Effect of Sishen pill on expression of type I and type III interferon in acute ulcerative colitis mice model

Jia-Min Dong, Tao Lu, Ke Li, Meng-Jia Li, Xu-Dan Wang, Dong-Yu Ge, Ying Wu,2✉

1. Beijing University of Chinese Medicine, Beijing, 102488, China

2. Liuzhou People's Hospital affiliated to Guangxi Medical University, Liuzhou, 545008, China

Keywords:TypeⅠinterferon Type Ⅲ interferon Ulcerative colitis Sishen pill

ABSTRACT Objective:To investigate the effects of Sishen pill on the expression of type I interferon(IFN)and type III interferon and their receptors in colonic tissues of mice with acute ulcerative colitis(UC). Methods: Male C57BL/6Cnc mice were randomly divided into control group,model group, sishenwan group and salazosulfapyridine group. The model was made with 0.2 mL 4% dextran sodium sulfate (DSS) for 5 days, and the control group was given 0.2mL normal saline by gavage. On the second day of modeling, sishen pill group was given 0.2mL 1.5 g·kg-1 sishen pill, and SASP group was given 0.2mL 0.25 g·kg-1 sulfasalazine, twice a day, for 7 days. During the administration period, the disease activity index (DAI) of mice was calculated every day. After administration, the histopathological changes of colon tissues of mice in each group were observed by hematoxylin eosin (HE) staining, and the histological scores were calculated. The expression of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 mRNAs in colon tissues of mice in each group were detected by qRT-PCR. The expression levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 in colon tissues of mice in each group were detected by ELISA. Western blot was used to detect the expression of interferon receptors IFNAR1,IFNAR2 and IFNLR1 in colon tissues of mice in each group. Results: Compared with the control group, the DAI of mice increased significantly (P＜0.001) in the model group. The inflammatory cells in colonic tissues infiltrated heavily, lymph nodes enlarged, colonic mucosal structure destroyed, crypt structure lost, inflammation involved a wide range, and the histological score increased significantly (P＜0.001). The levels of IFN-α, IFN-β and IFNλ2 mRNA were significantly decreased (P＜0.05, P＜0.05, P＜0.01). The expression levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 were significantly decreased (P＜0.01, P＜0.01,P＜0.001, P＜0.001). The levels of IFNAR1, IFNAR2 and IFNLR1 were significantly decreased(P＜0.01, P＜0.05, P＜0.01). Compared with the model group, the DAI decreased significantly(P＜0.001) in Sishen pill group, the infiltration of inflammatory cells in colon tissue were significantly reduced, the structural regeneration of colon mucosa was significantly recovered,the crypt structure was significantly recovered, the lymph nodes were significantly reduced, the range of inflammation involvement was reduced, and the histological score was significantly reduced (P＜0.001). The levels of IFN-α, IFN-β and IFN-λ2 mRNA were significantly increased (P＜0.01, P＜0.001, P＜0.001). The levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 were significantly increased (P＜0.01, P＜0.001, P＜0.01, P＜0.001). The levels of IFNAR1,IFNAR2 and IFNLR1 were significantly increased (P＜0.05, P＜0.001, P＜0.05). Conclusion:Sishen pill may alleviate the symptoms and signs of mice with acute ulcerative colitis by regulating the expression of type I and type III interferon and their receptors in colon tissues.

1. Introduction

2. Materials and methods

2.1 Animals

40 SPF grade male C57BL/6cnc mice, 6-7 weeks old, 18-22g,purchased from Beijing Weitonglihua Experimental Animal Technology Co., LTD, with animal Certificate No. scxk (Beijing)2016-0006, were raised in the animal room of Liangxiang campus of Beijing University of Traditional Chinese medicine, with temperature of 18 ～ 22 ℃ and relative humidity of 50% ～ 60%, The animal experiment was reviewed and approved by the experimental animal ethics committee of Beijing University of Traditional Chinese medicine (BUCM-4-2020091401-3049).

