Research Article | Open Access
Screening of Serum Protein Markers for Avascular Osteonecrosis of Femoral Head Differentially Expressed after Treatment with Yuanshi Shengmai Chenggu Tablets
Avascular necrosis of the femoral head (ANFH) is an a frequently occurring orthopaedic disease with high morbidity. Traditional Chinese Medicine (TCM) Yuanshi Shengmai Chenggu Tablet is a valid prescription for treating steroid-induced femoral head necrosis. However, there are rare investigations about the serum protein marker expression after the acting of drugs on hormone and TCM. In the present study, we aimed to systematically discover and validate the serum biomarkers expression difference in patients with steroid-induced avascular necrosis of femoral head (SANFH) after taking Yuanshi Shengmai Chenggu Tablets (SANFH-TCM), so as to reveal the action mechanism of TCM from the molecular level by using isobaric tags for relative and absolute quantification (iTRAQ) with multiple reaction monitoring quantification. Significant differences in fibrinogen alpha, fibrinogen beta, fibrinogen gamma, fibronectin, C-reactive protein, apolipoprotein A, apolipoprotein D, and apolipoprotein E were found among SANFH, SANFH-TCM, and healthy controls. Therefore, our study proposes potential biomarkers for SANFH diagnosis and for the prognosis of femoral head necrosis after Traditional Chinese Medicine treatment.
Avascular necrosis of the femoral head (ANFH) is a clinical common orthopaedic disease [1–3] with very high morbidity. It is difficult to cure and is one of the major medical problems that have not yet been overcome [4, 5]. Primary osteonecrosis of the femoral head is due to gene or gene mutation of patients. Secondary osteonecrosis of the femoral head could divide into traumatic and nontraumatic osteonecrosis of femoral head [6–8], in which traumatic osteonecrosis of the femoral head is the avascular necrosis of osteocyte caused by interruption of blood flow in the blood vessels in femoral head, which was due to trauma [9, 10]. Its etiology is still unclear. It was demonstrated that long-term and large dosage usage of hormone and drinking are two important factors that cause ANFN [4, 11]. In recent years, with the wide use of corticosteroids clinically, the cases with ANFN have also increased greatly [12, 13]. However, the pathogenesis of steroid-induced ANFN is still unknown. For developing new methods to prevent and treat ANFN, study on the pathogenesis of steroid-induced ANFN is particularly urgent [12, 14, 15].
Recent reports have shown that the occurrence of ANFN could be greatly decreased by an early intervention on high-risk crowds of ANFN who use hormones such as steroid and alcohol . However, the composition of the serum is very complex . It contains high-abundance proteins like albumin and immunoglobulins (mainly IgG), as well as low abundance proteins that are secreted by tissue or cells [18, 19]. Some of them are key proteins involved in signal transduction and regulation . Tan et al.  adopted two-dimensional electrophoresis technology and separated 7 differentially expressed proteins between patients with primary femoral head necrosis and normal subjects from 10 pairs serum samples. They found that four important proteins including tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), Crosslaps, and anti-p53 antibody were significantly changed and that all of them can be used as the diagnosis serum markers of nontraumatic femoral head necrosis. Although the pathogenesis of ANFN is still unclear and the relevance of this finding with the further clinical application was not reported, analysis of the differentially expressed proteins in the serum could provide useful information.
Traditional Chinese Medicine (TCM) Yuanshi Shengmai Chenggu Tablet is valid and specialty drug for steroid-induced ANFN treatment. Yuanshi Shengmai Chenggu Tablet has obtained the certificate of new medicine in China and has been applied in clinical . Its active ingredients are mainly flavonoids such as vitexin. By clinical studies, it was demonstrated the application of Yuanshi Shengmai Chenggu Tablet can significantly relieve the patients’ pain and accelerate the absorption of dead bone and formation of new bone, showing a relatively strong osteogenetic activity .
Liu et al.  extracted proteins in bone tissue from the femur and humerus bone in rat osteonecrosis model with or without Yuanshi Shengmai Chenggu Tablet TCM treatment and performed proteomics research. They reported that anticoagulating proteins heavy chain II B, phospholipid hydroperoxide glutathione peroxidase, and ubiquitin enzymes E2 (MW: 17 kD) are closely associated with steroid-induced bone necrosis, as well as the therapeutic efficacy of TCM.
In this study we aimed to investigate the differentially expressed protein in serum between steroid-induced ANFN patients with or without TCM treatment (Yuanshi Shengmai Chenggu Tablets). For this purpose, the proteomics method isobaric tags for relative and absolute quantification (iTRAQ) with multiple reaction monitoring (MRM) quantification was adopted in this study, so as to reveal the molecular mechanism of TCM treated the SANFN in the molecular level.
