Effects of Wenglian Jiedu decoction on expression of serum IL-18,TNF-α and MUC2 in colon tissue of ulcerative colitis rats

Yu-Ting Lu, Gui-Ye An, Xiao-Yan Zhang, Qian Yang, Yong-Li Huo✉, Dian-Gui Li

1. Hebei College of Traditional Chinese Medicine, Shijiazhuang 050000, China

2. Department of Spleen and Stomach Diseases, the First Affiliated Hospital of Hebei College of Traditional Chinese Medicine., Shijiazhuang 050000,China

Keywords:Wenglian Jiedu decoction Mesalazine Interleukin-18 Tumor necrosis factor-α Mucin-2

ABSTRACT Objective: To study the effect of Wenglian jiedu decoction on the expression of serum interleukin-18 (IL-18), tumor necrosis factor-α (TNF--α) and colonic mucin-2 (MUC2)in rats with ulcerative colitis of turbid toxin type, and to explore its mechanism. Methods:60 Wistar male rats were randomly divided into model group, Wenglian jiedu decoction low, middle and high dose groups, mesalazine group and blank group. Except for the blank group, the rat model of ulcerative colitis was established by trinitrobenzenesulfonic acid(TNBS) / ethanol compound method. The general condition, body weight, fecal properties and occult blood of rats were observed 2 weeks after administration. The disease activity index (Diseaseactivityindex,DAI) was scored, the content of MUC2 protein was detected by immunohistochemical method, and the contents of IL-18 and TNF- α in serum were detected by enzyme-linked immunosorbent assay (Elisa). Results: compared with the blank group,the DAI score increased, the contents of IL-18 and TNF-α increased significantly, and the content of MUC2 protein decreased in the model group. Compared with the model group,the DAI score, the content of IL-18 and TNF- α decreased and the content of MUC2 protein increased in each treatment group. Compared with the mesalazine group, the DAI score and the contents of IL-18 and TNF-α in the high and middle dose groups had no significant difference. Compared with the low dose group, the DAI score in the mesalazine group and the high and middle dose group decreased, and the contents of IL-18 and TNF- α decreased.Conclusion: Wenglian jiedu decoction can regulate the immune system of intestinal epithelium by regulating the levels of serum IL-18 and TNF- α and increasing the expression of MUC2 protein, so as to achieve the purpose of repairing intestinal mucosa.

1. Introduction

Ulcerative colitis (ulcerativecolitis,UC) is an inflammatory intestinal disease, which is characterized by chronic onset,procrastination and recurrence. It has been listed as a modern refractory disease by the World Health Organization [1]. The clinical symptoms of most patients are abdominal pain, diarrhea and mucous purulent and bloody stool. The incidence of this disease is high in western countries. According to epidemiological statistics,the incidence and prevalence rate of UC in China is also increasing year by year. At present, aminosalicylic acid drugs and steroids are mainly used in the treatment of UC by western medicine [2],but there are serious side effects. High recurrence rate and other shortcomings. Traditional Chinese medicine has played its unique advantage in the diagnosis and treatment of UC. Professor Li Shaogui, a master of traditional Chinese medicine, summed up more than 50 years of experience and gradually established the theory of turbid toxin [3]. Through clinical observation, turbid toxin was the initiating factor of UC, and the syndrome of internal accumulation of turbid toxin was its main pathogenesis [4]. Wenglian jiedu decoction was made to treat UC. Previous studies showed that its clinical effect was significant [5]. In addition, immune factors play an important role in the pathogenesis of UC, in which cytokines,tumor necrosis factor and the pathogenesis of UC have an important relationship. The purpose of this study was to investigate the effects of Wenglian jiedu decoction on the expression of IL-18 and TNFαin serum and MUC2 in colon tissue of UC rats.

2. materials and methods

2.1 Experimental animals

A total of 60 clean-grade Wistar male rats (animal certificate number: 2008220) with a body mass of (200 ±20) g were purchased in the Animal Center of Hebei Medical University. The scheme and operation procedure involved in this experiment have been approved by the Ethics Committee of Hebei College of traditional Chinese Medicine.

