Dynamic Evaluation of Compound Ento-PB for the Repair of Mucosal Ulcer in Dogs With Acetic Acid-induced Experimental Colitis

Background: Mucosal inammation and ulcer play important roles in the pathogenesis of ulcerative colitis. As as traditional Chinese medicine compound composed of Periplaneta americana and Taraxacum mongolicum, Ento-PB is always prescribed for the treatment of ulcer and inammatory diseases. As for the signicant role of P. americana in terms of promoting mucosal healing, the compatibility of the anti-inammatory drug T. mongolicum may enable Ento-PB to simultaneously play anti-inammatory and promote mucosal healing effects on the treatment of UC. Therefore, this study aimed to evaluate the therapeutic potential and possible mechanism of Ento-PB for UC by establishing an acetic acid-induced colitis model in dogs. Methods: Preliminary identication to the chemical components of compound Ento-PB was carried out through high performance liquid chromatography. A cross-bred dogs model of acetic acid-induced ulcerative colitis was established to evaluate the ecacy of compound Ento-PB. The expression levels of inammatory cytokines C-reactive protein (CRP), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10) in plasma were measured by carrying out enzyme-linked immunosorbent assay (ELISA). Results: With the extension of treatment time, Ento-PB could effectively improve clinical symptoms of UC cross-bred dogs. Colonoscopy displayed that mucosal redness, swelling and congestion decreased gradually, and obviously repaired after mucosal injury. The intestinal texture was gradually clear, and the colonoscopy score gradually reduced. Histopathological examination revealed that the structure of colon was restored signicantly, the inltration of inammatory cells was reduced, and the histological score was remarkably reduced. At the same time, the results of dynamic monitoring of inammatory cytokines in plasma proved that Ento-PB can gradually down-regulate the activity of CRP, iNOS and COX-2, reduce the

pathogenesis of colitis [4]. The damage to the intestinal mucosa causes microorganisms enter the lamina propria from lumen, which triggers an in ammatory response, thus leading to the overproduction of in ammatory cytokines, including TNF-α, IL-1β, and COX-2. These cytokines further exacerbate the damage of intestinal mucosa [2]. At present, UC clinical rst-line treatment drugs such as 5-ASA and steroid hormones can relieve clinical symptoms by inhibiting the production of pro-in ammatory cytokines and nitric oxide. With the availability of multiple treatment options in e cacy and safety pro les, there are considerable practices variability in the application of these drugs in the treatment of outpatients and inpatients with moderate-severe UC [5]. Variations in practices may have unintended negative consequences in patient outcomes. The disadvantages of traditional anti-in ammatory drugs such as 5-ASA in colonic mucosal healing become increasingly prominent. As a result, the treatment goal of UC changed from clinical symptom relief to endoscopic remission, namely endoscopic mucosal healing [6]. However, so far, none of the approved IBD drugs directly target this process. In fact, some drugs may also inhibit the repair of intestinal mucosal damage [7].
Previous clinical and experimental studies proved that adjuvant Traditional Chinese Medicine (TCM) treatments such as herbal medicine and acupuncture are bene cial to the relief of UC symptoms, with reliable e cacy, few side effects, and low recurrence rate [8]. Among them, the compound Ento-PB composed of Periplaneta Americana L. and Taraxacum mongolicum Hand.-Mazz. is a common prescription for the treatment of ulcer and in ammatory diseases in Yunnan folk. Modern pharmacological studies con rmed that P. Americana extract has signi cant advantages in wound repair and ulcer healing. As a raw material, Kangfuxin Liquid can improve the immune barrier of the gastrointestinal mucosa of patients and relieve in ammation in clinical treatment of gastrointestinal diseases in China, increase the gastric mucosa hexose and prostaglandin E2 levels, and promote the repair of gastrointestinal mucosa [9,10]. Li et al. found that P. Americana extract can improve colitis in rats with acetic acid-induced ulcerative colitis by reducing in ammation and enhancing the activity of broblasts [11]. T. mongolicum is the key component of Ento-PB and can exert its anti-in ammatory effect. T. mongolicum is commonly applied alone or combined with other Chinese herbal medicine to treat gastrointestinal in ammation in Chinese clinics [12]. It was discovered that dandelion root extract can protect NCM460 colonic cells and alleviate experimental colitis by blocking NF-κB signal transduction, inhibiting in ammation, and reducing oxidative stress [13]. The active ingredient, namely chicory acid, can regulate the in ammatory response of intestinal epithelial cells that are induced by LPS through regulating the in ammatory signal of NF-κB p65 and in ammatory factors such as COX-2 and IL-1β [14]. We speculated that Ento-PB may have anti-in ammatory and promote mucosal healing effects on the treatment of UC, and hope to prove it through experiments, because P. Americana and T. mongolicum has signi cant e cacy respectively in the clinical treatment of colitis in China. With reference to the clinical diagnosis and treatment requirements for UC mucosal healing, conventional small animals, including mice, rats and rabbits, cannot meet the requirements of colonoscopy. Therefore, this study selected the canine colitis model, evaluated the repairing effect of compound Ento-PB on the intestinal mucosal injury of colitis dogs through dynamic observation of colonoscopy, and comprehensively evaluated its e cacy based on the dynamic changes of serum in ammatory factors and histopathological examination.

