Protection of glycyrrhizic acid against AGEs-induced endothelial dysfunction through inhibiting RAGE/NF-κB pathway activation in human umbilical vein endothelial cells.
ETHNOPHARMACOLOGICAL RELEVANCE: Licorice (Glycyrrhiza uralensis roots) is used as a traditional medicine for the treatment of diabetes mellitus and its vascular complications. Glycyrrhizic acid (GA, also known as Glycyrrhizin), a triterpenoid saponin glycoside, is considered to be a bioactive component in Licorice and is beneficial to diabetic vascular complications. AIM OF STUDY: The present study was conducted to evaluate the potential protective activities on AGEs-induced endothelial dysfunction, including anti-apoptosis, antioxidant stress and anti-proinflammatory responses, and explore the underlying mechanism. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated and pre-treated with GA (10(-9)-10(-6)M) or RAGE-Ab (5μg/ml) in the presence or absence of 200μg/ml AGEs. AO/EB fluorescence staining assay was performed to evaluate anti-apoptosis activity. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in cell supernatant were detected by kits while the intracellular reactive oxygen species (ROS) generation was determined by 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) kit. Immunocytochemistry analysis was designed to determine transforming growth factor beta1(TGF-β1) protein expression while immunofluorescence analysis for RAGE and NF-kB. The protein expressions of TGF-β1, RAGE and NF-kB were analyzed by Western blot analysis. RESULTS: Pretreatment with GA at a concentration of 10(-8)-10(-6)M significantly reduced the AGEs-induced apoptosis in HUVECs. GA significantly increased antioxidant enzyme SOD activity and decreased peroxide degradation product MDA level in a dose-dependent manner. Furthermore, GA also remarkably inhibited the overgeneration of AGEs-induced ROS. Both immunocytochemistry analysis and western blot analysis showed that GA significantly decreased the protein expression of poinflammatory cytokine TGF-β1 in a similar manner which RAGE-Ab did. Additionally, AGEs-induced RAGE and NF-kB protein expressions were down-regulated significantly by the pretreatment with GA or RAGE-Ab. CONCLUSION: These findings provide evidences that GA possesses protective activity on AGEs-induced endothelial dysfunction, including anti-apoptosis, anti-inflammation and antioxidant stress, via inhibiting RAGE/NF-kB pathway. GA might be an alternative for the prevention and treatment of diabetic vascular complications in an appropriate dosage.
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MDA glycyrrhizic acid transforming growth factor beta1 TGF-β1 RAGE NF-kB TGF-β1 RAGE NF-kB SOD oxygen cytokine TGF-β1 RAGE RAGE NF-kB RAGE RAGE NF-kB RAGE superoxide dismutase 2,7-dichlorodihydrofluorescin diacetate SOD NF-κB RAGE DCFH-DA MDA Glycyrrhizic acid Glycyrrhizin triterpenoid saponin glycoside superoxide malondialdehyde