Lycopus lucidus inhibits high glucose-induced vascular inflammation in human umbilical vein endothelial cells

Abstract

Vascular inflammatory process has been suggested to play a key role in the initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Lycopus lucidus Turcz. has been used as an oriental traditional medicine including Korea and its crude drug is known to have an anti-inflammatory effect. Thus we investigated whether the aqueous extract of the leaves of L. lucidus Turcz. (ALT) suppresses vascular inflammatory process induced by high glucose in primary cultured human umbilical vein endothelial cells (HUVEC). Western blot analysis revealed that incubation of HUVEC with high glucose increased cell adhesion molecules (CAMs) expression levels. However, high glucose-induced increase of CAMs expression was significantly attenuated by pretreatment with ALT in a dose-dependent manner. The enhanced cell adhesion between monocyte and HUVEC induced by high glucose was also blocked by pretreatment with ALT. High glucose-induced hydrogen peroxide production and DCF-sensitive intracellular reactive oxygen species (ROS) formation. Pretreatment with ALT inhibited high glucose-induced ROS formation. In addition, ALT suppressed the translocation and promoter transcriptional activity of NF-κB increased in high glucose condition. Taken together, the present data suggested that ALT could suppress high glucose-induced vascular inflammatory process, which may be closely related with the inhibition of ROS and NF-κB activation in HUVEC.

Introduction

Macrovascular complications including atherosclerosis are the leading cause of morbidity and mortality in patients with diabetes mellitus (DM) (Kannel and McGee, 1979, Ruderman and Haudenschild, 1984). Hyperglycemia is thought to be an important regulator of vascular lesion development. Hyperglycemia-induced endothelial dysfunctions, along with hypercoagulable potential of diabetes mellitus, accelerate the process of atherothrombotic complications. Vascular disorders, through overexpression of adhesion molecules and cytokines, are thought to participate in the pathogenesis of atherosclerosis. In particular, endothelial cells in human atherosclerotic lesions have been shown to increase cell adhesion molecules (CAMs) expression such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial selectin (E-selectin) (Lopes-Virella and Virella, 1992, Quagliaro et al., 2005). Under these conditions, numerous leukocytes adhere to vascular endothelium, transmigrate the endothelium, and aggravate endothelial dysfunction and tissue injury. Endothelial cells and monocytes cultured in media of high glucose concentration exhibit increased adhesiveness, suggesting that leukocyte binding through adhesion molecules increases under conditions of high glucose concentration (Kwon et al., 2004). However, the underlying mechanisms between hyperglycemia and vascular disease remain unclear.

Oxidative injury, due to the high glucose concentration, plays a central role in the development of diabetic complications (Brownlee, 2001). Under oxidative stress, macrophages generate reactive oxygen species (ROS) such as superoxides, peroxynitrite, leading to LDL oxidation (Steinberg, 1997). The production of ROS together with inflammatory factors including chemokines, cytokines, and adhesion molecules has been shown to be increased in atherosclerotic lesions (Liao et al., 1994, Ham and Skoryna, 2004). Inflammatory responses, including inflammatory gene transcription, appear to involve free radicals or oxidative stresses, and thus anti-oxidants or free radical scavengers can suppress inflammatory gene expression. However, the effect of Lycopus lucidus Turcz. on high glucose-induced oxidative stress was not cleared in vascular endothelial cell.

Previous studies have shown that high glucose as well as TNF-α activate nuclear factor-κB (NF-κB), one of the transcription factors for pro-inflammatory genes. NF-κB is present in the cytoplasm as an inactive form bound to its inhibitor molecule, inhibitory factor of NF-κB-α (IκB-α). Translocation of NF-κB form the cytoplasm to the nucleus is preceded by the phosphorylation, ubiquitination and proteolytic degradation of IκB-α (Thurberg and Collins, 1998). It is hypothesized that high glucose-induced CAMs expression may depend upon activation of NF-κB.

The Labiatae plant, L. lucidus Turcz. has been used for centuries as an oriental traditional medicine. This crude drug is used for treatment of menstrual disorder and inflammatory disease (Park, 2004, Shin et al., 2005). L. lucidus Turcz. contains three phenolic compounds, rosmarinic acid, methyl rosmarinate, ethyl rosmarinate, and two flavonoids, luteolin, luteolin-7-O-beta-d-glucuronide methyl ester, and it has been known to have anti-oxidative and anti-inflammation effects (Woo and Paio, 2004). However, the anti-inflammatory action of L. lucidus Turcz. under the diabetic condition has not been well examined. Because we previously found that the aqueous fractions of L. lucidus Turcz. leaves have more potent anti-oxidant activities than ethanol soluble fraction (unpublished data 2006), we used aqueous extracts of L. lucidus Turcz. (ALT) Thus, we investigated whether ALT suppresses vascular inflammatory process induced by high glucose in primary cultured human umbilical vein endothelial cells (HUVEC).