This study found no differences in the alpha-richness and diversity indexes, which were consistent with some reports. Getachew et al. [24] reported no significant differences in bacterial diversity and species richness between saline and antidepressant drug ketamine groups. Furthermore, study by Zhang et al. had compared the gut microbiota of T2DM rats and rats treated with metformin, with no significant differences reported in alpha-richness and diversity indices [25]. It should be noted that the diversity of bacteria was affected by several factors, including health status, age, diet, medication and so on [26]. No difference in the alpha-richness and diversity may be explained in part by the consistency of age and diet among all samples. Part of the reasons may be that RbCl did not affect the diversity of the fecal microbial communities. In addition, we found that RbCl altered the structure of fecal bacterial communities, reflected in changes in fecal microbial composition. Wei et al. [27] observed that microbiota were significantly different between healthy rats and chronic diseased rats. Moreover, Zhang et al. [28] revealed that the microbiota structure changed significantly in response to high fat diet (HFD) feeding and berberine administration. Shifts of microbiota structure were also thought to occur in Crohn’s disease patients [29]. Thus, changes in fecal microbial composition have played an important role in the progression of human diseases.
In our study, we observed that RbCl maintained the abundances of Firmicutes, Bacteroidetes, Actinobacteria, Bacteroides and Helicobacter. Chen et al. reported that Firmicutes significantly reduced in intestinal lumen of patients with colorectal cancer (CRC) [30]. In Crohn’s disease, the abundance of Firmicutes was also significantly decreased [31, 32]. In addition, most works showed that Firmicutes was the higher abundant phylum in breast tissue [33-35]. In depression patients, it was also found that the relative abundance of Firmicutes significantly changed [36, 37], which was related to depression through inflammation [38]. Therefore, these findings indicated that changes of Firmicutes were closely associated with diseases. Anticancer and anti-depressant effects of RbCl might be mediated by maintaining the abundance of Firmicutes in the gut. Bacteroidetes was non-endospore-forming anaerobes with bile resistance, accounting for more than 25 % of gastrointestinal microbiota [39-41]. Proportions of Bacteroidetes were significantly lower in CRC rats than in healthy rats [42, 43]. Although the exact physiological implications of Bacteroidetes in CRC were not fully understood, it was likely that inflammatory bowel diseases were known risk factors for CRC, and a significant reduction of the phylum Bacteroidetes occurred in inflammatory bowel diseases [29, 44]. In addition, Jiang et al. [37] reported that Bacteroidetes were significantly more abundant in active-major depressive disorder subjects. The increase in Bacteroidetes was mainly promoted by Alistipes. Naseribafrouei et al. [45] reported increased abundance of Alistipes in the depressed subjects. Therefore, it can be inferred that changes of Bacteroidetes were closely associated with diseases. The Actinobacteria, which is comprised of gram-positive bacteria, includes 5 subclasses and 14 suborders [46]. Major depressive disorder (MDD) patients characterized by significant increase in the relative abundance of Actinobacteria [36]. Yang et al. [47] reported that the abundance of Actinobacteria was significantly higher in the depression mice. It was possible that enrichment of Actinobacteria was closely related to the development of depression. Exactly, RbCl did not significantly increase the abundances of Actinobacteria. Bacteroides is anaerobic, bile-resistant, non-spore-forming, gram-negative rods [48]. Changes of Bacteroides were assumed to be associated with metabolic diseases such as obesity and diabetes [49, 50]. In Type I diabetes mellitus patients, Bacteroides was significantly increased [51]. The Bacteroides, which was known to be associated with increased gut permeability and inflammation, was positively associated with β-cell autoimmunity. Moreover, Zhu et al. [43] reported greater genera Bacteroides abundance in colon cancer patients compared with controls. It was likely that Bacteroides produced a metalloprotease known as fragilysin, which might favor carcinogenesis. Taken together, these findings indicated that variations of Bacteroides were closely associated with diseases. It should be noted that RbCl did not change the proportion of Bacteroides. Lower abundance of Helicobacter was observed in gut microbiota of overall gastric cancer (GC) patients as compared to healthy controls [52]. It was possible that low proportion of Helicobacter contributed to the pathogenesis of GC. Exactly, RbCl did not change proportion of Helicobacter.
