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

Authors: Yunke Zhang; Junhong Yang; Yingzhe Chen; Jie Lv; Jing Zhang; Yingna Zhang; Xue Zhao; Hua Fang; Chongchong Liu; Qingyong Zhang; Xinzheng Cui; Xiaohan Wang; Feng Gao
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

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.


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## Body

## 1. Introduction

Myasthenia gravis (MG) is a disorder of neuromuscular transmission with an incidence of 0.3 to 2.8 cases per 100,000 people and an annual mortality of 0.06 to 0.89 per million people worldwide [1–3]. MG patients can generate autoantibodies against postsynaptic neuromuscular proteins and epitopes such as acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and lipoprotein receptor-related protein-4 (LRP4) to attack the body’s tissues [4–6]. 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 early-onset MG and atrophic thymus and fat tissue-replaced thymus as the frequent pathological manifestations of late-onset MG [8]. Moreover, AChR antibodies are common in patients with MG and thymoma [4]. The 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 [7–9]. 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]. The 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 “Bojungikki-tang” 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 [25–28]. 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-γ 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 Th17 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.


## 2. Methods

### 2.1. 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. The 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:Astragalus membranaceus (Fisch.) Bunge (50 g, Lot: 161101QF), Glycyrrhiza uralensis Fisch. (15 g, Lot: 160829), Bupleurum chinense DC. (15 g, Lot: 160601QF), Angelica sinensis (Oliv.) Diels (10 g, Lot: 160402QF), Codonopsis pilosula (Franch.) Nannf. (25 g, Lot: 161202), Atractylodes macrocephala Koidz. (15 g, Lot: 160701QF), Cimicifuga foetida L. (10 g, Lot: 160601), Citrus reticulata Blanco peel (10 g, Lot: 160101), Curculigo orchioides Gaertn. (15 g, Lot: 16101910), and Epimedium baiealiguizhouense S.Z.He & Y.K.Yang (15 g, Lot: 161101QF). All raw herbs were purchased from Zhang Zhongjing Pharmacy (Zhengzhou, China). For extraction of Jia Wei BZYQ decoction, 10 herbal medicines were boiled in water for a total of 4 hours with water. The extraction step was repeated once. Then, 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.Table 1
Basic clinical parameters of healthy volunteers and MG patients before treatment.
Group
Number
Age of admission
Sex
Disease duration (years)
Neostigmine test
RNS
Thymus pathology
Thymectomy
C
1
53
M
—
ND
ND
Nor
No
2
69
F
—
ND
ND
Nor
No
3
49
M
—
ND
ND
Nor
No
T1
4
54
M
1
+
+
Nor
No
5
49
F
0.2
+
+
Nor
No
6
44
F
10
+
+
Multiple nodules
No
T2
7
58
F
0.1
+
+
Thymoma
Yes
8
42
F
27
+
+
Nor
No
9
43
F
1
+
+
Nor
No
T3
10
58
F
0.1
+
+
Thymoma
Yes
11
44
F
1
+
+
Thymoma
Yes
12
52
M
0.6
+
+
Nor
No
Note: F = female; M = male; nor = normal; ND = no detected; “+” = positive; “−” = negative; RNS = repetitive nerve stimulation (a variant of the nerve conduction study where electrical stimulation is delivered to a motor nerve repeatedly several times per second).Table 2
Clinical features of MG patients before and after treatment, related treatment methods, and clinical outcomes.
Group
Number
Maximum severity of disease
AchR Ab (nmol/L)
Titin Ab (nmol/L)
Osserman classification
Outcome
Treatment
Before/after
Before/after
Before/after
Before/after
T1
1
Right blepharoptosis/unchanged
—/—
—/—
I/I
SD
—
2
Limb weakness, blepharoptosis with blurred vision, weak chewing, dysphagia/unchanged
1.8/4.5
—/—
IIA/IIA
PD
—
3
Right blepharoptosis with double vision/unchanged
8.5/7.3
—/—
I/I
SD
—
T2
4
Drooping eyes with squint, limb weakness/squint, weak limbs when tired
12.3/9.3
—/—
IIA/IIA
PR
PRE, PYR
5
Drooping eyelids, limb weakness, mild dysphagia/general fatigue, remaining symptoms disappear
16.3/11.7
—/—
IIA/IIA
PR
PRE, PYR
6
Blepharoptosis with double vision, limb weakness, mild mastication weakness/double vision, remaining symptoms disappear
—/—
—/—
IIA/I
PR
PRE
T3
7
Blepharoptosis with strabismus and weakness of limbs/strabismus, remaining symptoms disappear
12.3/8.4
—/—
IIA/I
PR
PRE, PYR, and Jia Wei BZYQ decoction
8
Weakness of limbs, blepharoptosis, mild masticatory weakness, dizziness/eyelids lift weakly when excessive use of the eyes, remaining symptoms disappear
11.3/8.9
8.7/1.9
IIA/I
PR
PYR and Jia Wei BZYQ decoction
9
Double blepharoptosis, double vision/symptoms disappear
3.6/1.3
3.4/2.2
I/I
PR
PYR and Jia Wei BZYQ decoction
Note: AchR Ab normal ranges: <0.4 nmol/L; Titin Ab normal ranges: < 1.0 nmol/L; “−” = negative; SD = stable disease; PD = progression disease; PR = partial remission; PRE = prednisone; PYR = pyridostigmine.


### 2.2. 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. Then, the plasma in the supernatants was collected and subpackaged in freezer tubes and stored at liquid nitrogen.


### 2.3. iTRAQ 2D-LC-MS/MS-Based Proteomic Analysis

Plasma samples were treated with 200μl triethylammonium bicarbonate (TEAB) solution and then subjected to ultrasonication and high-speed centrifugation (12,000 r/min, 20 min). Next, cell supernatants were precipitated using 4 volume exchanges of ice-cold acetone containing 10 mM dithiothreitol (DTT). After 2 h of incubation, cell precipitation was collected by centrifugation (12000 r/min, 20 min) and then resuspended in 800 μl ice-cold acetone containing 10 mM DTT. Next, protein precipitation was collected by high-speed centrifugation (12,000 r/min for 20 min), air-dried, and dissolved in 100 μl TEAB buffer. Protein concentration was determined by a Bradford microassay (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as the standard as previously described [40, 41]. Then, protein (100 μg/100 μl) was diluted using 500 μl NH4HCO3 (50 mM) and digested overnight at 37°C with 2 μg trypsin solution, followed by acidification using an equal volume of 0.1% fatty acid (FA). Next, the acidified solution was poured through a methanol-activated and 0.1% FA-balanced Strata-X C18 column 3 times. After washed twice with 0.1% FA + 5% acetonitrile, the column was eluted once with 1 ml 0.1% FA + 80% acetonitrile. Subsequently, 1 ml elution was freeze-dried and redissolved in TEAB solution. Next, the peptides were labeled with 8-plex iTRAQ reagents (SCIEX, Redwood City, CA, USA) following the protocols provided by the manufacturer. Next, samples were fractionated using a Durashell C18 column (5 μm, 100 Å, 4.6 × 250 mm, Agela Technologies, Tianjin, China) on a Thermo DINOEX Ultimate 3000 BioRS system (Thermo Scientific, Waltham, MA, USA). Subsequently, the peptides were analyzed by tandem mass spectrometry (MS/MS) on an AB SCIEX NanoLC-MS/MS instrument (Triple TOF 5600 plus, SCIEX).


### 2.4. 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.


### 2.5. Statistical Analysis

One-way analysis of variance (ANOVA) and Turkey’s post hoc test were used to measure the difference of multiple groups withP<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.


