清感冬饮通过调控NF-κB/iNOS/NO信号通路抑制LPS诱导的RAW264.7巨噬细胞炎症

Qinggan Dongyin inhibits LPS-induced inflammation of RAW264.7 macrophages by regulating NF-κB/iNOS/NO signaling pathway

[目的]通过构建脂多糖(LPS)诱导的RAW264.7巨噬细胞炎症模型,探讨清感冬饮(QGDY)的抗炎作用及其机制。[方法]采用CCK-8法和LDH法检测不同浓度的清感冬饮对HEK293T细胞和RAW264.7巨噬细胞的细胞活力及LDH漏出量的影响。分别建立抗氧化反应元件(ARE)和核因子-κB(NF-κB)双荧光素酶报告系统,考察清感冬饮对HEK293T细胞ARE、NF-κB表达情况的影响。建立LPS诱导的RAW264.7巨噬细胞体外炎症模型,将RAW264.7巨噬细胞随机分为空白对照组、LPS模型组、清感冬饮低、中、高剂量组(1、10、100μg/mL),倒置显微镜下观察各组细胞的形态差异;采用Griess法和ELISA法测定各组细胞上清中一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)的含量;采用qRT-PCR法检测各组细胞中TNF-α、IL-6和IL-1β的mRNA表达水平;利用免疫荧光法观察核因子-κB p65(p65)核移位情况;采用Western blot法检测各组细胞一氧化氮合酶(iNOS)和p65蛋白表达情况。[结果]0.01~10μg/mL清感冬饮对HEK293T细胞活力无显著影响,1~10μg/mL清感冬饮明显抑制TNF-α诱导的NF-κB启动子的活性,清感冬饮对ARE启动子的活性无显著影响。初步证明清感冬饮具有抗炎作用,而无明显抗氧化作用。0.01~100μg/mL清感冬饮干预24 h对RAW264.7巨噬细胞无细胞毒性作用。与模型组相比,1~100μg/mL清感冬饮显著抑制LPS诱导的RAW264.7巨噬细胞中NO及炎症因子TNF-α、IL-6和IL-1β的释放;10~100μg/mL清感冬饮显著下调TNF-α、IL-6和IL-1β的mRNA表达水平,抑制p65核移位;100μg/mL清感冬饮显著抑制总蛋白iNOS、核蛋白p65表达,显著促进浆蛋白p65表达。[结论]清感冬饮可抑制LPS诱导的RAW264.7巨噬细胞的炎症反应,该抗炎作用可能与调控NF-κB/iNOS/NO信号通路、抑制促炎因子释放有关。

[Objective]To investigate the anti-inflammatory effects and mechanism of Qinggan Dongyin(QGDY)by establishing lipopolysaccharide(LPS)-induced inflammation model in RAW264.7 macrophages.[Methods]CCK-8 assay and Cytotoxicity LDH Assay were used to determine the effects of different concentrations of QGDY on cell viability and LDH leakage in HEK293T cells and RAW264.7 macrophages.Antioxidant reaction element(ARE)and nuclear factor-κB(NF-κB)dual luciferase reporter systems were established to examine the effects of QGDY on the expression of ARE and NF-κB in HEK293T cells,respectively.LPS-induced RAW264.7 macrophages inflammatory model was established in vitro.RAW264.7 cells were randomly divided into control group,model group,and QGDY low-dose group,medium-dosegroup,high-dose group(1,10,and 100μg/mL).The morphological differences of cells in each group were observed by inverted microscope.The release of nitric oxide(NO),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in supernatant were determined by Griess assay and ELISA kits.The mRNA expression levels of TNF-α,IL-6 and IL-1βwere detected by qRT-PCR assay.The nuclear translocation of p65 was observed by immunofluorescence.The protein expressions of p65 and iNOS were detected in each group by western blot.[Results]The 0.01-10μg/mL QGDY had no significant effect on cell viability of HEK293T cells,1-10μg/mL QGDY obviously inhibited the activity of NF-κB promoter induced by TNF-α,and QGDY had no significant effect on the activity of ARE promoter,suggesting that QGDY had anti-inflammatory effect but no obvious antioxidant effect.QGDY(0.01-100μg/mL)had no cytotoxic effect on RAW264.7 macrophages after 24 h of intervention.Compared with the model group,the release of NO and inflammatory factors TNF-α,IL-6 and IL-1βwere significantly inhibited by 1-100μg/mL QGDY in LPS-induced RAW264.7 macrophages.Compared with model group,10-100μg/mL QGDY groups not only decresed the mRNA expression levels of TNF-α,IL-6 and IL-1β,but also inhibited the nuclear translocation of p65 in RAW264.7 macrophages,significantly.In addition,100μg/mL QGDY significantly inhibited the expression of iNOS and nuclear protein p65,and significantly promoted the expression of plasma protein p65.[Conclusion]QGDY could inhibit the inflammatory response of RAW264.7 macrophages induced by LPS,which might be related to the regulation of NF-κB/iNOS/NO signaling pathway and the inhibition of the release of pro-inflammatory factors.