Establishment and evaluation of specific antibiotic-induced Inflammatory bowel disease (IBD) model in rats

Background: Physical and chemical methods have been established for rat enteritis model, but antibiotic induction has been relatively rare. This article aims to establish and evaluate rat model of Inflammatory bowel disease (IBD) using antibiotics. Methods: Fourteen SD rats were divided into A-G group according to the dosage and method of antibiotics, among which group A was the control group. The drug was stopped on the 7th day, the modeling period was 1-7 days, and the recovery period was 8-15 days. Half of the animals were dissected on 11 th day and the other animals were dissected on 15 th day . Record the food and water intake, body weight, and fecal weight for 2 hours on different days. Nine intestinal flora were analyzed by bacterial culture and three strains were analyzed by quantitative PCR. TNF-α, IL1-β, IL-6 and CRP in abdominal aorta blood were detected and analyzed. Colon and rectal tissues were pathologically examined for inflammation and scored. Results: Rat weight, food intake, water intake, and two-hour feces were significantly different (P = .04, .016, <. 001, .009). Compared with group A, there were significant differences in 9 kinds of flora in the experimental group (all P <.001). Bacteroides , F aecalibacterium prausnitzii , and Dialister invisus concentrations were analyzed by Quantitative real time polymerase chain reaction (q-PCR) and showed significant differences in groups A, C, and F (p = .033). There were significant differences about TNF-α, IL1-β, IL-6 and CRP between the groups (P = .016, .042, .037, .012). The colonic and rectal pathological inflammation scores of other groups were significantly different from those of the control group (all P <.001). Conclusion : Specific antibiotic-induced IBD model in SD rats is feasible.

inducing the disease. [2,3] In order to have a deeper understanding of the disease, many scholars use animal models to study the disease. [4] Rats are good mammals for research. [5] Sprague Dawley (SD) rats are easy to raise and are easily controlled due to their mild temperament. They have been used for the establishment of various disease models. At present, rat enteritis models mostly use physical and chemical factors. [6,7] However, these methods easily lead to intestinal perforation and intestinal necrosis in rats, which can lead to failure of modeling. New modeling methods need to be studied and discussed. Because intestinal flora imbalance may cause enteritis, we thought of using flora imbalance for modeling. Antibiotics often cause imbalances in the intestinal flora.
Therefore, in this article, we used different doses of clindamycin (single use) and different doses of clindamycin plus ampicillin and streptomycin (combination) to cause intestinal flora disturbance in rats. The inflammatory factors in the abdominal aorta and the inflammation of the colon and rectum were analyzed in order to assess whether modeling is feasible.

Rats
Sprague Dawley (SD) female rats (n=14), obtained from Liaoning Changsheng Biotechnology Co., Ltd. ranged in size from 172.4 to 179.5 g. All animals were introduced to quarantine and adaptive feeding in the Specific Pathogen Free (SPF) barrier system of this institution for 9 days. All procedures were SD rats were anesthetized intraperitoneally with 2% pentobarbital sodium (0.2 ml / 100 g). After about 15 ml of arterial blood was drawn from the abdominal aorta using a syringe (of which 5 ml of arterial blood was collected for inflammatory factor detection), the rat's abdominal aorta stopped beating, the pupils were dilated and the SD rats were ensured to be euthanized.
We also used rat IL-1β, rat IL-6, rat TNF-α, and rat CRP reagents from Bioswamp (Wuhan, China). We purchased soil genomic DNA rapid extraction kit, rapid competent cell preparation kit (one-step method), SanPrep column plasmid DNA small amount extraction kit, SanPrep column DNA gel recovery kit from Sangon Biotech (Shanghai),purchased Taq

