苦参醇F调节肠道菌群及免疫应答对溃疡性结肠炎小鼠的改善作用机制研究

Improvement mechanism study of kushenol F on ulcerative colitis mice by regulating gut microbiota and immune response

目的探究苦参醇F(KSCF)治疗溃疡性结肠炎(UC)小鼠的作用机制。方法采用网络药理学与分子对接预测KSCF干预UC的潜在靶点。将C57BL/6J小鼠按体重分为模型组、阳性对照组(柳氮磺胺吡啶,703 mg/kg)、KSCF组(100 mg/kg)和正常组,每组6只;采用饮用葡聚糖硫酸钠溶液的方法建立小鼠UC模型;造模期间灌胃给药,每天1次,连续7 d。末次给药后,对小鼠疾病活动指数(DAI)进行评分;测量小鼠结肠长度;观察小鼠结肠组织病理形态学变化;检测小鼠血清中脂多糖(LPS)及结肠中髓过氧化物酶(MPO)、一氧化氮(NO)和超氧化物歧化酶(SOD)水平;检测小鼠结肠组织中闭合蛋白(occludin)、紧密连接蛋白1(ZO-1)表达水平;检测小鼠脾脏中CD3+T、CD4+T、CD8+T淋巴细胞比例及CD4+/CD8+值变化;采用16S rDNA测序法分析结肠微生物变化。结果网络药理学结果显示,KSCF可能调节磷脂酰肌醇3激酶/蛋白激酶B、核因子κB(NF-κB)等信号通路治疗UC。分子对接结果表明,KSCF与NF-κB p65蛋白结合最稳定。动物实验结果表明,与模型组比较,KSCF组小鼠结肠组织病理形态学特征得到改善;DAI评分、血清LPS水平、结肠组织中MPO及NF-κB p65磷酸化和NLRP3蛋白表达水平、脾脏中CD8+T淋巴细胞比例显著降低(P<0.05);小鼠体重,结肠组织中SOD水平、occludin和ZO-1表达水平,脾脏中CD3+T、CD4+T淋巴细胞比例及CD4+/CD8+值显著升高(P<0.05);肠道中厚壁菌门、放线菌门、阿克曼氏菌属和乳酸杆菌属丰度增加,变形菌门丰度减少,菌群结构向正常组趋近。结论KSCF通过修复肠道微生态失调、增强免疫应答来抑制结肠炎症反应,改善肠屏障完整性来缓解UC。

OBJECTIVE To explore the action mechanism of kushenol F(KSCF)in treating ulcerative colitis(UC)in mice.METHODS The potential targets of KSCF intervening in UC were predicted with network pharmacology and molecular docking.C57BL/6J mice were randomly divided by body weight into model group,positive control group(sulfasalazine,703 mg/kg),KSCF group(100 mg/kg),and normal group,with 6 mice per group.The UC model of mice was induced by dextran sulfate sodium solution.During the modeling period,the mice were given relevant medicine intragastrically,once a day,for 7 consecutive days.After the last administration,the disease activity index(DAI)of the mice was scored;the length of the mice’s colon was measured;pathological changes in the colon tissue of mice were observed;the levels of lipopolysaccharide(LPS)in serum,myeloperoxidase(MPO),nitric oxide(NO)and superoxide dismutase(SOD)in the colon were detected in mice;the expression levels of occludin and ZO-1 in colon tissue of mice were detected;the proportions of CD3+T,CD4+T,and CD8+T lymphocytes in the spleen and the ratio of CD4+/CD8+were detected;changes in colonic microbiota were analyzed by 16S rDNA sequencing.RESULTS Results of network pharmacology indicated that KSCF may treat UC by regulating signaling pathways such as phosphatidylinositol-3 kinase/protein kinase B(PI3K/AKT)and nuclear factor kappa B(NF-κB).Molecular docking results showed that KSCF bound most stably with NF-κB p65 protein.Animal experiment results demonstrated that,compared with the model group,the pathological characteristics of colon tissue in mice were improved in KSCF group.DAI scores,serum levels of LPS,the levels of MPO,NF-κB p65 phosphorylation and NLRP3 protein expression in the colon,and the proportion of CD8+T lymphocytes in the spleen were reduced significantly(P<0.05).Body weight,SOD levels,expression levels of occludin and ZO-1 in the colon,proportions of CD3+T and CD4+T lymphocytes,and the CD4+/CD8+ratio in the spleen were significantly increased(P<0.05);the abundance of Firmicutes,Actinobacteria,Akkermansia,and Lactobacillus genera were increased,while Proteobacteria decreased;the microbial community structure tended towards that of the normal group.CONCLUSIONS KSCF alleviates UC by restoring intestinal microbial imbalance,enhancing immune response,and inhibiting colonic inflammatory responses,thereby improving intestinal barrier integrity.