Objective: To observe the effects of icariin on S-nitrosogultathione(GSNO) induced endothelial cell apoptosis, and to explore the relative mechanisms.
Methods: EA.hy926 cell line was provided by Zhejiang University and cells were divided into blank control group, GSNO group (1 mmol/L GSNO), icariin (ICA) intervention group (GSNO+ different concentrations (high, medium and low: 10, 1 and 0.1 μmol/L ICA) and 1 μmol/L LY294002 pretreatment groups (AKT protease pathway inhibitor on top of ICA groups) by cluster random sample method. After 48 hours. EA.hy 926 cell survival was detected by thiazolyl blue tetrazolium bromicle (MTT) method. Lactate dehydrogenase (LDH) activity, malonaldehyde (MDA) content, reactive oxygen species (ROS) level, mitochondrial membrane potential were also measured. The protein expression of protein kinase B(AKT)/phosphorylation protein kinase B(p-AKT), people tumor-suppressor protein (protein 53, P53), cytochrome C (CYC), endothelial nitric oxide synthetase (eNOS)/phosphorylation endothelial nitric oxide synthetase (p-eNOS), procaspase-3/caspase-3 was detected by Western blot.
Results: (1) The cell survival rate was significantly lower in GSNO group than in the blank control group (P< 0.01), which was significantly higher in the high, medium and low concentration ICA groups than in the GSNO group (all P< 0.01). (2) The LDH activity was significantly higher in the GSNO group than in the blank control group ((142.65±5.56) U/L vs. (50.01±3.42) U/L, P< 0.05), which was significantly reduced by high, medium and low concentration ICA ((98.02±3.52), (105.29±6.89) and (117.16±4.27) U/L vs. (142.65±5.56) U/L, all P< 0.05) in a dose-dependent manner. (3) The MDA content was significantly higher in GSNO group than in the blank control group ((11.14±0.37) nmol/mg vs. (5.21±0.18) nmol/mg, P< 0.05), which could be reduced by pretreatment with high, medium and low concentration ICA ((6.60±0.41), (6.83±0.21) and (8.29±0.07) nmol/mg vs. (11.14 ±0.37) nmol/mg, all P< 0.05). (4) The ROS content was significantly higher in GSNO group than in the blank control group ((173.15±11.12)% (relative ratio to the blank control group), P< 0.05), which could be significantly reduced by pretreatment with high, medium and low concentration ICA ((122.56±8.09)%, (134.52±9.09)%, and(149.89±9.16)% (the ratio of the above are compared with the blank control group), P< 0.05). (5) The mitochondrial membrane potential was significantly higher in the GSNO group (0.84 ± 0.04) than in the blank control group (0.12 ±0.12), which could be significantly reduced by pretreatment with high, medium and low concentration ICA ((0.57±0.08), (0.63±0.02), (0.66±0.04) vs. (0.84±0.04), all P<0.05). (6) The expression of AKT/p-AKT was significantly lower in GSNO group than in the blank control group (P< 0.05), which could be significantly upregulated by pretreatment with high, medium and low concentration ICA in a concentration-dependent manner, above effects could be blocked by LY294002 (all P<0.05). (7) The expression of P53 was significantly higher in GSNO group than in the blank control group (P< 0.05), which could be significantly down regulated by pretreatment with high, medium and low concentration ICA in a concentration-dependent manner, above effects could be blocked by LY294002(all P<0.05). (8) The expression of CYC and caspase-3 was significantly reduced in the mitochondria and increased in cytoplasm post GSNO treatment compared to blank control group, which could be reversed by pretreatment with high, medium and low concentration ICA(all P<0.05). (9) The expression of eNOS/p-eNOS was similar between GSNO and the blank control group, while it was significantly upregulated by pretreatment with high, medium and low concentration ICA and this effect could be blocked by LY294002(all P<0.05).
Conclusion: Icariin could reduce GSNO induced endothelial cell apoptosis through activating AKT pathway and downregulating P53 activity.