2. Material and Methods
Patients diagnosed as ANFN in the First Affiliated Hospital of Traditional Chinese Medicine University of Guangzhou from February 2014 to February 2015 were included. The ANFN diagnosis was established by referring to standard of adult femoral head necrosis diagnosis expert consensus (2012 edition) and the diagnosis and treatment of avascular necrosis of the expert advice of diagnostic criteria. Patients in active period of ANFN, alcoholics who are simultaneously treated by long-term high dose of glucocorticoids (taken steroid > 10 mg/d longer than 3 years), or patients with combining chronic disease which needs prolonged treatment were excluded in present study. All participants gave written informed consent before being enrolled in the study (AE-2013012011).
2.2. Specimens and Groups
Patients with ANFN who have used long-term and high-dosage of steroid (SANFN) were further treated with TCM Yuanshi Shengmai Chenggu Tablet (6 tablets each time, 3 times per day, total 3 months; prepared by the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou, China). Serum samples () from patients with or without TCM were prospectively collected after obtaining written informed consent. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. Five healthy subjects were collected during the same period who were sex- and age-matched. Thus, the verification population was divided into 3 groups: steroid-induced avascular necrosis of femoral head (SANFH), SANFH-TCM treatment, and healthy controls.
All serum samples were centrifuged at 1250 for 5 min and then 13500 for 15 min at 4°C within 1 h of collection. All samples were then stored at −80°C until use.
2.3. iTRAQ Analysis of Serum Samples
iTRAQ labeling and mass spectrometry analysis were performed as previously described . Then, six iTRAQ labeled sample polls were generated (steroid-induced avascular necrosis of femoral head, SANFH-TCM treatment, and health controls, each for two subgroups). Briefly, high-abundance serum proteins such as albumin, IgG, and haptoglobin were removed by using the Human 14 Multiple Affinity Removal System (Agilent Technologies, Santa Clara, CA, USA). Then, 50 μg protein of each sample was concentrated and desalted, followed by digestion using trypsin before iTRAQ labeling. Six groups were labeled, including steroid-induced avascular necrosis of femoral head, iTRAQ reagent 113, 116; SANFH-TCM treatment 114, 117; and health controls 115, 118. The six sample groups were mixed, desalted, and dried.
The iTRAQ labeled peptides were separated by Strong Cation Exchange (SCX) chromatography (Bonna-Agela Technologies, Tianjin, China). SCX was carried out on a Polysulfoethyl 4.6 × 100 mm column (5 μm, 200 Å, PolyLC Inc., Maryland, USA). The peptides were eluted at the 45 min gradient from 100% buffer A (10 mM KH2PO4 pH 3.0, 25% acetonitrile) to 45% buffer B (10 mM KH2PO4 pH 3.0, 500 mM KCl, 25% acetonitrile) at the flow rate of 800 μL/min on Agilent 1210 LC system. All the fractions were analyzed by MALDI-TOF/TOF 5800 mass spectrometer (AB SCIEX, California, USA). Protein quantification and identification were performed with the Proteome Discoverer (version 1.3, thermos). The default bias correction was used and all quantitative variables were analyzed by the Proteome Discoverer 1.3. Peptide abundances were calculated based on the areas of the monoisotopic peaks. Protein ratios were the average ratios of all quantified peptides. Proteins with quantification value < 0.05 in at least two pairs (113 : 114, 113 : 115, 114 : 115; 116 : 117, 116 : 118, 117 : 118) and with the ratio > 1.2 (the average ratio of two repeat experiments) or ratio < 0.83 were considered as differentially expressed proteins, using a cutoff of 2 times standard deviation .
2.4. Bioinformatics Analysis
Biomarker candidates were then prioritized using scoring a system based on iTRAQ values from Proteome Discoverer analysis. The cellular component, molecular function, and biological process were analyzed through Gene Ontology (GO) database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping was performed by KEGG Mapper (http://www.genome.jp/kegg/mapper.html), and the enrichment analysis was performed by Blast2GO PRO software (https://www.blast2go.com/, version 2.8).
