摘要
目的通过体内外实验阐明苏木酮A基于核转录因子红系2相关因子2(Nrf2)/核因子κB(NF-κB)双通路协同调控的抗炎、抗氧化作用及其分子机制。方法体外培养RAW264.7巨噬细胞,分为对照组、脂多糖(LPS)组(1μg/mL)、LPS+苏木酮A(20μmol/L)组及LPS+苏木酮A(40μmol/L)组,通过LPS刺激RAW264.7巨噬细胞建立体外炎症模型,检测苏木酮A对炎症因子表达的影响。构建葡聚糖硫酸钠(DSS)诱导的小鼠急性结肠炎模型,随机分为对照组、DSS组、苏木酮A低剂量组(25 mg/kg)及高剂量组(50 mg/kg),于造模第4天开始药物干预。实验期间监测疾病活动指数(DAI)及体重变化,评估结肠长度和组织病理损伤。采用ELISA、qPCR及Western blot技术分别检测炎症因子水平、抗氧化通路[Nrf2/Keap1/血红素加氧酶-1(HO-1)]及炎症通路[NF-κB/人核因子κB抑制蛋白α(IκB-α)]相关分子表达。结果CCK-8检测显示,20和40μmol/L苏木酮A对细胞增殖无显著抑制作用,差异无统计学意义(t=1.25、1.89,P均>0.05)。体外实验中,与LPS组相比,40μmol/L苏木酮A显著抑制白细胞介素-6(IL-6)、IL-1β、肿瘤坏死因子-α(TNF-α)mRNA表达,并上调IL-10表达水平,差异均有统计学意义(t=5.23、4.89、6.12、3.97,P均<0.05)。Western blot证实,其剂量依赖性抑制环氧化酶-2(COX-2)和诱导型一氧化氮合酶(iNOS)蛋白表达,差异均有统计学意义(F=9.34、8.76,P均<0.05)。体内实验中,高剂量苏木酮A(50 mg/kg)显著改善DAI评分,体重下降、结肠缩短,改善脾脏指数及结肠病理,并上调紧密连接蛋白闭锁小带蛋白-1(ZO-1)、细胞间粘附分子1(E-cadherin)和闭合蛋白(Occludin)表达水平,差异均有统计学意义(P均<0.05)。血清IL-6、TNF-α、IL-1β水平呈剂量依赖性降低(P均<0.05)。机制上,苏木酮A增加HO-1表达水平,抑制p65和IκB-α磷酸化水平,且Nrf2沉默后该抑制作用显著减弱(P均<0.05)。结论苏木酮A通过协同调控Nrf2介导的抗氧化防御与NF-κB相关的炎症反应,发挥对实验性结肠炎的治疗作用。
Objective To elucidate the anti-inflammatory and antioxidant effects of sappanone A based on the coordinated regulation of nuclear transcription factor erythroid 2-related factor 2(Nrf2)/nuclear factor κB(NF-κB)dual pathways and their molecular mechanisms through in vitro and in vivo experiments.Methods RAW264.7 macrophages were cultured in vitro and divided into control group,lipopolysaccharide(LPS)group(1μg/mL),LPS+sappanone A(20μmol/L)group and LPS+sappanone A(40μmol/L)group.The in vitro inflammatory model of RAW264.7 macrophages was established by stimulating RAW264.7 macrophages with LPS,and the effect of sappanone A on the expression of inflammatory factors was detected.The acute colitis model of mice induced by dextran sulfate sodium(DSS)was established and randomly divided into control group,DSS group,low-dose sappanone A group(25 mg/kg)and high-dose group(50 mg/kg).Drug intervention began on the 4th day of modeling.During the experiment,the disease activity index(DAI)and body weight changes were monitored,and the colon length and tissue pathological damage were evaluated.ELISA,qPCR and Western blot were used to detect the levels of inflammatory factors,the expression of molecules related to the antioxidant pathway[Nrf2/Keap1/heme oxygenase-1(HO-1)]and the inflammatory pathway[NF-κB/inhibitor of nuclear factor kappa B alpha(IκB-α)].Results CCK-8 assay showed that 20μmol/L and 40μmol/L sappanone A had no significant inhibitory effect on cell proliferation,and the difference was not statistically significant(t=1.25,1.89;all P>0.05).In vitro experiments,40μmol/L sappanone A significantly inhibited the expression of interleukin-6(IL-6),IL-1β,and tumor necrosis factor-α(TNF-α)mRNA,and upregulated the expression level of IL-10,and the differences were statistically significant(t=5.23,4.89,6.12,3.97;all P<0.05).Western blot confirmed that sappanone A inhibited cyclooxygenase-2 in a dose-dependent manner.The expression of COX-2 and inducible nitric oxide synthase(iNOS)proteins was significantly different(F=9.34,8.76;all P<0.05).50 mg/kg sappanone A significantly improved DAI score(F=12.35,P<0.05),weight loss,colon shortening,spleen index and colon pathology(t=5.67,8.23,3.89,6.78;all P<0.05),and upregulated the expression levels of tight junction proteins zonula occludens-1(ZO-1),intercellular adhesion molecule 1(E-cadherin)and occludens(Occludin),with significant differences(all P<0.05).Serum IL-6,TNF-α,and IL-1β levels decreased in a dosedependent manner,with significant differences(all P<0.05).Mechanistically,sappanone A could increase the expression levels of HO-1 inhibit the expression level of p65 and IκB-α;the inhibitory effect was significantly weakened after Nrf2 silencing(all P<0.05).Conclusion Sappanone A might play a therapeutic role in experimental colitis by synergistically regulating Nrf2-mediated antioxidant defense and NF-κB-related inflammatory response.
作者
马睿
郑鹏
刘晓雅
张朝军
MA Rui;ZHENG Peng;LIU Xiaoya;ZHANG Chaojun(Medical School of Chinese PLA,Beijing 100853,China;Department of General Surgery,the First Medical Center of Chinese PLA General Hospital,Beijing 100853,China)
出处
《热带医学杂志》
2025年第7期881-887,924,F0004,共9页
Journal of Tropical Medicine
基金
国家自然科学基金(81972320)。
