iTRAQ-based quantitative proteomics and target-fishing strategies reveal molecular signatures on vasodilation of Compound Danshen Dripping Pills

Journal Pre-proof iTRAQ-based quantitative proteomics and target-fishing strategies reveal molecular signatures on vasodilation of Compound Danshen Dripping Pills Xin Wu, Xiujiang Han, Lili Li, Simiao Fan, Pengwei Zhuang, Zhen Yang, Yanjun Zhang PII:

S0009-2797(19)31123-8

DOI:

https://doi.org/10.1016/j.cbi.2019.108923

Reference:

CBI 108923

To appear in:

Chemico-Biological Interactions

Received Date: 2 July 2019 Revised Date:

26 November 2019

Accepted Date: 10 December 2019

Please cite this article as: X. Wu, X. Han, L. Li, S. Fan, P. Zhuang, Z. Yang, Y. Zhang, iTRAQ-based quantitative proteomics and target-fishing strategies reveal molecular signatures on vasodilation of Compound Danshen Dripping Pills, Chemico-Biological Interactions (2020), doi: https://doi.org/10.1016/ j.cbi.2019.108923. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Author Statement Investigation & Writing-Original Draft: Xin Wu

Xin Wu performed the experiment

and wrote the original draft. Formal analysis: Xiujiang Han Xiujiang Han applied the statistical and analyzed the data. Methodology: Lili Li and Simiao Fan Lili Li developed the methodology. Project administration: Pengwei Zhuang Pengwei Zhuang managed and coordinated the research activity planning and execution. Writing-Review & Editing: Zhen Yang Zhen Yang supervised the whole experiment and revised the manuscript Conceptualization & Supervision: Yanjun Zhang Yanjun Zhang designed the whole experiment and supervised it.

1

iTRAQ-Based

Quantitative

Proteomics

and

2

Target-Fishing Strategies Reveal Molecular Signatures

3

on Vasodilation of Compound Danshen Dripping Pills

4 5

Xin Wua,d,1, Xiujiang Hane,1, Lili Lia,b, Simiao Fana, Pengwei

6

Zhuanga,b, Zhen Yanga,b,c*, Yanjun Zhanga,b*

7

a

8

Tianjin, 300193, China

9

b

Chinese Materia Medica College, Tianjin University of Traditional Chinese Medicine,

Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of

10

Traditional Chinese Medicine, Tianjin, 300193, China

11

c

12

d

13

Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking

14

Union Medical College, Tianjin, 300192, China

15

e

16

300100, China

17

Beijing University of Chinese Medicine, Beijing, 100029, China Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,

Department of Cardiology, Tianjin Hospital of ITCWM Nankai Hospital, Tianjin,

*

Correspondence to:

18

Yanjun Zhang and Zhen Yang, Tianjin University of Traditional Chinese Medicine,

19

No.312 Anshanxi Road, Nankai District, Tianjin, 300193, China. Tel: +86-22-59596138.

20

E-mail: [email protected], [email protected]

21

1

The first two authors contributed equally to this work. 1

1

Abstract

2

Angina pectoris can be used as an early warning for coronary artery disease. Vasodilation

3

is a potential target for angina pectoris. Traditional Chinese medicine - Compound

4

Danshen Dripping Pill (CDDP) is widely used to improve the symptoms of

5

cardiovascular diseases (CVDs). To investigate the influence of vasodilation effect and

6

underlying pharmacological mechanisms of CDDP, we determined the vasodilation effect

7

of thoracic aorta ring on rat induced by norepinephrine (NE). Then targets-fishing method

8

was used to predict the potential mechanism of CDDP on vasodilation, based on the

9

structures of the main components. Then, iTRAQ-based quantitative proteomics analysis

10

was used for verification of the candidate target proteins and pathways to illustrate the

11

underlying pharmacological mechanisms. Furthermore, the differentially expressed

12

proteins in the enriched pathways were validated by western blotting. In this study, we

13

found that CDDP can significantly inhibit NE induced aortic contraction tension, and the

14

mechanism may be related to platelet activation, cGMP – PKG signaling pathway and

15

vascular smooth muscle contraction. The method of discovery and verification on

16

mechanism for vasodilation of CDDP provides a new way to analysis the mechanism of

17

drug, especially the multi-component herbal medicine.

18

Keywords: Vasodilation; Target protein; ITRAQ; Compound danshen dripping pill.

