异鼠李素通过调控NF-κB通路发挥镇痛作用

Isorhamnetin plays an analgesic role by regulating NF-κB pathway

目的研究异鼠李素的镇痛作用及潜在机制。方法采用小鼠热板致痛模型和小鼠醋酸扭体疼痛模型研究异鼠李素的镇痛作用。每种模型需选取50只SPF级昆明种小鼠,依据体质量分为5组,分别为空白对照组,阳性对照吲哚美辛组,异鼠李素高、中、低剂量组。Griess法和酶联免疫法检测小鼠免疫细胞RAW264.7中一氧化氮(NO)和肿瘤坏死因子(TNF-α)含量;RT-PCR法检测IL-1β和IL-6表达水平;Western blot检测NF-κB信号通路的变化。结果在体动物实验表明异鼠李素(25, 50, 100 mg/kg)可以剂量依赖性地提高小鼠对热板所致疼痛的痛阈值(P<0.05),并可显著减少醋酸所致的小鼠扭体数(P<0.05)。体外细胞实验表明异鼠李素(5,10,20μmol/L)显著抑制脂多糖(LPS)诱导的RAW264.7细胞中NO和TNF-α含量升高(P<0.05),降低IL-1β和IL-6的mRNA表达水平,并可以阻断RAW264.7细胞NF-κB信号通路的激活。预先给予NF-κB信号通路抑制剂可以与异鼠李素协同调节RAW264.7细胞NO的水平。结论异鼠李素具有一定的镇痛作用,其镇痛机制可能与抑制NF-κB通路,继而减少NO、TNF-α、IL-1及IL-6的表达、合成或释放有关。

Objective To investigate the analgesic effect of isorhamnetin and its underlying mechanisms.Methods The analgesic effect of isorhamnetin was measured by hot plate pain model and acetic acid torsion pain model in mice.Fifty SPF Kunming mice were selected in each model and then were randomly divided into 5 groups according to their body weight:blank control group,positive control indomethacin group,and isorhamnetin high,middle and low dose groups,and the analgesic effect of isorhamnetin was observed and recorded.In addition,the effects of isorhamnetin on the expression levels of pain-inducing factors and inflammatory factors were examined.The contents of NO and TNF-αin mouse macrophage RAW264.7 cells were detected by Griess assay and ELISA assay,respectively,and the mRNA levels of IL-1βand IL-6 in RAW264.7 cells were determined using RT-PCR.Finally,the activation of NF-κB pathway in LPS-induced RAW264.7 was determined by Western blotting.Results In vivo experiments showed that isorhamnetin(25,50 and 100 mg/kg)dose-dependently increased the pain threshold of mice induced by hot plate test(P<0.05),and significantly reduced the acetic acid-induced writhing response in mice(P<0.05).In vitro cell experiments suggested that isorhamnetin(5,10,20μmol/L)greatly inhibited the up-regulation of NO and TNF-αinduced by LPS(P<0.05),decreased the mRNA levels of IL-1βand IL-6,and blocked the activation of NF-κB signaling pathway in RAW264.7 cells.Pre-administration of the inhibitor of NF-κB pathway synergically enhanced the inhibitory effect of isorhamnetin on NO level in RAW264.7 cells.Conclusion Isorhamnetin has certain analgesic effect,and its mechanism may be associated with the suppression of NF-κB pathway and the subsequent reductions in expression,syntheses and releases of NO,TNF-α,IL-1βand IL-6.