Preparation of Ento-PB
According to the Yunnan folk Yi nationality prescription, take 70 g of P. americana and 30 g of T. mongolicum, add 10 times the amount of pure water, boil and extract twice, 2 hours each time, lter the extract with gauze. The twice ltrates were combined, placed in a rotary evaporator, and the ltrate was concentrated by rotating at 50 °C to a density of 1.15 g/mL. Add 95% ethanol to the concentrate, stir well, let it stand overnight, centrifuge at 3500 rpm/min for 10 min, take the supernatant and spin and concentrate to dryness at 50°C to obtain the compound Ento-PB extract, which was sealed and stored at -20 ℃ until required.

HPLC Analysis of Ento-PB
High performance liquid chromatography (HPLC) was used to further characterize the potentially biologically active components in Ento-PB. Firstly, the mixed standard solution containing uracil 1.0 mg/mL, hypoxanthine 1.0 mg/mL, uridine 1.0 mg/mL, adenosine 1.0 mg/mL, inosine 1.0 mg/mL, caftaric acid1.0 mg/mL, caffeic acid 1.0 mg/mL, cichoric acid 1.0 mg/mL was diluted with pure water into a standard mixed solution with concentration of 0.1 mg/mL. Accurately weigh 200.0 mg of Ento-PB sample, dissolve in an appropriate amount of ultrapure water, sonicate for 15 minutes, add ultrapure water to make Ento-PB sample solution with a nal concentration of 20.0 mg/mL. 0.5 mL of the standard mixture or Ento-PB sample solution is ltered through a 0.45 μm microporous membrane for chromatographic analysis. HPLC analysis was performed on Agilent 1260 machine (Agilent technologies, USA), equipped with autosampler and UV DAD detector. Samples were separated using an Agilent Reversed-phase C18 alkyl silica gel column (4.6×250 mm, 5 μm) (Agilent technologies, USA) with a preconnected column in-line lter at 25 °C. Mobile phase A was 0.1% tri uoroacetic acid-water solution, while mobile phase B was methanol. The gradient elution condition was set as follows: 0-10 min (0-0% B); 10- 38-60 min (35-95% B). Ento-PB was analyzed with ow rate at 0.5 mL/min, injection volume of 10 μL and UV detection wavelength at 254 nm.
Animal Sixteen male small cross-bred dogs aged 1-2 years old were provided by the Experimental Animal Center of Dali University, weighing about 8-12kg. All dogs were raised separately in the IVC observation room of the Experimental Animal Center of Dali University (temperature 22 ±2 °C, relative humidity 55 ±5%, light / dark cycle 12 minutes 12 hours). In the course of the experiment, they were allowed to eat and drink freely and were allowed to adapt to the environment for two weeks. All animal operations, including anesthesia, surgery, postoperative care and sacri ce, have been approved by the Committee on Animal Care and use of Dali University of China ((IACUC)). The ethical quali cation number is 2017-0741.
Establishment of acetic acid-induced UC cross-bred dogs model Before 16 male cross-bred dogs were used for induction of UC by 10% acetic acid, colonoscopy was evaluated by electronic endoscope. For this purpose, animals were kept fasting for 1 day and bowel preparation was performed on the day of induction in cross-bred dogs. All cross-bred dogs underwent enemas that rconsisted of 10 %, acetic acid (210 mg/kg, in distilled water) administered with a No.15 feeding tube to induce UC. Dynamic examination of colonoscopy and histopathological examination of colon to observe the repair of intestinal mucosa after injury.

