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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 8  |  Issue : 1  |  Page : 123-130

Effect of salivary antimicrobial factors on microbial composition of tongue coating in patients with coronary heart disease with phlegm-stasis syndrome


1 Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
2 Institute of Comparative Medicine, College of Veterinary Medicine, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China
3 Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine; Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China

Date of Submission05-Feb-2021
Date of Acceptance23-Feb-2021
Date of Web Publication21-Jul-2021

Correspondence Address:
Dr. Peng Cao
Nanjing University of Chinese Medicine Jiangsu Province Academy of Traditional Chinese Medicine, 100 Shizi Street, Hongshang Road, Nanjing 210028, Jiangsu
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_34_21

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  Abstract 


Objective: Phlegm-stasis syndrome is one of the most common traditional Chinese medicine (TCM) syndromes and found in 59% of patients with coronary heart disease (CHD) in routine TCM clinical practice in China. One of the diagnostic criteria of phlegm-stasis syndrome is its characteristic white-greasy and thin tongue coating. We have previously reported that different types of tongue coating have different microbiome characteristics that can be used as diagnostic markers. However, the microbial characteristics of tongue coating of CHD patients with phlegm-stasis syndrome, including coating feature and underlying reason of formation, have rarely been reported. Herein, we examined the characteristic microbiome of tongue coating, and discussed the cause of tongue coating formation via salivary proteins in patients with phlegm-stasis syndrome. Methods: We examined white-greasy and thin tongue coatings obtained from 10 patients with CHD having phlegm-stasis syndrome (n = 10), and compared with those of patients with almost no coating – patients with Qi-Yin deficiency syndrome (n = 10) – and healthy controls (n = 10). 16S rRNA sequencing of tongue coating microbiome and isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative analysis of salivary proteins were used to detect tongue coating and salivary protein separately. Salivary levels of sIgA, lysozyme, and amylase were detected by ELISA. Results: We identified Candidatus_Saccharimonas and Candidate_division_TM7_norank as the prominent members of tongue coating in patients with CHD having phlegm-stasis syndrome. Salivary proteins involved in biological processes, pentose phosphate pathway, and complement and coagulation cascades were among the differentially expressed proteins identified in patients with CHD having phlegm-stasis syndrome on iTRAQ analysis. Moreover, the formation of microbiota in tongue coating was associated with salivary sIgA, lysozyme, and saliva flow rate. Conclusions: We explored the characteristics of microbial composition of tongue coating patients with CHD having phlegm-stasis syndrome and identified correlations between salivary proteins and microbiome formation, providing a theoretical and mechanistic basis for tongue coating formation.

Keywords: Coronary heart disease; oral microbiome; salivary proteins; traditional Chinese medicine syndrome; tongue diagnosis


How to cite this article:
Ye J, Su KL, Xu YH, Yang Y, Zhou Q, Gao W, Cai XT, Wei QY, Cao M, Cao P. Effect of salivary antimicrobial factors on microbial composition of tongue coating in patients with coronary heart disease with phlegm-stasis syndrome. World J Tradit Chin Med 2022;8:123-30

How to cite this URL:
Ye J, Su KL, Xu YH, Yang Y, Zhou Q, Gao W, Cai XT, Wei QY, Cao M, Cao P. Effect of salivary antimicrobial factors on microbial composition of tongue coating in patients with coronary heart disease with phlegm-stasis syndrome. World J Tradit Chin Med [serial online] 2022 [cited 2022 May 18];8:123-30. Available from: https://www.wjtcm.net/text.asp?2022/8/1/123/336829




  Introduction Top


Syndrome differentiation (ZHENG classification) is an integral part of the clinical diagnosis and treatment in traditional Chinese medicine (TCM).[1] Tongue diagnosis lays the foundations for syndrome differentiation, and tongue coating is a decisive indicator of the occurrence, development, and prognosis of disease.[2] Microbial composition of tongue coating is a novel and holistic biomarker for characterization of patients' syndromes, namely hot and cold syndrome[3] or Gan-Dan (dampness-heat syndrome) and Gan-stagnancy (Qi-deficiency syndrome).[4] Several studies have assessed the correlation between differences in the microbiota of tongue coating microbiota and specific diseases.[5],[6] Changes in the microbiota of tongue coating can objectively manifest the disease state, which can help to differentiate syndromes, establish treatment methods, prescribe remedial measures, and determine disease prognosis.