We also found RbCl significantly inhibited the abundances of Tenericutes, Mollicutes, Anaeroplasmatales, Anaeroplasmataceae and Anaeroplasma lineages. Yang et al. [47] reported that the abundance of Tenericutes was significantly lower in the depression mice. RbCl did not improve reduction of Tenericutes, which was consistent with reports. A previous animal study demonstrated that antidepressant drug (R)-ketamine and (S)-ketamine also did not improve the reduced proportion of Tenericutes [47]. Additionally, Ketamine, known to induce antidepressant effects, also significantly reduced abundances of Tenericutes [24]. Tully et al. [53] reported that some species of Mollicutes were significant pathogens in human disease. A study also found that some Mollicutes were associated with diseases [54]. It was worth noting that the abundances of Mollicutes were significantly lower after treatment with RbCl. The reduction of Mollicutes could decrease the pathogenesis of depression and cancers. However, one study has reported a significant reduction in the relative abundance of Mollicutes in MDD patients [47]. As the physiological mechanism of Mollicutes in depression was unclear, further studies on the relationship between depression and Mollicutes are needed. Anaeroplasmatales is an order of Mollicutes bacteria which do not have a cell wall [55]. Song et al. [56] found Anaeroplasmatales significantly increased in depression group. In addition, ketamine, known to induce antidepressant effects, significantly reduced the abundance of Anaeroplasmatales [24]. Exactly, the abundances of Anaeroplasmatales were significantly lower in RbCl groups. Anaeroplasmataceae, which belongs to Class Mollicutes and Order Anaeroplasmatales, is strictly anaerobic wall-less bacteria [57]. The abundance of Anaeroplasmataceae was significantly higher in depression group [56]. Moreover, Anaeroplasmataceae significantly increased in patients with Crohn’s disease localized in the colon (CCD), but significantly decreased in patients with ulcerative colitis (UC) [58]. Interestingly, we observed that RbCl inhibited the proportion of Anaeroplasmataceae. The reduction of Anaeroplasmataceae could decrease the pathogenesis of depression. In the study of colon cancer, Zeng et al. [59] found that the abundance of Anaeroplasma increased in the HFD-azoxymethane (AOM) group. The Anaeroplasma bacteria is negative by Gram stain, which belongs to Mollicutes class, Tenericutes phylum. Anaeroplasma was opportunistic pathogens which elicited various host immune responses in numerous human diseases including colon cancer [60, 61]. Interestingly, the results of RbCl inhibited the proportion of the bacteria.
Expressions of sulfate-reducing bacteria (SRB) including Deltaproteobacteria, Desulfovibrionales, Desulfovibrionaceae and Desulfovibrio were significantly higher in RbCl groups. Deltaproteobacteria belonging to Proteobacteria is sulfate-reducing bacteria [62]. Hydrogen sulfide (H2S) produced by SRB was a process of sulfate reduction [63]. H2S could lead to chronic inflammation and imbalance between cellular proliferation, apoptosis and differentiation by damaging the intestinal epithelium [64]. Reports showed that Deltaproteobacteria was possibly associated with CRC [65, 66]. Jin et al. [67] reported that Deltaproteobacteria was commonly pathogenic bacteria in the intestine. Desulfovibrionales, belonging to Deltaproteobacteria, is also a sulfate-reducing bacteria that can reduce sulfur to produce hydrogen sulfide (H2S) [62]. Desulfovibrionaceae, which was the main biological source of hydrogen sulfate (H2S), involved in a wide range of physiological processes by influencing cellular signaling pathways and sulfhydration of target proteins [68, 69]. Zhang et al. [70] reported that the proportion of Desulfovibrionaceae was higher in animal models of metabolic syndrome. Desulfovibrio could also produce hydrogen sulfide (H2S) by reducing sulfate [71]. H2S derived from Desulfovibrio was associated with gastrointestinal disorders, such as UC, Crohn’s disease, and irritable bowel syndrome [68]. Besides, Hale et al. [72] also reported that Desulfovibrio produced metabolites such as secondary bile acids, which may catalyze the formation of colorectal cancer. However, it should be noted that the proportions of sulfate-reducing bacteria were promoted by RbCl. RbCl led to the enrichment of sulfate-reducing bacteria which could cause inflammation directly or indirectly in mice. It was likely that RbCl used as antigen in healthy mice which could elicit immune responses.