## 2.1. 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. The 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:Astragalus membranaceus (Fisch.) Bunge (50 g, Lot: 161101QF), Glycyrrhiza uralensis Fisch. (15 g, Lot: 160829), Bupleurum chinense DC. (15 g, Lot: 160601QF), Angelica sinensis (Oliv.) Diels (10 g, Lot: 160402QF), Codonopsis pilosula (Franch.) Nannf. (25 g, Lot: 161202), Atractylodes macrocephala Koidz. (15 g, Lot: 160701QF), Cimicifuga foetida L. (10 g, Lot: 160601), Citrus reticulata Blanco peel (10 g, Lot: 160101), Curculigo orchioides Gaertn. (15 g, Lot: 16101910), and Epimedium baiealiguizhouense S.Z.He & Y.K.Yang (15 g, Lot: 161101QF). All raw herbs were purchased from Zhang Zhongjing Pharmacy (Zhengzhou, China). For extraction of Jia Wei BZYQ decoction, 10 herbal medicines were boiled in water for a total of 4 hours with water. The extraction step was repeated once. Then, 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.Table 1
Basic clinical parameters of healthy volunteers and MG patients before treatment.
Group
Number
Age of admission
Sex
Disease duration (years)
Neostigmine test
RNS
Thymus pathology
Thymectomy
C
1
53
M
—
ND
ND
Nor
No
2
69
F
—
ND
ND
Nor
No
3
49
M
—
ND
ND
Nor
No
T1
4
54
M
1
+
+
Nor
No
5
49
F
0.2
+
+
Nor
No
6
44
F
10
+
+
Multiple nodules
No
T2
7
58
F
0.1
+
+
Thymoma
Yes
8
42
F
27
+
+
Nor
No
9
43
F
1
+
+
Nor
No
T3
10
58
F
0.1
+
+
Thymoma
Yes
11
44
F
1
+
+
Thymoma
Yes
12
52
M
0.6
+
+
Nor
No
Note: F = female; M = male; nor = normal; ND = no detected; “+” = positive; “−” = negative; RNS = repetitive nerve stimulation (a variant of the nerve conduction study where electrical stimulation is delivered to a motor nerve repeatedly several times per second).Table 2
Clinical features of MG patients before and after treatment, related treatment methods, and clinical outcomes.
Group
Number
Maximum severity of disease
AchR Ab (nmol/L)
Titin Ab (nmol/L)
Osserman classification
Outcome
Treatment
Before/after
Before/after
Before/after
Before/after
T1
1
Right blepharoptosis/unchanged
—/—
—/—
I/I
SD
—
2
Limb weakness, blepharoptosis with blurred vision, weak chewing, dysphagia/unchanged
1.8/4.5
—/—
IIA/IIA
PD
—
3
Right blepharoptosis with double vision/unchanged
8.5/7.3
—/—
I/I
SD
—
T2
4
Drooping eyes with squint, limb weakness/squint, weak limbs when tired
12.3/9.3
—/—
IIA/IIA
PR
PRE, PYR
5
Drooping eyelids, limb weakness, mild dysphagia/general fatigue, remaining symptoms disappear
16.3/11.7
—/—
IIA/IIA
PR
PRE, PYR
6
Blepharoptosis with double vision, limb weakness, mild mastication weakness/double vision, remaining symptoms disappear
—/—
—/—
IIA/I
PR
PRE
T3
7
Blepharoptosis with strabismus and weakness of limbs/strabismus, remaining symptoms disappear
12.3/8.4
—/—
IIA/I
PR
PRE, PYR, and Jia Wei BZYQ decoction
8
Weakness of limbs, blepharoptosis, mild masticatory weakness, dizziness/eyelids lift weakly when excessive use of the eyes, remaining symptoms disappear
11.3/8.9
8.7/1.9
IIA/I
PR
PYR and Jia Wei BZYQ decoction
9
Double blepharoptosis, double vision/symptoms disappear
3.6/1.3
3.4/2.2
I/I
PR
PYR and Jia Wei BZYQ decoction
Note: AchR Ab normal ranges: <0.4 nmol/L; Titin Ab normal ranges: < 1.0 nmol/L; “−” = negative; SD = stable disease; PD = progression disease; PR = partial remission; PRE = prednisone; PYR = pyridostigmine.


## 2.2. 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. Then, the plasma in the supernatants was collected and subpackaged in freezer tubes and stored at liquid nitrogen.


## 2.3. iTRAQ 2D-LC-MS/MS-Based Proteomic Analysis

Plasma samples were treated with 200μl triethylammonium bicarbonate (TEAB) solution and then subjected to ultrasonication and high-speed centrifugation (12,000 r/min, 20 min). Next, cell supernatants were precipitated using 4 volume exchanges of ice-cold acetone containing 10 mM dithiothreitol (DTT). After 2 h of incubation, cell precipitation was collected by centrifugation (12000 r/min, 20 min) and then resuspended in 800 μl ice-cold acetone containing 10 mM DTT. Next, protein precipitation was collected by high-speed centrifugation (12,000 r/min for 20 min), air-dried, and dissolved in 100 μl TEAB buffer. Protein concentration was determined by a Bradford microassay (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as the standard as previously described [40, 41]. Then, protein (100 μg/100 μl) was diluted using 500 μl NH4HCO3 (50 mM) and digested overnight at 37°C with 2 μg trypsin solution, followed by acidification using an equal volume of 0.1% fatty acid (FA). Next, the acidified solution was poured through a methanol-activated and 0.1% FA-balanced Strata-X C18 column 3 times. After washed twice with 0.1% FA + 5% acetonitrile, the column was eluted once with 1 ml 0.1% FA + 80% acetonitrile. Subsequently, 1 ml elution was freeze-dried and redissolved in TEAB solution. Next, the peptides were labeled with 8-plex iTRAQ reagents (SCIEX, Redwood City, CA, USA) following the protocols provided by the manufacturer. Next, samples were fractionated using a Durashell C18 column (5 μm, 100 Å, 4.6 × 250 mm, Agela Technologies, Tianjin, China) on a Thermo DINOEX Ultimate 3000 BioRS system (Thermo Scientific, Waltham, MA, USA). Subsequently, the peptides were analyzed by tandem mass spectrometry (MS/MS) on an AB SCIEX NanoLC-MS/MS instrument (Triple TOF 5600 plus, SCIEX).


## 2.4. 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.


## 2.5. Statistical Analysis

One-way analysis of variance (ANOVA) and Turkey’s post hoc test were used to measure the difference of multiple groups withP<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.


## 3. Results

### 3.1. 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, 115, 86, and 158, respectively (Table3). Additionally, 67, 53, 67, 64, 38, and 82 proteins were upregulated (ratio ≥ 1.5) and 43, 64, 76, 51, 48, and 76 proteins were downregulated (ratio ≤ 0.67) in the groups of T1 vs. C, T2 vs. C, T2 vs. T1, T3 vs. C, T3 vs. T1, and T3 vs. T2, respectively (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)).Table 3
Protein number analysis of different treatment groups.
Type
T1 vs. C
T2 vs. C
T2 vs. T1
T3 vs. C
T3 vs. T1
T3 vs. T2
The number of quantified proteins
447
447
447
447
447
447
The number of upregulated proteins
67
53
67
64
38
82
The number of downregulated proteins
43
64
76
51
48
76
The number of differently expressed proteins
110
117
143
115
86
158