2.2 Main reagents and instruments

Sishen pills (Batch No.20080016, Beijing Tongrentang (Group)Co., LTD), Sulfasalazine enteric-coated tablet (Batch No.09190911,Shanghai Xinyi Tianping Pharmaceutical Co., LTD), Low molecular sodium dextran sulfate (Batch No. CAM6550, Wako Pharmaceutical Industry Co., LTD), Qualitative test Kit for o-toluidine method fecal occultation blood (Batch No.L18S11G125170, Shanghai Yuanyue Biotechnology Co., LTD), Hematoxylin-eosin (HE) staining kit,high efficiency RIPA tissue cell lysate, BCA protein concentration determination Kit, SDS-PAGE Gel preparation kit(Beijing Solebo Technology Co., LTD), The miRNeasy Mini Kit(Qiagen), Reveraid First Strand cDNA Synthesis Kit(ThermoFisher), Power STBR®Green PCR Master Mix(Applied Biosystems), PCR primers(Sangong Bioengineering (Shanghai) Co., LTD), Mouse IL-28B (IFN lambda 3) Uncoated ELISA kit, Recombinant Mouse IL-28 R alpha/IFNlambda R1 Fc Chimera, CF(Invitrogen), VeriKineTMMouse IFN Alpha ELISA Kit, VeriKineTMMouse IFN Beta ELISA Kit(PBL Assay Science), Mouse IL-28A SimpleStep ELISA®Kit(Abcam),Mouse IFN- /β R2 Antibody(R&D), HRP-conjugated Affinipure Rabbit Anti-Goat IgG (H+L), ECL Luminescent Chemical Detection Kit(Proteintech), Rabbit Anti-Mouse IFN- /β R1 Antibody(Sino Biological Inc, Beijing, China).

Paraffin embedding machine(HistoCore Aracadia H), research grade high-resolution microanalysis system, Leica HI1210,LeicaHI1220, automatic tissue dehydrator histocore pearl, cooled histocore Arcadia C, full-automatic rotary slicer, multifunctional dyeing machine ST5020, RM 2255(Leica microsystems Co.,LTD), Cryogenic Centrifuge 5417R(Eppendorf), Multifunctional fluorescent molecular imaging system, electrophoresis instrument,electrotransfer apparatus(Bio-Rad), SpectraMax® i3x(MD),QuantStudio™ Real-Time PCR(ThermoFisher).

2.3 Methods

2.3.1 Disease activity index score of Sishen Pill in acute UC mice

Male C57BL / 6cnc mice were randomly divided into control group (Control), dextran sodium sulfate model group (DSS), Sishen pill group (SSW) and sulfasalazine group (SASP). 4% DSS was administered by gavage for modeling, 0.2 ml/d for 5 consecutive days. After the day of modeling, mice in SSW group were given 0.2ml 1.5 g·kg-1Sishen pill, mice in SASP group were given 0.2ml 0.25 g·kg-1SASP, Control group and DSS group were given equal volume normal saline, twice a day for 7 consecutive days.After modeling, the body weight of mice was recorded and the body weight fraction was calculated every day, the viscosity of feces was observed, fecal occult blood was detected by o-toluidine method, the DAI score of mice was calculated according to body weight fraction,fecal viscosity and fecal occult blood fraction.

2.3.2 Effect of Sishen Pill on pathological changes of colonic tissue in acute UC mice

After the last administration, mice were fasted for 1 day. The mice were sacrificed by neck removal, and the colon was cut out.After cleaning, the colon was removed about 3 cm and fixed with 10% neutral formalin solution for 24h, and fully immersed in tap water. Staining was performed by dehydration, paraffin embedding,sectioning, and HE staining. Observation was performed under a microscope and histological score was calculated.

