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Proteomic analysis of differential protein expression in rat platelets treated with notoginsengnosides.

Abstract

Sanqi, the root of Panax notoginseng, is a popularly used traditional Chinese medicine with cardiovascular effects. Notoginsengnosides (NG) isolated from Sanqi could inhibit ADP-induced platelet aggregation of rat washed platelets. To identify the possible target proteins of NG in platelets, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after NG treatment were identified by MALDI-TOF MS/MS. Treatment of 200 [micro]g/ml NG caused regulation of the levels of 12 proteins, which play important roles in platelet activation, oxidative stress and cytoskeleton. In the NG-treated platelets, there were increase in the levels of growth factor receptor-bound protein 2 (Grb2), thrombospondin 1, tubulin alpha 6 and decrease in the levels of thioredoxin, Cu-Zn superoxide dismutase, DJ-1 protein, peroxiredoxin 3, thioredoxin-like protein 2, ribonuclease inhibitor, potassium channel subfamily V member 2, myosin regulatory light chain 9 and laminin receptor 1. The change in the levels of these proteins caused by NG treatment might contribute to the inhibitive effect of NG on platelet aggregation. Furthermore, analysis of the reactive oxygen species (ROS) level indicated that NG could decrease the ROS level in platelets. The regulation of ROS level might play important role in the effect of NG on platelets.

[c] 2008 Elsevier GmbH. All rights reserved.

Keywords: Panax notoginseng; Sanqi; Notoginsengnosides; Platelet aggregation; Proteomics

Introduction

Sanqi, the root of Panax notoginseng, is widely used in China and other Asian countries for treatment of cardiovascular disorders. The first detailed description of Panax notoginseng could be found in the Chinese Materia Medica classics, Shen Nong Ben Cao Jing (dated 206 BC-8 AD). Presently, Sanqi is officially listed in the Chinese Pharmacopeia (Chinese Pharmacopoeia Commisson, 2005). Previous reports indicated that Sanqi had cardiovascular effects including protecting cardiomyocytes against ischemic injury, vasorelaxation, anti-inflammatory effects, etc. (Chen et al., 1995; Ng, 2006). Interestingly, Sanqi was considered in traditional Chinese medicine to have dual-direction effects on blood coagulation. The traditional description of Sanqi was "San Yu Zhi Xue", which suggested it had both antithrombus and anti-bleeding effects. So, Sanqi was used clinically as both anti-bleeding agent (Wei et al., 2007) and anti-thrombus agent (Wang et al., 2004). The mechanism for the dual-direction effects of Sanqi is still unclear. However, in in vitro study, lots of reports showed that saponins from Sanqi had inhibitive effect on platelet aggregation (Kimura et al., 1988; Kuo et al., 1990; Pan et al., 1993; Shi et al., 1990; Wang et al., 2004; Yang et al., 2004). Our previous study also indicated that notoginsengnosides (NG), which contained mainly ginsenoside Rgl, Rb1, Rd and notoginsenoside R1, exhibited inhibitory effects on ADP-induced aggregation of rat washed platelets (Yao et al., 2008).

To study the mechanism of the inhibitive effect of NG on platelet aggregation, a proteomic approach was used in the present study to comprehensively analyze the possible molecular targets of NG in platelets. It is well known that platelets do not contain nucleus but keep active translation and protein synthesis. Therefore, the proteomic technology was particularly suitable for platelet-related study (Lindemann and Gawaz, 2007). Proteomic methods had been successfully used in studying the signal cascades of platelets (Gnatenko et al., 2006). We used two-dimensional gel electrophoresis (2-DE)-based comparative proteomic assay in the present paper. Proteins from rat washed platelets treated with 200 [micro]g/ml NG (similar to the IC.sub.50] value in inhibition of ADP-induced platelet aggregation) were extracted and 2-DE was conducted. Then, differentially expressed proteins in NG-treated group compared with control were identified by MALDI-TOF MS/MS. Furthermore, the ROS level in platelets treated with NG was also checked.

Materials and methods

Extraction and chemical characterization of NG from Panax notoginseng

NG was isolated from Sanqi, roots of Panax notoginseng, by the laboratory of TCM chemistry, Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, as reported before (Yao et al., 2008). Briefly, the dried roots of Panax notoginseng were pulverized into powder and then extracted with water. The water extract was concentrated under vacuum and then the pH value of the said syrup was adjusted to 10. After 12 h, the suspension was subjected to a D-101 macro-porous resins column and eluted with de-ionized water, 30%, 50% and 70% ethanol. The 70% ethanol elution was collected and eliminated the solvent under vacuum to yield NG. The HPLC-fingerprint of the NG was showed in our previous report (Yao et al., 2008). Briefly, NG contained mainly ginsenoside Rgl, Rbl, Rd and notoginsenoside R1 with contents of about 40.5%, 31.3%, 8.4%, 8.5%, respectively. For treatment of platelets, NG was dissolved in natural saline.

