iTRAQ-Based Proteomics Analysis of Plasma of Myasthenia Gravis Patients Treated with Jia Wei Bu Zhong Yi Qi Decoction

Myasthenia gravis (MG) is an autoimmune disease. A proportion of MG patients did not get satisfactory results after treatment with pyridostigmine and prednisone. Jia Wei Bu Zhong Yi Qi (Jia Wei BZYQ) decoction, a water extract from multiple herbs, has been demonstrated to be effective in the treatment of multiple “Qi deficiency type” diseases including MG in China. In this text, we investigated protein alterations in the plasma from healthy volunteers (C), MG patients without any treatment (T1), MG patients with routine western medical treatment (T2), and MG patients with combined treatments of Jia Wei BZYQ decoction and routine western medicines (T3) and identified some potential proteins involved in the pathogenesis and treatment of MG. iTRAQ (isobaric tags for relative and absolute quantitation) and 2D-LC-MS/MS (two-dimensional liquid chromatography-tandem mass spectrometry technologies) were employed to screen differentially expressed proteins. The identification, quantification, functional annotation, and interaction of proteins were analyzed by matching software and databases. In our project, 618 proteins were identified, among which 447 proteins had quantitative data. The number of differentially expressed proteins was 110, 117, 143, 115, 86, and 158 in T1 vs. C, T2 vs. C, T2 vs. T1, T3 vs. C, T3 vs. T1, and T3 vs. T2 groups, respectively. Functional annotation results showed that many differentially expressed proteins were closely associated with immune responses. For instance, some key proteins such as C-reactive protein, apolipoprotein C-III, apolipoprotein A-II, alpha-actinin-1, and thrombospondin-1 have been found to be abnormally expressed in T3 group compared to T1 group or T2 group. Interaction network analyses also provided some potential biomarkers or targets for MG management.

