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.


| INTRODUC TI ON
Ulcerative colitis (UC) is a chronic, non-specific intestinal inflammation, which is a refractory intestinal disease with the main pathological features of local ulcers and chronic inflammation of the colon. UC is difficult to cure, and it is prone to repeated attacks. Inducing and maintaining clinical remission and mucosal healing, improving quality of life, and preventing complications are considered to be the therapeutic targets of UC. 1 At present, the main drugs for UC treatment are 5-ASA drugs, steroid, immunosuppressants, biological preparations, etc.

Previous clinical and experimental studies have proven that adjuvant
traditional Chinese medicine treatments such as herbal medicine and acupuncture are beneficial to the relief of UC symptoms, with reliable efficacy, few side effects and low recurrence rate. 2,3 QingBai Decoction (QBD) can be used to treat UC based on traditional Chinese medicine (TCM) theory. Previous studies 4 have found that QBD can effectively relieve intestinal symptoms such as diarrhoea, mucous bloody stool and fever for the treatment of mild-to-moderate active UC in clinic. The endoscopic examination after 8 weeks of treatment revealed that the mucosal healing was achieved, which improved the patient's quality of life.
Recent studies have shown that UC submucosal inflammation affects the integrity of the intestinal mucosal barrier structure, which leads to changes in permeability of the intestinal mucosa, and thus aggravates submucosal abnormal immune responses. 5,6 Therefore, objectively enhancing the function of the intestinal mucosal barrier may be a therapeutic solution for relieving intestinal inflammation. Our previous experiments have showed that QBD can improve the clinical symptoms caused by local inflammation, such as bloody purulent stool and local mucosal ulcers, but the mechanism is not yet clear. Therefore, in this study, the mechanism of QBD in UC treatment was studied from two aspects of inflammatory reaction and intestinal mucosal barrier function through the mouse model, and the mechanism of signal transduction to the pathway was discussed.

| Colitis model construction and treatment
Wild-type C57BL/6 mice were purchased from the Experimental Animal Center of fourth military Medical University [Certificate SCXK2012-0007], twenty-eight mice(weight, 21 ± 2 g) were randomly divided into four groups: the water group(treatment with water) (n = 8);the control group (n = 8); the QBD group (n = 8); and the mesalazine group (n = 8). Except for the control group, Six-to eight-week-old mice were administered 5% DSS in drinking water for 5 days, then mice were killed 3 days after recovery with water.
According to the conversion factor of experimental animals and clinical administration dose, the mice in QBD group and mesalazine group were administered with QBD enema (0.0195 mL/g per day, the suitable dose in our previous experiment, which show in supplementary information) or mesalazine enema (0.008 mL/g per day) by enemata for 8 day. The control group and the DSS group mice were administered normal saline by enemata. The colour, activity, faeces condition and weight were observed daily during modelling and drug treatment. FOB was tested and the severity of colitis was assessed daily using the disease activity index (DAI).  Table 2 shows the sequences of reverse and forward primers.

| Immunohistochemistry analysis
Briefly, paraffin-embedded slides were deparaffinized, and antigen unmasking was carried out by microwave heating in a citrate buffer for 20 min. After washing with PBS, slides were incubated with 3% H2O2 and then goat serum for 10 min and 15 min at room temperature, respectively. Primary antibodies were added to slides for incubating at 4°C overnight. Biotinylated secondary anti-rabbit antibodies were added and incubated at room temperature for 15 min.
After 15 min with streptavidin-HRP, sections were stained with DAB substrate for 5 min. According to previous reports, the score of immunostaining intensity was performed.

| Western blot analysis
Tissue was extracted using RIPA lysis buffer with the protease inhib- buffer for 1 h at room temperature, followed by incubation with primary antibodies at 4°C overnight. After incubation with a secondary antibody for 1 h at room temperature, proteins were detected using ECL reagent (Millipore). Three independent replicates were conducted for each.

| Statistical analysis
One-way ANOVA was used to analyse the data expressed as mean ± SD for comparison between multiple groups and least significant difference t test for internal group comparison. All statistical analyses were performed using SPSS version 19 (Standford, CA, USA). The threshold of statistical significance was set to P < 0.05. GraphPad Prism version 6.02 was used to generate histograms.

