Over the past 40 years, obesity has become a substantial health economics issue because of its high-risk factor of dyslipidemia, type 2 diabetes, hypertension, and cardiovascular disease (CVD) (2016; Grieve et al., 2013; O'Neill and O'Driscoll, 2015). However, not all obese individuals exhibit characteristics of metabolic disorders. Some obese people are highly sensitive to insulin, have normal blood pressure, average blood glucose, and normal lipid levels. They are often referred to as metabolically healthy obesity (MHO) (Hinnouho et al., 2013; Stefan et al., 2013). A meta-analysis of data from 12 cohorts and seven intervention studies found that almost one-third of obese individuals were metabolically healthy (Lin et al., 2017). Therefore, accurate identification of metabolic abnormalities in obese patients, or those at risk for metabolic abnormalities, and individualized prevention are essential. Over the years, in many independent clinical studies, investigators have typed obesity. One category is based on obesity phenotypes such as BMI, waist circumference, waist-to-hip ratio, and visceral fat content; the other category is based on concomitant metabolic markers such as blood pressure, fasting glucose, triglycerides, (TG), and high-density lipoprotein cholesterol (HDL-C); in addition, some scholars have used homeostasis models to assess insulin resistance or insulin sensitivity (Matthias, 2020). The diversity of obesity typing methods reflects not only the importance of this clinical phenomenon but also the difficulty of typing studies and the need to expand new perspectives in order to get an earlier and more accurate picture of the population at risk for metabolic disorders of obesity.

Traditional Chinese medicine (TCM) is typically individualized medicine, which emphasizes the idea of “tailoring to the individual” and determines the phenotype by summarizing the signs and symptoms exhibited by the patient to guide the choice of treatment. As early as in the Inner Classic of Yellow Emperor, the foundation of Chinese medicine, it is recorded that obesity can be divided into “lipid-fat”, “oily-fat”, and “muscular-fat” based on body shape and lipid muscle distribution. Among them, oily-fat people are similar to the abdominal obese people described in modern medicine and more susceptible to various metabolic diseases. The discipline of TCM constitution (TCMC), which inherits the TCM idea of “tailoring to the individual”, divides people into nine types, including one BC and eight unbalanced constitutions (qi-deficiency constitution, yang-deficiency constitution, yin-deficiency constitution, phlegm-dampness constitution, dampness-heat constitution, blood stasis constitution, qi stagnation constitution, and inherited special constitution), these different constitutions with different susceptibility to diseases (Wang, 2019). PDC is caused by the dysfunction of water metabolism in the body and the coalescence of phlegm and dampness, which manifests itself as fat and flabby abdomen; sticky feeling in the mouth; phlegm in the chest; sweaty, oily forehead; bloated pouch; and thick tongue coating. Many factors influence the formation of phlegm-damp constitution, including heredity or excessive consumption of greasy and sweet foods, as well as long-term handling of humid environments, etc., resulting in impaired spleen function and imbalance of body fluid metabolism. TCM believes that the spleen plays a significant role in the digestive system that produces Qi and blood from digested foods and governs water transportation in the body, similar to the stomach and the small or large intestine. Several studies have shown that PDC is a high risk factor for metabolic diseases (Wang et al., 2011), as the “Common Soil” for cerebrovascular accidents, coronary heart disease, diabetes, hypertension, metabolic syndrome, polycystic ovary syndrome, and sleep apnea syndrome. An epidemiological study (Zhu et al., 2010) based on the relationship between TCMC and overweight/obesity in 18,805 adults in China showed that the risk of obesity was significantly higher in the PDC population (OR=2.05, 95% CI, 1.79-2.35). Multi-omics studies have found that PDC populations present metabolic disorder-related single-nucleotide polymorphisms (Wang et al.) (SNPs), transcriptomic features, DNA methylation modifications (Yao, 2016), and metabolomic features (Li et al., 2016). Further studies revealed significant differences in transcriptome expression profiles and metabolome profiles between obese populations with or without PDC. Obese individuals with PDC exhibit a more significant molecular profile related to metabolic disorders and higher insulin resistance, inflammatory response, and oxidative stress levels. Thus, identifying PDC may be a viable approach to screening high-risk subgroups in obese communities.