In addition, RbCl significantly increased the abundances of Rikenellaceae, Alistipes and Clostridium XlVa. Wu et al. [73] found that the abundance Rikenellaceae decreased in the colitis-associated colorectal cancer (CAC) group compared with control group. Alkadhi et al. [74] also reported that the proportion of Rikenellaceae reduced in CAC mice. In addition, the report found that Rikenellaceae was overrepresented in healthy control subjects [36]. Following RbCl treatment, the abundance of Rikenellaceae increased in the present study. Therefore, the increase in Rikenellaceae abundance could accelerate the antitumor efficacy of RbCl. Alistipes, which belongs to Bacteroidetes, is present in the human intestinal tract [75]. Alistipes was indole-positive and may thus influence tryptophan availability [76]. In our results, RbCl promoted the abundance of Alistipes. As tryptophan was also the precursor of serotonin, enrichment of Alistipes might affect serotonergic system by interfering with tryptophan metabolism. Clostridium XlVa, belonging to Firmicutes phylum, produces short-chain fatty acids (SCFAs) [77]. The SCFAs produced in the gut are mainly acetate, butyrate and propionate [78]. SCFAs could modulate cell functions either by inhibiting histone deacetylase activity, or through the activation of ‘metabolite-sensing’ G-protein coupled receptors (GPCRs) such as GPR43 and protect the integrity of epithelial barrier [79-81]. RbCl promoted the abundance of Clostridium XlVa. The increase in abundance Clostridium XlVa could alleviate the pathogenesis of depression and cancers. Clostridium XlVa was significantly lower in CRC patients [77]. Clostridium XIVa was overrepresented in healthy control subjects [36, 82].
Regarding the composition of archaea, the abundances of Crenarchaeota, Thermoprotei, Sulfolobales, Sulfolobaceae and Sulfolobus lineages significantly increased in RbCl groups. Crenarchaeota was originally considered to grow in habitats characterized by high temperature, high salinity, or an extreme pH. Later studies found that Crenarchaeota also seem to occur ubiquitously in temperate or cold aquatic [83] and terrestrial environments [84]. The presence of Crenarchaeota in intestinal tracts was reported by Friedrich et al [85]. In addition, Rieu-Lesme et al. [86] suggested that Crenarchaeota was present in the microbiota of the human digestive ecosystem. Thermoprotei, the crenarchaeal class, consists solely of obligate thermophiles. Thermophiles were well-known for participating in rampant lateral gene transfer (LGT) [87, 88]. It was likely that the nature of their extreme environments encouraged the exchange of genetic material. Thermoprotei mostly occurred in the marine environment [89]. However, report showed that Thermoprotei was observed to have an appreciably higher representation in healthy child [90]. Interestingly, the proportion of Thermoprotei was promoted by RbCl in this study. Sulfolobales, a monophyletic group within the Crenarchaeota, is thermophilic sulfur-metabolizing archaea [91]. The report found that Sulfolobales was present in human feces sample [86]. The family Sulfolobaceae is composed of extreme thermoacidophiles that are found in terrestrial environments [92]. The Sulfolobaceae could produce bacteriocin, which played an important role in microbial interaction or microbe-environment interactions, and therefore improved their adaptation in extreme environments [93]. Enrichment of Sulfolobaceae promoted by RbCl may be beneficial in combating disease-related adverse environments. The genera Sulfolobus, which belongs to Sulfologaceae, grows at low pH (2-3) and high temperature (70-85 ℃) [94, 95]. The acidophilic and thermophilic properties of Sulfolobus offered many obvious advantages for industrial applications [96, 97]. In addition, Sulfolobus was able to reduce ferric iron when growing on elemental sulfur as an energy source [98].
