Name: effects of desmodium caudatum on gastrointestinal hormones and intestinal flora in rats with gastritis
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This work was funded by the Key R&#38;D projects of the Science and Technology Department of Sichuan Province (2020YFS0539).

The procedures for the care and use of animals were approved by the Ethics Committee of Mianyang Normal University, and all relevant institutional and governmental regulations concerning the ethical use of animals were strictly followed. For the present study, SPF rats (Kunming species, both male and female, weighing 180-220 g) were utilized. The animals were obtained from a commercial source (see Table of Materials). All animals were housed in a pathogen-free environment and provided ad libitum access to food.
1. Preparation of reagents
<ol>
	<li>Prepare a 5% sodium salicylate solution by weighing 2.5 g of sodium salicylate powder (see Table of Materials) with an electronic scale. Dissolve the powder in a small amount of distilled water and dilute it to a total volume of 50 mL.</li>
	<li>Prepare the decoction of D. caudatum (12.8 g/kg). Weigh 128 g of D. caudatum (see Table of Materials), place it into a round-bottom flask containing 1000 mL of laboratory distilled water, and concentrate it to a final volume of 100 mL.</li>
</ol>
2. Grouping and administration
<ol>
	<li>Divide the rats into different groups and intragastrically administer the required solutions. 
	NOTE: For the present study, 18 rats (9 males, 9 females) were divided into three groups: the control group (Group C), the model group (Group M), and the treatment group (Group T), with 6 rats in each group. The control group received saline solution, and the model group received a 5% sodium salicylate solution for 5 weeks at a dose of 10 mL/kg/day15. The treatment group was pretreated with a 5% sodium salicylate solution for 5 weeks at a dose of 10 mL/kg/day, followed by administration of the D. caudatum decoction at a dosage of 1 mL/100 g for 10 days15,16.</li>
	<li>Collect feces from the rats in dry and sterile microcentrifuge tubes using the tail-carrying method17 on the last day of the experiment and freeze them in a liquid nitrogen ultra-low-temperature freezer.</li>
	<li>After the end of sampling, sacrifice the rats by cervical dislocation (following institutionally approved protocols). Open the abdominal cavity with scissors to expose the abdominal aorta.
	<ol>
		<li>Insert the needle (23-25 G) almost parallel into the abdominal aorta, collect the serum, and preserve it in an ultra-low-temperature freezer. Take out the stomachs from the abdominal cavity16 and soak in a 10% formalin solution for preservation. 
		NOTE: In Group M, the chronic gastritis model is considered successful if the gastric mucosa is dilated and congested with regular glandular morphology15, while observing a small amount of inflammatory cell infiltration in the lamina propria.</li>
	</ol>
	</li>
</ol>
3. Pathological section of gastric wall
<ol>
	<li>Take the gastric wall samples from rats in each group for pathological sections. Subsequently, perform routine hematoxylin-eosin staining to observe and analyze the pathological changes18.</li>
</ol>
4. Determination of serum gastrointestinal hormones
<ol>
	<li>In the serum sample, detect the contents of gastrin (GAS) and malondialdehyde (MDA)19 using ELISA following the manufacturer&#39;s instructions (see Table of Materials). Utilize a microplate reader for the analysis.</li>
</ol>
5. Fecal total DNA extraction and PCR amplification and purification
<ol>
	<li>Perform microbial DNA extraction utilizing the commercially available DNA isolation kit (see Table of Materials) and determine the concentration and purity using UV-Vis spectrophotometer16. Subsequently, assess DNA integrity through 1% agarose gel electrophoresis18.</li>
	<li>Amplify the bacteria 16S rRNA gene with primers 338F (5&#8242;-ACT CCT ACG GGA GGC AGC AG-3&#8242;) and 806R (5&#8242;-GAC TAC HVG GGG GTW TCT AT-3&#8242;) using a thermocycler PCR system17. The primers are obtained from commercial sources (see Table of Materials). 
	NOTE: Follow the PCR program: denaturation (3 min, 95 &#176;C), followed by 27 cycles (30 s, 95 &#176;C; 30 s, 55 &#176;C; 45 s, 72 &#176;C), and a final extension (10 min, 72 &#176;C). Conduct PCR reactions with the following components: 4 &#181;L of 5&#215; DNA polymerase buffer, 2 &#181;L of 2.5 mM dNTPs, 0.8 &#181;L of each primer (5 &#181;M), 0.4 &#181;L of DNA polymerase, and 10 ng of template DNA (see Table of Materials).</li>
	<li>Extract, purify, and quantify the PCR products sequentially using 2% agarose gel, commercially available DNA gel extraction kit, and fluorometer, respectively17.</li>
</ol>
6. Sequencing
NOTE: Step 6 and step 7 are performed following previously published methods20,21.
<ol>
	<li>Utilize the Illumina platform for sequencing. 
	NOTE: One end of the DNA fragment complements the base of the connector embedded in the chip, securing it onto the chip. The other end randomly complements the base of another adjacent buried joint, forming a &#34;bridge.&#34;</li>
	<li>Perform PCR amplification to generate DNA clusters. Linearize DNA amplifiers into single strands.</li>
	<li>Introduce an altered DNA polymerase alongside deoxyribonucleotide triphosphates (dNTPs) featuring four distinct fluorescent markers. Synthesize only one base per cycle.</li>
	<li>Utilize a laser to scan the surface of the reaction plate and determine the nucleotide types polymerized during the initial reaction of each template sequence.</li>
	<li>Chemically cleave the &#34;fluorescence group&#34; and &#34;termination group&#34; to reestablish the 3&#39; sticky end. Subsequently, advance to polymerizing the second nucleotide.</li>
	<li>Obtain the sequence of template DNA fragments by analyzing the fluorescence signal results collected in each round.</li>
</ol>
7. Processing of sequencing data
<ol>
	<li>Initially, trim the reads of raw fastq data at any site with a score &#60; Q20 and eliminate reads with uncertain bases. Additionally, permit two mismatches in precisely matched primers. Subsequently, merge the sequences with an overlap &#62;10 bp.</li>
	<li>Cluster operational taxonomic units (OTUs) with 97% similarity using UPARSE, and remove chimeric sequences with UCHIME20,21.</li>
	<li>Employ the RDP Classifier algorithm to analyze the taxonomy of each 16S rRNA gene sequence against the Silva (SSU123) 16S rRNA database, using a confidence threshold of 70%.</li>
	<li>Perform Alpha diversity analysis and Beta diversity analysis using Mothur and Qiime, respectively (see Table of Materials).</li>
</ol>
8. Statistical analysis
<ol>
	<li>Analyze the data in this study using statistical analysis software (see Table of Materials). Employ one-way analysis of variance (ANOVA) for comparisons among multiple groups and utilize the least-significant-difference (LSD) test for comparisons between two groups.</li>
	<li>Consider P &#60; 0.05 as statistically significant in all comparisons. P &#60; 0.01 is regarded as extremely significant.</li>
</ol>

