3.1. Overall structural profile from phylum to genus in microbiota composition
In order to profile the specific distribution characteristics of the gastrointestinal microbial community in case of chronic atrophic gastritis (CAG) rats after medicine and antibiotic intervention, the relative abundance of the top taxa bacterial were identified from phylum to genes among all treatments (Fig. 1). At the phylum level, Firmicutes was most dominant phylum and accounting for 85.1%-90.5% in antibiotic-free treatments. Relatively higher relative abundance (RA) of Firmicutes were detected in medicine added group and QHY has richest abundance compare with control. The RA of Proteobacteria and Actinobacteria obviously increased in M and similar in other treatments. The RA of Bacteroidetes showed a certain level of reduction in medicine group compare with control (2.7–1.2%). While, distinct microbial distribution was descripted for antibiotic added treatments, the RA of Firmicutes was observed significantly decreased till 21.9%-68.5%. The RA of Proteobacteria showed a clearly increased with richest RA presented in M_ATB and V_ATB. Considerable enhance of Bacteroidetes richness was illustrated in QHY_ATB and V_ATB (37.7 and 30.7%).
At the class level, Clostridia was predominant in C, V, YHF M, QHY (45.6–71.7%), followed by Erysipelotrichia and richness in M and QHY, the RA of Bacilli was slightly increased in QHY and YHF (19.6 and 19.2%) than control (10.9%). On the contrary, Erysipelotrichia and Bacteroidia were dominant in antibiotic treatments, the proportion of Clostridia significantly reduced (25.7–4.8%) and richest in M_ATB, the RA of Erysipelotrichia and Bacteroidia were sharply increased and richest in QHY_ATB and YHF_ATB. While the RA of Bacilli was decreased and similar abundance of Gammaproteobacteria and Deltaproteobacteria were increased compare with non-antibiotic treatments. At the order level, Clostridiales was dominant and richest in YHF, followed by Lactobacillales and relatively increased in medicine added treatments, Erysipelotrichales was richer in M and DHY. In contrast, antibiotic treatments were dominated by Erysipelotrichales and Bacteroidales that richness in QHY_ATB and YHF_ATB, while Clostridiales was dramatically reduce and the richness of Enterbacteriales and Desulfovibrionales were increased. At the family level, Ruminococcaceae (29.9–35.5%), Lachnospiraceae (20.8–26.1%) and Lactobaciillaceae (18.5–17.6%) were dominant in antibiotic-free treatments. While the RA of Erysipelotrichaceae was clearly increased in antibiotic treatments and richest in YHF_ATB and QHY_ATB. The proportion of Enterobacteriaceae and Bacteroidacea were increased and Lactobacteriaceae was decreased (< 2%). When come to genus level, unidentified and Lactobacillus (11.7–18.5%) were dominant in non-antibiotic treatments. While antibiotic treatments were dominated by Allobaculum and Bacteroides, especially richness in YHF_ATB (33.5 and 26.9%) and QHY_ATB (55.7 and 33.1%), respectively.
Consistently, Fig. 2 shows the distribution alteration of dominant bacterial in each treatment, there is obvious aggregation effect in the transformation of different medicine intervention. In the case of apply antibiotic, the major proportion of bacterial from Firmicutes convert to Firmicutes, Bacteroidetes and Proteobacteria at phylum. Major class from Clostridia and Bacilli were convert to Bacteroidia, Gammaproteobacteria and Erysipelotrichia. For order, Clostridiales and Lactobacillales were convert to Bacteroidales, Enterobacteriales and Erysipelotrichales. While Lachnospiraceae, Lactobacillaceae and Ruminococcaceae convert to Enterobacteriaceae, Bacteroidaceae and Erysipelotrichaceae at family level. Superior genus from Lactobacillus and unidentified convert to Bacteroides, Allobalulum and Escherichia-Shigella, respectively.
Therefore, the heat map of bacterial RA altered by medicine and antibiotic treatment showed the significantly different distribution pattern of bacterial composition. Firmicutes and Bacteroidetes were two major phyla in rats and humans (Wei et al., 2018). The phylum of Actinomycetes were generally regarded as beneficial bacteria (e.g. Bifidobacterium) (Zareef et al., 2020). The phylum of Bacteroides including potential pathogen that could be disturb the immune function, and reduced in QHY treated rats that result revealed the inhabitation of potential pathogen. Faecalibacterium and Roseburia were involved butyrate generating, Faecalibacterium was known as functionally bacterial derived from salicylic acids and butyrate and anti-inflammatory molecule producing (Ou et al., 2019). Lactobacilli was considered and well-known as probiotic and the RA were significantly decreased in antibiotic feed model rats that would be impair intestinal metabolism, similar with Jaan et al. (2020) and Iino et al. (2018).
Chinese and western medicine-treated rats showed a slight difference in bacterial diversity while antibiotic caused a marked depletion in bacterial diversity and richness, which confirmed previous report (Fröhlich et al., 2016). However, analysis through all sequences detected, some distinct bacterial were show increased patterns under reaction exposure on antibiotics in present study. Antibiotic group shown lower RA of beneficial bacteria while higher RA of pathogenic bacterial like Proteobacteria that same with Zhang et al. (2018). Rat in QHY has highest population of beneficial bacterial of Ruminococcus, Lactobacillus and Bacteroides, while decreased abundance of Desulfovibrio and Proteobacteria that suggesting QHY might be maintain the microbiota homeostasis.
