title: [Effect of moxibustion on colonic TNF-alpha 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].
abstract: OBJECTIVE
  To observe the effect of moxibustion on colonic tumor necrosis factor (TNF)-alpha 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 ocoludin, claudin-1 and zonula occiludens (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 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-a content of the colonic supernatant was detected by enzyme linked immunosorbent assay.
  
  
  RESULTS
  Compared with the normal group, colonic TNF-alpha content was remarkably increased in the model group (P < 0.01). In comparison with the model group, colonic TNF-acx 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).
  
  
  CONCLUSIONS
  Both MWM and HPM can downregulate colonic mucosal TNF-alpha 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.
authors.affiliation: Ahang-Hai Institute of Acu-moxibustion and Meridians, Shanghai 200030, China. flysy0636@hotmail.com
authors.forename: Yin
authors.initials: Y
authors.lastname: Shi
authors.forename: Chun-hui
authors.initials: CH
authors.lastname: Bao
authors.forename: Huan-gan
authors.initials: HG
authors.lastname: Wu
authors.forename: Xiao-peng
authors.initials: XP
authors.lastname: Ma
authors.forename: Li-qing
authors.initials: LQ
authors.lastname: Yu
authors.forename: Rong
authors.initials: R
authors.lastname: Zhang
authors.forename: Wei-feng
authors.initials: WF
authors.lastname: Chen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D062445:Claudin-1; D003106:Colon / Q000737:chemistry* / Q000378:metabolism; D003424:Crohn Disease / Q000378:metabolism / Q000628:therapy*; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism; D007413:Intestinal Mucosa / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D009071:Moxibustion; D062793:Occludin; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*; D062826:Zonula Occludens-1 Protein
chemical_list: D062445:Claudin-1; C568114:Cldn1 protein, rat; D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D010750:Phosphoproteins; D012333:RNA, Messenger; C569044:Tjp1 protein, rat; D014409:Tumor Necrosis Factor-alpha; D062826:Zonula Occludens-1 Protein