Abstract

This study aimed to investigate the effect of Baohe pill decoction on the bacterial diversity and community structure of the intestinal mucosa of mice with diarrhea caused by feeding HFD and HPD, and provides a scientific basis for the efficacy of Baohe pill decoction in treating this condition. Specific pathogen-free Kunming male mice were randomized into three equal groups, i.e. the control (ftcm), natural recovery (ftmm), and Baohe pill decoction treatment (fttm) groups. The ftcm group was fed a general feed diet, and the ftmm and fttm groups were treated with HFD and HPD. Following the successful induction of diarrhea, the mice in the fttm group were gavaged with Baohe pill decoction, and the mice in the ftmm group were given equal amounts of sterile water. The intestinal mucosal microbiota characteristics were analyzed by 16S ribosomal ribonucleic acid Pacbio single-molecule, real-time full-length gene sequencing. The operational taxonomic unit (OTU) number and alpha diversity in the intestinal mucosal flora of the fttm group were higher than in the ftcm and ftmm groups (P > 0.05), and the composition of the intestinal mucosa bacteria differed between the ftmm and fttm groups. The ftmm group showed a higher abundance of Proteobacteria, Actinobacteria, and Streptophyta, while the Firmicutes, Bacteroidetes, and Verrucomicrobia in the fttm group were higher than that in the other two groups. Compared with the ftmm group, certain beneficial bacteria (Lactobacillus) showed an increased abundance, and pathogenic bacteria (Bacillus, Bradyrhizobium, and Ralstonia) showed a decreased abundance. Bacterial species, such as Lactobacillus (Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus acidophilus and Lactobacillus intestinalis) were significantly higher in the fttm and closely matched those in the ftcm group. Furthermore, various opportunistic pathogens caused by feeding an HFD and HPD were not detected in the fttm group. The current study results indicated that Baohe pill decoction improved diarrhea symptoms by regulating the balance of mucosal microbiota. Specifically, Baohe pill decoction increased the abundance of probiotic Lactobacillus and decreased the abundance of the opportunistic Ralstonia pathogen.

1. Introduction

Gut microbiota is an essential part of the intestinal microbial ecosystem, which plays a vital role in the body's digestion and absorption, energy supply, construction of the intestinal mucosal barrier, and maintaining an intestinal microecological balance ¹. The resident bacteria of the gut microbiota are closely arranged on the intestinal mucosa's surface and constitute the intestinal mucosa's physical barrier, which significantly reduces the opportunity for pathogenic bacteria to invasion. Diarrhea is closely related to intestinal microecology ². When the balance between the host's microbiota is disrupted, and the intestinal flora becomes imbalanced, the risk of developing diseases such as irritable bowel syndrome, antibiotic-associated diarrhea, and ulcerative colitis (UC) will be significantly increased ³. A gut microbiota imbalance clinically manifests as abdominal discomfort, bloating, abdominal pain, diarrhea, and other symptoms; diarrhea, a standard clinical condition, is often a primary symptom in these cases ^{4, 5}.

Dietary proteins and fats are not only primary nutrients needed for the body's daily requirements but also sources of intestinal microbial metabolites ^{6, 7}. Daily protein supplementation is beneficial to human health, and a high-protein diet (HPD) is often used to achieve weight loss and improve muscle function and mass ⁸. The demand for protein-rich foods is expected to reach $90 billion by 2021 ⁹. However, research ¹⁰ suggests that a long-term HPD may damage gut health and inflammation. A new study published in M Systems, a journal of the American Society for Microbiology, showed that a high-fat diet (HFD) and HPD accelerated the proliferation of Clostridium difficile in the guts of mice and inhibited the growth of beneficial bacteria, such as Lactobacillus and Bifidobacteria. Clostridium difficile is closely correlated with diarrhea, which can produce toxin A, toxin B, and binary toxins, leading to diarrhea or pseudomembranous colitis; approximately 20% of antibiotic-associated diarrhea is primarily caused by Clostridium difficile ¹¹. Previously conducted animal experiments ¹² showed that an HFD could increase the number of Bacteroidetes, Actinomycetes, and Proteobacteria in mice and decrease the number of Firmicutes. Moreover, an HPD promoted the proliferation of opportunistic pathogens, such as Clostridium perfringens, but inhibited the growth of Firmicutes ¹³. When the homeostasis of intestinal mucosal flora is disrupted, the growth of beneficial bacteria is inhibited, and pathogenic bacteria multiply ¹⁵, thereby decreasing the intestinal mucosal barrier function. As a result, foreign or transit bacteria colonize and multiply in the gut, becoming dominant and resulting in diarrhea ^{15, 16}. Therefore, one of the critical approaches for treating diarrhea is to restore balance in the gut microbiota.

