ZHANG Huixiang,WANG Limei,GUO Jipeng,WANG Jiai,ZHANG anqian,WANG Yutao,LIU Xun,ZHANG Lihuan,SHI Lanlan,WU Hongxiang,CAO Xue

ZHANG Huixiang,Institute of Neuroscience,Basic Medical College,Kunming Medical University,Kunming 650500,China

WANG Limei,ZHANG Lihuan,Department of Laboratory Animal Science,Kunming Medical University,Kunming 650500,China

CAO Xue,Department of Laboratory Animal Science,Kunming Medical University,State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,Yunnan University,Kunming 650500,China

WU Hongxiang,Faculty of Rehabilitation Medicine,Kunming Medical University,Kunming 650500,China

GUO Jipeng,School of Electrical and Information Technology,Yunnan Minzu University,Kunming 650500,China

WANG Jiai,School of Medicine,Kunming University,Kunming 650214,China

ZHANG Qianqian,WANG Yutao,LIU Xun,Basic Medical College,Kunming Medical University,Kunming 650500,China

SHI Lanlan,Department of Anatomy,Histology and Embryology,Basic Medical College,Kunming Medical University,Kunming 650500,China

Abstract OBJECTIVE: Yang-deficiency constitution (YADC) is a common unbalanced constitution that predisposes individuals to certain diseases.However,not all people with YADC manifest develop diseases.This calls for delineation of the underlying molecular mechanisms.Previous studies suggested that the gut microbiota and gene differential expression should be considered.METHODS: In the present study,we compared profiles of gut microbiota between four healthy YADC individuals and those of five healthy balanced constitution (BC)counterparts,based on 16S rRNA gene sequence analysis.Furthermore,YADC relevant genes identified by comparing 62 healthy YADC and 58 healthy BC individuals in total to perform intersection analysis,functional clustering and pathway enrichment analyses.RESULTS: The levels of harmful gut microbiota(Prevotellaceae,LDA score ＞ 4.0,P= 0.0141) and beneficial gut microbiota (Ruminococcaceae,LDA score ＞ 4.0,P= 0.0025,Faecalibacterium,LDA score ＞4.0,P= 0.0484) were both elevated in healthy YADC individuals.Also,we found that the specific metabolic pathway with 2,6-Dichloro-p-hydroquinone 1,2-Dioxygenase (PcpA) as the core in gut microbiota and the glutathione transferase activity has been enriched by YADC relevant genes in healthy YADC individuals were both responsible for the detoxification of halogenated aromatic hydrocarbon substances.CONCLUSIONS: Both beneficial and harmful factors had been detected in healthy YADC individuals,functionally,they may have triggered homeostasis to maintain the health of individuals with YADC.The homeostasis may be maintained by beneficial and harmful factors from gut flora and genes.Future studies are expected to focus on halogenated aromatic hydrocarbons and their detoxification processes.

Keywords: Yang deficiency;physical constitution theory;health promotion;gastrointestinal microbiome;genes;hydrocarbons,aromatic;homeostasis