2.5. Validation of Differential Expressed Protein by Multiple Reaction Monitoring (MRM) Quantification
To validate the expression of biomarker candidates, MRM quantifications were performed as previously described . Briefly, 30 μg protein of each sample was digested using trypsin before being desalted. Then, desalted peptide mixtures were loaded onto an Acclaim PePmap C18-reversed phase column (100 Ǻ, Thermo Scientific, Massachusetts, USA) and separated with reversed phase C18 column (300 Ǻ, Bonna-Agela Technologies) mounted on a Dionex ultimate 3000 nano-LC system. Peptides were eluted using a gradient of 5–80% (v/v) acetonitrile in 0.1% formic acid over 45 min at a flow rate of 300 nL/min combined with a Q Exactive mass spectrometer (Thermo Scientific, Massachusetts, USA), and then the eluates were directly entered in Q Exactive MS (Thermo Scientific, Massachusetts, USA), setting in positive ion mode and data-dependent manner with full MS scan within 350–2000 , full scan resolution at 70000, MS/MS scan resolution at 17500, and MS/MS scan with minimum signal threshold , isolation width at 2 Da. To evaluate the performance of this mass spectrometry on the iTRAQ labeled samples, two MS/MS acquisition modes and higher collision energy dissociation (HCD) were employed. And to optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined, stepped 20%. Each MS/MS spectrum was searched against a mascot database (Uniprot_2015_human database, 20194 protein entries) and a decoy database for FDR analysis (programmed in the software). The search parameters were as follows: sample type, iTRAQ 8-plex (Peptide Labeled); cysteine modification by methyl methane-thiosulfonate; digestion, trypsin enzyme; proteins with, at least, two peptides with a high confidence score (>95%) and a low FDR (estimated local FDR of 5%) were considered positively identified.
2.6. Statistical Analysis
All studies to identify biomarkers by iTRAQ/MRM LC-MS/MS were performed on three separate occasions. Statistical analysis was performed using R (version 3.4.2, Bell Laboratories, USA). Analysis of variance (ANOVA) was performed for groups comparison. A value < 0.05 was considered as statistical significantly.
A total of 26 patients were included in the present study. Demographic characteristics of present population were summarized in Table 1. All of them were diagnosed as Association Research Circulation Osseous (ARCO) II stage SONFH, and the time windows of being illness were from 6 to 34 months. The mean age was 39.5 years old and 11 (42.3%) of them were males, suggesting that the patients SONFH were younger. Primary cause of 50% of patients was systemic lupus erythematosus, indicating a high risk of long-term high dose of steroid in systemic lupus erythematosus.
3.2. Protein Identification and Differentially Abundant Proteins
Serum proteins of steroid-induced ANSF (SANFH) patients, SANFH-TCM treatment patients, and health subjects were screened using iTRAQ. The experiment was repeated twice and detected 399 proteins. Among them, 61 proteins were differentially expressed between SANFH and healthy controls (Table 2), including 35 significantly upregulated proteins (>1.21-fold, ) and 26 significantly downregulated proteins (<0.83-fold, ). The top four upregulated proteins in SANFH compared to healthy controls were serum amyloid A-2 (SAA2), Ig lambda, sodium/potassium-transporting ATPase subunit alpha-3 (ATP1A3), and calcium-binding mitochondrial carrier protein Aralar1 (SLC25A12) with fold change values of 4.57, 2.64, 2.07, and 1.95. The top four downregulated proteins were properdin, keratin type I cytoskeletal 9 (KRT9), apolipoprotein (a) (LPA), and tropomyosin alpha-4 (TPM4) with fold change values of −1.77, −1.57, −1.65, and −1.61, respectively.
A total of 74 proteins were differentially expressed between SANFH-TCM and healthy controls (Table 3), including 45 significantly upregulated proteins (>1.21-fold, ), and 29 significantly downregulated proteins (<0.83-fold, ). The top four upregulated proteins in SANFH-TCM compared to healthy controls were ATP-binding cassette subfamily B member 9 (ABCB9), fibrinogen alpha, fibrinogen gamma, and fibrinogen beta with fold change values of 17.47, 13.05, 12.67, and 12.11. The top four downregulated proteins were C-reactive protein (CRP), Tubulin alpha-1A (TUBA1A), fibronectin, and LPA with fold change values of −1.73, −1.73, −1.52, and −1.48, respectively.
In addition, a total of 81 proteins were differentially expressed between SANFH-TCM and SANFH (Table 4), including significantly 44 upregulated proteins (>1.21-fold, ) and 37 significantly downregulated proteins (<0.83-fold, ). The top four upregulated proteins in SANFH-TCM compared to SANFH were ABCB9, IQ, and AAA domain-containing protein 1 (IQCA1), fibrinogen alpha, and fibrinogen beta, which showed the fold change values of 21.82, 13.86, 13.66, and 13.64, respectively. The top four downregulated proteins were serum amyloid A-2 (SAA2), Ig lambda, CRP, and collagen alpha-1 with fold change values of −2.36, −2.26, −2.24, and −2.04, respectively.