PCOS is an endocrine metabolic disorder with multiple causes and polymorphic clinical symptoms. Chinese medicine can obviously improve the clinical symptoms of obese patients with PCOS, with small side effects, without drug dependence, and with other advantages [19]. BZYQ is an effective prescription for the treatment of obese patients with PCOS and SPSD [20], but its mechanism on the intestinal environment has not been reported yet. In this study, the impact of BZYQ on the gut microbiota and fecal metabolites of obese patients with PCOS and SPSD were discussed through the study of intestinal microecology and nontargeted metabolomics.
Gut microbiota is a general term for sojourn microorganisms in the human intestine, which has physiological functions, such as participating in the body’s nutritional metabolism, antagonizing pathogenic microorganisms, immunity, and maintaining the balance of the internal environment [21]. In TCM theory, the gut microbiota is closely related to the physiological function of the “spleen” [22]. When the gut microbiota is disturbed, gastrointestinal discomfort, decline in digestive and absorption function, and other clinical manifestations will be manifested, similar to the spleen deficiency syndrome in TCM [23]. Obese patients with PCOS often have spleen deficiency symptoms, including obesity, fatigue, loss of appetite, and thin stool. In recent years, increasing studies have confirmed that spleen-invigorating TCM compounds help regulate the gut microbiota and maintain the balance in intestinal microecology [24]. BZYQ is one of the spleen-invigorating TCM prescriptions and have a remarkable impact on recovering the gut microbiota of the host. The regulation of gut microbiota may be one of the mechanisms of the treatment for the spleen deficiency syndrome.
The results of the 16S rRNA high-throughput sequencing demonstrated that the composition structure of the gut microbiota in obese patients with PCOS and SPSD at the phylum and genus levels had changed significantly. At the phylum level, the abundance of Spirochaetae increased significantly after BZYQ treatment, but its abundance in the sample was too small, and the clinical significance was small. At the genus level, the bacteria in the top 20 abundances were compared. After BZYQ treatment, the abundances of [Eubacterium]_rectale_group, Escherichia-Shigella, unclassified_f__Lachnospiraceae, and Fusicatenibacter increased significantly, whereas the abundance of Megamonas decreased significantly. [Eubacterium] _rectale_group is a bacterium that produces butyrate (an anti-inflammatory compound) and plays a key role in fighting inflammation [25]. Cattaneo et al. [26] found that the serum levels of proinflammatory cytokines IL-1β, NLRP3, and CXCL2 in the elderly with cognitive impairment cerebral amyloidosis were negatively correlated with the abundance of [Eubacterium]_rectale_group. The increased abundance of [Eubacterium] _rectale_group in patients with inflammatory bowel disease indicates the enhancement of the anti-TNF-α [27].
PCOS is a chronic inflammatory disease, and chronic nonspecific inflammatory factors affect follicle development, resulting in infertility and adverse pregnancy outcomes by influencing ovarian function, androgen synthesis in vivo, and insulin resistance [28, 29]. The present study suggested that BZYQ may improve chronic inflammation in obese patients with PCOS and SPSD by improving the abundance of [Eubacterium]_rectale_group]. Megamonas could produce short-chain fatty acids (SCFA) [30], which are converted from indigestible carbohydrates by the gut microbiota [31]. Meanwhile, den Besten et al. [32] found that SCFA could activate peroxisome proliferator-activated receptor-γ in the liver and muscle, thereby regulating uptake of glucose and oxidation of fatty acid. In addition, the gut microbiota could influence the insulin sensitivity by SCFA-mediating inflammatory responses [33]. In the present study, the abundance of Megamonas in the patients decreased after BZYQ treatment, which was beneficial for reducing intestinal permeability and maintaining intestinal homeostasis. The Lefse multi-level differential analysis of species showed that the characteristic genera were Dialister, Holdemania, Megamonas, Ruminiclostridium_9, and vadinBC27_wastewater_sludge_group 5 species before treatment, but the characteristic genera after treatment were Fusicatenibacter, Blautia, and Dorea. Studies have confirmed that lipopolysaccharides produced by Gram-negative bacteria are key molecules involved in the early development of inflammation and metabolic diseases, and these bacteria have an endotoxin effect. Gram-negative bacteria could stimulate the production of many inflammatory factors and produce chronic systemic inflammation by binding to the CD14-toll receptor 4 complex on the surface of innate immune cells [34]. These bacteria could also promote insulin resistance via the phosphorylation of insulin receptor substrate 1 through signaling pathways, such as nuclear factor κB. Thus, Dialister was speculated to be associated with chronic inflammation and insulin resistance in obese patients with PCOS and SPSD.
