Lycopus lucidus inhibits high glucose-induced vascular inflammation in human umbilical vein endothelial cells
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).
Section snippets
Materials
d-glucose, l-glucose, d-mannitol, β-actin and goat anti-mouse IgG conjugate fluorescein isothiocyanate (FITC) were purchased from Sigma Chemical Company (St. Louis, MO). Lipofectamine LTX was purchased from Invitrogen Inc (Carlsbad, CA). Mouse monoclonal antibody to ICAM-1 was obtained from Zymed LALTratories Inc (San Francisco, CA). Mouse monoclonal antibodies to VCAM-1 and E-selectin were obtained from R&D systems Inc (Minneapolis, MN). NF-κB and IκB-α were purchased from Cell Signaling
Effect of high glucose on vascular inflammation
The concentration responses of high glucose on CAMs expression such as ICAM-1, VCAM-1, and E-selectin were determined by cell ELISA. Exposure to the primary cultured HUVEC to high glucose significantly increased expression of ICAM-1, VCAM-1, and E-selectin after incubation with 25 mM d-glucose (Fig. 1A. High glucose concentration can result in altered medium osmolarity. To rule out the involvement of hyperosmolarity in 25 mM glucose-induced increase of ICAM-1, VCAM-1, and E-selectin expression,
Discussion
This report presents evidence suggesting that ALT blocked the high glucose-induced vascular inflammation via inhibition of ROS and NF-κB in primary cultured HUVEC. Over the past decade, a prominent role of inflammation has been appreciated in the pathogenesis of atherosclerosis. One of the earliest events in vascular inflammation process is the adhesion of monocytes to the endothelium, which is followed by their infiltration and differentiation into macrophages. This key step is mediated by an
Acknowledgements
This study was supported by the grants of the Oriental Medicine R&D Project (03-PJ9-PG6-SO02-0001) to Dr. HS Lee funded by Ministry of Health and Welfare and Technology and a grant [L06010] to Dr. DG Kang funded by Korea Institute of Oriental Medicine.
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