ABSTRACT
AIM:To investigate the effect of microRNA-429 (miR-429) on the expression of occludin (Ocln) in intestinal epithelial cells ( IECs) and intestinal epithelial barrier function in diabetic mice.METHODS:Diabetes mel-litus mouse model was induced by intraperitoneal injection of streptozocin.The expression of miR-429 in IECs of diabetic mice was inhibited by antagomiRNA-429 injected via tail vein.The expression of miR-429 and mRNA expression of Ocln were detected by real-time PCR.The protein expression of Ocln was determined by Western blotting and immunohistochem-istry.The urinary lactulose/mannitol ratio was measured by gas chromatography.The plasma LPS concentrations in the mice were measured by chromogenic end-point TAL kit.RESULTS:The results of real-time PCR confirmed that the ex-pression of miR-429 in IECs of diabetic mice was remarkably inhibited by tail-vein administration of antagomiRNA-429, and resumed to similar level of normal mice on the 6th day after the administration.After suppressing the level of miR-429, the expression of Ocln in IECs of diabetic mice increased significantly (P<0.05), while the urinary lactulose/mannitol ra-tio and the plasma LPS concentration decreased obviously ( P<0.05 ) .CONCLUSION:AntagomiRNA-429 effectively suppresses miR-429 expression in IECs of diabetic mice, and then enhances the expression of Ocln and partially resumes the intestinal epithelial barrier function.
ABSTRACT
Objective To investigate the role of Rho kinase (ROCK) in the protective effects of hydrogen on intestinal epithelial barrier function in sepsis. Methods Caco-2 cells were cultured routinely, and divided into 6 groups randomly (n=3):control group (C group), hydrogen-rich medium group (H group), lipopolysaccharide (LPS)-treatment group (L group), hy?drogen+LPS-treatment group (HL group), Rho kinase inhibitor (Y-37632) treatment group (Y group) and Rho kinase inhibi?tor Y-27632+LPS-treatment group (YL group). H group was treated with 0.6 mmol/L hydrogen-rich media. The concentra?tion of LPS and Y-27632 were 50 mg/L and 25μmol/L separately. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured regularly. When the TEER value reached 800Ω·cm2, the treatment was administered. Then TEER values were measured at 6 h, 12 h and 24 h, and FITC-dextran permeability was de?tected at 24 h. Cells were seeded on 6-well plates. After cell density reached 80%-90%, treatments were given randomly. The real time-polymerase chain reaction (RT-PCR) was conducted to assess mRNA levels of ZO-1 and ROCK mRNA. ZO-1 and ROCK protein expression levels were detected by Western blot assay. Results Compared with C group, TEER values were elevated in 12 h and 24 h in H group (P protein expression levels of ZO-1 and ROCK between C group and H group (P>0.05). TEER values were elevated at 6 h, 12 h and 24 h in Y group (P 0.05). The mRNA expression of ZO-1 increased and mRNA expression of ROCK decreased in Y group (P <0.05). The TEER values reduced at 6 h, 12 h and 24 h in L group. The FITC-dextran permeability increased significantly, mRNA and protein expressions of ZO-1 significantly decreased, mRNA and protein expressions of ROCK significantly in?creased in L group (all P<0.05). Compared with L group, TEER values increased significantly at 6 h, 12 h and 24 h in YL group, FITC-dextran permeability decreased, mRNA expressions of ZO-1 increased, mRNA expressions of ROCK de?creased in YL group (P<0.05). Compared with L group, TEER values increased at 6 h, 12 h and 24 h in HL group, FITC-dextran permeability reduced markedly, protein expressions of ZO-1 increased at each time point, protein expressions of ROCK decreased at each time point in HL group (P<0.05). Conclusion Hydrogen can protect intestinal barrier function against sepsis, ameliorate the integrity and permeability of intestinal epithelium and increase the expressions of intercellular tight junction proteins. The suppression of Rho kinase over-expression induced by LPS may be involved in these protective effects of hydrogen.