19 20

1. Introduction

2

1

Coronary heart disease (CHD), one of the major forms of CVDs, has become a

2

primary health concern in the general population in recent years. According to the World

3

Health Organization, CVDs are the leading cause of death in non-communicable diseases

4

and accounted for approximately one-third of all deaths worldwide (Zhu et al, 2016).

5

Angina pectoris could be conceptualized as a warning and death signal for coronary

6

artery disease and impending death and show signs of coronary artery disease (Börjesson,

7

1999). The patient with angina pectoris is at high risk of myocardial infarction and

8

sudden death (Morikawa et al, 2013). Angina pectoris is typically relieved by rest and

9

nitroglycerin (Kloner and Chaitman, 2017). Several recent studies showed that angina

10

pectoris is a predictor of major adverse cardiac events. Several factors have been

11

considered to play roles in the mechanism of angina pectoris. Coronary artery spasm

12

(CAS) has been demonstrated to be the primary pathophysiologic mechanism responsible

13

for angina pectoris (Parrinello et al, 2014; Hamabe et al, 2001). Coronary

14

vasoconstriction is another important mechanism but has received little attention and yet

15

is a potential therapeutic mechanism(Maseri et al, 2009).

16

Compound Danshen Dripping Pill (CDDP; Fufang Danshen Diwan in Chinese), a

17

proprietary pharmaceutical preparation consisting of Radix salviae miltiorrhizae

18

(Danshen), Radix notoginseng (Sanqi), and Borneolum syntheticum (Bingpian) was

19

widely used for the treatment of coronary arteriosclerosis, angina pectoris, and other

20

cardiovascular diseases in China and some other countries including Russia, Cuba, North

21

Korea, and Saudi Arabia (Xu et al, 2014; Yao et al, 2015). The phase II clinical trial of 3

1

CDDP for treating chronic stable angina (http://clinicaltrials.gov/, NCT00797953) was

2

completed in the USA in 2010 (Luo et al, 2013). CDDP has pleiotropic effects including

3

protecting endothelial function, increasing nitric oxide bioavailability, antioxidant,

4

anti-inflammatory effects and vasodilator properties (Xin et al, 2013; Yi et al, 2014; Deng

5

et al, 2013). These cardiovascular benefits are likely to be applicable to the chronic stable

6

angina pectoris. However, there is limited investigation and evidence supporting their use

7

in cardiovascular disorders.

8

Therefore, we hypothesized that treatment with CDDP for chronic stable angina and

9

modulate key early events in atherosclerosis may relate to vasodilation effect. In this

10

study, we validated the new target of CDDP and investigated more deeply mechanism. So,

11

we first confirm the vasodilation effect of CDDP in the thoracic aorta of rats. Then, target

12

proteins and pathways related to vasodilation effect of CDDP were predicted based on

13

target fishing. Additionally, isobaric tags for relative and absolute quantitation (iTRAQ)

14

followed by two-dimensional gel electrophoresis and mass spectrometry were used to

15

identify proteins that were differentially expressed of the CDDP. Differential proteins

16

related to vasodilation effect are screened via bioinformatics analysis combining with

17

predicted proteins and pathways.

18

2. Material and methods

19

2.1. Sample preparation

20

CDDP extracts were provided by Tianjin Tasly Pharmaceutical Co. Ltd., Tianjin,

21

China. To eliminate the effects of batches on drug quality, we randomly sampled three 4

1

batches of CDDP extracts (No.20150403; No.20150302; No.20140902). Batches of

2

extracts were mixed equally as standard CDDP extract.

3

2.2. Animal treatment

4

Male Sprague-Dawley (SD) rats (weighting 250 ± 20 g), were supplied by Tianjin

5

University of Traditional Chinese Medicine (Tianjin, China). The rats were strictly take

6

care according to the Care and Use of Laboratory Animals of Institutional Animal Care

7

and the study was approved by the steering committee and conducted under the

8

guidelines for clinical studies of Tianjin University of Traditional Chinese Medicine.

9

Animals were housed in standard cages with free access to water and standard diet and

10

acclimatized to the facilities for one week. The room was controlled temperature at (25 ±

11

2) oC, relative humidity of (50 ± 5) %, and 12/12 h dark-light cycle. Prior to the

12

experiment, all animals were fasted for 12 h, but with free access to water.