Baohe pill decoction is derived from the Dan xi Xin Fa and is a classical Chinese medicine prescription for treating food accumulation syndrome caused by an improper diet and excessive dietary intake. Modern pharmacological studies have shown that Baohe pill decoction is antibacterial and can improve digestive enzyme activity, enhance gastrointestinal motility and deliver anti-ulcer properties ¹⁷. You et al. ¹⁸ treated dextran sulfate sodium-induced UC-model mice with Baohe pill decoction and found that it could treat diarrhea by increasing the abundance of intestinal probiotic Akkermansia muciniphila and repair damaged intestinal mucosa. The authors’ research group also found that Baohe pill decoction could treat diarrhea caused by an HFD and HPD by reducing the levels of Lactobacilli and Escherichia coli ¹⁹. These results indicated that Baohe pill decoction had a specific inhibitory effect on gut microbiota; less is known about the effect of Baohe pill decoction on global intestinal mucosal bacteria in mice with diarrhea induced by an HFD and HPD.

The present study was conducted to investigate the effect of Baohe pill decoction on intestinal mucosal bacteria in mice with diarrhea caused by feeding an HFD and HPD using high-throughput sequencing. The therapeutic mechanism of Baohe pill decoction for treating diarrhea was interpreted from a microecological perspective of the intestines to promote the application of this traditional Chinese medicine formula in treating diarrhea.

2. Materials and Methods

Fifteen four-week-old specific pathogen-free Kunming mice (all male), weighing 18–22 g each, were purchased from Hunan Slaccas Jingda Laboratory Animal Co., Ltd. (SCXK [Xiang] 2016-0002). Feeding occurred under stable conditions (temperature 23°C–25°C, relative humidity of 50%–70%, 12 h light/dark cycles, and free access to food and water) in the Laboratory Animal Center of the Hunan University of Chinese Medicine. The study was approved by the Animal Ethics and Welfare Committee of the Hunan University of Chinese Medicine.

The regular fodder used in this study was a 100% basal feed (protein, 20%; fat, 4%) provided by the Laboratory Animal Center of the Hunan University of Chinese Medicine. The HFD and HPD comprised Nestlé milk powder (300 g/bag, product execution standard nr., GB 19644; 30% protein and 20% fat), Huiyi soybean powder (350 g/bag, product execution standard nr., GB/T18738; 33% protein and 18% fat), Huiyi low gluten flour (1,000 g/bag, product execution standard nr., GB/T 8608; 13% protein and 2% fat), and Anhui Lizheng meat pine (500 g/bag; 30% protein and 25% fat) in a ratio of 1:2: 1:1.

For the Baohe pill decoction, 18 g hawthorn (Hebei, nr., HY 20081206), 6 g medicated leaven (Sichuan, nr., 190503), 9 g pinellia ternata (Sichuan, nr., 2004260132), 9 g Poria cocos (Hunan, nr., CK20072102), 6 g forsythia suspensa (Shanxi, nr., HH20070604), 6 g pinellia ternata (Sichuan, nr., 2004260132), and 6 g radish seed (Anhui, nr., 1909180412) were weighed. The ingredients were purchased from the First Affiliated Hospital of the Hunan University of Chinese Medicine. The above ingredients were placed into a porcelain pot with 500 mL water, boiled for 30 min, and then filtered. Then, 400 mL water was added to the residue, and the mixture was boiled for a further 15-20 minutes; the two decoctions were then mixed, evaporated, and concentrated into 180 mL (0.28 g/mL crude drug) and preserved at 4°C.

Following adaptive feeding for two days, 15 Kunming mice were randomly divided into three groups with five males in each group, i.e. the control (ftcm), natural recovery (ftmm), and Baohe pill decoction treatment (fttm) groups. The diarrhea model was established according to the method explored in existing studies ²⁰. The mice in the ftmm and fttm groups were fed with a combination of HFD and HPD, then gavaged with vegetable oil (0.4 mL) from day four, twice a day for three days, and the mice in the ftcm group were gavaged with the same amount of sterile water. When mice in the ftmm and fttm groups successfully developed diarrhea, the fttm group was gavaged with 0.35mL Baohe pill decoction at a dose of 6.67 g·kg⁻¹ day⁻¹ twice a day. The mice in the ftcm and ftmm groups were gavaged with an equal amount of sterile water. After four days of treatment, all the mice were sacrificed by cervical dislocation. The small intestine mucosa in each group was collected under sterile conditions ²¹. After extruding the intestinal contents, the small intestine wall was washed with sterile saline to remove all the adhering content and fat, and the intestinal mucosa was scraped using coverslips and cooled in a –80°C refrigerator.

