Qu-Yu-Jie-Du Decoction Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice by Modulation of Neutrophils and Macrophage Infiltration

Background Inflammatory bowel disease (IBD) is becoming a global disease. A percentage of IBD patients will not react to therapy or will lose their response. Qu-Yu-Jie-Du Decoction (QYJD) is a traditional Chinese medicine formula commonly used for intestinal diseases. It has been reported that QYJD has an anti-inflammatory effect, but the mechanism is not fully understood. In this study, we mainly evaluated the anti-inflammatory effect of QYJD and explored the possible mechanisms. Methods Twenty-four BALB/c mice were randomly divided into 4 groups according to their body weight, namely, the control group, the dextran sulfate sodium (DSS) group, the DSS + QYJD group, and the QYJD group. Mice were given 3% DSS drinking water freely, and at the same time, mice were given normal saline or QYJD (4.44 mg/g/d), respectively. Mental state, faeces, and weight were recorded every day. On the 10th day, the mice were sacrificed and collected for investigation. The length of the mice colon was measured. Histological analysis was used to detect the morphological changes in the colon. Immunohistochemistry was used to measure the infiltration of macrophages (F4/80, CD163) and neutrophils (Ly6G). Colorimetry was used to detect the myeloperoxidase (MPO) activity of colon tissues. ELISA was utilized to detect associated inflammatory cytokines and chemokines in colon tissues. Results QYJD alleviated the weight loss and colitis symptoms of mice caused by DSS. QYJD fought against the shortening of the intestine caused by DSS; that is, it improved the decline of intestinal compliance in mice and had a protective effect on colon tissues. The mechanisms were related to downregulating macrophages and neutrophils in colon tissues of infiltration. Besides, QYJD simultaneously reduced the activity of myeloperoxidase activity (MPO) and the contents of IL-1β, IL-6, TNF-α, TGF-β, CCL2, and CXCL2 in colon tissues. Conclusions QYJD can ameliorate DSS-induced colitis in mice and the mechanism is connected with a reduction in neutrophil and macrophage infiltration.


Introduction
Infammatory bowel disease (IBD) is becoming a global disease in the twenty-frst century. According to statistics, there are more than 2 million IBD patients in North America, 3.2 million in Europe, and millions more around the world. Te incidence rates of IBD in newly industrialized countries have been on the rise in recent years with the westernization of lifestyle [1]. Although the etiology of IBD is not completely clear, it may result from predisposing genetic mutations, complex environmental factors, gut microbiota, and immune responses [2][3][4]. Crohn's disease (CD) and ulcerative colitis (UC) are known as the two major forms of this disease. CD leads to transmural infammation and occurs in any part of the gastrointestinal tract continuously. CD commonly results in complications such as abscesses, fstulas, and strictures. UC is appertaining to mucosal infammation and is limited to the colon in an uninterrupted pattern [4]. IBD presents with chronic infammation, a relapsing and remitting clinical course, the requirement for lifelong medication or even surgery, and decreased quality of life. It is well recognized that patients with IBD are at an increased risk of developing colorectal cancer (CRC) and extra-intestinal malignancies, primarily secondary to chronic infammation state and immunosuppressive therapies [5,6]. Te main treatments for IBD are aminosalicylates, short courses of steroids for severe fares, and escalation to immunomodulators. Despite the fact that there are numerous therapeutic alternatives for the treatment of IBD, a portion of patients fail to respond or lose their response to medication [7]. Meanwhile, healthcare costs are dramatically increased. Tus, new therapeutic strategies for IBD are urgently needed.
Neutrophils and macrophages are essential innate immune cells playing a variety of roles in the host's pathogen defense [8]. Tey are able to generate efector molecules such as granular proteins, oxidants, and cytokines, among others [9,10]. When infammation occurs, neutrophils are the frst to be recruited and are outftted with powerful microbicidal activity. Neutrophils can release chemoattractants such as cathepsin G and azurocidin, which aid in the recruitment of other immune cells [11]. Ten, macrophages are recruited, which have the ability to digest antigens and present them to other immune cells, enabling them to interact with the adaptive immune system [12]. Neutrophil chemoattractants such as CXCL1, CXCL2, and monocyte chemoattractant protein-1(MCP-1) are produced when tissue-resident macrophages are activated [13,14]. Evidently, neutrophils and macrophages organize a successful immune response against pathogens [15].
Dextran sodium sulfate (DSS), a water-soluble sulfated polysaccharide, is the most widely used to induce a mouse model of colitis. Te molecular weight of DSS used for induction of colitis is 36-50 kD. DSS is toxic to the colon epithelium and can induce erosion that compromises barrier integrity, resulting in increased permeability of the colon epithelium and difusion of proinfammatory intestinal substances, such as bacteria and their items, into the underlying tissues. In addition, DSS has an anticoagulant character that exacerbates intestinal bleeding. Te DSS colitis model is widely used in IBD research because of its quickness, efortlessness, and numerous similarities with human UC [7,17,18]. In this current study, we validated that QYJD can ameliorate acute colitis in dextran sulfate sodium (DSS)-induced mouse models. We also demonstrated that the alleviation of colitis by QYJD was related to the modulation of neutrophils and macrophage infltration.  Table 1. According to the prescription, we weighed the crude drugs and added water to exceed 5 cm of the drug surface to soak for 30 minutes. We boiled the mixture with gentle heat for 40 minutes and then collected the decoction. We added water to the drug residue to 1 cm above the surface and boiled it gently for 30 minutes again. We obtained the QYJD extract by collecting and mixing the two decoctions. Te extract was concentrated to 1.8 g/mL by a rotary evaporator. Te concentrates were made into a freeze-dried powder with a vacuum freeze-drying machine in a cold trap at −50°C. Te freeze-dried powder was sealed at −20°C. Te fnal weight ratio of crude drug to lyophilized powder was 9.756. According to the LC/MS chromatogram of QYJD, matrine and oxymatrine are the two main components of QYJD [16].