### 3.2. 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 Figure1, 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). In immune responses, C-reactive protein (CRP) ranked in first place among downregulated proteins in T1 vs. C group, while it was the most upregulated protein in T2 vs. T1 and T3 vs. T1 groups. KEGG analysis on the top 10 upregulated and downregulated pathways disclosed that pathogen (e.g., Escherichia coli and Staphylococcus aureus) infection-related pathways occupied a larger percentage in T1 vs. C, T2 vs. T1, T3 vs. T1, and T3 vs. T2 groups (Figures 2(a)–2(d)). In T1 vs. C group, the most upregulated protein in Complement and coagulation cascade pathway was plasma kallikrein (KLKB1), while Immunoglobulin heavy variable 6-1 (IGHV6-1) was the most upregulated protein in the pathways of Systemic lupus erythematosus, primary immunodeficiency, amoebiasis, phagosome, Staphylococcus aureus infection, Fc gamma R-mediated phagocytosis, Fc epsilon RI signaling pathway, Dilated cardiomyopathy, and Hematopoietic cell lineage. In T1 vs. C group, the most downregulated protein was keratin, type I cytoskeletal 16 (KRT16) in Pathogenic Escherichia coli infection and Staphylococcus aureus infection pathways with 14-3-3 protein epsilon (YWHAE) in Cell cycle, Neurotrophin signaling pathway, and Oocyte meiosis pathway, von Willebrand factor (VWF) in Focal adhesion pathway, and Immunoglobulin heavy variable 2-26 (IGHV2-26) in the pathways of Phagosome, Viral myocarditis, and Systemic lupus erythematosus. In T2 vs. T1 group, the most upregulated protein was tubulin beta chain (TUBB) in Phagosome and Pathogenic Escherichia coli infection pathways with VWF in Focal adhesion pathway, vitamin K-dependent protein C in Complement and coagulation cascades, cofilin-1 in Regulation of actin cytoskeleton pathway, tropomyosin alpha-4 chain in Dilated cardiomyopathy, integrin alpha-IIb (ITGA2B) in Hematopoietic cell lineage pathway, Ig gamma-4 chain C region (IGHG4) in Primary immunodeficiency pathway, haptoglobin in Staphylococcus aureus infection pathway, and alpha-actinin-1 in Systemic lupus erythematosus pathway. In T2 vs. T1 group, the most downregulated protein was complement component C9 in Complement and coagulation cascades, Systemic lupus erythematosus, Prion diseases, and Amoebiasis pathways with complement C1q subcomponent subunit C (C1QC) in Staphylococcus aureus infection pathway, Ig gamma-1 chain C region (IGHG1) in pathways of Phagosome, Dilated cardiomyopathy, Primary immunodeficiency, Fc gamma R-mediated phagocytosis, and Hematopoietic cell lineage. In T3 vs. T1 group, the most upregulated protein was vitamin K-dependent protein C in the pathway of Complement and coagulation cascades with KRT16 in Staphylococcus aureus infection and Pathogenic Escherichia coli infection pathways, histone H4 (HIST1H4A) in Systemic lupus erythematosus pathway, IGHG4 in Phagosome and Primary immunodeficiency pathways, VWF in Focal adhesion pathway, hemoglobin subunit alpha (HBA1) in Malaria pathway, and apolipoprotein C-III (APOC3) in PPAR signaling pathway. In T3 vs. T1 group, the most downregulated protein was complement C4-A (C4A) in the pathways of Complement and coagulation cascades, Staphylococcus aureus infection, and Systemic lupus erythematosus with thrombospondin-1 (THBS1) in Phagosome and Focal adhesion pathways, tropomyosin alpha-4 (TPM4) in Dilated cardiomyopathy and Hypertrophic cardiomyopathy pathways, vinculin (VCL) in Amoebiasis and Regulation of actin cytoskeleton pathways, and immunoglobulin heavy variable 6-1 (IGHV6-1) in Hematopoietic cell lineage. In T3 vs. T2 group, the most upregulated protein is C9 in the pathways of Complement and coagulation cascades, Amoebiasis, Prion diseases, and Systemic lupus erythematosus, with complement factor H-related protein 1 (CFHR1) in Staphylococcus aureus infection pathway, complement C1r subcomponent (C1R) in Phagosome pathway, Ig alpha-2 chain C region (IGHA2) in Primary immunodeficiency, Dilated cardiomyopathy, and Fc gamma R-mediated phagocytosis pathways, and phospholipid transfer protein (PLTP) in PPAR signaling pathway. The most downregulated protein is THBS1 in Phagosome and Focal adhesion pathways, with C4A in Complement and coagulation cascades and Staphylococcus aureus infection pathways, ITGA2B in Hematopoietic cell lineage pathway, alpha-actinin-1 (ACTN1) in Systemic lupus erythematosus pathway, myosin-9 (MYH9) in Regulation of actin cytoskeleton pathway, TPM4 in Dilated cardiomyopathy pathway, tubulin alpha-4A chain (TUBA4A) in Pathogenic Escherichia coli infection pathway, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Metabolic pathway.Figure 1
The number of proteins with functional annotation in different databases.Figure 2
KEGG analysis of the top 10 upregulated and downregulated pathways in (a) MG patients without treatment (T1) vs. healthy volunteers (C); (b) the group of MG patients with routine western medical treatment (T2) vs. MG patients without treatment (T1); (c) MG patients with combined treatments of Jia Wei BZYQ decoction and routine western medicines (T3) vs. MG patients without treatment (T1); (d) the group of MG patients with combined treatments of Jia Wei BZYQ decoction and routine western medicines (T3) vs. MG patients with routine western medical treatment (T2).
(a)
(b)
(c)
(d)