13

2.3. Effect of CDDP on vascular tension induced by NE

14

The rats were randomly divided five groups and each group was six. Rats were

15

administered CDDP through gastric garage according weight for seven days, 40 mg/kg,

16

80 mg/kg, 160 mg/kg, 320 mg/kg, respectively. The control group was given equal

17

amount of water. After seven days of drug administration, rats were sacrificed. The

18

thoracic aorta was immediately isolated and transferred to culture dishes containing

19

pre-cold Krebs Henseleit (K-H) (NaCl 18 mM, KCl 4.75 mM, MgSO4·7H2O 1.2 mM,

20

KH2PO4 1.2 mM, CaCl2 2.5 mM, NaHCO3 25 mM and glucose 11 mM). The surrounding

21

fat and connective tissues of thoracic aorta were removed cautiously. The aorta was 5

1

subsequently cut into 4 mm rings that were suspended horizontally between two parallel

2

stainless hooks which were immersed in the organ bath filled with warmed (37°C) and

3

oxygenated (95% O2 and 5% CO2) K-H solution, while the other connected to a force

4

displacement transducer (AD Instruments Pty Ltd., Australia). A computer-assisted data

5

acquisition system (Power Lab/4SP; AD Instruments, Australia) recorded the changes in

6

isometric tension during the experiments. The aorta rings were equilibrated at a based

7

tension of 2.0 g for 60 minutes, and the K-H solution was changed every 15 minutes.

8

Every experiment started with a repeated KCl treatment to test the contractility, after the

9

rings rising with a pre-warmed and oxygenated K-H solution and the muscle tension

10

returning to the basal level. Then added the NE solution into the bath tube and recorded

11

vascular tension curve.

12

2.4. Prediction of the potential mechanism of CDDP for vasodilation

13

The chemical components of each herb in CDDP were collected from traditional

14

Chinese medicine database (TCMD), which is an authoritative database of Chinese herbal

15

ingredients. The structures of the main components of each herb were downloaded and

16

put into PharmMapper server to fish their possible targets. PharmMapper server is online

17

server for predicting potential drug targets by pharmacophore mapping. Then the top 300

18

targets were selected based on the predicted fit value, and were functionally annotated

19

with KEGG and Wikipedia. Because we only focus on the vasodilative role of CDDP, the

20

targets related to vasodilation in the 300 targets were selected for pathway enrichment to

21

elucidate the potential mechanism.

22

2.5. Protein sample preparation

6

1

The rats were randomly divided two groups, CDDP group and control group. The

2

control group was administrated with water. CDDP group was orally administered with

3

CDDP with 160 mg/kg for seven days. After administrating for the last time, rats were

4

sacrificed. The thoracic aortas were immediately isolated and washed away blood by

5

normal saline. Then they were snapped frozen in liquid nitrogen and stored at -80 °C until

6

use.

7

Samples were added with SDT lysate and ultrasonic, then bathed in boiling water for

8

15 minutes. The samples were centrifuged at 14000 g for 15 minutes and the supernatant

9

was used for analysis. Quantification of protein was measured by BCA method. Protein

10

samples were stored at -80 °C until use.

11

2.6. 2D LC-MS/MS analysis and I-TRAQ labeling

12

The protein samples were added to 5×sample buffer (10%SDS, 0.5% bromophenol

13

blue, 50% glycerol, 500mM DTT, 250mM Tris-HCl, pH6.8), bathing by boiling water for

14

5 minutes, 12% SDS-PAGE electrophoresis (constant voltage 250V, 40 minutes), with

15

Coomassie brilliant blue staining.

16

30 µL protein samples were added to DTT, obtaining the final concentration of

17

100mM and bathing by boiling water for 5 minutes. Added 200 µL UA buffer after

18

cooling to room temperature and mixed it into the 30kD ultrafiltration centrifuge tube to

19

centrifuge 14000 g for 15 minutes with discarding filtrate. Then buffer was exchanged

20

with 600 rpm oscillating and centrifugation 14000 g for 15min, and repeated this step for

7

1

many times. The filtrate was collected and the peptide was quantified. Each sample was

2

taken 100 g peptide segments and labeled according to AB SCIEX iTRAQ labeling kit.

3

2.7. ITRAQ analysis

4

The labeled peptides were mixed and graded by Agilent 1260 infinity II HPLC

5

system with an Xbridge Peptide BEH C18 column (5 µm, 130 Å, 4.6 mm × 100 mm,

6

Waters). The solvent A was 10mM HCOONH4, 5% ACN, pH 10, solvent B was 10mM

7

HCOONH4, 85% ACN, pH 10. The chromatographic column was balanced by solvent A,

8

and the sample was separated from the manual injector to the chromatographic column.

9

The flow rate was 1 mL/min. The liquid phase gradient was as follows: 0min-25min, B

10

0%; 25min-30min, B 0%-7%; 30min-65min, B 7%-40%; 65min-70min, B 40%-100%;

11

70min-85min, B was maintained at 100%.