BZYQ also exhibited implications on the fecal metabolites of obese patients with PCOS and SPSD. The contents of taurocholic acid and xanthine were upregulated after BZYQ treatment, while eight differential metabolites, such as palmitic acid, stearic acid, and sphingosine, were downregulated according to the nontargeted metabolomic studies. Taurocholic acid is a primary bile acid that binds amide to the amino group of the cholic acid carboxyl group and taurine. Previous studies [35] have proven that taurocholic acid has a significant inhibitory effect on acute and chronic inflammations, and its mechanism of action is related to its inhibition of macrophage infiltration and the production of pro-inflammatory adipokines. Palmitic acid is a long-chain saturated fatty acid, which is an important component of blood lipids. Some studies [36] have found that free palmitic acid can induce stress of the endoplasmic reticulum and then induce β cell apoptosis and inhibit insulin synthesis and secretion when glucose concentration is too high. Moreover, in the macrophages, palmitic acid induces the inflammatory responses by increasing FABP4/aP2 protein expression [37]. BZYQ may improve the disorder of glucose metabolism and chronic inflammation of obese patients with PCOS and SPSD by reducing palmitic acid abundance. Sphingosine and its metabolic enzymes are key mediators in the human body. Sphingosine kinase and its lipid product, namely, sphingosine 1-phosphate, are involved in signal transduction and diseases, especially in chronic inflammatory diseases and autoimmunity. These molecules play an important role in the occurrence and development of the disease. In the present study, the abundance of sphingosine in obese patients with PCOS and SPSD was reduced after BZYQ treatment, and this event may help reduce the inflammatory response in the body.
In addition, the differential metabolites were significantly enriched in 14 KEGG pathways, such as the biosynthesis of unsaturated fatty acids, fatty acids, cutin, and wax. Most enrichment pathways were lipid metabolism enrichment pathways. The pathogenesis of abnormal lipid metabolism was related to ApoA1, the related regulators of lipid metabolism, adiponectin, leptin, and endogenin [38]. Obirikorang et al. [39] found that a decrease in total blood adiponectin levels can induce IR, obesity, and type 2 diabetes. The gut microbiota is affected by sex hormones and also has an impact on the serum the sex hormone levels [40]. Through the correlation analysis of the gut microbiota–fecal metabolites-serum sex hormones and HOMA-IR values, the abundances of the different bacterial groups after BZYQ treatment were adjusted in different directions, and most of the metabolites involved in KEGG showed an upward trend. Among these bacteria, the abundance of Paraprevotella in obese patients with PCOS and SPSD was positively correlated with serum LH levels, and the correlation was significant. The abundances of Lachnospiraceae_NC2004_group and Faecalibacterium were negatively correlated with the serum DHEAS and T levels, whereas the abundance of [Ruminococcus]_gnavus_group was positively correlated with the aforementioned parameters. The abundance of Blautia was positively correlated with HOMA-IR values. Published studies have proven that Blautia contributes to maintaining glucose stability and its dysregulation impairs the intracellular insulin signaling [41]. The abundance of [Eubacterium]_ventriosum_group was positively correlated with tetracosanoic_acid, but this acid was negatively correlated with serum DHEAS levels. The abundance of Bacteroides was positively correlated with serum T levels but negatively correlated with serum levels. A linear relationship existed among the gut microbiota, fecal metabolite, and hyperandrogenemia. BZYQ may have improved the disorder of hyperandrogen by regulating the abundance of Bacteroides, [Eubacterium]_ventriosum_group, and their differential fecal metabolites.
Although the number of subjects was relatively small, the authors complied strictly by controlling the inclusion and exclusion criteria and excluded the most factors with potential impact on the gut microbiota. In addition, the authors conducted diet guidance and trained sampling method for subjects before sampling. Hence, heterogeneity was greatly reduced in the group, so the authors are confident that the results are greatly meaningful. In this study, fecal metabolomics combined with gut microbiota was used to explore the relationship between the intestinal environment and clinical parameters in obese patients with PCOS and SPSD who were treated with BZYQ.