Desmodium caudatum Decoction's Impact on Intestinal Microbiota of Rats

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The authors have nothing to disclose.

D. caudatum, a commonly used folk medicine by the Qiang nationality12, has shown significant efficacy in treating gastrointestinal diseases. With the advancement of modern pharmacological research, the imbalance of flora resulting from gastrointestinal microecology imbalance is identified as a key factor in acute and chronic gastrointestinal diseases22,23. Certain microorganisms in the intestinal tract play a crucial role in maint...

Chronic gastritis (CG), one of the most common clinical diseases, is characterized by chronic and persistent inflammatory changes in the gastric mucosa epithelium, which is frequently and repeatedly attacked by various pathogenic factors1. The incidence rate of CG ranks first among all types of stomach diseases, accounting for 40% to 60% of the outpatient service rate in the Department of Gastroenterology2. Moreover, the incidence rate generally increases with age, especially in individuals who are middle-aged and older3. Undoubtedly, CG significantly reduces people&#39;s quality of life, emphasizing the critical need to discover new therapeutic agents.
Numerous studies have reported that the occurrence and development of CG are linked to the secretion of gastrointestinal hormones, such as gastrin (GAS)4,5. GAS, a common gastrointestinal peptide hormone in the digestive tract, stimulates cells to secrete gastric acid by promoting the release of histamine from eosinophils. Additionally, it improves the nutrition and blood supply of the gastric mucosa, promoting the proliferation of gastric mucosa and parietal cells6. Therefore, gastrin can be utilized as an indicator to evaluate the development level of CG. Furthermore, lipid peroxidation products triggered by reactive oxides can activate inflammatory cells, leading to CG. Malondialdehyde (MDA), a lipid peroxidation marker, is a commonly used indicator to measure the degree of oxidative stress. It reflects the level of free radicals in the gastric mucosa to a certain extent. The MDA level can indicate the attack of unsaturated fatty acids in the local gastric mucosa caused by free radicals7,8.
Intestinal microecology consists of millions of microorganisms residing in the host gut, playing a vital role in maintaining host health and regulating host immunity. A healthy intestinal flora, characterized by high richness, diversity, and stable microbiota function, acts as a protective barrier against the invasion of pathogenic microorganisms by participating in metabolism. Disturbances in the intestinal flora make individuals more susceptible to acute and chronic gastrointestinal diseases9,10. In recent years, microbiota therapy for gastrointestinal diseases has progressed rapidly and demonstrated significant efficacy11. In summary, the consideration of intestinal flora is crucial in understanding and addressing gastrointestinal diseases.
As an indispensable component of the treasure trove of traditional Chinese medicine, folk medicinal materials hold great significance for clinical practice and the modernization of national medicine. The roots and the entire parts of Desmodium caudatum (Thunb.) DC. has been widely used for relieving stomach discomfort in the Beichuan Qiang area of Mianyang City for an extended period. Some articles indicate the scientific basis for treating gastrointestinal diseases with D. caudatum, citing its effects such as hemostasis, anti-oxidation, and gastrointestinal protection12,13,14. However, due to the lack of in-depth research, no clinical pharmacodynamics mechanism has been established. This article aims to study the effects of D. caudatum in treating gastritis based on gastrointestinal hormones and intestinal flora, providing a basis for its rational clinical application.

<table><tbody><tr><td>Alpha diversity analysis</td><td>Mothur 1.30.2</td><td></td><td></td></tr><tr><td>AxyPrep deoxyribonucleic acid (DNA) gel extraction kit&amp;nbsp;</td><td>Axygen Biosciences&amp;nbsp;</td><td>AP-GX-50</td><td></td></tr><tr><td>Beta diversity analysis</td><td>Qiime 1.9.1</td><td></td><td></td></tr><tr><td>Cryogenic refrigerator</td><td>Forma-86C ULT freezer</td><td></td><td></td></tr><tr><td>Desmodium caudatum</td><td>The Traditional Chinese Medicine Hospital of Beichuan Qiang Autonomous County</td><td></td><td></td></tr><tr><td>E.Z.N.A. soil kit</td><td>Omega Bio-tek</td><td>D5625-01</td><td></td></tr><tr><td>Illumina MiSeq Platform</td><td>Illumina Miseq PE300/NovaSeq PE250</td><td></td><td></td></tr><tr><td>Multiskan Spectrum</td><td>spectraMax i3</td><td></td><td></td></tr><tr><td>OTU clustering</td><td>Uparse 7.0.1090</td><td></td><td></td></tr><tr><td>OTU statistics</td><td>Usearch 7.0</td><td></td><td></td></tr><tr><td>PCR instrument</td><td>TransGen AP221-02</td><td></td><td></td></tr><tr><td>PCR instrument</td><td>ABI GeneAmp 9700</td><td></td><td></td></tr><tr><td>QuantiFluor-ST double-stranded DNA (dsDNA) system</td><td>Promega Corp.</td><td></td><td></td></tr><tr><td>Sequence classification annotation</td><td>RDP Classifier 2.11</td><td></td><td></td></tr><tr><td>Sodium salicylate</td><td>Sichuan Xilong Chemical Co., Ltd</td><td>54-21-7</td><td></td></tr><tr><td>SPF rats</td><td>Chengdu Dashuo Experimental Animal Co., Ltd</td><td></td><td></td></tr><tr><td>SPSS 18.0</td><td>IBM</td><td></td><td></td></tr></tbody></table>

effects of desmodium caudatum on gastrointestinal hormones and intestinal flora in rats with gastritis

In order to preliminarily explore the effects of Desmodium caudatum on gastritis and intestinal flora in rats, a chronic gastritis rat model was established using the classic sodium salicylate method. Eighteen SPF rats were divided into three groups: the control group (Group C), the model group (Group M), and the treatment group (Group T). Pathological sections of the gastric wall were taken from rats in each group. Furthermore, the concentrations of gastrin and malondialdehyde in the serum of rats in each group were determined by ELISA. Additionally, the effects of D. caudatum on the intestinal flora of rats with gastritis were explored through a detailed comparison of gut bacterial communities in the three groups, employing Illumina-based 16S rRNA gene sequencing. The results indicated that D. caudatum decoction could reduce the malondialdehyde content and increase the gastrin content. Moreover, D. caudatum decoction was found to enhance the diversity and abundance of intestinal flora, exerting a positive impact on the treatment of gastritis by regulating and restoring the intestinal flora.

The results of the pathological section of the stomach wall are depicted in Figure 1. In comparison to Group C, Group M exhibited mild gastric wall atrophy and mild inflammation. However, when compared to Group M, Group T showed no evident inflammation, intestinal metaplasia, or atrophy. This suggests that D. caudatum decoction can effectively improve gastritis.
The serum gastrointestinal hormone assay results are presented in Figure ...

Watch this Scientific Journal Video about Author Spotlight: Exploring the Mysteries of Sichuan's Herbal Medicine in Chinese Medicine Research at JoVE.com

Author Spotlight: Exploring the Mysteries of Sichuan's Herbal Medicine in Chinese Medicine Research

The present protocol describes the method for analyzing intestinal flora using Illumina-based 16S rRNA gene sequencing and provides a framework for evaluating the effectiveness of herbal decoctions.

Effects of Desmodium caudatum on Gastrointestinal Hormones and Intestinal Flora in Rats with Gastritis

In order to preliminarily explore the effects of Desmodium caudatum on gastritis and intestinal flora in rats, a chronic gastritis rat model was ...

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Research

JoVE Journal

Medicine

472 Views.  Mianyang Normal University. The present protocol describes the method for analyzing intestinal flora using Illumina-based 16S rRNA gene sequencing and provides a framework for evaluating the effectiveness of herbal decoctions.

Video: Author Spotlight: Exploring the Mysteries of Sichuan's Herbal Medicine in Chinese Medicine Research

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

Cancer Research

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Biology

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Immunology and Infection

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Experimental Rat Model of Pouchitis for Studying Ulcerative Colitis Pathogenesis and Treatment

A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing lifelong morbidity. When medical treatment is ineffective, especially in cases of massive gastrointestinal bleeding, perforation, toxic megacolon, or carcinogenesis, surgery becomes the last line of defense to cure UC. Total colorectal resection and ileal pouch-anal anastomosis (IPAA) offer the best chance for long-term treatment. Pouchitis is the most common and troublesome postoperative complication. In this investigation, microsurgery is employed to create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat model of pouchitis is established by inducing inflammation of the ileal pouch with dextran sulfate sodium (DSS). The successful establishment of rat pouchitis is validated through analysis of postoperative general status, weight, food and water intake, fecal data, as well as pouch tissue pathology, immunohistochemistry, and inflammatory factor analysis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and treatment of the condition.

Author Spotlight: Developing a Rat Model for Pouchitis Research and Treatment

This protocol describes the method for establishing a rat model of pouchitis. The ileal pouch model was created by performing ileal pouch-anal anastomosis (IPAA) surgery using microsurgical techniques. After the surgery, the rat was treated with 4% dextran sulfate sodium (DSS) for 4 days.

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing ...

Protocol for Studying Gastrointestinal Dynamics and Drug Effects on Motility

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug digestion and absorption and plays a crucial role in treating these diseases. The intestinal tube movement experiment, a common and essential in vitro method, is utilized to study gastrointestinal dynamics. This includes the preparation of the isolated intestinal tube, as well as the suspension of the prepared intestinal tube in the bath and its connection to a signal detector. This is followed by the recording and analysis of a series of parameters, such as tension, which can be used to assess intestinal motor function, as well as considerations for keeping the intestinal tube active in vitro. The standardized program from sampling to data collection greatly improves the repeatability of the experimental data and ensures the authenticity of the recording of intestinal tension after physiological, pathological, and drug intervention. Here we present the key problems in experimental operation and a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

Author Spotlight: Reliable and Reproducible In Vitro Assessment of Drug Impact on Rat Intestinal Tubes

We present a method for isolating rat intestinal tubes and assessing the impact of drugs on their tension, frequency, and amplitude in vitro. This method offers a valuable approach for researchers investigating intestinal tubes.

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug ...

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in alleviating colitis remain undefined. Most analysis methods rely on limited diversity and abundance of gut microorganisms. Therefore, observational studies cannot establish causation. In this study, we applied antibiotic-induced pseudo-germ-free mice to investigate the role of gut microbiota in regulating the probiotic effects of Bacillus&#160;cereus (B. cereus)&#160;on dextran sulfate sodium (DSS)-induced colitis in mice. This process allows for evaluating the bidirectional regulating effect of B. cereus&#160;supplementation on health and provides stable and reproducible results. Here, the detailed protocols for B. cereus cultivation, gavage operation, stool collection, and antibiotic clearance treatment on colitis mice are provided. The optimization methods are also applicable for other chronic inflammatory-associated disorders. The results showed that B. cereus administration decreased body weight loss, colon length shortening, disease activity index, and histopathological scores. However, treatment with antibiotics suppressed the positive effect of B. cereus on colitis. These results indicate that gut microbiota are needed for the alleviating effects of B. cereus on colitis. Therefore, exploring the beneficial effects of probiotics in this research is a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory-associated disorders.

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Here, we present a protocol to establish the antibiotic-induced pseudo-germ-free dextran sulfate sodium-induced colitis mouse model to investigate the role of gut microbiota in regulating the positive effects of Bacillus cereus on colitis.

Effects of Desmodium caudatum on Gastrointestinal Hormones and Intestinal Flora in Rats with Gastritis

Summary

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Chapters in this video

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Gastrointestinal Motility Monitor (GIMM)

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation