Polyclonal anti-vWF antibody was purchased from DAKO

ication by an investigator in a blinded manner and the apoptotic index was calculated. with enhanced chemiluminescence and captured on X-ray film. Optical density of the bands was measured using the BandScan imaging analysis system. Statistical analysis Renal HO-1 and Nrf2 immunohistochemical assays Paraffin-embedded renal sections were stained using the streptavidin-biotin complex immunohistochemistry technique for HO-1 and Nrf2 detection. Brown staining in the cytoplasm and/or nucleus was considered as an indicator of positive expression. Results were evaluated semi-quantitatively with Image-ProH plus version 6.0 software according to optical density values correlating with positive expression. Mean 6 SEM values were calculated to summarize all outcome measurements. One-way analysis of variance and the Duncan’s multiple range method were used to compare significant differences among the groups. The level of significance was set at P,0.05 for all statistical tests. Results Histopathological assessment of intestines In Fig. 1A-F, normal villi were observed in the Sham and ATRA + Sham groups. By contrast, the IIR and ATRA + IIR groups demonstrated edema in the villi and apparent inflammatory cells infiltration, and many intestinal villi were severed and denuded. In addition, the gap between epithelial cells significantly increased and capillaries and lymph 518303-20-3 vessels were markedly dilated. Significant amelioration of histological injury was observed in the RB1-treated groups, while the ATRA + RB1 group exhibited the same extent of injury as seen in the IIR group. In parallel with the mucosal morphologic changes, Chiu’s score in the IIR group was higher than that in the Sham group. This increase was significantly reduced by administration of 18347191 RB1. However, there was no statistically Western blot analysis Cytoplasmic and nuclear proteins were extracted from frozen renal tissues with a nuclear 22523636 extraction kit according to the manufacturer’s instructions. An equal amount of protein was loaded on to 12% SDS-PAGE at 100 V for 3 h. After electrophoresis, proteins were transferred onto PVDF membranes at 200 mA for 2 h. The transferred membranes were incubated overnight at 4uC with rabbit anti-mouse polyclonal antibodies for HO-1, Nrf2, Bcl-2 or Bax in Tris phosphate-buffered saline containing 5% skimmed milk. After washing three times in TBS-T, membranes were incubated with anti-rabbit IgG conjugated to horseradish peroxidase at a dilution of 1:2,000 in TBS-T containing 5% skimmed milk for 2 h at room temperature. The immunoreactive bands were visualized Ginsenoside Rb1 against Renal Injury significant difference in Chiu’s score between the IIR group and the ATRA + RB1 group . group. Pre-treatment with ATRA eliminated the effects of RB1 in reducing the expression ratio of Bcl-2/Bax . Evaluation of intestinal mucosal injury DAO is an enzyme synthesized primarily by gastrointestinal mucosal cells, and the serum level of DAO has been used as an indicator of the integrity and functional mass of the intestinal mucosa. The serum DAO activity was increased in groups IIR, ATRA+IIR and ATRA+RB1 and was decreased in groups treated with RB1 . Effects of RB1 on HO-1 and Nrf2 expression in renal tissues assessed by immunohistochemical assay Analysis of the expression of HO-1 in the Sham group showed sparse brown immunostaining in the cytoplasm while there was significant, positive expression of HO-1, as indicated by dense brown staining in the cytoplasm in the II