3.1. Overall structural profile from phylum to genus in microbiota composition
In order to identify the profile of specific group among 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 position and accounting for 85.1%-90.5% in antibiotic-free treatments. Relatively higher relative abundance (RA) of Firmicutes were detected in medicine intervened group and QHY has richest abundance compare with control. The RA of Proteobacteria and Actinobacteria were obviously increased in M while similarly 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%-58.5%. The RA of Proteobacteria showed a clearly increased with richest RA presented in M_ATB and V_ATB. Considerably enhance of Bacteroidetes richness was illustrated in QHY_ATB (37.7%). The phylum of Firmicutes and Bacteroidetes were two major phyla in rats and humans and Actinomycetes was generally regarded as beneficial bacteria (e.g. Bifidobacterium) (Wei et al., 2018; Zareef et al., 2020). The phylum of Bacteroides including potential pathogen that could be disturbed the immune function, and reduced richness in QHY treated rats revealed the inhabitation of potential pathogen of Chinese medicine.
At the class level, Clostridia was predominant in C, V, M, and QHY (45.6-71.7%), followed by Erysipelotrichia and richness in M and QHY. The RA of Bacilli was slightly increased in QHY (19.6%) than control (10.9%). On the contrary, Erysipelotrichia and Bacteroidia were dominant in antibiotic treatments, and 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. While the RA of Bacilli was decreased and similar abundance of Gammaproteobacteria and Deltaproteobacteria were increased as compare with non-antibiotic treatments. At the order level, Clostridiales was dominant in antibiotic-free treatments and QHY show lowest richness, followed by Lactobacillales was relatively increased in medicine added treatments, Erysipelotrichales was richer in QHY. In contrast, antibiotic-added treatments were dominated by Erysipelotrichales and Bacteroidales that richest in QHY_ATB while Clostridiales was dramatically reduced and the richness of Enterbacteriales and Desulfovibrionales were significantly 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. The proportion of Enterobacteriaceae and Bacteroidacea were increased and Lactobacteriaceae was decreased (<2%) while the RA of Erysipelotrichaceae was clearly increased in antibiotic-added treatments and richest in QHY_ATB. When come to genus level, unidentified and Lactobacillus (11.7-18.5%) were dominant in antibiotic-free treatments. While antibiotic-added treatments were dominated by Allobaculum and Bacteroides, especially richness in QHY_ATB (55.7%). The genus of Faecalibacterium was known as functionally bacterial derived from salicylic acids and butyrate as well as anti-inflammatory molecule producing, Faecalibacterium and Roseburia were involved butyrate generating (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).
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 antibiotic-added treatments, the major proportion of bacterial phylum from Firmicutes convert into Bacteroidetes and Proteobacteria. Major class from Clostridia and Bacilli convert into Bacteroidia, Gammaproteobacteria and Erysipelotrichia. For order, Clostridiales and Lactobacillales were convert into Bacteroidales, Enterobacteriales and Erysipelotrichales. While Lachnospiraceae, Lactobacillaceae and Ruminococcaceae convert into Enterobacteriaceae, Bacteroidaceae and Erysipelotrichaceae at family level. Superior genus from Lactobacillus and unidentified convert into Bacteroides, Allobalulum and Escherichia-Shigella. Therefore, the heat map of dominant bacterial was altered by medicine intervene and antibiotic-added treatment showed the significantly different distribution pattern of bacterial community.
Chinese and western medicine-treated rats showed a certain level difference in bacterial diversity while antibiotic amendment caused a marked depletion in bacterial diversity and richness, which confirmed in previous report (Fröhlich et al., 2016). However, some specific bacterial were show increased patterns under reaction exposure on antibiotics in present study through all sequences detected. Antibiotic-added treatments shown lower RA of beneficial bacteria while higher RA of pathogenic bacterial like Proteobacteria that similar with Zhang et al. (2018). Treatment of 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 micro-biota homeostasis.
3.2. Classification display 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 bacterial mainly 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 phylum of Cyanobacteria was occupied by Veillonellaceae and Gastranaerophilales. It can be observed that the distribution of the bacterial community from distinct treatments were varied through discriminant transformation effect. Among the superior micro-biome phylotypes modulated by Chinese medicine and antibiotic-added intervene in rats. In this study results, we observed several putative beneficial genera containing Lactobacillus, Paraprevotella and Eubacterium. A pronounced depletion of Clostridiales and recovery of Bacteroidales were detected in antibiotic-added treatments. The RA of Enterobacteriales and Verrucomicrobiales were boosted at antibiotic-added treatment. Rats with antibiotic has obviously higher abundance of Bacteroides and lower Clostridium that may palliation the opportunities for pathogenic infection (Zhang et al., 2015ac; Lv et al., 2017).
The remarkably proportion of Lactobacillus in antibiotic-free treatments than antibiotic-added group that might be related with the protective effect of Chinese medicine. Since long time, Chinese medicine has been considered as an important therapy to ameliorate adverse antibiotic reaction comprised diarrhea and dysbiosis (Xu et al., 2017; 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. Various member of Lactobacillus has beneficial or positive effect on colitis and gastroenteritis model test (Rodríguez 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 micro-biota components owing to enable to suppress the metabolic and proliferation of micro-biota 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 especially for beneficial microorganisms of CAG was observably strengthened by Chinese medicine.
