Protection Of Chrysin On Vascular Endothelial Dysfunction Induced By Oxidative Stress

Objective:1.To observe whether the acute administration of Chrysin could relieve oxidative stress injury of HUVEC induced by H₂O₂;2.To investigate whether Chrysin could reduce the damage of HUVEC induced by high glucose and improve the rat isolated aortic vasodilator response.Methods:1.?1?Human umbilical vein endothelial cells?HUVECs?were used to observe the effects of different concentrations of H₂O₂ on cell viability and cell membrane stability by MTT and LDH examnation to find the optimal condition as the cell model;Divide the cultured cells into several groups as follows: the group of control,the group of H₂O₂,the groups of Chrysin?25 ?M,50 ?M,100 ?M?,and the cell viability and cell membrane stability was examed;?2?The concentration of MDA and SOD activity of HUVECs were examed;?3?The generation of cellular reactive oxygen species?ROS?of HUVECs was obtained by chemiluminescence method;?4?The release of NO of HUVECs was obtained by Griess method;?5?The synthesis of nitric oxide synthase?NOS?in HUVEC cells was determined by colorimetric method;?6?The gene levels of ICAM-1 and VCAM-1 were detected by RT-PCR;?7?The protein expression of endothelial nitric oxide synthase?e NOS?in HUVEC cells was detected by Western Blot.2.?1?HUVECs were used to observe the effects of different concentrations of glucose?11.1mM,22.2mM,33.3mM,44.4mM?on cell viability by MTT,and the model of endothelial cells demaged by high glucose was established;Divide the cultured cells into several groups as follows:the group of normal glucose,the group of high glucose,the group of mannitol control,the groups of Chrysin?25 ?M,50 ?M,100 ?M?,and the cell viability was examed;The production of ROS of HUVECs was obtained by chemiluminescence method;The liberation of NO of HUVECs was detected by Griess method;The activity of nitric oxide synthase?NOS?in HUVEC cells was determined by colorimetric method;?2?The rat isolated aortic vascular rings was used to exam the intervention effects of Chrysin on the endothelium-dependent vasodilator reactivity induced by ACh in high glucose K-H solution.Results:1.The cell viability of HUVECs was gradually decreased after 2 hours treatment with different concentrations of H₂O₂,And there was significantly difference when the concentration reach to 400 ?M,the cell viability was?65.73 ± 4.06?%;2.There was significantly difference compared to the group of H₂O₂ damage model after incubation with Chrysin,The cell viability was respectively increase to?71.94 ± 4.68?%,?82.17 ± 4.65?%,?86.24 ± 3.44?%at the concentration of 25 ?M,50 ?M,100 ?M;3.The content of MDA was increased to?8.23 ± 0.09??M and the activity of SOD was decreased to?0.59 ± 0.07?U/mgprot after incubation with H₂O₂ 2 hours.And there was significantly difference compared to the group of H₂O₂ when the cell was pretreated with Chrysin 12 hours,the content of MDA was decrease to?7.91 ± 0.09??M,?7.65 ± 0.16??M,?7.63 ± 0.18??M respectively,and the activity of SOD was?0.79 ± 0.06?U/mgprot,?1.37 ± 0.04?U/mgprot at the concentration of 50 ?M,100 ?M;4.The fluorescence of ROS was significantly enhanced when incubated with H₂O₂ 2 hours,and Chrysin could alleviate the fluorescence induced by H₂O₂ in the concentration of 25 ?M,50 ?M,100 ?M;5.The release of NO was significantly reduced when incubated with H₂O₂ 2 hours,And there was significantly difference compared to the group of H₂O₂ when the cell was pretreated with Chrysin 12 hours,the content of NO was increase to?10.97 ± 0.65??M,?11.43 ± 0.85??M respectively;6.The activity of TNOS and e NOS was reduced otherwise the activity of i NOS was increased after in the incubation of H₂O₂ 2 hours and there was significantly difference compared to the control group,the activity was?0.81 ± 0.01?U/mgprot,?0.05 ± 0.05?U/mgprot,?0.82 ± 0.16?U/mgprot repectively;There was statistical difference compared with H₂O₂ damage group in the activity of TNOS and e NOS which was relatively increased and the activity of i NOS was lowered after pretreated with Chrysin,the activity of TNOS was?0.97 ± 0.10?U/mgprot,?1.09 ± 0.12?U/mgprot,?1.12 ± 0.13?U/mgprot repectively;the activity of e NOS was?0.20 ± 0.09?U/mgprot,?0.38 ± 0.12?U/mgprot and?0.41 ± 0.13?U/mgprot repectively;the activity of i NOS was?0.84 ± 0.09?U/mgprot,?0.70 ± 0.10?U/mgprot,?0.71 ± 0.03?U/mgprot repectively;7.RT-PCR test results showed that different concentrations of Chrysin preincubated 12 hours could significantly suppress the m RNA expression of pro-inflammatory factor such as ICAM-1 and VCAM-1 which were induced by H₂O₂ and its inhibition was dose related;8.Western blot experiments results suggest that after preprocessing different concentration of Chrysin 12 hours could reduce the e NOS protein expression that was induced by H₂O₂,and there was significantly difference compared to H₂O₂ damage group;9.After incubated with high glucose,the cell viability was decreased and there was significantly difference between the group of high glucose and the group of normal glucose;there was no significantly difference between the mannitol group and the normal glucose group;After preprocessing with chrysin 2 hours,the cell survival rate was significantly rised to?103.40 + 4.78?%,?107.60 + 11.58?% and?121.80 + 7.40?% respectively;10.After incubated with high glucose 48 hours,the intracellular ROS fluorescence intensity significantly enhanced,and the ROS fluorescence decreased when pretreated with Chrysin 2 hours;11.There was significantly difference in the content of NO of HUVEC cell after incubated with high glucose compared with normal glucose group;And the release of NO in mannitol group have no significantly difference with normal glucose group;After different concentrations of Chrysin pretreatment 2 hours,intracellular content of NO statistically increased compared to high glucose group,the level of NO increased to?4.03 ± 0.69??M,?4.07 ± 0.98??M respectively at the concentration of 50 ?M,100 ?M;12.After preprocessing different concentration of Chrysin 2 hours could enhance the intracellular activity of TNOS and e NOS and could reduce the activity of i NOS that was induced by high glucose,and there was significantly difference compared to high glucose group,the activity of TNOS was?1.35 ± 0.31?U/mgprot,?1.66 ± 0.38?U/mgprot,?1.71 ± 0.25?U/mgprot repectively;the activity of e NOS was?0.22 ± 0.58?U/mgprot,?0.70 ± 0.25?U/mgprot,?1.03 ± 0.37?U/mgprot repectively;the activity of i NOS was?1.13 ± 0.29?U/mgprot,?0.96 ± 0.13?U/mgprot,?0.69 ± 0.15?U/mgprot repectively;13.After incubated with high glucose 4 hours,there were significantly difference in ACh-induced vascular relaxation between the normal glucose group and the high glucose group.The Emax was?31.78 ± 3.36?%,and the EC50 value was 81.10 ?M of high glucose group.The endothelium-independent relaxation induced by SNP was not significantly different between the two groups.And Chrysin could reverse the decline of ACh-induced vasorelaxation response induced by high glucose.The Emax was?70.53± 3.69?%,and the EC50 value was 1.05 ?M.Conclusion:1.Chrysin could reduce the cell oxidative stress injury induced by H₂O₂,and could increase the release of NO;2.The chrysin could alleviate the endothelial oxidative stress damage caused by high glucose and ameliorate the blood vessel endothelial-dependent relaxation.