12

Each sample was separated by an Easy-nLC system (Thermo Fisher Scientific,

13

Waltham, MA, USA). The buffer A solution was 0.1% formic acid (v/v), and the B

14

solution was 0.1% formic acid, 80% acetonitrile (v/v) and gradient was set as follows:

15

0min-5min, B was from 0%-6%; 5min-45min, B was from 6%-28%; 45min-50min, B

16

was from 28%-38%; 50min-55min, B was from 38%-100%; 55min-60min, B was

17

maintained at 100%, and the flow rate was 300 nL/min.

18

The samples were separated by chromatography and analyzed by Q-Exactive Plus

19

mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The analysis time

20

was 60 min at positive ion. The parent ion scan range of 350 - 1800 m/z, resolution was

21

70000 MS, AGC target 3e6, 50 ms maximum ion time. The quality of polypeptides and 8

1

polypeptide fragments collection were in accordance with the following methods:

2

collecting 10 pieces of MS2 scan after each full scan (MS2 Activation Type HCD,

3

Isolation window was 2 m/z, MS2 resolution 17500, Microscans 1, maximum ion time

4

45ms, Normalized Collision Energy 30eV).

5

2.8. Protein identification and bioinformatics analysis

6

Analyze inventory identification and quantitation were by software Mascot2.5 and

7

Proteome Discoverer 2.1. The related parameters and instructions are in the

8

supplementary materials. For data analysis, we defined the differentially expressed

9

protein (DEP) as the protein quantification data with a fold change of >1.2 or < 0.8 and

10

correlated p value < 0.05.

11

To get an insight of the DEPs and to explore the potential mechanism of CDDP on

12

vasodilation, protein-protein interaction (PPI) analysis, pathways enrichment and GO

13

annotation were performed. String database (https://string-db.org/cgi/input.pl) was used

14

for the PPI analysis. After PPI analysis, the targets were used for pathways enrichment,

15

by using DAVID bioinformatics resources 6.8 (https://david.ncifcrf.gov/). Gene Ontology

16

functional annotation: The process of GO annotation of target protein can be summarized

17

into four steps: Blast, GO entry mapping, GO annotation, and Annotation Augmentation.

18

First, the set of target proteins was aligned with the appropriate protein sequence

19

database using the NCBI BLAST + (ncbi-blast-2.2.28 -win32.exe) and selected the top

20

10 sequences with E-value <= 1e-3 for follow-up analysis. Next, proteins were enriched

21

using the Blast2GO Command Line and mapped to the associated sequences 9

1

(www.geneontology.org). Annotation with proteins from the mapping process was

2

performed by comprehensively considering the similarity of protein sequence and

3

alignment sequence, the reliability of the GO source. After the completion of annotation,

4

in order to further improve the efficiency and accuracy of annotation, we searched for the

5

conserved motif matched with the target protein in the EBI database and annotated the

6

motif related functional information to further supplement the annotation information.

7

2.9. Western blotting

8

The differentially expressed proteins in the enriched pathways were selected for

9

verification by Western blotting. The protein of the thoracic aorta tissue was extracted by

10

RIPA lysis buffer (Beyotime, China), and its concentration was measured by BCA Protein

11

Assay Kit (Beyotime Institute of Biotechnology, China). Then it was separated by

12

sodium-dodecyl-sulfate polyacrylamide gel electrophoresis, and transferred onto

13

polyvinylidene fluoride membranes. The membranes were blocked by 5% skimmed milk,

14

and incubated overnight at 4 ℃ with the following primary antibodies: anti-GAPDH

15

(1:5000, Bioss, China) or anti-Endothelin B Receptor antibody (1:1000, Abcam, USA);,

16

anti-Rasgrf1 Antibody (1:1000, Abcam, USA), antibody anti-PP1α (1:1000, cell signaling

17

technology, USA). Anti-goat IgG and anti-rabbit IgG secondary antibody (1:1000,

18

Beyotime, China) was diluted to identify the corresponding primary antibodies.

19

Immunoreactive bands were visualized using chemiluminescent HRP Substrate

20

(Millipore Corporation, USA) and were quantified by ImageJ software (National Institute

10

1

of Mental Health, Bethesda, MD, USA). The total protein was normalized to GAPDH

2

and the CDDP was normalized to control.

3

2.10.

Statistical analysis

4

All data were presented as means ± SD and statistically analyzed by one-way

5

ANOVA (multiple groups) or unpaired t-test using the GraphPad Prism 5 software. A p <

6

0.05 was considered as the significant difference.