Animals and reagents

Twenty inbred SD rats (male, weight 280 [+ or -] 20 g, age 8-10 weeks) were fed and kept in Shanghai Experimental Animal Laboratory, Chinese Academy of Sciences. All experiments related to using of animals were performed under the control of the Institutional Animal Care and Use Committee. All reagents used in 2-DE were the products of Bio-Rad Laboratories (Hercules, CA, USA).

Preparation of rat washed platelets

Rat washed platelets were prepared as reported before (Yao et al., 2008). Briefly, blood samples were taken from the ventral aorta of rats and treated with trisodium citrate as anticoagulant. Platelet rich plasma was obtained by centrifugation of blood at 200g for 10 min at room temperature. Then, after centrifugation at 1000g for 10 min, the deposited platelets were washed twice with Tyrode buffer (136 mM NaCl, 2.7 mM KCl, 12 mM NaHC[O.sub.3], 0.42 mM Na[H.sub.2]P[O.sub.4], 0.2 mM MgS[O.sub.4], 5.0mM glucose) containing 0.2mM EGTA, pH 6.5. Washed platelets were finally re-suspended in Tyrode buffer containing 1.8 mM CaC[l.sub.2], pH 7.4 and the platelet number was adjusted to 3 x [10.sup.8] cells/ml for using in the NG treatment experiment.

Preparation of platelet protein samples

For NG treatment, washed platelets were incubated with natural saline (solvent control) or NG at 200 [micro]g/ml at 37 [degrees]C for 10 min. Then, protein samples for 2-DE analysis were prepared as reported before (Ma et al., 2008; Yue et al., 2008). Briefly, platelets were washed three times with ice-cold PBS and subsequently centrifuged for 10 min at 2500g. The platelets were then dissolved in lysis buffer and subjected to ultrasonication on ice. The lysed platelets were then centrifuged and the supernatant containing the solubilized proteins was used for 2-DE analysis.

2-DE and MALDI-TOF MS/MS identification of proteins

2-DE was carried out similar to previous report (Ma et al., 2008; Yue et al., 2008). Briefly, the Ready-Strip IPG Strips, 17 cm, pH 4-7 (Bio-Rad) were used and protein samples (each 150 [micro]g) were applied for 2-DE analysis. The strips were placed into a Protein IEF cell (Bio-Rad) for IEF and then applied on to SDS-PAGE gels for electrophoresis using a PROTEIN II xi Cell system (Bio-Rad). The gels were then silver stained with Bio-Rad Silver Stain Plus kit reagents (Bio-Rad). Triplicate electrophoresis was performed for each pair of protein samples (control and NG-treated) from three independent experiments to ensure reproducibility. The scanned 2-DE images were analyzed using PD-Quest software (Bio-Rad). Protein spots with two fold or more increased or decreased intensity between control and NG-treated group and with p<0.05 (Student's t-test) were considered as significantly differentially expressed proteins. The protein spots were cut from the gels and used for MALDI-TOF MS/MS identification. Briefly, the proteins were digested with trypsin and then the peptides were analyzed using an ABI 4700 Proteomics Analyzer with delayed ion extraction (Applied Biosystems). The obtained MS data were investigated using the MASCOT search engine (Matrix Science) against the NCBI protein sequence database. Proteins with protein score more than 50 were accepted.

Determination of intracellular ROS level of platelets

Intracellular ROS levels of platelets treated by NG were measured using the 2'7'-dicholorodihydrofluorescein diacetate ([H.sub.2]DCFDA, Molecular Probes, Eugene, OR), which is a non-polar compound that is hydrolyzed to a non-fluorescent polar derivative ([H.sub.2]DCF) by cellular esterases after diffusing into the cells (Wang and Joseph, 1999). [H.sub.2]DCF is membrane impermeable and rapidly oxidized to a highly fluorescent 2'7'-dichlorofluorescein (DCF) by intracellular ROS. Washed platelets were incubated with 20 [micro]M [H.sub.2]DCFDA for 20 min. Then, after centrifugation to remove [H.sub.2]DCFDA, cells were washed twice with and sequentially treated with natural saline (solvent control) or NG at different concentrations. After incubation for 10 min, the DCF fluorescence was detected by a GENios Microplate Reader (Tecan Inc., Research Triangle Park, NC) with excitation at 485 nm and emission at 538 nm. The results from three independent experiments (each with triplicate repeats) were used for statistical analysis.

Statistical analysis

Presented data are the mean [+ or -] S.D values. Non-paired Student's t-test (GraphPadPrism) was used to check significance of difference between groups. p<0.05 was accepted as significant.

Results

2-DE of control and SA-treated platelets

To investigate the possible target-related proteins of NG in platelets, protein profiles of control and NG-treated platelets were studied by comparative proteomic analysis. Representative 2-D gel images for control and NG-treated platelets are shown in Fig. 1. Up to 600 protein spots could be resolved from each gel. Nine down-regulated protein spots and three up-regulated protein spots in NG-treated group compared with control were found. The differentially expressed proteins are indicated by the arrowed spots in Fig. 1 and by the expanded plots in Fig. 2. The average intensity values and standard deviations of the spots, the statistical assay results and the fold differences between control and NG-treated group are shown in Table 1. The fold difference indicated the ratio of the intensity value of NG-treated group to the value of control group.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Table 1. Summary of differentially expressed proteins in the NG-treated
platelets

Spot Pair of
 gel numbers
 (n) Spot volume (ppm)

 Control (mean [+ or -] NG-treated (mean [+ or -]
 SD) SD)

 1 9 1019.4 [+ or -] 181.7 487.0 [+ or -] 112.0
 2 9 1405.7 [+ or -] 371.2 667.4 [+ or -] 130.4
 3 9 323.7 [+ or -] 92.8 125.2 [+ or -] 49.7
 4 9 372.8 [+ or -] 44.4 133.8 [+ or -] 48.7
 5 9 7.9 [+ or -] 7.8 394.6 [+ or -] 139.5
 6 9 983.3 [+ or -] 304.1 433.0 [+ or -] 144.7
 7 9 1294.9 [+ or -] 407.1 116.3 [+ or -] 78.4
 8 9 636.2 [+ or -] 274.5 1307.8 [+ or -] 436.8
 9 9 76.2 [+ or -] 45.4 462.9 [+ or -] 151.3
10 9 1413.9 [+ or -] 314.7 687.9 [+ or -] 258.5
11 9 1486.4 [+ or -] 446.0 718.3 [+ or -] 108.9
12 9 1186.9 [+ or -] 207.4 498.0 [+ or -] 153.7

Spot Fold difference p-Value

 1 0.48 <0.05
 2 0.47 <0.05
 3 0.39 <0.05
 4 0.36 <0.05
 5 49.77 <0.05
 6 0.44 <0.05
 7 0.09 <0.05
 8 2.06 <0.05
 9 6.07 <0.05
10 0.49 <0.05
11 0.48 <0.05
12 0.42 <0.05


Identification of the differentially expressed proteins

Table 2 shows the MALDI-TOF MS/MS analysis result of the proteins. The NCBI accession number, theoretical molecular weight and pI of each protein spot were showed. The protein score, coverage and the best ion score of each spot are also included in Table 2. Furthermore, the reported biological functions of the proteins are also listed. The MALDI-TOF MS/MS analysis result of spot 11 is shown in Fig. 3 as an example.
Table 2. The results of protein identifications of differentially
expressed proteins using MALDI-TOF MS/MS

Spot Target protein NCBI accession Theoretical
 number Mr (kDa)/PI

 1 Thioredoxin 16758644 11.7/4.80

 2 Potassium channel, subfamily V, 27684873 64.1/6.08
 member 2

 3 Myosin regulatory light chain 9 2498032 19.7/4.80

 4 Cu-Zn superoxide dismutase 203658 15.6/5.89

 5 Tubulin alpha 6 58865558 49.9/4.96

 6 DJ-1 protein 16924002 20.0/6.32

 7 Peroxiredoxin 3 11968132 28.3/7.14

 8 Growth factor receptor-bound protein 914957 23.5/6.31
 2 (Grb2)

 9 Thrombospondin 1 61556835 129.6/4.74

10 Thioredoxin-like protein 2 78187979 37.8/5.51

11 Ribonuclease inhibitor 77416905 49.9/4.67

12 Laminin receptor 1 8393693 32.8/4.80

Spot Protein Sequence Best ion Function
 score coverage (%) score

 1 69 32 37 Oxidative stress

 2 57 14 32 Potassium ion permeability of
 membrane

 3 66 12 42 Cytoskeleton

 4 179 39 37 Oxidative stress

 5 100 17 36 Cytoskeleton

 6 56 39 31 Oxidative stress

 7 87 11 35 Oxidative stress

 8 88 22 33 Platelet activation

 9 96 17 57 Platelet activation

10 65 24 35 Oxidative stress, PKC inhibition

11 142 26 65 Ribonuclease inhibitor, oxidative
 stress

12 119 23 44 Cell adhesion


[FIGURE 3 OMITTED]