2.2 Experimental drugs and preparation

Wenglian jiedu decoction was composed of Huanglian 6g,Baitouweng 15g, Sophora flavescens 6g, dandelion 15g, Rhodiola,Radix Paeoniae Alba, Radix Paeoniae Alba, Phellodendron,Cortex Phellodendri, Cortex Sanguisorba, Ulmus officinalis,Pueraria lobata, tangerine peel, Poria cocos 15g, fried Atractylodes macrocephala 6g, Coix seed 20g, licorice 6g. Traditional Chinese medicine is the formula decoction-free granule (Guangdong Party Pharmaceutical Co., Ltd.). The decoction-free granules of traditional Chinese medicine were stirred and dissolved in aseptic drinking water at 80 ℃. According to the equivalent dose ratio of body surface area between human body and rats, the daily doses of rats in high, middle and low dose groups were 43.8g, 21.9g and 10.95g per kilogram body weight, respectively. The corresponding doses of decoction-free particles were diluted into 2ml aseptic drinking water suspension with the corresponding concentrations of 5.92g/ml, 2.96g/ml and 1.48g/ml, respectively. The prepared solution was stored in refrigerator at 4410.Mesalazine enteric-coated tablets (Huidi manufacturer is sunflower pharmaceutical group Jiamusi Luling Pharmaceutical Co., Ltd.,specification: 0.25g, batch number: 201222). According to the equivalent dose ratio, the dosage of mesalazine enteric-coated tablets is 0.36g/ (kg ·d), add 2ml aseptic drinking water into 0.045g/ml concentration solution, label notes, put refrigerated storage.

2.3 main reagents and instruments

2.3.1 Kit

Quantitative enzyme-linked detection kit for IL-18 and TNFα(batch number: 20180319, Shanghai Senxiong Science and Technology Industry Co., Ltd.); ABC immunohistochemical kit(PK-4000,V ector Laboratories,Inc.); hematoxylin eosin (HE)staining kit (serial number: C0105, Solebo Technology Co., Ltd.);trinitrobenzenesulfonic acid (batch number: SL-BG2566V, sigma Co., Ltd.)

2.3.2 testing instrument

Enzyme-linked immunosorbent assay (MK3FC,ThomerFisher);microscopic image analysis system (HMIAS-2000, Wuhan Qianping Image Technology Co., Ltd.); Gel Imaging system (LeicaDFC295,Olympus, Japan); High-speed freezing centrifuge (115KDemery Thermo); paraffin slicer (Leica, Germany); Optical microscope(Zeiss Optical Instruments (Shanghai) Co., Ltd.)

2.4 Modeling and grouping

60 rats were randomly divided into blank group and model group,high, middle and low dose groups of traditional Chinese medicine,and mesalazine group, with 10 rats in each group. The UC rat model was induced by TNBS--ethanol compound. The rats were adaptively fed for one week. The general condition, body weight,fecal characteristics and occult blood of the rats were recorded before modeling. The rats began to make models 24 hours after fasting (during the period of fasting), and the rats were anesthetized with ether before the modeling began. After complete anesthesia,according to the dose of TNBS100mg/kg, TNBS- ethanol solution of 0.6mL was injected into the anus for enema. After the colonic mucosa was fully contacted with the mixture, the rats were placed in a cage. At the same time, the blank group was injected with the same volume of normal saline through the anus. After the establishment of the model, all the rats were waiting for the rats to wake up, and all the rats returned to normal diet and drinking water after awakening.After 3 days of modeling, the changes of fecal characteristics of rats were observed, such as sparse stool and purulent blood. 2 rats were randomly selected (model group and high-dose traditional Chinese medicine group) and killed, and the colonic tissue was dissected to observe the congestion and edema of colonic mucosa and the formation of ulcer to confirm the successful establishment of UC model. At the end of the experiment, one rat died in the high-dose group, the middle-dose group and the mesalazine group, of which 2 were caused by accidental infusion of gastric juice into the trachea,and the cause of death was unknown.

