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Background: Herb-partitioned moxibustion (HPM) at Tianshu (ST25) and Qihai (RN6) has been used to treat Crohn's disease (CD). Injury to intestinal epithelial tight junctions (TJs) is the leading cause of CD onset with under expression of TJ-related proteins such as occludin, claudin-1, and zonula occludens protein-1 (ZO-1). This study aimed to investigate whether HPM can change the permeability of the intestinal epithelial barrier by affecting the expression of colonic epithelial TJ-related proteins in vitro. Methods: Forty-eight male Sprague-Dawley rats were randomly divided into four groups of twelve rats: normal control (NC) group; model control (MC) group; herb-partitioned moxibustion (HPM) group; and mesalazine control (MESA) group. The rats in the latter three groups were given trinitrobenzene sulfonic acid (TNBS) enemas to establish CD models. The HPM group was treated with HPM at Tianshu (ST25) and Qihai (RN6) once daily for 14 consecutive days, while the MESA group was given mesalazine solution (at the proportion of 0.018:1) by lavage twice daily for the same period. After the treatment period, the colon tissues from all groups were partly processed for macroscopic damage assessment and histological observation, and partly purified and cultured in vitro to examine the permeability of the intestinal epithelial cell barrier by trans-epithelial electrical resistance (TEER). Western blot and fluorescence quantitative polymerase chain reaction (FQ-PCR) analyses were performed to observe the expression of occludin, claudin-1, and ZO-1 proteins and mRNAs, respectively. Results: In the HPM and MESA groups, the typical CD macroscopic damage, i.e., inflammatory cell infiltration in colonic mucosa and submucosa, submucosal lymphoid follicular hyperplasia, hyperemia and edema, and morphological changes were improved to different degrees in the colonic tissues (HPM, MESA vs. MC for macroscopic score of colonic damage: all P < 0.001). The decreasing tendencies were minor for colonic TEER values (HPM, MESA vs. MC: all P < 0.001), and expression of intestinal epithelial TJ-related proteins (HPM, MESA vs. MC: all P < 0.05) and mRNAs (HPM, MESA vs. MC: all P < 0.05), especially in the HPM group (HPM vs. MESA for TEER values: P < 0.001). Conclusions: HPM at Tianshu (ST25) and Qihai (RN6) upregulated the expression of occludin, claudin-1, and ZO-1 in TNBS-induced CD model rats. [ABSTRACT FROM AUTHOR]