ROS level of platelets treated with NG

2-DE assay results showed that the protein levels of six antioxidant proteins, i.e. thioredoxin, Cu-Zn superoxide dismutase, DJ-1 protein, peroxiredoxin 3, thioredoxin-like protein 2 and ribonuclease inhibitor, were all down-regulated in NG-treated platelets. The detected change in antioxidant protein levels induced us to check the ROS level in platelets after NG treatment. Interestingly, as shown in Fig. 4, NG (25, 50, 100 and 200 [micro]g/ml) dose-dependently decreased ROS level in platelets.

[FIGURE 4 OMITTED]

Discussion

As reported in our previous reports (Yao et al., 2008), NG isolated from Sanqi could inhibit ADP-induced aggregation of rat washed platelets with an I[C.sub.50] value of 189[+ or -]72([micro]g/ml). The inhibitive effect on platelet aggregation might be one of the important bases for the cardiovascular actions of saponins from Sanqi. Understanding the mechanism of the effect of NG on platelet would be helpful for the study of Sanqi. Previous reports only suggested that the effect of Sanqi on platelets might be related to the inhibition on TX[A.sub.2] production (Yang et al., 2004) and inhibition on the elevation of cytosolic free calcium concentration (C[a.sup.2+] ) induced by adrenaline and thrombin (Kimura et al., 1988). In the present study, the differentially expressed proteins in platelets treated with NG were checked using the proteomic method, which had been shown to bevery useful in the study of platelet functions. Our proteomic study found three proteins with up-regulated expression and nine proteins with down-regulated expression in NG-treated platelets compared with control. Based on their biological functions, these total 12 proteins were classified into following four categories: (1) platelet activation; (2) oxidative stress; (3) cytoskeleton structure and others.

Platelet activation

Proteins including growth factor receptor-bound protein 2 (Grb2) and thrombospondin 1 are directly involved in platelet activation. The Grb2 is well known to be involved in cell proliferation via the Ras signalling pathway. In platelets, Grb2 was shown to play an important role in the inside-out signaling after activation by thrombin. Grb2 is composed of two Src homology 3 (SH3) and one SH2 domains. Inhibition of Grb2-SH3 interactions with signal transduction proteins would down-regulate thrombin-induced plate- let activation but also potentiate Fc receptor- and alphallb-beta3-mediated platelet activation (Saci et al., 2002). And, Grb2 was shown to be closely related to ERK1/2 and tyrosine kinase activation (Wang and Reiser, 2003; Lowenstein et al., 1992). Thrombospondin 1 is a large homotrimeric glycoprotein of approximately 450 kDa, which could be synthesized by several cell types. It is abundantly stored in platelet alpha-granules and could be secreted into plasma during platelet activation. The complex multidomain structure of thrombospondin 1 enables it to interact with many cell-adhesive receptors, including CD36, several integrins, integrin-associated protein, GPIb/IX/V complex, heparan sulfate and collagen. It has been shown that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiological levels of nitric oxide (Isenberg et al., 2008; Bonnefoy et al., 2006). On the contrary, there were also reports showing that recombinant peptides of thrombospondin could inhibit platelet aggregation as well as platelet macroaggregate formation (Rabbi-Sabile et al., 1996; Legrand et al., 1994).

Oxidative stress

Proteins including thioredoxin, Cu-Zn superoxide dismutase, DJ-1 protein, peroxiredoxin 3, thioredoxin-like protein 2, ribonuclease inhibitor were involved in the antioxidant system of platelets. Thioredoxin could participate in various redox reactions through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol--disulfide exchange reactions. Cu-Zn superoxide dismutase is a well-known antioxidant protein. DJ-1 is an atypical peroxiredoxin-like peroxidase and may function as a redox-sensitive chaperone and as a sensor for oxidative stress (Andres-Mateos et al., 2007). Peroxiredoxin 3 belongs to the peroxiredoxins family, which control the constitutive level of [H.sub.2][O.sub.2] in the cell and protect against ROS-induced damage by catalyzing the reduction of [H.sub.2][O.sub.2] into water (Mukhopadhyay et al., 2006). Thioredoxin-like protein 2 belongs to the thioredoxin family (Sadek et al., 2003) and is also a protein kinase C-interacting protein (Witte et al, 2000). Ribonuclease inhibitor was expressed in platelets (Nadano et al., 1995) and had recently been shown to scavenge several reactive oxygen species (ROS) (Wang and Li, 2006).