3.3. Diversity analysis based on alpha and beta
In order to comprehensively identify the changes of intestinal bacterial composition in 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 operational taxonomic units (OTUs) in the bacterial community was present in chao1 (Fig. 4a), 998, 765, 754, 965, 157, 122 and 146 were detected in C, M, QHY, V, M_ATB, QHY_ATB and V_ATB. The actually number of OTUs observed with the increase of sequencing depth was shown in observed species (Fig.4b), 795, 587, 596, 773, 88, 72, and 81 were identified in the above treatments. The pedigree diversity based bacterial abundance and evolutionary distance were performed in PD whole tree (Fig. 4c). The result shown 58, 46, 45, 51, 13, 9, and 12 were discovered in C, M, QHY, V, M_ATB, QHY_ATB and V_ATB. As well as shannon demonstrated 7.5, 5.9, 5.6, 6.4, 3.5, 3.2, and 4 were present in above treatments (Fig. 4d). Therefore, bacterial diversity of antibiotic-added treatments was obviously decreased. The Venn figure also verified the consistent results (Fig. S2) and indicated the metabolism conspicuously weakened by antibiotic intervention.
The beta diversity aspect of non-metric multi-dimensional scaling (Fig. 5a) and principal component analysis (Fig. 5b) were carried out to compare the bacterial community distribution, further demonstrated an obviously separation among different treatments of overall bacterial structure in rat after medicine and antibiotics treated. The principal components were account for 58.68% and 12.41% of the total variation respectively. Treatments of C and V were clustered one group, M and QHY were gathered while M_ATB distributed individually one group, QHY_ATB gather together and have intersection with V_ATB. In addition, weighted (Fig. 5c) and un-weighted unifrac (Fig. 5d) also demonstrated the differences among all treatments. The degree of dispersion between the medicine and the antibiotic-added 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; Jaan et al., 2020). The diversity of gastrointestinal microbial composition enables to resist the adverse altered in the environment and recover equilibrium after perturbation (resistance and resilience), which was positively facilitated by Chinese medicine.
3.4. Correlation analysis among superior genus based on network
The intestinal microecology plays essential role aspect of health and fight disease and ecological disorders accounting for great proportion in pathophysiology. Interestingly, microorganisms play an essential contribution in maintain the immune system homeostasis steady state and the niche-specific microbial network can be reflected the intestinal microenvironment (Coker et al., 2017; Jaan et al., 2020). Coker et al. (2017) observed the co-occurrence and co-exclusion interaction between GC enrichment and replacement bacteria were increasing with disease progression and external factor intervention. To be specific, present study was indicated that intricate correlations among richness genus, Lactobacillus was strongly positive with Ruminococcaceae_UCG_005 and Christensenellaceae_R7, negatively with Subdoligranulum, Klebsiella and Lachnoclostridium. The bacterial of Bacteroides was positively with Klebsiella, Akkermansia and Subdoligranulum, negatively 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, negatively with Klebsiella, Akkermansia and Subdoligranulum. Akkermansia has positively related with Bilophila, Subdoligranulum, and Morganella, negatively 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 is same with present study and the RA was obviously increased in M-treated. The genus of Akkermansia as a mucus-degrading bacterial that exists in the mucus layer, which enhance the integrity of the intestinal barrier and regulate intestinal metabolism (Everard et al., 2013; Ottman et al., 2017). The genus of 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. 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 and indicated that the selective specific bacterial community correlated with inflammation has altered in case of antibiotic application (Tang et al., 2018).
A Chinese medicine intake has been shown to trigger gut dysbiosis, increase intestinal permeability and alter gut microbiota composition (Xiao et al., 2014; Su et al., 2019). 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 (Liu et al., 2020; Li et al., 2020). Zhou et al. (2016) verified Chinese medicine ginseng stimulated the growth of crucial probiotics Lactobacillus and Bacteroides. The increased number of beneficial bacterial in the intestines Lactobacillus was great significance for inhibiting the proliferation of spoilage bacterial and enhancing the immunity as well as resistance of the body. 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 multiplies that is related to the survival requirement and colonization mechanism of the organism (Manuel et al., 2014). When long-term use of antibiotics might be caused gastrointestinal diseases and showing a state of intestinal micro-ecosystem disorders, owing to pathogens such as Escherichia coli will proliferate while probiotics like Bifidobacterium, Lactobacillus, and Bacteroides were reduced significantly. 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.
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, while the application of QHY can recover new balance and more optimized by improving the diversity of the intestinal microbial. Therefore, this phenomenon possibly has great potential to contributed for the alleviation of clinical inflammation and intestinal microenvironmental homeostasis, which conductive to understanding and apply the effective and promising approach of microbial-specific targeted therapy while its metabolic pathway of action needs to be further studied.