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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 2 | Page : 141-149
Effect of herb-partitioned moxibustion on structure and functional prediction of gut microbiota in rats with irritable bowel syndrome with diarrhea
Xia Liu1, Jia-Nan Cao2, Tao Liu3, Huan Zhong2, Mi Liu2, Xiao-Rong Chang2, Qiong Liu2
1 Department of Traditional Chinese Medicine, Chongqing Three Gorges Medical College, Changsha, China
2 College of Acupuncture and Tuina and Rehabilitation, Hunan University of Chinese Medicine, Changsha, China
3 Department of Acupotomy, Jiangsu Integrated Traditional Chinese and Western Medicine Hospital, Nanjing, China
Correspondence Address:
Dr. Qiong Liu
College of Acupuncture and Tuina and Rehabilitation, Hunan University of Chinese Medicine, Changsha 410208
China
Prof. Xiao-Rong Chang
College of Acupuncture and Tuina and Rehabilitation, Hunan University of Chinese Medicine, Changsha, 410208
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.373586
Objective: The objective of this study was to observe the effect of herb-partitioned moxibustion (HPM) on the gut microbiota of rats with diarrhea-predominant irritable bowel syndrome (IBS-D). Materials and Methods: A total of 48 male rats were randomly divided into a normal control group and an irritable bowel syndrome (IBS) model group. Using acetic acid irrigation and constraint stress, an IBS-D rat model was developed. After the model was made, the IBS rats were divided into IBS, HPM group, and pinaverium bromide (PB) group. The HPM received HPM for 20 min every day, while the PB was given gastric perfusion once a day for 14 days. After modeling and treatment, the abdominal withdrawal reflex, fecal character score, and fecal water content of rats were scored, and a 16S rRNA sequencing analysis was performed on the gut microbiota. Results: After treatment, the fecal character score and fecal water content in the HPM increased significantly, while visceral sensitivity decreased. Investigation of 16S rDNA sequencing revealed that α-diversity was reduced in the IBS, and HPM could increase the diversity of flora. The flora structure of IBS-D rats changed. HPM can increase the abundance of probiotics such as Akkermansia and reduce the abundance of opportunistic pathogens such as Bacteroides and Prevotella. Functional prediction analysis showed that the HPM was mainly related to the bacillary secret system, tricarboxylic acid cycle, and other pathways. Conclusion: HPM can regulate the gut microbiota of rats with IBS-D.
Keywords: Functional prediction, gut microbiota, herb-partitioned moxibustion, irritable bowel syndrome, rats
Irritable bowel syndrome (IBS) is a persistent functioning digestive disorder, characterized mostly by abdominal pain and changes in bowel habits,[1] with an incidence rate as high as 45%.[2] Since there is no specific disease biomarker, clinical manifestations play a pivotal role in the identification of IBS.[3] According to the clinical symptoms of IBS, it can be separated into four categories: diarrhea IBS (IBS-D), constipation IBS, mixed IBS, and undefined IBS. IBS-D is the most common subtype, accounting for 28%–46% of all IBS.[4] Its primary symptoms are diarrhea, abdominal pain, and abdominal distension.[5] The etiology of IBS is not completely clear and may involve various factors, including psychological factors, infection, diet, genetics, immunity, brain–gut axis, and alterations in gut microbiota.[6] In recent years, the role of gut microbiota in IBS has gained increasing attention. Growing evidence shows that the diversity and structural composition of gut microbiota in IBS patients have changed compared with that in healthy people.[7],[8]
Currently, the treatment of IBS mainly aims to minimize the causes and alleviate the symptoms; however, consequences from drug abuse compel patients to explore alternative treatments. Herb-partitioned moxibustion (HPM) is practiced by first affixing an herbal cake to the desired acupoints, and then setting a moxa cone on the herbal cake. Through the thermal effect of moxibustion, the effective ingredients of the medicine penetrate the acupoints through the skin. The physical factors of the moxibustion burning, the effect of herbal cake, and the special function of the acupoints produce a “comprehensive effect” that dredges the meridians, harmonizes the qi and blood, and regulates the viscera. HPM has been proven to effectively regulate gut microbiota and make it develop toward a more normal flora.[9] To investigate the potential mechanism by which HPM helps IBS-D, this research employed 16S rRNA high-throughput sequencing technology to discover alterations in the gut microbiota of IBS-D rats.
