The effect of olsalazine of chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt in BALB/c mice

ABSTRACT Purpose: To explore effect and mechanism of olsalazine of Chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt (DSS) in BALB/c mice. Methods: The mouse model of ulcerative colitis was induced by free drinking of 3% (w/v) DSS aqueous solution for seven days. The mice were treated with olsalazine (0.6 g·kg-1) of Chinese generic drugs. The therapeutic effect of olsalazine on ulcerative colitis mice was evaluated by measuring disease activity index (DAI), colonic mucosal injury index (CMDI), histopathological score (HS), and detected the expression levels of interleukin (IL)-2, IL-10, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-1β in serum and IL-7, IL-17, IL-22, epidermal growth factor (EGF), transforming growth factor β1 (TGF-β1) in colonic homogenate of mice. Results: Olsalazine significantly increased the contents of IL-2, IL-10, IL-22, TGF and EGF in ulcerative colitis rats, and significantly decreased the scores of DAI, CMDI, HS and the contents in IL-7, IL-17, TNF-α, IL-1β and IFN-γ when compared with the model group. It improved the degree of colonic lesion in ulcerative colitis mice. Conclusions: It was suggested that olsalazine has a therapeutic effect on ulcerative colitis induced by DSS in mice, and the mechanism may be related to the increase of IL-2, IL-10, IL-22, TGF, and EGF and the decrease of the expression of IL-7, IL-17, TNF-α, IL-1β, and IFN-γ.

The effect of olsalazine of chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt in BALB/c mice The animal models used for anti-UC drug screening mainly include chemical stimulation animal model, genetic engineering animal model, tissue or cell transplantation animal model, spontaneous animal model, and traditional Chinese medicine (TCM) syndrome animal model at present. The dextran sulfate sodium salt (DSS) of chemical reagent stimulation animal model is widely used in experimental research because of its advantages, such as easy to make, low cost, strong maneuverability, good repeatability, among others.
DSS was used to induce acute UC in BALB/c mice in this study, and the treatment of UC mice was by intragastric administration of olsalazine of Chinese generic drug. It was observed the effect of olsalazine on UC mice and to explore the mechanism of olsalazine on DSS-induced UC in BALB/c mice from the changes of general condition, body weight, disease activity index (DAI) score, organ index, colon colonic mucosal injury index (CMDI) score, serum antibody, colon pathological, and biochemical indexes.

Preparation of main reagents
Preparation of 3%DSS solution 120-g DSS were dissolved in 4-L hot-pressed ultra-pure water to obtain 3% DSS solution (currently used).

Establishment and grouping of models
Seventy-two healthy male BALB/c mice were fed adaptively for one week. Twenty-four mice were randomly selected as the normal group to drink ultra-pure water, and to the rest 3% (w / w) DSS solution was given freely for seven days, and all the mice were scored by DAI. The mice with very mild inflammation were excluded, and the other mice were randomly divided into model group and olsalazine group (0.6g kg -1 ), with 24 mice in each group. At the end of the model, the treatment group was given olsalazine according to 0.1 mL·kg -1 ·d -1 , while the model control group and normal control group were given normal saline 0.1 mL kg -1 ·d -1 intragastrically for 10 days.

Observation of indicators
Observation of general state The changes of body mass, drinking water, diet, and fecal characteristics of mice in each group were observed and recorded during the experiment.

The score of disease activity index
On the first, fourth, seventh, eighth, tenth, 14 th , and 17th day of the experiment, the normal group and the model group referred to the Hamamoto standard 10 . The mice were weighed, the fecal traits and fecal occult blood were observed, and the DAI scores of the mice were evaluated. The disease activity was evaluated by DAI on the eighth, 11th, 14th, and 17th day in the olsalazine group, and the DNA score criteria are shown in Table 1. Fecal traits and occult blood score are shown in Fig. 1.  The effect of olsalazine of chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt in BALB/c mice

Colonmucosa damage index
The removed colon was laid flat on white paper, and the colonic mucosal injury was observed and scored according to standards of Ekström et al. 11 and Luk et al. 12 in Table 2.

Organ index in mice
The liver, spleen, lung, thymus, and colon were taken, and the organ index was calculated (Eq. 1).

Organ index = Organ mass (mg) / Mouse body weight (g) 1
Observation on the pathomorphology of colon The longitudinally sectioned colon was cut into two halves, wrapped, fixed, and preserved in 10% neutral formalin solution. The pathological sections were observed under light microscope after hematoxylin and eosin (HE) stained, and the colon histopathological score (HS) was performed according to Ekström 11 standards ( Table 3). The HS re was diagnosed by Yan Changbao, an attending physician in the Department of Pathology of the people's Hospital of Dali Bai Autonomous Prefecture in China. Loss of a small number of crypt cells The infiltration penetrated into the muscular layer of the mucosa, accompanied by mucosal thickening and edema 4 Massive loss of crypt cells Infiltrate into the submucosa *Colonic histopathological score: "epithelial cell" score + "inflammatory cell infiltration" score. Source: Ekström et al. 11 .

