文献类型：期刊文献

中文题名：柔肝止泻方对腹泻型肠易激综合征肝气乘脾证大鼠模型的干预效应及机制研究

英文题名：Effect and Mechanism of Rougan Zhixie Formula on IBS-D Rat Model with Liver Qi Attacking Spleen

作者：符博雅[1];刘俊宏[2];方慧[3];王淼蕾[2];李林蔓[3];李欣瑜[3];牛媛媛[3];李明[3];赖学倩[3];李亚静[3]

第一作者：符博雅

机构：[1]河西学院附属张掖人民医院,甘肃张掖734000;[2]甘肃中医药大学附属医院,甘肃兰州730020;[3]甘肃中医药大学,甘肃兰州730101

第一机构：河西学院附属张掖人民医院,甘肃张掖734000

年份：2024

卷号：52

期号：7

起止页码：19

中文期刊名：中医药学报

外文期刊名：Acta Chinese Medicine and Pharmacology

收录：CSTPCD

基金：国家自然科学基金地区基金项目(81660773);甘肃省自然科学基金项目(23JRRG0039);甘肃省中医药管理局科研项目(GZKZ-2021-6);甘肃省中西医结合消化病临床医学研究中心(21JR7RA682);兰州市科技局项目(2021-1-98);国家中医药管理局基金项目——国医大师周信有传承工作室(国中医药规财发[2017]28号)。

语种：中文

中文关键词：腹泻型肠易激综合征;肝气乘脾证;柔肝止泻方;肠黏膜通透性;肠道炎症

外文关键词：Diarrhea-predominant irritable bowel syndrome;Liver qi attacking spleen;Rougan Zhixie formula;Intestinal mucosal permeability;Intestinal inflammation

摘要：目的:从肠黏膜通透性及肠道炎症角度探讨柔肝止泻方对肝气乘脾型腹泻型肠易激综合征的干预效应及机制。方法:50只SPF级Wistar雌性大鼠中随机选取8只作为空白组,其余制备肝气乘脾型腹泻型肠易激综合征(IBS-D)病证结合大鼠模型。造模后将40只成模大鼠随机分为模型组、匹维溴铵组及柔肝止泻方低、中、高剂量组,每组8只,分别给予生理盐水、匹维溴铵及不同剂量的柔肝止泻方(0.86、1.72、3.44 g·kg-1),灌胃14 d后取材。记录体质量、粪便含水量,并行相关行为学实验;光镜下观察HE染色后结肠组织形态,并于透射电镜下观察肠黏膜超微结构变化;采用ELISA法检测大鼠血浆中D-乳酸和血清中TNF-α、IL-6、IFN-γ的含量;采用Western blot法及RT-qPCR法检测大鼠结肠组织中MMP9、p38MAPK、MLCK、occludin蛋白以及mRNA的表达水平。结果:与空白组比较,模型组大鼠体质量下降、粪便含水量增加及新奇抑制摄食潜伏期延长(P<0.01);与模型组比较,柔肝止泻方各剂量组大鼠体质量增加、粪便含水量减少及新奇抑制摄食潜伏期缩短(P<0.05,P<0.01)。HE染色显示,各组大鼠结肠组织结构完整,未见明显的病理性改变。透射电镜可见,模型组肠上皮细胞间隙显著增宽,紧密连接及缝隙连接结构模糊,桥粒显著减少,绒毛稀疏、不齐,柔肝止泻方各剂量组肠上皮细胞间隙明显缩小,顶端连接复合体结构清晰,绒毛整密。与空白组比较,模型组大鼠D-乳酸及炎症因子含量增加(P<0.05,P<0.01),结肠组织中MMP9、p38MAPK、MLCK的蛋白及mRNA表达水平均升高(P<0.05,P<0.01),occludin的表达水平下降(P<0.05,P<0.01);与模型组比较,柔肝止泻方各剂量组D-乳酸及炎症因子含量降低(P<0.05,P<0.01),结肠组织中MMP9、p38MAPK、MLCK的蛋白及mRNA表达水平均下调(P<0.05,P<0.01),occludin水平上调(P<0.01)。结论:柔肝止泻方可改善肝气乘脾型IBS-D大鼠模型肠黏膜通透性及肠道炎症,可能是通过减少炎症因子,抑制MMP9激活p38MAPK/MLCK信号通路发挥干预效应。
Objective:To explore the intervention effect and mechanism of Rougan Zhixie formula(RZF)on diarrhea predominant-irritable bowel syndrome(IBS-D)rat model with liver qi attacking spleen from the perspective of intestinal mucosal permeability and intestinal inflammation.Methods:Eight female Wistar rats were randomly selected from 50 SPF grade Wistar rats as blank group,and the rest were used to prepare the rat model of IBS-D with liver qi attacking spleen.After modeling,40 model rats were randomly divided into model group,pinaverium bromide group and RZF low,medium and high dose groups,with 8 rats in each group,and were gavaged with normal saline,pinaverium bromide and RZF(0.86,1.72,3.44 g·kg-1)respectively for 14 days,and then samples were taken.Body mass and fecal water content were recorded,and related behavioral experiments were performed.The morphology of colon after HE staining was observed under light microscope,and the ultrastructure of intestinal mucosa was observed under transmission electron microscope.Enzyme linked immunosorbent assay was used to detect D-lactic acid in plasma and TNF-α,IL-6,IFN-γin serum.The expression levels of MMP9,p38MAPK,MLCK,and occludin proteins and mRNA in the colonic tissues of rats were detected by Western blot and RT-qPCR.Results:Compared with blank group,body mass decreased,fecal water content increased and novel inhibition feeding latency prolonged in model group(P<0.01).Compared with model group,body weight increased,fecal water content decreased and novelty inhibition feeding latency shortened in RZF groups(P<0.05,P<0.01).HE staining showed that the colonic structure of rats in each group was intact,and no obvious pathological changes were observed.Transmission electron microscopy showed that the intestinal epithelial cell gap in the model group was significantly widened,the structure of tight junctions and gap junctions was blurred,desmosomes were significantly reduced,and villi were sparse and uneven.The intestinal epithelial cell gap in the RZF groups was significantly reduced,the structure of the apical junction complex was clear,and the villi was dense.Compared with blank group,the contents of D-lactic acid and inflammatory factors in model group increased(P<0.05,P<0.01),the protein and mRNA expression levels of MMP9,p38MAPK and MLCK in colon tissue increased(P<0.05,P<0.01),and occludin expression level decreased(P<0.05,P<0.01).Compared with model group,the contents of D-lactic acid and inflammatory factors decreased(P<0.05,P<0.01),the protein and mRNA expression levels of MMP9,p38MAPK and MLCK in colon tissues decreased(P<0.05,P<0.01),and occludin level was up-regulated(P<0.01).Conclusion:Rougan Zhixie formula can improve intestinal mucosal permeability and intestinal inflammation in rat model of IBS-D with liver qi attacking spleen,possibly by reducing inflammatory factors and inhibiting the intervention effect of MMP9 activation of p38MAPK/MLCK signaling pathway.