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Phosphocreatine protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway

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Abstract

Endothelial apoptosis triggered by oxidized low-density lipoprotein (oxLDL) can accelerate the progression of endothelial dysfunction atherosclerosis. Phosphocreatine (PCr) is a natural compound, which has been used in cardiac disease and cardiopulmonary resuscitation. However, its protective effects on atherosclerosis and its mechanism have not been clarified. In the present study, we investigated the anti-apoptotic effect of phosphocreatine in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. HUVECs were pre-treated with 10–30 mM PCr and then stimulated with oxLDL. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, CCK assay, and flow cytometry respectively. Levels of Bax, Bcl-2, protein expression of protein kinase B (Akt), eNOS and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Lactate dehydrogenase (LDH), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) contents were determined by spectrophotometer. Our results showed that PCr dose-dependently prevented oxLDL associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, LDH and MDA leakage and loss of SOD, decrease of Bcl-2/Bax protein ratio, activation of caspase-3 and 9, and ROS generation. In addition, the antiapoptotic effect of PCr was partially inhibited by a PI3K inhibitor (LY294002) and also enhanced p-Akt/Akt protein ratio, eNOS activation and NO production. In conclusion, our data show that the inhibition of oxLDL-induced endothelial apoptosis by PCr is due, at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.

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Abbreviations

PCr:

Phosopho creatine

oxLDL:

Oxidative low density lipoprotein

FBS:

Fetal bovine serum

HUVEC:

Human umbilical vein endothelial cell

LDH:

Lactate dehydrogenase

MDA:

Malondialdehyde

SOD:

Superoxide dismutase

ROS:

Reactive oxygen species

MMP:

Mitochondrial membrane potential

Akt:

Protein kinase B

eNOS:

Endothelial nitric oxide synthase

NO:

Nitric oxide

NAC:

N-Acetyl cysteine

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Acknowledgments

This work was supported by the Natural Science Foundation of China (No. 30772601) and the University Innovation Team Project Foundation of Education Department of Liaoning Province (No. LT2013019).

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Authors and Affiliations

  1. Department of Pharmacology, Dalian Medical University, West Section 9, South Road of Lushun, Dalian, 116044, China

    Anil Ahsan, Guozhu Han, Shumin Liu, Peng Chu, Zhengwu Sun, Zonghui Zhang, Bin Sun, Jingjun Wu, Yuan Lin, Jinyong Peng & Zeyao Tang

  2. Shanghai Huatuo Pharmaceutical Technology Development Limited Company, No. 590, Building 7, Layer 4, Road Ruiqing, Pudong New Area, Shanghai, China

    Junfang Pan

  3. Biochemistry and Molecular Biology Department, Dalian Medical University, Dalian, China

    Arshad Ahmed Padhiar

  4. Dalian University of Foreign Language, Dalian, China

    Aisha Irshad

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  1. Anil Ahsan
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  2. Guozhu Han
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  4. Shumin Liu
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Correspondence to Zeyao Tang.

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Ahsan, A., Han, G., Pan, J. et al. Phosphocreatine protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway. Apoptosis 20, 1563–1576 (2015). https://doi.org/10.1007/s10495-015-1175-4

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