Tongue color and coating are the two main characteristics in ZHENG diagnosis of TCM.[7] Healthy individuals are considered to have a thin coating, whereas thick coating is indicative of exuberance of pathogenic factors, frequently because of infection due to exogenous febrile disease-causing pathogens, or phlegm and Qi-dampness or Qi-stagnation. Absence of tongue coating is considered a sign of weakness, indicative of Wei Qi deficiency and Qi and Yin insufficiency. Furthermore, white tongue coating is observed in healthy people and patients with exterior syndromes or cold syndromes. Yellow tongue coating is a sign of hot syndrome or interior syndrome. Abnormal color and shape of the sublingual vessel primarily indicate underlying obstruction of Qi and blood.[8] We previously identified Bacillus as a potential biomarker of yellow tongue coating.[9] However, the microbial characteristics of other variants of tongue coating, including coating feature and underlying reason of formation, have rarely been reported.

Coronary heart disease (CHD) is a life-threatening disease.[10] CHD with pathological characteristics of atherosclerotic coronary artery and myocardial ischemia is more likely to cause phlegm and blood stasis. Phlegm-stasis syndrome is one of the most common TCM syndromes and found in 59% of patients with CHD in routine TCM clinical practice in China.[11] Recently, the diagnosis and treatment of phlegm-stasis syndrome have become the thrust areas of research in the field of TCM syndromes. Tongue coating plays a crucial role in the diagnosis of phlegm-stasis syndrome, with patients presenting a typically white, greasy, and thick coating.[12] Saliva can provide a “window” into the health of an individual[13] and plays an important role in many biological functions, including the maintenance of a balanced oral cavity ecosystem.[14] It also inhibits the growth of exogenous organisms by nonspecific (lysozyme, lactoferrin, and histatins, which have bactericidal and fungicidal actions) and specific (immunoglobulin A [IgA]) defense factors present in the saliva.[15] Studies have reported the association of salivary lysozyme[16] and IgA[17] with CHD. Thus, we hypothesize that salivary proteins influence the formation of microbial composition of white, greasy, and thick tongue coating in patients with CHD having phlegm-stasis syndrome. Thus, we first explored the correlation between saliva and tongue coating and provided a theoretical basis for tongue coating formation.


  Methods Top


Ethical approval

This study was approved by the Jiangsu Province Hospital on Integration of Chinese and Western Medicine Institutional Ethics Review Board (2019 LWKYS-027). Clinical examinations and sample collection were performed after approval from the hospital management. Informed consent was obtained from all individual participants included in the study.

Study population

The study population comprised 20 patients with CHD (mean age, 69.9 ± 4.2 years) and 10 healthy controls (mean age, 53 ± 7.5 years). The syndrome and tongue coating classification followed the “National TCM Diagnosis Principle”[18] established by TCM doctors. Inclusion criteria of patients with CHD were prior myocardial infarction (>1 month before randomization), previous percutaneous coronary intervention, coronary artery bypass graft, or multivessel CHD confirmed by coronary angiography.[19],[20] Exclusion criteria included participants not taking antibiotics and no oral disease within 3 months.

Tongue coating samples

Tongue coating samples were collected and photographed between 08:00 and 11:00 h in the morning by a single examiner. All participants gargled thrice using 10 mL of sterile water each time to remove residual food before sampling. Tongue coating samples were collected as described previously.[9] The samples were stored in liquid nitrogen immediately after acquisition.

Amount of unstimulated saliva

We collected saliva samples from the participants between 08:00 and 11:00 in the morning. They were advised to avoid eating or smoking 1 h before sample collection. With the free-flow method, whole saliva was collected into a 50-mL test tube for 15 min after initial swallowing. Whenever possible, saliva was centrifuged (10 min, 12,000 g) and analyzed immediately. For subsequent use, the centrifuged supernatants were frozen and stored at − 80°C.