Materials and MethodsExperimental animals
Forty-eight SPF male Sprague-Dawley (SD) rats (4 weeks old, weighing 180 ± 10 g) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number: SCXK (Xiang) 2016-0002. The rats were raised in the Experimental Animal Center of Hunan University of Chinese Medicine. The laboratory room temperature was maintained at 22°C ± 1°C, the humidity was 40%–60%, and the alternation of day and night was 12 h/12 h. This study was conducted in accordance with the Animal Experiment Center's requirements for the ethical examination of animal welfare.
Herb-partitioned moxibustion and drug preparation
HPM: The following is the herbal powder composition that was employed during the HPM treatment: Baizhu (Rhizoma atractylodis) (500 g), Baishao (Radix paeoniae alba) (500 g), Chenpi (Pericarpium citri reticulatae) (500 g), and Fangfeng (Radix saposhnikoviae divaricatae) (500 g). Then, they were made into powder and mixed. The powder was combined with vinegar to form a paste, and then, an herbal cake measuring 3 mm thick and 1 cm in diameter was pressed out using a 10 ml syringe. The moxa cone was then placed on the herbal cake for moxibustion.
Drug
Pinaverium bromide (PB) tablets (Abbott Healthcare SAS, France) were used. The daily clinical dose of adults is 150 mg/day, and the drug was made with a concentration of 13.5 mg/kg (150 × 0.018 × 5 = 13.5 mg/kg), which corresponds to the dosage based on body surface area. The intragastric dose was calculated as 10 ml/kg for rats.
Model preparation
After 1 week of adaptive feeding, the rats were divided into two groups: normal control (NC) (12 rats) and IBS (36 rats). The IBS rats were prepared by inserting a silicone tube connected with a syringe through the anus (smeared with paraffin), about 8 cm away from the anus, injecting 1 mL of 4% acetic acid into the anus, slowly pulling out the catheter, pressing the anus with hands and raising the tail of the rats for 30 s, then washing the colon with 1 mL of 0.01 mol/L PBS, and performing restraint stress 7 days later.[10],[11] The rats were then put in a self-made cage for 1 h/day for 2 weeks. The NC rats were given 1 mL of normal saline enema in the same way. IBS was evaluated from Bristol fecal character score, fecal water content, and visceral sensitivity. After verifying the model, the rats in the original IBS were randomly divided into IBS, PT, and HPM, with 12 rats in each group.
Intervenient measures
The NC rats were not given any intervention, and the IBS rats were bound without treatment.
Herb-partitioned moxibustion
After the rats were fixed on the self-made fixator, the hair of the rats near the acupoint was shaved, and the herbal cake was placed directly on the acupoint. Then, the moxa cone was placed on the herbal cake, lit for moxibustion, and after it was completely burned, another moxa cone was used. Each acupoint underwent continuous moxibustion with 45 moxa cones (about 20 min). Five acupoints were divided into two groups. The first group comprised Ganshu (BL18), Pishu (BL20), and Zusanli (ST36), and the second group comprised Zhangmen (LR13) and Qimen (LA14). Two groups of acupoints were alternately treated with moxibustion for 2 weeks. The location of the acupoint was referred to as the “Experimental Acupuncture and Moxibustion Science” combined with an anthropomorphic analogy.
Ganshu: below the 9th thoracic vertebra and 5 mm lateral to the posterior midline.
Pishu: below the 12th thoracic vertebra and 5 mm lateral to the posterior midline.
Zusanli: 5 mm below the fibular head and lateral to the anterior tubercle of the tibia.
Zhangmen: in front of the 11th rib.
Qimen: in the 6th intercostal space.
PT group: PB tablets (Dicetel) were given by gavage once a day for 2 weeks.