2.3.3 The levels of IFN-α, IFN-β, IFN-λ2 and IFNλ3 mRNAs in colon tissues of mice were detected by qPCR

And after Catherine had gone her way her lady s Destiny went to find her sister, and said to her, Dear sister, has not Catherine suffered enough? It is surely time for her good days to begin? And the sister answered, To-morrow you shall bring her to me, and I will give her something that may help her out of her need

100 mg colon tissue was added to liquid nitrogen for grinding, and tissue homogenization was carried out with a homogenizer. RNA was extracted according to the instructions of miRNeasy Mini Kit,then RNA concentration was determined, and cDNA was prepared by reverse transcription. Establishment of 20 μL qPCR reaction system, Power SYBR Green PCR Master Mix 10 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, high purity water without nuclease 7.2 μL, cDNA (5 times diluted) 2 μL.95 ℃ for 10 min, and 95 ℃ 5 s, 60 ℃ 30 s, 40 cycles, 95 ℃ for 15 s, 60 ℃ for 1 min, 95 ℃ for 15 s.

Table 1 Sequence primers

2.3.4 The levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 in colon tissues were detected by ELISA

100 mg colon tissue was added to 1ml normal saline for grinding,transfer the grinding solution to 1.5ml EP tube, placed on ice for 20 min, shaken every 5 min, centrifuged at 12000 rpm for 5 min, and the supernatant was absorbed. Protein concentration was determined by BCA method, ELISA kit was used to determine the expression levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3.

2.3.5 The expression of IFNAR1, IFNAR2 and IFNLR1 in colon tissues were detected by Western Blot

Cut a small section of colon tissue, add high-efficiency RIPA tissue and cell lysate, fully lyse the tissue, then suck the tissue lysate into 1.5 ml EP tube and centrifuge at 12000 rpm for 5 min. Absorb the supernatant, determine the protein concentration by BCA and denature it. The protein samples were electrophoretic with SDSPAGE gel, then closed for 1 h and added to IFN- /β R2, IFNLR1,IFN- /β R1, incubate overnight at 4 ℃, wash with PBST for three times, 5-10 min/time, add secondary antibody, incubate for 1 h, wash with PBST for three times, 5-10 min/time. Add ECL luminescent solution and expose it with multifunctional fluorescent molecular imaging system.

2.3.6 Statistical method

The data were processed by Graphpad Prism 8.0.2, and the differences among groups were compared by one-way ANOVA.When the variances were uneven, the differences among groups were compared by tamhance's T2 test. When the variance is homogeneous, LSD test was used to compare among the groups.P＜0.05 was considered statistically significant. Compared with Control mice,*P＜0.05,**P＜0.01,***P＜0.001, compared with DSS mice,#P＜0.05,##P＜0.01,###P＜0.001.

3. Results

3.1 Sishen pill can reduce DAI level in UC mice

Compared with DSS group, the body weight fraction of mice treated with Sishen pill and SASP decreased significantly (P＜0.001,P＜0.001), the diarrhea score decreased significantly (P＜0.001,P＜0.01), the hematochezia score decreased significantly (P＜0.01,P＜0.01). Compared with the control group, DAI of mice in DSS group increased significantly (P＜0.001), and the DAI of mice in Sishen pill group and SASP group also increased significantly(P＜0.001, P＜0.001). After treatment with Sishen pill or SASP, the DAI of mice decreased significantly (P＜0.001, P＜0.001)(Table 2 and Figure 1).

Figure 1 Changes of DAI of mice in each group

3.2 Sishen pill can restore the pathological changes of colon tissue

He staining of colonic tissues showed that Sishen pill could reduce inflammatory cell infiltration, restore colonic mucosa and recess structure, shrink lymph nodes and effectively reduce tissue lesions.Compared with the control group, the histological scores of colon tissues in DSS group, Sishen pill group and SASP group were significantly increased (P＜0.001, P＜0.01, P＜0.001). After treatment with Sishen pill or SASP, the histological scores of colon tissue decreased significantly (P＜0.001, P＜0.001)(Table 3 and Figure 2).