### 3.3. The Identification of Key Proteins by Protein Association Network Analyses

Additionally, Toll-like receptor (TLR), p53 signaling, nucleotide oligomerization domain- (NOD-) like receptor, mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptor (PPAR), and transforming growth factor-beta (TGF-beta) signaling pathways have been identified as essential players in immunity and host defense against pathogen infections. In this study, we selected these immune pathway-related proteins and the top 10 upregulated or downregulated proteins in each group, which were integrated and are listed in ExcelS1. The association network of proteins in Excel S1 was further established via the STRING database by assessing the possibility of protein-protein interactions based on the combined score (Figure 3). The interacted proteins with a combined score ≥0.9 are presented in Table 4. Moreover, we further analyzed the number of proteins that could interact with a random protein (Table 5) to identify key proteins in the protein association network. Results suggested that some proteins (e.g., alpha-2-HS-glycoprotein (AHSG), apolipoprotein A-II (APOA2), apolipoprotein A-IV (APOA4), CRP, ceruloplasmin (CP), complement C4-A (C4A), histidine-rich glycoprotein (HRG), apolipoprotein A-V (APOA5), von Willebrand factor (VWF), apolipoprotein C-III (APOC3), insulin-like growth factor-binding protein 1 (IGFBP1), inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), vitamin K-dependent protein C (PROC), insulin-like growth factor-binding protein 3 (IGFBP3), endoplasmin (HSP90B1), and serotransferrin (TF)) might play vital roles in MG progression (Table 5). Data for these proteins were collected and are summarized in Table 6 and Excel S2.Figure 3
The association network of proteins.Table 4
Interacted proteins with combined score ≥0.9.
Node1
Node2
Homology
Coexpression
Experimentally_determined_interaction
Database_annotated
Automated_textmining
Combined_score
MYL6
MYH9
0
0.64
0.858
0.9
0.711
0.998
APOA2
APOC3
0
0.796
0
0.9
0.86
0.996
TUBB
TUBA1A
0.92
0.227
0.948
0.9
0.591
0.995
AHSG
APOA2
0
0.918
0
0.9
0.455
0.995
APOA5
APOC3
0
0.466
0
0.9
0.86
0.991
TUBB
TUBA4A
0.92
0.189
0.881
0.9
0.679
0.99
APOA4
APOC3
0
0.565
0
0.9
0.789
0.99
AHSG
ITIH2
0
0.841
0
0.9
0.439
0.99
AHSG
HRG
0
0.67
0
0.9
0.644
0.987
AHSG
TF
0
0.68
0
0.9
0.571
0.985
AHSG
IGFBP1
0
0.788
0
0.9
0.25
0.982
TF
APOA2
0
0.675
0
0.9
0.502
0.982
TUBA1A
TUBA4A
0.986
0
0.809
0.9
0.58
0.98
CD14
LBP
0
0.063
0.379
0.9
0.712
0.98
ILK
RSU1
0
0.265
0.925
0
0.674
0.98
PF4
PPBP
0.886
0.786
0
0.9
0.922
0.98
VWF
THBS1
0
0.063
0
0.9
0.785
0.978
C1R
C4A
0
0.183
0.05
0.9
0.746
0.977
AHSG
PROC
0
0.742
0
0.9
0.128
0.975
APOA2
ITIH2
0
0.728
0
0.9
0.128
0.974
THBS1
HRG
0
0
0.379
0.9
0.609
0.973
SERPINA5
PROC
0
0.177
0.435
0.9
0.416
0.969
APOA5
APOA2
0
0.399
0
0.9
0.526
0.969
C1R
C1QC
0
0.119
0
0.9
0.666
0.968
PF4
VWF
0
0.061
0
0.9
0.685
0.967
TF
ITIH2
0
0.578
0
0.9
0.297
0.967
HIST1H4A
HIST3H3
0
0.107
0.494
0.9
0.371
0.967
TF
CP
0
0.181
0
0.9
0.636
0.967
PPBP
VWF
0
0.061
0
0.9
0.669
0.966
MYL6
TPM4
0
0.185
0.185
0.9
0.546
0.965
APOA2
IGFBP1
0
0.645
0
0.9
0.101
0.965
ACTN1
TLN1
0
0.116
0.083
0.9
0.622
0.965
APOA2
APOA4
0
0.115
0
0.9
0.626
0.964
AHSG
CP
0
0.157
0
0.9
0.609
0.964
ILK
HSP90AA1
0
0
0.161
0.9
0.584
0.962
C8A
C9
0.781
0.341
0.379
0.9
0.576
0.96
APOA5
AHSG
0
0.556
0
0.9
0.189
0.96
C4A
C1QC
0
0.16
0
0.9
0.551
0.959
ITIH2
PROC
0
0.593
0
0.9
0.05
0.957
C4A
APOA2
0
0.083
0.379
0.9
0.308
0.955
PF4
THBS1
0
0.06
0
0.9
0.561
0.955
ITIH2
IGFBP1
0
0.554
0
0.9
0
0.953
ADIPOQ
CRP
0
0
0
0
0.952
0.952
TUBB
HSP90AA1
0
0.129
0.13
0.9
0.411
0.949
HSP90AA1
TUBA1A
0
0.062
0.305
0.9
0.305
0.948
APOA2
PON1
0
0.141
0
0.72
0.794
0.946
APOA2
CP
0
0.115
0
0.9
0.44
0.946
MYL6
TLN1
0
0.427
0
0.9
0.135
0.946
APOA5
TF
0
0.175
0
0.9
0.406
0.946
C4A
TF
0
0.159
0
0.9
0.405
0.945
PF4
HRG
0
0
0
0.9
0.471
0.944
ILK
ACTN1
0
0.064
0.133
0.9
0.38
0.943
PF4
PROC
0
0
0.379
0.9
0.164
0.943
TF
IGFBP1
0
0.214
0.313
0.9
0.073
0.943
CP
PROC
0
0.111
0.379
0.9
0.09
0.943
PLTP
APOA2
0
0
0.379
0.72
0.7
0.943
APOA5
IGFBP1
0
0.394
0
0.9
0.126
0.942
ITIH2
CP
0
0.24
0
0.9
0.289
0.941
APOA5
ITIH2
0
0.428
0
0.9
0
0.94
C4A
CP
0
0.159
0
0.9
0.35
0.94
FLNA
VWF
0
0.061
0.085
0.9
0.38
0.939
ITIH2
FETUB
0
0.916
0
0
0.31
0.939
C1R
CRP
0
0.061
0
0.9
0.399
0.938
APOC3
CETP
0
0.06
0
0.72
0.785
0.938
APOA5
C4A
0
0.106
0
0.9
0.363
0.938
C4A
AHSG
0
0.148
0
0.9
0.322
0.937
APOA5
APOA4
0.681
0.197
0
0.9
0.802
0.937
IGFBP3
IGFBP1
0.755
0.214
0
0.9
0.929
0.936
VWF
PROC
0
0
0
0.9
0.392
0.936
PPBP
THBS1
0
0.062
0
0.9
0.363
0.935
EFEMP1
FBLN1
0.72
0.16
0.12
0.9
0.709
0.935
TF
IGFBP3
0
0.096
0.313
0.9
0.057
0.933
HSP90AA1
TUBA4A
0
0.055
0
0.9
0.342
0.932
C4A
ITIH2
0
0.177
0
0.9
0.237
0.931
APOA5
PROC
0
0.338
0
0.9
0
0.931
SERPINA5
VWF
0
0
0
0.9
0.31
0.928
C4A
PROC
0
0.128
0.05
0.9
0.231
0.927
VWF
HRG
0
0.06
0
0.9
0.292
0.927
TLN1
VWF
0
0.055
0
0.9
0.282
0.926
APOA2
PROC
0
0.275
0
0.9
0.062
0.926
IGFBP1
PROC
0
0.287
0
0.9
0
0.925
AHSG
IGFBP3
0
0.152
0
0.9
0.197
0.925
TPM4
TLN1
0
0.089
0
0.9
0.247
0.925
APOA5
CETP
0
0.061
0
0.72
0.735
0.924
APOA2
CETP
0
0
0
0.72
0.738
0.923
IGFBP1
CP
0
0.146
0
0.9
0.164
0.922
C4A
C8A
0
0.127
0
0
0.914
0.922
PPBP
HRG
0
0
0
0.9
0.25
0.921
ITIH3
ITIH4
0.896
0.18
0
0.9
0.718
0.92
ACTN1
THBS1
0
0.098
0
0.9
0.186
0.92
C1QC
CRP
0
0
0
0.9
0.236
0.92
SAA1
PF4
0
0.061
0
0.9
0.195
0.917
SAA1
PPBP
0
0.063
0
0.9
0.195
0.917
ACTN1
VWF
0
0.061
0.17
0.9
0.066
0.917
IGFBP3
APOA2
0
0.141
0
0.9
0.107
0.916
AHSG
THBS1
0
0
0
0.9
0.189
0.915
TF
HSP90B1
0
0
0
0.9
0.168
0.913
TF
PROC
0
0.147
0.067
0.9
0
0.913
C4A
IGFBP1
0
0.116
0
0.9
0.096
0.913
PON1
CETP
0
0
0
0.72
0.703
0.913
AHSG
PF4
0
0
0
0.9
0.161
0.912
AHSG
VWF
0
0.057
0
0.9
0.144
0.912
APOA5
CP
0
0.116
0
0.9
0.071
0.91
APOA2
HSP90B1
0
0
0
0.9
0.135
0.909
IGFBP3
ITIH2
0
0.112
0
0.9
0
0.907
C4A
IGFBP3
0
0.095
0
0.9
0.061
0.907
AHSG
PPBP
0
0
0
0.9
0.111
0.907
HSP90B1
CP
0
0.077
0
0.9
0.08
0.907
IGFBP3
CP
0
0.076
0
0.9
0.076
0.907
ACTN1
PF4
0
0
0
0.9
0.116
0.907
ACTN1
PPBP
0
0
0
0.9
0.111
0.907
ITIH2
HSP90B1
0
0
0
0.9
0.104
0.906
IGFBP3
HSP90B1
0
0
0
0.9
0.093
0.905
APOA5
IGFBP3
0
0.069
0
0.9
0.061
0.904
AHSG
HSP90B1
0
0
0
0.9
0.072
0.903
C4A
HSP90B1
0
0.061
0
0.9
0.042
0.902
IGFBP3
PROC
0
0.062
0
0.9
0
0.902
AHSG
ACTN1
0
0
0
0.9
0.058
0.901
HSP90B1
IGFBP1
0
0
0
0.9
0.058
0.901
HSP90B1
PROC
0
0
0
0.9
0
0.9
APOA5
HSP90B1
0
0
0
0.9
0
0.9
ACTN1
HRG
0
0
0
0.9
0
0.9Table 5
Number of proteins that could interact with a random protein in ExcelS1.
Protein name
The numbers of interacted protein
Alpha-2-HS-glycoprotein GN = AHSG
26
Apolipoprotein A-II GN = APOA2
21
Apolipoprotein A-IV GN = APOA4
21
C-reactive protein GN = CRP
21
Ceruloplasmin GN = CP
20
Complement C4-A GN = C4A
20
Histidine-rich glycoprotein GN = HRG
20
Apolipoprotein A-V GN = APOA5
19
von Willebrand factor GN = VWF
18
Apolipoprotein C-III GN = APOC3
17
Insulin-like growth factor-binding protein 1 GN = IGFBP1
17
Inter-alpha-trypsin inhibitor heavy chain H2 GN = ITIH2
17
Vitamin K-dependent protein C GN = PROC
17
Insulin-like growth factor-binding protein 3 GN = IGFBP3
16
Endoplasmin GN = HSP90B1
15
Serotransferrin GN = TF
15
Alpha-actinin-1 GN = ACTN1
13
Complement component C8 alpha chain GN = C8A
13
Serum amyloid A-1 protein GN = SAA1
13
Inter-alpha-trypsin inhibitor heavy chain H3 GN = ITIH3
12
Adiponectin GN = ADIPOQ
11
Serum paraoxonase/arylesterase 1 GN = PON1
11
Thrombospondin-1 GN = THBS1
11
Cholesteryl ester transfer protein GN = CETP
10
Complement component C9 GN = C9
10
Fetuin-B GN = FETUB
10
Heat-shock protein HSP 90-alpha GN = HSP90AA1
10
Complement C1r subcomponent GN = C1R
9
Inter-alpha-trypsin inhibitor heavy chain H4 GN = ITIH4
9
Platelet factor 4 GN = PF4
9
Filamin-A GN = FLNA
8
Myosin-9 GN = MYH9
8
Phospholipid transfer protein GN = PLTP
8
Proplatelet basic protein GN = PPBP
8
Stromelysin-1 GN = MMP3
8
Talin-1 GN = TLN1
8
Apolipoprotein D GN = APOD
6
Complement C1q subcomponent subunit C GN = C1QC
6
Lumican GN = LUM
6
Sex hormone-binding globulin GN = SHBG
6
Heat-shock protein beta-1 GN = HSPB1
5
Integrin-linked protein kinase GN = ILK
5
Plasma serine protease inhibitor GN = SERPINA5
5
Tropomyosin alpha-4 chain GN = TPM4
5
Tubulin alpha-1A chain GN = TUBA1A
5
Tubulin alpha-4A chain GN = TUBA4A
5
Myosin light polypeptide 6 GN = MYL6
4
Tubulin beta chain GN = TUBB
4
Fibulin-1 GN = FBLN1
3
Inter-alpha-trypsin inhibitor heavy chain H1 GN = ITIH1
3
Lipopolysaccharide-binding protein GN = LBP
3
Complement factor H-related protein 1 GN = CFHR1
2
Histone H3.1t GN = HIST3H3
2
Histone H4 GN = HIST1H4A
2
Insulin-like growth factor-binding protein 2 GN = IGFBP2
2
Monocyte differentiation antigen CD14 GN = CD14
2
Phosphatidylinositol-glycan-specific phospholipase D GN = GPLD1
2
Ras suppressor protein 1 GN = RSU1
2
EGF-containing fibulin-like extracellular matrix protein 1 GN = EFEMP1
1
Thrombospondin-4 GN = THBS4
1
FERM and PDZ domain-containing protein 1 GN = FRMPD1
0
Keratin, type I cytoskeletal 16 GN = KRT16
0
Neutrophil defensin 3 GN = DEFA3
0Table 6
Upregulated or downregulated ratio of the top 16 proteins in Table5 in different groups.
Gene name
T1 vs. C ratio
T2 vs. C ratio
T2 vs. T1 ratio
T3 vs. C ratio
T3 vs. T1 ratio
T3 vs. T2 ratio
C-reactive protein GN = CRP
0.05
9.54
27.77
2.90
Alpha-2-HS-glycoprotein GN = AHSG
1.82
0.13
0.07
1.73
13.16
Serotransferrin GN = TF
0.33
3.23
4.29
Apolipoprotein C-III GN = APOC3
0.20
3.72
0.46
Apolipoprotein A-II GN = APOA2
4.28
0.12
0.22
1.83
von Willebrand factor GN = VWF
0.26
1.63
6.19
0.66
2.53
0.41
Histidine-rich glycoprotein GN = HRG
4.46
0.54
4.31
1.90
Insulin-like growth factor-binding protein 1 GN = IGFBP1
0.24
0.14
0.61
4.68
Ceruloplasmin GN = CP
2.41
0.13
0.06
0.31
5.59
Apolipoprotein A-IV GN = APOA4
0.37
0.38
1.61
4.42
4.31
Complement C4-A GN = C4A
0.37
0.17
0.13
0.37
Apolipoprotein A-V GN = APOA5
1.86
3.65
1.56
Endoplasmin GN = HSP90B1
2.06
0.66
Insulin-like growth factor-binding protein 3 GN = IGFBP3
0.28
0.21
5.78
Vitamin K-dependent protein C GN = PROC
0.28
1.88
7.66
4.17
0.53
Inter-alpha-trypsin inhibitor heavy chain H2 GN = ITIH2
10.60
0.28
5.28
0.51
1.80Data in Table6 suggested that some proteins such as CRP, TF, APOC3, and VWF might inhibit MG development, while others (e.g., APOA2 and ITIH2) have potential promotional effects on MG progression. Furthermore, our results indicated that routine western medicines alone or along with Jia Wei BZYQ decoction might alleviate MG by regulating the targets in Table 6. The routine treatment plus Jia Wei BZYQ decoction group might experience stronger therapeutic effects due to the upregulation of proteins such as CRP compared with routine treatment group (Table 6). Additionally, our data suggested a link between therapeutic inefficiency and these genes (Table 6). Furthermore, proteomic analyses showed that combined treatment of Jia Wei BZYQ decoction and routine western medicines resulted in abnormal expression of some proteins including myosin heavy chain 9 (MYH9), filamin A (FLNA), tubulin alpha-4A (TUBA4A), thrombospondin-1 (THBS1), tropomyosin alpha-4 (TPM4), Ras suppressor protein 1 (RSU1), ACTN1, and PPBP compared with healthy control group, untreated group, and routine treatment group, indicating that Jia Wei BZYQ decoction might exert its therapeutic effects for MG by regulating these proteins (Table 7).Table 7
Altered proteins in the T3 vs. C, T3 vs. T1, and T3 vs. T2 groups.
Gene name
T3 vs. C ratio
T3 vs. T1 ratio
T3 vs. T2 ratio
Histone H4 GN = HIST1H4A
10.05
9
5.75
Myosin-9 GN = MYH9
0.34
0.27
0.09
Filamin-A GN = FLNA
0.33
0.23
0.07
Alpha-actinin-1 GN = ACTN1
0.27
0.33
0.12
Tubulin alpha-4A chain GN = TUBA4A
0.18
0.26
0.07
Thrombospondin-1 GN = THBS1
0.17
0.08
0.05
Proplatelet basic protein GN = PPBP
0.17
0.12
0.06
Talin-1 GN = TLN1
0.16
0.19
0.07
Tropomyosin alpha-4 chain GN = TPM4
0.11
0.21
0.06
Ras suppressor protein 1 GN = RSU1
0.11
0.16
0.06We also found that monocyte differentiation antigen CD14 expression, related to TLR pathway, was markedly upregulated in T1 vs. C group (ratio: 2.662247514), but was notably downregulated in T2 vs. T1 (ratio: 0.306661468) and T3 vs. T1 (ratio: 0.438830803) groups. Furthermore, TLR pathway-related lipopolysaccharide-binding protein (LBP) expression was noticeably reduced in T1 vs. C group (ratio: 0.102162533) but was remarkably increased in T2 vs. T1 group (ratio: 5.087579717).