Sample preparation
After 2 weeks of therapy, two fecal samples of rats were collected using a sterile EP tube, rapidly frozen in liquid nitrogen, and kept in a refrigerator at −80°C for the identification of intestinal flora. The rats in each group fasted overnight before being anesthetized the following day with an intraperitoneal dose of 1% pentobarbital sodium (50 mg/kg). The section of the colon at a distance of 6 cm from the anus was used for pathological examination.
Observation indicators
Stool observation and score
The rats' feces were examined and assessed using the Bristol Stool Form scale before and after the treatment [Table 1].
Fecal water content
After modeling and treatment, feces were collected from rats in each group for a whole day. The weight of the feces was measured using a tray electronic analytical balance, which measured the wet weight of feces. Next, the feces were placed into a drying oven for 20 min, weighed, and recorded. The process was repeated, with the feces being placed in the drying oven for an additional 5 min each time until the weight of the feces was constant. This final weight represented the dry weight of the feces. Fecal water content was calculated as follows: water content (%) =100 × (wet weight − dry weight)/wet weight.
Abdominal withdrawal reflex score
After modeling and treatment, a paraffin-coated urinary catheter (8F) with an airbag was inserted approximately 6 cm into the anus of the rats while they were awake, and the catheter was fixed at the root of the tail of the rats with medical adhesive tape. After that, the rats' behavior was studied by placing them in a transparent, permanently attached box. The rats were restricted to a single direction of movement inside the box. Water was slowly injected into the airbag when the rats were calm, and the amount of water injected was observed when the abdominal retreat reflex score of the rats was 3 (indicating strong abdominal and back muscle contraction and lifting of the abdomen off the ground). Each experiment lasted for 30 s and was repeated three times per rat at an interval of 4 min.
Pathological examination of the colon
The samples were fixed with 4% paraformaldehyde, dehydrated with ethanol at all levels, and then treated with xylene to achieve transparency. After paraffin embedding and sectioning, h and e staining was performed, and the pathological changes of the colon were observed under a light microscope.
16S rDNA sequencing analysis of rat fecal samples
Fecal samples were sequenced with 16S rDNA technology (completed by Xiamen Anjie Zhishan Medical Data Technology Co., Ltd., project number: AJ1803149811a). Following the protocol in the QIAamp Fast DNA Stool Mini Kit's user manual, the complete genomic DNA of the fecal flora was extracted from 200 mg of rat feces. Multiskan™ GO microplate analyzer was used to quantify DNA concentration and detect DNA purity. The DNA integrity was examined by agarose gel electrophoresis. Primers 341F and 806R were used to amplify the 16S V3-V4 region, and the 16S database was constructed using the Illumina method. Each sample was quantified with Qubit 3.0 and mixed in corresponding proportions according to the requirements. The first detection and measurement of library concentration were performed using Qubit 3.0. After ensuring that the library was fit for sequencing, data were generated using the Illumina HiSeq/MiniSeq high-throughput sequencing technology. After obtaining data from the sequencing platform, Flash software was used for splicing and quality control to obtain high-quality clean reads, and then, chimeric filtering was performed. Finally, a table showing the abundance of OTUs in each sample was generated by clustering using QIIME software. Taxonomic analysis was carried out on the samples at each taxonomic level (mainly phyla and genus) to obtain the community structure diagram of each sample. Picrust 2.0 software was used for function prediction analysis.
Statistical analysis
SPSS 22.0 statistical software (IBM Corp., Armonk, NY, USA) was used for data analysis. The data were expressed as mean ± standard deviation (x¯ ± s). Multigroup comparisons were performed by one-way ANOVA. Two groups were compared using the least significant difference test. P <0.05 demonstrated a statistically significant difference.
ResultsModel evaluation indicators
The scores of fecal scale and fecal water content of rats with IBS were considerably higher than those of rats in the NC group (P < 0.01), suggesting that the diarrhea symptoms of rats with IBS were more severe. The amount of water injected in the IBS group was significantly lower than that in the NC group when the abdominal wall retreat reflex score was 3 (P < 0.01), which was closely related to the increased visceral sensitivity of the IBS [Table 2].