Table 2 Changes of disease activity index of mice in each group (±s)

Table 2 Changes of disease activity index of mice in each group (±s)

Group n Weight Score Diarrhea Hematochezia DAI Control 10 0.71±0.58 0 0 0.76±0.52 DSS 10 3.06±0.59*** 1.72±0.59*** 2.36±1.00*** 5.48±1.43***SSW 10 1.46±0.52### 0.74±0.30### 0.96±0.46## 2.82±0.85###SASP 10 1.82±0.50### 0.77±0.41## 0.93±0.24## 2.89±0.46###Statistical value F=25.6 F=26.29 F=23.93 F=36.82 P value 0.000 0.000 0.000 0.000

Table 3 Changes of histological score of mice in each group (±s)

Table 3 Changes of histological score of mice in each group (±s)

Group n HE Stain Control 10 0 DSS 10 12.5±0.93***SSW 10 1.00±0.89###SASP 10 1.29±0.76###Statistical value F=563.6 P value 0.000

Figure 2 HE staining of mice in each group (×200)

3.3 Effects of Sishen pill on expression of IFN-α, IFN-β, IFNλ2 and IFN-λ3 mRNAs in colon tissue of acute UC mice

Compared with the control group, the levels of IFN-α, IFN-β and IFN-λ2 mRNA in colon tissue of DSS group decreased significantly (P＜0.05, P＜0.05, P＜0.01), but the levels of IFN-λ3 mRNA decreased without statistical significance (P＞0.05), the levels of IFN-α, IFN-β and IFN-λ2 mRNA in Sishen pill group increased (P＜0.01, P＜0.001, P＜0.001), while the IFN-λ3 mRNA levels were increased but not statistically significant (P＞0.05), the levels of IFN-α, IFN-λ2 and IFN-λ3 mRNA in SASP group increased significantly (P ＜0.001, P＜0.01, P＜0.05), but the IFN-β mRNA levels were not statistically significant (P＞0.05). After treatment, compared with DSS group, Sishen pill group had higher IFN-α, IFN-βand IFN-λ2 mRNA levels increased significantly(P＜0.01, P＜0.001, P＜0.001), but there was no statistical significance in the increase of IFN-λ3 mRNA levels (P＞0.05), the mRNA levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 in SASP group were increased (P＜0.001, P＜0.05, P＜0.01, P＜0.05) (Table 4).

Table 4 Changes of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 mRNAs of mice in each group (±s)

Table 4 Changes of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 mRNAs of mice in each group (±s)

Group n IFN-αmRNA IFN-βmRNA IFN-λ2 mRNA IFN-λ3 mRNA Control 3 1 1 1 1 DSS 3 0.40±0.26* 0.88±0.05* 0.69±0.07** 0.35±0.50 SSW 3 2.37±0.52## 3.69±0.11### 3.82±0.55### 3.76±2.34 SASP 3 12.46±0.14### 12.19±7.03# 10.08±2.23## 5.45±2.57#Statistical value F=1097 F=6.902 F=42.95 F=5.531 P value 0.000 0.013 0.000 0.024

3.4 Effects of Sishen pill on the levels of IFN-α, IFN-β,IFN-λ2 and IFN-λ3 in colon tissue of acute UC mice

ELISA method was used to detect the expression of IFN-α,IFN-β, IFN-λ2 and IFN-λ3 in colon tissue homogenate supernatant, it was found that the levels of IFN-α, IFN-β, IFNλ2 and IFN-λ3 in DSS group were significantly lower than control group (P＜0.01, P＜0.01, P＜0.001, P＜0.001), the levels of IFN-β in Sishen Pill group were increased (P＜0.01), but there was no statistical significance in the levels of IFN-α, IFN-λ2 and IFNλ3, the levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 in SASP group were significantly increased (P＜0.001, P＜0.01, P＜0.001,P＜0.001). After treatment, compared with DSS group, Sishen Pill group had higher IFN-α, IFN-β and IFN-λ2 and IFN-λ3 levels increased significantly (P＜0.01, P＜0.001, P＜0.01, P＜0.001), the levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 in SASP group were significantly increased (P＜0.001, P＜0.01, P＜0.001, P＜0.001)(Table 5).

