Rosuvastatin inhibit ox-LDL-induced platelet activation by the p38/MAPK pathway

Abstract

OBJECTIVE:

This study intends to explore the effects of Rosuvastatin on ox-LDL induced platelet activation and its molecular mechanism.

METHODS:

Platelet aggregation rate was detected by aggregometer. ELISA kit was used to detect the levels of cAMP. Immunofluorescence staining was used to detect the platelet adhesion. The expression levels of platelet surface markers CD62p and PAC-1 were detected by flow cytometry. The protein levels of p-p38, p-IKKa and p-IKKB in platelets were detected by western blot.

RESULTS:

We found that rosuvastatin significantly inhibited platelet aggregation and increased the level of cAMP in a dose-dependent manner. Immunofluorescence staining results showed that rosuvastatin could inhibit platelet adhesion. Flow cytometry results showed that rosuvastatin could reduce the expression of platelet activation markers. Western blot results showed that rosuvastatin could down-regulate the expression levels of p-p38, p-IKKa and p-IKKb.

CONCLUSION:

Our study revealed the rosuvastatin could inhibit the aggregation, adhesion and activation of platelet induced by ox-LDL, its mechanism may be related to inhibition of p38/MAPK signal pathway.

Keywords

Rosuvastatin platelet aggregation adhesion p38/MAPK

1 Introduction

Cardiovascular disease is the leading cause of death in the world [1]. It is reported that about 17.3 million people die of cardiovascular disease every year, and the number is expected to reach approximately 23.6 million by 2030 [2]. Thrombotic complications are the fundamental manifestations of cardiovascular diseases, such as myocardial infarction, venous embolism, and hypertension [3, 4]. Under physiological conditions, platelets can participate in hemostasis and wound healing [5]. Under pathological conditions, multiple signaling pathways could activate platelets, leading to thrombus formation and promoting atherosclerosis [6, 7]. Moreover, activated platelets could release various inflammatory factors, activate immune responses, interact with endothelial cells and participate in the pathological changes of atherosclerosis [8]. Oxidative stress, especially ox-LDL, may actively bind to low-density lipoprotein receptor 1, causing lipid deposition in the artery, platelet activation endothelial cell dysfunction, thus accelerating the development of atherosclerosis [9 –11]. The research on the mechanism of platelet activation mediated by ox-LDL may provide potential guidance for clinically treatment of atherosclerosis.

Statins are inhibitors of hydroxyl methylglutaryl coenzyme A (HMG CoA) reductase, which can competitively inhibit endogenous HMG CoA and block the metabolic pathway of cellular hydroxyl pentanoic acid, thus reducing the synthesis of cholesterol in cells and regulating blood lipids [12]. It has been reported that statins can be used in the prevention of myocardial infarction patients and the treatment of hypertension. Rosuvastatin plays a protective role in coronary artery endothelial cells by activating JAK/STAT3 signaling pathway and inhibiting apoptosis [13]. Xie et al reported [14] that pretreatment with Rosuvastatin effectively diminishes the occurrence of cardiovascular events among patients undergoing percutaneous coronary intervention (PCI) by suppressing the miR-155/SHIP-1 signaling pathway, without inducing microbleeds [15]. The mechanism of rosuvastatin in regulating ox-LDL-induced platelet activation, aggregation, and adhesion remains unclear.

In this study, we aim to investigate the effect of rosuvastatin on ox-LDL-induced platelet activation, including platelet aggregation, adhesion, and activation, as well as its potential molecular mechanism.

2 Materials and methods

2.1 Platelet-rich plasma preparation

Peripheral blood samples were collected from 30 healthy volunteers, and informed consent was signed. This study was approved by the medical ethics committee of The Second Hospital of Hebei Medical University. Whole blood (5 mL) was stored directly in plastic tubes containing 1 : 10 sodium citrate. Platelet-rich plasma was obtained by centrifugation of 300×g of the supernatant at room temperature and was reserved for use.

2.2 Groups and treatment

The platelet-rich plasma was divided into 5 groups in equal volumes: the control group (platelet-rich plasma treated with the vehicle, DMSO); ox-LDL group (platelet-rich plasm treated with 50μg/ml ox-LDL for 5 min); ox-LDL+Rosu 0.3μM group; ox-LDL+Rosu 3μM group; ox-LDL+Rosu 30μM group.

2.3 Platelet aggregation

Platelet aggregation was measured with a platelet Lumi-Aggregometer (Payton, Canada) as previously reported [16]. Platelets (3×10⁸ platelets/μL) were induced with 5μM ADP, the maximum platelet aggregation rate was calculated.

2.4 ELISA kit

According to the previously reported method, ELISA kit was used to determine the content ofcAMP.