1.INTRODUCTION

Constitution is a Traditional Chinese Medicine (TCM)term,which describes the physical and physiological character of the human body.It is believed that constitution is partly genetically determined and partly acquired.1In fact,the TCM constitutional theory is widely used in disease prevention and treatment.Epidemiological studies have classified the Chinese Han population into nine constitutions,comprising one balanced constitution and eight unbalanced constitutions.2,3Generally,a balanced constitution refers to an overall healthy state of an individual,manifested by fine lustrous complexion,good sleep,good appetite,and good defecation.4On the other hand,an unbalanced constitution denotes a dysfunctional/sub-health status that does not fulfill or achieve diagnostic criteria for a specific disease.2Generally,individuals with unbalanced constitutions are more prone to certain diseases than those in a balanced state.5Yang-deficiency constitution(YADC) is a common unbalanced constitution which is mainly characterized by cold intolerance,such as a cold body and limbs,resulting from an insufficiency of“Yang-Qi” which fails to warm the body.2According to the TCM theory,“Yang-Qi” is the power (energy) that promotes blood circulation and maintains physiological functions of the human body.Consequently,YADC is linked to insufficient warmth and a lack of vigour.Previous studies have implicated it in a high proportion of the constitution types of patients with gastrointestinal cancer,lung and breast cancers,as well as hyperlipidemia,allergic rhinitis,and hypothyroidism,indicating that people with this constitution are susceptible to these chronic diseases and malignancies.6,7According to the TCM theory,constitution related diseases can be prevented or treated by conditioning the constitution.8However,a lack of clarity regarding the actual mechanism of YADC susceptibility of YADC to certain diseases,has limited the identification of targets and detection indexes,thereby affecting the efficacy of TCM.So,it is imperative to evaluate health maintenance of YADC from the “Preventive Treatment of Disease”perspective.Previous studies have shown that individuals with BC are less prone to diseases,compared to those with YADC,since the former condition manifests an overall healthy state.4In addition,several studies seeking to elucidate the molecular basis of YADC,by comparing it with BC,have revealed varying profiles of gut microbiota,as well differential expression of single nucleotide polymorphisms (SNPs),genes,miRNAs and proteins in YADC compared with BC.These differential changes have been related to energy metabolism,immune function,and disease-related pathways.2,4,5,8-10Despite these breakthroughs,the role of these differentially expressed factors in health maintenance of YADC has not been elucidated.

Huanget al10compared gut microbiota between 30 YADC and 30 BC healthy subjects in 20-45-year-old and found only a small amount of differential floras between two groups,as well the gut microbiota was heterogeneous among YADC individuals.They inferred that might be related to a wide age range of subjects.According to the classical TCM theory,viscera functions become strong,and bodies are fully developed,without all invisible declines,at the ages of about 21 and 24 years in men and women,respectively.11The present study investigated the role played by differential gut microbiota and genes in maintenance of a healthy state,between YADC and BC individuals.To ensure accuracy,we selected healthy YADC subjects near 19-24-year-old for analysis of gut microbiota,alongside age-matched healthy BCs as controls.We also analysed the differential expression genes between healthy YADC and BC individuals,using bioinformatics analysis.

2.MATERIALS AND METHODS

2.1.Ethics

Research activities were approved by the local ethics committee of Kunming Medical University,with informed consent obtained from all subjects before enrollment in the study.

2.2.Study subjects

We recruited a group of healthy university student volunteers,from Kunming,Yunnan,China,aged between 19 and 24 years (ethnic Han) in October 2018.Four healthy YADC subjects (the male-female ratio is 1∶1),as well as five healthy balance constitution (BC)counterparts (the male-female ratio is 2 ∶3),were selected in based on the Chinese Traditional Medicine Association standard (ZYYXH/T157-2009) (Table S1 and S2).Their status was confirmed by TCM clinical doctor.Subjects did not take antibiotics,steroid hormones,or Chinese herbal medicine,including oral,intramuscular,or intravenous injections within the three months prior to collection of faecal samples.

2.3.Faecal sample collection,processing,RNA sequencing and data processing

Fresh fecal samples were collected within 24 h of diagnosis,and stored at－80 ℃ immediately.DNA was extracted as previous research described.12The V4 region of 16S rRNA genes was sequenced using standard procedures at the Novogene Bioinformatics Technology Co.,Ltd.,Tianjin,China.13The barcodes and primers,low-quality reads and chimera sequences were removed using the Cutadapt (V1.9.1) and UCHIME software to obtain clean reads.14-16The original sequencing data have been submitted to NCBI Sequence Read Archive(accession number: PRJNA646012).High-quality reads were analyzed with QIIME,17then similarities among samples between the groups estimated using principal coordinates analysis plots (PCoA),and displayed by the ggplot2 package in R software (Version 2.15.3).The bacterial taxa with differential abundance between groups were identified by the linear discriminate analysis(LDA) effect size (LEfSe) method,18with a LDA threshold value of 4.0 and MetaStats at 95% confidence interval，respectively.Potential functional contributions of the observed microbes were inferred using Tax4Fun.19A Venn diagram was generated by Venn Diagram package in R whereas metabolic pathways were visualized using the Interactive pathways (Ipath)explorer v2.

