脾阴虚证大鼠模型中水通道蛋白及肠道菌群的变化分析  

Analysis on Changes of Aquaporin and Intestinal Flora in a Rat Model with Spleen-Yin Deficiency Syndrome

作　　者：黄红侠 卢雪凤 周权 方红 邓夏夏 孙有智[1] 赵益[1] HUANG Hongxia;LU Xuefeng;ZHOU Quan;FANG Hong;DENG Xiaxia;SUN Youzhi;ZHAO Yi(Research Center for Differentiation and Development of Traditional Chinese Medicine(TCM)Basic Theory,Jiangxi Key Laboratory of TCM Etiopathogenisis,Formula-pattern Research Center,Jiangxi University of Chinese Medicine,Nanchang 330004,China)

机构地区：[1]江西中医药大学中医基础理论分化发展研究中心,江西省中医病因生物学重点实验室,方-证研究中心,南昌330004

出　　处：《中国实验方剂学杂志》2023年第23期131-139,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81860777);江西省重大科技研发专项(20203ABC28W019);江西省中医药骨干人才培养项目(赣中医药科教字[2021]4号);江西省“十四五”期间一流大学和一流学科建设项目(赣教研字[2022]1号);江西中医药大学校级科技创新团队发展计划项目(CXTD22007);江西中医药大学校级研究生创新专项(JZYC22S39)。

摘　　要：目的:以脾阴虚证模型大鼠为研究对象,探析脾阴虚对胃肠吸收、水液代谢及肠道菌群的影响。方法:采用荤素饮食不节、负重疲劳游泳和温热伤阴药灌胃复合因素建立脾阴虚证大鼠模型,观察空白组和模型组大鼠的体质量、进食量、饮水量、游泳持续时间的变化;苏木素-伊红(HE)染色观察胃和结肠组织病理损伤情况;间苯三酚法检测尿D-木糖排泄率;酶联免疫吸附测定法(ELISA)检测血清中胃泌素(GAS)含量;蛋白免疫印迹法(Western blot)检测胃组织中血管活性肠肽(VIP)、水通道蛋白3(AQP3)、AQP4的相对表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测胃组织中VIP、AQP3、AQP4的mRNA相对表达水平;16S rDNA测序分析其肠道菌群的变化。结果:与空白组比较,一般体征结果显示,模型组大鼠体质量和进食量均明显降低,饮水量明显增多(P<0.05,P<0.01),游泳持续时间显著减少(P<0.01);病理检测结果显示,模型组大鼠胃组织黏膜出现排列不齐,结肠组织黏膜可见明显变薄或有残缺,上皮细胞出现坏死甚至脱落现象;与空白组比较,模型组大鼠尿D-木糖排泄率显著降低(P<0.01),血清GAS含量明显降低(P<0.05);Western blot结果显示,与空白组比较,模型组大鼠胃组织中VIP蛋白的相对表达水平显著降低(P<0.01),AQP4、AQP3蛋白的相对表达水平显著增加(P<0.01);Real-time PCR结果显示,与空白组比较,模型组大鼠胃组织中VIP mRNA的相对表达水平显著降低(P<0.01),AQP3和AQP4 mRNA的相对表达水平明显增加(P<0.05,P<0.01);肠道菌群分析结果显示,与空白组比较,模型组操作分类单元(OTU)数目和α多样性增加,β多样性明显降低,紫单胞菌科丰度明显增加,产丁酸菌种丰度明显降低(P<0.05);斯皮尔曼计算法(Spearman)相关分析显示,紫单胞菌科与AQP4蛋白水平呈明显正相关(P<0.05),产丁酸菌种与VIP蛋白水平呈明显正相关(P<0.05),与AQP3、AQP4蛋白水平呈�Objective:To investigate the effects of spleen-Yin deficiency on gastrointestinal absorption,water metabolism and intestinal flora in rats with spleen-Yin deficiency syndrome.Method:A rat model of spleen-Yin deficiency syndrome was established by using the composite factors,including irregular meat and vegetable diet,weight-bearing fatigue swimming and gavage with warm-heat injury-Yin drugs.The changes of body weight,food intake,water intake and duration of swimming in the blank and model groups were observed.Hematoxylin-eosin(HE)staining was used to observe the histopathological damage of the stomach and colon.Urinary excretion rate of D-xylose was determined by phloroglucinol method.The content of gastrin(GAS)in serum was determined by enzyme-linked immunosorbent assay(ELISA).The relative expression levels of vasoactive intestinal peptide(VIP),aquaporin 3(AQP3)and AQP4 in gastric tissues were detected by Western blot.The relative mRNA expression levels of VIP,AQP3 and AQP4 in gastric tissues were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and the changes of intestinal flora were analyzed by 16S rDNA sequencing.Result:Compared with the blank group,the results of general physical signs showed that the body weight and food intake of rats in the model group were significantly decreased,the water intake was significantly increased(P<0.05,P<0.01),and the duration of swimming was significantly decreased(P<0.01).Pathological examination results showed that in the mucosa of gastric tissues of rats in the model group appeared to be misaligned,the mucosa of colonic tissues could be seen to be obviously thinned or mutilated,and the epithelial cells appeared to be necrotic or even exfoliated.Compared with the blank group,the urinary D-xylose excretion rate of rats in the model group was significantly decreased(P<0.01),and the serum GAS content was significantly decreased(P<0.05).Compared with the blank group,Western blot results showed that the relative expression level of VIP p

关 键 词：脾阴虚证 血管活性肠肽(VIP) 水通道蛋白(AQP) 水液代谢 肠道菌群 相关性分析 聚合酶链式反应(PCR) 

分 类 号：R22[医药卫生—中医基础理论] R574.4[医药卫生—中医学] R28[生物学—微生物学] Q939