基于线粒体氧化应激损伤探讨白杨素对溃疡性结肠炎大鼠的保护作用及机制

Study on the Protective Effect and Mechanism of Chrysin in Ulcerative Colitis of Rats Based on Mitochondrial Oxidative Stress Damage

目的:探究白杨素对溃疡性结肠炎的作用及其机制。方法:60只SD大鼠随机分为正常对照组、模型组、美沙拉嗪(200 mg/kg)阳性对照组及白杨素低(25 mg/kg)、中(50 mg/kg)、高(100 mg/kg)剂量组,每组10只。3%葡聚糖硫酸钠喂养1 w联合2%冰醋酸直肠灌注1次制备UC大鼠模型。对各组大鼠进行DAI评分,HE染色观察结肠组织病理学变化,透射电镜观察结肠线粒体超微结构。ELISA法检测血清TNF-α、IL-1β、IL-6及结肠组织4-HNE水平,比色法检测结肠组织MDA水平,Western Blot检测结肠组织TNF-α、IL-1β、IL-6、Bcl-2、Bax、cleaved Caspase-3、SIRT3、OGG1、p-IκBα、p-NF-κB P65蛋白表达及NF-κB P65核转移情况。体外培养结肠上皮细胞(NCM460),实验设正常对照组、白杨素(100μmol/L)对照组、模型组及白杨素1、10、100μmol/L组。DCFH-DA荧光探针检测细胞ROS水平,比色法检测细胞MDA水平,Western Blot和(或)免疫荧光法检测细胞Bcl-2、Bax、cleaved Caspase-3、SIRT3、OGG1、p-IκBα、p-NF-κB P65的蛋白表达及NF-κB P65核转移情况。结果:体内实验中,与模型组比较,各给药组大鼠体质量显著升高,DAI评分显著降低(P<0.05或P<0.01),结肠病理损伤明显缓解;血清TNF-α、IL-1β、IL-6水平与结肠组织TNF-α、IL-1β、IL-6蛋白表达显著降低,结肠组织Bcl-2蛋白表达显著升高,Bax、cleaved Caspase-3蛋白表达显著降低(P<0.05或P<0.01);结肠线粒体损伤明显减轻,SIRT3、OGG1蛋白表达显著升高,MDA、4-HNE水平显著降低,IκBα、NF-κB P65磷酸化水平与NF-κB P65核转移程度显著降低(P<0.05或P<0.01)。体外实验中,与模型组比较,白杨素各组ROS、MDA、4-HNE、TNF-α、IL-1β、IL-6水平显著降低,SIRT3、OGG1蛋白表达显著升高,IκBα、NF-κB P65磷酸化程度及NF-κB P65核转移水平显著降低(P<0.05或P<0.01),线粒体损伤减轻。结论:白杨素对UC具有一定的缓解作用,其机制可能与减轻线粒体氧化应激损伤,抑制NF-κB信号通路活化,降低炎症反应和细胞凋亡有关。

Objective:To investigate the protective effect and mechanism of chrysin on ulcerative colitis(UC).Methods:Sixty SD rats were randomly divided into normal control group,model group,mesalazine(200 mg/kg)model group,chrysin low(25 mg/kg),medium(50 mg/kg)and high(100 mg/kg)dose groups,with 10 rats in each group.The UC rat model was prepared by rectal infusion of 3%dextran sulfate sodium for 1 week combined with 2%glacial acetic acid.DAI score was used,the pathological changes of colonic tissues were observed by HE staining and ultrastructure of colonic mitochondria was observed by transmission electron microscopy.Serum levels of TNF-α,IL-1β,IL-6 and 4-HNE in colon tissues were detected by ELISA,MDA levels in colon tissues were detected by colorimetry.Protein expressions of TNF-α,IL-1β,IL-6,Bcl-2,Bax,cleaved Caspase-3,SIRT3,OGG1,p-IκBα,p-NF-κB P65 in colon tissue and nuclear transfer of NF-κB P65 were detected by Western Blot.Colonic epithelial cells(NCM460)were cultured in vitro,and normal control group,chrysin(100μmol/L)control group,model group and chrysin(1,10,100μmol/L)groups were set up.The ROS level was detected by DCFH-DA fluorescent probe,and the MDA level was detected by colorimetry.The protein expressions of Bcl-2,Bax,cleaved Caspase-3,SIRT3,OGG1,p-IκBα,p-NF-κB P65 and nuclear transfer of NF-κB P65 were detected by Western Blot and(or)immunofluorescence assay.Results:In vivo experiment,compared with model group,the body mass of rats in each administration group was significantly increased,DAI score was significantly decreased(P<0.05 or P<0.01),and colon pathological injury was significantly relieved.TNF-α,IL-1β,IL-6 levels in serum and their protim expressions in colon tissnes were significantly decreased,colon tissue Bcl-2 protein expression was significantly increased,Bax,cleaved Caspase-3 protein expressions were significantly decreased(P<0.05 or P<0.01);The mitochondrial damage of colon was significantly reduced,the protein expressions of SIRT3 and OGG1 were significantly increased,and the levels of MDA and 4-HNE were significantly decreased.The phosphorylation levels of IκBαand NF-κB P65 and the nuclear transfer degree of NF-κB P65 were significantly decreased(P<0.05 or P<0.01).In vitro experiments,compared with model group,the levels of ROS,MDA and 4-HNE,TNF-α,IL-1β,IL-6 in all chrysin groups were significantly decreased,the protein expressions of SIRT3 and OGG1 were significantly increased,and the phosphorylation degree of IκBαand NF-κB P65 and nuclear transfer level of NF-κB P65 were significantly decreased(P<0.05 or P<0.01).The mitochondrial damage was mitlyated.Conclusion:Chrysin has a certain alleviating effect on UC,which may be related to reducing mitochondrial oxidative stress damage,inhibiting the activation of NF-κB signaling pathway,reducing inflammation and apoptosis.