Intestinal mucosal bacterial metagenomic deoxyribonucleic acid (DNA) was extracted using the cetyl trimethylammonium bromide or sodium dodecyl sulfate method; the full-length bacterial 16S ribosomal ribonucleic acid gene sequence was amplified using the extracted DNA as a template. Primer pair 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5 '-ACCTTGTTACGACTT-3') were provided by Shanghai Pesseno Biotechnology Co., Ltd. (Fig. https://www.personalbio.cn/). The amplification program proceeded as follows: pre-denaturation at 98°C for 2 min; 25–30 cycles (denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s), and a final extension at 72°C for 5 min. The amplification system comprises the following: The polymerase chain reaction (PCR) amplification system, including 2.0 µL deoxyribonucleotide triphosphate (2.5 mmol L−1), 5.0 µL 5 × Q5 reaction buffer, 5.0 µL 5 × Q5 high enhancer, 1.0 µL forwarding primer (10 µmol L−1), 1.0 µL reverse primer (10 µmol L−1), 0.25 µL Q5 polymerase (5 U µL−1), 2.0 µL template DNA (0.2 ng µL−1), and 8.75 µL sterilized ddH₂O. We measured the PCR products by 2% agarose gel electrophoresis. The target fragment was recovered using an AxyPrep DNA gel extraction kit (Oxygen) and quantified using a QuantiFluor^TM-ST handheld fluorometer with a blue channel (Promega).

Selected sequences were clustered into operational taxonomic units (OTUs) with a threshold similarity of 97% and were rowed into one OTU. The OTU table used for analysis was subsequently obtained ²². The abundance of bacteria was reflected via the Chao1, ACE, Simpson, and Shannon indexes, which reflect community diversity. Principal component analysis (PCA), multidimensional scaling (NMDS), and clustering analysis were carried out using the R package (https://www.R-project.org/) to analyze the main distribution characteristics and the similarities between community samples.

The SPSS Statistics 21.0 (IBM Corporation, Armonk, NY, USA) software was used to conduct statistical analysis. Data were expressed as mean ± SE. Significant differences in parameter variance were evaluated either using analysis of variance or a Kruskal–Wallis test, depending on the data normality distribution; P < 0.05 was considered statistically significant.

3. Results

Mice in the ftcm group showed a regular food intake, stable mental state and weight, brown stools, and smooth coat color. The mice in the ftmm and fttm groups showed darkened fur, poor mental condition, abdominal distension, wet and soft feces, and a dirty perianal area; half of the mice successfully developed diarrhea after three days following the feeding of an HFD and HPD. Compared with the ftmm group, the mental status and feces of the mice in the fttm group gradually returned to normal after intervention with Baohe pill decoction for four days.

As shown in Figure 1a, the ftcm, ftmm, and fttm groups had 192, 185, and 212 OTUs, respectively. Among them, 96 were identical. The number of OTUs observed at the bacterial phylum, class, order, family, genus, and species levels in the fttm was higher than those in the ftcm and ftmm , the number of OTUs at each taxonomic level of the ftmm was closely matched that of the ftcm (Figure 1b). These indicated that HFD and HPD can reduce the number of bacterial OTUs in the intestinal mucosa and reduce the bacterial species in mice and Baohe Pill decoction has a certain recovery effect on it. After stopping the HFD and HPD diet intervention, the diversity of intestinal mucosal bacteria gradually returned to normal levels under the self-regulating effect.

Using a sparse curve, the number of OTUs was compared in different samples using the same sequencing depth to measure the diversity of each sample. The smoothness of the curve in the sparse curve reflected the effect of sequencing depth on the diversity of each sample. As shown in Figure 2, the sparse curve fattens after 100 bps. Each curve tended to gradually flatten out, indicating that the amount of sequencing data in our study was sufficient for estimating the vast majority of microbial species information in the sample. The alpha diversity indexes that were applied included the Chao1, ACE, Simpson, and Shannon indexes, which estimated the intestinal mucosal bacteria richness and diversity of intestinal mucosal bacteria ²³. The Chao1 and ACE indexes evidenced the abundance of intestinal mucosal flora (Figure 3a and Figure 3b), while the Shannon and Simpson indexes reflected the community diversity (Figure 3c and Figure 3d). As shown in Figure 3, compared with the ftcm group, the alpha diversity increased in the ftmm and fttm groups; however, there were no significant changes among the three groups (P > 0.05). These results indicated that an HFD, HPD, and Baohe pill decoction treatment could increase the diversity of intestinal mucosal bacteria in mice.