Grouping and treatment
After 24 hours of acetic acid induction, colonoscopy was performed on all cross-bred dogs, and the degree of in ammatory damage was scored. According to the colonoscopy score, the cross-bred dogs were randomly divided into 4 groups: model group, Jiechangning group (178 mg/kg), Ento-PB low-dose group (Ento-PB-L, 35.6 mg/kg), Ento-PB high-dose group (Ento-PB-H, 71.2 mg/kg), 4 animals/group. Blood samples were collected from veins and then the drugs were administered by enema, once a day for 10 consecutive days.

Colonoscopy
As shown in the experimental design ow chart in Fig. 1a, dogs were subjected to colonoscopy within a certain period of time to dynamically evaluate the repair effect of Ento-PB on intestinal mucosa injury. The speci c scoring standards refer to the Baron endoscopy scoring and Mayo colonoscopy scoring standards commonly used in clinical UC patients [15,16], with certain modi cations ( Table 1).

Detection of in ammatory cytokines in plasma
As shown in the ow chart of the experimental design in Fig. 1a, during the speci c time of the experiment, the venous blood samples were collected and placed in the EP tube containing anticoagulants, acentrifuged at 4°C, 3500 rpm/min for 10 minutes, and plasma was taken. According to the instructions of the manufacturer, the ELISA kits were used to detect the levels of IL-1β, IL-10, COX-2, CRP, TNF-α and INOS in dog plasma. Histopathology 24 hours after the last administration, the dogs were anesthetized with serazine hydrochloride injection (0.02 mL/kg) and the colon segment was removed. The surrounding mesenteric adipose tissue was removed, the colon cavity was longitudinally opened, and the contents were slowly washed with ice normal saline. samples were provided from mucosa in 10 cm proximal to the anal verge and moved to 4% paraformaldehyde (4% PFA) for histological studies. After xing the colon tissue (4% PFA, 12-24 h), it was dehydrated by an automatic dehydrator and embedded in para n. The coronal section of colon (5 μm) was prepared. The sections were subsequently stained with hematoxylin and eosin (H&E). According to the criteria of Mehrabani et al [16,17]. (Table 2), a professional pathologist who is not clear about the grouping of this study scored the pathological changes of the canine colon.

Statistical analysis
The results of in ammatory cytokines were statistically analyzed by the difference before and after modeling (difference = measured value at each time point after modeling -measured value before modeling). The experimental data is expressed as the mean ± standard deviation, and SPSS 24.0 software was used for statistical analysis. The in ammatory factor difference analysis and colonoscopy score are performed by repeated measures analysis of variance. The histopathology score is performed by one-way ANOVA. The comparison between groups was performed by Dunnett t test. P < 0.05 was considered statistically signi cant.

Identi cation of chemical constituents by HPLC in Ento-PB
The HPLC chromatogram of Ento-PB and mixed standard compunds at 254 nm is shown in additional le 1: Fig. s1. By comparing with the HPLC chromatograms of the mixed standard, eight compounds were preliminarily identi ed from Ento-PB: uracil, hypoxanthine, uridine, adenosine, inosine, caftaric acid, caffeic acid and chicoric acid from A to H, respectively. This is consistent with the results reported in the literatures [18,19].
Colonoscopy score of dog colitis was improved by Ento-PB As shown in g. 1b, before acetic acid induction, the colon surface of each group was smooth, the vascular structure was clear, and there were no pathological manifestations such as hyperemia, edema, ulcer and so on. On the rst day of administration, colonoscopy showed that the mucosal surface was rough, blood vessel structure disappeared, edema, redness, bleeding, and ulcers were obvious. On the 4th day of administration, colonoscopy showed large areas of ulcers, mucosal congestion and redness in the model group; small areas of ulcers and mucosal congestion and redness were found in the administration group. On the 7th day of administration, colonoscopy showed obvious congestion, edema, roughness, a few spontaneous bleeding points and small area ulcers in the mucosa of the model group; swelling, roughness, and small area ulcers in the mucosa of the Jiechangning group and the low-dose Ento-PB group; Ento-PB high-dose group mucosal redness and swelling subsided signi cantly and the surface was smoother. 24 hours after the last administration, colonoscopy showed that the mucosal surface of the model group was still rough, small areas of ulcers and redness; the Jiechangning group and the low-dose Ento-PB group had mucosal redness and swelling, the hyperemia subsided signi cantly, and the surface was smoother; In the Ento-PB high-dose group, the mucosal surface was smooth, the structure of part of the intestine was restored, a small area was hyperemia, and no obvious ulcers were observed.