Illumina MiSeq sequencing and data analysis

16S rRNA sequencing and basic analysis have been described previously on Illumina MiSeq (Illumina, Inc., USA).[9] Correlation analysis was performed using the differential species obtained by Metastat (3 groups of comparison results; 19 species in total) and ELISA data. The correlation was calculated using the Spearman coefficient analysis performed in Cytoscape software, and subsequently plotted using R.

Isobaric tags for relative and absolute quantitation-based quantitative analysis

For isobaric tags for relative and absolute quantitation (iTRAQ) analysis, tryptic digest of peptides from each group of technical duplicates was labeled with iTRAQ reagents for an 8-plex reaction according to the manufacturer's instructions (Applied Biosystems, Foster City, USA). Other steps were performed as described previously.[21]

Measurement of salivary proteins

Commercially available ELISA kits were used to detect the levels of salivary sIgA, amylase, and lysozyme, according to the manufacturer's instructions. All ELISA kits were supplied from Cusabio Company, China.

Statistical analysis

A Student's t-test was performed to test the significance compared to the healthy control group. P < 0.05 is considered statistically significant.


  Results Top


Characteristics of patients with coronary heart disease and healthy controls

We collected tongue coating and saliva of 30 participants and allocated the samples to three groups, namely healthy group (n = 10) and patients with CHD having phlegm-stasis syndrome group (n = 10) and patients with CHD having Qi-Yin deficiency syndrome group (n = 10). The demographics and characteristics of patients are shown in [Table S1]. In TCM theory, white, thin tongue coating is characteristic of healthy individuals [Figure 1]a, almost no coating is associated with Qi and Yin deficiency [Figure 1]b, and white, greasy, and thin tongue coating is characteristic of phlegm- and blood-stasis syndrome [Figure 1]c. The body mass index (BMI) of patients with CHD having phlegm-stasis syndrome was higher (H vs. T, P = 0.0075; Q vs. T, P = 0.0006; H, healthy control; Q, patients with CHD having Qi-Yin deficiency syndrome; T, patients with CHD having phlegm-stasis syndrome) than those in the other groups [Figure 1]d, and there was no difference observed in the different groups regarding age- or sex-specific effects.
Figure 1: Classification of typical tongue coat appearance. (a) A healthy individual: A thin layer of a whitish coating, (b) a CHD patient with Qi-Yin deficiency syndrome: Little tongue coating, (c) a CHD patient with phlegm-stasis syndrome: Greasy and thick tongue coating, (d) BMI between the three groups. H: Healthy control, Q: CHD patients with Qi-Yin deficiency syndrome, T: CHD patients with phlegm-stasis syndrome. **P < 0.01, and ***P < 0.001. CHD: Coronary heart disease, BMI: Body mass index

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Characteristics of tongue coating and microbiome communities in patients with coronary heart disease having phlegm-stasis syndrome