3.2. Classification based comparison of phylum and genus levels
The abundance and evolutionary relationship of genus are visually displayed in phylogeny and cladogram (Fig. 3 and Fig. S1). The chiefly bacterial distributed in Firmicutes, Bacteroidetes and Proteobacteria that consist with Aggeletopoulou et al. (2019), while the composition of bacterial among distinct treatments presented a remarkably variations. To be specific, the dominant phylum of Firmicutes was richness with Ruminococcaceae, Peptostreptococcaceae, Lachnospiraceae, Clostridiaceae_1, Lactobacillaceae and Staphylococcaceae. The Bacteroidetes was richen in Bacteroidaceae and Prevotellaceae. The Proteobacteria was mainly distributed by Enterobacteriaceae, Desulfovibrionaceae, Helicobacteraceae, Alcaligenaceae and Campylobacterales. The phylum of Actinobacteria was distributed by Coriobacteriaceae. The Cyanobacteria was occupied by Veillonellaceae and Gastranaerophilales. It can be observed that the distribution of the bacterial from distinct treatments were different through discriminant transformation effect. Among the superior microbiome phylotypes modulated by Chinese medicine and antibiotic in rats, we observed several putative beneficial genera containing Lactobacillus, Paraprevotella and Eubacterium. A pronounced depletion of Clostridiales and recovery of Bacteroidales were detected in antibiotic treatments, the RA of Enterobacteriales and Verrucomicrobiales were boosted at antibiotic treatment. Rats with antibiotic has obviously higher abundance of Bacteroides and lower Clostridium that may palliation the opportunities for pathogenic infection (Zhang et al., 2015ab; Lv et al., 2017).
The remarkably proportion of Lactobacillus in medicine group than antibiotic treated rats that might be related with the protective effect of Chinese medicine. Chinese medicine has been considered as an important therapy to ameliorate adverse antibiotic reaction comprised diarrhea and dysbiosis (Jaan et al., 2020). The main way for Chinese medicine to promote the proliferation of gut microbial is to play key role of prebiotic-like and selectively stimulating the metabolism of symbiotic beneficial bacterial, such as Lactobacillus, Bacteroides and Bifidobacterium (Xu et al., 2017). Various member of Lactobacillus has beneficial or positive effect on colitis and gastroenteritis model test (Rodríguez-Nogales et al., 2017; Chang et al., 2017; Szajewska et al., 2019). In addition to having beneficial effects on host physiology, some component of symbiotic bacterial could be secrete molecules to limit microbial metabolism and proliferation (Suez et al., 2018). Turroni et al. (2014) found the alteration of Lactobacillales and Bifidobacteriales might be related to microbiota components owing to enable to suppress the metabolic and proliferation of microbiota through decreasing the number or diversity of observed genus and regulate microbial community structure (Suez et al., 2018). Present finding obviously illustrated the intestinal biotransformation of CAG was observably strengthened by Chinese medicine.
3.3. Diversity analysis based on alpha and beta
In order to comprehensively identify the changes in the composition of the intestinal bacterial of CAG rat after interventions with normal saline, Chinese and western medicines as well as antibiotics, alpha and beta diversity analysis were performed to determine whether the community structure and steady state have changed (evaluated from the perspective of diversity and uniformity). The bacterial richness index based on the number of OTUs in the bacterial community was present in Chao1 (Fig. 4a), 1092, 865, 854, 1005, 994, 146, 157, 122 and 84 were detected in C, M, QHY, V, YHF, V_ATB, M_ATB, QHY_ATB and YHF_ATB, respectively. The actually number of OTUs observed with the increase of sequencing depth was shown in observed_species (Fig. 4b), 795, 627, 626, 804, 797, 88, 101, 72, and 57 were identified in the above treatments, respectively. The pedigree diversity based bacterial abundance and evolutionary distance were performed in PD_whole_tree (Fig. 4c). The result shown 58, 46, 45, 51, 54, 13, 12, 9 and 8 were discovered in C, M, QHY, V, YHF, V_ATB, M_ATB, QHY_ATB and YHF_ATB, respectively. As well as Shannon (Fig. 4d) demonstrated 6, 5, 5, 6, 3, 3, 3 and 2 were present in above treatments, respectively. Therefore, bacterial diversity of antibiotic apply treatments were obviously decreased. The Venn figure also verified the same results (Fig. S2) that indicated the metabolism conspicuously weakened by antibiotic intervention.
