VC+VD3 Promotes Recovery of DSS Induced Colitis in Guinea Pigs
Adaptive feeding of guinea pigs for 5 weeks, to induce acute colitis, guinea pigs were treated 2%DSS for 5 days. At the beginning of the experiment, the guinea pigs in each group had normal mental state and good health. There was no significant difference in body weight between the guinea pigs. In the low, medium and high dose VC group, the weight of the guinea pigs began to decrease on the first day after drinking 2% DSS solution. From the second day, the guinea pigs gradually showed signs of apathy, decreased mobility, loose stools, mucus, bloody stools, etc. It is aggravating, indicating that ulcerative colitis model is successful. After modeling, the body weight of each group of guinea pigs showed a downward trend, but there was no significant difference in body weight between the groups (P > 0.05)(Figure 1A).Compared with the control group, the colonic gross morphology score and histopathological score of the other groups were significantly increased (P < 0.05). Compared with the medium dose VC group, the colon length of the high dose VC group was significantly increased (P < 0.05)(Table 1).Compared with the control group, the H&E staining showed epithelial crypt damage and severe mucosal inflammation in the other group. In the low-dose VC group, a small amount of epithelial cells were exfoliated in the colon, the mucosal surface was finger-like, and the crypt opening was widened, but it was improved compared with the medium-dose VC group. The medium-dose VC group had severe epithelial cell shedding in the colon of the guinea pigs, and the mucosal surface the villous changes, the crypt shrinkage, the spacing increased, and the connective tissue in the submucosa was loose. The mucus in the colonic goblet cells of the guinea pigs in the high-dose VC group decreased, the crypts were branched and twisted, but the epithelial cell shedding was better than the medium-dose VC group (Figure 1B).
VC+VD3 Attenuate DSS-induced Epithelial Permeability through Regulates Colonic Tight Junction.
DSS model would not only affect the integrity of the mucosal barrier, but also regulate colonic tight-junctions. Damage to intestinal permeability may be due to dysregulation of the mucus or epithelial junction complex, which enhances susceptibility to colitis.
The previous reports believed that both vitamin D3 and vitamin C play a role in maintaining the integrity of the intestinal mucosal barrier and repairing the mucosal barrier [12, 13, 15].According to our results，there was no significant change in the ultrastructure of colonic epithelial cells in the control group (Figure 2A). The low, medium and high doses of the guinea pig colonic epithelial cells in the VC group showed focal reduction, shortening and sparseness in different degrees. Among them, the medium dose VC group was the most severely damaged (Figure 2C). Compared with the control group, the tight junction structure between the colonic epithelial cells of the medium-dose VC group was severely damaged, and the cell gap became larger (Figure 2C), while the tight junction gap between the colonic epithelial cells in the low-dose and high-dose VC group was slightly widened, but the tight joint structure remains essentially intact (Figure 2B, D).
VC+VD3 Regulate Notch Signaling in the Colon of DSS-treated Mice
Notch signaling pathway plays a key role in the fate of intestinal epithelial cells ，which regulate a lot of genes including Hes-1 to promote the repair process of damaged epithelium .Therefore, Notch signaling pathway may play an important regulatory role in the maintenance of intestinal mucosal homeostasis by regulating the proliferation and differentiation of intestinal epithelial cells.
The results of this experiment showed that the expression levels of Notch1 and Hes1 mRNA were significantly higher than those of the control group (P<0.05), but the expression levels of the middle and high dose groups were lower than those of the low dose group (P<0.05). The expression distribution of tight junction complexes is closely related to apothecary endothelial cell permeability. The results of this experiment showed that compared with the control group, the expression level of ZO-1 mRNA in the high dose group was significantly increased (P<0.05) (Table 2). However, the mRNA expression level of claudin-2 was not significantly different among the groups.
Repair Effect of VD3 Combined VC on Tight Connection Damage between SW480 Cells Induced by Lipopolysaccharide by Notch Signal Pathway
Cell Scratch Test
The cell scratch test is a method for determining the migration and repair ability of cells. Therefore, in order to observe the effect of different concentrations of vitamin C combined with vitamin D3 on the migration of SW480 cells, the experiment was performed using a cell scratch test.
Compared with cell scratches 0h, cell migration was observed in all groups 24h after cell scratching. Among them, 0.1VC+VD3 group had the most significant cell migration compared with the control group, while 10VC+VD3 group had the least cell migration. Obviously; compared with the VD3 group, the cell migration and repair ability of the 0.1VC+VD3, 1VC+VD3, and 5VC+VD3groups was significantly improved (Figure 3).
VC+VD3 Attenuate LPS-induced Tight Junction Structure Damage in SW480 Cells
Tight junctions is essential construction for mucosal barrier, therefore, in this experiment, the LPS in vitro model was used to induce cell membrane barrier destruction, and the effects of different vitamin C and vitamin D3 on cell tight junction were observed. As marker of tight junction structure, the distribution and expression of claudin-2 and ZO-1 were detected by western blot. Compared with the control group, The expression level of claudin-2 protein was significantly decreased in LPS, 0.1VC+VD3, 1VC+VD3 and 10VC+VD3 groups (P< 0.05), and the expression level of claudin-2 protein in group VD3 was significantly higher than that in LPS group (P< 0.05), and the expression level of claudin-2 protein in the VC+VD3 group was significantly lower than that in the VD3 group (P< 0.05). Western blot analysis demonstrated decreased expression of ZO-1 in other groups vs control group (P<0.05) (Figure 4). Thus, the role of VC+VD3 in the intestinal epithelium may enhance epithelial tight junctions in SW480 cells.