## 3.1. 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, 115, 86, and 158, respectively (Table3). Additionally, 67, 53, 67, 64, 38, and 82 proteins were upregulated (ratio ≥ 1.5) and 43, 64, 76, 51, 48, and 76 proteins were downregulated (ratio ≤ 0.67) in the groups of T1 vs. C, T2 vs. C, T2 vs. T1, T3 vs. C, T3 vs. T1, and T3 vs. T2, respectively (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)).Table 3
Protein number analysis of different treatment groups.
Type
T1 vs. C
T2 vs. C
T2 vs. T1
T3 vs. C
T3 vs. T1
T3 vs. T2
The number of quantified proteins
447
447
447
447
447
447
The number of upregulated proteins
67
53
67
64
38
82
The number of downregulated proteins
43
64
76
51
48
76
The number of differently expressed proteins
110
117
143
115
86
158


## 3.2. 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 Figure1, 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). In immune responses, C-reactive protein (CRP) ranked in first place among downregulated proteins in T1 vs. C group, while it was the most upregulated protein in T2 vs. T1 and T3 vs. T1 groups. KEGG analysis on the top 10 upregulated and downregulated pathways disclosed that pathogen (e.g., Escherichia coli and Staphylococcus aureus) infection-related pathways occupied a larger percentage in T1 vs. C, T2 vs. T1, T3 vs. T1, and T3 vs. T2 groups (Figures 2(a)–2(d)). In T1 vs. C group, the most upregulated protein in Complement and coagulation cascade pathway was plasma kallikrein (KLKB1), while Immunoglobulin heavy variable 6-1 (IGHV6-1) was the most upregulated protein in the pathways of Systemic lupus erythematosus, primary immunodeficiency, amoebiasis, phagosome, Staphylococcus aureus infection, Fc gamma R-mediated phagocytosis, Fc epsilon RI signaling pathway, Dilated cardiomyopathy, and Hematopoietic cell lineage. In T1 vs. C group, the most downregulated protein was keratin, type I cytoskeletal 16 (KRT16) in Pathogenic Escherichia coli infection and Staphylococcus aureus infection pathways with 14-3-3 protein epsilon (YWHAE) in Cell cycle, Neurotrophin signaling pathway, and Oocyte meiosis pathway, von Willebrand factor (VWF) in Focal adhesion pathway, and Immunoglobulin heavy variable 2-26 (IGHV2-26) in the pathways of Phagosome, Viral myocarditis, and Systemic lupus erythematosus. In T2 vs. T1 group, the most upregulated protein was tubulin beta chain (TUBB) in Phagosome and Pathogenic Escherichia coli infection pathways with VWF in Focal adhesion pathway, vitamin K-dependent protein C in Complement and coagulation cascades, cofilin-1 in Regulation of actin cytoskeleton pathway, tropomyosin alpha-4 chain in Dilated cardiomyopathy, integrin alpha-IIb (ITGA2B) in Hematopoietic cell lineage pathway, Ig gamma-4 chain C region (IGHG4) in Primary immunodeficiency pathway, haptoglobin in Staphylococcus aureus infection pathway, and alpha-actinin-1 in Systemic lupus erythematosus pathway. In T2 vs. T1 group, the most downregulated protein was complement component C9 in Complement and coagulation cascades, Systemic lupus erythematosus, Prion diseases, and Amoebiasis pathways with complement C1q subcomponent subunit C (C1QC) in Staphylococcus aureus infection pathway, Ig gamma-1 chain C region (IGHG1) in pathways of Phagosome, Dilated cardiomyopathy, Primary immunodeficiency, Fc gamma R-mediated phagocytosis, and Hematopoietic cell lineage. In T3 vs. T1 group, the most upregulated protein was vitamin K-dependent protein C in the pathway of Complement and coagulation cascades with KRT16 in Staphylococcus aureus infection and Pathogenic Escherichia coli infection pathways, histone H4 (HIST1H4A) in Systemic lupus erythematosus pathway, IGHG4 in Phagosome and Primary immunodeficiency pathways, VWF in Focal adhesion pathway, hemoglobin subunit alpha (HBA1) in Malaria pathway, and apolipoprotein C-III (APOC3) in PPAR signaling pathway. In T3 vs. T1 group, the most downregulated protein was complement C4-A (C4A) in the pathways of Complement and coagulation cascades, Staphylococcus aureus infection, and Systemic lupus erythematosus with thrombospondin-1 (THBS1) in Phagosome and Focal adhesion pathways, tropomyosin alpha-4 (TPM4) in Dilated cardiomyopathy and Hypertrophic cardiomyopathy pathways, vinculin (VCL) in Amoebiasis and Regulation of actin cytoskeleton pathways, and immunoglobulin heavy variable 6-1 (IGHV6-1) in Hematopoietic cell lineage. In T3 vs. T2 group, the most upregulated protein is C9 in the pathways of Complement and coagulation cascades, Amoebiasis, Prion diseases, and Systemic lupus erythematosus, with complement factor H-related protein 1 (CFHR1) in Staphylococcus aureus infection pathway, complement C1r subcomponent (C1R) in Phagosome pathway, Ig alpha-2 chain C region (IGHA2) in Primary immunodeficiency, Dilated cardiomyopathy, and Fc gamma R-mediated phagocytosis pathways, and phospholipid transfer protein (PLTP) in PPAR signaling pathway. The most downregulated protein is THBS1 in Phagosome and Focal adhesion pathways, with C4A in Complement and coagulation cascades and Staphylococcus aureus infection pathways, ITGA2B in Hematopoietic cell lineage pathway, alpha-actinin-1 (ACTN1) in Systemic lupus erythematosus pathway, myosin-9 (MYH9) in Regulation of actin cytoskeleton pathway, TPM4 in Dilated cardiomyopathy pathway, tubulin alpha-4A chain (TUBA4A) in Pathogenic Escherichia coli infection pathway, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Metabolic pathway.Figure 1
The number of proteins with functional annotation in different databases.Figure 2
KEGG analysis of the top 10 upregulated and downregulated pathways in (a) MG patients without treatment (T1) vs. healthy volunteers (C); (b) the group of MG patients with routine western medical treatment (T2) vs. MG patients without treatment (T1); (c) MG patients with combined treatments of Jia Wei BZYQ decoction and routine western medicines (T3) vs. MG patients without treatment (T1); (d) the group of MG patients with combined treatments of Jia Wei BZYQ decoction and routine western medicines (T3) vs. MG patients with routine western medical treatment (T2).
(a)
(b)
(c)
(d)