2.4.Differential gene functional analysis

YADC relevant SNP dataset (30 YADC ∶30 BC individuals),20protein dataset (12 YADC ∶12 BC individuals)8and miRNAs dataset (5 YADC∶5 BC individuals)9was collected from previous literature,respectively.Then the target genes of those miRNAs were predicted using miRTarBase with 0.01 as the cutoff.Moreover,YADC-related datasets GSE87474 (12 YADC∶8 BC individuals）and GSE56116 (3 YADC∶3 BC individuals) were downloaded from Gene Expression Omnibus (GEO) database,and performed differentially expressed analysis by GEO2R using correctedP-value ≤ 0.000001/P-value ≤ 0.01 as a cutoff.Combined all above datasets as a YADC relevant gene set and performed the functional clustering and pathway enrichment analysis by DAVID(https://david.ncifcrf.gov/).21

2.5.Statistics

The Student’st-test,permutation test,non-factorial parametric Kruskal Wallis (KW) sum-rank test and Wilcoxon signed-rank test analysis were performed using R (Version 2.15.3).AllP-values reported in the study were from two-tailed tests in clinical data analysis.P-values less than 0.05 was considered to indicate statistical significance both in clinical data analysis and bacterial microbiome analyses.

3.RESULTS

3.1.Composition and function of gut microbiota in YADC and BC subjects

We found a good separation in the gut microbial communities between YADC and BC individuals,consistent with TCM classification (Figure 1A).Specifically,8 bacterial taxa,including Negativicutes,Selenomonadales,Ruminococcaceae,Prevotellaceae,Veillonellaceae,Faecalibacterium,unidentified_Prevotellaceae,unidentified _Ruminococcaceae,were enriched in the YADC group following LEfSe analysis(LDA score ＞ 4.0) (Figure 1B).On the other hand,MetaStats analysis revealed 39 bacterial taxa with significant differences,between the groups,including rare species (relative abundance ＜ 1%).Among these taxa,21 and 18 were higher in the YADC and BC groups,respectively (P ＜0.05) (Table S3,Figure 1C).

Five unique Kyoto Encyclopedia of Genes and Genomes(KEGG) orthologs (KOs) were found in the YADC group (P ＜0.05) (Figure 1D).Among them,we found a special metabolic pathway that centered on 2,6-Dichloro-p-hydroquinone 1,2-Dioxygenase (PcpA)(Figure 1E).PcpA,encodes an aromatic ring-cleaving dioxygenase and is found at the end of the degradation pathway (Figure 1F),plays an important role in degrading isoforms insecticide pentachlorophenol (PCP).Previous studies have also reported production of intermediate products,such as tetrachlorobenzoquinone(TCBQ),tetrachlorohydroquinone (TCHQ),2,6-dichlorohydroquinone (2,6-DCHQ),during the degradation process.22

3.2.Differential genes function in YADC

Transforming all YADC related SNP,miRNA and proteins to their corresponding gene.We got 38 SNP,84 protein and 70 miRNA relevant genes,and 152 different expression genes.We found only one intersection between miRNA and expression,protein and expression,respectively (Figure 1G).342 YADC relevant genes were acquired from combining SNP,miRNA,expression and protein relevant genes.These genes were enriched at 189 different pathways (Table S4),such as some of the genes were enriched together in glutathione transferase activity pathway (GO: 0004364,P ＜0.05),whereas some were enriched together in other pathways related to diseases (Table 1).Glutathione transferase (GST)activity pathway (GO: 0004364) mainly functions to catalyse the degradation of halogenated aromatic compounds,using the following catalytic reaction: RX +glutathione=HX+RS-glutathione (R can be an aliphatic,aromatic or heterocyclic group;X can be a sulfate,nitrile or halogen group).Additionally,PCP and its intermediate metabolites mentioned above also belonged to halogenated aromatic compounds.Furthermore,we found enrichment of several YADC relevant genes in pathways related to microorganisms,including antifungal humoral response (Table 1).

Figure 1 The intestinal flora and genes related to YADC are different from those of BC,which may be related to the disease susceptibility of YADC

4.DISCUSSION

Despite the numerous essential contributions by the human intestinal microbial community to human physiology and health,23the actual role of gut microbiota and their interactions with YADC relevant genes in maintaining healthy status of YADC subjects remain unclear.Here,we compared the gut microbiota between YADC and BC individuals,than analyzed the YADC relevant genes to explore role of two factors in health maintenance in YADC individuals.