Repeated measurement analysis of variance was performed on colonoscopy scores of dogs with acetic acid-induced experimental colitis at each time point. Therefore, the Greenhousc-Geisser method is used to correct the degrees of freedom and analyze the results. The results showed that there was statistical signi cance between different time points (F=305.130, P=0.000<0.01), and there was an interaction effect between time and grouping (F=4.435, P=0.001<0.01); the difference between groups was statistically signi cant (F=7.209, P=0.005<0.01). As shown in g. 1c, compared with the model group, after controlling for the in uence of different measurement points, the colonoscopy scores of the Jiechangning group and the Ento-PB low and high dose groups were signi cantly reduced (P<0.05 or P<0.01).
The results of colon anatomy (Fig. 1d) showed that the model group's colon was obviously widened and thickened, with different widths, and the mucosa showed roughness, redness and swelling, scattered bleeding spots, small area ulcer, and disordered structure; In Jiechangning group and Ento-PB low-dose group, a small amount of scattered bleeding spots and punctate ulcers were found, and the colon was widened; the colon of the Ento-PB high-dose group had clear structure, uniform thickness and width, and no obvious bleeding spots and ulcers.
Ento-PB treatment alleviated the histopathological symptoms of acetic acid-induced colitis in dogs.
As shown in g. 2a, the model group showed epithelial necrosis and shedding of mucosal tissue, largescale ulcer formation, enlarged glands surrounding the ulcer, proliferation of glandular epithelial cells, disappearance of goblet cells and crypt cells in the mucosal layer, and obvious in ammatory cell in ltration in the submucosa. And there is extensive edema; In Jiechangning group, the structure of mucosal layer was disordered, some goblet cells and crypt cells were lost, small area ulcers and epithelial hyperplasia could be observed, and there were obvious in ammatory cell in ltration and tissue edema in submucosa; In Ento-PB high dose group, goblet cells and crypt cells were partially lost, but the basic structure of colon was still found. there was a small amount of ulcer surface, and epithelial hyperplasia could be seen on the ulcer surface. In addition, a small number of in ammatory cells in ltrated into the submucosa, and the submucosal edema was obviously alleviated; In the low dose Ento-PB group, a small amount of mucosal epithelial cells fell off, forming a small area ulcer. Besides, partial loss of goblet cells and crypt cells, obvious in ammatory cell in ltration and tissue edema could be obeserved in the submucosa. It could be found from g. 3b-h that compared with the model group, ulcers, mucosal atrophy, in ammatory cell in ltration, and total HS scores were signi cantly decreased after treatment with Jiechangning and Ento-PB (P<0.05 or P<0.01). In dog colon tissue, the ulcer was signi cantly improved, mucosal atrophy and submucosal edema were effectively relieved, and the degree of in ammatory cell in ltration was signi cantly reduced.
Ento-PB treatment reduced the contents of CRP, COX-2 and iNOS in plasma of dogs with acetic acidinduced colitis Repeated measurement analysis of variance was performed on the difference between the level of CRP in plasma at each time point after modeling and the level of CRP in plasma before modeling. The results of Mauchly sphericity test showed that the data conformed to the sphere hypothesis (P=0.244>0.05). The analysis of the results showed that the difference between different time points was statistically signi cant (F=121.174, P=0.000<0.01), there was an interaction effect between time and grouping (F=6.052, P=0.000<0.01), and the difference between groups was statistically signi cant (F=4.084, P=0.033<0.05). As shown in g. 3a, compared with the model group, after controlling for the effects of different measurement points, the levels of CRP in the Ento-PB low and high dose groups were signi cantly reduced (P<0.05), and the levels of CRP in the Jiechangning group had a decreasing trend, but there was no statistical signi cance (P > 0.05).