7

3. Results

8

3.1. The Vasorelaxation of CDDP on Thoracic Aorta Induced by NE

9

According to the results of previous experiments, the concentration 10-7 and 10-6 M

10

were chosen as the inducer and the results were shown in Supplementary Information.

11

Vascular tension results in rats after oral administration of different concentrations of

12

CDDP, compared with the control group, each group can significantly inhibit NE induced

13

aortic contraction tension and shows a concentration dependent manner. As shown in

14

Figure 1, the concentration of 160 mg/kg is the best concentration of oral administration.

15

It also can be concluded that CDDP has certain relaxing effect on blood vessel and has a

16

concentration effect positive correlation.

11

1 2

Figure 1 Vascular tension in rats of 40 mg/kg, 80 mg/kg, 160 mg/kg, 320 mg/kg of CDDP

3

induced by NE, and control groups. The concentration of NE is 1×10-7mmol/L in Fig. 1A,

4

1×10-6mmol/L in Figure 1B. Compared with the control group, ** P <0.01. Values are

5

presented as the mean ± SD.

6

3.2. Prediction of the potential mechanism of CDDP for vasodilation

7

The high-content and major components of each herb in CDDP were collected. We

8

collected 24 components in Danshen, 17 components in Sanqi and 7 components in

9

Bingpian (Table S3). The structures of the collected components are put into

10

PharmMapper server to fish their possible targets. Based on the functionally annotation

11

of the top 300 targets, 51 targets related to vasodilation were obtained for Danshen, 45

12

targets related to vasodilation were received for Sanqi and 34 targets were obtained for

13

Bingpian (Figure 2A). To elucidate the roles of CDDP on vasodilation, the corresponding

14

pathways and regulating pathways involved were considered to be the potential

15

mechanism. VEGF signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling

16

pathway, MAPK signaling pathway, Platelet activation, cAMP signaling pathway, 12

1

vascular smooth muscle contraction, Renin secretion, Renin-angiotensin system and

2

cGMP-PKG signaling pathway were associated to those fished targets of components in

3

CDDP (Figure 2).

4 5

Figure 2 The predicted potential mechanism of CDDP on vasodilation. A: The predicted

6

targets of the main components of each herb and the predicted targets belong to which

7

enriched pathway. 51 targets related to vasodilation were obtained for Danshen, 45 targets

8

related to vasodilation were received for Sanqi and 34 targets were obtained for Bingpian. 13

1

The predicted targets were presented in pink and the enriched pathways were presented in

2

blue. B: The pathways were enriched of the predicted targets of components in CDDP.

3

The radius of the circle represents the number of genes enriched in this pathway. The

4

color represents the p value.

5

3.3. Identification of Proteins Affected by CDDP Based on Quantitative Proteomic

6

Analysis

7

In the iTRAQ analysis, 4935 proteins were successfully identified. There are 42

8

different proteins between CDDP administration group and control group, and among

9

them, 17 proteins were up-regulated and 25 proteins were down-regulated. Details are

10

shown in Table 1.

11

As Figure 3A shows, samples in control group and CDPP group were used to draw

12

volcanic maps together with two factors, Fold change and P value, which were used to

13

show significant differences between the two groups of sample data. The transverse

14

coordinates are the differential multiple (the logarithmic transformation with the bottom

15

of the 2), the ordinate is P value (the logarithmic transformation with the bottom of the

16

10). Therefore, the points in the picture present identified proteins fold change and P

17

values. Among these points, the red ones are the different expression proteins.

18

Hierarchical cluster analyses of protein expression changes were depicted of

19

heatmap in Figure 3B. Each row in the figure represents a protein, each column

20

represents a comparative group. The color bar designates protein expression fold changes

14

1

and the colors change from green to red with increasing values. Red indicates upregulated

2

proteins and green indicates downregulated proteins.

3 4

Figure 3 The samples of control group and CDPP group were used to draw volcanic maps

5

together with Fold change and P value values. The point in the picture present identified

6

proteins fold change and P values, and the red point is the different expression proteins

7

(Figure 3A). Hierarchical cluster analyses of protein expression changes were depicted of

8

heatmap (Figure 3B).

