基于PI3K/Akt/NF-κB通路探讨人参败毒散、榆瑞灌肠液内外合治干预溃疡性结肠炎大鼠肠黏膜损伤的作用机制  

Renshen Baidusan Combined with Yurui Enema Treats Intestinal Mucosal Injury in Rat Model of Ulcerative Colitis via PI3K/Akt/NF-κB Pathway

作　　者：刘兴隆[1] 张培旭 熊珮宇 钟春 陈旭 贾波[1] LIU Xinglong;ZHANG Peixu;XIONG Peiyu;ZHONG Chun;CHEN Xu;JIA Bo(School of Basic Medical Sciences,Chengdu University of Traditional Chinese Medicine,Chengdu 610075,China)

机构地区：[1]成都中医药大学基础医学院,成都610075

出　　处：《中国实验方剂学杂志》2023年第19期42-51,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82074327)。

摘　　要：目的:从磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/核转录因子-κB(NF-κB)相关通路指标变化,探讨内治方人参败毒散、外治方榆瑞灌肠液及二方合用对溃疡性结肠炎(UC)大鼠肠黏膜损伤的作用机制。方法:将50只SPF级SD大鼠,随机分为空白组、模型组、人参败毒散组(内治15.6 g·kg^(-1))、榆瑞灌肠液组(外治25 g·kg^(-1))、内外合治组(内治15.6 g·kg^(-1)+外治25 g·kg^(-1)),每组10只。除空白组外,采用2,4,6-三硝基本磺酸(TNBS)/乙醇诱导大鼠建立UC动物模型,造模后第8天给药,日1次,连续14 d。苏木素-伊红(HE)染色观察结肠组织病理学变化;采用酶联免疫吸附测定法(ELISA)检测结肠组织肿瘤坏死因子-α(TNF-α)、γ干扰素(IFN-γ)、白细胞介素-4(IL-4)、白细胞介素-10(IL-10)含量;采用原位末端标记法(TUNEL)检测结肠上皮细胞凋亡情况;采用免疫组化法检测结肠组织B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、TNF-α、IL-6定位及表达情况;采用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测结肠组织PI3K/Akt/NF-κB通路相关mRNA和蛋白表达。结果:与正常组比较,模型组大鼠结肠组织HE染色后镜下可见黏膜及黏膜下层大量炎性细胞浸润,结肠组织TNF-α、IFN-γ含量增高,IL-4、IL-10含量降低(P<0.05),结肠组织上皮细胞凋亡率增高(P<0.05),结肠组织Bax、TNF-α、IL-6阳性表达增高,Bcl-2阳性表达降低(P<0.05),结肠组织PI3K、Akt、NF-κB mRNA表达升高(P<0.05),PI3K、磷酸化(p)-PI3K、Akt、p-Akt、p65、p-p65、Bax、活化(cleaved) Caspase-3蛋白表达、Bax/Bcl-2、cleaved Caspase-3/Caspase-3表达增高(P<0.05),NF-κB抑制蛋白α(IκBα)、Bcl-2、Caspase-3蛋白表达降低(P<0.05)。与模型组比较,给药后内治组、外治组、合治组大鼠结肠黏膜损伤均有一定程度的改善,结肠组织TNF-α、IFN-γ含量降低,IL-4、IL-10含量升高(P<0.05),结肠细胞凋亡率明显降低(P<0.05Objective:To explore the mechanisms of internal treatment(Renshen Baidusan),external treatment(Yurui Enema),and combination of the two methods in treating intestinal mucosal injury in the rat model of ulcerative colitis(UC)from the changes of phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/nuclear factor-κB(NF-κB)pathway.Method:Fifty SPF-grade SD rats were randomized into blank,model,Renshen Baidusan(15.6 g·kg^(-1)),Yurui Enema(25 g·kg^(-1)),and combined treatment(15.6 g·kg^(-1) Renshen Baidusan+25 g·kg^(-1) Yurui Enema)groups(n=10).The rat model of UC was established in other groups except the blank group by 2,4,6-trinitrosulfonic acid(TNBS)/ethanol.The rats were administered with corresponding drugs once a day for 14 consecutive days since the 8th day after modeling.The histopathological changes of colon were observed by hematoxylin-eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the levels of tumor necrosis factor(TNF)-α,interferon(IFN)-γ,interleukin(IL)-4,and IL-10 in the colon tissue.The apoptosis of colon epithelial cells was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL).The location and expression of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),TNF-α,and IL-6 in the colon tissue were examined by immunohistochemistry.Real-time quantitative fluorescence polymerase chain reaction(Real-time PCR)and Western blot were employed to determine the mRNA and protein levels,respectively,of the proteins in the PI3K/Akt/NF-κB pathway in the colon tissue.Result:In the model group,HE staining showed a large number of inflammatory cell infiltration in the mucosa and submucosa.Compared with the blank group,the model group showed elevated levels of TNF-αand IFN-γand lowered levels of IL-4 and IL-10 in the colon tissue,increased apoptosis rate of colon epithelial cells,increased positive expression of Bax,TNF-α,and IL-6,and decreased positive expression of Bcl-2(P<0.05).Moreover,the model group showed up-regulated mRNA le

关 键 词：人参败毒散 榆瑞灌肠液 内外合治 溃疡性结肠炎 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/核转录因子-κB(NF-κB)通路 

分 类 号：R2-0[医药卫生—中医学] R33R289R318.14R574.1