| Objective:The purpose of the present study was to explore the repair mechanism of Huang Qi Jian Zhong Pellet(?)(HQJZ) against2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colonic mucosal injury in rats.Methods:Colonic mucosal injury was induced by TNBS in Sprague-Dawley rats followed by treatment with HQJZ for14days. The therapeutic effects of HQJZ were evaluated by macroscopic observation and histological examination, the status of colonic microcirculation and blood flow were observed by inverted intravital microscopy and Laser-Doppler perfusion imager, respectively. The level of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and productions of myeloperoxidase (MPO), interleukin-6(IL-6) and interleukin-10(IL-10) were measured by enzyme linked immunosorbent assay (ELISA), while MPO expression in colonic mucosa tissues was stained by immunohistochemistry. Expression of CD11b in blood and tumor necrosis factor-a (TNF-a) in colonic mucosa were assessed by flow cytometry analysis. The immunofluorescence staining of F-actin and Occludin in colon tissues were observed by confocal microscopy. The expression of AMP-activated protein kinase-α (AMPK-α), phospho-AMPK-α (p-AMPK-α), ATP5D, RhoA, Rho kinase-Ⅰ (ROCK-Ⅰ), phospho-myosin light chain (p-MLC), Claudin-5, Occludin, Rac-1, p21-activated kinase-1(PAK-1) proteins were analyzed by Western blotting.Results:After treatment for14days, compared with TNBS-induced rats without treatment, HQJZ effectively decreased colonic weight, colonic weight index, macroscopic and histological scores, shortened colonic length, and finely improved colonic microcirculation disturbances, as well as increased colonic blood flow and functional capillary density, inhibited albumin leakage, leukocyte rolling on and adhesion to chorion and mucous layer. Moreover, to restore damaged colonic mucosa, HQJZ remarkably enhanced activity of AMPK-α, phospho-AMPK-α and ATP5D which resulted in increase the level of ATP and decrease of the ratio of ADP/ATP and AMP/ATP, and restored F-actin polymerization in TNBS-induced rats treated by HQJZ. Meanwhile HQJZ effectively inhibited increased activity of RhoA and ROCK-I, decreased expession of Rac-1and PAK-1in damaged colonic mucosa of rats induced by TNBS, and restrained MLC phosphorylation, enhanced expression of Occludin, and decreased productions of CD11b, MPO, TNF-a and IL-6, heighted expression of IL-10.Conclusions:The present study demonstrated that HQJZ effectively enhanced ATP level and improved energy status to restore damaged colonic mucosa induced by TNBS, which was attributed to activation of AMPK resulted in balance of Rho/Rac and improvement of colonic microcirculatory disturbances. |
PDF Full Text Request