摘要
目的观察脾阴虚状态下肠黏膜屏障的改变及滋补脾阴方药的调控作用。方法将SPF级雄性Wistar大鼠随机分为正常组(简称C组)6只和模型组(简称M组)14只。采用饮食不节与劳倦过度结合耗伤阴液法建立脾阴虚大鼠模型。脾阴虚造模后将M组随机分为脾阴虚组(简称S组)和滋补脾阴方药组(简称Z组)。每周收集粪便进行16S rRNA测序。采用qPCR技术检测空肠Claudin-1、Occludin、ZO-1 mRNA水平;Western Blot法检测空肠Claudin-1、Occludin、ZO-1蛋白表达;ELISA法检测肠道炎症因子水平。结果在门水平上,S组较C组厚壁菌门比例增加(P<0.05),拟杆菌门下降(P<0.05),Z组较S组厚壁菌门减少(P<0.01),拟杆菌门增加(P<0.01);在属水平上,S组较C组乳杆菌属和Paraprevotella属比例升高(P<0.05,P<0.01),瘤胃球菌属和粘放线菌属下降(P<0.01,P<0.05);Z组比较于S组乳杆菌属比例明显减少(P<0.0001),瘤胃球菌属和粘放线菌属增加(P<0.05,P<0.01),Paraprevotella属呈下降趋势(P>0.05)。与C组比较,S组空肠Claudin-1 mRNA和蛋白表达下降(P<0.05,P<0.01),Occludin mRNA和蛋白表达降低(P<0.01),ZO-1 mRNA和蛋白表达呈降低趋势(P>0.05);与S组比较,Z组Claudin-1 mRNA和蛋白水平升高(P<0.01),Occludin mRNA和蛋白表达增加(P<0.05),ZO-1 mRNA和蛋白水平呈上升趋势。S组空肠IFN-γ和IL-17含量较C组增加(P<0.001),sIgA、TGF-β、IL-4与IL-10水平下降(P<0.01,P<0.001);Z组较S组IFN-γ、IL-17含量减少(P<0.001,P<0.01),sIgA、TGF-β、IL-4与IL-10含量升高(P<0.01,P<0.001)。结论滋补脾阴方药能够调节肠道菌群中厚壁菌门、拟杆菌门、瘤胃球菌属、乳酸杆菌属与粘放线菌属比例,增加Claudin-1、Occludin mRNA和蛋白表达,并调节Th1/Th2、Th17/Treg细胞分泌的细胞因子水平,改善脾阴虚大鼠肠黏膜生物屏障、机械屏障及免疫屏障的异常变化。
Objective To observe the changes of intestinal mucosal barrier and the regulatory effect of Zibu Piyin Recipe(ZBPYR)in Pi-yin deficiency(PYD)model rats.Methods SPF male Wistar rats were randomly divided into a normal group(C,n=6)and a model group(M,n=14).SYD model was established by improper diet and overwork combined with exhaustion of yin fluid.After ending the PYD model,rats in Group M were randomly divided into PYD group(S)and ZBPYR group(Z).Fecal samples were collected every week for 16 S rRNA sequencing.mRNA levels of Claudin-1,Occludin,and ZO-1 in the jejunum were detected by qPCR.The protein expressions of Claudin-1,Occludin,and ZO-1 in the jejunum were detected by Western Blot.The levels of intestinal inflammatory factors were detected by ELISA.Results Compared with Group C,the level of Firmicutes increased(P<0.05),while Bacteroides decreased in Group S(P<0.05).Compared with Group Z,the level of Firmicutes decreased(P<0.01),while the level of Bacteroides increased in Group S(P<0.01).At the genus level,the proportions of Lactobacillus and Paraprevotella in Group S were higher than those in Group C(P<0.05,P<0.01).And the proportions of Ruminococcus and Adlercreutzia decreased more in Group S than in Group C(P<0.01,P<0.05).Compared with Group S,the proportion of Lactobacillus significantly decreased(P<0.0001),while the proportions of Ruminococcus and Adlercreutzia increased in Group Z(P<0.05,P<0.01).The level of Paraprevotella had a downward trend(P>0.05).Compared with Group C,jejunum Claudin-1 mRNA and protein expressions decreased(P<0.05,P<0.01),Occludin mRNA and protein expressions decreased(P<0.01),and ZO-1 mRNA and protein expressions showed a decreasing trend in Group S.Compared with Group S,Claudin-1 mRNA and protein levels increased(P<0.01),Occludin mRNA and protein expressions increased(P<0.05),and ZO-1 mRNA and protein levels showed an upward trend in Group Z.Compared with Group C,the levels of IFN-γand IL-17 in jejunum increased(P<0.001),while the levels of sIgA,TGF-β,IL-4 and IL-10 decreased(P<0.01,P<0.001)in Group S.Compared with Group S,the levels of IFN-γand IL-17 were reduced(P<0.001,P<0.01),and contents of sIgA,TGF-β,IL-4 and IL-10 increased(P<0.01,P<0.001)in Group Z.Conclusion ZBPYR improved abnormal changes of intestinal mucosal biological barrier,mechanical barrier,and immune barrier in PYD rats by regulating the proportions of intestinal flora(Firmicutes,Bacteroidetes,Ruminococcus,Lactobacillus,Actinobacteria),increasing mRNA and protein expressions of Claudin-1 and Occludin,and adjusting cytokines secreted by Th1/Th2 and Th17/Treg cells.
作者
孙晓霞
周雯
战丽彬
SUN Xiao-xia;ZHOU Wen;ZHAN Li-bin(School of Traditional Chinese Medicine&School of Integrated Traditional Chinese and Western Medicine,Modern Research Laboratory of Spleen Visceral Manifestations Theory,Nanjing Uni-versity of Chinese Medicine,Nanjing,210023)
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2020年第10期1233-1240,共8页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家重点研发计划项目(No.2018YFC1704400)
国家重点基础研究发展计划(973计划)项目(No.2013CB531704)
江苏省研究生科研与实践创新计划项目(No.KYCX18_1534)。
关键词
脾阴虚
肠黏膜屏障
滋补脾阴方药
Pi-yin deficiency
intestinal mucosal barrier
Zibu Piyin Recipe