Furthermore, RbCl maintained the abundances of archaea Euryarchaeota, Thermoplasmata, Thermoplasmatales, Ferroplasmaceae, Acidiplasma lineages. Euryarchaeota, one of the four major divisions of archaea, contributed substantially to global energy cycling [99]. Euryarchaeota was detected in marine picoplankton [100, 101] and in coastal salt marsh and continental shelf sediments [102]. Methanobrevibacter smithii, which belonged to Euryarchaeota phylum, was a major archaeal player in human gut system [103]. A few studies confirmed that M. smithii was probably involved in inflammatory bowel disease (or Crohn’s disease), irritable bowel syndrome, colorectal cancer, and obesity [104, 105]. Methanobrevibacter oralis, belonging to Euryarchaeota phylum, was the predominating methanogenic species in the oral cavity [103]. M. oralis was identified in apical periodontitis [106]. Therefore, these findings proved that Euryarchaeota might play key roles for human health and disease. However, the proportions of Euryarchaeota did not change after RbCl treatment. Thermoplasmata was affiliated with Euryarchaeota phylum. Auguet et al. [107] showed that Thermoplasmata represented important component of soil microbial communities. In the human body, Li et al. found that Thermoplasmata was not the predominant archaeons in the subgingival dental plaque and Thermoplasmata was closely correlated with chronic periodontitis [108]. Following RbCl treatment, the abundance of Thermoplasmata did not change. Horz et al. found that Thermoplasmatales existed in the human oral cavity [109]. He et al. reported that Thermoplasmatales was also observed in healthy subjects, but the abundance of Thermoplasmatales increased in individuals with periodontitis [110]. It was possible that enrichment of Thermoplasmatales contributed to the pathogenesis of periodontitis. Exactly, RbCl inhibited the enrichment of Thermoplasmatales. The Ferroplasmaceae is represented by cell wall-deficient, acidophilic, facultatively anaerobic and iron-oxidizing archaea [111]. As iron oxidizers, the family Ferroplasmaceae may contribute to the cycle of iron and sulfur [112]. It was likely that Ferroplasmaceae was involved in the pathogenesis of diseases through oxidizing iron. Thus, further studies on the relationships between diseases and Ferroplasmaceae are needed. Interestingly, RbCl did not change the abundance of Ferroplasmaceae.Acidiplasma, which belongs to the family Ferroplasmaceae, order Thermoplasmatales, phylum Euryarchaeota, is a novel acidophilic, cell-wall-less archaeon [113]. The genera Acidiplasma included two species, namely Acidiplasma aeolicum and Acidiplasma cupricumulans [112]. Acidiplasma aeolicum and Acidiplasma cupricumulans were isolated from the hydrothermal pool located on Vulcano Island (Italy) and chalcocite/copper-containing heaps (Myanmar), respectively [113]. It should also be noted that there were no reports on the relationships between Acidiplasma and diseases. In our results, Acidiplasma was observed in stool samples and its abundances were not affected by RbCl. These data showed that fecal archaea were profoundly altered by RbCl, which might provide direct evidence of the relationship between Rb and diseases.
Some reports found Rb could be used as anticancer and anti-depressant drugs. The mechanisms of Rb against cancer and neurological disease remain unclear. Microbiota may participate in the pathogenesis of depression through the brain-gut-microbiota axis [114]. Serotonin (5-HT) is a critical signaling molecule in the brain-gut-microbiota axis [115]. The accumulation of 5-HT and the rate of synthesis of 5-HT in the brain were enhanced by intraperitoneal administration of RbCl [116]. In the present study, Clostridium XlVa, SCFAs producing bacteria, was significantly promoted by RbCl. SCFAs could promote colonic 5-HT production [117, 118]. Enrichment of Alistipes promoted by RbCl might disrupt the intestinal serotonergic system by affecting tryptophan metabolism. Therefore, anticancer and anti-depressant effects of RbCl might be partly mediated by modifying brain-gut-microbiota axis. Nonetheless, additional studies elucidating the relationship between the gut microbiota–brain axis and the anticancer or anti-depressant effects of RbCl are needed.