Painong San, a Traditional Chinese Compound Herbal Medicine, Restores Colon Barrier Function on DSS-Induced Colitis in Mice

Abstract

Objective: The intestinal barrier decreases in colitis and restores the integrity of the mucosal barriers that could be used for the treatment of colitis. Painong San (PNS), a traditional Chinese compound herbal medicine originally recorded in "Jingui Yaolve" by Zhongjing Zhang in the Later Han Dynasty, is often used in China and Japan to treat various purulent diseases including intestinal carbuncle. This study was to investigate the effect of PNS on mucosal barrier function in mice with DSS-induced colitis and its related mechanisms.

Methods: BALB/C mice were given 3% DSS to induce colitis. The body weight and stool status of the mice were recorded daily, and the histopathological changes of the colon were observed after execution. The permeability of the intestinal mucosa was measured by fluorescein isothiocyanate-dextran 4000, the change of intestinal microbiota was measured by 16S rDNA, and the tight junction-related proteins and Muc-2 were investigated by immunohistochemical or immunofluorescence. The possible signaling pathways were detected by western blot.

Results: Compared with the control group, the composition of the microbiota in the PNS group was close to that of the normal group, the number of goblet cells was improved, and the mucosal permeability was significantly reduced. PNS could upregulate the expression of tight junction-related proteins (ZO-1, claudin-1, and occludin) and Muc-2, and at the same time, regulate the Notch pathway.

Conclusion: PNS could effectively improve the mucosal barrier function through multiple ways, including restoring the balance of intestine flora, enhancement of the mucous layer barrier, and mechanical barrier function. These protective effects may relate to inhibiting the Notch signaling pathway activated by DSS.

Figure 1

The UPLC chromatogram of PNS.

Figure 2

PNS promotes recovery of DSS‐induced colitis in mice. (a) Daily changes in body weight of each group during the course of the disease; (b) evaluation of the DAI scores of each group after execution; (c) observation of the colon; (d) measurement of the length of the colon in each group; (e) H&E staining of tissues, n = 12. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, versus the control group, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus the DSS group.

Figure 3

PNS affects the composition of the microbiota in mice with colitis. (a) Diversity index diagram of each group based on OTU level; (b) phylum level analysis of intestinal bacteria in different groups of mice; (c) PNS treatment of DSS induction colitis causes 50 OTUs changes in the heatmap. The phylum, family, and genus names of OTUs are displayed on the right panel; (d) sample similarity tree of each group; (e) primary coordinate analysis (PCoA) of each group of samples , using weight-unifrac distance algorithm; n = 5. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, versus the normal group, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus the DSS group.

Figure 4

PNS improves intestinal epithelial barrier function. (a) Colon tissue section (FITC-4 kDa submucosal infiltration, Claudin-1 IHC staining, and representative photos of ZO-1 and occludin-1 IF staining), secondary antibody was used and observed by fluorescence microscopy (red staining). Nuclei were stained with 4,6‐diamidino-2-phenylindole (DAPI; blue staining); (b) the fluorescence intensity of FITC-D in serum at 4 h after administration (n = 9); (c) statistical histogram of Claudin-1 positive expression; (d) statistical histogram of positive expression of ZO-1; (e) statistical histogram of positive expression of occludin-1; the histogram data is calculated by selecting 10 random fields of view for each mouse, n = 3 mice for each group. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, versus the normal group, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus the DSS group.

Figure 5

PNS improves GC consumption and mucus barrier destruction caused by DSS. (a) Colon tissue section (representative photos of AB staining, PAS staining, and Muc-2 IHC staining); (b) statistical histogram of the number of goblet cells; (c) statistical histogram of the size of goblet cells; (d) statistical histogram of Muc-2 positive expression. The histogram data is calculated by selecting 10 random fields of view for each mouse, n = 3 mice for each group. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, versus the normal group, ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 versus the DSS group.

Figure 6

PNS can inhibit Notch signaling. (a) Representative western blot images for Notch1, Hes1, and Math1. (b) The protein expressions of Notch1, Hes1, and Math1. The histogram is the result of 3 independent experimental data. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, versus the normal group, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus the DSS group.