Patients with multidrug-resistant tuberculosis (MDR-TB) who are resistant to at least both rifampicin and isoniazid, lack effective treatment options in clinic. The gold standard for the diagnosis of MDR-TB is drug sensitivity test, which is time-consuming and has a relatively low positive detection rate. Screening early diagnostic biomarker for MDR-TB is urgent need in clinical practice. A total of 33 patients with MDR-TB, healthy controls and drug-sensitive tuberculosis (DS-TB) were included in this study. Total plasma exosomal RNA was extracted from the subjects, and the MDR-TB plasma-specific exosomal miRNAs were obtained by Illumina sequencing. There were 644 and 647 differentially expressed miRNAs in the plasma exosomes of MDR-TB patients obtained by sequencing and biogenic analysis compared with DS-TB patients and healthy controls, respectively. Differential miRNAs are mainly involved in the biological function of regulation of transcription and protein binding, and enriched in the pathways in cancer and MAPK signaling pathway. Moreover, seven plasma exosomal miRNAs in MDR-TB patients were significantly different from those in DS-TB patients and healthy controls. Among them, three of the miRNAs (hsa-miR-122-5p_R-1, hsa-miR-23b-3p_R + 1, and hsa-miR-15a-5p_R-1) were found to be in target relationship with MDR-TB related genes (NTRK2, KIDINS220, NCKAP1, MAPK9, NFAT5, ATF6 and SLC11A2) by target gene prediction analysis. Further the bioinformatic analysis showed that hsa-miR-122-5p_R-1 targets the protein PGLYRP2, a diagnostic biomarker identified in our previous study. We suggest that hsa-miR-122-5p_R-1, hsa-miR-23b-3p_R + 1, and hsa-miR-15a-5p_R-1 are closely related to MDR-TB.

Yin-deficiency-heat (YDH) syndrome is a very common subhealth status in Traditional Chinese Medicine. However, currently, there is no unified standard for diagnosing YDH syndrome. We applied the iTRAQ-2D LC-MS/MS method to explore the potential of serum protein profiles as biomarker for YDH syndrome. A total of 120 differentially expressed proteins (79 downregulated and 41 upregulated) were identified by the proteomic profiling. The results of KEGG pathway analysis showed that the functions of the differentially expressed proteins were mainly involved in complement and coagulation cascades. The clinical data showed that YDH syndrome was closely related to inflammation and coagulation, compared with the healthy controls. The ELISA validation results indicated that the expression levels of ALB, CFI, and KLKB1 were downregulated in the YDH syndrome group (p < .05). Moreover, we established a decision tree model based on the combination of these three proteins and achieved a sensitivity of 87.5%, a specificity of 84.4%, and AUC of 0.891. The results indicated that the combination of ALB, CFI, and KLKB1 may serve as potential biomarkers for diagnosing YDH syndrome. Our study can provide a new method for YDH syndrome diagnosis, and may also provide an experimental basis to understand the molecular mechanism of YDH syndrome.

Patients with multidrug-resistant tuberculosis (MDR-TB) tend to have a long course of anti-TB treatment and severe side effects. Traditional Chinese Medicine (TCM) has a synergistic effect in attenuation of MDR-TB. However, the lack of objective biological standards to classify and diagnose MDR-TB TCM syndromes could result in less effective TCM treatment. Therefore, in this study, we identified differentially expressed proteins (DEPs) in serum of individuals with MDR-TB TCM syndromes by applying isobaric tags for relative and absolute quantification coupled with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) method and bioinformatics analysis. The functional analysis of DEPs was also performed. Additionally, DEPs among three different TCM syndromes of MDR-TB were validated by enzyme-linked immunosorbent assay (ELISA). Finally, a receiver operating characteristic (ROC) curve was performed to estimate the diagnostic ability of DEPs. A total of 71 DEPs were identified in the three different MDR-TB TCM syndrome groups such as the pulmonary Yin deficiency (PYD) syndrome group, the Hyperactivity of Fire due to Yin deficiency (HFYD) syndrome group, and the deficiency of Qi and Yin (DQY) syndrome group. The results showed that the expression level of transforming growth factor-beta-induced protein ig-h3 (TGFBI) was lower in the PYD syndrome group (p = .002), the proprotein convertase subtilisin/kexin type 9 (PCSK9) was overexpressed in the HFYD syndrome group (p < .0001), and the C-C motif chemokine ligand 14 (CCL14) expression level was reduced in the DQY syndrome group (p = .004). Our study demonstrated that serum TGFBI, PCSK9, and CCL14 may serve as potential novel biomarkers for PYD syndrome, HFYD syndrome and DQY syndrome of MDR-TB, respectively. The study provides a biological basis for MDR-TB TCM syndromes classification and can be of great significance for the treatment of different TCM syndromes.