It is generally accepted that the human metabolic phenotype is determined by the human genome inherited from the parents. Still, in recent years there is increasing evidence that human commensal bacteria, especially commensal gut flora, has a regulatory effect on the metabolic phenotype of the host (Guarner and Malagelada, 2003). A lot of evidence supports an essential role for the gut microbiota in the progression of obesity and its complications (Boulangé et al., 2016; Peters et al., 2018). Patients with obesity exhibit marked alterations in the structure and composition of the gut microbiota, but the richness (Cotillard et al., 2013) of the gut microbiota decreases with the severity of metabolic complications (Aron-Wisnewsky et al., 2019). A European MetaCardis cohort study showed that gut bacterium 2 (bact2) enrichment in patients with severe obesity was associated with inflammatory markers (Vieira-Silva et al., 2020). Some researchers found that (Gao et al., 2018) obese patients with acanthosis nigricans (AN) had worse metabolic status and a lower microbiota diversity than patients without AN. The above studies suggest that the gut microbiota plays a vital role in obesity development and is also a sensitive indicator for identifying metabolic disorders complicating obesity.

Based on the above studies, we speculate that obese people with PDC may have different microbial composition and structure from those with non-PDC. This differential gut microbiota may contribute to the occurrence of severe metabolic disorders. Our study analyzed the gut microbial characteristics of obese people with or without PDC through pyrosequencing of the 16S rRNA gene, compared with healthy individuals with the BC. We compared their gut microbiota and predicted the functional potential of the bacterial community to explore the association between gut microbiota in obese populations with PDC/non-PDC and obesity complications. In addition, gut microbial abundance was used to construct a disease classifier to distinguish people with PDC from obese individuals. This study helps reveal the microecological mechanisms of the obesity-related constitutions. It has important implications for developing individualized intervention programs targeting intestinal flora to prevent and treat metabolic disorders effectively.

Obesity is a complex condition commonly associated with metabolic abnormalities. To date, there are various methods to measure obesity subtypes, but they cannot accurately predict the metabolic risk associated with obesity. Therefore, to achieve precise control of obesity-associated metabolic abnormalities, new obesity typing methods need to be explored. Phlegm-damp constitution, a concept specific to TCM, is considered an obese subtype with high metabolic risk, and the risk of metabolic abnormalities associated with obesity can be reduced by specific interventions for phlegm-damp constitution. In the present study, we aimed to further understand the role played by PDC in obesity and its complications by exploring PDC-related microbiome changes in obese patients with phlegm-dampness.

Overall, although we observed no significant differences in the abundance of gut bacteria between groups according to the alpha diversity analysis, the beta diversity analysis showed that significant differences in microbial composition in both the PDC group and the non-PDC group/BC group. However, there was no significant difference in beta diversity between the non-PDC group and BC, suggesting variation in PDC-associated gut microbiota compared to obese non-PDC and healthy individuals, but the non-PDC obese population are similar to healthy individuals. Furthermore, we focused on specific taxa changes associated with PDC. In our study, the Firmicutes/Bacteroidetes ratio was decreased in the PDC group. This ratio was elevated in obesity (Chang et al., 2015), hypertension (Li et al., 2017), and autism (Dan et al., 2020), and decreased in a Hypoxia Induced Factor 1α (HIF-1α) induced alcoholic liver disease mouse model (Shao et al., 2018). Bacteroidetes, which are enriched in PDC, are the most abundant bacterial phylum in the human intestine. It is involved in the fermentation of polysaccharide, the utilization of nitrogenous substances, the production of propionates, and is often portrayed as a beneficial bacterium in previous studies (Hayden et al., 2020; Sequeira et al., 2020). However, it is also involved in the release of toxic substances during proteolysis, which promotes inflammation (Jia et al., 2021). In our study, the Bacteroidetes enriched in PDC, which was positively correlated with serum C-reactive protein and systolic blood pressure and negatively correlated with HDL, plays a negative factor for obesity and its complications. Firmicutes depleted in PDC plays an important role in polysaccharide breakdown and production of short-chain fatty (SFCA) acids, many different species of bacteria within several families belonging to the phylum Firmicutes have been identified as SCFAs producers, mainly acetate, propionate, and butyrate, which are anti-inflammatory and improve metabolism effects through reduction of pro-inflammatory factors in the blood, such as LPS (Fernández et al., 2016). Interestingly, the results of the correlation analysis between Firmicutes and clinical phenotypes were opposite to those of Bacteroidetes, with the former enriched with PDC associated with lower SBP, triglycerides, and uric acid, and higher HDL-C. In other words, Firmicutes played a probiotic role in this cohort.

