| Part1. Screening for the ethyl acetate extracts of edible-medicinal mushrooms of protective effects on vascular endothelial cells injury induced by high glucose or hydrogen peroxideObjectiveThe decline of proliferation activity is one change of injured cells. This study was to investigate the effects of the ethyl acetate extracts of Cordyceps gunnii(Berk.) Berk (CGBE), Lepista sordida (Schum.:Fr.) Sing (LSSE), Lactarius deliciosus (LDE), Lentimts striguellus (LSE), Cordyceps militarys (CME), Fistulina hepaticaon (FHE) on proliferation activity of high-glucose or hydrogen peroxide treated human umbilical vein endothelial cells (HUVECs) in order to screen for the effective fungus with endothelial protective activity.MethodsEndothelial cells of vein from newborn cord were isolated using collagenase, The cultured cells were verified as HUVECs by morphology and Immunocytochemical staining. The HUVECs injured models were established by incubation with high glucose (40mmol/L) for48h and72h or hydrogen peroxide (0.1mmol/L) for48h in vitro. Cell proliferation was determined by the methylthiazolyl tetrazolium (MTT) assay. The extract of Cordyceps Sinensis (CSE)(12.5-100μg/mL) was treated as a positive control, and then CGBE, LSSE, LDE, LSE, CME, FHE(12.5-100μg/mL) were treated to observe their effects on endothelial cells injury.ResultsThe culture cells had a cobblestone appearance, and the purity of endothelial cells was more than95%identified by Immunocytochemical staining for VIH factor. HUVECs MTT absorbance value (A value) in high glucose group after incubating for 48h was remarkably lower (P<0.05or P<0.01) compared with the normal control group. Compared with high-glucose group, A value treatment with CGBE, LSSE, LDE, LSE, or FHE for48h were showed to further lower in a dose-dependent manner (P<0.05or P<0.01). However, A value with treated CME (25-50ug/mL) were significantly increased (P<0.05or P<0.01). After incubated for72h, compared with the normal control group, A value in high glucose group was remarkably lower (P<0.01); compared with high-glucose group, treatment with CGBE, LSSE, LDE, or LSE for72h further lowered cells A value in a dose-dependent manner (P<0.05or P<0.01); while CME (12.5-50ug/mL) treatment significantly increased HUVECs A value (P<0.05or P<0.01); FHE(12.5ug/mL) treatment significantly increased HUVECs A value (P<0.01). After incubated for48h, compared with the normal control group, HUVECs A value in hydrogen peroxide group was remarkably lower (P<0.01); compared with hydrogen peroxide group, treatment with CGBE, LSSE, LDE, LSE, or FHE for48h further lowered cells A value in a dose-dependent manner (P<0.01); while CME (25-100ug/mL) treatments significantly increased cells A value (P<0.05or P<0.01).ConclusionThe HUVECs injured models were successfully established by incubation with high glucose or hydrogen peroxide in vitro.the ethyl acetate extract of Cordyceps militarys effectively protect high-glucose or hydrogen peroxide induced injury in HUVECs. The ethyl acetate extract of Fistulina hepaticaon has doubtful effect on injured endothelial cells, and needs further observation. The ethyl acetate extracts of Cordyceps gunnii(Berk.) Berk, Lepista sordida (Schum.:Fr.) Sing, Lactarius deliciosus, or Lentimts striguellus have no protective effect on high-glucose or hydrogen peroxide induced injury in HUVECs. Part2. Effect of the extract of cordyceps militaris on senescence in cultured human umbilical vein endothelial cells treated with high glucoseObjectiveVascular endothelial senescence, as one form of endothelial cell injury, is thought to play a important role in cardiovascular diseases such as atherosclerosis. The aim of this study was to determine the effect of the ethyl acetate extract of cordyceps militaris (CME) on senescence in cultured human umbilical vein endothelial cells (HUVECs) treated with high glucose, initially investigating the mechanism of its anti-senescence and anti-injury activities.MethodsThe HUVECs senescence model was established by incubation with high glucose (40mmol/L), the extract of Cordyceps Sinensis (CSE)(50μg/mL) was treated as a positive control, and then CME (12.5-100μg/mL) was treated to observe its effect on cell senescence. Cell proliferation was determined by the methylthiazolyl tetrazolium (MTT) reduction assay, SA-β-galactosidase (SA-β-gal) positive cells were detected by X-gal, phase distribution of cell cycles and cellular reactive oxygen species (ROS) were detected by flow cytometry, and pi6, p21, SirT1, and Bcl-2genes mRNA expression levels were detected by Real-time fluorescent quantitative RT-PCR.ResultsCompared with unintervented cells, cell A value significantly decreased in cells treated with high glucose for24h,48h, and72h (P<0.01); The percentage of cells positive for SA-β-gal, the percentage in G0/G1phase, ROS levels, and p21gene mRNA expression notably increased after cells incubated by high glucose for96or48h (P<0.01), while SirTl gene mRNA expression notably decreased. Compared with high glucose intervention cells, cell A value significantly increased in cells intervented by CME (25-50ug/mL) for48h or72h (P<0.05); CME (12.5-100μg/mL) stimulating for96h significantly lowered the percentage of cells positive for SA-β-gal (P<0.01); CME (12.5-100μg/mL) incubation for48h significantly lowered the percentage of G0/G1phase of HUVECs (P<0.01) and ROS production (P<0.05); Meanwhile, CME (50μg/mL) incubation for48h significantly lowered p21gene mRNA expression (P<0.01), and increased SirTl gene mRNA expression (P<0.01); All these affects of CME25,50ug/mL groups were better than12.5,100μg/mL groups (P<0.05), and were similar to CSE50μg/mL grouop (P>0.05).ConclusionThe ethyl acetate extract of cordyceps militaris may promote the HUVECs growth and proliferation, protecting from high glucose-stimulated vascular endothelial senescence. These may be associated with its effects of inhibiting reactive oxygen species, alleviating oxidative stress injury, decreasing p21gene mRNA expression, and increasing SirTl gene mRNA expression. |
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