Beta diversity can affect the comparative analysis of the microbial community composition of different samples. To measure beta diversity, principal PCA was used to reflect the differences in each group and the variances in sample composition ²⁴. An unweighted uniFrac measure was used to determine the presence/absence of bacteria or OTUs to compare community composition ²⁵. Figure 4 shows that most of the samples in the ftmm group were close to each other and could be significantly distinguished. Compared with the ftmm group, each sample in the ftcm group was more scattered, suggesting that the difference within the samples may have been relatively significant. This may have been the impact of individual differences in animals, anatomical sampling, DNA extraction and sequencing, and other factors. The results showed that the difference was the largest between the ftcm and fttm groups. Little difference was observed between the ftcm and ftmm groups, consistent with the OTU classification results and the alpha diversity results.

The microbial composition of each group at the phylum level is shown in Figure 5a. Overall, 9 phyla were identified. Proteobacteria was the most predominant phylum in the ftcm, ftmm, and fttm groups, accounting for 55.6%, 59.94%, and 45.73%, respectively. Firmicutes accounted for 32.95%, 25.80% and 41.71%, respectively. In addition, Bacteroidetes and Verrucomicrobia had a higher abundance in the fttm than the ftmm group. In contrast, Proteobacteria and Streptophyta were more abundant in the ftmm than in the ftmm group.

At the genus level, 75 genera of bacteria were detected from samples taken from the three groups. Figure 5b shows the relative abundance of the top 10 among them. The dominant (relative abundance >1%) genera in the ftcm, ftmm and fttm groups were Ralstonia (53.32% vs. 56.05% vs. 43.32%), Lactobacillus (12.27% vs. 8.67% vs. 11.03%), and Afipia (1.76% vs. 1.71% vs. 1.68%). Ralstonia had the highest abundance in the ftmm group and was the lowest in the fttm group. The abundance of Lactobacillus in the ftcm and fttm groups was significantly higher than in the ftmm group, while the abundance of Afipia was not significantly different among the three groups. Remarkably, Pelomonas, Acinetobacter, Phocaeicola, Pseudomonas, and other conditional pathogenic bacteria in the intestinal mucosal flora in the modeling stage were not detected in the intestinal mucosal flora of mice in the treatment stage.

134 species were detected from the samples taken from the three groups at the species level. Figure 5c shows the relative abundance of the top 20 among these. Compared with the fttm, the relative abundance species of reduction in the ftmm group were a filamentous bacterium, Lactobacillus johnsonii, Limosilactobacillus reuteri, Lactobacillus murinus, Limosilactobacillus sp. BG-AF3-A, Lactobacillus intestinalis, Limosilactobacillus sp. c9Ua_26_M, Pelomonas saccharophila and bacterium cL10-2b-4. The species that showed an increase in the ftmm group were Ralstonia insidiosa, bacterium MNFS-9, Afipia geno sp. 1, Ralstonia sp. 1F2, the Bacillus cereus group, Bradyrhizobium sp., Ralstonia pickettii, Solanum nigrum and Aerococcus urinaeequi.

Table 1 shows the main genera that substantially contributed to the Baohe pill decoction experiment involving the intestinal mucosal microbiota of diarrhea mice induce by HFD and HPD. These included Ralstonia (0.006607), Lactobacillus (0.003315) and Bradyrhizobium (0.002598). The related species were primarily Ralstonia pickettii, Ralstonia insidiosa, Ralstonia sp. 1F2, Lactobaci llusmurinus, Lactobacillus johnsonii, Lactobacillus intestinalis and Bradyrhizobium sp.