Repeated measurement analysis of variance was performed on the difference between the content of COX-2 in plasma at each time point after modeling and the level of COX-2 in plasma before modeling. The Mauchly sphericity test results showed that the data did not conform to the sphericity hypothesis (P=0.006<0.05), so the Greenhousc-Geisser method is used to correct the degrees of freedom and analyze the results. The analysis of the results showed that the difference between different time points was statistically signi cant (F=132.147, P=0.000<0.01), there was an interaction effect between time and grouping (F=3.977, P=0.007<0.01), and the difference between groups was statistically signi cant (F=5.064, P=0.017<0.05). As shown in g. 3b, compared with the model group, after controlling for the in uence of different measurement points, the content of COX-2 in the Jiechangning group and the Ento-PB high-dose group was signi cantly reduced (P<0.05 or P<0.01), the content of COX-2 in the Ento-PB low-dose group had a decreasing trend, but it was not statistically signi cant (P>0.05).
Repeated measurement analysis of variance was performed on the difference between the level of iNOS in plasma at each time point after modeling and the level of iNOS in plasma before modeling. The Mauchly sphericity test results showed that the data did not conform to the sphericity hypothesis (P=0.001<0.05), so the Greenhousc-Geisser method is used to correct the degrees of freedom and analyze the results. The analysis of the results showed that the difference between different time points was statistically signi cant(F=44.892, P=0.000<0.01). There was no interaction effect between time and grouping (F=0.196, P=0.973>0.05), and there was no statistical difference between groups (F=0.052, P=0.983>0.05); However, it can be found from g. 3c, compared with the model group, the Jiechangning group and Ento-PB low and high doses group could reduce the level of iNOS in plasma of colitis dogs in different degrees. Although the inhibitory effect mediated by Ento-PB is not statistically signi cant, it at least shows the trend of suppression.

Ento-PB regulated the expression of in ammatory cytokines in plasma of dogs with acetic acid-induced colitis
Repeated measurement analysis of variance was performed on the difference between the level of TNF-α in plasma at each time point after modeling and the level of TNF-α in plasma before modeling. The Mauchly sphericity test results showed that the data did not conform to the sphericity hypothesis (P=0.000<0.05), so the Greenhousc-Geisser method is used to correct the degrees of freedom and analyze the results. The analysis of the results showed that the difference between different time points was statistically signi cant (F=47.960, P=0.000<0.01), there was an interaction effect between time and grouping (F=0.589, P=0.005<0.01), and the difference between groups was statistically signi cant (F=6.026, P=0.010<0.05). As shown in g. 4a, compared with the model group, after controlling the in uence of different measurement points, the level of TNF-α in the high dose group of Ento-PB decreased signi cantly (P<0.01), while the level of TNF-α in the Jiechangning group and the low dose group of Ento-PB decreased, but there was no statistical signi cance (P>0.05).
Repeated measurement analysis of variance was performed on the difference between the level of IL-1β in plasma at each time point after modeling and the level of IL-1β in plasma before modeling. The results of Mauchly sphericity test showed that the data conformed to the sphere hypothesis (P=0.238>0.05). The analysis of the results showed that the difference between different time points was statistically signi cant (F=86.332, P=0.000<0.01), there was an interaction effect between time and grouping (F=3.668, P=0.001<0.01), and the difference between groups was statistically signi cant (F=43.653, P=0.044<0.05). As shown in g. 4b, compared with the model group, after controlling the in uence of different measurement points, the level of IL-1β in the high dose group of Ento-PB decreased signi cantly (P<0.05), while the level of IL-1β in the Jiechangning group and the low dose group of Ento-PB decreased, but there was no statistical signi cance (P>0.05).