Detection of related biochemical factors in serum and colonic mucosa
The blood extracted from eyeballs of mice was placed at 4°C for 4 hours, and the supernatant was obtained by centrifugation at 3,000 rpm (4°C) for 10 min. The other half of the longitudinally dissected colon was made into 10% homogenate in an ice bath, and the supernatant was obtained by centrifugation at 3,000 rpm (4°C) for 10 min. According to the instructions, the contents of related biochemical factors in serum and colonic mucosa were determined by ELISA method, and the data were calculated by ELISACalc.

Determination of lymphocyte subsets
One-hundred-μL spleen cell suspension (10 6 spleen cells) was added to the flow tube, and the corresponding mixed fluorescent antibody was added for FACS Calibur detection. The ratio of T lymphocytes (CD3 + ) to single spleen cells and the proportion of Th cells (CD3 + CD4 + CD8 -), Tc cells (CD3 + CD4 -CD8 + ) and Treg cells (CD3 + CD4 + CD25 + ) to T lymphocytes in spleen were analyzed.

Statistical analysis
Statistical Package for the Social Sciences (SPSS) 23.0 and GraphPad Prism 5.0 were used to describe and analyze the data. Rank sum tested was used to compare the rank data, and the concentrated trend and discrete trend are expressed by median (M) and quartile (QR) spacing, respectively. The measurement data are expressed as X _ ± S, the data onto normal distribution and uniform variance are analyzed by t-test and one-way analysis of variance (ANOVA), and the data that are not consistent with normal distribution are tested by rank sum test. The continuous data were analyzed by repeated analysis of variance and univariate analysis of variance. Least-significant difference (LSD) tested was used for pairwise comparison between groups. P < 0.05 was used as the standard of statistically significant difference.

Changes in general physical signs
In the first three days of the experiment, the body weight of the model mice changed little, and on the third day, mice began to develop bloody stools, loose stools, severe weight loss and reduced water consumption. On the seventh day, mice showed mental malaise, hematochezia or diarrhea, curled up in piles and dull color. The intestinal wall of the mice was swollen and congested, but no obvious ulcer was found in the naked eye. On the tenth day, the body weight of the mice slowly recovered, the drinking water returned to normal, the symptoms of diarrhea were mild, the hematochezia decreased or even disappeared, but the activity did not increase. The activity and body weight on the 14th day increased, and the coat color returned to a lighter luster. At the end of the model, after administration of oxalazine (0.6 g·kg -1 ), the weight loss of mice slowed down, followed by weight gain, coat color gradually returned to luster, and the amount of exercise increased.

Impact of disease activity index score
The feces of mice in the normal group were basically formed, and the feces of some mice were loose or weakly positive for occult blood. However, with the increase of days of drinking DSS, thin stools, bloody stools, and weight loss occurred; weight gain, stool score, occult blood score decreased after administration. The scores of DSS-induced acute UC mice increased gradually on the first, fourth, seventh, eighth, and tenth day DAI. The 10 th -day DAI score reached the peak in the experiment. Compared with the normal group, the DAI score of each model group was significantly higher than that of the normal group at different time points (P < 0.01). The DAI score of the mice in the drug group was significantly lower than that in the model group at different time periods of treatment (P < 0.01). The results are shown in Fig. 2.

Changes of colonic length and colonic mucosal injury index
The colon length of mice in the normal group changed little, but the colon length in the model group decreased gradually with the increase of days of drinking DSS, and the length of colon recovered from the eigthth day (Fig. 3a). On 0 day, the general morphology of mouse colon was normal, the mucosa was smooth, and no mucosal hyperemia, edema, erosion, and ulcer were found. With the increase of days of drinking DSS, colonic inflammation, colonic mucosal congestion and edema, intestinal wall thickening and erosion, ulcer, oval ulcer, marginal hyperemia, and edema, CMDI score also showed an upward trend. After stopping drinking DSS, it gradually recovered and reached the peak on the tenth day CMDI (Fig. 3c). Compared with the model group, when oxalazine was given for on, four and seven days, the CMDI of mice decreased significantly (P < 0 01). At the 17 th day, there was no significant difference in CMDI between the model group and each treatment group (P > 0.05), and there was no significant change in colon length from the beginning to the end (Figs. 3b and 3d).