Induction of Colitis and Treatment.
24 BALB/C mice were randomly divided into 4 groups according to their body weight: (1) control group (without DSS and QYJD); (2) DSS group; (3) DSS + QYJD group; and (4) QYJD group (without DSS). Colitis was induced by the administration of 3% (w/v) DSS in drinking water for 9 days. At the same time, mice of the control group and DSS group were given intragastric administration of normal saline once daily. Mice of the DSS + QYJD group and QYJD group were simultaneously given intragastric administration of 4.44 mg/g QYJD once daily. Te volume of gavage was 10 μL/g. Te mental state of the mice was observed every day, including whether there was back arching, slow activity, or reduced eating. Te weight of the mice was measured, and the stool situation and hematochezia of the mice were recorded daily. If the mice did not defecate immediately, continue to observe for at least 15 minutes. On the 10th day, the mice were sacrifced, and colon tissue samples were gathered.

Disease Activity Index (DAI).
Te DAI was comprehensively evaluated by weight change, stool texture, and hematochezia of mice, refecting the severity of colitis [17,19]. DAI of mice was calculated according to Table 2. Te disease activity index � (score of weight loss + score of consistency + score of bleeding)/3.

2.7.
Immunohistochemistry. Te slides were dewaxed using xylene, diferent concentrations of ethanol, and rinsed with water. Ten, the slides were placed into citric acid antigen repair bufer and heated in a pressure cooker for tissue antigen repair. We put the slices in 3% H 2 O 2 solution away from light for 25 minutes at room temperature to block endogenous peroxidase. 3% BSA was dropped onto the tissue to seal. Remove the blocking solution and drop the primary antibody onto the glass slide (dilution ratios of antibodies: F4/80 antibody 1 : 500; CD163 antibody 1 : 250; ly6G antibody 1 : 400; CCR2 antibody 1 : 250). Te slides were placed in a wet box and incubated overnight at 4°C. And then, the slides were incubated with HRP-labeled goat anti-rabbit IgG for 50 minutes at room temperature. DAB color solution was added to develop color. Te nucleus was restained with hematoxylin for 3 minutes. Te slides were dehydrated and sealed. We used Image-Pro Plus 6.0 software to select the brown-yellow color with positive expression as the standard to judge the positivity of all photos, analyze the cumulative optical density (IOD) of the images and the pixel Area of the tissues, and calculate the areal density. Areal density � IOD/Area.