2.5 Animal administration

On the 4th day, the rats in the western medicine group were given mesalazine 0.36g/kg, the low, middle and high dose Wenglian jiedu decoction groups were given 10.95g/kg, 21.9g/kg and 43.8g/kg respectively, and the blank group and model group were given the same volume of normal saline once a day for 2 weeks.

2.6 Collection and treatment of specimens

After the last administration, the rats fasted for 24 hours, during which water could not be avoided. All rats were anesthetized with chloral hydrate, and the blood 4ml was quickly taken from the right femoral artery, coagulated and centrifuged for 15 minutes. The supernatant was marked and stored in the refrigerator at-20 ℃. The rats were killed, cut along the longitudinal axis of the mesentery, and the 8cm colon tissue above the anus was removed, the intestines were washed with normal saline, and the water was absorbed by the filter paper. after naked eye observation, the rats were sealed and protected from light in the paraformaldehyde fixed solution of encoded 40g/L. 24 hours later, the lesions were cut, paraffin embedded sections,routine HE staining, sealing slices, and microscopic observation.

2.7 Index detection

2.7.1 The general status and DAI score of rats in each group were observed

Feces were collected after anal disinfection, and the occult blood of feces was measured by o-toluidine method [8]. According to the DAI score standard [9], DAI= (body mass decline score + stool trait score+ hematochezia score) / 3. See Table 1.

Table 1 standard for evaluation of DAI in rats

2.7.2 The colonic tissue of rats was observed under microscope after routine HE staining

And the structure of colon was observed under light microscope.

2.7.3 biochemical indicators were detected according to the standards specified in the kit instructions

The serum contents of IL-18 and TNF- α were detected by enzyme linked immunosorbent assay (Elisa), the protein content of MUC2 in colon tissue was detected by immunohistochemical method, and the average photometric value was measured in cell measurement mode.2.8 Statistical methods SPSS23.0 software was used to analyze the data. The measurement data are expressed as "". The data were tested for normality and homogeneity of variance. If the normality and homogeneity of variance were satisfied, one-way analysis of variance ((One-WayANOVA)) was used for comparison between groups, and rank sum test was used for heterogeneity of variance. It was considered that there was statistical difference according to the standard of P ＜ 0.05.

3. Results

3.1 General condition of rats

The rats in the blank group have always been energetic, responsive,normal diet, significantly increased body mass, shiny hair, no loose stool, no purulent blood and mucus in stool. After the establishment of the model, the rats in the other groups showed reduced food intake, weight loss, lying still and less movement, poor spirit,unformed stool with purulent and bloody stool and so on. After intragastric administration, the activity, appetite and body mass of the rats in each treatment group began to increase, and the general condition of the rats in the high and middle dose groups and mesalazine group was significantly improved. the general condition of the rats in the model group was always worse, depressed in spirit,dull hair, reduced diet, decreased physique and perianal filth.

3.2 Results of rat DAI score

Compared with the blank group, the DAI score of rats in the model group and each administration group increased, while the DAI score in the low, middle and high dose groups and mesalazine group decreased compared with the model group, but there was no significant difference between the high and middle dose groups and the mesalazine group. In the administration group, the DAI score was the highest in the low dose group. See Table 2.

Table 2 Comparison of Dai scores of rats in each group (score,±s)

Table 2 Comparison of Dai scores of rats in each group (score,±s)

Compared with blank group, *P＜0.05;Compared with model group,△P＜0.05;Compared with mesalamine group, ◇P ＞ 0.05;Compared with the low dose group, #P＜0.05

groupnDAI score The blank group100.00±0.00 Model group93.28±0.49*Chinese medicine high dose group91.56±0.44*△◇#Chinese medicine medium dose group91.59±0.52*△◇#Chinese medicine low dose group102.81±0.44*△Mesalamine group F91.62±0.50*△#309.790