9

3.4. Bioinformatics analysis

10

In this study, we used Gene Ontology functional annotation to analyze the cellular

11

functions involved in target proteins. Three main types of annotations were obtained from

12

the gene ontology cellular component, metabolic functions, and biological processes. The

15

1

details of proteins Gene Ontology functional annotation were shown in the Figure 4. The

2

enrichment analysis of GO annotation on the target protein pool was performed by

3

Fisher's Exact Test to compare the distribution for better explanation biological

4

significance to the different proteins. Our result in Figure 5 showed the top three GO

5

terms played important roles in the vasorelaxation of CDDP. The top three are protein

6

functions are localization to cell periphery, establishment of protein localization to

7

plasma membrane and mRNA binding. The numbers of identification proteins and

8

different expression proteins in the go terms were shown in Figure 5A. The percentages

9

of their respective accounts were shown in the Figure 5B.

10 11

Figure 4 Gene Ontology functional annotations of target proteins from gene ontology

12

cellular component, metabolic functions, and biological processes. 16

1

2 3

Figure 5 The top three GO terms played important roles in the vasodilation of CDDP, the

4

numbers of identification proteins and different expression proteins in the go terms

5

(Fig.5A), the percentages of their respective accounts (Fig.5B).

6

To further explore the potential mechanism of CDDP on vasodilation,

7

protein-protein interaction (PPI) analysis was performed. After PPI analysis, the targets

8

were used to pathways enrichment. Platelet activation, cGMP – PKG signaling pathway

9

and vascular smooth muscle contraction were associated with vasodilation (Figure 6).

10

These three pathways were consistent with the predicted mechanism, which indicated that

11

CDDP played the role of vasodilation mainly through these three pathways. The genes

12

enriched in the three pathways were Ppp1ca, Ppp1cb, Ppp1r14a, Ednrb, Gnaq, which

13

related the different expression proteins PP1α, Ednrb and Rasgrf1.

17

1 2

Figure 6 The potential mechanism of CDDP on vasodilation. The results based on

3

proteomics. The radius of the circle represents the number of genes enriched in this

4

pathway. The color represents the p value.

5

3.5. Validation of the mechanism of CDDP on vasodilation

6

The different expression proteins enriched in the three pathways was validated by

7

western blotting. The protein levels of PP1α and Rasgrf1 were largely decreased and the

8

protein level of Ednrb was largely increased (Figure 7). The results indicated that the

9

vasodilation of CDDP was associated with Platelet activation, cGMP – PKG signaling

10

pathway and vascular smooth muscle contraction.

18

1 2

Figure 7 Relative protein expressions of Rasgrf1 (B), Ednrb (C) and PP1α(D) in rat

3

thoracic aorta. Expression level was normalized to GAPDH. Compared with the control

4

group, *P< 0.05 ,** P <0.01. Values are presented as the mean ± SD.

5

4. Discussion

6

Angina pectoris is triggered to decreased myocardial oxygen supply or increased

7

myocardial oxygen demand. Angina pectoris can be relieved by rest or nitroglycerin

8

(Shao et al, 2015; Tobin, 2010). Nitroglycerin is one of the most commonly prescribed

9

drugs for symptoms improvement of coronary atherosclerotic heart diseases and angina

10

pectoris due to its potent vasodilator capacity (Divakaran et al, 2017). CDDP has benefits

11

for the angina pectoris and has been widely used in China as a supplement to

12

nitroglycerin (Jia et al, 2012). We concluded that CDDP benefits to angina pectoris that 19

1

may be mainly responsible for its vasodilation. Therefore, our proposal in the present

2

study is that CDDP could relieve symptom of angina pectoris by dilating blood vessels.

3

Target proteins also determined combing targets fishing and quantitative proteomics to

4

illustrate underlying mechanism.

5

We first confirm the vasodilation of CDDP oral administration using thoracic aorta

6

ring. Our result shows CDDP had a repressive effect on the contraction induced by NE.

7

As one of major compositions of CDDP, Danshen (Salvia miltiorrhiza) exhibits

8

significant vasodilation effect. Danshensu is the major marker for the antioxidant and

9

vasodilation effects of Danshen (Zhou et al, 2012). The vasodilation effect of CDDP

10

depends on both endothelium and non-endothelium (Zhan et al, 2008). In recent years,

11

another composition of CDDP that Sanqi (Panax notoginseng) also has been reported

12

vasodilation effects. Panax notoginseng saponins, the major component of Sanqi, have a

13

relaxing effect on the thoracic aorta, coronary arteries, mesenteric artery and tail artery

14

(Wang et al, 2016).

15

Endothelin-1 (ET-1) is a major regulator of vascular function, acting via both

16

endothelin receptor type A (ETAR) and type B (ETBR). Although the role of ETAR in

17

vascular smooth muscle (VSM) contraction has been studied, little is known about ETBR.