Measurement of Myeloperoxidase (MPO) in Colon.
Te activity of myeloperoxidase (MPO) in colon tissue was determined by colorimetry to evaluate the number and function of neutrophils in colon tissues. A homogenate of 5% was prepared by adding homogenate medium at a ratio of 1 : 19 (w/v). We added reagents according to the manufacturer's instructions and then used a spectrophotometer to detect the absorbance of each tube at 460 nm. Te MPO activity is evaluated by the following formula: Te MPO activity (U/tissue wet weight (g)) � (determination OD value−control OD value)/(11.3 × sample weight (g)).

Determination of Cytokines and Chemokines Levels in
Colon. Te cytokines and chemokines levels in the colon were measured by ELISA kits according to the manufacturer's instructions. Te tissue homogenates were prepared by adding lysate at a ratio of 1 : 100 (100 mg tissue and 1 mL tissue lysate). Homogenates were centrifuged (12000 rpm, 4°C, 20 minutes) to collect the supernatants. Supernatants were employed to estimate cytokines and chemokines. Draw the standard curve according to the concentration of the

QYJD Attenuated DSS-Induced Colitis.
In order to study the therapeutic efect of QYJD on DSS-induced colitis in BALB/c mice, we evaluated from weight change, disease activity index (DAI), colon length, and histological analysis. As shown in Figure 1(a), the body weight of the DSS group decreased signifcantly compared with the control group, P < 0.001. Te weight loss degree of the DSS + QYJD group was less than that of the DSS group, P � 0.015. Tese results demonstrate that DSS could cause weight loss in mice, QYJD could reduce the degree of weight loss in mice caused by DSS, while QYJD alone had no signifcant efect on the weight of mice. Te severity of colitis was evaluated daily using the disease activity index (DAI) scoring system, which combined weight loss, stool consistency, and bleeding scores. Te DAI of the DSS group was signifcantly higher than that of the control group, P < 0.001; Te DAI of the DSS + QYJD group was signifcantly lower than that of the DSS group, P < 0.001. Tere was no statistical diference between the control group and the QYJD group, P � 0.941 (Figure 1(b)). Colon length, an indicator of intestinal permeability, was signifcantly shortened in the DSS group than that in the control group, P < 0.001. While the colon length of mice in the DSS + QYJD group was signifcantly greater than the DSS group, P � 0.041 (Figures 1(c) and 1(d)). QYJD could alleviate DSS-induced colon length shortening.
To determine the status of the colon, a histological analysis was performed after colon tissue was stained with hematoxylin and eosin. As shown in Figure 1(e), in the DSS group, large area ulcers were found in colon tissue, loss of mucosal epithelial cells, loss of intestinal gland structure, loss of goblet cells, hyperplasia of connective tissue, and a small amount of blood vessel congestion, injury, and invasion of the submucosa, and a large number of infammatory cells were found in the mucosal layer and submucosa. In the DSS + QYJD group, the mucosal layer of colon tissue was damaged, the local intestinal gland structure disappeared, connective tissue hyperplasia was accompanied by a small amount of infammatory cell infltration, and the damage did not invade the submucosa. Mice in the control group and QYJD group had a clear structure of each layer of colon tissue, complete epithelium of mucosal layer, abundant and compact intestinal gland, normal cell morphology, and no obvious infammatory reaction in colon tissue. Furthermore, the histological score of the DSS + QYJD group was lower than the DSS group (P � 0.056), but the diferences were not signifcant (Figure 1(f )).

QYJD Decreased Neutrophil Infltration and the Activity of Myeloperoxidase Activity (MPO).
In order to evaluate the efect of QYJD on neutrophils in the colon tissue of DSSinduced colitis mice, we used immunohistochemistry to detect the expression of Ly6G in colon tissue. As shown in Figure 2(a), the expression of Ly6G in the DSS + QYJD group was signifcantly lower than that in the DSS group (P � 0.001). Te activity of myeloperoxidase (MPO) represents the infltration of neutrophils and stress intensity to injury. Compared with the control group, the MPO activity of colon tissue in the DSS group was signifcantly increased (P < 0.001). Te MPO activity in the DSS + QYJD group was signifcantly lower than that in the DSS group (P < 0.001) (Figure 2(b)).

