Diosmetin alleviates intestinal epithelial barrier damage in vitro and in vivo
As shown in Fig. 2A, B compared to the control group, the expression levels of tight junction proteins (Occludin and Claudin) in the mice of DSS only group were observably decreased, however, diosmetin and SASP were able to markedly increase the expression of Occludin and Claudin compared to the model group. In addition, the results of immunofluorescence analysis of ZO-1 as shown in Fig. 2C, D, the expression levels of ZO-1 in colon sections were markedly decreased after DSS treatment in vivo compared to the control groups. These levels were significantly up-regulated when the cells were pre-treated with diosmetin and SASP. Next, we analyzed the expression of tight junction proteins, Occludin and Claudin, by Western blot after 24 h exposure to different concentrations (0.1–30μg/ml) of LPS in caco2 and IEC-6 cells. As shown in Fig. 3A, the levels of expression of Occludin and Claudin were obviously decreased in a dose-dependent way for the caco2 and IEC-6 cells in LPS-treated group. Furthermore, the expression level of Occludin and Claudin administrated with LPS (30 μg) was significantly decreased.After pretreatment with diosmetin (25-100 ng/mL) for 24 h, the levels of expression of Occludin and Claudin were obviously increased in caco2 and IEC-6 cells (Fig. 3B). As shown in Fig. 3C, D, the immunofluorescence analysis of ZO-1 exhibited similar results.
Diosmetin reduced pro-inflammatory cytokines in vivo
The effects of diosmetin on the expression of inflammation related genes were studied at mRNA level shown in Fig. 4. diosmetin (50 mg/kg) significantly inhibited the expression of IL-1β, IL-6, cyclooxygenase-2 (COX-2) and IFN-γ (p<0.05) Compared to 3% DSS only group (Fig4 A-D). Compared to 5% DSS only group, diosmetin (50 mg/kg) significantly inhibited the expression of IL-1β, IL-6, cyclooxygenase-2 (COX-2) and IFN-γ (p<0.05) (Fig. 4E- H).