18

ETBR, which is coding by Ednrb gene, is important for the control of vascular reactivity.

19

The unique anatomical location of endothelial ETBR favors both the release of

20

endothelium-derived vasodilators, which counteract the protracted vasoconstrictor effects

21

of ET-1/ETAR, and clearance of ET-1 from the circulation. ETBRs are also present in 20

1

VSM and share several intracellular signaling pathways with the ETAR (Lind et al, 2013;

2

Mazzuca et al, 2012). RasGRF2 promotes exchange activities on both Ras and Rac

3

GTPases; activation of either pathway can have profound effects on actin remodeling

4

(Ma et al, 2014). In cells, actin-filament-based structures control diverse activities such as

5

polarized intracellular trafficking, cytokinesis, migration and adhesion (Benz et al, 2009).

6

RAS molecular signaling and its effect on blood pressure as well as its relationship to

7

renal function and cardiovascular disease are significant. Some of these targets in the

8

signaling way respond to exercise, stimulating nitric oxide synthesis and endothelial

9

vasodilation (Petriz et al, 2013). The myosin light chain phosphatase (MLCP) is a

10

cytoskeleton-associated protein phosphatase-1 (PP1) holoenzyme. Vascular smooth

11

muscle contraction is tightly coupled to the phosphorylation of the 20-kDa myosin light

12

chain (MLC20), which is regulated by the opposing activities of myosin light chain

13

kinase (MLCK) and myosin light chain phosphatase (MLCP) (Khasnis et al, 2014).

14

Phosphorylation and dephosphorylation of MLC lead to smooth muscle contraction and

15

relaxation, respectively and are mediated by MLCK and MLCP, respectively (Lin et al,

16

2011). MLCP dephosphorylated MLC and relaxed vascular smooth muscle independent

17

of intra cellular Ca2+ level. MLCP activity depends largely upon the phosphorylation

18

status. MLCP will be activated to dephosphorylate MLC and induce relaxation (Matsuo

19

et al, 2011). We have shown that CDDP mediated coronary relaxation involves inhibitory

20

effect on MLCP, resulting in decreased phosphorylation of MLC. Many proteins are

21

involved in the regulation of vasodilation and different drugs have a variety of targets. 21

1

However, based on target fishing and quantitative proteomics, three pathways (platelet

2

activation, cGMP – PKG signaling pathway and vascular smooth muscle contraction)

3

were found more closely related to vasodilation of CDDP, and the related differentially

4

expressed proteins found in iTRAQ were Endothelin receptor type B (Ednrb), RasGRF1

5

and MLCP (PP1α). Since traditional Chinese medicine complex, its mechanism is not

6

clear. This study provides a solution to solve the mechanism reveal of tradition Chinese

7

Medicine.

8

5. Conclusions

9

Vasodilation is a mechanism for the treatment of angina pectoris. In this study, the

10

results showed that CDDP can significantly inhibit NE induced aortic contraction tension.

11

Furthermore, we investigated the target protein of vasodilation effect of CDDP via

12

fishing target to prediction and quantitative proteomics for verification. The results

13

showed that the mechanism of CDDP presenting obvious vasodilatory effect may be

14

achieved by mediating platelet activation, cGMP – PKG signaling pathway and vascular

15

smooth muscle contraction. We believe that this method can provide a strategy for target

16

protein determination of traditional Chinese medicine and lay a foundation for the

17

discovery of innovative traditional Chinese medicine.

18

Conflict of interest

19 20

The authors have no conflicts of interest to declare. Acknowledgments

22

1

This work was supported by the National standardisation project of traditional

2

Chinese Medicine (ZYBZH-C-TJ-55), and the Program for Changjiang Scholars and

3

Innovative Research Team in University (PCSIRT IRT_14R41).

4

Supporting Information

5

Supplementary Figure 1 Inducer NE concentration-effect curve.

6

Supplementary Table S1 and Table S2: The related parameters and instructions of

7

qualitative and quantitative software Mascot2.5 and Proteome Discoverer2.1.

8

Table S3 The major components of each herb in CDDP.