Repeated measurement analysis of variance was performed on the difference between the level of IL-10 in plasma at each time point after modeling and the level of IL-10 in plasma before modeling. The Mauchly sphericity test results showed that the data did not conform to the sphericity hypothesis (P=0.022<0.05), so the Greenhousc-Geisser method is used to correct the degrees of freedom and analyze the results. The analysis of the results showed that the difference between different time points was statistically signi cant (F=235.087, P=0.000<0.01) there was an interaction effect between time and grouping (F=8.623, P=0.000<0.01), and the difference between groups was statistically signi cant (F=4.012, P=0.010<0.05). As shown in g. 4c, compared with the model group, after controlling the in uence of different measurement points, the level of IL-10 in the high dose group of Ento-PB increased signi cantly, while the level of IL-10 in the Jiechangning group and the low dose group of Ento-PB increased, but there was no statistical signi cance (P>0.05). Discussion P. americana in Ento-PB can invigorate spleen, dispel blood stasis, and promote the subsidence of swelling, wound healing sore muscle and repair. T. mongolicum can clear away heat dry dampness, purge intense heat and dsintoxicate. The two drugs are applied together to regulate viscera, replenish qi to invigorate spleen, promote granulation and the removal of necrotic tissues, and eliminate in ammation and edema. Jiechangning (enema) is a Chinese herbal compound and approved by CFDA for the clinical treatment of ulcerative colitis [20]. The main components are Typhae and Polygonum syringae. It was proved that, combined with mesalazine, Jiechangning retention enema can effectively relieve UC symptoms in moderate active stage, improve intestinal mucosal permeability, and is superior to dexamethasone and mesalazine alone when promoting the proliferation of probiotics and restoring the diversity of intestinal ora [21]. Jiechangning could decrease the level of IFN -γ and increase the level of IL-10 in serum and colonic mucosa, regulate the balance of Th1/Th2, improve immunity, and improve DSS-induced acute colitis in rats [20]. Based on the reliability of Jiechangning in clinical and experimental colitis, we selected Jiechangning (enema) as the positive control drug.
Acetic acid-induced colitis can cause severe in ammation of colon and rapid formation of ulcer, and be accompanied by diarrhea, hematochezia, and weight loss, which is similar to the situation of acute ulcerative colon in ammation that caused by abnormal arachidonic acid metabolism in human colitis [22,23]. Mucosal ulcer is an early event in the occurrence mechanism of UC, and repair after mucosal injury is used as an important index to evaluate the e cacy of drugs [24]. Colonoscopy is currently an important method to clinically evaluate the healing of intestinal mucosal injury after the treatment of UC patients [25,26]. In this study, we referred to the clinically common Baron endoscopy score and Mayo enteroscopy score for the pathological manifestations of experimental colitis, focused on mucosal damage and intestinal hemorrhage, redness, and developed a colonoscopy score criterion for experimental colitis animal model, so as to evaluate the repairing effect of Ento-PB after intestinal mucosal injury in experimental colitis dogs. The results illustrated that after the treatment with Ento-PB, the color of the intestinal mucosa of dogs with colitis gradually changed from hyperemia and redness to lighter, the surface changed from granular to smooth, the ulcers signi cantly reduced or disappeared, and the colonoscopy score gradually decreased. By anatomy visible, the colon morphology was normal. Histopathological examination demonstrated that mucosal epithelial cells were intact, and in ammatory cell in ltration signi cantly reduced. In addition, CRP is a representative biomarker of acute in ammatory response, and has high sensitivity to human in ammatory response. Meanwhile, it is positively correlated with endoscopic activity of patients, and is frequently adopted to verify the extent of remission and mucosal healing of IBD [27,28]. Our results displayed that after the acetic acid induction, the level of CRP in plasma increased signi cantly, and Ento-PB could gradually decrease its expression level, which was consistent with the results of colonoscopy and histopathological score. All these suggested that Ento-PB could improve colonic mucosal ulcers in dogs, effectively relieve mucosal atrophy and submucosal edema, reduce the degree of in ammatory cell in ltration, and effectively promote the healing of intestinal mucosa.
