| BackgroundIn recent years,the incidence of diabetes mellitus is increasing in all age groups all over the world.Diabetes is characterised by hyperglycaemia,deranged metabolism and sequelae predominantly affecting the vasculature.Nowadays,cardiovascular complication has become the main cause of death in diabetic patients.Many research shows that the hyperglycaemia-induced oxidative stress in vascular endothelium is closely related to the diabetes cardiovascular complications.Therefore,inhibiting the hyperglycaemia-induced oxidative stress may be a new target for the therapy of diabetes complications.Puerarin(daidzein 8-C-glucoside),the main isoflavone glycoside found in the root of P.lobata,has been reported to possess potential antioxidant property.Now little is known about whether puerarin could protect against high glucose-induced dysfunction of vasculature. Objectives(1) To investigate the protective effect of puerarin on vascular reactivity subsequent to high glucose stress.(2) To explore the role of heme oxygenase-1(HO-1) in the protective mechanism of puerarin.Methods(1) Measurement of endothelial-dependent relaxant function in rat aorta rings: The thoracic aortic rings with endothelium from male SD rats were mounted in an organ bath.Aortic rings were precontracted with 10-6 mol/L PE.When the maximal contractile plateau was reached,ACh(3×10-8,10-7,3×10-7,10-6,3×10-6, 10-5,3×10-5 mol/L) added to the bath cumulatively.Relaxant responses were expressed as the percentage decreases of the magnitude of the contraction induced by PE before the application of ACh.(2) Cell apoptosis evaluation:Human umbilical vein endothelial cells(HUVECs) were harvested and stained with PI(100 mg/ml) for 30 min.Cell apoptosis was measured using the percentage of PI-stained nuclei in the subdiploid peak by flow cytometric analysis.(3) Calpain and caspase-3 activity assay:Calpain and caspase-3 activity in HUVECs was measured following the procedure described by Promega technical bulletin.(4) HO-1 mRNA expression and HO activity evaluation:HO-1 mRNA expression was analyzed by RT-PCR.HO activity was detected with bi-wavelength method and expressed as picomoles of bilirubin formed per milligram of protein per hour.Results(1) Effects of puerarin on high glucose-induced acute relaxation dysfunction: Compared with the control group,incubation with high glucose(44mmol/L) for 2 h decrease the Ach-induced relaxation.However,coincubation of aortic rings with pueratin(10-9 or 10-8 mol/L) and high glucose for 2 h inhibited the high glucose-induced decrease in Ach-induced relaxation.(2) Effects of puerarin on high glucose-induced apoptosis:Compared with the normal glucose(5.5 mmol/L) group,exposure of HUVECs to high glucose(33 mmol/L) for 48 h significantly increased apoptosis rate and caspase-3 activity of endothelial cells.Puerarin(10-6 or 10-4 mol/L) treatment prevented the increased apoptosis rate and caspase-3 activity observed in high glucose condition.(3) Effects of puerarin on high glucose-induced enhancement of calpain activity: Treatment of HUVECs with high glucose for 48 h induced an obvious rise in calpain activity.But puerarin reduced high glucose-induced calpain activity in a concentration-dependent manner.(4) Role of HO-1 in the protective mechanism of puerarin:Results of RT-PCR showed a significant decrease of HO-1 mRNA level and HO activity in HUVECs treated with high glucose for 48 h.Compared with high glucose group, co-incubation of endothelial cells with puerarin and high glucose induced an increase in the expression of HO-1 mRNA and HO activity.Meanwhile HO-1 inhibitor ZnPP also abolished the inhibitive effect of puerarin on high glucose-induced calpain activation and apoptosis.ConclusionThe present study provides evidence that puerarin protects against high glucose-induced endothelial-dependent relaxation dysfunction and endothelial cells apoptosis by a mechanism involving upregulation of HO-1 expression and inhibition of calpain activity.
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