More interesting were the dysbiosis patterns. We found that many of the microbial taxon with altered abundance in the PDC group were associated with the production of SCFAs, plasma bile acid, and anti-inflammatory protection according to several independent studies. Ruminococcaceae depleted in PDC could ferment fiber and other plant components of the diet such as inulin and cellulose, producing short-chain fatty acids (SCFA) which can both be utilized by the host for energy and display anti-inflammatory properties in the gut (Astbury et al., 2020). Cold exposure reduced high-fat diet-induced obesity in mice Their gut microbiotawere characterized by increased levels of Ruminococcaceae, which may be associated with increased iBAT thermogenesis and a plasma bile acid profile (Ziętak et al., 2016). In obese individuals, Prevotellaceae enrichment was elevated, producing circulating succinate, which is a potential microbiota-derived metabolite related to CVD risk (Serena et al., 2018). Notably, and this is consistent with our study, Prevotellaceae were enriched in PDC and were positively correlated with neck circumference and negatively correlated with HDL-C. At the genus level, Faecalibacterium, which is involved in producing intestinal epithelial nutrition, producing short-chain fatty acid, achieving anti-inflammatory effects, and strengthening intestinal barrier functions (Hiippala et al., 2018; Chen et al., 2020), were depleted in obese patients with PDC. Further exploration revealed the depleted Faecalibacterium_prausnitzii in the PDC group, which was found to exert anti-inflammatory activity both in vitro and in vivo ( Llopis et al., 2016; Quévrain et al., 2016). In our study, Prevotella enriched in PDC is one dominant genus of Bacteroidetes, however, it had contradictory results on host metabolic effects in previous studies. Elevated abundance of Prevotella is usually associated with dietary fiber-rich dietary interventions, and enrichment of Prevotella can promote weight loss, lower cholesterol levels, and improve glucose metabolism (Kovatcheva-Datchary et al., 2015; Ortega-Santos and Whisner, 2019). By contrast, another study found that Prevotella copri, was identified as the primary species driving the association between biosynthesis of branched-chain amino acids (BCAAs) and insulin resistance, inducing insulin resistance, exacerbating glucose intolerance, and increasing circulating BCAAs levels (Pedersen et al., 2016), and the abundance of Prevotella copri potentially attenuate the protective effect of the Mediterranean diet on reduction of insulin resistance and cardiometabolic disease risk (Meslier et al., 2020; Wang et al., 2021).Based on the above analysis, we hypothesize that bacteria involved in the synthesis of SFCAs and bile acids are in reduced abundance in PDCs, thus promoting inflammation and increasing the risk of metabolic disorders. However, the above ideas need to be validated by metagenomic sequencing studies with larger sample sizes.

This is emphasized by the predicted gene function based on 16srDNA amplicon sequencing data. The PICRUSt analysis demonstrated that several microbial functions were significantly over- or underrepresented between groups, due to important differences in bacteria composition. Compared with non-PDC people, gut microbiota in PDC individuals had a depleted abundance of genes involved in metabolic pathways such as arginine and proline metabolism, flavone and flavonol biosynthesis, glycolysis/gluconeogenesis, and primary bile acid biosynthesis. Conversely, there was an increase in genes related to arachidonic acid metabolism, glycine, serine and threonine metabolism, the lipopolysaccharide biosynthesis pathway, related to inflammation and immune response. The relative abundance of genes associated with a given pathway may indicate an increased metabolic capacity of the gut microbiota with regard to this pathway. Disturbances in the metabolism of arginine and proline associated with urinary phthalate exposure may contribute to the development of overweight and obesity in school-aged children (Xia et al., 2018). Flavonoids, plant-derived polyphenolic compounds, have been linked with health benefits. For the gastrointestinal tract, it can modulate the secretion of gut hormones, immune system, shape microbiota composition and function, and maintain the intestinal barrier integrity. Importantly, flavonoid actions at the GI tract can have an impact systemically, e.g., on glucose homeostasis, lipid and energy metabolism, or cardiovascular risk factors (Oteiza et al., 2018). Arachidonic acid metabolism has a higher proportion in the PDC group, which is an unsaturated fatty acid, which entails risk for developing intestinal inflammation, obesity, type 1 diabetes, and alcoholic fatty liver (Leiva-Gea et al., 2018; Miyamoto et al., 2019; Mayr et al., 2020; Sun et al., 2020). LPS, a microbial cell wall fraction, is capable of disrupting the intestinal barrier and triggering systemic low-grade inflammation associated with metabolic syndrome, visceral fat mass, and type 1 diabetic nephropathy (Leiva-Gea et al., 2018).