Score of colonic histopathology in mice
As shown in Fig. 4, a small number of goblet cells were lost, and a small number of inflammatory cells infiltrated in normal mice. In the model group, with the increase of modeling days, there was a large area loss of goblet cells, different degrees of crypt defect, and a large area of inflammatory cells infiltrated into the mucous membrane and submucosa. After discontinuation of DSS, the defects of goblet cells and crypt cells gradually recovered, and the degree of inflammatory cell infiltration decreased, but it was still significantly different from that of the normal group. The HS showed that, compared with 0 day, the colonic HS of mice at each time point increased in varying degrees, and the HS of the eighth-, tenth-, 14th-, and 17th-day model mice was significantly higher than that of 0 day (P < 0.01). On the 10th day HS reached the peak, and then showed a downward trend, as shown in Table 4    Compared with the model group, the mice in the treatment group showed crypt blood stool, goblet cell repair and less inflammatory cell infiltration, which showed that the scores of HS decreased with the increase of administration days. The HS decreased significantly on the 14th day, but there was no significant difference on the 17th day, as shown in Figs. 4 and 5.  The effect of olsalazine of chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt in BALB/c mice

Changes of T cell subsets in model mice at different time points
CD3+ T lymphocytes were selected and analyzed by CD4, CD8 and CD25 staining. As shown in Fig. 6, with the increase of days of drinking DSS, the proportion of CD3+ decreased gradually (P < 0.01). Compared with the mice on the 0th day, the proportion of CD4+ gradually increased, the proportion of CD3+CD8+ cells did not change significantly, and the proportion of CD3+CD4+/CD3+CD8+ gradually increased and peaked on the 10th day. However, when drinking ultrapure water, the proportion gradually recovered. The proportion of CD3+CD4+CD25+/CD3+CD4+ decreased gradually before the 10th day, reached the lowest value on the 10th day, and then gradually recovered.

Effects of olsalazine on the contents of TNF-α, IL-1β, IL-2, IL-10 and IFN-γ in serum of ulcerative colitis mice
Compared with the normal group, the contents of TNF-α, IL-1β and IFN-γ in the serum of the model group were significantly increased, while the contents of IL-2 and IL-10 in the serum of the model group were significantly decreased (P < 0.01 or P < 0.05). Compared with the model group, the contents of TNF-α, IL-1β and IFN-γ in serum of olsalazine group decreased significantly, while the contents of IL-2 and IL-10 increased in different degrees (P < 0.01 or P < 0.05) as shown in Fig. 7.  Compared with the normal group, *P < 0.05, **P < 0.01. Compared with the model group, ∆ P < 0.05, ∆∆ P < 0.01.

Effects of olsalazine on the contents of IL-7, IL-17, IL-22, EGF and TGF in the colon of ulcerative colitis mice
Compared with the normal group, the contents of IL-17 and IL-7 in the colonic tissue of the model group increased significantly, while the contents of IL-22, TGF and EGF decreased significantly. Compared with the model group, the content of IL-7 and IL-17 in the colon of olsalazine group decreased significantly, while the contents of TGF and EGF increased significantly, as shown in Fig. 8.  Compared with the normal group, *P < 0.05, **P < 0.01. Compared with the model group, ∆ P < 0.05, ∆∆ P < 0.01.

Discussion
The purpose of this study was to investigate the therapeutic effect and mechanism of olsalazine on UC in mice. It will cause a series of immune responses when the antigen invades the intestinal mucosal barrier. Intestinal epithelial cells and dendritic cells absorb the antigen and present it to immature T lymphocytes (T-lymphocyte), stimulating T cells to proliferate and differentiate into helper T cells, effector T cells and regulatory T cells. B lymphocytes are also activated, The effect of olsalazine of chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt in BALB/c mice as well as secrete antibodies 13,14 . Activated T cells secrete a large number of cytokines, such as IL-1β, TNF-α, IFN-γ, etc., which promote the chemotaxis of neutrophils and monocytes / macrophages to gather in the inflammatory site, secrete inflammatory factors, and aggravate inflammation 15 . Some immunomodulatory cells in the body, such as Treg, Th3 and Tr1 cells, reduce the secretion of pro-inflammatory factors by IL-2 and IL-10 cells through contact inhibition in the acute stage of inflammation at the same time 16 .
The results of this experiment showed that the DAI score, CMDI score and colon HS of mice increased at first and then decreased at each time point after DSS modeling. On the 14th day, slight congestion, edema, and no obvious ulcer were observed in the colon of mice. CMDI scores and colon HS were still significantly higher than those on the first day (P < 0.01), indicating that the model was successfully replicated and stable. Colonic congestion, shortening, narrowing, and thickening of intestinal wall were caused by colonic mucosal barrier injury in mice after modeling. Colonic index, colonic CMDI scores and colonic HS in the model group were significantly higher than those in the normal group

Conclusion
Sufficient and reliable data were obtained by exploring the therapeutic effect of olsalazine on DSS-induced UC in mice in this study. It was proved that olsalazine can promote the expression of IL-2, IL-22 and IL-10 by inhibiting the expression of TNF-α, IFN-γ, IL-1β, IL-17 and IL-7 in UC mice, and accelerate the repair of intestinal injury by enhancing the expression of EGF and TGF, so as to achieve the purpose of treating UC.

Conflict of interest
Nothing to declare.

Data availability statement
All dataset were generated or analyzed in the current study.

Funding
Yunnan Provincial Major Science and Technology Special Project Grant no. 202002AA1000053