16S rRNA sequences were assigned to species-level operational taxonomic units (OTUs) using a distance-based similarity of >97%. A total of 183 OTUs were identified from 30 samples by 16S rRNA sequencing. Initial analyses examined the global microbial diversity of the entire microbial population. To observe the tongue coating sample clusters of patients with phlegm-stasis syndrome separately from the healthy controls, we performed a principal coordinate analysis of the log-normalized abundance of the 183 OTUs; results indicated that based OTUs, the CHD groups and healthy controls are distinguishable, but phlegm-stasis syndrome and Qi-Yin deficiency syndrome could not [Figure 2]a. Diversity estimates for species richness and evenness[22] were lower in patients with Qi-Yin deficiency syndrome than in healthy controls and those with phlegm-stasis syndrome [Figure 2]b and [Figure 2]c. Examination of the results of RDP classification algorithm[23] at the phylum level showed that microbes belonging to 7 phyla dominated the tongue coating microbiota of the two groups. Comparison of samples from each group using the Metastat method revealed statistically significantly different in the genus of the microbiota [[Table S2]; P < 0.05]. At genus level, total 87 genera were calculated [Figure 2]d. Using the Statistical Analysis of Metagenomic Profiles method, we compared the abundance of microbiome in the three groups and found that Candidatus_Saccharimonas [Figure 2]e and Candidate_division_TM7_norank [Figure 2]f showed significant differences among the three groups.
Figure 2: Characters of the tongue coating microbial communities in tongue coatings of patients with CHD having phlegm-stasis syndrome. Principal component analysis plots of the tongue coating microbiome diversity (a), richness (b), and evenness (c) for the operational taxonomic units observed in this study. 16S ribosomal ribonucleic acid sequencing analysis and taxonomy classification of the tongue coating microbiome at genus level (d) and Statistical Analysis of Metagenomic Profiles analysis (e and f) between samples from patients with CHD having phlegm-stasis syndrome, Qi-Yin deficiency syndrome, and healthy controls. H: Healthy control, Q: Patients with CHD having Qi-Yin deficiency syndrome, T: Patients with CHD having phlegm-stasis syndrome. CHD: Coronary heart disease

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Characteristics of salivary protein in the tongue coating of patients with coronary heart disease having phlegm-stasis syndrome

Quantitative proteomic analysis of whole saliva samples was performed by iTRAQ-coupled liquid chromatography with tandem mass spectrometry method. Equal amounts of salivary proteins from the three groups (100 μg for each group), along with technical duplicates, were pooled for iTRAQ analysis. For protein quantification, the Pearson correlation analysis of the normalized tag intensities of spectra was used, which revealed high reproducibility with correlation coefficients >0.93 between technical duplicates from the three groups. Many differentially expressed genes were identified in each group [Figure 3]a. On comparison with the data from the healthy control group, 29 genes were identified to be upregulated and 35 genes were downregulated genes in the saliva samples of patients with phlegm-stasis syndrome; 19 genes showed high expression and 40 genes had low expression in the samples from patients with Qi-Yin deficiency syndrome. When compared with the phlegm-stasis syndrome group, 37 genes were upregulated and 25 genes were downregulated in the Qi-Yin deficiency syndrome group. Gene ontology (GO) terms were further assigned to differentially expressed proteins according to the cellular components, molecular functions, biological processes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Majority of the differentially expressed proteins in all the groups [Figure 3]b, [Figure 3]c, [Figure 3]d were involved with biological processes. In the KEGG pathway enrichment analysis, pentose phosphate pathway, Staphylococcus aureus infection, complement and coagulation cascades, and salivary secretion were the top pathways in which the differentially expressed proteins were present in both patients with CHD and healthy controls [P < 0.01; [Figure 3]b]. In addition, the top pathways in which the differentially expressed proteins in the T versus Q group were involved were the complement and coagulation cascades and amoebiasis pathway [P < 0.01; [Figure 3]c]. Carbon metabolism, proximal tubule bicarbonate reclamation pathway, pentose phosphate pathway, and focal adhesion pathway were the significantly upregulated pathways in Q versus T groups [P < 0.01; [Figure 3]d]. An integrated analysis of proteins expressed at different levels among the three comparison groups, with successful validation, was performed using the tools of the STRING database; it excluded proteins that had no information in the STRING database. The protein–protein interaction network confirmed the results [Figure 3]e, [Figure 3]f, [Figure 3]g.
Figure 3: Characters of saliva proteins in CHD tongue coating with phlegm-stasis syndrome. Totally up- or downregulated genes and their corresponding biological functions (a), GO (b-d), and KEGG (e-g) analysis between samples from patients with CHD phlegm-stasis syndrome, Qi-Yin deficiency syndrome, and healthy controls. H: Healthy control, Q: CHD patients with Qi-Yin deficiency syndrome, T: CHD patients with phlegm-stasis syndrome. CHD: Coronary heart disease, KEGG: Kyoto Encyclopedia of Genes and Genomes, GO: Gene ontology

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Verification of candidate salivary proteins with enzyme-linked immunosorbent assay

To further validate the reliability of the iTRAQ results, salivary levels of sIgA, lysozyme, and amylase were detected by ELISA. In addition, the saliva flow rate was detected by results of iTRAQ analysis. Saliva flow rate was significantly lower in patients with CHD [Figure 4]a, in T versus H, and Q versus H, but not T versus Q [Figure 4]b. Noticeably, lysozyme level was lower in the H group than the CHD group [Figure 4]c, with both the Q and T groups totally lower than the H group [Figure 4]d. However, sIgA level was higher in the CHD group [Figure 4]e, which increased in T compared with H [Figure 4]f. Furthermore, salivary amylase level was lower in the CHD group [Figure 4]g, but a significant difference was only observed in Q versus H [Figure 4]h.
Figure 4: Characters of salivary proteins verified by ELISA. The saliva flow rate (a), lysozyme (c), sIgA (e), and amylase (g) levels between CHD patients and healthy controls, the saliva flow rate (b), lysozyme (d), sIgA (f), and amylase (h) levels between samples from patients with CHD phlegm-stasis syndrome, Qi-Yin deficiency syndrome, and healthy controls. H: Healthy control, Q: CHD patients with Qi-Yin deficiency syndrome, T: CHD patients with phlegm-stasis syndrome. *P < 0.05, **P < 0.01, and ***P < 0.001. CHD: Coronary heart disease

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Correlation analysis between salivary protein and microbiota of tongue coating

Correlation analysis was performed using the different species obtained by Metastat analysis (3 groups of comparison results; a total of 19 species) and ELISA data. The correlation was calculated by Cytoscape software using the Spearman coefficient, and subsequently, heatmap was drawn using R software. Candidate_division_TM7_norank and Candidatus_Saccharimonas showed a positive correlation with saliva flow rate. No microbial species correlated with sIgA, although it was significantly elevated in patients with CHD on ELISA. Among these microbial species that showed statistical differences, those which have not been cultured or reported previously in intestinal or vaginal flora were excluded. Actinobacillus correlated negatively with lysozyme and amylase levels, and may play a role in the pathogenesis of acute coronary syndrome. Lachnospiraceae correlated positively with amylase levels and salivary flow rate and has been previously associated with diabetes in mice [Figure 5].
Figure 5: Heatmap of correlation of microbial taxa of the characterized tongue coating microbiome of salivary proteins and saliva flow rate (*P < 0.05, **P < 0.01, and ***P < 0.001)

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  Discussion Top


Tongue color and coating are the two main characteristics of the tongue diagnosis in the ZHENG classification of TCM. Previously, we reported that yellow tongue coating may have a characteristic microbiota; however, whether other types of tongue coating have characteristic microbiota and the underlying mechanism of tongue coating formation are unknown.

In this study, we collected the tongue coating and saliva samples of 20 patients with CHD (10 with phlegm-stasis syndrome and 10 with Qi-Yin deficiency syndrome) and 10 healthy controls. Illumina MiSeq sequencing of 16S ribosomal RNA, gene expression analysis of the microbiome constituting the tongue coating microbiome, and isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative analysis of the salivary proteome identified Candidatus_Saccharimonas and Candidate_division_TM7_norank as the primary microbes comprising the thick, greasy tongue coating of patients with CHD having phlegm-stasis syndrome. Formation of tongue coat microbiota was associated with the salivary contents and saliva flow rate.

Herein, the greasy, thick tongue coating of patients with CHD having phlegm-stasis syndrome was examined. By combining 16S rRNA sequencing and iTRAQ-based proteomics approach, we found that Actinobacillus, a periodontal pathogen, has a high abundance in the tongue coating of patients with CHD and may be involved in the pathogenesis of acute coronary syndrome.[24] Recent epidemiological studies identify periodontitis as an important risk factor for CHD. Moreover, a systemic antibody response was associated with CHD. These results indicate that microbiota in the tongue coating of patients with phlegm-stasis syndrome may be a potential risk factor for CHD.[25] Interestingly, Actinobacillus correlated negatively with salivary lysozyme and amylase levels. Saliva plays a major role in maintaining a healthy oral environment.[26] Salivary components, particularly the antimicrobial factors, exert significant selective pressures on the oral microbiota and control the resident microbial communities. Salivary amylase belongs to the glycoside hydrolase family of enzymes; it hydrolyses complex carbohydrates like starch into simple sugars, such as glucose or maltose, by cleaving the alpha-1,4-glycosidic bonds.[14] Further breakdown of starch is mediated by pancreatic amylase during digestion. Salivary amylase can also bind to oral streptococci aids in their clearance from the oral cavity.[27] Studies have associated salivary lysozyme with the prevalence of CHD.[16] Taken together, we believe that salivary lysozyme and amylase would affect the microbiome of greasy, thick tongue coating in patients with CHD having phlegm-stasis syndrome.

Moreover, we found a positive correlation of Lachnospiraceae with salivary amylase levels and salivary flow rate; this bacterial genus has been previously associated with diabetes in mice. According to the results of iTRAQ analysis, pentose phosphate pathway, S. aureus infection, complement and coagulation cascades, and salivary secretion pathway were the pathways with the most differentially expressed proteins in patients with CHD having phlegm-stasis syndrome. These observations are congruent with existing TCM theory that CHD is associated with abnormal glucose and lipid metabolism, and has a high risk of phlegm and blood stasis.

Interestingly, saliva flow rate was significantly low in the CHD with phlegm-stasis syndrome group. This observation was consistent with the results of mass spectrometry and KEGG analysis. Saliva flow rate is a vital index for human oral health. Saliva removes fermentable substrate from the mouth, maintains a pH that is conducive for the growth of beneficial oral bacteria, neutralizes acidic conditions needed for the enrichment of cariogenic bacteria, and promotes remineralization of the enamel.[28] Recent in silico modeling has shown that maintenance of a predominantly beneficial microbiota aids salivary buffering and prevention of plaque pH from falling below the critical values.[29] Saliva secretion is disordered in patients with CHD having phlegm-stasis syndrome; this explains the thick and greasy consistency of tongue coating in patients with CHD having phlegm-stasis syndrome. Therefore, saliva flow rate may be one of the most critical factors in oral health.

The oral cavity is a complex dynamic environment with site-specific and temporal differences in microbial populations, mediated by their local, microenvironmental conditions that are specific to the systemic health status, diet, genetic predisposition, and salivary antimicrobial susceptibility of the host. We explored the microbial characteristic of white-greasy and thin tongue coating of patients with CHD having phlegm-stasis syndrome; we found that salivary factors influence the formation and microbial composition of tongue coating.

Nevertheless, we need to increase the sample numbers in future research since TCM syndrome itself is a very complicated state. Moreover, establishment of animal models of TCM syndromes has always been one of the important factors hindering the development of TCM, so conduct phlegm-stasis syndrome animal models to validate the results is a potential direction in tongue diagnosis study.

In summary, holistic view of TCM treatment is one of the vital principles in TCM theory, and tongue coating, like a mirror, used for tongue diagnosis is deemed that could reflect human healthy states of the whole body. Our research provides a scientific and theoretical basis for tongue diagnosis of TCM.


  Conclusions Top


In TCM clinical practice, white-greasy tongue coating is deemed as a typical tongue coating with CHD having phlegm-stasis syndrome which is found in 59% of patients with CHD in China. However, its biological material base and the cause of formation are not clear. We explored the characteristics of microbial composition of white-greasy tongue coating in patients with CHD having phlegm-stasis syndrome and identified correlations between salivary proteins and microbiome formation, providing a theoretical and mechanistic basis for tongue coating formation, thus providing a new idea for the objective study of tongue diagnosis of TCM.

Financial support and sponsorship

This study was supported by the Key R&D Program of Jiangsu Province (BE2017768).

Conflicts of interest

There are no conflicts of interest.



 
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