Objective To observe the effect of moxibustion on colonic tumor necrosis factor (TNF)-o level in Crohn's Disease (CD) rats and the effect of colonic supernatant of CD 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. Methods Sixty SD rats were ran-domized into normal control, model, moderately warm moxibustion (MWM), herbs-partitioned moxlbustion (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" (ST25) and "Qihai" (CV6) 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-a content of the colonic supernatant was detected by enzyme linked immunosorbent assay. Results Compared with the normal group, colonic TNF-a content was remarkably increased in the model group (P<0. 01). In comparison with the model group, colonic TNF-a contents were significantly decreased in the medication, MWM and HPM groups (P<0. 01), and those of the MWM and HPM groups were markedly lower than that of the medication group (P<0. 05). The expression levels of the cultured normal colonic epithelial occludin, claudin-1 and ZO-1 proteins and their mRNAs in the medication, MWM and HPM groups were remarkably increased compared with those in the model group(P<0. 01, P<0. 05). The expression levels of colo-nic epithelial occludin, claudin-1 and ZO-1 proteins and their mRNAs were significantly higher in the MWM and HPM groups than in the medication group (P<0. 05). Conclusion Both MWM and HPM can downregulate colonic mucosal TNF-a content in CD rats, and the colonic supernatant of rats undergoing MWM and HPM may upregulate the expression of colonic epithelial occludin, claudin-1 and ZO-1 proteins and their mRNAs in the cultivated colonic epithelial cells, which may contribute to the effect of moxibustion in relieving colonic epithelial barrier defect.%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嘧溶液灌胃,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嘧治疗后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.ccludin,claudin 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.
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