Yin-deficiency-heat (YDH) syndrome is a common sub-health state of the human body in traditional Chinese medicine (TCM). However, due to the lack of objective quantitative diagnostic indicators, patients with early-stage YDH syndrome cannot be treated in time and can develop a pathological (disease) state. Therefore, it is necessary to apply modern diagnostic techniques in order to identify the biological markers for the diagnosis of early-stage YDH syndrome. In the present study, we performed Solexa sequencing and non-targeted metabolomics analysis using high-performance liquid chromatography coupled with mass spectrometry to screen differentially expressed mRNAs and differential metabolites in individuals with early-stage YDH syndrome and healthy controls. Bioinformatics methods were used to perform enrichment analysis of differentially expressed mRNAs and differential metabolites for biological functions and signaling pathways. Furthermore, we found that differentially expressed mRNAs and differential metabolites were related to energy metabolism. Real-time PCR was used to validate the mRNA expression in the serum of subjects with early-stage YDH syndrome. We found that the mitochondrially encoded NADH dehydrogenase 2 (MT-ND2) mRNA was differentially expressed in the serum of individuals with early-stage YDH syndrome. Receiver operating characteristic (ROC) curve and logistic regression analysis were used to evaluate the efficacy of the diagnostic model based on eight differential metabolites. We combined the three metabolites such as Glycine, Sphingomyelin, and Isocitrate to establish the diagnostic model with a sensitivity of 0.853 and a specificity of 0.800. The combination of the above three metabolites may serve as a potential biomarker for the diagnosis of early-stage YDH syndrome. Our study reveals potential biomarker for the diagnosis of early-stage YDH syndrome and also provides a new method for the quantification and objectification of TCM syndromes.

BACKGROUND:Yin-deficiency-heat (YDH) syndrome is a subhealth state of the individual, mainly manifested as oral ulcers, dry mouth, constipation, and other symptoms. Zhibai Dihuang granule (ZDG), as a classic traditional Chinese medicine, is effective in treating YDH syndrome. We screened the potential biomarkers for diagnosing YDH syndrome, and explored the mechanisms of the therapeutic effect of ZDG.METHODS:Plasma samples from the Pinghe (PH, healthy control) group, the Shanghuo (SH, YDH syndrome) group, and the ZDG treated group (therapeutic group) were analyzed by using metabolomics profiling. The data were analyzed by multivariate statistical and bioinformatics analyses.RESULTS:We screened four differential metabolites such as, decanoylcarnitine, dodecanoylcarnitine, phosphatidylcholine (PC), and Aspartate (Asp) Arginine (Arg) Proline (Pro) in the SH group and the PH group. The results showed that the combination of above four metabolites could serve as a potential biomarker for the early diagnosis of YDH syndrome. The metabolites decanoylcarnitine and glucose were found to be differentially expressed in the YDH syndrome group and tended to be normalized after ZDG treatment.CONCLUSION:The increased levels of four differential metabolites (decanoylcarnitine, dodecanoylcarnitine, PC, and Asp Arg Pro) revealed that individuals with YDH syndrome may have increased energy metabolism in the body, which could lead to disorders of fatty acids β-oxidation and immune function. The levels of two differential metabolites including decanoylcarnitine and glucose returned to normal after ZDG treatment, indicating that ZDG could treat YDH syndrome by regulating glucose metabolism and fatty acids β-oxidation. Our study provides a new method for the diagnosis of YDH syndrome, and may provide theoretical basis for novel therapeutic strategies of YDH syndrome.