MG with autoantibodies against AChR (AChR-MG) is the most common MG subtype, accounting for about 70%-80% of all MG cases [7]. MuSK antibodies are found in 1-10% of MG patients, and LRP4 antibodies can be detected in approximately 7% of MG patients without antibodies against AChR and MuSK [8]. AChR antibodies mainly occur in generalized and ocular MG (both early-onset and late-onset) with thymic hyperplasia as the common feature of earlyonset MG and atrophic thymus and fat tissue-replaced thymus as the frequent pathological manifestations of lateonset MG [8]. Moreover, AChR antibodies are common in patients with MG and thymoma [4]. e concentration of total AChR antibody was not directly related to MG severity, whereas AChR antibody concentration is increased when the condition for MG patients is exacerbated [7,8]. MG patients with AChR or MuSK antibodies usually develop more severe symptoms (51-52% MGFA I-II at onset) compared with LRP4 antibody-positive subgroup [7][8][9]. Moreover, MG patients with double-positive autoantibodies of AChR/LRP4 or MuSK/LRP4 have more severe symptoms relative to any single-positive MG subgroup [9]. It is presumed that thymus is not related to the pathogenesis of MG in MG patients with MuSK antibodies, and extremely rare MuSK antibodies are found in MG patients with thymoma [4]. MG patients with positive LRP4 antibodies usually have ocular or mild generalized symptoms (85% with MGFA grade I or II at disease onset), and some have thymic changes (31% hyperplasia, 29% involuted thymus, 7% atrophy, 33% normal thymus, and none with thymoma) [9]. e average age for MG patients is 33.4 years for females and 41.9 years for males at disease onset [9].
MG is characterized by multiple clinical symptoms such as muscle weakness, drooping eyelids, double vision, trouble talking, and trouble walking [10,11]. Most MG patients have good prognosis due to great advances in diagnostic techniques, epidemiologic methodologies, and treatment for MG over the past several decades [2,10,12]. However, only a few MG patients have complete remission and most patients need sustained treatment to alleviate related symptoms [13]. Moreover, MG diagnosis is often different at the beginning of disease due to its heterogeneity [14]. Hence, it is imperative to explore MG pathogenesis and search for potential biomarkers or targets for MG management.
Recently, mass spectrometry-(MS-) based iTRAQ (isobaric tag for relative and absolute quantification) technique has become prominent in proteomics research requiring relative quantification [15,16]. Emerging study shows that iTRAQ is an effective chemical tagging strategy that offers a deep insight into the molecular mechanisms implicated in disease progression and drug responses [16,17]. For instance, iTRAQ-based quantitative proteomic analyses identified key proteomic changes and critical metabolic pathways in arsenic-induced liver fibrosis rat models [18]. Additionally, Wang et al. pointed out that iTRAQ-based proteomic analysis may reveal the molecular targets of drugs and bioactive small molecules [19].
Immunotherapy with glucocorticosteroids and symptomatic treatments with acetylcholinesterase inhibitors are the bedrock for MG management [2,12]. Pyridostigmine and prednisone have been widely used as the first-line therapeutic drugs for MG patients [2]. However, the uneven absorption and side effects of pyridostigmine and prednisone limited their clinical applications [2]. Traditional Chinese medicine (TCM) is a holistic medical system for diagnosis, prevention, and treatment of diseases and has been an integral part of Asian cultures for thousands of years [20]. Some studies pointed out that combined therapy of TCM and western medicines could markedly improve clinical outcomes of MG patients with reduced side effects [21,22].
Bu Zhong Yi Qi (BZYQ) decoction (also called "Bojungikkitang" or "Hochu-ekki-to"), a water extract from multiple herbs, has been widely used to treat "Qi deficiency type" or "Yang deficiency type" diseases in Asia [23,24]. BZYQ decoction can enhance immunological responses, improve nutritional status, ameliorate chronic fatigue syndromes, and reduce cytotoxicity of chemotherapeutic drugs [25][26][27][28]. BZYQ decoction has therapeutic effects on multiple diseases such as tumors [29,30], chronic obstructive pulmonary disease [25], and MG [31]. In this study, two herbs (Curculigo orchioides Gaertn and Epimedium baiealiguizhouense S.Z.He & Y.K.Yang) that can ameliorate kidney-Yang deficiency syndromes and replenish kidney essence were added into the formula of BZYQ decoction to generate Jia Wei BZYQ decoction [32,33]. Clinical studies over many years in China presented that Jia Wei BZYQ decoction was effective and safe to treat MG [34,35]. Moreover, our antecedent finding revealed that Jia Wei BZYQ decoction could markedly decrease acetylcholine receptor antibody (AChR-Ab) serum level and reduce concentrations of IL-2, IL-6, IL-17A, and IFN-c in thymus and spleen tissue fluid in experimental autoimmune MG rat models [36]. Also, our researchers found that Jia Wei BZYQ decoction in combination with pyridostigmine bromide was more effective to alleviate the clinical symptoms, reduce serum AChR-Ab level, and decrease 17 cell proportion without obvious side effects compared with pyridostigmine bromide alone in the treatment of MG patients with spleen and kidney deficiency syndromes [37,38]. However, the pharmacological basis for Jia Wei BZYQ decoction in the treatment of MG has not been well defined at present.
In the present study, iTRAQ and 2D-LC-MS/MS technologies as well as bioinformatics approaches were used to investigate potential plasma biomarkers in MG patients treated with routine western medicines (prednisone and/or pyridostigmine) alone or along with Jia Wei BZYQ decoction. In addition, we identify some vital proteins associated with MG etiology.