3.3 pathological observation of colon tissue

After the pathological sections were stained by HE, the clear structure of colonic mucosa, normal morphology of glands and crypts, regular arrangement, abundant goblet cells and no inflammatory cell infiltration were observed in the blank group.Compared with the blank group, the glands in the model group were seriously damaged, the structure of the crypt was disordered, a large number of goblet cells were lost, and infiltrating inflammatory cells could be seen in the mucous membrane and submucosa. Compared with the model group, the colonic mucosa of rats in high dose group, middle dose group and mesalazine group were repaired and improved, the glandular structure was basically intact, and the infiltration of inflammatory cells was significantly reduced. In the low dose group, the structure of glandular mucosa was slightly less clear, and a small amount of inflammatory cell infiltration could be seen. Figure 1

Figure 1 histopathological changes in each group (HE staining,× 200)

3.4 biochemical index test results

3.4.1 ELISA test resultsCompared with the blank group, the contents of IL-18 and TNFαin the model group were significantly higher than those in the blank group. Compared with the model group, the contents of IL-18 and TNF- α in all treatment groups decreased, but the contents of IL-18 and TNF- α in high dose group, middle dose group and mesalazine group were lower than those in low dose group (P ＜0.05). See Table 3

Table 3 IL-18 and TNF- of rats in each group α Comparison (pg/ml,±s )

Compared with blank group, *P＜0.05;Compared with model group, △P＜0.05;Compared with mesalamine group, ◇P ＞ 0.05;Compared with the low dose group, #P＜0.05.

GroupnIL-18TNF-α The blank group10142.31±27.3313.70±2.23 Model group9199.42±70.32*20.51±4.25*Chinese medicine high dose group9172.85±22.82*△◇#14.69±4.43*△◇#Chinese medicine medium dose group9174.38±31.97*△◇#14.86±5.37*△◇#Chinese medicine low dose group10181.79±29.46*△16.13±3.18*△Mesalamine group F9173.15±23.50*△#14.54614.55±3.15*△# 39.495

3.4.2 Immunohistochemical results

Compared with the blank group, the expression level of MUC2 in colon epithelial tissue of rats in model group was significantly decreased (P＜0.05);Compared with model group, the expression level of MUC2 in all treatment groups was increased (P＜0.05);There was no significant difference between the high dose and medium dose groups and the mesalazine group (P ＞ 0.05). The expression level of MUC2 in the low dose group was lower than that in the other groups, and the distribution range of positive cells was lower than that in the other groups (P＜0.05).See Table 4 and Figure 2.

Table 4 Comparison of MUC2 protein integral optical density (IOD) in each group of rats(±s)

Table 4 Comparison of MUC2 protein integral optical density (IOD) in each group of rats(±s)

Compared with blank group, *P＜0.05;Compared with model group,əP＜0.05;Compared with mesalamine group, ЖP ＞ 0.05;Compared with the low dose group, #P＜0.05.

GroupnMUC2 The blank group1020853.12±842.16 Model group98469.28±1035.49*Chinese medicine high dose group916996.39±868.28*△◇#Chinese medicine medium dose group 916895.16±869.39*△◇#Chinese medicine low dose group1014369.25±907.35*△Mesalamine groupF9 16495.69±813.55*△#228.736

Figure 2 Effect of Weng Lian Jie Du Decoction on expression of MUC2 protein in colonic epithelium of rats with ulcerative colitis(×200)

4. Discussion

UC is a chronic and idiopathic intestinal inflammatory disease.Because the pathogenesis of UC is not completely clear, there is no specific treatment for this disease. Modern medicine is mainly composed of hormones, aminosalicylic acid preparations and immunosuppressants. Mesalazine belongs to aminosalicylic acid preparation, which is widely used in the treatment of mild to moderate UC. Although the curative effect is worth affirming, there are many adverse reactions, easy to repeat and other problems,which reduce the quality of life of patients. Traditional Chinese medicine has a multi-system, multi-target overall regulatory role in the treatment of UC, and has its unique advantages in the clinical treatment of UC patients. In particular, the compound preparation of traditional Chinese medicine plays an important role in regulating immunity, antioxidation, regulating inflammatory factors, etc.,so that the condition tends to be stable and the curative effect is enhanced [10].