Animal grouping and modeling
Group A was the control group, group B was the low-dose clindamycin group (250 mg/kg), group C was the middle-dose clindamycin group (500 mg/kg), and group D was the high-dose clindamycin group (750 mg/kg). Group E was the low-dose triple antibiotic group (clindamycin, ampicillin, and streptomycin; 250 mg/kg, 272.1 mg/kg, and 136.1 mg/kg, respectively). Group F was the mediumdose triple antibiotic group (clindamycin, ampicillin, and streptomycin; 500 mg/kg, 563.7 mg/kg, and 281.8 mg/kg, respectively). Group G was the high-dose triple antibiotic group (clindamycin, ampicillin, and streptomycin; 750 mg/kg, 835.8 mg/kg, and 417.9 mg/kg, respectively). The experiment was divided into two stages: the modeling period (days 1-7) and the recovery period (days 8-15). The administration volume was 10 ml/kg once a day through stomach feeding by oral needle during the modeling period between 8:30-10:00 AM. The intragastric administration was stopped at 8 th day. The weight, food-intake volume, water-intake volume, and stool samples were taken on days 1, 3, 5, 7, 9, 11, and 14 within 2 hours were collected. For each rat, the fecal microbial flora on the 1 st , 4 th , 8 th , 11 th , and 14 th days were examined. On day 11 and day 15, half of each animal was dissected with 2% pentobarbital sodium (0.2 ml/100 g) by intraperitoneal anesthesia injection. After SD rats were euthanized, we quickly removed the colon and rectal tissue for next experiments. Rat (D2300) abnormally died on day 13 and we failed to collect fecal volume within 2 hours of antibiotic use, food intake and water intake on days 13 and 14, and blood on day 15. (Fig.1A-C)
We Mixed 1 g of feces with 9 ml of tryptone soy broth, diluted to the appropriate concentration, and took 20 ul of the sample and spread it evenly on the agar medium using a coating bar. Organisms were plated onto mannitol sodium chloride agar medium plates, EMB, and CATC agar plates under aerobic conditions at 37 o C for 48 hours. Organisms cultured on TPY agar medium plates, BBE agar plates, reinforced Clostridium medium plates, anaerobic agar plates, and lactobacillus selective agar plates were cultured at 37 o C for 48 hours in anaerobic conditions. Organisms inoculated on DRBC agar plates were cultured for 5 days at 28 o C in aerobic conditions. Colonies were enumerated using the following formula: number of colonies (cfu/g) = number of plate colonies × 50 × dilution factor, with x 10 6 (E6) as a uniform unit.

Real-time quantitative PCR analysis
Soil genomic DNA rapid extraction kit (B518233, Shengong Biological Co., Ltd., Shanghai) was used to extract fetal DNA from SD rats. The process is as follows:1. Weigh 400 mg of SD rat feces, add 400 µl of 65 ° C pre-warmed Buffer SCL, mix by shaking, and place in 65 ° C water bath for 5 min.2.
Centrifuge at 12,000 rpm for 3 minutes at room temperature. Pipette 350µl of the supernatant into a Faecalibacterium prausnitzii, Dialister invisus in rats fecal were collected and analyzed by PCR. After PCR, electrophoresis analysis was performed using 1.5% agarose syrup electrophoresis map.

Analysis of inflammatory factors
On the 11th and 15th days, half of the rats were dissected in each group with 2% pentobarbital sodium (0.2 ml/100 g) by intraperitoneal anesthesia injection, and the abdomen was cut, and 5mL blood was drawn from the abdominal aorta into the blood collection tube using syringe. TNF-α, IL-1β, IL-6, and CRP was detected in the blood serum without diluting by an enzyme-linked immune sorbent assay (ELISA).

Colon and rectal pathological inflammation assessment
0 points: no inflammation; 1 point: a small amount of multifocal neutrophil infiltration (<10 per HPF); 2: a moderate multifocal neutrophil infiltration (more submucosal involvement) (10 50 / HPF); 3 points: a large number of multifocal and even aggregated neutrophils infiltration (more submucosa involvement and muscle layer) (> 50 / HPF); 4 points: the lesions involved the same 3 points, but abscesses or more extensive muscle layer involvement occurred.

Ethics
This study follows the Basel Declaration 2010 and Institutional Animal Care and Use Committee (IACUC) of Xi'an United Nations Quality Detection Technology CO.,Ltd. We use animals to a minimum in terms of animal welfare principles and without affecting the accuracy of the experiment. All applicable international ,national and/or institutional guidelines for care and use of animals were followed.

Statistical analysis
Primer Premier 5.0 software was used for sequencing primer design. SPSS 21 software (IBM, USA)was utilized to analyze all data. Measured data were analyzed by ANOVA and F-test. Crosstabs were also used for measurement data analysis. Categorical variables were used by crosstabs and chi-square test. The independent sample T-test was used for comparative analysis between two sets of measured variables.

Comparison of basic indice
The average starting weight of all rats was 17.26 ± 2.49 g, and there was no significant difference in weight between groups A-G (P <0.05). There were significant differences from groups A (control) to G (treated) in weight, food intake, water intake, and stool 2 hours post antibiotic use (P =0.04, 0.016, <0.01, and 0.009, respectively). Means and standard deviations are shown in Table 1 and Fig.2(A-D).

