Chu YG, Shi J, Hu YH, Wu HQ, Liu GJ, Hu CJ, Li YZ, Li Y, Chen ZJ, He Q. J Chin Integr Med. 2009; 7(7): 629-635. Received February 14, 2009; accepted April 14, 2009; published online July 15, 2009. Indexed/abstracted in and full text link-out at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao. Free full text (HTML and PDF) is available at www.jcimjournal.com. Forward linking and reference linking via CrossRef. DOI: 10.3736/jcim20090706

Correspondence: Prof. Yuan-hui HU; Tel: 010-88001018; E-mail: huiyuhui@yahoo.com.cn

    The core of traditional Chinese medicine (TCM) is syndrome differentiation treatment, and syndrome is a pathological manifestation with a certain regulation. By observing the patient's abnormal manifestation, one can grasp the nature of the disease. External pathological manifestation is bound to its material basis, which can be explained by proteomics study. After the study of proteomics relating to TCM syndrome of essential hypertension (EH) patients, the basis of the proteomics resulting from TCM syndrome of the EH patients was researched, and the syndrome-protein expression profiling was established to reveal the scientific meanings of TCM syndrome, and to further supply the basis and methods of the objectiveness of TCM syndrome differentiation.

1   Clinical data and methods
1.1   Clinical data
1.1.1   Study objects   From June to November 2008, 52 EH patients in Department of Cardiology, Guang'anmen Hospital, including 39 EH patients with abundant phlegm-dampness and 20 EH patients with non-abundant phlegm-dampness were included in the study, and another 30 healthy persons were also included.
1.1.2   Diagnostic criteria   The diagnosis of hypertension followed A Draft of Chinese Guidelines for Hypertension Prevention and Treatment^［1］. The diagnosis of TCM syndrome followed National Standards of People’s Republic of China: Syndrome Part of Terminology of Traditional Chinese Medicine Clinical Diagnosis and Treatment^［2］ and Guidelines for Clinical Research on Chinese New Herbal Medicines^［3］. The main common symptoms of abundant phlegm-dampness syndrome including sticky sputum, sputum expectoratant, limb heaviness, stuffiness and oppression in chest and epigastrium, poor appetite, greasy taste in the mouth, white and greasy tongue coating, moderate and soggy pulse or slippery pulse. Patients meeting the hypertension diagnosis filled TCM syndrome scale, and then two associate chief physicians performed the syndrome differentiation according to the TCM scale.
1.1.3   Including criteria   The patients who met with the diagnostic criteria were included, and they were asked to fill the TCM scale. The blood biochemical tests including blood glucose, blood lipid, liver and kidney function, and uric acid, and the necessary clinical examinations including electrocardiogram, ambulatory blood pressure, and carotid artery ultrasound were performed.
1.1.4   Excluding criteria   1) Patients with chronic diseases such as secondary hypertension, hyperlipidemia, diabetes, cerebral infarction, cerebral hemorrhage, angina pectoris, myocardial infarction, abnormal lipid metabolism and atherosclerosis diagnosed by carotid artery ultrasound; 2) Patients with cancer history; 3) Patients with liver and kidney dysfunction, hypothyroidism, hypoproteinemia, alcoholism and mental diseases.
1.2   Study methods
1.2.1   Materials and instruments   Weak cation-exchange (WCX) nano-magnetic beads were from SELDI Bio-engineering Technology Co., Ltd., Beijing. Tris (hydroxymethyl) aminomethane (Tris), 3-cyclohexylamino-1-propanesulfonic acid (CHA-PS), acetonitrile (ACN), dithiothreitol (DTT), and sinapic acid (SPA) were purchased from Sigma Co., Ltd. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) PBS-Ⅱc protein chip reader (Ciphergen Inc., USA), Hitachi-SCR20BA high-speed refrigerated centrifuge (Hitachi Co., Ltd., Japan), and Sanyo-MDF-U73V Ultra-low temperature refrigerator (Sanyo Electric, Japan).
1.2.2   Methods of obtaining and processing samples   Three milliliters of fasting blood were collected in the morning, contained in BD vacuum test tube without anticoagulant, stored for 1 hour at 4 ℃, and centrifuged at the speed of 3 000 r/min for 10 minutes, and then the blood serum was split into 5 tubes, each tube 100 μL and stored in －86 ℃ refrigerator. For analysis, took the serum out from refrigerator, melt the serum with ice-bath and then centrifuged for 10 minutes with 2 000×g at 4 ℃. The serum (10 μL) was taken and added to 20 μL U9 buffer solution (9 mol/L urea, 20 g/L CHAPS, 10 g/L DTT, 50 mmol/L Tris-HCl, pH 9.0), well mixed, till centrifuged for 30 minutes at 4 ℃, and 100 mmol/L NaAc 370 μL (pH 4.0) was added and mixed immediately.
1.2.3   Activation of weak cation-exchange nano-magnetic beads   WCX nano-magnetic beads (10 g/L, 50 μL) was added to each PCR tube, and separated for 2 minutes on the magnetic separation plate. The supernatant part was discarded, and then 100 mmol/L NaAc 100 μL was added for activating two times, 5 minutes each time.
1.2.4   Serum protein captured by activated nano-magnetic beads   A total of 100 μL prepared serum sample was added into the activated magnetic beads, and then oscillated and incubated for 1 hour. After two-minute separation on the magnetic separation plate, the supernatant part was discarded and 100 mmol/L NaAc 100 μL was added and eluted for 2 times, 5 minutes each time. The protein was eluted and combined with the magnetic beads for 5 minutes by 5 mL/L trifluoroacetic acid (TFA) 10 μL. The protein eluent (5 μL)was mixed with 5 μL saturated SPA, and then absorbed 2 μL protein mixture spotting on the Au-chip (just as vector effect; Au-chip, Ciphergen Biosystems Inc.), dried naturally, and the protein fingerprint was detected by PBS-Ⅱc protein chip reader.
1.2.5   Chip detection   The parameters of chip reading instrument were set to the maximum detection range of 5 000, optimized scope of 2 000-10 000, laser intensity of 205, and detection sensitivity of 8. Before testing, adjusted the instrument using all-in-one polypeptide standard chip, and system quality deviation was less than or equal to 0.1%. The original data were processed by ProteinChip Software 3.1 in standardization and protein peak of 4 091 m/z (mass to charge ratio) was used as internal standard to correct.
1.2.6   Establishment and verification of model   Linear classification analysis was made to the peaks of differentially expressed proteins with same relative molecular masses between the abundant phlegm-dampness group and the control groups (EH patients with non-phlegm-dampness and healthy persons) by Biomarker Pattern software (BPS). With further optimization of the experiment para-meters, we determined the best classification model and output original results, and then carried out cross-validation and exported the results by BPS. The model is based on the neural network decision tree, and a best training sample was determined first. The choice of computing equation is the default choice of classification, if no logistic regression would be chosen, and the core operational idea is Bayes criteria. Classification function was used to classify and the higher score Yi function belongs to i kinds. The core operational idea of independent variable was applied for all possible set method in multiple regressions after t test. The neural network decision tree was used for drawing the decision tree after the determination of types and equation. All results were automatically formed by BPS software.
1.2.7   Perspective evaluation   Blind test (perspective evaluation) was performed to establish abundant phlegm-dampness model by testing the protein fingerprint and the sensitivity, specificity, false positive rate, false negative rate as well as Youden's index of the model for abundant phlegm-dampness were obtained.
1.3   Statistical analysis   Statistical treatment of the protein fingerprint was performed by using Ciphergen ProteinChip software and BioMarker Wizard software. T-test was applied for the contrast between the abundant phlegm-dampness group and the control group (selected together, operated respectively and then summed up) with the data expressed as `x±s and α=0.05 as statistical standard. After the mass spectrogram of differential protein peak was formed, BPS 5.0 was used to identify and diagnose the best marker of abundant phlegm-dampness and establish the diagnostic model of abundant phlegm-dampness. SPSS was used to perform Chi-Square and ANOVA analysis for the general information (gender, age) of the objects.