Clinical Information.
Blood samples were collected from healthy volunteers (n � 3, C group, labeled 113, 18-70 years old) and ambulant or hospitalized primary MG patients (n � 9, 18-70 years old) at the First Affiliated Hospital of Henan University of Traditional Chinese Medicine and the affiliated hospital of Henan Medical Science Research Institute between April 2016 and January 2017. MG patients were diagnosed according to western medicine criteria as previously described [39]. MG severity was assessed following the modified Osserman classification standard as described in a previous document [39]. MG patients with a stable disease status and a modified Osserman I or IIA subtype were enrolled in our project. In addition, MG patients need to have main symptoms and at least one minor symptom of spleen and kidney deficiency based on TCM diagnostic criteria. Main symptoms include (i) ptosis or diplopia; (ii) articulation, chewing and swallowing difficulties, and choking and coughing while drinking water; and (iii) fatigability of the whole body. Minor symptoms contain some features of Qi deficiency and Yang deficiency. Qi deficiency is characterized by shortness of breath, sluggish eyes, complexion chlorosis, body fatigue, loss of control over bowel movements, light or dark red tongue with a thin white coating, and a weak pulse. e characteristics of Yang deficiency are cold limbs, fear of cold, abdominal pain, borborygmus, light or dark red tongue with a thin white coating, and a weak pulse. Moreover, MG patients with other diseases, patients underwent thymectomy or plasma exchange therapy, and patients in prenatal and suckling periods were excluded from our study. All participants signed written informed consent documents, and our study was approved by the Ethical Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine.
Nine MG patients were divided into 3 groups as follows: untreated group (n � 3, T1, labeled 114), a routine western medicine treatment group (n � 3, T2, labeled 115), and combined treatment group of routine western medicine plus Jia Wei BZYQ decoction (n � 3, T3, labeled 116). MG patients in T2 group were treated with prednisone or pyridostigmine bromide tablets, alone or in combination. At the beginning, 60-80 mg prednisone was administered once daily to MG patients for 20 days; the dose was gradually decreased at a rate of 2.5 mg/15 days according to the status of patients. Pyridostigmine bromide tablets were given to MG patients at a dosage of 60 mg × 4 times/day for 20 days; the dose was gradually reduced by 5 mg/ 15 days according to the status of patients. MG patients in T3 group were given Jia Wei BZYQ decoction orally for 2 months with twice daily (1 pack each time) on the basis of the routine western medicine treatment as T2 group.
Jia Wei BZYQ decoction was generated by the First Affiliated Hospital of Henan University of Chinese Medicine from a boiled water extraction of 10 traditional Chinese herbal medicines with the prescription as below: en, the two extracts were mixed and subpackaged into bags with 200 ml in each pack. Basic clinical parameters of healthy volunteers and MG patients before treatment are displayed in Table 1. Clinical features of MG patients before and after treatment along with related treatment methods and clinical outcomes are displayed in Table 2.

Blood Sample Collection.
Blood samples (10 ml) were collected using EDTAK2 anticoagulation tubes on the second day after enrolling and two months later after treatment and then centrifuged at 4000 r/min for 5 min at room temperature. en, the plasma in the supernatants was collected and subpackaged in freezer tubes and stored at liquid nitrogen.

Database Searching and Protein
Identification. MS data were analyzed via MASCOT and Protein Pilot software to identify and quantify corresponding proteins in different treatment groups. Interaction of identified proteins was analyzed using the STRING database. Functional annotations on identified proteins were performed by Gene Ontology (GO) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, and Clusters of Orthologous Groups of proteins (COGs) database.

Statistical Analysis.
One-way analysis of variance (ANOVA) and Turkey's post hoc test were used to measure Evidence-Based Complementary and Alternative Medicine the difference of multiple groups with P < 0.05 as the threshold for statistical significance. Proteins were considered as differentially expressed when satisfying the following two conditions simultaneously: (a) P value <0.05 and (b) upregulated ratio ≥1.5 or downregulated ratio ≤0.67.

Protein Identification from Plasma Samples.
To screen MG-related protein markers and explore potential therapeutic mechanisms of Jia Wei BZYQ decoction and routine western medicines for MG, iTRAQ and 2D-LC-MS/ MS technologies were used to analyze differentially expressed plasma proteins in MG patients treated with or without routine western medicines alone or along with Jia Wei BZYQ decoction. In our study, a total of 618 proteins were identified with 447 proteins having quantitative results. Quantitative results revealed that the number of differentially expressed proteins in the groups of T1 vs. C, T2 vs. C, T2 vs. T1, T3 vs. C, T3 vs. T1, and T3 vs. T2 was 110, 117, 143,   (Table 3). Moreover, the names and fold changes of differentially expressed proteins in T1 vs. C and T3 vs. T2 groups are presented in a heat map ( Supplementary Figures 1(A) and 1(B)).

Functional Annotation of Identified Proteins.
Due to the limitations of annotation databases, the number of proteins with annotation information was disparate in different databases. As presented in Figure 1, 567, 216, and 432 annotated proteins were available for GO, COG, and KEGG functional annotation databases, respectively. GO analysis results showed that the pathogenesis of MG was closely related to abnormal immune responses (data not presented).

