| Part I Pathological Analysis and Screening of Specific Protein for Pulmonary Tuberculosis in Traditional Chinese Medicine by iTRAQ-2DLC-MS/MSBackground:Pulmonary tuberculosis (TB) is the leading death cause among the single infections. The routine anti-TB treatment is Western chemotherapy. However, it last at least for 6 months, and may lead to drug-resistant TB and severe side effects. For example, isoniazid, rifampicin and pyrazinamide could cause hepatotoxicity, and may even lead to liver failure and death. All these increased the TB recurrence and generation of drug-resistant TB. TB is divided into four distinctive syndromes based on Traditional Chinese medicine (TCM) theory:pulmonary Yin deficiency (PYD) syndrome, hyperactivity of fire due to Yin deficiency (HFYD) syndrome, deficiency of Qi and Yin (DQY) syndrome, and deficiency of Yin and Yang (DYY) syndrome. Choosing appropriate prescriptions based on the TCM syndromes, could have better effects of inhibiting the mycobacterium, strengthening the body immune, alleviating the pulmonary toxicity and reducing side effects of anti-TB chemotherapy. However, syndrome differentiation depends heavily on subjective observation, clinical experience, and knowledge of practitioners with the lack of scientific researches and objective tests. Thus, the clinical application of integrated Chinese-Western therapy for TB is greatly influenced.Methods:Pulmonary TB cases were divided into four distinctive TCM syndromes: PYD syndrome, HFYD syndrome, DQY syndrome, and DYY syndrome. The serum samples from 214 pulmonary TB patients were collected, and the clinical and pathological data was analyzed by bioinformatics methods. The differentially expressed proteins were screened and tested by using iTRAQ stable isotope labeling (iTRAQ), two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) and validated by ELISA. Only 5 patients with DYY syndrome were recruited in 3 years, which were not enough for further research.Results:PYD cases had more proliferative lesions, such as tuberculous nodules, patch and stripping shadows. HFYD cases were identified as having more degenerative pulmonary lesions, compared with the PYD and DQY cases (P=0.0427). DQY cases had multiple pulmonary lesion areas with mixed pulmonary lesions and showed highest incidence of miliary TB, compared with the PYD and HFYD cases (P=0.0013). Besides, the DQY cases had higher erythrocyte sedimentation rate (ESR) compared to the PYD and HFYD cases (P=0.0178).94.44%(12 PYD,18 HFYD, and 4 DQY before anti-TB treatment) of 36 treated TB cases were transformed to PYD accompanied with the reduction of ESR and absorption of pulmonary lesions. A total of 39 differentially expressed proteins were found among the three TCM syndromes. Proteomic studies revealed that gamma-glutamyl hydrolase (GGH), Ig gamma-3 chain C region (IGHG3), and haptoglobin (HPT) were specifically over-expressed in PYD (P<0.01), HFYD (P<0.001), and DQY cases (P<0.01), respectively. Furthermore, GGH was significantly higher in PYD cases compared to the HFYD and DQY cases (P<0.01,P<0.001, respectively), whereas IGHG3 was significantly higher in HFYD cases than PYD and DQY cases (P<0.001, P<0.01, respectively).Conclusions:(1) The results of statistical analysis suggest that TCM syndromes are significantly correlated with the pulmonary lesions and ESR. (2) Serum proteins GGH, IGHG3 and HPT were acquired by iTRAQ-2DLC-MS/MS and ELISA, and were specifically over-expressed in PYD, HFYD, and DQY patients, respectively. (3) The present study provides new biological basis to understand the pathological changes and proteomic differences of TB syndromes.Part II Study of Serum Potential Protein Biomarkers for Pulmonary Tuberculosis by iTRAQ-2DLC-MS/MSBackground:Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) poses great threat to human health, especially after the emergence of HIV-associated TB and multi-drug resistant TB. China has the second largest number of TB cases in the world. Delay in the diagnosis of TB may increase the severity and mortality of the disease as well as increase the risk of