The occurrence and development of UC are largely due to chronic in ammation, and massive in ltration of immune cells, and accompanied by increased production of pro-in ammatory mediators, including IL-1β, TNF-α, and COX-2, and decreased production of anti-in ammatory factors such as IL-10 [29,30]. Many evidences indicated that cytokines TNF-α and IL-1β play important roles in intestinal in ammation and barrier dysfunction [31]. Elevated levels of TNF-α and IL-1β increase neutrophil in ltration, damage the intestinal barrier, and drive diarrhea symptoms of UC [2]. The mucosal biopsy of IBD patients and the acetic acid-induced rat colitis model also con rmed that the expression level of TNF-α was positively correlated with the degree of colitis [32]. TNF-α could trigger the phosphorylation of myosin light chain, destroy the intestinal barrier and tight junction proteins, and can up-regulate the expression of iNOS and COX-2, activate the NF-κB pathway and other in ammatory signals, and initiate the cascade of in ammation reaction, thus resulting in sustained damage to the intestinal mucosa [2]. COX-2 catalyzes the release of many kinds of PGs (such as PGE4) and leukotrienes by arachidonic acid 33]. These in ammatory mediators can cause redness, swelling, pain, edema and in ammatory cell in ltration of intestinal mucosa, which in turn affect intestinal transport, intestinal activity and immune regulation, thus aggravating the existing in ammation [34]. The high expression of a variety of pro-in ammatory cytokines stimulates the up-regulation of iNOS expression and leads to the synthesis of a large number of NO, thus further aggravating the injury of intestinal mucosa [35]. As an effective anti-in ammatory cytokine, IL-10 can protect the host from excessive in ammation and immune response by exerting the immunosuppressive characteristics of negative feedback regulation that is related to TNF-α and IL-1β signal transduction [36,37]. Its level is strongly down-regulated in UC. Therefore, the development of colonic in ammation can be inhibited by regulating the balance of in ammatory cytokines. The results of dynamic monitoring of in ammatory cytokines in dogs with acetic acid-induced colitis suggested that Ento-PB treatment could gradually reduce the levels of pro-in ammatory cytokines COX-2, iNOS, TNF-α, and IL-1β, and gradually increase the level of anti-in ammatory cytokines IL-10, indicating that Ento-PB could reduce the acetic acid-induced colon in ammation in UC dogs by reducing the level of proin ammatory cytokines and increasing the level of anti-in ammatory cytokines.
The chemical constituents of P. americana and T. mongolicum were reported in many pharmacological studies, and many potential anti-UC bioactive chemical components were described, such as polypeptides [38], and amino acids [11] in P. americana, and caffeic acid [39], chicory acid [14], dandelion polysaccharides [40] in T. mongolicum. In this study, we preliminarily determined the presence of uracil, hypoxanthine, uridine, adenosine, inosine, caftaric acid, caffeic acid and cichoric acid in Ento-PB by HPLC analysis. Lu et al. proposed that the adenosine extract of P. americana can accelerate the remodeling of epithelium after skin injury through macrophage-TGF-β1-Smad signal, and the repair of skin injury [41].
Caftaric acid, caffeic acid and cichoric acid all have signi cant antioxidant, anti-in ammatory and antitumor activities, among which cichoric acid could also extremely inhibit the growth of colon cancer cells [42,43]. Thus, the combination of all these identi ed compounds could be directly or indirectly associated with the prevention of ulcerative colitis, and it could be argued that these components contribute, at least in part, to the con rmation of the anti-ulcerative colitis effect on Ento-PB.
Although current studies revealed that Ento-PB has signi cant intestinal mucosal repair and good antiin ammatory levels in dogs with acetic acid-induced colitis, there are still certain limitations that need to be considered. We have determined some chemical components in Ento-PB by HPLC, but we cannot determine which components can provide main support for Ento-PB anti-UC. Therefore, the main pharmacodynamic material basis of Ento-PB and the mechanism of anti-UC need to be further explored, so as to provide strong evidence support for the better development of ethnic experience prescription Ento-PB.

Conclusion
In conclusion, this study demonstrated that Ento-PB could promote the healing of colon ulcer in a dose and time-dependent manner, reduce the in ammatory response that is caused by the in ltration of neutrophils and other immune cells, and prevent the development of acetic acid-induced canine colitis. At the same time, the signi cant repairing effect of Ento-PB on intestinal mucosal injury attracted our great attention. If being used with anti-in ammatory drugs together, it will be possible to make up the low healing rate of mucosal injury after anti-in ammatory drug treatment. Most importantly, this study also proved that the folk TCM compound Ento-PB has the potential to treat ulcerative colitis.   The mucosa is still smooth, but a small area of hyperemia, redness and the refractive index enhanced 2 The mucosa is hyperemia, edema, and granularity. The mucosa is friability with contact bleeding 3 The mucosa is obviously hyperemic, edema, rough, and a few spontaneous bleeding points or contact bleeding. There are more in ammatory secretions, multiple erosions and small ulcers.

4
The mucosa becomes extensively congested and rough mucus membrane with mucosal edema, marked and spontaneous bleeding or contact bleeding. Multiple punctate erosion and large area ulcer. Representative photos of colonoscopy in each group; c. Colonoscopy score; d. Representative photos of colon in each group. *P<0.05, **P<0.01, vs. model group.