Table 2: Comparison of fecal character score, fecal water content, and water injection of rats in each group after modelingComparison of fecal scale scores, fecal water content, and visceral sensitivity of rats in each group after treatment
The NC rats had clean anuses, and their feces were formed, whereas the rats in the IBS group had soft or unformed feces with more feces adhesion at the anus, and the score of fecal properties was significantly increased (P < 0.01). The rats in the HPM and the PT had rounded, soft stools, and their scores on a measure of stool features were significantly lower than those of rats with IBS (P < 0.01). Rats with IBS had a substantially higher fecal water content than the control group (P < 0.01), whereas rats with HPM and PT had lower fecal water contents than the control group (P < 0.01). Compared with the control group, when the abdominal withdrawal reflex (AWR) score of rats with IBS was 3, the water injection volume was lower, which was related to the increased visceral sensitivity of rats with IBS. However, after treatment with HPM and PB, the water injection volume increased, indicating that HPM and PB had a good effect on the visceral hypersensitivity of IBS-D rats [Table 3].
Table 3: Comparison of fecal scale scores, fecal water content, and water injection of rats in each group after treatmentPathological section detection of rat colon
The H and E staining results of each group showed that the colonic tissue structure was normal, with intact mucosal epithelium and no obvious hyperemia, edema, ulcer, inflammatory cell infiltration, or other histopathological changes. This may be due to the fact that IBS-D is considered a functional gastrointestinal disease [Figure 1].
Figure 1: H and E staining of colon. (a) NC, (b) IBS, (c) PT, (d) HPM. The morphology of colonic mucosa of rats in each group is intact, the epithelial cells are arranged in order, there is no obvious inflammation under the mucosa and no edema in the stroma, and there is no obvious organic change in the morphology and structure. NC: Normal control, IBS: Irritable bowel syndrome, HPM: Herb-partitioned moxibustion, PT: Pinaverium bromideAnalysis of gut microbiota in each group
Alpha-diversity analysis
Alpha-diversity analysis was used to analyze the complexity of microbial community composition in the sample, which can be reflected by Shannon and Simpson indices. In this study, compared with that of the control group, the Shannon index of the IBS decreased (P < 0.01), while the Shannon index of the HPM group increased compared with that of the IBS group (P < 0.05). Although there was a declining trend in the IBS Simpson index, the difference was not statistically significant [Table 4].
Beta-diversity analysis
Beta-diversity analysis is the examination of the composition of microbial communities in various samples. Each dot in the PCoA analysis graph represents a sample, and the various colors stand for distinct groups. The closer the distance between the points, the more similar the samples are. Abscissa PC1 contributed 12.63% and vertical sitting PC2 contributed 10.04%; this may help differentiate the gut microbiota of rats among treatment groups [Figure 2].