Background: The lack of rapid and efficient diagnostic methods has been one of the most frustrating challenges in controlling the pulmonary tuberculosis (TB) epidemic. This study was aimed to identify novel non-invasive biomarkers for pulmonary TB. Methods: The subjects in this study were divided into four groups: the pulmonary TB group, the community-acquired pneumonia (CAP) group, the lung cancer (LC) group, and the normal control (NC) group. Plasma small molecule metabolites were investigated in each group by using ultra-high performance liquid chromatography coupled with Q Exactive mass spectrometry. Multivariate statistical methods and bioinformatics were used to analyze the data. Results: We identified three differential plasma metabolites such as, Xanthine, 4-Pyridoxate and d-glutamic acid in the pulmonary TB group, compared to the other groups (CAP, LC and NC). The pathway enrichment analysis indicated that the energy source in pulmonary TB was multi-center, which might be involved in maintaining the reproductive ability and virulence of Mycobacterium tuberculosis. Conclusion: The results suggested that Xanthine, 4-Pyridoxate, and d-glutamic acid may serve as potential biomarkers for pulmonary TB. The present study provides experimental basis for developing potential biomarkers of pulmonary TB.

[目的]探讨血清磷脂酶A2组ⅡA(phospholipase A2 groupⅡA,PLA2G2A)和凝血因子Ⅸ(coagulation factorⅨ,F9)对耐多药肺结核病(multidrug-resistant tuberculosis,MDR-TB)气阴两虚(deficiency of Qi and Yin,DQY)证的潜在诊断价值.[方法]选择MDR-TB肺阴虚(pulmonary Yin deficiency,PYD)证、MDR-TB阴虚火旺(hyperactivity of fire due to Yin deficiency,HFYD)证、MDR-TB DQY证患者和健康对照(healthy control,HC)者各15例,以ELISA检测每组PLA2G2A和F9的蛋白浓度,并利用受试者工作特征(receiver operating characteristic,ROC)曲线分析PLA2G2A和F9作为MDR-TB DQY证潜在生物学标志物的临床价值.[结果]MDR-TB证候分布以DQY证患者数最多,其次是HFYD证,再者是DQY证,而阴阳两虚(deficiency of Yin and Yang,DYY)证患者数最少.DQY证组PLA2G2A的蛋白浓度均明显高于PYD证组、HFYD证组和HC组,差异有统计学意义(P<0.05);DQY证组F9的蛋白浓度明显低于HFYD证组,差异有统计学意义(P<0.05).ROC曲线结果显示,血清PLA2G2A有鉴别诊断MDR-TB DQY证的作用.[结论]血清PLA2G2A和F9在诊断MDR-TB DQY证方面具备一定临床价值,血清PLA2G2A可作为MDR-TB DQY证的潜在生物学标志物.

Lack of laboratory standards for cured tuberculosis (TB) can lead to early discharge of untreated TB patients from the hospital, resulting in increased risk of TB spread and of developing drug resistant Mycobacterium tuberculosis (Mtb). We used ultra-high performance liquid chromatography coupled with mass spectrometry (LC–MS) to detect heparin anticoagulant in plasma of untreated TB patients, two-month treated TB patients, cured TB subjects, and healthy controls. Screening of differentially expressed metabolites resulted in identification of four differentially expressed metabolites such as, l-Histidine, Arachidonic acid (AA), Biliverdin, and l-Cysteine-glutathione disulfide after 6 months of TB treatment. Among them, l-Cysteine-glutathione disulfide and AA could be identified after 2 months of TB treatment. We established a cured TB model with an area under the curve (AUC) of 0.909 (95% CI, 0.802–0.970), 86.2% sensitivity, and 85.2% specificity. The diagnostic model fitted from the four differential metabolites in combination (l-Histidine, AA, Biliverdin, and l-Cysteine-glutathione disulfide) can be used as potential biomarkers for cured TB. Our study provided laboratory standards for hospital discharge of TB patients, as well as experimental basis for evaluating the efficacy of anti-TB drugs.