transmission. Therefore, early diagnosis and treatment are significant for the control of TB. In clinic, the detection of TB is still dependent heavily on sputum smear, sputum culture, chest radiography (chest X-ray and CT scan) and clinical symptomatology. However, the sputum smear positive rate is low, and the Mtb culture is time consuming. The radiological features and clinical symptoms of TB in the early stage of infection are not specific, and it is difficult to distinguish them from other pulmonary diseases, such as pneumonia, and chronic obstructive pulmonary disease (COPD). Emphasis on biomarker discovery for rapid and accurate diagnosis of TB has become a focal point.Methods:A total of 423 subjects were included in this study, including 138 untreated active pulmonary TB patients,67 healthy controls,73 pneumonia patients,73 COPD patients, and 72 treated TB patients. To screen and identify differentially expressed proteins of TB, serum proteomics of the untreated TB patients, healthy controls, pneumonia patients and COPD patients were compared by iTRAQ-2DLC-MS/MS, and validated by ELISA in a larger sample number. Clinical data of TB cases and their correlation with differentially expressed serum proteins were analyzed by statistical methods. A diagnostic model was constructed by logistic regression analysis, and the accuracy, specificity and sensitivity of the model was analyzed by receiver operating characteristic (ROC) curve.Results:79 abnormal proteins were discovered in TB patients compared with controls, 47 of them were also significantly different compared with pneumonia and COPD patients. Serum amyloid A (SAA), vitamin K-dependent protein Z (PROZ), and C4b-binding protein β chain (C4BPB) in untreated TB patients (n=138) were significantly higher than healthy controls (n=67) (P<0.0001,P<0.0001,P<0.0001), and significantly lower than pneumonia patients (n=73) (P<0.0001,P<0.0001,P<0.0001) and COPD patients (n=73) (P<0.0001,P<0.0001, P=0.0016). After 6 months treatment, SAA and PROZ were significantly increased in treated TB cases (n=72) (P=0.022, P<0.0001). While, C4BPB was significantly decreased (P=0.0038), but was still significantly higher than controls (P<0.0001). Meanwhile, significantly increased SAA and PROZ were observed in cavitary TB patients (P=0.006, P=0.03) compared with noncavitary patients. SAA were also higher in TB patients with double lung lesions than TB patients with single lung lesions (P=0.003). The serum SAA levels were also increased in smear positive TB patients than smear negative TB patients (P=0.02). Clinical analysis showed that clotting and lipids indices of untreated TB patients were significantly different in controls, pneumonia, COPD, and treated TB patients (P<0.05). Correlation analysis revealed that PROZ was significantly correlated with prothrombin time international normalized ratio (INR) (rs=0.414, P=0.044), and C4BPB was significantly correlated with FIB (rs=0.617, P=0.0002) in TB patients. Receiver operating characteristic (ROC) curve analysis revealed that the area under the ROC curve (AUC) value of the diagnostic model combined by SAA, PROZ, and C4BPB was 0.971,0.895,0.895 and 0.872 to discriminate untreated TB patients from healthy controls, pneumonia, COPD, and cured TB patients.Conclusions:(1) In this study,3 potential diagnostic biomarkers (SAA, PROZ and C4BPB) for TB were acquired by iTRAQ-2DLC-MS/MS and ELISA. (2) The combination of SAA, PROZ and C4BPB had good specificity and sensitivity to discriminate untreated TB patients from healthy controls, pneumonia, COPD, and treated TB patients, and the AUC value was 0.971,0.895,0.895, and 0.872, respectively. (3) Bioinformatics analysis demonstrated that the clotting index of untreated TB patients was abnormal. A significant positive correlation was also observed between PROZ and INR, and a significant positive correlation was also observed between C4BPB and fibrinogen. However, the exact mechanisms of PROZ and C4BPB involved in the disturbance of blood coagulation in TB patients remain to be elucidated. |
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