The Crude Extracts Of Edible-medicinal Fungus Of Protective Effects On High-glucose-induced Endothelial Injury: Screening And Mechanism Research

Part1.Screening for the crude extracts of edible-medicinal fungus of protectiveeffects on vascular endothelial cells injury induced by high glucoseObjective：We investigated the effects of crude extracts of Cordyceps gunnii (CGE), Lepistalentinus (LLE), Cordyceps sinensis (CSE) and Lentinus striguellus (LSE) onproliferation activity of high-glucose-treated human umbilical vein endothelial cells(HUVECs)， in order to sieve out the effective fungus with endothelial protectiveactivity.Methods：Endothelial cells of vein from newborn cord were isolated using collagenase,The cultured cells were verified as HUVECs by morphology andimmunocytochemical staining. The cultured HUVECs were treated with as followedrespectively: normal control group (added M199cultrue medium), high glucose group(33mmol/L glucose M199cultrue medium) and4crude extracts of edible-medicinalfungus (CGE, LLE, CSE and LSE) intervention groups (added differentconcentrations of every crude extract at12.5、25、50、100ug/ml in high glucose M199culture medium). Cell proliferation was evaluated by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide （MTT) assay.Results：1.The culture cells showed a cobblestone appearance, and the purity ofendothelial cells were more than95%identified by immunocytochemical staining forⅧ factor.2The OD value of cell MTT absorbance induced by high glucose wasremarkably lower compared with the normal control group (P<0.05). Compared withhigh-glucose group, treatment with the crude extracts of Lepista Lentinus andLentinus striguellus significantly decreased cell absorbance value (P<0.05), and the inhibition effects was showed in a dose-dependent manner. However, cordycepsgunnii has no the effect (P>0.05). Crude extracts of cordyceps sinensis(12.5-50ug/mL) significantly enhanced cell proliferation activity.Conclusion:The crude extracts of cordyceps sinensis may effectively protect from HUVECsinjury induced by high-glucose.The crude extracts of Cordyceps gunnii (CGE),Lepista lentinus (LLE) and Lentinus striguellus (LSE) have no protectiveeffectory on.HUVECs injury simulated by high-glucose.Part2. Study on effect and mechanism of the crude extracts of Cordycepssinensis on protection of human umbilical vein endothelial cells induced by highglucoseObjective:Diabetes and its cardiovascular complications is a serious threat to humanhealth.Previous studies showed that high glucose induced endothelial dysfunction bydirect or inderect mean,which play a important role in cardiovascular complicationssuch as atherosclerosis. The aim of this study was to determine the effect of the crudeextracts of Cordyceps sinensis (CSE) on injury in cultured human umbilical veinendothelial cells (HUVECs) treated with high glucose.Methods:Endothelial cells of vein from newborn cord were isolated using collagenase.TheHUVECs injury model was established by incubation with highglucose(40mmol/L).The Cultured HUVECs were divided into groups as followed:normal control group(added M199cultrue medium), high glucose group (40mmol/Lglucose M199cultrue medium) and the crude extracts of Cordyceps Sinensis（CSE）intervention groups (added different concentrations at12.5、25、50、100ug/ml in highglucose M199culture medium). Cell proliferation was evaluated by MTT assay, phasedistribution of cell cycles and cellular reactive oxygen species (ROS) were detectedby flow cytometry, the nitric oxide （NO）、superoxide dismutase （SOD） and malondialdehyde （MDA）content in medium were evaluated by Spectrophotometricmethod; and Nox4, eNOS, SirT1, endothelin-1and E-selectin genes mRNAexpression levels were detected by Real-time fluorescent quantitative RT-PCR.Results:1、Compared with the normal control group, HUVECs MTT absorbance value inhigh glucose group was remarkably lower (P<0.05), the percentage of G0/G1stagewas significantly decreased (P<0.05), while the percentage of S+G2/M wassignificantly increased (P<0.05), and ROS level significantly increased (P<0.05).Highglucose markedly decrease NO and SOD contents,and increase MDAcontents(P<0.05). Meanwhile, treatment with high-glucose up-regulated the level ofNox4,endothelin-1and E-selectin mRNA，and down-regulated the level of eNOS,SirT1mRNA（P<0.01）。2、Compared with high-glucose group, treatment with the crude extracts ofCordyceps sinensis, HUVECs MTT absorbance value in high glucose group wasremarkably higher (P<0.05), the percentage of G0/G1stage was significantlyincreased (P<0.05), while the percentage of S+G2/M was significantly decreased(P<0.05), and ROS level significantly decreased (P<0.05).High glucose markedlyincrease NO and SOD contents,and decrease MDA contents(P<0.05).Meanwhile,treatment with Cordyceps sinensis down-regulated the level of Nox4,endothelin-1and E-selectin mRNA，and up-regulated the level of eNOS, SirT1mRNA（P<0.01）.Conclusion:Crude extracts of Cordyceps sinensis may promote the HUVECs growth andproliferation, and inhibits apotosis; and then it may increase the secretion of NO,and down regulate the level of E-selectin and endothelin-1mRNA to improveendothelial function.These protective effect on endothelial cell may be associatedwith its effects of inhibiting reactive oxygen species, alleviating oxidative stressinjury, and up-regulating Sirt1-mRNA.