QYJD Reduced Macrophage
Infltration. F4/80 and CD163 were used as markers of macrophage. Te expression level of F4/80 in the DSS + QYJD group was lower than that in the DSS group in trend, but there was no signifcant statistical diference (P � 0.095) (Figure 3(a)). Te expression level of CD163 in the DSS + QYJD group was signifcantly lower than that in the DSS group (P � 0.001) (Figure 3(b)).

QYJD Regulated the Expression of Infammation Cytokine.
To further investigate the mechanism of QYJD on DSSinduced colitis in mice, we measured macrophage-related and neutrophil-related infammation cytokines in the colon tissues of mice. Compared with the control group, DSS could signifcantly increase the protein levels of infammation cytokines IL-1β, IL-6, TNF-α, TGF-β, and IL-12 in colonic tissues. As shown in Figure 4, the protein levels of Il-1β, TGF-β, TNF-α, and IL-6 in the DSS + QYJD group were signifcantly lower than those in the DSS group.

QYJD Regulated the Chemokines Expression.
Compared with the control group, DSS could signifcantly increase the protein levels of CCL2 and CXCL2. Te content of chemokines CCL2 and CXCL2 in the DSS + QYJD group  were signifcantly lower than those in the DSS group. Te results showed that QYJD could reduce the content of chemokines CCL2 and CXCL2 in colonic tissue of mouse models of colitis induced by DSS. While there was no signifcant diference in CXCL1 expression among all groups ( Figure 5).

Discussion
Our study indicated that QYJD could ameliorate DSSinduced colitis in mice by reducing the infltration of neutrophils and macrophages. As for our result, we assessed the anti-infammatory efect of QYJD by observing the degree of weight loss, the DAI score, colon length, and histopathology. Te activity of myeloperoxidase (MPO) represents the infltration of neutrophils and stress intensity to injury [23,24]. Impressively, DSS can signifcantly increase MPO activity in the colon tissue of mice, and QYJD can efectively reduce MPO activity in the colon tissue of mice.
To study the anti-infammatory mechanism of QYJD, we frst observed the efect of QYJD on congenital immune cells such as macrophages and neutrophils in colon tissue. Macrophages play a central role in maintaining intestinal homeostasis by engulfng necrotic cell debris and pathogens [25]. Neutrophils are important efectors of antimicrobial immunity, secreting matrix metalloproteinases and myeloperoxidase during infammation. Te infltration of macrophages and neutrophils in the colon tissues of each group was detected by immunohistochemistry. F4/80 is a surface marker protein of macrophages, representing the infltration of macrophages. CD163 was highly expressed in M2 macrophages. Ly6G is mainly expressed on the membrane surface of neutrophils. We found that compared with the DSS group, QYJD could signifcantly reduce the expression levels of CD163 and Ly6G in colon tissues. Te expression of F4/80 in the treatment group of QYJD showed a downward trend, but there was no signifcant diference between the two groups, which might be caused by the small sample size.
When the colonic mucosa is damaged by DSS and other pathogenic factors, many infammatory cytokines are increased. Among these cytokines, IL-1β, IL-6, and TNF-α play essential roles in intestinal infammation [26]. Furthermore, we also measured TGF-β, which plays important roles in growth and development, infammation and repair, and immunity [27]. In this study, we used ELISA to detect the contents of cytokines IL-1β, IL-6, TNF-α, IL-12, and TGF-β in mouse intestinal tissue, and we found that QYJD could signifcantly reduce the contents of IL-1β, IL-6, TNFα, and TGF-β in colon tissue of mice with colitis.
To further consolidate the efect of QYJD in regulating neutrophils and macrophages to block infammation cascades, we tested related chemokines. Chemokines belong to the G protein-coupled receptor superfamily of cytokines, which are related to the migration and activation of leukocytes and play a major role in maintaining infammation [28]. CCL2 is also called monocyte chemoattractant protein-1(MCP-1). CCL2/MCP-1 is produced by dendritic cells, macrophages, fbroblasts, keratinocytes, and other cells. Te function of CCL2/MCP-1 is to lead monocytes to leave the blood and diferentiate into macrophages in tissues, promote the release of histamine from basophils, and improve T2 activity [29,30]. In addition, CCL2 has chemotactic efects on T cells, mast cells, basophils, and natural killer cells Te protein levels of cytokines were quantifed by ELISA kits. * P < 0.05, * * P < 0.01, and * * * P < 0.001. [31,32]. Elevated CCL2 can be detected in the intestinal mucosal tissue of patients with IBD [33]. CXCL2 is a protein encoded by proto-oncogenes derived from monocytes, fbroblasts, and endothelial cells. CXCL2 has the function of chemotaxis to neutrophils, initial T lymphocytes, and fbroblasts. In our study, we found that QYJD down-regulated the expression of CCL2 and CXCL2 in colon tissues, resulting in less locally infltrated neutrophils and macrophages. Strengths of our study include that the mice were randomly assigned by weight, and the basic condition of mice in each group was the same. Besides, we observed the efects of QYJD on a variety of cytokines and chemokines in the colon tissues of mice. Tis refects the characteristics of multipathway and multitarget of the TCM, providing ideas for follow-up studies and having certain reference values, although there are still some shortcomings, such as insufcient mechanism digging. But we have tested it multiple times and proved that QYJD could reduce DSS-induced intestinal infammatory response in mice. Infammation afects cancer invasion, epithelial-mesenchymal transformation, and cell migration at many levels [34]. Infammation, infammatory cytokine release, and infammatory cell infltration are regarded as major contributors to the initiation and development of UC and its associated cancer [35]. Tis study ofered an approach to studying the efect of QYJD on the malignant tumor. In future studies, we will verify by enlarging the sample size and exploring from the perspective of neutrophils and macrophages, considering its signifcant changes and the important role of the infammatory process.