Comparison of nine species bacterial between groups
Staphylococcus aureus, Bifidobacterium, yeast, Bacteroides, Clostridium, anaerobic bacteria, E. coli, Enterococcus, Lactobacillus were cultured and counted using special medium. (Fig.3). All collected bacterial loads for these 9 species according to above methods were counted and compared between A group to B-G groups. The unit of bacteria in the stool is (CFU / g).The details were as followed: ( Table 3). Figure 5 (A-C) showed amplification plot of the three strains, and the agarose syrup electrophoresis picture is shown in Figure 5 Table 4.

Comparison of pathological inflammation scores
Colonic and rectal tissues were scored and compared for pathological inflammation according to the

Discussion
This article has made an important discussion on antibiotic-induced rat IBD model. It is important to choose the appropriate animal model of IBD to evaluate the non-clinical anti-inflammatory effects of the drug. Some scholars have conducted comparative research on rat IBD models caused by different chemical factors, and believe that each has its own advantages and disadvantages. [6] A highly reversible and reliable IBD rat model has broad application prospects for new drug treatment of IBD.
Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders that affect individuals throughout life. Although the etiology and pathogenesis of IBD are largely unknown, studies with animal models of colitis indicate that dysregulation of host/microbial interactions are requisite for the development of IBD. [8] The modeling method of this article is from the 2015 master's thesis of Wendi Zhang of Southern Medical University. [9] In her paper, he compared and analyzed the imbalance of rat enteritis flora induced by antibiotics. Although we used her modeling method, the research focuses are different.
We focused on the changes of 9 kinds of flora and the changes of inflammatory factors in the blood.
At the same time, we compare and analyze the colon and rectal pathological inflammation in the two stages of model recovery period.
In our paper, we analyzed the changes of 9 intestinal flora during the modeling period (days 1 and 4) and the recovery period (days 8, 11 and 14). Staphylococcus aureus is a major human pathogen that causes a wide range of clinical infections. [10,11] we noticed that as the dose of the antibiotic increased, the increase in bacterial load was not obvious, indicating that the drug inhibited the strain.
Bifidobacterium are defined as a group of living microorganism supplements that confer health benefits on the host when administered in adequate amounts. [12] The beneficial bacterium was significantly reduced in B, D,E,F group relative to the control group. Yeast cells are often employed in industrial fermentation processes for their ability to efficiently convert relatively high concentrations of sugars into ethanol and carbon dioxide. [13] Yeast is not a common intestinal bacterium , and it is absent in the control group. It was only found in the low-and middle-doses of the triple-agent groups.
Bacteroide is a gram-negative, non-spore, obligate anaerobic bacillus. Our study showed a significant reduction in the high-dose medication group, indicating that it was sensitive to high-dose and combination drugs and it was relative with type 2 diabetes. [14] Clostridium species are anaerobic, Gram-positive, rod-shaped, endospore-forming bacteria belonging to the phylum Firmicutes, and they constitute both a class and a genus in the phylum. [15] Clostridium decreased in the single-agent group and the combined drug group increased. Anaerobic bacteria have pivotal roles in the microbiota of humans and they are significant infectious agents involved in many pathological processes. [16] Pathogenic variants of E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide. [17] Enterococcus strains that adhere strongly to the intestinal epithelium, form biofilms and possess antioxidant defence mechanisms seem to have the greatest influence on the inflammatory process. [18] The genus Lactobacillus consists of 173 species and many genomes are available to study taxonomy and evolutionary events. [19] In another article, we elaborate on the changes of these nine floras. In general, with the increasing dose of antibiotics and the enhanced degree of combination, the beneficial bacteria decreased significantly, while the pathogenic bacteria increased significantly.
We performed real-time quantitative PCR analysis on three strains of Bacteroides, Faecalibacterium prausnitzii, and Dialister invisus (control group, medium-dose alone group, and medium-dose combined group). The results showed that the lower the number of copies, the higher the dose and the combination. Bacteroides species make up a significant fraction of the human gut microbiome, and can be probiotic and pathogenic, depending upon various genetic and environmental factors.
These can cause disease conditions such as intra-abdominal sepsis, appendicitis, bacteremia, endocarditis, pericarditis, skin infections, brain abscesses and meningitis. [20][21][22] There is an increasing interest in Faecalibacterium prausnitzii, one of the most abundant bacterial species found in the gut.
F.prausnitzii phylogroups can be found within this species and their distribution is different between healthy subjects and patients with gut disorders. It also remains unknown whether or not there are other phylogroups within this species, and also if other Faecalibacterium species exist. [23,24] Dialister invisus is reported to be low or not expressed in IBD patients. [25] Our research showed that Dialister invisus is little expressed in the model.
Our study found that TNF-α and IL-6 were significantly higher than the control group except for group D. However, there was no significant relationship with the dose administered and cytokine levels.
TNF-α has been reported as a potent stimulator of IL-6 production. [26][27][28] Inflammation induces IL-1β production in Kupffer cells and hepatocytes. [29] In our study, IL-1β was significantly increased compared with the control group, showing elevated value in dose administered group. CRP is currently a hot spot for studying inflammation and related diseases. [30][31][32] Our results showed that elevated CRP was associated with those groups that received antibiotic compared with the control group.
Pathological examination is an important method to judge the degree of tissue inflammation. Colon and rectal tissues were scored based on the degree of neutrophil invasion. Our study found that the degree of inflammation of the intestinal mucosa of rats dissected on day 11 was more severe than that of rats dissected on day 15, which may be related to the self-recovery of the intestinal flora.Deregulation of host-microbiota interactions in the gut is a pivotal characteristic of Crohn's disease. It remains unclear, however, whether commensals and/or the dysbiotic microbiota associated with pathology in humans are causally involved in Crohn's pathogenesis. [33] We investigated the degree of inflammation of colon and rectal tissues in antibiotic-induced SD rat enteritis models with drug doses and combined doses significantly increasing.. And we found that the greater the degree of flora disturbance, the more severe the inflammation of the colon and rectal tissues. This may be related to the release of inflammatory factors by the intestinal disorder flora. [34] It may also be related to the damage of intestinal mucosa caused by the release of metabolites by pathogenic bacteria to induce humoral and cellular immunity. [34,35] .
However, there are some shortcomings in this paper, such as not enough experiment rats, not deep discussion about inflammation factors with intestinal mucosa necrosis.