Rapid diagnosis of pulmonary tuberculosis is an effective measure to prevent the spread of tuberculosis. However, the grim fact is that the new, rapid, and safe methods for clinical diagnosis are lacking. Moreover, although auto-lysosome is critical in clearing Mycobacterium tuberculosis, the pathological significance of microRNAs, as biomarkers of tuberculosis, in autophagosome maturation is unclear. Here, these microRNAs were investigated by Solexa sequencing and qPCR validation, and a potential diagnostic model was established by logistic regression. Besides that, the mechanism of one of the microRNAs involved in the occurrence of tuberculosis was studied. The results showed that the expression of miR-423-5p, miR-17-5p, and miR-20b-5p were significantly increased in the serum of patients with tuberculosis. The combination of these three microRNAs established a model to diagnose tuberculosis with an accuracy of 78.18%, and an area under the curve value of 0.908. Bioinformatics analysis unveiled miR-423-5p as the most likely candidate in regulating autophagosome maturation. The up-regulation of miR-423-5p could inhibit autophagosome maturation through suppressing autophagosome-lysosome fusion in macrophages. Further investigations showed that VPS33A was the direct target of miR-423-5p, and the two CUGCCCCUC domains in VPS33A 3'-UTR were the direct regulatory sites for miR-423-5p. In addition, an inverse correlation between VPS33A and miR-423-5p was found in peripheral blood mononuclear cells of patients with tuberculosis. Since the inhibition of autolysosome formation plays a critical role in tuberculosis occurrence, our findings suggests that miR-423-5p could suppress autophagosome-lysosome fusion by post-transcriptional regulation of VPS33A, which might be important for the occurrence of active tuberculosis.

Background: Zhibai Dihuang Granule (ZDG) is a traditional Chinese medicine which has been used to treat Yin-deficiency- heat (YDH) syndrome for thousands of years in China. However, little work has been conducted to explore the molecular mechanism of ZDG in YDH syndrome, and the processes of YDH syndrome prevention and treatment have been developed slowly. The present study was aimed to explore the therapeutic mechanism of ZDG on YDH syndrome. Methods: The YDH syndrome rats were induced by hot Chinese herbs, then treated by ZDG orally for 1 week. Body weight was measured every 2 days. After sacrifice, blood samples were collected and the thymus, adrenal glands, spleen, and liver were immediately removed and weighed. iTRAQ-based proteomics approach was applied to explore the serum protein alterations with the treatment of ZDG, and to investigate the underlying mechanism of ZDG in treating YDH syndrome. Results: The body weights of YDH syndrome rats were significantly decreased compared with control group, and increased in ZDG treated rats. The relative weights of thymus in YDH syndrome rats were increased compared with the control rats, and significantly decreased in after ZDG treatment. In the proteomic analyses, seventy-one proteins were differentially expressed in the YDH syndrome group and the ZDG treated group, including 10 up-regulated and 61 down-regulated proteins. Gene ontology analysis revealed that the differentially expressed proteins were mostly related to immune response, and pathway enrichment analysis showed that these proteins were enriched in coagulation and complement cascades. Enzyme-linked immunosorbent assay was performed to detect the protein levels in coagulation and complement cascades, and the results showed that complement component 5 levels were significantly increased, while fibrinogen gamma chain levels were significantly decreased in the ZDG treated group. Conclusions: We found that ZDG treatment could lead to proteins alteration in immune response, especially in coagulation and complement cascades. ZDG can up-regulate the proteins in the complement cascade to eliminate pathogens, and down-regulate the proteins in the coagulation cascade to suppress inflammation. Our study provides experimental basis to understand the therapeutic mechanism of ZDG and revealed that ZDG can regulate coagulation and complement cascades in treating YDH syndrome.