Figure 2: Scatter diagram of PCoA analysis. A = NC, B = IBS, C = PT, D = HPM. HPM. Each dot represents a single sample; different colors indicate different groups; and the closer the points are, the more similar the sets are. PCoA was analyzed using the distance matrix calculated from the species composition of the sample. The horizontal and vertical axes represent the contribution rates of the PC1 and the PC2, respectively. NC: Normal control, IBS: Irritable bowel syndrome, HPM: Herb-partitioned moxibustion, PC1: First principal component, PC2: Second principal component, PT: Pinaverium bromideAnalysis of species composition of gut microbiota
Through the analysis of species composition, researchers may examine the gut microbiota on many taxonomic levels (phylum, class, order, family, genus, and species) to determine which species predominate and their abundance. Taking the phylum and genus levels as examples, at the level of phylum, the composition of the bacterial community structure of the four groups of samples showed high similarity, mainly including Firmicutes, Bacteroides, Proteobacteria, Actinobacteria, and Cyanobacteria. The proportion of Firmicutes was the highest, and the relative abundance ratio in each group was: NC (69.72%), IBS (65.52%), PT (67.79%), and HPM (68.16%), followed by Bacteroides with a relative abundance ratio in each group of NC (22.48%), IBS (26.54%), PT (23.88%), and HPM (24.48%). At the genus level, the bacterial community structure of the four groups of samples also showed high similarity, mainly including unclassified Clostridium (un_f_Clostriales), unclassified S24-7 (un_f_S24-7), unclassified Ruminococcaceae (un_f_Ruminococcaceae), Oscillospira, Ruminococcus, Prevotella, Bacteroides, and Lactobacillus. The relative abundance of unclassified Clostridiales in each group was NC (25.14%), IBS (26.37%), PT (24.86%), and HPM (25.45%). The relative abundance of unclassified S24-7 (un_f_S24-7) in each group was NC (14.86%), IBS (13.31%), PT (16.47%), and HPM (17.72%). The relative abundance of Prevotella in each group was NC (2.84%), IBS (4.04%), PT (3.08%), and medicated cake moxibustion group (3.01%). Finally, the relative abundance of Bacteroides in each group was NC (1.92%), IBS (1.59%), PT (1.51%), and medicated cake moxibustion group (1.21%) [Figure 3].
Figure 3: Relative abundance of each group of gut. Microbiota at the level of phylum and genus, A = NC, B = IBS, C = PT, D = HPM. The left figure is the relative abundance of each group of phylum-level flora, the right figure is the relative abundance of each group of genus-level flora, and the icons on the right of each figure are the flora structure at the classification level. NC: Normal control, IBS: Irritable bowel syndrome, HPM: Herb-partitioned moxibustion, PT: Pinaverium bromideAnalysis of species differences
Species whose abundances vary significantly across groups may be identified using linear discriminant analysis (LDA) effect size analysis, which enables the comparison of numerous groups at once. The LDA value distribution circle chart illustrates the species with an LDA score >2, with the length of the histogram showing the impact of significantly different species. The different bacteria in the HPM include Akkermansia of the Verrucomicrobia phylum, and Peptostreptococcaceae. The different bacteria in the PT include Rikenellaceae and α-Alphaproteobacteria, whereas the different bacterial groups of the IBS include Bacteroides and Prevotella, among others. The dominant bacterial species of the NC include Proteobacteria, Flexispira, Helicobacteraceae, Bacteroides, and Desulfovibrionaceae. Further inter-group comparisons showed that compared with the control group, the levels of Prevotella and Bacteroides in IBS were significantly higher, whereas the levels in the drug-cake moxibustion group were lower. Compared with the control group, the digestive streptococcal IBS decreased, while the HPM increased. Akkermansia bacteria was the dominant bacterial group in the HPM, which was significantly higher than in other groups [Figure 4].
Figure 4: The LEfSe multilevel species difference discriminant analysis chart of gut microbiota in each group. A = NC, B = IBS, C = PT, D = HPM. The left figure shows the distribution of LDA values. It shows the species with LDA scores greater than the set value and the species with significant differences in abundance in different groups. The column length represents the impact of significantly different species. The diagram on the right shows the evolution relationship. The circle from the inside to the outside represents the classification level from the door to the genus (or species). A small circle represents a species. The larger the diameter of the circle, the higher the relative abundance. The species with no significant difference in abundance are marked in yellow, and those with a significant difference are marked in the color of corresponding groups. The upper right corner shows the species name in English letters in the corresponding circle. NC: Normal control, IBS: Irritable bowel syndrome, HPM: Herb-partitioned moxibustion, LDA: Linear discriminant analysis, LEfSe: LDA effect size, PT: Pinaverium bromidePrediction and analysis of gut microbiota function
In this study, PICRUSt2 was used to make predictions and conduct analyses on the role of gut microbiota. Based on the findings of the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional categorization, the gut microbiota of each group was mainly related to various metabolic and signal pathways such as lipopolysaccharide biosynthesis proteins, adipocytokine signaling pathway, and lipoic acid metabolism. Further inter-group analysis and comparison showed that compared with that in the control group, the relative abundance of the bacterial secretion system, membrane and intracellular structural molecules, citrate cycle, and transcription machinery functional pathways in the IBS group was increased, while the relative abundance of HPM decreased after the intervention [Figure 5].