From ancient times to the present, various physicians'understanding of the disease has been continuously improved.Traditional medicine classifies the disease as "intestinal opening","stagnation", "intestinal wind" and "dysentery". As early as in the Internal Classic of Huangdi, there is a discussion on the etiology and pathogenesis of "intestinal opening". The book holds that the etiology and pathogenesis of "intestinal opening" is "disharmony between yin and yang, evil qi and yin". The incidence of this disease is mainly related to improper diet, emotional disorders, weakness of spleen and stomach, blood collaterals and intestinal collaterals[11]. Professor Li Shaogui, a master of modern Chinese medicine,pioneered the theory of "turbid toxin" and understood ulcerative colitis from the point of view of turbid toxin. He believed that turbid toxin was the main pathogenesis of UC, the lesion was located in the intestine, and mainly damaged the intestinal collaterals [12]. Its development mechanism was that factors such as feeling external evil, or unclean diet acted on the weak body of spleen and stomach,the movement of spleen and stomach was powerless, resulting in dampness-heat generating turbid evil, cementing in intestinal viscera, and becoming poison for a long time. Then the evil of turbid toxin damages the blood collaterals of intestinal lipid membrane,turns into pus, and ulcers inside [13]. According to the data statistics,the distribution of TCM syndromes of ulcerative colitis in Beijing-Tianjin-Hebei region is mainly turbid toxin internal accumulation syndrome [14]. Wenglian jiedu decoction was established based on the pathogenesis of turbid toxin, with the treatment of resolving turbidity and detoxification, cooling blood and stopping dysentery.The prescription group consists of Coptis chinensis, Pulsatilla chinensis, Sophora flavescens, dandelion, Rhodiola, Rhizoma Cortex, Radix Paeoniae Alba, Fangfeng, Cortex Phellodendri, Cortex officinalis, Ulmus officinalis, Pueraria lobata, tangerine peel, Poria cocos, fried Atractylodes macrocephala, Coix seed and licorice.Coptis chinensis has the function of clearing heat, dryness and stopping diarrhea, and berberine, the main chemical component of Coptis chinensis, has obvious anti-inflammatory effect, which can balance pro-inflammatory cells and anti-inflammatory cells. some studies have shown that berberine has a good therapeutic effect on colonic inflammation, and its widespread use in Chinese population also shows that it has a good tolerance. Pulsatilla chinensis has the effect of clearing heat and detoxification, eliminating carbuncle and dispelling carbuncle. Among them, in the pharmacological research of traditional Chinese medicine, the saponins in Pulsatilla chinensis have the potential of regulating pro-inflammatory factor and tumor necrosis factor-α, and have immunomodulatory effect, inhibit the proliferation of cancer cells and induce their apoptosis at the same time, so it can effectively inhibit the risk of carcinogenesis of UC which can not be cured repeatedly. Sophora flavescens, turbid detoxification, dampness and dampness, Phellodendron flavescens,Rhizoma Atractylodis and Coix seed are used together to clear the damp-heat of the intestine; Poria cocos, Atractylodes macrocephala and Coix seed are used to remove spleen dampness, invigorate spleen and stop diarrhea; dandelion and big blood vine clear heat,detoxify and activate blood circulation, not only clear large intestine turbid toxin, but also activate blood circulation and stop dysentery;Rhodiola can activate blood stasis, detoxify and relieve pain, so that intestinal blood stasis can be dissipated, spleen and stomach can be transported, turbid toxin can not be contained. Ground elm clearing heat and cooling blood, astringent hemostasis, compatible with Pulsatilla chinensis, astringent in both, is one of the commonly used drugs for the treatment of blood dysentery; Qinpi slightly bitter,slightly astringent, cold, clearing heat and astringent intestines to stop dysentery; Pueraria flavour Xin Shengfa, can increase the qi of Qingyang, encourage the qi of spleen and stomach, and play the function of stopping diarrhea. Fangfeng, as a wind medicine, can be used, can be made, can be introduced, Atractylodes macrocephala can remove dampness and stop diarrhea, white peony soften the liver to relieve pain, need to float with the help of wind medicine, help the growth trend, remove turbidity and detoxification, and grill licorice to reconcile all kinds of drugs. All kinds of medicines are used together to cool the blood and stop dysentery, dissolve turbid and detoxify.