The beta diversity aspect of principal component (Fig. 5a) and non-metric multi-dimensional scaling analysis (Fig. 5b) were carried out to compare the bacterial community structures, further demonstrated an obviously separation among different treatments of overall bacterial structure in rat after Chinese medicine and antibiotics treated. The principal components were account for 60.27% and 10.57% of the total variation respectively. Treatments of C, YHF and V were clustered one group, M and QHY gathered together, V_ATB and M_ATB distributed one group, QHY_ATB and YHF_ATB gather together and have intersection. In addition, weighted (Fig. 5c) and unweighted_unifrac (Fig. 5d) also demonstrated the difference among treatments. The degree of dispersion between the medicine added and the antibiotic group were different significantly. The west and Chinese medicine group has an overlap and large dispersion with antibiotic group. The affection of antibiotics is mainly focus on reduce the diversity and richness of beneficial intestinal microorganisms, aggravate the intestinal microbial imbalance and increase the probability of the intestinal pathogen invasion and attachment, further caused more serious damage to the intestinal barrier function (Brugiroux et al., 2016). The diversity of gastrointestinal microbial composition enables to resist the adverse altered in the environment and recover equilibrium after perturbation (resistance and resilience) (Jaan et al., 2020), which was positively facilitated by Chinese medicine.
3.4. Correlation analysis among superior genus based on network
The niche-specific microbial network can be reflected the disease microenvironment. Coker et al. (2017) observed the co-occurrence and co-exclusion interaction between GC enrichment and replacement bacteria were increasing with disease progression. Disease-related bacterial can interact and build a synergistic network that may contributions to the diseases. To be specific, present study was presented intricate correlations among richness genus, Lactobacillus was strongly positive with Ruminococcaceae_UCG_005 and Christensenellaceae_R7, negative with Subdoligranulum, Klebsiella and Lachnoclostridium. The bacterial of Bacteroides was positively with Klebsiella, Akkermansia and Subdoligranulum, negative with Eubacterium_coprostanoligenes and Lachnospiraceae_NK4A136. The genus of Bilophila with Subdoligranulum, Akkermansia and Eubacterium_nodatum shown a strong negative correlation. In addition, genus of Blautia was positively correlated with Ruminococcaceae_UCG_014, Ruminococcaceae_NK4A214, Lachnospiraceae_NK4A136 and Eubacterium_coprostanoligenes, negative with Klebsiella, Akkermansia and Subdoligranulum. Akkermansia was positive related with Bilophila, Subdoligranulum, Morganella, negative related with Ruminococcaceae and Lachnospiraceae (Fig. 6).
In view of Blautia can be convert carbohydrates and proteins to acetic acid and further responsible for energy supply. While many researches demonstrated the RA of Blautia was increased in diseases condition such as diabetes, irritable bowel syndrome due to it can be activate inflammatory cytokines (Yan et al., 2016), which was same with present study and the RA was obviously increased in M-treated. Clostridium is the main bacteria producing butyric acid and further fermentation convert to short-chain fatty acids that play an important role in maintaining host health and disease prevention. It can provide the host colonic epithelial cells and promote the growth of intestinal epithelial cells, accelerate the repair of damaged intestinal mucosa, also physiologically regulate the gene expression of intestinal epithelial cells that effectively inhibit the occurrence of enteritis and colorectal cancer. Akkermansia as a mucus-degrading bacterial that exists in the mucus layer (Everard et al., 2013), which enhance the integrity of the intestinal barrier and regulate intestinal metabolism (Ottman et al., 2017).
Interestingly, Chinese medicine intervention restored the microbial community perturbed by CAG holistically that consistent with Zhou et al. (2016) and the mechanisms involved can be intricate. After the intervention of antibiotics, the specific bacterial such as Enterococcus spp. multiplies that is related to the survival requirement and colonization mechanism of the organism (Manuelf et al., 2014). When long-term use of antibiotics might be caused gastrointestinal diseases, pathogens such as Escherichia coli will proliferate while probiotics like Bifidobacterium, Lactobacillus, Bacteroides were reduced significantly, showing a state of intestinal microecosystem disorders. At the same time, the imbalance of microbial will in turn affect the absorption of nutrients, reduce the immunity, weaken the intestinal barrier function, and further aggravate the disease. The genus of Enterococcus can produce tyramine that was related to tyrosine metabolism and can enhance the adhesion of bacteria to the intestinal, further improving colonic mucosal adhesion (Jaan et al., 2020). The RA of Desulfovibrio and Bilophila were increased that indicated the selective specific bacterial community correlated with inflammation has altered in case of antibiotic application (Tang et al., 2018).
The mechanism of Chinese medicine action on the weakened spleen and stomach CAG was mainly to protect the gastric mucosa and reduce inflammation, improve gastric mucosal secretion and regulate gastrointestinal motility, the curative effect is accurate and the safety is high. Zhou et al. (2016) also verified Chinese medicine ginseng stimulated the growth of crucial probiotics Lactobacillus and Bacteroides. The increase in the number of beneficial bacteria in the intestines Lactobacillus is of great significance for inhibiting the proliferation of spoilage bacteria and enhancing the immunity and resistance of the body. This study suggests that the total number of bacteria and Lactobacillus during the treatment of QHY have shown a growth trend, chronic atrophic gastritis disrupts the balance of the normal microbial community, the application of Chinese medicine can recover new balance and more optimized by improving the structure of the intestinal microbial diversity, while its metabolic pathway of action needs further study.