2.5 Western blot

Protein samples were extracted from platelets using RIPA lysates. Western blot assays were performed as previously reported, in which specific primary antibodies included p38, phospho-p38, phospho-IKKb, phospho-IKKa, and GAPDH. Finally, image J software was used to measure the gray value of protein bands to evaluate the relative expression level of proteins.

2.6 Flow cytometry

As previously reported, flow cytometry was used to detect the expression levels of platelet surface markers CD62p and PAC-1. Briefly, 25μL HEPES buffer, 5μL anti-CD62p or PAC-1 (1 : 100) were added to 5μL whole blood. After 15 min of incubation at room temperature in the dark, 1 mL of PBS containing 1% paraformaldehyde was added. The samples were analyzed on a flow cytometer.

2.7 Platelet adhesion

Platelet-rich plasma was centrifuged at 200×g for 10 min. This was followed by gentle washing with Hepes buffer and resuspended. Slides were coated overnight with 10μg/ml fibrinogen and incubated for 1 h at room temperature with the addition of 2×10⁸ platelet suspension. Nonadherent platelets were aspirated and discarded and washed twice with PBS. The plates were fixed with 4% paraformaldehyde and stained with phalloidin for 30 min. Finally, the morphology of platelets was observed under a fluorescence microscope.

2.8 Statistical analysis

Data in this study are presented as mean±SD. SPSS22.0 software package was used for statistical analysis. The t test was used to compare the data between the two groups. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. A P value less than 0.05 was considered statistically significant.

3 Results

3.1 Comparison of platelet aggregation and cAMP level in each group

To detect the effect of rosuvastatin on platelet aggregation, ADP-induced platelet aggregation rates were measured using a platelet aggregometer. It (Fig. 1AB) showed that compared with control group, the platelet aggregation was increased significantly in ox-LDL group. After rosuvastatin treatment, the platelet aggregation rate was reduced significantly in a dose-dependent manner. ELISA kit results (Fig. 1C) showed that cAMP levels increased significantly in the ox-LDL group, and this trend was significantly reversed in the rosuvastatin treatment group in a dose-dependent manner. The results indicated that rosuvastatin could inhibit platelet aggregation and reduce cAMP levels.

Fig. 1

Comparison of platelet aggregation and cAMP level in each group (A) Morphology of ADP-induced platelet aggregation. (B) Aggregometer was used to detect the platelet aggregation. (C) The levels of cAMP in the groups were determined using ELISA kit. ^**P < 0.01, vs control group; ^#P < 0.05, ^##P < 0.01, vs ox-LDL group, n = 3.

3.2 Comparison of platelet adhesion in each group

Next, the morphology of platelet skeleton was detected by immunofluorescence staining and the adhesion of platelet was evaluated. As shown in Fig. 2, it was found that Compared with the control group, the adhesion of platelets in ox-LDL group was significantly increased. The adhesion of platelets was significantly reduced in the rosuvastatin treatment group, with the most significant difference in the 30μM rosuvastatin group. It suggested that rosuvastatin could inhibit the platelet adhesion.

Fig. 2

Comparison of platelet adhesion in each group. The skeleton morphology of adherent platelets was detected by immunofluorescence staining, ^***P < 0.001, vs control group; ^##P < 0.01, ^###P < 0.001, vs ox-LDL group, n = 3.

3.3 Comparison of the expression of platelet activation marker (CD62p and PAC-1) in each group

CD62p and PAC-1 were the markers of platelet activation. Then, the positive rate of CD62p and PAC-1 was detected by flow cytometry to evaluate platelet activation. As shown in Fig. 3A, it was found that compared with control group, the positive rate of PAC-1 in ox-LDL group was significantly increased and the difference was significant

Fig. 3

Comparison of the expression of platelet activation marker (CD62p and PAC-1) in each group. The levels of CD62p (A) and PAC-1 (B) on platelet surface were detected by flow cytometry, ^***P < 0.001, vs control group; ^##P < 0.01, vs ox-LDL group, n = 3.

The positive rate of PAC-1 was significantly reduced in the rosuvastatin treatment group, with the most significant difference in the 30μM rosuvastatin group. Similarly, the expression level of CD62p showed the same trend. Taken together, we found that rosuvastatin could significantly inhibit platelet activation.