Table 5 Changes of IFN- , IFN-β, IFN-λ2 and IFN-λ3 of mice in each group (±s)

Group n IFN-α IFN-β IFN-λ2 IFN-λ3 Control 3 67.96±2.94 78.85±2.67 76.38±3.00 233.70±20.08 DSS 3 58.74±1.25** 41.00±19.11** 15.02±5.79*** 56.86±14.37***SSW 3 74.49±5.32## 135.90±23.76### 64.59±19.62## 242.80±32.76###SASP 3 78.39±1.17### 212.60±61.87## 102.00±5.27### 390.80±31.86###Statistical value F=29.60 F=18.72 F=46.86 F=110.7 P value 0.000 0.000 0.000 0.000

3.5 Effects of Sishen pill on expression of IFNAR1, IFNAR2 and IFNLR1 in colon tissue of acute UC mice

Compared with the control group, the levels of IFNAR1, IFNAR2 and IFNLR1 in DSS group decreased significantly (P＜0.01, P＜0.05,P＜0.01), the level of IFNAR2 increased in Sishen pill group(P＜0.001), while the levels of IFNAR1 and IFNLR1 increased without statistical significance, the levels of IFNAR2 and IFNLR1 increased in SASP group (P＜0.001, P＜0.05), but there was no significant increase in IFNAR1 level. After treatment, the levels of IFNAR1, IFNAR2 and IFNLR1 in Sishen pill group and SASP group were significantly higher than those in DSS group (P ＜ 0.05,P ＜ 0.001, P ＜ 0.05)(Table 6 and Figure 3).

Table 6 Changes of IFNAR1, IFNAR2 and IFNLR1 of mice in each group (±s)

Table 6 Changes of IFNAR1, IFNAR2 and IFNLR1 of mice in each group (±s)

Group n IFNAR1 IFNAR2 IFNLR1 Control 3 0.95±0.06 0.52±0.05 1.04±0.06 DSS 3 0.60±0.11** 0.37±0.03* 0.57±0.14**SSW 3 1.14±0.20# 0.84±0.03### 1.41±0.36#SASP 3 0.93±0.08# 1.18±0.01### 0.90±0.05#Statistical value F=10.46 F=338.50 F=9.275 P value 0.003 0.000 0.005

Figure 3 Expression levels of IFNAR1, IFNAR2 and IFNLR1 in colon tissues of mice in each group

4. Discussion

UC is a spontaneous chronic disease that has been described as beginning in the rectum and extending into the adjacent colon as a continuous lesion. With the continuous improvement of people's living standards, the change of living environment, living habits and diet structure, the incidence rate of UC in China is increasing year by year. UC has a long course of disease, and conventional drugs (such as aminosalicylic acid drugs, immunosuppressants and steroids)can only relieve symptoms and are difficult to cure completely. In addition, some patients have adverse reactions to conventional drug treatment, which is easy to develop into refractory UC[14]. Therefore,seeking more effective methods to treat UC has become a top priority.