## 3.3. The Identification of Key Proteins by Protein Association Network Analyses

Additionally, Toll-like receptor (TLR), p53 signaling, nucleotide oligomerization domain- (NOD-) like receptor, mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptor (PPAR), and transforming growth factor-beta (TGF-beta) signaling pathways have been identified as essential players in immunity and host defense against pathogen infections. In this study, we selected these immune pathway-related proteins and the top 10 upregulated or downregulated proteins in each group, which were integrated and are listed in ExcelS1. The association network of proteins in Excel S1 was further established via the STRING database by assessing the possibility of protein-protein interactions based on the combined score (Figure 3). The interacted proteins with a combined score ≥0.9 are presented in Table 4. Moreover, we further analyzed the number of proteins that could interact with a random protein (Table 5) to identify key proteins in the protein association network. Results suggested that some proteins (e.g., alpha-2-HS-glycoprotein (AHSG), apolipoprotein A-II (APOA2), apolipoprotein A-IV (APOA4), CRP, ceruloplasmin (CP), complement C4-A (C4A), histidine-rich glycoprotein (HRG), apolipoprotein A-V (APOA5), von Willebrand factor (VWF), apolipoprotein C-III (APOC3), insulin-like growth factor-binding protein 1 (IGFBP1), inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), vitamin K-dependent protein C (PROC), insulin-like growth factor-binding protein 3 (IGFBP3), endoplasmin (HSP90B1), and serotransferrin (TF)) might play vital roles in MG progression (Table 5). Data for these proteins were collected and are summarized in Table 6 and Excel S2.Figure 3
The association network of proteins.Table 4
Interacted proteins with combined score ≥0.9.
Node1
Node2
Homology
Coexpression
Experimentally_determined_interaction
Database_annotated
Automated_textmining
Combined_score
MYL6
MYH9
0
0.64
0.858
0.9
0.711
0.998
APOA2
APOC3
0
0.796
0
0.9
0.86
0.996
TUBB
TUBA1A
0.92
0.227
0.948
0.9
0.591
0.995
AHSG
APOA2
0
0.918
0
0.9
0.455
0.995
APOA5
APOC3
0
0.466
0
0.9
0.86
0.991
TUBB
TUBA4A
0.92
0.189
0.881
0.9
0.679
0.99
APOA4
APOC3
0
0.565
0
0.9
0.789
0.99
AHSG
ITIH2
0
0.841
0
0.9
0.439
0.99
AHSG
HRG
0
0.67
0
0.9
0.644
0.987
AHSG
TF
0
0.68
0
0.9
0.571
0.985
AHSG
IGFBP1
0
0.788
0
0.9
0.25
0.982
TF
APOA2
0
0.675
0
0.9
0.502
0.982
TUBA1A
TUBA4A
0.986
0
0.809
0.9
0.58
0.98
CD14
LBP
0
0.063
0.379
0.9
0.712
0.98
ILK
RSU1
0
0.265
0.925
0
0.674
0.98
PF4
PPBP
0.886
0.786
0
0.9
0.922
0.98
VWF
THBS1
0
0.063
0
0.9
0.785
0.978
C1R
C4A
0
0.183
0.05
0.9
0.746
0.977
AHSG
PROC
0
0.742
0
0.9
0.128
0.975
APOA2
ITIH2
0
0.728
0
0.9
0.128
0.974
THBS1
HRG
0
0
0.379
0.9
0.609
0.973
SERPINA5
PROC
0
0.177
0.435
0.9
0.416
0.969
APOA5
APOA2
0
0.399
0
0.9
0.526
0.969
C1R
C1QC
0
0.119
0
0.9
0.666
0.968
PF4
VWF
0
0.061
0
0.9
0.685
0.967
TF
ITIH2
0
0.578
0
0.9
0.297
0.967
HIST1H4A
HIST3H3
0
0.107
0.494
0.9
0.371
0.967
TF
CP
0
0.181
0
0.9
0.636
0.967
PPBP
VWF
0
0.061
0
0.9
0.669
0.966
MYL6
TPM4
0
0.185
0.185
0.9
0.546
0.965
APOA2
IGFBP1
0
0.645
0
0.9
0.101
0.965
ACTN1
TLN1
0
0.116
0.083
0.9
0.622
0.965
APOA2
APOA4
0
0.115
0
0.9
0.626
0.964
AHSG
CP
0
0.157
0
0.9
0.609
0.964
ILK
HSP90AA1
0
0
0.161
0.9
0.584
0.962
C8A
C9
0.781
0.341
0.379
0.9
0.576
0.96
APOA5
AHSG
0
0.556
0
0.9
0.189
0.96
C4A
C1QC
0
0.16
0
0.9
0.551
0.959
ITIH2
PROC
0
0.593
0
0.9
0.05
0.957
C4A
APOA2
0
0.083
0.379
0.9
0.308
0.955
PF4
THBS1
0
0.06
0
0.9
0.561
0.955
ITIH2
IGFBP1
0
0.554
0
0.9
0
0.953
ADIPOQ
CRP
0
0
0
0
0.952
0.952
TUBB
HSP90AA1
0
0.129
0.13
0.9
0.411
0.949
HSP90AA1
TUBA1A
0
0.062
0.305
0.9
0.305
0.948
APOA2
PON1
0
0.141
0
0.72
0.794
0.946
APOA2
CP
0
0.115
0
0.9
0.44
0.946
MYL6
TLN1
0
0.427
0
0.9
0.135
0.946
APOA5
TF
0
0.175
0
0.9
0.406
0.946
C4A
TF
0
0.159
0
0.9
0.405
0.945
PF4
HRG
0
0
0
0.9
0.471
0.944
ILK
ACTN1
0
0.064
0.133
0.9
0.38
0.943
PF4
PROC
0
0
0.379
0.9
0.164
0.943
TF
IGFBP1
0
0.214
0.313
0.9
0.073
0.943
CP
PROC
0
0.111
0.379
0.9
0.09
0.943
PLTP
APOA2
0
0
0.379
0.72
0.7
0.943
APOA5
IGFBP1
0
0.394
0
0.9
0.126
0.942
ITIH2
CP
0
0.24
0
0.9
0.289
0.941
APOA5
ITIH2
0
0.428
0
0.9
0
0.94
C4A
CP
0
0.159
0
0.9
0.35
0.94
FLNA
VWF
0
0.061
0.085
0.9
0.38
0.939
ITIH2
FETUB
0
0.916
0
0
0.31
0.939
C1R
CRP
0
0.061
0
0.9
0.399
0.938
APOC3
CETP
0
0.06
0
0.72
0.785
0.938
APOA5
C4A
0
0.106
0
0.9
0.363
0.938
C4A
AHSG
0
0.148
0
0.9
0.322
0.937
APOA5
APOA4
0.681
0.197
0
0.9
0.802
0.937
IGFBP3
IGFBP1
0.755
0.214
0
0.9
0.929
0.936
VWF
PROC
0
0
0
0.9
0.392
0.936
PPBP
THBS1
0
0.062
0
0.9
0.363
0.935
EFEMP1
FBLN1
0.72
0.16
0.12
0.9
0.709
0.935
TF
IGFBP3
0
0.096
0.313
0.9
0.057
0.933
HSP90AA1
TUBA4A
0
0.055
0
0.9
0.342
0.932
C4A
ITIH2
0
0.177
0
0.9
0.237
0.931
APOA5
PROC
0
0.338
0
0.9
0
0.931
SERPINA5
VWF
0
0
0
0.9
0.31
0.928
C4A
PROC
0
0.128
0.05
0.9
0.231
0.927
VWF
HRG
0
0.06
0
0.9
0.292
0.927
TLN1
VWF
0
0.055
0
0.9
0.282
0.926
APOA2
PROC
0
0.275
0
0.9
0.062
0.926
IGFBP1
PROC
0
0.287
0
0.9
0
0.925
AHSG
IGFBP3
0
0.152
0
0.9
0.197
0.925
TPM4
TLN1
0
0.089
0
0.9
0.247
0.925
APOA5
CETP
0
0.061
0
0.72
0.735
0.924
APOA2
CETP
0
0
0
0.72
0.738
0.923
IGFBP1
CP
0
0.146
0
0.9
0.164
0.922
C4A
C8A
0
0.127
0
0
0.914
0.922
PPBP
HRG
0
0
0
0.9
0.25
0.921
ITIH3
ITIH4
0.896
0.18
0
0.9
0.718
0.92
ACTN1
THBS1
0
0.098
0
0.9
0.186
0.92
C1QC
CRP
0
0
0
0.9
0.236
0.92
SAA1
PF4
0
0.061
0
0.9
0.195
0.917
SAA1
PPBP
0
0.063
0
0.9
0.195
0.917
ACTN1
VWF
0
0.061
0.17
0.9
0.066
0.917
IGFBP3
APOA2
0
0.141
0
0.9
0.107
0.916
AHSG
THBS1
0
0
0
0.9
0.189
0.915
TF
HSP90B1
0
0
0
0.9
0.168
0.913
TF
PROC
0
0.147
0.067
0.9
0
0.913
C4A
IGFBP1
0
0.116
0
0.9
0.096
0.913
PON1
CETP
0
0
0
0.72
0.703
0.913
AHSG
PF4
0
0
0
0.9
0.161
0.912
AHSG
VWF
0
0.057
0
0.9
0.144
0.912
APOA5
CP
0
0.116
0
0.9
0.071
0.91
APOA2
HSP90B1
0
0
0
0.9
0.135
0.909
IGFBP3
ITIH2
0
0.112
0
0.9
0
0.907
C4A
IGFBP3
0
0.095
0
0.9
0.061
0.907
AHSG
PPBP
0
0
0
0.9
0.111
0.907
HSP90B1
CP
0
0.077
0
0.9
0.08
0.907
IGFBP3
CP
0
0.076
0
0.9
0.076
0.907
ACTN1
PF4
0
0
0
0.9
0.116
0.907
ACTN1
PPBP
0
0
0
0.9
0.111
0.907
ITIH2
HSP90B1
0
0
0
0.9
0.104
0.906
IGFBP3
HSP90B1
0
0
0
0.9
0.093
0.905
APOA5
IGFBP3
0
0.069
0
0.9
0.061
0.904
AHSG
HSP90B1
0
0
0
0.9
0.072
0.903
C4A