Conclusions
QYJD has the efect of inhibiting colitis in mice, and its mechanism is related to the regulation of the infammatory immune system. QYJD can mitigate the weight loss and enteritis symptoms of mice caused by DSS. It fghts against the shortening of the intestine brought about by DSS, improves the decline of intestinal compliance in mice, and has a protective efect on colon tissue. Te mechanism is related to the regulation of IL-1β, TGF-β, IL-6, TNF-α, and other cytokines in colon tissue by QYJD, and the reduction of chemokine CCL2 and CXCL2, as well as the reduction of neutrophils and macrophages infltration in colon tissue.

Data Availability
Data are available upon request by sending an e-mail to the author Hongwei Zhao (zhaohongwei129@163.com).

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Supplementary Materials
Te authors did a pilot experiment earlier, which was a dosedependent design. Details are as follows: 25 BALB/c mice were randomly divided into 5 groups according to their body weight: (1) control group (0.9% NaCl, without DSS and QYJD); (2) DSS group (0.9% NaCl); (3) DSS + QYJD × 1/2 group (the concentration of QYJD was 1.11 mg/g/d); (4) DSS + QYJD × 1 group (the concentration of QYJD was 2.22 mg/g/d); (5) DSS + QYJD × 2 group (the concentration of QYJD was 4.44 mg/g/d). Colitis was induced by the administration of 3% (w/v) DSS in drinking water for 7 days. At the same time, mice were given intragastric administration of normal saline or diferent doses of QYJD once a day. Te volume of gavage was 10 μL/g. Te mental state of the mice was observed daily, including whether there was  Evidence-Based Complementary and Alternative Medicine slow activity, reduced eating, and back arching. Te weight was measured; the stool situation and hematochezia of the mice were recorded every day. According to the scores of the disease activity index (DAI) and weight loss (%) of the mice, we found that twice the equivalent dose of QYJD had a better efect on attenuating DSS-induced colitis. Terefore, twice the equivalent dose (4.44 mg/g/d) of QYJD was used in the subsequent formal experiment ( Figure S1). (Supplementary  Materials)