QingBai decoction regulates intestinal permeability of dextran sulphate sodium-induced colitis through the modulation of notch and NF-κB signalling

Abstract

Objective: Chinese Herb QingBai decoction (QBD) has been approved affective in the treatment of IBD patients in clinic. However, the underlying mechanism remains unknown. We aim to investigate the effect of QBD on the mouse model of ulcerative colitis and its possible mechanism.

Methods: C57/bL mice were given 5% DSS to induce colitis and were divided as QBD and mesalazine group. Weight, faeces and mental status were recorded each day and the histopathological changes (goblet cells etc) of the colon were observed after sacrificed. Fluorescein isothiocyanate-dextran 4000 was measured to reflect the intestinal mucosal permeability. In addition, cell junction-related proteins and possible signal pathways were investigated.

Results: QingBai decoction could significantly alleviate the inflammation and the protection effect of colitis is comparable as those in mesalazine enema group. It was found that the permeability reduced significantly with QBD treatment vs the control group, while no significant difference between the mesalazine and QBD groups. QBD treatment could upregulate the expression of tight junction complex（ZO-1, claudin-1 and occludin）and muc-2 expression. It significantly reduced the production and secretion of serials proinflammatory cytokines (IL-1β, IL-6, Kc and TNF-α) compared with the control group. Meanwhile, NF-κB and Notch pathways were regulated.

Conclusion: QingBai decoction can effectively alleviate intestinal inflammation and mucosal barrier function in colitis mice, and the mechanism may be related to the inhibition of inflammatory cascade as well as enhanced mucus layer barrier and mechanical barrier function by NF-κB and Notch signalling.

Keywords: MMP-9; Mucin-2; Notch; QingBai decoction; intestinal permeability; ulcerative colitis.

Figure 1

QBD promotes recovery of DSS‐induced colitis in mice. Except the water group， mice in other groups were given DSS (5% w/v) for 5 days and the mice in the QBD and were performed enema with QBD or mesalazine, respectively. (A) Body weight change (the change of body weight in each group of mice was compared with the body weight in the same group of mice on the 1st day); Disease activity index（QBD group vs control group）； and (B) Length of colon (from the appendix to the anus); (C) Histopathologic scoring of the inflammation, depth of inflammation and crypt damage. Data are represented as the percentage of mice per group with the indicated score; (D) Histologic images of mice colon (H and E, magnification ×100). A: water group; B: control group; C: QBD group; D: SASP group; (E) Macroscopic appearance. *P < 0.05 vs water group **P < 0.01 vs water group ***P <0.001 vs water group, ^# P < 0.05 vs control group ^## P < 0.01 vs control group ^### P < 0.001 vs control group. Water group: treatment with water; SASP group: treatment with mesalazine; Control group: treatment with Dextran sulphate sodium; QBD: treatment with QingBai decoction

Figure 2

QBD inhibits apoptosis and promotes proliferation in the colon of DSS‐treated mice. Apoptosis and proliferation in the colon from both groups (day‐8) were examined (A) Immunohistochemistry staining for Ki67; and (B) Immunohistochemistry staining for active caspase‐3; (C) The score of Ki67 immunohistochemistry; (D) Representative western blots of total caspase‐3 and active caspase‐3; (E) Quantitative analysis of active caspase‐3. *P < 0.05 vs water group **P < 0.01 vs water group ***P < 0.001 vs water group, ^# P < 0.05 vs control group ^## P < 0.01 vs control group ^### P <0.001 vs control group. Water group: treatment with water; SASP group: treatment with mesalazine; Control group: treatment with Dextran sulphate sodium; QBD: treatment with QingBai decoction

Figure 3

QBD ameliorates the damage of junctional complex induced by DSS through regulates the expression of MMP‐9. QBD preserves intestinal barrier function in DSS‐treated mice. Paraffin‐embedded colon tissues were used to determine zonula occludens‐1 (ZO‐1) and claudin‐1 distribution by immunohistochemistry using an anti‐ZO‐1 antibody and anti‐claudin‐1 antibody， then FITC‐labelled secondary antibody were used and observed by fluorescence microscopy (red staining). Nuclei were stained with 4,6‐diamidino‐2‐phenylindole (DAPI; blue staining) (A). The distribution of ZO‐1 in the colon from both groups; (B). The distribution of claudin‐1 in the colon from both groups (C). Immunohistochemistry staining for MMP‐9 in colon. *P < 0.05 vs water group **P < 0.01 vs water group ***P < 0.001 vs water group, ^# P < 0.05 vs control group ^## P < 0.01 vs control group ^### P <0.001 vs control group. Water group: treatment with water; SASP group: treatment with mesalazine; Control group: treatment with Dextran sulphate sodium; QBD: treatment with QingBai decoction

Figure 4

QBD attenuate DSS‐induced epithelial permeability through enhances the function of mucus layer barrier and mechanical barrier. QBD preserves epithelial permeability in DSS‐treated mice.(A) submucosal infiltration of FITC‐dextran was examined 4 h after administration. The frozen sections of colons observed by fluorescence microscopy;(B) Immunohistochemistry staining for PAS and Muc‐2 in colon;(C) the expression of MMP‐9, ZO‐1, claudin‐1 and occludin were detected by Western blot analysis; (D) the fold changes of FITC from each groups. *P < 0.05 vs water group **P < 0.01 vs water group ***P < 0.001 vs water group, ^# P <0.05 vs control group ^## P <0.01 vs control group ^### P <0.001 vs control group. Water group: treatment with water; SASP group: treatment with mesalazine; Control group: treatment with Dextran sulphate sodium; QBD: treatment with QingBai decoction

Figure 5

QBD inhibits Notch signalling in the colon of DSS‐treated mice. (A) Immunohistochemistry staining for NICD in colon tissue samples; (B) Immunoblot analysis to determine the expression of NICD, p‐ERK1/2 and total ERK1/2 in colon tissue samples; (C), (D) real‐time PCR for Hes‐1 and ATOH1 in colon tissue of each groups. *P <0.05 vs water group **P <0.01 vs water group ***P <0.001 vs water group, ^# P < 0.05 vs control group ^## P < 0.01 vs control group ^### P < 0.001 vs control group. Water group: treatment with water; SASP group: treatment with mesalazine; Control group:treatment with Dextran sulphate sodium; QBD: treatment with QingBai decoction

Figure 6

QBD inhibited the NF‐κB pathway, regulated the cytokine expression of IL‐1β, IL‐6, Kc and TNF‐α mRNA levels in colon tissue. (A) Immunoblot analysis to determine the expression of p‐p65 and total p65 in colon tissue samples;(B) real‐time PCR for IL‐1β， IL‐6， Kc and TNF‐α in colon tissue of each groups. *P < 0.05 vs water group **P < 0.01 vs water group ***P < 0.001 vs water group, ^# P < 0.05 vs control group ^## P <0.01 vs control group ^### P < 0.001 vs control group. Water group: treatment with water; SASP group: treatment with mesalazine; Control group: treatment with Dextran sulphate sodium; QBD: treatment with QingBai decoction