Yin and Yang are the two counter‐balancing aspects in ancient Chinese philosophy. In traditional Chinese medicine, Yin deficiency syndrome (YDS) is a common sub‐health state with complex causes. While the syndrome may be treated to various degrees of effectiveness with traditional Chinese medicine, efficient modern methods are yet to be developed for diagnosing and treating the YDS. Here we performed a metabolomics study on YDS in rats. Serum metabolites in rats were analyzed using ultra‐performance liquid chromatography‐mass spectrometry (UPLC–MS) method to identify potential biomarkers for YDS. The rats were divided randomly into the healthy control group, the untreated YDS group, and the anemarrhena treated YDS group. Compared with the control group, significant increase in the metabolites such as dihydrotestosterone (DHT) and 5β‐DHT, 4‐imidazolone‐5‐propanoate, 4‐(L‐alanin‐3‐yl)‐2‐hydroxy‐cis,cis‐muconate 6‐semialdehyde, and 5‐(L‐alanin‐3‐yl)‐2‐hydroxy‐cis,cis‐muconate 6‐semialdehyde were observed in the serum of untreated YDS group, which returned to normal in the anemarrhena treated group. Therefore, these metabolites may serve as potential biomarkers for YDS, and may facilitate the diagnosis and treatment of YDS.

Ethnopharmacological relevance: Zhibai Dihuang Granule (ZDG), a traditional Chinese medicine (TCM) made from eight Chinese herbs, has been classically used to treat Yin-deficiency-heat (YDH) syndrome. ZDG is well known with the therapeutic efficacy of nourishing Yin and decreasing internal heat in clinic, but the mechanism of ZDG's therapeutic effect is still not clear. Materials and methods: High doses of triiodothyronine (T3) were given intraperitoneally to induce Hyperthyroid YDH syndrome in SD rats. The animals were then treated with ZDG for one week. The iTRAQ-coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique was used to screen the differentially expressed serum proteins between ZDG treated rats and YDH syndrome rats. The differentially expressed proteins were analyzed by bioinformatics method and were verified by enzyme-linked immunosorbent assay (ELISA). Results: A total of 55 differentially expressed proteins were identified, including 23 up-regulated proteins (> 1.25 fold, p < 0.05) and 32 down-regulated proteins (< 0.80 fold, p < 0.05). Among the differentially expressed proteins, 26 proteins returned to normal after ZDG treatment. Bioinformatics analysis showed that these proteins were mainly involved in immune response, including regulation of immune system process, complement activation, and humoral immune response mediated by circulating immunoglobulin. ELISA revealed significantly increased levels of Zinc-alpha-2-glycoprotein (Azgpl), L-selectin, C-reactive protein (Crp), Plasminogen (Pig), Kininogen 1 (Kngl), and significantly decreased levels of Mannose binding lectin 2 (Mb12) and Complement Clqb chain (Clqb) in ZDG treated rats compared with YDH syndrome rats. Bioinformatics analyses indicated that Azgpl participated in antigen processing and presentation, Crp, Clqb, and Mb12 were involved in complement activation, while L-selectin, Plg, and Kngl were involved in regulating the inflammatory response. Conclusions: Our study provides experimental evidence to understand the therapeutic mechanism of ZDG in YDH syndrome. The results suggested that ZDG may regulate the complement activation and inflammatory response, and promote the ability to recognize antigens to alleviate YDH syndrome.

This research aimed to discover potential biomarkers for evaluating the therapeutic efficacy of intensive therapy in pulmonary tuberculosis (TB). Protein profiles in 2-months intensively treated TB patients, untreated TB patients, and healthy controls were investigated with iTRAQ-2DLC-MS/MS technique. 71 differential proteins were identified in 2-months intensively treated TB patients. Significant differences in complement component C7 (CO7), apolipoprotein A-IV (APOA4), apolipoprotein C-II (APOC2), and angiotensinogen (ANGT) were found by ELISA validation. CO7 and ANGT were also found significantly different in sputum negative patients, compared with sputum positive patients after intensive treatment. Clinical analysis showed that after 2-months intensive treatment several indicators were significantly changed, and the one-year cure rate of sputum negative patients were significantly higher than sputum positive patients. Diagnostic models consisting of APOC2, CO7 and APOA4 were established to distinguish intensively treated TB patients from untreated TB patients and healthy controls with the AUC value of 0.910 and 0.935. Meanwhile, ANGT and CO7 were combined to identify sputum negative and sputum positive TB patients after intensive treatment with 89.36% sensitivity, 71.43% specificity, and the AUC value of 0.853. The results showed that APOC2, CO7, APOA4, and ANGT may be potential biomarkers for evaluating the efficacy of intensive anti-TB therapy.