4. Discussion

Dietary habits have a significant impact on the formation of intestinal flora patterns, and changes in the composition and function of intestinal flora have gradually become potential new targets for the prevention and treatment of diarrhea. Microbial composition affects dietary energy intake. In turn, diet shapes the microbial community structure and activity. Existing studies have shown that an HFD can reduce gut microbiota diversity ²⁶, while protein intake correlates with increased diversity ²⁷. The present study found that an intervention using both an HFD and HPD and treatment using Baohe pills could enrich the alpha diversity of intestinal mucosal bacteria in mice. Further analysis found that an HFD and HPD mainly increased the relative abundance of Proteobacteria (Afipia, Ralstonia, and Bradyrhizobium sp.). Some species of the abovementioned genera are disease-related, particularly concerning infections ^{28, 29, 30}. However, Baohe pill decoction mainly increased the relative abundance of Firmicutes (Lactobacillus). The species and abundance of Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus acidophilus, Lactobacillus intestinalis, Lactobacillus sp. C30An22 and Lactobacillus sp. C30An7 was significantly increased, similar to the results found for the ftcm group. Among these, Lactobacillus johnsonii had a solid resistance to gastric and bile acid, respectively, and could inhibit the adhesion of pathogens to the host’s intestinal mucosa ³¹. Lactobacillus murinus could reduce the number of TH17 cells related to the inflammatory response ³². Both an HFD and HPD and treatment using a Baohe pill decoction could increase the diversity of intestinal mucosal flora in mice, but the focus of the two methods was significantly different. An HFD and HPD mainly increased the types and numbers of various opportunistic pathogens, while Baohe pill treatment could increase the abundance of beneficial flora and genera.

The pathogenesis of diarrhea is complex and often involves a disordered gut microbiota, HFD and HPD interfering with carbohydrates and bile acid metabolism, and the direct effects of HFD and HPD deits ²³. Lactobacillus is one of the dominant bacteria in the gut and promotes the development of helper T cells, thereby inducing cytokine production, enhancing cellular immune function, and inhibiting intestinal pathogens ³⁴. In the authors’ experiment, an HFD and HPD suppressed the abundance of Lactobacillus, which implied colonization resistance and a weakened intestinal mucosal barrier, a situation that will provide niche opportunities for opportunistic pathogenic bacteria (e.g., Afipia, Ralstonia, and Bradyrhizobium sp.). These results implied that diarrhea induced by an HFD and HPD might result from intestinal infections. Inhibiting the development of pro-inflammatory flora in diarrhea may be one of the therapeutic mechanisms of Baohe pill decoction.

Random forest analysis showed that Ralstonia, Akkermansia, Helicobacter, Duncaniella, Parasutterella, and Muribaculum contributed significantly to the diarrhea mice model induced by an HFD and HPD. Ralstonia, Lactobacillus, and Bradyrhizobium contributed considerably to the Baohe pill decoction treatment. The genus in which the two methods intersected was Ralstonia. Studies have found the Ralstonia genus to be an opportunistic pathogen capable of causing bacteremia, meningitis, and septic arthritis ³⁵. More importantly, based on whether a modeling or treatment experiment, respectively, is used, some Ralstonia species showed a higher abundance after feeding an HFD and HPD, e.g., Ralstonia insidious and Ralstonia pickettii, which are waterborne bacteria that were recently considered as emerging pathogens of infectious diseases, in particular, as the pathogens responsible for nosocomial infection in immunocompromised patients ³⁶. Therefore, it is speculated that Ralstonia may be a pivotal genus in the case of diarrhea caused by an HFD and HPD. Additional in-depth validation experiments are needed to establish the characteristic bacteria associated with diarrhea caused by an HFD and HPD. Concurrently, Helicobacter, Akkermansia, and Lactobacillus should be included in such studies.

5. Conclusions

Our analysis indicated that Baohe pill decoction had a modulating effect on the diversity and constitution of the bacterial community in diarrhea mice induced by HFD and HPD. The prescription inhibited the growth of opportunistic pathogen Ralstonia and promoted the growth of probiotic Lactobacillus, and restoring the balance of flora. This study provides a reference for treating diarrhea caused by an HFD and HPD using Baohe pill decoction from the perspective of gut microbiota; it may also provide a theoretical basis for the use of traditional Chinese medicine to provide new ideas for clinical treatment of diarrhea.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81874460).

Conflict of Interest

The authors declare that there is no confict of interests regarding the publication of this paper

Author Contributions

Author contributions were as follows: study design (Zhoujin Tan), data collection (Tao Zheng), statistical analysis (Lili Huang, Yawei Liu), data interpretation (Lili Huang, Tan Guo), manuscript preparation (Lili Huang), and funds collection (Zhoujin Tan, Tan Guo). All authors have read and approved the final manuscript.

Human and Animal Rights Statement

The study was approved by the Animal Ethics and Welfare Committee of Hunan University of Chinese Medicine (LL2020062302).

References