Berberine improves endothelial function by reducing endothelial microparticles-mediated oxidative stress in humans

https://doi.org/10.1016/j.ijcard.2012.03.090Get rights and content

Abstract

Background

Circulating endothelial microparticles (EMPs) lead to endothelial dysfunction by increasing oxidative stress. Berberine has a beneficial effect on endothelial function, but no data are available on the EMP-mediated oxidative stress. The present study tests the hypothesis that berberine contributes to the improvement of endothelial function in humans via inhibiting EMP-mediated oxidative stress in vascular endothelium.

Methods

Twelve healthy subjects received a 1-month berberine therapy and eleven healthy subjects served as control. Endothelium-dependent and -independent function in the brachial artery was assessed by flow-mediated vasodilation (FMD) and sublingual nitroglyceride-mediated vasodilation (NMD). Circulating EMPs and serum malondialdehyde (MDA) were measured before and after therapy. Furthermore, in vitro human umbilical vein endothelial cells (HUVECs) were stimulated by EMPs with or without presence of anti-oxidant compound apocynin or berberine. Intracellular reactive oxygen species (ROS), nitric oxide (NO) production and NADPH oxidase 4 (Nox4) protein expressions were examined, respectively.

Results

The levels of serum MDA and circulating CD31 +/CD42 − MPs were significantly reduced in the berberine group compared with the control group, which were associated with improvement of FMD. The EMPs in vitro facilitated ROS production and Nox4 protein expression and reduced NO synthesis in HUVECs. These alterations can be reversed by the presence of apocynin or berberine, respectively.

Conclusion

The present study demonstrated for the first time that EMP-induced upregulation of Nox4 expression may enhance ROS production in HUVECs. Berberine treatment contributes to the amelioration of endothelial function through a partially reducing oxidative stress of vascular endothelium induced by circulating CD31 +/CD42 − microparticles in humans.

Introduction

Endothelial dysfunction leads to the pathogenesis of cardiovascular disease (CVD) [1], [2], [3]. Recently, it has been reported that alteration of circulating endothelial microparticles (EMPs), mostly defined as CD31 +/CD42 − microparticles, emerged as a sensitive marker for endothelial injury in response to various stimuli [4], [5], [6], [7]. Accumulating evidence indicates that elevated EMPs per se actively contribute to deterioration of endothelial homeostasis partially via increasing oxidative stress in endothelial cells [8], [9], [10]. However, the detailed mechanisms underlying EMP-mediated endothelial dysfunction and its relation to oxidative stress are not completely understood. The generation of reactive oxygen species (ROS) derived from nicotine adenine dinucleotide phosphate (NADPH) oxidase is considered as one of the major sources of oxidative stress in vessel wall [11], [12]. It has been reported that EMPs impair endothelial cells via increasing NADPH oxidase activity [13]. Therefore, reduced oxidative stress induced by EMPs probably via diminishing NADPH oxidase activity may be a novel therapeutic strategy for the maintenance of endothelial homeostasis.

Berberine is an alkaloid extract from herb that has been used for long in oriental medicine to treat gastrointestinal infections and diarrhea, but not until recent decades its beneficial effects on cardiovascular system have been realized [14], [15]. Previous studies showed that berberine has multiple beneficial effects on vascular homeostasis [15], [16], [17], [18], [19]. We recently reported that the reduction in circulating EMPs by berberine treatment improves endothelial function in healthy subjects without notable side effects [20]. It raises the possibility that berberine may be a novel therapeutic regimen on patients with CVD, whose endothelial function is markedly impaired. However, the exact molecular mechanisms underlying the salutary effect of berberine on vascular endothelium remain largely unknown. Given the close association between oxidative stress and endothelial function, we hypothesize that berberine may protect endothelial function at least in part via reducing oxidative stress induced by elevated EMPs in endothelium.

To address this assumption, we recruited healthy middle-aged subjects who received 1-month berberine therapy. The change of circulating CD31 +/CD42 − microparticles and serum malondialdehyde (MDA) was measured before and after berberine therapy. Endothelium-dependent and -independent function in the brachial artery was assessed by flow-mediated vasodilation (FMD) and sublingual nitroglyceride-mediated vasodilation (NMD). To further investigate the effect of EMPs on oxidative stress, in vitro cultured human umbilical vein endothelial cells (HUVECs) were stimulated by EMPs with or without the presence of berberine and apocynin, an anti-oxidant compound which primarily acts through inhibiting NADPH oxidase activity. The alterations in ROS, protein expression of NADPH oxidase 4 (Nox4), and nitric oxide (NO) production were examined, respectively. The present study may provide valuable information on our understanding the novel mechanism of berberine-mediated endothelial protection and help to develop a potential therapeutic strategy to counteract the endothelial dysfunction in CVD patients.

Section snippets

Materials

Berberine hemisulfate was from Alexis (Switzerland). PE-conjugated monoclonal antibody against PECAM-1(PE-CD31) and FITC-conjugated monoclonal antibody against Platelet Glycoprotein Ib (FITC-CD42b) was used to identify endothelial microparticles. All the flow cytometry labeling reagents including PE- and FITC-conjugated monoclonal IgGs were obtained from Immunotech (France). Flowcount fluorospherical beads used to determine microparticles' absolute values and 1-μm calibrant beads used to define

Berberine improved FMD, which was associated with decreased circulating CD31 +/CD42 − microparticles and serum MDA

The characteristics of the recruited healthy subjects before and after therapy were shown in Table 1. As Fig. 2 shows, berberine significantly improved flow-mediated vasodilation (before, 7.30 ± 20.81%; after, 12.04 ± 0.99% [p < 0.05]) (Berberine group, 12.04 ± 0.99%; Control group, 8.02 ± 1.44% [p < 0.05]) but not nitroglyceride-mediated vasodilation (before, 27.32 ± 3.38%; after, 30.92 ± 2.14%) (Berberine group, 30.92 ± 0.99%; Control group, 26.76 ± 5.89%). At the same time, berberine decreased circulating CD31 

Discussion

The major findings of the present study are as follows: 1) Berberine therapy in healthy subjects reduces serum MDA and circulating CD31 +/CD42 − microparticles. In parallel, flow-mediated vasodilation in the brachial artery was improved but nitroglyceride-mediated vasodilation kept unchanged. 2) The reduction of serum MDA and circulating CD31 +/CD42 − microparticles were both closely related to flow-mediated vasodilation improvement. 3) The EMP stimulation in vitro resulted in increased ROS and

Acknowledgments

This study is financially supported by the grants from the National Natural Scientific Foundation of China (330973535 and 81170131), 973 program (2012CB517802) of China and Guangzhou Scientific Project Foundation (2007Z3-E0241). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.

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