岭南山竹子提取物oblongifolins A的体外抗炎作用及机制研究

Study on in vitro anti-inflammatory effects and mechanisms of oblongifolins A extracted from Garcinia oblongifolia

目的研究岭南山竹子提取物oblongifolins A(OA)的体外抗炎作用及机制。方法以RAW264.7细胞为研究对象,将细胞分为对照组(0.5%二甲基亚砜),脂多糖(LPS)组(1μg/mL),地塞米松(DEX)组(10μmol/L DEX+1μg/mL LPS),OA低、中、高浓度组(7.5、15、30μmol/L OA+1μg/mL LPS)。除对照组外,其余各组细胞先用LPS刺激1 h后,再与药物混合培养24 h。观察各组细胞形态变化,检测各组细胞中一氧化氮(NO)、活性氧(ROS)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β、IL-4和IL-10含量,TNF-α、IL-6、IL-1βmRNA表达水平,核因子κB(NF-κB)和核转录因子红系2相关因子2(Nrf2)通路相关蛋白的表达水平以及对照组、LPS组、OA高浓度组细胞中NF-κB p65和Nrf2蛋白的核易位情况。结果与LPS组比较,OA各浓度组纺锤形和不规则细胞逐渐减少,细胞中NO、ROS(OA低浓度组除外)、TNF-α、IL-6、IL-1β含量,TNF-α、IL-6(OA低浓度组除外)、IL-1βmRNA表达水平,磷酸化NF-κB p65(p-NF-κB p65)、磷酸化核因子κB抑制蛋白α(p-IκBα)、Kelch样环氧氯丙烷相关蛋白1(Keap1)蛋白表达水平均显著降低(P<0.05);IL-4、IL-10含量,IκBα、Nrf2(OA低、中浓度组除外)、血红素加氧酶1(HO-1)(OA低浓度组除外)、醌NADH脱氢酶1(NQO1)蛋白表达水平均显著升高(P<0.05);高浓度OA可抑制NF-κB p65蛋白核易位,促进Nrf2蛋白核易位。结论OA能够抑制LPS诱导的RAW264.7细胞炎症,其作用机制可能与抑制NF-κB通路、激活Nrf2通路、减少ROS和炎症因子的过度释放有关。

OBJECTIVE To investigate the in vitro anti-inflammatory effects and mechanisms of oblongifolins A(OA)extracted from Garcinia oblongifolia.METHODS RAW264.7 cells were used as the research subject and divided into control group(0.5%DMSO),lipopolysaccharide(LPS)group(1μg/mL),DEX group(10μmol/L DEX+1μg/mL LPS),and low-,medium-,and high-concentration groups of OA(7.5,15,30μmol/L OA+1μg/mL LPS).Except for the control group,the remaining groups were first stimulated with LPS for 1 hour and then mixed with drugs for 24 hours.The morphological changes of cells were observed in each group.The contents of nitric oxide(NO),reactive oxygen species(ROS),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β,IL-4 and IL-10 were detected in cells of each group;mRNA expression levels of TNF-α,IL-6 and IL-1βwere measured.The expression of key proteins in the nuclear factorκB(NF-κB)and nuclear factor-erythroid 2-related factor 2(Nrf2)signaling pathways in each group,as well as the nuclear translocation of NF-κB p65 and Nrf2 proteins in control group,LPS group and OA high-concentration group,were detected.RESULTS Compared to the LPS group,the number of spindle-shaped and irregular cells gradually decreased in OA groups,the contents of NO,ROS(except for OA low-concentration group),TNF-α,IL-6 and IL-1β,the mRNA expressions of TNF-α,IL-6(except for OA low-concentration group)and IL-1βas well as the protein expressions of phosphorylated NF-κB p65(p-NF-κB p65),p-IκBα,and Kelch-like ECH-associated protein 1(Keap1)were decreased significantly(P<0.05).The contents of IL-4 and IL-10,protein expressions of IκBα,Nrf2(except for OA low-and medium-concentration groups),HO-1(except for OA low-concentration group)and NQO1 were all increased significantly(P<0.05).OA of high concentration could inhibit NF-κB p65 protein nuclear translocation and promote Nrf2 protein nuclear translocation.CONCLUSIONS OA can suppress LPS-induced inflammation in RAW264.7 macrophages.The underlying molecular mechanism likely entails the inhibition of the NF-κB signaling pathway,the activation of the Nrf2 signaling pathway and the reduction of ROS and inflammatory factor release.