Conclusion
Specific antibiotic-induced IBD model in SD rats is feasible.

Ethics approval and consent to participate
This study follows the Basel Declaration 2010.Most of the authors of this article have been trained in animal experiments and have obtained a certificate of competency. We use animals to a minimum in terms of animal welfare principles and without affecting the accuracy of the experiment. Xi'an United Nations Quality Detection Technology laboratory was commissioned to perform our experiments under his IACUC permission. All applicable international ,national and/or institutional guidelines for care and use of animals were followed.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author.

Competing interests
The contents of this manuscript have not been copyrighted or published previously. There are no directly related manuscripts or abstracts, published or unpublished, by any authors of this manuscript. The authors indicated no conflicts of interests.

Funding
This study was supported by Project 2018C37090 to Guojun Tong. The fund is mainly used for experiment, data collection, experimental indice detection, statistical analysis, expert communication and so on.      Figure 1 Study flow and method. A: Fourteen SD rats of similar body weight were randomly divided into A-G 7 groups, 2 in each group, of which group A was the control group, B-D group was the low-dose, middle-dose, and high-dose clindamycin administration group alone; E-G The group was a low-dose, middle-dose, high-dose clindamycin, ampicillin, and streptomycin combination administration group. B: SD rats were anesthetized with 2% pentobarbital sodium (0.2 ml / 100 g) by intraperitoneal injection on day 11 and day 15, and 5 ml of abdominal aortic blood was taken. Colon and rectal tissues were taken at the same time.C:

Authors' contribution
The schematic diagram is the modeling process, 1-7 days is the model period for feeding specific antibiotic, and 8-15 is the recovery period after specific antibiotic withdrawal.

Figure 2
Basic indicators of the SD rat feeding process. A: High-low maps of animal weights comparison in each group on1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 14th days. There were statistical differences between the groups, P = .04; B: Histograms of the food intake of the animals in each group on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 14th days. There was statistical difference between the groups, P <.016; C: Heat maps of animals in each group on1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 14th days. There was a statistical difference between the groups, P <.001; D: Bar graphs of animals in each group for 2 hours on day 1, 3,5,7,9,9,11,13,14 There was a statistical difference between the groups, P = .009.  Colonic HE staining pathological picture (* 100), the left is the dissection of the rat colon tissue on the 11th day, and the right is the dissection of the rat colon tissue on the 15th day. In group A, there was no obvious neutrophil infiltration and mucosal edema; in group B-G, neutrophil infiltration became more and more serious, mucosal edema became more and more obvious, and even mucosal necrosis and focal ulcer formed.

Figure 7
Rectal tissue HE staining pathological picture (* 100), the left side is the dissection of rat rectum tissue on the 11th day, and the right side is the dissection of rat rectum tissue on the 15th day. In group A, there was no obvious neutrophil infiltration and mucosal edema; in group B-G, neutrophil infiltration became more and more serious, mucosal edema became more and more obvious, and even mucosal necrosis and focal ulcer formed.

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