葛根素对LPS诱导的RAW264.7细胞炎症反应的作用机制研究

Anti-inflammatory effects and mechanisms of Puerarin on LPS-induced RAW264.7 macrophages

目的探讨葛根素(puerarin,PUE)在脂多糖(lipopolysaccharide,LPS)诱导的RAW264.7巨噬细胞中的抗炎作用,并揭示其分子机制。方法采用CCK-8比色法分别设置不同浓度的LPS和PUE,观察对RAW264.7细胞活性的影响,筛选出合适的LPS造模浓度及PUE的给药浓度。将细胞分为正常对照组、模型组(1μg/mL)、PUE给药组(12.5、25、50μmol/L)。Griess法检测一氧化氮(nitric oxide,NO)释放量;ELISA法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-6(interleukin-6,IL-6)的含量;qRT-PCR法检测环氧化酶-2(cyclooxygenase-2,COX-2)mRNA水平;Western blot法检测诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、COX-2、p-IκBα、p-p65蛋白表达水平。结果与正常对照组比较,不同浓度的LPS诱导24 h,RAW264.7细胞存活率降低,但对细胞增殖无影响(P>0.05),PUE在浓度100μmol/L及以下时对RAW264.7细胞增殖也无影响(P>0.05);与模型组相比,PUE给药组(12.5、25、50μmol/L)NO、TNF-α、IL-1β及IL-6的释放量显著降低(P<0.01),COX-2的蛋白及mRNA表达水平显著降低(P<0.05),i NOS、p-IκBα、p-p65的蛋白表达水平呈浓度依赖性降低(P<0.01)。结论PUE的体外抗炎作用机制与抑制核因子κB(nuclear factor-κB,NF-κB)信号通路相关。

Objective To explore the anti-inflammatory potential of Puerarin(PUE)in lipopolysaccharide(LPS)-induced RAW264.7 macrophages and reveal its potential molecular mechanisms.Methods The effects of different concentrations of LPS and PUE on the viability of RAW264.7 cells were determined by CCK-8 colorimetric method,and the appropriate LPS modeling concentration and PUE administration concentration were screened.Cells were divided into normal control group,model group(1μg/mL),and PUE administration group(12.5,25,50μmol/L);Griess assay was used to assess the nitric oxide(NO)concentration;tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)content was measured by ELISA;cyclooxygenase-2(COX-2)mRNA levels were detected by qRT-PCR;the protein expression levels of inducible nitric oxide synthase(iNOS),COX-2,p-IκBα,and p-p65 were determined by Western blot.Results Compared with the normal control group,the survival rate of RAW264.7 cells decreased after 24 h of LPS induction at different concentrations,and there was no effect on cell proliferation(P>0.05),PUE has no significant cytotoxic effects at or below the concentrations of 100μmol/L(P>0.05);Compared with the model group,PUE administration group(12.5,25,50μmol/L)displayed favorable inhibitory effects on the release level of NO,TNF-α,IL-1β and IL-6(P<0.01),and the protein and mRNA expression levels of COX-2 significantly decreased(P<0.05)in LPS-induced RAW264.7 cells,and it is the same for the expression levels of iNOS,p-IκBα,p-p65(P<0.01).Conclusion The anti-inflammation mechanisms of PUE is related with nuclear factor-κB(NF-κB)signaling pathway.