Figure 5: Functional prediction of gut microbiota. A = NC, B = IBS, C = PT, D = HPM. The left side of the figure shows the distribution of the KEGG pathway differing significantly between the two categories (different colors). The column length represents the abundance of functional genes in the metabolic pathway of the corresponding sample. The right side of the figure shows the size of the P value of the ANOVA test. A higher color difference indicates a more substantial distinction between categories. NC: Normal control, IBS: Irritable bowel syndrome, HPM: Herb-partitioned moxibustion, PT: Pinaverium bromide Discussion IBS is a common, chronic, functional gastrointestinal disease that can cause symptoms including abdominal pain and changes in stool frequency or character. In this experiment, compared with that in the IBS group, the score of stool characteristics and the fecal water content in the HPM group decreased after the intervention, indicating that the HPM significantly improves diarrhea symptoms. High visceral sensitivity is one of the primary pathophysiological mechanisms of IBS-D. In this experiment, the water injection at the AWR score of 3 points was employed as the visceral sensitivity measurement scale of the IBS-D rat model. The findings demonstrate that the IBS-D rat model presented visceral hypersensitivity, and HPM could benignly regulate it. Evidently, HPM has a significant improvement effect on IBS-D symptoms.
Gut microbiota is crucial to the pathogenesis and development of IBS. Acupuncture and moxibustion can regulate gut microbiota. Qi et al.[12] showed that moxibustion can improve the diversity of gut microbiota in rats with ulcerative colitis (UC), enhance the abundance of Firmicutes, and reduce the level of Bacteroides and Proteus. Wang et al.[13] observed in rats with UC that moxibustion can increase beneficial bacteria such as bifidobacteria and lactobacilli, while reducing colitis-related Escherichia coli and Bacteroides fragilis, indicating that HPM can regulate the disorder of intestinal flora in UC rats. In this work, we employed the 16S rRNA high-throughput sequencing approach to investigate the impact of HPM on the gut microbiota of rats with IBS. The findings demonstrated that HPM could regulate the gut microbiota of IBS-D rats, change their diversity, and regulate their composition.
Alpha-diversity analysis mainly reflects the diversity of microbial communities in the sample, which can be evaluated by Shannon and Simpson indexes. One study has shown that the Simpson index of IBS model rats decreased compared with that of the NC.[9] Zhang et al. demonstrated that the Shannon index was lower in the IBS rats than in the control group, indicating that the bacterial diversity declined.[14] Therefore, most of the current research results show that the diversity of bacteria in IBS patients decreased. In this study, the Shannon index of the IBS decreased compared with that of the NC (P < 0.01), while the Shannon index of the HPM increased compared with that of the IBS (P < 0.05), which is consistent with the previous research results, indicating that the intestinal flora diversity of the IBS-D rat model decreased, while the HPM has positive effects on regulating the gut microbiota diversity. Although the Simpson index of the IBS has a downward trend, the difference is not statistically significant, which may be related to the insufficient quantity of subjects in this study.