HSP90B1
0
0.061
0
0.9
0.042
0.902
IGFBP3
PROC
0
0.062
0
0.9
0
0.902
AHSG
ACTN1
0
0
0
0.9
0.058
0.901
HSP90B1
IGFBP1
0
0
0
0.9
0.058
0.901
HSP90B1
PROC
0
0
0
0.9
0
0.9
APOA5
HSP90B1
0
0
0
0.9
0
0.9
ACTN1
HRG
0
0
0
0.9
0
0.9Table 5
Number of proteins that could interact with a random protein in ExcelS1.
Protein name
The numbers of interacted protein
Alpha-2-HS-glycoprotein GN = AHSG
26
Apolipoprotein A-II GN = APOA2
21
Apolipoprotein A-IV GN = APOA4
21
C-reactive protein GN = CRP
21
Ceruloplasmin GN = CP
20
Complement C4-A GN = C4A
20
Histidine-rich glycoprotein GN = HRG
20
Apolipoprotein A-V GN = APOA5
19
von Willebrand factor GN = VWF
18
Apolipoprotein C-III GN = APOC3
17
Insulin-like growth factor-binding protein 1 GN = IGFBP1
17
Inter-alpha-trypsin inhibitor heavy chain H2 GN = ITIH2
17
Vitamin K-dependent protein C GN = PROC
17
Insulin-like growth factor-binding protein 3 GN = IGFBP3
16
Endoplasmin GN = HSP90B1
15
Serotransferrin GN = TF
15
Alpha-actinin-1 GN = ACTN1
13
Complement component C8 alpha chain GN = C8A
13
Serum amyloid A-1 protein GN = SAA1
13
Inter-alpha-trypsin inhibitor heavy chain H3 GN = ITIH3
12
Adiponectin GN = ADIPOQ
11
Serum paraoxonase/arylesterase 1 GN = PON1
11
Thrombospondin-1 GN = THBS1
11
Cholesteryl ester transfer protein GN = CETP
10
Complement component C9 GN = C9
10
Fetuin-B GN = FETUB
10
Heat-shock protein HSP 90-alpha GN = HSP90AA1
10
Complement C1r subcomponent GN = C1R
9
Inter-alpha-trypsin inhibitor heavy chain H4 GN = ITIH4
9
Platelet factor 4 GN = PF4
9
Filamin-A GN = FLNA
8
Myosin-9 GN = MYH9
8
Phospholipid transfer protein GN = PLTP
8
Proplatelet basic protein GN = PPBP
8
Stromelysin-1 GN = MMP3
8
Talin-1 GN = TLN1
8
Apolipoprotein D GN = APOD
6
Complement C1q subcomponent subunit C GN = C1QC
6
Lumican GN = LUM
6
Sex hormone-binding globulin GN = SHBG
6
Heat-shock protein beta-1 GN = HSPB1
5
Integrin-linked protein kinase GN = ILK
5
Plasma serine protease inhibitor GN = SERPINA5
5
Tropomyosin alpha-4 chain GN = TPM4
5
Tubulin alpha-1A chain GN = TUBA1A
5
Tubulin alpha-4A chain GN = TUBA4A
5
Myosin light polypeptide 6 GN = MYL6
4
Tubulin beta chain GN = TUBB
4
Fibulin-1 GN = FBLN1
3
Inter-alpha-trypsin inhibitor heavy chain H1 GN = ITIH1
3
Lipopolysaccharide-binding protein GN = LBP
3
Complement factor H-related protein 1 GN = CFHR1
2
Histone H3.1t GN = HIST3H3
2
Histone H4 GN = HIST1H4A
2
Insulin-like growth factor-binding protein 2 GN = IGFBP2
2
Monocyte differentiation antigen CD14 GN = CD14
2
Phosphatidylinositol-glycan-specific phospholipase D GN = GPLD1
2
Ras suppressor protein 1 GN = RSU1
2
EGF-containing fibulin-like extracellular matrix protein 1 GN = EFEMP1
1
Thrombospondin-4 GN = THBS4
1
FERM and PDZ domain-containing protein 1 GN = FRMPD1
0
Keratin, type I cytoskeletal 16 GN = KRT16
0
Neutrophil defensin 3 GN = DEFA3
0Table 6
Upregulated or downregulated ratio of the top 16 proteins in Table5 in different groups.
Gene name
T1 vs. C ratio
T2 vs. C ratio
T2 vs. T1 ratio
T3 vs. C ratio
T3 vs. T1 ratio
T3 vs. T2 ratio
C-reactive protein GN = CRP
0.05
9.54
27.77
2.90
Alpha-2-HS-glycoprotein GN = AHSG
1.82
0.13
0.07
1.73
13.16
Serotransferrin GN = TF
0.33
3.23
4.29
Apolipoprotein C-III GN = APOC3
0.20
3.72
0.46
Apolipoprotein A-II GN = APOA2
4.28
0.12
0.22
1.83
von Willebrand factor GN = VWF
0.26
1.63
6.19
0.66
2.53
0.41
Histidine-rich glycoprotein GN = HRG
4.46
0.54
4.31
1.90
Insulin-like growth factor-binding protein 1 GN = IGFBP1
0.24
0.14
0.61
4.68
Ceruloplasmin GN = CP
2.41
0.13
0.06
0.31
5.59
Apolipoprotein A-IV GN = APOA4
0.37
0.38
1.61
4.42
4.31
Complement C4-A GN = C4A
0.37
0.17
0.13
0.37
Apolipoprotein A-V GN = APOA5
1.86
3.65
1.56
Endoplasmin GN = HSP90B1
2.06
0.66
Insulin-like growth factor-binding protein 3 GN = IGFBP3
0.28
0.21
5.78
Vitamin K-dependent protein C GN = PROC
0.28
1.88
7.66
4.17
0.53
Inter-alpha-trypsin inhibitor heavy chain H2 GN = ITIH2
10.60
0.28
5.28
0.51
1.80Data in Table6 suggested that some proteins such as CRP, TF, APOC3, and VWF might inhibit MG development, while others (e.g., APOA2 and ITIH2) have potential promotional effects on MG progression. Furthermore, our results indicated that routine western medicines alone or along with Jia Wei BZYQ decoction might alleviate MG by regulating the targets in Table 6. The routine treatment plus Jia Wei BZYQ decoction group might experience stronger therapeutic effects due to the upregulation of proteins such as CRP compared with routine treatment group (Table 6). Additionally, our data suggested a link between therapeutic inefficiency and these genes (Table 6). Furthermore, proteomic analyses showed that combined treatment of Jia Wei BZYQ decoction and routine western medicines resulted in abnormal expression of some proteins including myosin heavy chain 9 (MYH9), filamin A (FLNA), tubulin alpha-4A (TUBA4A), thrombospondin-1 (THBS1), tropomyosin alpha-4 (TPM4), Ras suppressor protein 1 (RSU1), ACTN1, and PPBP compared with healthy control group, untreated group, and routine treatment group, indicating that Jia Wei BZYQ decoction might exert its therapeutic effects for MG by regulating these proteins (Table 7).Table 7
Altered proteins in the T3 vs. C, T3 vs. T1, and T3 vs. T2 groups.
Gene name
T3 vs. C ratio
T3 vs. T1 ratio
T3 vs. T2 ratio
Histone H4 GN = HIST1H4A
10.05
9
5.75
Myosin-9 GN = MYH9
0.34
0.27
0.09
Filamin-A GN = FLNA
0.33
0.23
0.07
Alpha-actinin-1 GN = ACTN1
0.27
0.33
0.12
Tubulin alpha-4A chain GN = TUBA4A
0.18
0.26
0.07
Thrombospondin-1 GN = THBS1
0.17
0.08
0.05
Proplatelet basic protein GN = PPBP
0.17
0.12
0.06
Talin-1 GN = TLN1
0.16
0.19
0.07
Tropomyosin alpha-4 chain GN = TPM4
0.11
0.21
0.06
Ras suppressor protein 1 GN = RSU1
0.11
0.16
0.06We also found that monocyte differentiation antigen CD14 expression, related to TLR pathway, was markedly upregulated in T1 vs. C group (ratio: 2.662247514), but was notably downregulated in T2 vs. T1 (ratio: 0.306661468) and T3 vs. T1 (ratio: 0.438830803) groups. Furthermore, TLR pathway-related lipopolysaccharide-binding protein (LBP) expression was noticeably reduced in T1 vs. C group (ratio: 0.102162533) but was remarkably increased in T2 vs. T1 group (ratio: 5.087579717).


