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.
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.
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 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.
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
2. Grouping and administration
3. Pathological section of gastric wall
4. Determination of serum gastrointestinal hormones
5. Fecal total DNA extraction and PCR amplification and purification
6. Sequencing
NOTE: Step 6 and step 7 are performed following previously published methods20,21.
7. Processing of sequencing data
8. Statistical analysis
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 2. The content of malondialdehyde (MDA) was significantly higher in Group M than in Group C. After treatment with D. caudatum decoction, it was significantly reduced. Regarding the content of gastrin (GAS), Group M showed significantly lower levels than Group C, but Group T exhibited a significant increase. These results indicate that D. caudatum decoction may regulate the level of gastrointestinal hormones to treat gastritis.
Alpha diversity analysis, as shown in Table 1 and Figure 3, reveals a significant difference between the communities of Group M and Group T. The number of bacteria in Group C and Group T is higher than that in Group M, suggesting that the number and types of intestinal bacteria in rats gradually tend to a normal level after treatment.
Species composition analysis
According to Figure 4A, the community barplot illustrates that Lactobacillus and norank_f__Muribaculaceae account for the largest proportion in Group C. Group M contains a higher abundance of Clostridium_sensu_stricto_1 and some Helicobacter. The community composition of Group T is more similar to that of Group C, with important components being Lactobacillus and Romboutsia. Additionally, the Spearman correlation heatmap (Figure 4B) combined with the circos diagram (Figure 4C) reveals significant differences in flora composition between Group C and Group M, with distinct variations in dominant flora. After treatment, Group T tends to return to a normal flora state. Furthermore, Figure 5 indicates extreme differences in community composition between Group M and Group C, with significant changes occurring in the structure and composition of intestinal flora in rats with gastritis. After treatment, there are similarities in flora composition between Group C and Group T, as well as some similarities between Group T and Group M. These results indicate that D. caudatum decoction could restore the intestinal flora of rats with gastritis to a normal or close-to-normal state.
Species difference analysis
From the multi-species comparison column chart in Figure 6A, it can be observed that Norank_f_Muribaculaceae is abundant in Group C and significantly differs from Group M and Group T. After treatment, the number of Norank_f_Muribaculaceae in Group T shows an increasing trend. Additionally, Clostridium_sensu_stricto_1, abundant in Group M, exhibits resilience in harsh environments. After treatment, the number decreases significantly, suggesting that D. caudatum decoction improves the survival environment of intestinal flora to some extent. On the other hand, the LEfSe multi-level species hierarchy tree analysis in Figure 6B indicates 44 species with significant differences between groups. Norank_f_muribaculaceae, Clostridium_sensu_stricto_1, and Romboutsia were found to be abundant in Group C, Group M, and Group T, respectively.
Figure 1: The results of the pathological section of the stomach wall. (A) Pathological section of the stomach in Group C. (B) Pathological section of the stomach in Group M. (C) Pathological section of the stomach in Group T. Please click here to view a larger version of this figure.
Figure 2: The contents of serum gastrointestinal hormones. (A) Bar analysis chart of MDA (malondialdehyde) determination results. (B) Bar analysis chart of GAS determination results. *P < 0.05, **P < 0.01. Please click here to view a larger version of this figure.
Figure 3: Column gram of diversity index T. Red bar represents Group C, blue represents Group T, while green represents Group M.*P < 0.05, **P < 0.01. Please click here to view a larger version of this figure.
Figure 4: Community composition analysis. (A) Community histogram analysis. (B) Community heatmap analysis on the level of Genus. (C) Circos diagram analysis of relationship between samples and species. Please click here to view a larger version of this figure.
Figure 5: PLS-DA analysis on the level of Genus. Red represents Group C, blue represents Group T, and green represents Group M. Please click here to view a larger version of this figure.
Figure 6: Species difference analysis. (A) Kruskal-Wallis H test bar plot. (B) Tree map of LefSe multi-level species. Red represents Group C, blue represents Group T, and green represents Group M. Please click here to view a larger version of this figure.
Group | Mean value | Standard deviation |
C | 3.5201 | 0.63641 |
M | 3.2755 | 0.28494 |
T | 3.5388 | 0.29945 |
Table 1: Alpha diversity index results.
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 maintaining dynamic balance in the body, and hormones in the serum reflect the physiological and pathological state of the body. Therefore, the study focuses on intestinal flora and gastrointestinal hormones.
16S rRNA high-throughput gene sequencing is a primary method for detecting intestinal microorganisms. It enables the detection of microorganism types and functions in collected samples, allowing for accurate and quantitative analysis of each species of intestinal microorganisms. In recent years, 16S rRNA high-throughput gene sequencing has rapidly developed and been widely used for microbial diversity analysis in various ecological environments24,25,26.
The effects of D. caudatum on the intestinal flora of rats with gastritis were investigated through a detailed comparison of gut bacterial communities in three groups based on Illumina-based 16S rRNA gene sequencing. Modern scientific research indicates that Lactobacillus, norank_f__Muribaculaceae, Romboutsia, and Bifidobacterium are probiotics, each with specific health benefits such as reducing the risk of multiple malignant tumors, producing vitamin K with anti-inflammatory potential, promoting polysaccharide metabolism, and improving diarrhea and constipation27,28,29. Clostridium_sensu_stricto_1, a pathogenic bacterium in intestinal injury, can cause inflammation and bacteremia and is closely linked to blood and gastrointestinal tumors30. Chronic gastritis and atrophic gastritis contribute significantly to gastric tumors31.
Beta analysis indicates significant changes in the contents of Lactobacillus, norank_f__Muribaculaceae, Romboutsia, Bifidobacterium, and Clostridium_sensu_stricto_1 after treatment. The contents of Lactobacillus, norank_f__Muribaculaceae, and Romboutsia increased significantly, while Bifidobacterium and Clostridium_sensu_stricto_1 decreased significantly after D. caudatum treatment. Additionally, the community barplot suggests that the community composition of Group T is more similar to that of Group C. These results indicate that D. caudatum could improve the prevalence of gastritis in rats. However, the specific mechanisms behind the reduction in the content of the probiotic Bifidobacterium by D. caudatum require further research.
The ELISA kit method possesses high sensitivity, strong specificity, simplicity in operation, and suitability for small sample sizes32. In this study, it was utilized to detect the contents of malondialdehyde (MDA) and gastrin (GAS) in rat serum. MDA reflects the degree of lipid peroxidation, indirectly indicating the extent of cell damage. In rats with gastritis, the MDA level was significantly higher than that in normal rats, suggesting damage to gastric parietal cells. D. caudatum treatment significantly decreased the MDA level, indicating its potential in treating gastritis. Conversely, D. caudatum increased the reduced GAS level, contributing to the repair or treatment of gastritis.
However, there are limitations to both 16S rRNA high-throughput gene sequencing and the ELISA kit method. In 16S rRNA high-throughput gene sequencing, low resolution may make it challenging to distinguish strains or genera with highly similar sequences, leading to difficulties in classification at higher taxonomic levels such as genus, family, or phylum33. For the ELISA kit method, sample pretreatment steps like dilution and washing are usually required, introducing potential errors and variability34.
This study suggests that D. caudatum may treat gastritis by regulating gastrointestinal hormones and intestinal flora, providing insights into its broad clinical applications. However, future research should delve into other gastrointestinal hormones, the mechanisms of regulating intestinal flora, and the effective chemicals involved.
The authors have nothing to disclose.
This work was funded by the Key R&D projects of the Science and Technology Department of Sichuan Province (2020YFS0539).
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 |