Cytoskeleton structure and others

Proteins including myosin regulatory light chain 9 and tubulin alpha 6 were cytoskeleton-related proteins. Myosin regulatory light chain 9 is a regulatory protein and belongs to the myosin light chains family, which regulate diverse cell activities, including shape change, secretion, contractile functions and cytokinesis. In patients with impaired platelet aggregation associated with a heterozygous mutation in transcription factor CBFA2, the expression of myosin regulatory light chain 9 was selectively decreased about 77-fold (Sun, K et al., 2007; Sun, L et al., 2007). Tubulin alpha 6 belongs to the tubulin family, which is a major constituent of micro tubules. It is well known that the reorganization of cytoskeleton structure is necessary and important for activation of platelets (Hartwig, et al., 1999). Thus, the regulation of cytoskeleton-related protein levels might also affect platelet aggregation. Besides, laminin receptor 1 is a protein that play important roles in several physiologic and pathologic processes, including cell differentiation, growth, migration and cancer invasion (Wang et al., 2007). Potassium channel subfamily V member 2 belongs to the potassium voltage-gated channels, which mediate the potassium ion permeability of platelet membrane (Mahaut-Smith et al., 1990).

In summary, NG treatment could change the levels of 12 proteins, which play important roles in platelet activation, oxidative stress, cytoskeleton structure and others, in rat washed platelets. Importantly, two proteins closely related to platelet activation, i.e. Grb2 and thrombospondin 1, were regulated in NG-treated platelets. The roles of these two proteins in the effect of NG need further study. To date, this study is the first to employ the proteomic technique to search globally for the possible target-related proteins of NG in platelets. Twelve proteins that might be possible target-related proteins of NG in platelet were identified. However, the identified proteins might not be the only targets of NG in inhibition of ADP-included platelet aggregation. Further study is necessary for both confirming the roles of these identified proteins in the effect of NG and finding other targets of NG during the process of platelet aggregation.

In the present study, NG was also shown to significantly decrease the ROS level in platelets. As far as we know, the inhibitive effect of NG on ROS level in platelets had not been reported before. However, our result was consistent with previous reports about the effect of main constituents of NG, such as notoginsengnoside R1 and ginsengnoside Rg1, on ROS level in other types of cells. For example, notoginsengnoside R1 was shown to inhibit NADPH oxidase-mediated ROS generation and directly scavenge ROS in smooth muscle cells. Notoginsengnoside R1's direct [H.sub.2][O.sub.2] scavenging property might be related to its chemical structure, which includes a multiply substituted phenol ring. The reaction of the phenol ring with [H.sub.2][O.sub.2] results in the formation of a phenolderived free radical that is relatively stable and unreactive (Zhang and Wang, 2006). Besides, notoginsengnoside R1 and ginsengnoside Rg1 both could inhibit [H.sub.2][O.sub.2] release from LPS-induced neutrophils (Sun, K et al., 2007; Sun, L et al., 2007). The decrease of ROS level might play an important role in the effect of NG on platelets.

Acknowledgements

The present work was partly supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (SIMM0709QN-14), The Ministry of Science and Technology of the People's Republic of China (2006DFB31860) and The Science and Technology Commission of Shanghai Municipality (064319052).

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[c] 2008 Elsevier GmbH. All rights reserved.

* Corresponding authors. Tel./fax: 8621 50271516.

E-mail addresses: lillianliucn@yahoo.com.cn (X. Liu), gda5958@163.com (D.-A. Guo).

0944-7113/$-see front matter (c) 2008 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2008.06.013

Yan Yao (a), (b), Wan-Ying Wu (a), Shu-Hong Guan (a), Bao-Hong Jiang (a), Min Yang (a), Xiao-Hui Chen (b), Kai-Shun Bi (b), Xuan Liu (a), (*), De-An Guo (a), (*)

(a) Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China

(b) Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, PR China
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Author:Yao, Yan; Wu, Wan-Ying; Guan, Shu-Hong; Jiang, Bao-Hong; Yang, Min; Chen, Xiao-Hui; Bi, Kai-Shun; Li
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Oct 1, 2008
Words:4745
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