[目的]寻找阴虚上火的新型诊断标志物,深化对阴虚上火的认识.[方法]对34例受试者血清样本的临床资料进行统计分析,运用ELISA法检测血清单核细胞分化抗原(monocyte differentiation antigen)CD14、激肽原-1(Kininogen-1,KNG-1)、载脂蛋白B(apolipoprotein B-100,APOB)和载脂蛋白A2(apolipoprotein A2,APOA2)的浓度,并运用ROC曲线对上述蛋白诊断上火的效果进行评估.[结果]阴虚上火患者CD14与KNG-1显著上调,APOB与APOA2显著下调,与健康对照组比较,差异具有统计学意义(P<0.001,P<0.05,P<0.05,P<0.01).将CD14、APOA2、KNG-1进行组合构建诊断模型,ROC曲线显示组合物的曲线下面积(area under curve,AUC)值为0.934,敏感度和特异度分别为100％和71.40％,具有较好的准确性.[结论]阴虚上火与能量代谢、炎症反应密切相关.CD14、APOA2、KNG-1的组合可作为临床诊断阴虚上火的潜在的生物学标志物,有助于中医疾病诊断的客观化.

Pulmonary tuberculosis (TB) is among the diseases with the highest morbidity and mortality worldwide. Effective diagnostic methods for TB are lacking. In this study, we investigated long non-coding RNAs (lncRNAs) in plasma using microarray and the potential diagnostic value of lncRNAs for TB. We found a total of 163 up-regulated lncRNAs and 348 down-regulated lncRNAs. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and coding-noncoding co-expression (CNC) analyses showed that functions of differentially expressed lncRNAs were mainly enriched in the regulation of alpha-beta T cell activation and the T cell receptor signalling pathway. Four differentially expressed lncRNAs, NR_038221 (fold change = 3.79, P < 0.01), NR_003142 (fold change = 1.69, P < 0.05), ENST00000570366 (fold change = 3.04, P < 0.05), and ENST00000422183 (fold change = 2.11, P < 0.001), were verified using RT-qPCR. Among those, NR_038221, NR_003142, and ENST00000570366 were found to be up-regulated, while ENST00000422183 was down-regulated. The value of the area under the curve (AUC) for the diagnostic model consisting of the four lncRNAs was 0.845 (sensitivity = 79.2%, specificity = 75%). We further predicted 85 mRNAs and 404 miRNAs that potentially interact with these lncRNAs. Our study revealed the potential value of lncRNAs as biomarkers for early diagnosis of TB and the underlying mechanisms of these abnormally expressed lncRNAs in the pathogenesis of TB.

Objective:To explore the therapeutic mechanism of Zhibai Dihuang Granule ( ZDG) in Yin-deficiency-heat ( YDH) syndrome.Methods:ITRAQ-2DLC-MS/MS technique was applied to screen the differentially expressed serum proteins between ZDG treated rats and YDH syndrome rats.The serum protein profiles were analyzed by bioinformatics tools,such as Gene Ontology (GO) database,Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.Compared with normal rats,47 differen-tially expressed proteins were shown in the serum of YDH syndrome rats,including 20 up-regulated proteins and 27 down-regulated proteins.Nineteen proteins returned to normal after ZDG treatment,which mostly involved in immune reaction and metabolism.Complement C4 (C4),coagulation factor X (F10),and Src kinase-associated phosphoprotein 2 (Skap2) participated in immune response,while L-lactate dehydrogenase A chain (Ldha),Fructose-bisphosphate aldolase A (Aldoa),Arachidonate 12-lipoxyge-nase (Alox12),Rho GDP-dissociation inhibitor 1 (Arhgdia),and Myosin light polypeptide 6 (Myl6) mainly related to metabolic process.Conclusion:YDH syndrome is highly associated with the disturbance of immune function and metabolism,and ZDG treats YDH syndrome mostly through regulating immune activity and metabolic process.