3.4 Effects of Rosuvastatin on p38 MAPK pathway in ox-LDL-activated platelets

It has been reported that p38 MAPK signaling pathway is closely related to platelet activation. In this study, we investigated the effect of rosuvastatin on p38MAPK signaling pathway in ox-LDL-activated platelets. Western blot results (Fig. 4A) showed that compared with the control group, the phosphorylation level of p38 MAPK in ox-LDL group was significantly up-regulated. While the phosphorylation level of p38 MAPK was significantly reduced in the rosuvastatin treatment group. Furthermore, western blot results (Fig. 4B) showed that phosphorylation levels of p38, IKKa, and IKKb were significantly increased in the rosuvastatin treatment group compared with the ox-LDL group, and the same trend was shown in the SB203580 (p38 MAPK inhibitor) group. In conclusion, it indicated that Rosuvastatin could inhibit p38 MAPK signaling pathway in ox-LDL-activatedplatelets.

Fig. 4

Effects of Rosuvastatin on p38 MAPK pathway in ox-LDL-activated platelets. (A) The protein expression levels of p-p38 and p38 were detected by western blot. (B) western blot was used to detect the protein expression of p-p38, p38, p-Ikkb and p-Ikka. ^*P < 0.05, ^**P < 0.01, vs control group; ^#P < 0.05, ^##P < 0.01, vs ox-LDL group, n = 3.

3.5 p38 MAPK inhibitor could inhibit ox-LDL-Induced platelet activation

It was further verified that p38MAPK signaling pathway was involved in OX-LDL-induced platelet activation. As shown in Fig. 5A, compared with ox-LDL group, platelet aggregation rate in p38 MAPK inhibitor group was significantly reduced. Immunofluorescence staining results (Fig. 5B) showed that platelet adhesion was significantly reduced in p38MAPK inhibitor group compared with ox-LDL group. Flow cytometry results (Fig. 5CD) showed that the positive rates of PAC-1 and CD62p in the p38 MAPK inhibitor group were significantly reduced compared with ox-LDL group. The results confirmed that p38 MAPK inhibitors could significantly inhibit platelet activation, aggregation and adhesion.

Fig. 5

p38 MAPK inhibitor could inhibit ox-LDL-induced platelet activation. (A) Platelet aggregation (B) Platelet adhesion was detected by immunofluorescence staining; The expression of platelet surface marker P-selectin (C) and GPIIb/IIIa (D) was detected by flow cytometry.

4 Discussion

As a statin, rosuvastatin possesses diverse biological effects, chiefly inhibiting the synthesis of LDL-C and reducing proinflammatory cytokines [17]. It is extensively employed in clinical practice to lower blood lipids [18] and blood pressure [19]. Extensive clinical data has substantiated [20, 21] that rosuvastatin could effectively relieve the progressive development of atherosclerosis in the realm of cardiovascular diseases. Rosuvastatin has demonstrated various pharmacological actions in both clinical and animal studies, yet there are limited reports regarding its regulatory mechanism on platelet activation. In the study, we found that rosuvastatin significantly inhibited ox-LDL-induced platelet aggregation, adhesion, and activation. Our study validated that rosuvastatin could regulate platelet activation, thereby supporting its potential intervention in the o [22]ccurrence of cardiovascular events.

Antiplatelet therapy has significantly reduced the mortality of cardiovascular patients, and platelet inhibition drugs have become the first-line adjuvant drugs for the prevention and treatment of cardiovascular diseases [23, 24]. cAMP, a crucial messenger for platelet activation, plays a pivotal role in suppressing platelet activity [25]. Our study confirmed that rosuvastatin significantly elevates cAMP levels, thereby inhibiting platelet aggregation, adhesion, and activation.

Reports showed [26] that platelet activation by multiple signaling pathways, such as P2Y12 signaling pathway mediates platelet activation function in sepsis. PDK1 promotes collagen-induced increase of intracellular Ca+and mediates Rac1/PLCγ2 signaling pathway to activate platelet activation [27]. It has reported [28] that TLR4 could mediate platelet activation through MyD88 cGMP-PKG signaling pathway. In inflammatory response, antigen-antibody complexes could activate platelet activation through Fcγ-RIIA-ITAM pathway and participate in thrombosis [22]. Chen et al reported [29] that Fruitfow could inhibit platelet spreading and activation by suppressing p38/MAPK/AKT signaling pathway. Our findings showed that rosuvastatin significantly inhibited the phosphorylation of p38, IKKa and IKKb. Moreover, p38 inhibitor could reverse the inhibitory effect of rosuvastatin on platelet activation. We hypothesized that rosuvastatin could inhibit platelet activation, via inhibition of the p38 MAPK signaling pathway.

In summary, our study revealed the inhibitory effect of rosuvastatin on platelet activation and its potential molecular mechanism. Our findings may provide theoretical support for expanding the clinical application of rosuvastatin, especially in the field of cardiovascular system diseases.

Footnotes

Acknowledgments

We are grateful to all participants for their contributions for the present study.

Conflict of interest

All authors declare that there is no any conflict of interest.

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