Intestinal microbes, intestinal mucus layer, intestinal epithelium and immune cells interact to maintain gastrointestinal homeostasis[15,16].In UC, this dynamic balance is completely broken. The integrity of intestinal epithelium was decreased and the permeability of mucosal tissues increased in UC patients[17], the innate immune system is disordered, and the overexpression of many inflammatory cytokines aggravates inflammatory damage, such as IL-1β, IL-8, TNF-α,IL-6, IL-23, IL-12, IL-17, etc[18]. At the same time, the balance of T cells is also broken, and the excessive increase of helper T cells(Th1 and Th2) leads to the imbalance in the number of Th1 and Th2 cells and regulatory T cells (Treg)[19]. Activated T cells produce and release inflammatory factors, further aggravating persistent inflammation[20]. Recent studies have found that interferon system also plays an important role in the process of UC. Interferon is a cytokine found to interfere with virus replication[21], which is divided into type I, type II and type III IFN. Type I IFN, including IFN and IFNβ, functions through heterodimer receptor (IFN βR), activating JAK/STAT signaling pathway and forming interferon stimulating gene factor (ISGF)3[22]. Under normal conditions, the content of type I IFN in intestine or other tissues is very low[23]. Studies have shown that type I IFN signaling has a two-sided effect on UC,the symptoms of colitis in mice with type I IFN signal deficiency are aggravated, and the expression of type I IFN can maintain the intestinal immune balance[7]. Other studies have found that increased type I IFN aggravates intestinal inflammation[6,24]. Type II interferon(IFN-γ) mainly plays a pro-inflammatory role and destroys the integrity of colonic mucosa, leading to the occurrence of UC[25].Type III IFN (including IFNλ1, 2, 3, 4) uses the same JAK/STAT signaling pathway as Type I IFN[8], but its receptors are mainly composed of IFN-λR1 and IL-10R2 subunits[26]. Compared with type I IFN, type III IFN may interact more closely with intestinal epithelial cells in the transmission of immune signals. IFN-λ can reduce the excessive inflammatory response without weakening the body's defense function. Rauch et al. reported that after DSS induced colitis, the expression of CXCL10 in mice without type III interferon receptor was significantly upregulated compared with normal mice,which resulted in significant weight loss in mice and aggravated intestinal inflammatory damage[27].

In the theory of traditional Chinese medicine, UC is mostly caused by exogenous pathogenic factors, unclean diet, improper diet, internal emotional injury and deficiency of spleen and kidney,resulting in Qi stagnation, blood stasis, damp heat, phlegm turbidity and so on. UC is based on deficiency of spleen and kidney yang and marked by damp heat and blood stasis[10]. The treatment principle of spleen kidney yang deficiency syndrome is to warm and tonify the spleen and kidney, collect astringency and solidify detachment, and it is mostly treated with Sishen pill[28,29]. Many studies have shown that Sishen pill can alleviate the symptoms and signs of UC patients and control inflammation[30,31,32]. Sishen pill can effectively reduce the levels of protein kinase B (Akt), phosphatidylinositol-3 kinase(PI3K) and target of rapamycin (mTOR) mRNAs in colon tissues of UC rats with spleen and kidney Yang deficiency, can increase the expression level of IL-10, decreased the expression levels of p-PI3K,p-mTOR and p-Akt proteins[29]. Sishen pill can effectively reduce the expression level of IL-1β, increase the expression levels of IL-4 and IL-13, indicating that the immune mechanism of Sishen pill in UC rats may be related to regulating the immune balance of Th1/Th2, and inhibiting excessive immune response[33]. In this study,Sishen pill was used to treat DSS induced acute UC model in mice,which achieved good results and confirmed the therapeutic effect of Sishen pill. However, the immunological role of interferon system in the treatment of UC by Sishen pill is not clear at present. Our study found that the expression levels of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 and their receptors IFNAR1, IFNAR2 and IFNLR1 in type I and type III interferon pathways were significantly reduced in the colon tissues of the model group. These results suggest that DSS-induced acute mice UC model may inhibit type I and type III IFN signaling pathways, leading to abnormal immune regulation,and thus aggravating inflammatory injury of colonic mucosa. After treatment with Sishen pill, the expression levels of the above factors increased significantly, indicating that Sishen pill may regulate the immune response of the colon mucosa of mice by promoting the expression of type I and type III IFN and their receptors, thus inhibiting the inflammatory damage caused by excessive immune response and restoring the normal colonic mucosal structure of mice.When Sishen pill was used to treat acute UC in mice, the expression changes of type I and type III IFN alleviated the symptoms and signs of UC mice, reduced inflammatory damage and made UC mice tend to recover. This finding lays a foundation for further exploring the role of interferon signaling pathway in the treatment of UC by Sishen pill.

All authors' Conflict of interest Statements: All authors declare that there is no conflict of interest.

Author contribution: Dong Jiamin, Li Ke and Li Mengjia carried out experimental operation, collected and sorted out data and wrote articles. Lu Tao, Wang Xudan, Ge Dongyu, Wu Ying gave guidance to experimental design, data analysis and statistics, drawing and article writing.