Discussion
MG is both clinically and pathologically heterogeneous disease with multiple targets, treatment responses, and clinical manifestations [42,43]. Recently, immunotherapy has emerged as an effective approach to improve clinical outcomes for MG patients [44,45]. Inflammation and immunity-related pathways such as TLRs have been found to be implicated in the pathogenesis of MG [46,47]. Previous studies showed that BZYQ decoction could reduce side effects of chemotherapeutic drugs and enhance their therapeutic efficiency [48,49] and improve immune responses by regulating inflammation and immunity-related pathways and molecules [50][51][52]. For instance, BZYQ decoction inhibited T helper 2 ( 2) responses and promoted interleukin 12 (IL-12) release from macrophages by increasing TLR4 expression in murine allergic rhinitis models [53].  Evidence-Based Complementary and Alternative Medicine Table  4: Interacted proteins with combined score ≥0.9.      upregulation of these pathological pathway-related proteins in T3 vs. T2 group might result from the inhibitory effect of Jia Wei BZYQ decoction on prednisone-mediated antiimmunity responses. On the other hand, the network of cellular signal regulatory pathways is very complex and above pathological pathway-related proteins may participate in the regulation of multiple pathways with increased expression in one pathway and reduced or unchanged expression in other ways. In addition, the sample number is relatively small in our study and it is imperative to   e interaction of proteins plays important roles in coordinating biological behaviors of organisms. KEGG analysis is an effective approach to identify proteins implicated in vital metabolism and signaling transduction pathways. Regarding the vital roles of immune responses in MG etiology and Jia Wei BZYQ decoction treatment, we selected proteins implicated in immune pathways (e.g., PPAR, MAPK, p53, TGF-beta, Wnt, NOD-like receptor, and TLR) by KEGG function annotation analysis in T1 vs. C, T2 vs. T1, T3 vs. T1, and T3 vs. T2 groups. Moreover, the top 10 upregulated and downregulated proteins in T1 vs. C, T2 vs. T1, T3 vs. T1, and T3 vs. T2 groups were picked out, which are presented in Excel S1. CRP, a member of the pentraxin superfamily and a highly conserved acute-phase plasma protein in humans, has been recognized as a regulator of inflammation and autoimmunity [56][57][58]. In addition, CRP has been found to be implicated in the pathogenesis of multiple diseases such as inflammatory diseases, cardiovascular disease, and cancers [57,59]. Moreover, prior studies showed that steroid therapy before thymectomy resulted in a marked reduction in serum CRP concentration in MG patients compared with the nonsteroid treatment group [60]. Our data showed that CRP expression was markedly downregulated in T1 vs. C group, but was notably upregulated in T2 vs. T1 and T3 vs. T1 groups, hinting that CRP might inhibit the progression of MG and Jia Wei BZYQ decoction might enhance the therapeutic effect of routine western medicines for MG cases by increasing CRP expression. Additionally, our data indicated that proteins such as TF and VWF might hinder MG development, whereas proteins such as APOA2, ITIH2, and CP might promote MG progression. Proteomic analyses also suggested that Jia Wei BZYQ decoction might exert therapeutic effects for MG by regulating some vital protein expression. Among the proteins we detected, antigen uptake-and presentation-related gene THBS1 has been reported to be highly expressed in thymus tissues of MG patients [61].

Node1
Collectively, our data identified some potential biomarkers to facilitate the development of MG diagnosis and treatment. Moreover, our data provided insight into the therapeutic mechanisms of Jia Wei BZYQ decoction and routine western medicines for MG, deepening our understanding of MG pathogenesis. Although our study elucidated potential roles of some proteins in the etiology and pharmacopathology of MG, further in vitro and in vivo experiments are necessary to confirm our results of the iTRAQ-based plasma proteomics analysis in MG. Additionally, the sample size in our study was small, which is a limitation that should be considered with our results.

Conclusions
e treatments of Jia Wei BZYQ decoction and routine western medicines resulted in many protein alterations in the plasma of MG patients. Our data presented a valuable resource for diagnosis and treatment for MG.

Supplementary Materials
All upregulated (ratio ≥ 1.5) and downregulated proteins (ratio ≤0.67) in the groups of T1 vs. C, T2 vs. C, T2 vs. T1, T3 vs. C, T3 vs. T1, T3 vs. T2 are presented in Additional file 1; immune pathway-related proteins and the top 10 upregulated or downregulated proteins in each group were picked out and are presented in Excel S1. e 15 core protein in the protein interaction network ( Figure 3) and matching statistical analysis of fold changes in T1 vs. C, T2 vs. C, T2 vs. T1, T3 vs. C, T3 vs. T1, and T3 vs. T2 groups are presented in Excel S2. Protein interaction in Excel S1 was analyzed by STRING database and interacted proteins with combined score >0.