## 4. 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 [50–52]. For instance, BZYQ decoction inhibited T helper 2 (Th2) responses and promoted interleukin 12 (IL-12) release from macrophages by increasing TLR4 expression in murine allergic rhinitis models [53]. Our study demonstrated that CD14 was abnormally upregulated in MG patients, and routine western medicines alone or along with Jia Wei BZYQ decoction may treat MG by reducing CD14 expression. Prior studies showed that CD14 induced NF-kappa-B activation, cytokine secretion, and immune and inflammatory response by activating TLR4 signaling and TLR signaling cascade [54, 55], suggesting that BZYQ decoction might treat MG by inhibiting CD14/TLR4 signaling pathway. Furthermore, differences in LBP expression suggested that pyridostigmine and/or prednisone might exert therapeutic effects by upregulating LBP expression in MG cases. MG is an autoimmune disease. Pathogenic pathways such as Staphylococcal aureus infection, Complement and coagulation cascades, Fc gamma R-mediated phagocytosis, Fc epsilon RI signaling pathway, and some inflammatory factors are usually upregulated in MG. Hence, some proteins among these pathogenic pathways were highly expressed in T1 vs. C group. Prednisone could suppress abnormal immunity responses in MG patients, so the expression of these pathological pathway-related proteins was downregulated in T2 vs. T1 group. The 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 anti-immunity 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 investigate the roles of identified proteins in MG progression through other experiments.The 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 ExcelS1. 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 [56–58]. 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].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, furtherin 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.


## 5. Conclusions

The 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.



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*Source: 9147072-2019-12-13.xml*