艾灸对克罗恩病大鼠结肠肿瘤坏死因子-α含量及艾灸鼠结肠上清液对培养的结肠上皮细胞内紧密连接蛋白及其基因表达的影响  

Effect of Moxibustion on Colonic TNF-α Content and Influence of Colonic Supernatant of Crohn's Disease Rats Undergoing Moxibustion on Expression of Occludin,Claudin-1 and Zonula Occludens-1 Proteins and Genes in Cultured Colonic Epithelial Cells

作　　者：施茵[1] 包春辉[1] 吴焕淦[1] 马晓芃[1] 虞莉青[2] 张榕[1] 陈巍峰[3] 

机构地区：[1]上海市针灸经络研究所,上海200030 [2]上海中医药大学附属龙华医院,上海200032 [3]复旦大学医学院附属中山医院,上海200032

出　　处：《针刺研究》2011年第4期235-241,共7页Acupuncture Research

基　　金：国家重点基础研究发展计划(“973”计划)资助(No.2009CB522900);国家自然科学基金资助项目(No.30772831);国家中医药管理局针灸免疫效应重点研究室资助项目

摘　　要：目的:通过观察温和灸、隔药灸对肿瘤坏死因子-α(TNF-α)体外诱导大鼠结肠上皮细胞紧密连接——咬合蛋白(occludin)、闭合蛋白1(claudin-1)和闭锁蛋白1(zonula occludens-1,ZO-1)及其基因表达的影响,探讨艾灸对肠上皮屏障损伤的修复/保护机制。方法:将SD大鼠随机分成正常组、模型组、温和灸组、隔药灸组和西药组,各12只。采用三硝基苯磺酸制备克罗恩病(Crohn’s Disease,CD)大鼠模型。温和灸组与隔药灸组选择"天枢""气海"穴分别予以温和灸和隔药灸,每日1次,共治疗14次;西药组以柳氮磺胺吡啶溶液灌胃,每日2次,共14次。治疗结束后剖取各CD组大鼠结肠,制备结肠黏膜上清液;正常组纯化并培养结肠上皮细胞建立肠上皮屏障体外模型。将各CD组结肠黏膜上清液分别与正常大鼠结肠上皮细胞混合培养1周,采用ELISA法检测各组结肠黏膜上清液TNF-α含量,Western blot和荧光定量PCR法观察其对CD大鼠体外结肠上皮紧密连接occludin、claudin-1和ZO-1及其mRNA表达的影响。结果:CD模型组较正常组大鼠结肠黏膜上清液中TNF-α含量有显著增高(P<0.01),温和灸、隔药灸和柳氮磺胺吡啶治疗后TNF-α含量显著降低(均P<0.01),其中温和灸、隔药灸组优于西药组(均P<0.05);与此同时,温和灸、隔药灸和西药组结肠黏膜TNF-α上清液体外诱导大鼠结肠上皮紧密连接occludin、claudin-1和ZO-1及其mRNA表达较模型组有显著增高(P<0.01,P<0.05),且隔药灸、温和灸两组也优于西药组(均P<0.05)。结论:艾灸(隔药灸、温和灸)可能是通过降低CD大鼠结肠黏膜异常增高的TNF-α,进而促进或调节结肠上皮紧密连接occludinc、laudin-1和ZO-1及其mRNA表达,达到修复/保护肠上皮屏障损伤的目的。Objective To observe the effect of moxibustion on colonic tumor necrosis factor （TNF）-α level in Crohn＇s Disease （CD） rats and the effect of colonic supernatant of OD rats experiencing moxibustion on the expression of the tight junction proteins occludin, claudin-1 and zonula occludens （ZO）-1 and their genes in the cultivated colonic epithelial cells derived from CD rats, so as to reveal its underlying mechanism in resisting colonic epithelial barrier defects. Metbods Sixty SD rats were randomized into normal control, model, moderately warm moxibustion （MWM）, herbs-partitioned moxibustion （HPM） and medication （salazosulfapyridine, SASP） groups （n = 12）. CD model was established by intra-annual perfusion of trinitrobenzene sulfonic acid （TNBS） solution （TNBS ： 50% alcohol= 2 ： 1, 0.5 mL/kg）. For rats of the HPM and MWM groups, moxibustion was given to ＂Tianshu＂ （ST 25） and ＂Qihai＂ （CV 6） once daily for 14 d. For rats of the medication group, intragastric perfusion of SASP solution （0.0405 g/3 mL） was given twice daily for 14 d. After the treatment, all the rats including those of normal group were killed for preparing the supernatant of colonic mucosa tissue （6 - 8 cm superior to the anus）. The colonic epithelial cells of the normal group were purified and cultivated in DMEM culture fluid containing the prepared supernatant of normal group to establish an intestinal epi-thelial barrier defect model, and also cultured separately in the media containing the prepared supernatants of the model, medication, HPM and MWM groups. One week after the culture, the expression levels of occludin, claudin-1 and ZO-1 proteins and their genes in the cultured colonic epithelial cells were detected by Western blot and fluorescent quantitative polymerase chain reaction assay respectively. TNF-α content of the colonic supernatant was detected by enzyme linked immunosorbent assay. Results Compared with the normal group, colonic TNF-α content was remarkably increase

关 键 词：艾灸 肿瘤坏死因子-Α 结肠上皮细胞 紧密连接蛋白和基因 咬合蛋白 闭合蛋白 闭锁蛋白 

分 类 号：R245.81[医药卫生—针灸推拿学]