肠病药方调控PI3K/AKT/NF-κB信号通路改善溃疡性结肠炎的研究

Mechanism of changbing decoction in improving ulcerative colitis via regulating PI3K/AKT/NF-κB signaling pathway

目的探讨肠病药方调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子kappa B(NF-κB)信号通路对抗溃疡性结肠炎(UC)的作用机制。方法42只C57BL/6小鼠随机分为正常对照组(n=6,Control组,自由饮去离子水)、模型组(n=12,DSS组)、美沙拉嗪组(n=12,MES组)及肠病药方组(n=12,CBD组),后3组小鼠自由饮用3%葡聚糖硫酸钠(DSS)溶液7 d诱导UC模型,后两组同期灌胃美沙拉嗪或肠病药方,前两组灌胃等体积去离子水;观察实验期间小鼠体质量改变,评测小鼠疾病活动指数(DAI);造模结束后麻醉处死小鼠,测量结肠长度、观察结肠内容物及黏膜,采用苏木素-伊红(HE)染色观察各组小鼠结肠病理组织学变化,酶联免疫吸附试验(ELISA)检测各组小鼠结肠组织匀浆白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,蛋白免疫印迹法(WB)检测各组小鼠结肠组织中磷酸化PI3K(p-PI3K)-p85、磷酸化AKT1(p-AKT1)、磷酸化NF-κB(p-NF-κB)-p65蛋白表达,实时荧光定量PCR技术(qRT-PCT)检测各组小鼠结肠组织PI3K、AKT、NF-κB信使RNA(mRNA)表达水平。结果与DSS组比较,CBD组治疗后,有效缓解UC小鼠症状,粪便性状及便血情况改善,结肠黏膜炎症浸润减少,体质量下降有所缓解,DAI评分降低及结肠长度接近正常,结肠组织匀浆中IL-6、TNF-α表达降低(P<0.05),p-PI3K-p85、p-AKT1、p-NF-κB-p65蛋白及mRNA表达明显减少(P<0.05)。结论肠病药方可缓解UC小鼠的症状、减轻结肠黏膜损伤,其机制可能与调解结肠组织中PI3K/AKT/NF-κB信号通路中相关蛋白质及mRNA表达、降低结肠组织炎症因子表达有关。

Objective To investigate the mechanism of Changbing decoction in combating ulcerative colitis(UC)via regulating phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/nuclear factor-kappa B(NF-κB)signaling pathway.Methods Forty-two C57BL/6 mice were randomly divided into normal control group(n=6,Control group,freely drinking deionized water),model group(n=12,DSS group),mesalazine group(n=12,MES group)and Changbing decoction group(n=12,CBD group).Model,DSS and CBD groups were given with 3%dextran sulfate sodium(DSS)for 7 days to establish UC model.At the same time,DSS and CBD groups were intragastrically administrated with mesalazine and Changbing decoction,respectively,while control and model groups were intragastrically administrated with equal volume of deionized water.Mouse body mass changes were observed during the experiment.Mouse disease activity index(DAI)was evaluated.After the completion of modeling,the mice were anesthetized and euthanized.Colon lengths were measured.The contents and mucosa of the colons were observed.Hematoxylin eosin(HE)staining was used to observe the histopathological changes of mouse colons in each group.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in mouse colon tissue homogenates in each group.Western blot was used to detect phosphorylated-PI3K(p-PI3K p85),phosphorylated-AKT1(p-AKT1),and phosphorylated-NF-κB(p-NF-κB p65)in mouse colon tissues in each group.Real-time fluorescence quantitative PCR(qRT-PCT)was used to detect mRNA expression levels of PI3K,AKT,and NF-κB in mouse colon tissues.Results When compared with DSS group,CBD group exhibited alleviated UC symptoms,improved fecal traits and stool blood,reduced colon mucosal inflammatory infiltration,alleviated body mass,decreased DAI score,colon lengths close to normal,decreased IL-6 and TNF-αexpressions in colon tissue homogenates after treatment(P<0.05).The protein and mRNA expressions of p-PI3K-p85,p-AKT1,and p-NF-κB P65 were significantly decreased(P<0.05).Conclusion Changbing decoction can alleviate the symptoms and colon mucosal injury of UC mice,and its mechanism may be related to regulating the expression of PI3K/AKT/NF-κB signaling pathway and reducing the expression of inflammatory factors in colon tissues.