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Autophagic effects of Hibiscus sabdariffa leaf polyphenols and epicatechin gallate (ECG) against oxidized LDL-induced injury of human endothelial cells

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Abstract

Purpose

Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury. Hibiscus sabdariffa leaf polyphenols (HLP), rich in flavonoids, have been shown to possess antioxidant and antiatherosclerotic activities. In this study, we examined the protective role of HLP and its main compound (−)-epicatechin gallate (ECG) in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL in vitro.

Methods

In a model of ox-LDL-impaired HUVECs, assessments of cell viability, cytotoxicity, cell proliferation, apoptosis, and autophagy were detected. To highlight the mechanisms of the antiapoptotic effects of HLP and ECG, the expressions of molecular proteins were measured by Western blotting, real-time PCR, and so on.

Results

HLP or ECG improved the survival of HUVECs from ox-LDL-induced viability loss. In addition, HLP or ECG showed potential in reducing ox-LDL-dependent apoptosis. Next, the ox-LDL-induced formation of acidic vesicular organelles and upregulation of the autophagy-related genes were increased by HLP or ECG. The HLP-triggered autophagic flux was further confirmed by increasing the LC3-II level under the pretreatment of an autophagy inhibitor chloroquine. Molecular data indicated the autophagic effect of HLP or ECG might be mediated via class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt cascade signaling, as demonstrated by the usage of a class III PI3K inhibitor 3-methyladenine (3-MA) and a PTEN inhibitor SF1670.

Conclusions

Our data imply that ECG-enriched HLP upregulates the autophagic pathway, which in turn led to reduce ox-LDL-induced HUVECs injury and apoptosis and provide a new mechanism for its antiatherosclerotic activity.

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Abbreviations

LDL:

Low-density lipoprotein

ox-LDL:

Oxidized LDL

HUVECs:

Human umbilical vein endothelial cells

LC3:

Microtubule-associated protein 1 light chain 3

Atg:

Autophagy-related genes

mTOR:

Mammalian target of rapamycin

PKB:

Protein kinase B

PI3K:

Phosphatidylinositol 3-kinase

HLP:

Hibiscus sabdariffa leaf polyphenols

ECG:

(−)-Epicatechin gallate

CQ:

Chloroquine

3-MA:

3-Methyladenine

TBARS:

Thiobarbituric acid-reactive substances

MTT:

3-(4,5-Dimethylthiazol-zyl)-2,5-diphenyltetrazolium bromide

LDH:

Lactate dehydrogenase

BrdU:

Bromodeoxyuridine

DAPI:

4,6-Diamidino-2-phenylindole

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TBS:

Tris-buffered saline

ECL:

Enhanced chemiluminescence

AO:

Acridine orange

PTEN:

Phosphatase and tensin homolog

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Acknowledgments

This work was supported by the Grant from the National Science Council (NSC99-2632-B-040-001-MY3), Taiwan.

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Authors and Affiliations

  1. Department of Nutrition, Chung Shan Medical University, Taichung City, Taiwan

    Jing-Hsien Chen & Cheng-Chin Hsu

  2. Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung City, 40201, Taiwan

    Ming-Shih Lee, Chi-Ping Wang & Hui-Hsuan Lin

  3. Clinical Laboratory, Chung Shan Medical University Hospital, Taichung City, Taiwan

    Ming-Shih Lee, Chi-Ping Wang & Hui-Hsuan Lin

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  1. Jing-Hsien Chen
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  2. Ming-Shih Lee
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Chen, JH., Lee, MS., Wang, CP. et al. Autophagic effects of Hibiscus sabdariffa leaf polyphenols and epicatechin gallate (ECG) against oxidized LDL-induced injury of human endothelial cells. Eur J Nutr 56, 1963–1981 (2017). https://doi.org/10.1007/s00394-016-1239-4

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