The occurrence of UC is the result of immune disorder, host susceptibility and other factors, and the persistent chronic inflammation of the intestinal tract is an important feature. The key factor in the pathogenesis of UC is the release of a large number of pro-inflammatory cytokines such as interleukin and tumor necrosis factor-α [19]. IL-18 and TNF- αare recognized as pro-inflammatory factors, which play a key regulatory role in immune response. Under normal conditions, anti-inflammatory and pro-inflammatory factors are in a state of dynamic balance. If the secretion of IL-18, TNF and other pro-inflammatory factors increases, this dynamic balance will be broken, and the body will start the immune system, mediate the release of a large number of pro-inflammatory factors, inhibit the apoptosis of inflammatory cytokines, and produce inflammatory and toxic reactions, thus forming ulcers. It is through the overall regulation of multi-targets and multi-systems that traditional Chinese medicine plays an important role in maintaining its dynamic balance[5]. The intestinal mucosal layer has a direct protective effect on the intestinal tract, and the MUC2 in the mucosal layer has a direct protective effect on the external antigenic defense. Therefore, the expression of MUC2 is decreased, the mucosal barrier function is damaged, and intestinal antigen and endotoxin can induce immune response and form inflammation [20].

The results showed that compared with the model group, the secretion of IL-18 and TNF- α decreased and the expression of MUC2 protein increased in each dose group, and there was no significant difference between the high and middle dose groups and the mesalazine group, and the content of IL-18 and TNF- α in the low dose group was the highest and the expression of MUC2 protein was the lowest in the treatment group. The results showed that different doses of Wenglian jiedu decoction had different therapeutic effects on UC. The curative effects of high and middle dose groups of traditional Chinese medicine were better than those of low dose groups, but there was no significant difference between high dose group and middle dose group, indicating that when the dose of traditional Chinese medicine reached a certain blood concentration,the curative effect of Wenglian jiedu decoction did not increase with the increase of dose, which provided a basis for clinical application.There was no significant difference in curative effect between high dose group and mesalazine group, but a large number of clinical trials showed that mesalazine had obvious adverse reactions in liver,kidney, gastrointestinal tract and other systems. under the same effect of traditional Chinese medicine group and mesalazine group,the traditional Chinese medicine group has the advantages of low recurrence rate and small adverse reactions.

To sum up, Wenglian jiedu decoction can significantly reduce the contents of IL-18 and TNF- α in UC model rats, up-regulate the expression of MUC2, inhibit the release of inflammatory factors,protect intestinal mucosa, promote the repair of injured mucosa,and play the role of immune regulation, anti-inflammation and antioxidation in the treatment of UC, which provides a basis for clinical application in the later stage.

There are still some limitations in this study. 1. Due to the influence of budget, time, site and other factors, the sample size of each experimental group is small, resulting in a large bias in this study.In the later study, the sample size can be expanded to enhance the credibility of this study. 2. The purpose of this study is to explore the mechanism of Wenglian jiedu decoction on pro-inflammatory factors IL-18, TNF- α, the observation index is less, the later study can increase anti-inflammatory factors and other indicators to more fully clarify the mechanism of Wenglian jiedu decoction.

Author contribution: Lu Yuting: operation and data analysis of animal experiments, writing papers; an Guiye: participation in experimental implementation; Zhang Xiaoyan: data collation and analysis; Yang Qian: person in charge of the project (prevention and treatment of major and difficult diseases (ulcerative colitis) by traditional Chinese medicine; Huo Yongli: scientific research and design, revision of articles; Li Diangui: theoretical guidance. There is no conflict of interest in this article.