达显著上调(P<0.01);与LPS组相比,益气养阴逐瘀方各给药组可明显下调M1型巨噬细胞促炎症因子基因表达(P<0.05,P<0.01),对M2型mRNA表达无影响。Western blot显示,与空白组比较,LPS组TNF-α,IL-6,IL^-1β,iNOS蛋白的表达显著上调(P<0.01);与LPS组相比,益气养阴逐瘀方各给药组TNF-α,IL-6,IL^-1β,iNOS蛋白的表达均下调,且于2.0g·L^-1下调最显著(P<0.01)。结论:益气养阴逐瘀方可以有效抑制LPS诱导的RAW264.7细胞炎症。其诱导已经分化的促炎亚型M1型巨噬细胞向抗炎亚型M2型巨噬细胞转化不明显,其抗炎机制可能与通过抑制巨噬细胞向M1亚型方向极化从而发挥免疫调节作用,以减少NO,TNF-α,IL-6等相关炎症因子分泌相关。

Objective: To investigate the effect of Yiqi Yangyin Zhuyu recipe on expressions of inflammatory factor and transformation of classically activated macrophages(M1)/alternatively activated macrophages(M2) inflammatory phenotype in lipopolysaccharide(LPS)-induced RAW 264. 7 cells. Method:Methyl-thiazdyl-tetrazolium(MTT) reduction assay was used to detect the effect of different concentrations of Yiqi Yangyin Zhuyu recipe on the proliferation of the cells. The release of nitric oxide was detected by the Griess method. Enzyme linked immunosorbent assay(ELISA) was used to detect the release of M1/M2 inflammatory cytokines in cell supernatant. The expressions of the pro-inflammatory factor genes of M1-macrophages and the antiinflammatory factor genes of M2-macrophages were detected by Real-time PCR. The protein expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1,nitric oxide synthase(i NOS) were detected by Western blot. Result: Results of MTT showed that Yiqi Yangyin Zhuyu recipe with the concentration of 2. 0 g·L-1 and below had no effect on the cell proliferation. Results of Griess indicated that compared with blank group,the release of nitric oxide of LPS-induced group was increased(P < 0. 01);compared with LPS-induced group,Yiqi Yangyin Zhuyu recipe group could reduce the release of NO(P < 0. 01). Results of ELISA showed that compared with blank group,the release of M1/M2 inflammatory cytokines of LPS-induced group were both up-regulated(P <0. 01);compared with LPS-induced group,the secretion of M1-macrophages inflammatory cytokines was downregulated in Yiqi Yangyin Zhuyu recipe group(P < 0. 01),but with no change in M2-macrophages. Results of Real-time PCR indicated that compared with blank group,the expressions of M1/M2 were both increased(P <0. 01,P < 0. 05);compared with LPS-induced group,the expression levels of M1-macrophages were downregulated in traditional Chinese medicine group(P < 0. 01);anti-inflammatory factor genes of M2-macrophages had no significant change. Results of Western blot showed that compared with blank group,expression levels of TNF-α,IL-6,IL-1β,i NOS were up-regulated(P < 0. 01);compared with LPS-induced group,the expressions of TNF-α,IL-6,IL-1β,i NOS were down-regulated in Yiqi Yangyin Zhuyu recipe group,especially at the concentration at2. 0 g·L-1(P < 0. 01). Conclusion: Yiqi Yangyin Zhuyu recipe could effectively inhibit the inflammatory reaction induced by LPS. The anti-inflammatory mechanism of Yiqi Yangyin Zhuyu recipe may be related to inhibition of macrophages to M1 phenotype polarization,so as to play the role of regulating immune and reducing the release of inflammatory cytokines,like NO,TNF-α,IL-6,IL-1β.