In the present study,PCoA of gut bacterial communities revealed a that good separation between YADC and BC individuals,which was contrary to previous study,10possibly because of differences in ages of enrolled subjects in the studies.Results from LEfSe and MetaStats analysis both revealed upregulation of 8 same bacterial taxa in the YADC group.Among them,Prevotellaceae,which comprises of four genera,including Prevotella,has previously been implicated in bodily harm,owing to its association with opportunistic pathogens that exacerbate intestinal infections.24,25We also found elevated levels ofFaecalibacterium,that belongs to the Ruminococcaceae family and a key producer of butyrate (BA).Functionally,BA plays a positive role in suppression of inflammation and anticancer,26and implying that enrichment of these bacterial taxa may be beneficial to the body.It is worth noting that the beneficial and harmful bacteria also increase simultaneously in the YADC in Huang′s research.10In their research,the levels ofButyricimonas，Leuconostocwhich might be benefit for body andRothiawhich might be harmful for body were elevated in YADC (Table S5).It is possible that homeostasis exists between beneficial bacteria and harmful bacteria in healthy YADC individuals.

Table 1 Partial differential gene enrichment pathways

Table 2 Variation in the relative abundance of differential gut microbiota in diseases to which YADC are susceptibility

Functional analysis revealed presence of a special metabolic pathway,centered around 2,6-Dichloro-phydroquinone 1,2-Dioxygenase (PcpA) in YADC cases.PcpA is one of the important enzymes that catalyse PCP degradation.On the other hand,glutathione transferase(GST) activity pathway (GO: 0004364) mainly functions to catalyse degradation of halogenated aromatic compounds.In the present study,functional enrichment analysis showed that some of the YADC relevant genes were enriched in GST activity pathway,indicating their involvement in catalysis of degradation of halogenatedaromatic compounds.Several halogenated aromatic hydrocarbons are reportedly toxic to the body.Specifically,they act as agonists of the body′s aromatic hydrocarbon receptor,whose activation has been shown to play a key role in carcinogenesis.27,28For example,previous studies have shown that the PCP and its degradation intermediates can reduce the body′s immunity and promote tumourdevelopment.29It is therefore possible that another kind of homeostasis exists between halogenated aromatic hydrocarbons and their degradation pathway in healthy YADC individuals.

Some of YADC relevant genes were also enriched in pathways related to microorganisms,suggesting a possible association with human microbiota.Based on this result,we hypothesized that the two types of homeostasis formed by gut microbiota and YADC relevant genes might not exist independently,but work synergistically.Our results also showed few intersections among YADC relevant genes,indicating a high heterogeneity among these genes in YADC,in a similar fashion to gut microbiota.We attributed this heterogeneity to age of subjects enrolled in the present study,relative to a wide range in previous reports (18-50-year-old).Therefore,the expression profiles here in related to the detoxification pathway,and the gut floras may be easier influenced by age due to the lack of “Yang-Qi” in YADC than BC individuals,which leads to easy breaking of the homeostasis.As a result,the antiinflammatory and detoxification process have been hindered,then diseases occur.For example,we found the beneficial flora was down-regulated in diseases which YADC are susceptible to,while the harmful flora was significantly elevated (Table 2).These results indicated existence of a homeostasis between beneficial and harmful substances from gut microbiota and differential genes,which subsequently helps in maintaining healthy status in YADC individuals.An interruption in homeostasis might lead to the occurrence of diseases.

In conclusion,our results provide a molecular explanation of maintenance of health status in YADC individuals,and provide a new perspective for research to guide disease prevention.

However,the study had some limitations.Firstly,we could not infer the source of toxic substances,types of floras involved in detoxification,as well as some other toxic substances and detoxification pathways,owing to the limitation in 16S rRNA sequencing technology.Secondly,our sample size used for experimental analysis of gut microbiota was small,due to limited research funds,and this might have certain limitations on our results.Future studies are expected to increase the number of subjects,as well as combine metagenomic and metabolomics analysis to validate the key homeostasis components.