The comparative analysis of gut microbiota composition and differential flora shows that, as the dominant flora of the HPM, Akkermansia is a representative Gram-negative bacteria in the microbiota of verruca in the human gastrointestinal tract. It was first isolated from human feces in 2004 and can degrade mucin in the human gut.[15]Akkermansia stimulates goblet cells to produce more mucin by degrading it, thus reducing protein deposition in the intestine. In turn, the new mucin stimulates the growth of Akkermansia. The interaction between the two plays a beneficial role in protecting the intestine. There is a close relationship between Akkermansia and gastrointestinal diseases. Some studies have shown that the number of Akkermansia in patients with IBS decreased compared to that in healthy people.[16] In addition, Akkermansia has a very close relationship with the host's immune system. A study published in Science on lung and kidney cancer showed that Akkermansia is a probiotic, and patients with such probiotics can better receive immunotherapy.[17] The content of Akkermansia in the IBS group decreased while the content in the HPM group increased significantly in this study, indicating that the HPM has beneficial regulatory impacts on Akkermansia. Furthermore, the content of Prevotella in the IBS group increased in this study, which is consistent with previous research results. The research results of Su et al.[18] showed that the number of Prevotella in IBS-D patients continues to increase, indicating that Prevotella is associated with an increased risk of IBS-D. On the one hand, Prevotella may interact with other microflora to aggravate the symptoms of IBS by promoting carbohydrate fermentation to induce visceral hypersensitivity. On the other hand, Prevotella is related to its pro-inflammatory effect. Intervention with Prevotella in mice with colitis induced by dextran sulfate sodium (DSS) will aggravate their symptoms.[19] The abundance of Prevotella in the HPM group decreased significantly in this study, indicating that the HPM has a certain benign adjustment effect on Prevotella. In this study, the content of Bacteroides in the IBS group was higher than that in the NC, but it decreased after the intervention of HPM. This aligned with Zhuang et al.'s findings in their study, where they demonstrated that the content of Bacteroides in IBS-D patients was higher than that in healthy people.[20] However, there are also reports that are inconsistent with the findings of this investigation. Ding et al. revealed that, compared with healthy mice, the spleen deficiency IBS-D mice were characterized by decreased Bacteroidetes.[21] Jeffery's research results on 37 patients with various types of IBS showed that the proportion of Bacteroides in the intestinal flora of IBS patients was lower than that in the intestinal flora of healthy individuals,[22] and its mechanism needs further study. Peptostreptococcaceae are typical Gram-positive bacteria found in the oral cavity, upper respiratory system, intestinal flora of humans, and the female reproductive tract. In this study, the proportion of Peptostreptococcaceae in the IBS group was lower than in the NC but increased after the intervention of HPM in this study. Marie-Claude Denis showed that the content of Peptostreptococcaceae in DSS-induced inflammatory bowel disease increased.[23] However, these results differ from the results of this study, which may be due to the differences in the experimental models selected (C57BL6 mice in the Denis experiment and SD rats in this study), or differences in the reagents used in modeling. The functional prediction investigations revealed that the intestinal flora in each group was closely related to lipopolysaccharide biosynthetic protein, adipocyte factor signal pathway, and lipoic acid metabolism. In this experiment, compared with that in the IBS group, the HPM had a positive effect on tricarboxylic acid (TCA) circulation. In addition to the close relationship between the TCA cycle and energy metabolism, intermediate products of the TCA cycle can modify protein structure, change signal pathways, and regulate inflammation and immune responses.[24] However, further studies are needed to determine how HPM affects intestinal flora through the above channels to achieve a therapeutic effect.
ConclusionTo sum up, HPM has a positive effect on diarrhea symptoms, visceral sensitivity, intestinal microflora structure composition, and metabolic pathway of IBS-D rats. The research results show that HPM can regulate the variability of gut microflora in IBS-D rats by regulating the bacterial flora of Akkermansia, Prevotella, and Bacteroides and then ease the discomfort of IBS-D by affecting the degradation of mucin, immune regulation, visceral hypersensitivity, and intestinal inflammation. This may be one of the important mechanisms of HPM in the treatment of IBS-D, providing an important method for the clinical prevention and treatment of this disease. However, the flora involved in the pathogenesis and progression of IBS-D disease is complex and has not been fully described in this study. Therefore, the specific mechanism of intestinal flora needs further study.
Financial support and sponsorship
This study was financially supported by the National Natural Science Foundation Project (No. 81674084), Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN202002714, KJQN202102708), University Level Project of Hunan University of Chinese Medicine (2021XJJ013), Natural Science Project of Chongqing Three Gorges Medical College (2019XZYB07), and Chongqing Key Discipline of Traditional Chinese Medicine (Basic Theory of Traditional Chinese Medicine) Construction Project (YTCM (2021) No. 16).
Conflicts of interest
There are no conflicts of interest.
References
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