| QingBai decoction promotes recovery of DSSinduced colitis in mice
To induce acute colitis, mice were treated with 5% DSS for 5 days.
Increased epithelial injury and production of inflammatory cy- ing compared to the model group (P < 0.05) ( Figure 1B), However, no statistically significant difference was found between these two groups (P > 0.05). We also observed less congestion and oedema in the colon of QBD and mesalazine groups compared to the control groups ( Figure 1E). The H&E staining showed epithelial crypt damage and severe mucosal inflammation in the control group, which were less severe in QBD and mesalazine groups ( Figure 1D). Apart represented by the inflammation, depth of inflammation and crypt damage ( Figure 1C). These data suggest that QBD exerts therapeutic effects on severe colitis associated with intestinal epithelial cell injury probably.

| QingBai decoction inhibits apoptosis and promotes proliferation in the colon of DSS-treated mice
The previous reports believed that DSS is directly toxic to gut epi-  Figure 4A). In addition, the number of muc-2 and goblet cells positive cells increased obviously in colon after the treatment of QBD or mesalazine ( Figure 4B). As marker of tight junction structure, the distribution and expression of ZO-1 and claudin-1 were detected using immunostaining and immunoblotting, respectively.
The redistribution and depletion of these two proteins caused by DSS were reversed by QBD and mesalazine ( Figure 3A,B). We also found that ZO-1, claudin-1 and occludin protein levels increased significantly in the QBD and mesalazine groups vs control group(P < 0.05)( Figure 4C). Therefore, we determined the ex-

| QingBai decoction inhibits Notch signalling in the colon of DSS-treated mice
The Thus, our results suggested that QBD prevent the loss of muc-2 expression in DSS-treated mice by inhibiting Notch signalling probably.

| QingBai decoction inhibited the NF-κB pathway, regulated the cytokine expression of IL-1β, IL-6, Kc and TNF-α mRNA levels in colon tissue
NF-κB served as a major regulator of innate immunity and inflammatory responses 9 ， which regulates the production of proinflammatory cytokine. The effects of QBD in the DSS-induced colitis model were further studied. We compared mRNA expression levels of the key inflammatory cytokines, TNF-α, IL-1β, IL-6 and chemokine KC/CXCL1 using q-PCR. The control group showed significantly increased cytokine production including TNF-α, IL-1β, IL-6 and chemokine KC after DSS induction (P < 0.01) ( Figure 6B).
Compared with control group, a significantly reduced cytokine production including TNF-α(P < 0.01), IL-1β(P < 0.001), IL-6(P < 0.001), and chemokine KC (P < 0.01) was observed in QBD group. We also detected the protein expression of P-65 and P-P65 using immunoblotting. The QBD and mesalazine group showed significantly decreased in the protein level of P-P65 compared with control group (P < 0.05) ( Figure 6A), while it was no significant difference for QBD and mesalazine groups. These data suggest that regulation of signalling pathways involved in proinflammatory cytokine production may be another mechanism by which QBD inhibits proinflammatory responses in DSS-induced colitis model.

| D ISCUSS I ON
Ulcerative colitis is a chronic inflammatory disease， which results 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 of MMP-9, which may destroy tight junction complex and intestinal epithelial cells.
The abnormal activation of the NF-κB signalling pathway in the mucosal layer can lead to increased levels of inflammatory cytokines such as TNF-α, IL-1β and IL-6. 9 which induces an inflammatory cascade, and a large number of aggregated neutrophils can induce a series of pathological changes such as intestinal epithelial cell injury, crypt abscesses and small-vessel vasculitis. 21 Meanwhile, Intestinal permeability can be increased physiologically in response to pathologically by mucosal immune cells and cytokines. 22 Our studies using qPCR analysis showed that the expression of proinflammatory cytokine known to be upregulated by NF-κB signalling is decreased in QBD group. Moreover, the increased intestinal epithelial cell apoptosis caused by severe inflammatory may destroy tight junction proteins, and lead to a decreased barrier function and increased permeability. 23,24 While activated NF-κB can also induce apoptosis of intestinal epithelial cells by mediating caspase-3 activation, thereby to increase the permeability of intestinal mucosa. 9, 25 We observed the expression of active caspase-3 is significantly decreased in QBD group (compared to the control group). Combined, we suggest that QBD decreased the intestinal permeability by inhibiting NF-κB signalling to regulate mucosal inflammation.
Taken together, central outcome from our current study is that QBD alleviated mucosal inflammation in colitis model may through inhibiting NF-κB signalling and Notch activity to rescue the integrity of intestinal permeability from damage.

ACK N OWLED G EM ENTS
We would like to thank our colleagues in the Institute of Department

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.