In addition, it is important to mention that waist circumference, hip circumference, and BMI were higher in PDC individuals compared to non-PDC obese individuals, which is similar to previous studies that abdominal and pear-shaped obese individuals who hoard more white fat are at higher risk for metabolic disease (Quan et al., 2020). However, PDC is not the same as abdominal obesity. The constitution is a comprehensive classification method that includes three dimensions of body form, physiological function, and psychological state, and excessive abdominal fat is only one of the characteristics of PDC (Li et al., 2021). In our study, Ruminococcaceae and several ASVs annotated as Faecalibacterium, depleted in PDC group, were negatively correlated with BMI, the other PDC-associated ASVs and bacterial species were not correlated with BMI and waist circumference, the two keys to the diagnosis of obesity. Most of the bacterial species associated with PDC were more strongly correlated with indicators of metabolic disease diagnosis such as TG, HDL, hs-c, uric acid. Notably, ASVs enriched in PDC group had a stronger correlation with HDL, whereas ASVs reduced in PDC had a stronger correlation with TG, LDL, and uric acid. Bacteroidetes and Firmicutes were correlated with HDL, TG, hs-c and Uric acid, and were not associated with obesity-related indicators. These results suggest that changes in PDC-associated bacterial taxa carry a higher metabolic risk rather than more severe obesity.

In general, evidence that obese patients with PDC have a higher risk of inflammatory and metabolic diseases can be observed from the perspective of intestinal microbiota. Our study suggests that gut microbial characteristics can also be used as biomarkers to microscopically differentiate between obese individuals with PDC or non-PDC.

Our study has some limitations. Although our study showed that the gut microbiota of obese patients with PDC may have a higher risk of metabolic disorders, we only assessed microbial characteristics at the same time and did not monitor dynamic changes, which need to be further explored in future longitudinal studies. Second, the effects of many confounding factors were not recorded, such as diet and lifestyle, which may lead to bias in the correlation analysis. In addition, data interpretation may be limited by the relatively small sample size and amplicon sequencing-based analysis of the gut microbiome, and conclusions need to be validated by larger metagenomic studies.

Taken together, our study suggests that the composition of the gut microbiota differs between obese individuals with PDC and non-PDC subjects. Alterations in the gut microbiota of the obese population with the two PDCs were associated to a greater extent with more metabolic disturbances rather than more severe obesity. Such alterations in the gut microbiota may lead to systemic metabolic disturbances by affecting bacterial inflammatory and immune responses, polysaccharide, carbohydrate, and amino acid metabolism, etc. In addition, specific bacterial signatures have shown potential diagnostic value in differentiating obese individuals with different constitutions. These findings may provide a microbiome component to the TCM view that obese individuals with PDC are at high risk for metabolic disease, and also provide a new perspective on obesity typing to aid in more precise prevention and treatment of obesity.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, accession ID: PRJNA810767.

The studies involving human participants were reviewed and approved by the Ethics Committee of Beijing University of Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.

JS, TL, and LZ: study design, data collection, analysis, and writing. QW: study design, give direction to the paper. XL: manuscript writing and revising. YNL and SZ: data collection and analysis. XW, YSL and LL: study design, give direction to the paper. All authors contributed to the articles and approved the submitted version.

This work was supported by the State Key Program of National Natural Science of China (81730112 to QW), Beijing Nova Program (Z201100000820027 to LL), and Innovation Team Project of Beijing University of Chinese Medicine (2019-JYB-TD010 to LL).

Author YNL was employed by School of Traditional Chinese Medicine, Beijing University of Chinese Medicine. YNL was employed by People’s Medical Publishing House Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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