2   Results
2.1   Baseline data   There was no significant difference in general information between the abundant phlegm-dampness group and the control group (P>0.05). Detailed information and clinical grouping are listed in Table 1. To find out the special protein in the EH patients with abundant phlegm-dampness, the EH patients with non-phlegm-dampness and another group of healthy persons were combined as the control.
2.2   Fingerprint of proteins and the establishment of abundant phlegm-dampness model   A total of 102 significantly differentiated protein peaks were tested in the abundant phlegm-dampness group and the control group (2 000-5 000 m/z), among which 20 kinds of proteins were higher in the abundant phlegm-dampness group than in the control group, while the other 82 kinds of proteins were lower in the abundand-dampness group than in the control group (P<0.05). The information was shown in Table 2.
    

3   Discussion
   In recent years, with the rapid development of proteomics technology, proteomics study methods have been applied to clinical research^{［4, 5］}. A growing number of scholars carried out research on the nature of TCM syndrome by using the technology of proteomics. Xiao et al^［6］ carried out the research for the serum protein in 23 hypertension cases of hyperactivity of liver yang syndrome, contrasted with the normal. A total of approximately 500 protein spots were detected, and there were 16 significantly differentiated proteins by group comparison. Then peptide mass fingerprinting (PMF) was made by using MALDI-TOF-MS. It was found that n-methyl-aspartyl acid receptor, serum ceruloplasmin, transferrin, vitamin D-binding protein, apolipoprotein were up-regulated in hyperactivity of liver yang group, and the expression of glucoprotein reduced. Xiong et al^［7］ studied the serum protein differences between liver-yang transforming into wind and hyperactivity of liver-yang of hypertensive cerebral hemorrhage. Forty cases of hypertensive cerebral hemorrhage were divided into 17 cases of liver-yang transforming into wind and 23 cases of hyperactivity of liver-yang syndrome. The images were processed by using 2-DE electrophoresis, scanner and PDQuest V7.3.1 software, and the average protein points of serum map were 113 103 and 94 in liver-yang transforming into wind and hyperactivity of liver-yang syndrome of hypertensive cerebral hemorrhage as well as healthy persons respectively. Five proteins were preliminary identified, which were serum amyloid precursor, ceruloplasmin, vitamin D-binding protein, apo lipoprotein cⅢ and transfermin. These 5 proteins might be related to liver-yang transforming into wind syndrome of hypertensive cerebral hemorrhage.