23

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Table 1 Differentially expressed proteins of quantitative results Gene Name

1

2

Description

MW [kDa]

Sodium-dependent phosphate Slc17a2 48.268 transport protein 2A Immunoglobulin Jchain 17.773 joining chain

CDDP

Ratio

P value

78.200 121.800 1.558

0.037

82.933 117.067 1.412

0.035

Protein Vps18

110.116

86.933 113.067 1.301

0.028

4

Uncharacterized protein

14.274

87.267 112.700 1.291

0.010

5 Gnpnat1

Gnpnat1 protein

20.808

87.600 112.400 1.283

0.005

87.967 112.067 1.274

0.010

88.067 111.900 1.271

0.047

88.400 111.600 1.262

0.028

3

Vps18

Control

Putative sodium-coupled 6 Slc38a10 118.723 neutral amino acid transporter 10 Putative Lipt2 lipoyltransferase 25.181 7 2, mitochondrial Endothelin B 8 Ednrb 49.422 receptor 9 Tmbim1

Protein Tmbim1

34.271

88.433 111.567 1.262

0.041

10 Dopey2

Protein Dopey2

257.592

88.967 111.067 1.248

0.028

11

Nf2

Merlin

69.16

89.367 110.633 1.238

0.005

12

Cmc1

Protein Cmc1

12.563

89.600 110.400 1.232

0.002

13

Rbm3

RNA-binding protein 3

16.845

89.767 110.233 1.228

0.026

14

Numbl

Numb-like protein 68.584

89.800 110.167 1.227

0.001

30

15

Cpsf6

Cleavage and polyadenylation specific factor 6

16

Clec2g

Protein Clec2g

17

18

19

20

21

59.143

90.800 109.233 1.203

0.003

25.021

90.800 109.200 1.203

0.015

60.753

109.200 90.800

0.832

0.045

38.308

109.200 90.800

0.832

0.002

27.765

109.200 90.800

0.832

0.014

37.488

109.367 90.600

0.828

0.042

73.284

109.467 90.567

0.827

0.032

181.62

109.467 90.533

0.827

0.036

BET1-like protein 12.402

109.467 90.533

0.827

0.048

41.91

109.767 90.200

0.822

0.024

24.581

109.800 90.167

0.821

0.016

60.469

110.167 89.833

0.815

0.004

38.568

110.467 89.500

0.810

0.026

62.874

110.533 89.467

0.809

0.038

EH Ehd3 domain-containing protein 3 3-hydroxybutyrate Bdh1 dehydrogenas RWD Rwdd1 domain-containing protein 1 Serine/threonine-p rotein phosphatase Ppp1ca PP1-alpha catalytic subunit ATP-dependent DDX3Y RNA helicase DDX3Y

22

Prex2

23

Bet1l

Protein Prex2

Endothelial cell-selective 24 Esam adhesion molecule ADP-ribosylation factor-like protein 25 Arl6ip6 6-interacting protein 6 Sialate Siae 26 O-acetylesterase Antigen-presentin g glycoprotein 27 Cd1d1 CD1d Transmembrane 28 Tmem209 protein 20

31

5-demethoxyubiqu inone 29 Coq7 20.113 hydroxylase, mitochondrial LOC10255 Protein 30 10.81 0385 LOC102550385 31 32

33

34

35

36

37

Myo1g

110.633 89.367

0.808

0.018

111.033 88.967

0.801

0.024

116.625 111.100 88.900

0.800

0.007

27.486

111.200 88.800

0.799

0.043

22.644

111.300 88.733

0.797

0.016

35.152

111.500 88.533

0.794

0.047

47.301

111.700 88.333

0.791

0.016

42.232

112.467 87.500

0.778

0.040

73.852

112.767 87.233

0.774

0.034

33.15

115.067 84.900

0.738

0.010

Parvalbumin alpha 11.918

120.967 79.000

0.653

0.013

170.652 122.367 77.600

0.634

0.019

0.565

0.031

0.557

0.030

Protein Myo1g

Centromere protein V ADP-ribosylation Arfrp1 factor-related protein 1 rRNA adenine Dimt1 N(6)-methyltransf erase RILP-like protein Rilpl1 1 RNA-binding Rbmx motif protein, X chromosome Similar to ATP-binding cassette, Abcg3l4 sub-family G (WHITE), member 3 Cenpv

38 Gimap9 39

Pvalb

40

Aqr

GIMAP9

Aqr protein

N-alpha-acetyltran sferase 35, NatC 83.155 127.767 72.233 41 Naa35 auxiliary subunit Ras-specific guanine 42 Rasgrf2 135.741 128.433 71.600 nucleotide-releasi

32

ng factor 2

1

33

Highlights

1. CDDP can inhibit norepinephrine induced aortic contraction tension. 2. Target-fishing combined iTRAQ revealed the mechanism of CDDP on vasodilation. 3. PP1α, Ednrb and Rasgrf1 were the regulating proteins of CDDP.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: