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

Summary

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.

Abstract

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.

Introduction

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'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 radicals^7,8.

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 diseases^9,10. 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 protection^12,13,14. 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.

Protocol

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

1. 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.
2. 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.

2. Grouping and administration

1. 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 days^15,16.
2. 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.
3. 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.
   1. 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.

3. Pathological section of gastric wall

1. 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.

4. Determination of serum gastrointestinal hormones

1. In the serum sample, detect the contents of gastrin (GAS) and malondialdehyde (MDA)¹⁹ using ELISA following the manufacturer's instructions (see Table of Materials). Utilize a microplate reader for the analysis.

5. Fecal total DNA extraction and PCR amplification and purification

1. 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.
2. Amplify the bacteria 16S rRNA gene with primers 338F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GAC TAC HVG GGG GTW TCT AT-3′) 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 °C), followed by 27 cycles (30 s, 95 °C; 30 s, 55 °C; 45 s, 72 °C), and a final extension (10 min, 72 °C). Conduct PCR reactions with the following components: 4 µL of 5× DNA polymerase buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of DNA polymerase, and 10 ng of template DNA (see Table of Materials).
3. Extract, purify, and quantify the PCR products sequentially using 2% agarose gel, commercially available DNA gel extraction kit, and fluorometer, respectively¹⁷.

6. Sequencing

NOTE: Step 6 and step 7 are performed following previously published methods^20,21.

1. 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 "bridge."
2. Perform PCR amplification to generate DNA clusters. Linearize DNA amplifiers into single strands.
3. Introduce an altered DNA polymerase alongside deoxyribonucleotide triphosphates (dNTPs) featuring four distinct fluorescent markers. Synthesize only one base per cycle.
4. 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.
5. Chemically cleave the "fluorescence group" and "termination group" to reestablish the 3' sticky end. Subsequently, advance to polymerizing the second nucleotide.
6. Obtain the sequence of template DNA fragments by analyzing the fluorescence signal results collected in each round.

7. Processing of sequencing data

1. Initially, trim the reads of raw fastq data at any site with a score < Q20 and eliminate reads with uncertain bases. Additionally, permit two mismatches in precisely matched primers. Subsequently, merge the sequences with an overlap >10 bp.
2. Cluster operational taxonomic units (OTUs) with 97% similarity using UPARSE, and remove chimeric sequences with UCHIME^20,21.
3. 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%.
4. Perform Alpha diversity analysis and Beta diversity analysis using Mothur and Qiime, respectively (see Table of Materials).

8. Statistical analysis

1. 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.
2. Consider P < 0.05 as statistically significant in all comparisons. P < 0.01 is regarded as extremely significant.

Results

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 ...

Discussion

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 diseases^22,23. Certain microorganisms in the intestinal tract play a crucial role in maint...

Materials

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