| Objective:Oxidative stress is an important biochemical mechanism that cause chronic damage to vascular endothelium and lead to coronary heart disease or angina pectoris.In this article,the clinical drug Guanxinning injection was used as the research object.The oxidative stress damage model was established in EA.hy926 vascular endothelial cells induced by tert-butyl hydroperoxide(t-BHP),and the protective effect of Guanxinning injection and its main active ingredients on damaged cells were studied in vitro.The molecular mechanism of Kelch-like ECH associated protein 1(Keap1)-Nuclear factor erythroid 2-related factor 2(Nrf2)-Antioxidant response element(ARE)signaling pathway regulating oxidative stress damage in cell was investigated,and the mechanism of protecting the damaged vascular endothelial cells by Guanxinning injection was revealed.Methods:(1)The non-toxic concentration of Guanxinning injection and the appropriate concentration of t-BHP were measured with MTT assay in the oxidative damage of EA.hy926 cells induced by t-BHP.The effect of Guanxinning injection on the viability of EA.hy926 cells induced by t-BHP at different doses and time were measured with MTT assay.The influence of lactate dehydrogenase(LDH),malondialdehyde(MDA)and superoxide dismutase(SOD)levels on the antioxidant indexes treated with Guanxinning injection were detected by spectrophotometry,and the level of reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe method.(2)MTT assay was used to detect the viability and protection rate of ferulic acid,caffeic acid,rosmarinic acid,protocatechualdehyde,salvianic acid A sodium,and salvianolic acid B in Guanxinning injection on EA.hy926 cells induced by t-BHP.Used Graphpad 7.0 to fit the curve of protection rate and calculated the effect concentration 50(EC50).The LDH release assay was used to detect the ability of the main ingredients to release LDH,and the Real-Time PCR was used to detect the expression of active ingredient to the key antioxidant enzyme Heme oxygenase-1(HO-1)in the oxidative pathway.Finally,the main effective ingredients of Guanxinning injection were screened out.(3)The effects of Guanxinning injection on Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1),HO-1 mRNA were detected by Real-Time PCR.Western Blot method was used to detect the expression of Keap1,HO-1 and NQO1 protein in Guanxinning injection and salvianolic acid B.Nuclear export of Nrf2 was characterized by immunofluorescence.At the same time,the positive control was introduced by the inhibitor of Nrf2 ML385.Western Blot method was used to detect the influence of Guanxinning injection on the expression of downstream key proteins HO-1 and NQO1.Furthermore,the mechanism of Guanxinning injection through Keap1-Nrf2-ARE signaling pathway to regulate oxidative stress-induced endothelial cell injury was described.Results:(1)t-BHP induced cells at 800 ?mol/L concentration for 4 h were used as damage model.After 6,12,24 h treated,Guanxinning injection(1.25,2.5,5,10 ?L/mL)reduced the cell death and effectively improved viability rate.Guanxinning injection with concentration of 10 ?L/mL after 12 h treated showed the most significant protective ability.Guanxinning injection could up-regulate the activity of SOD and down-regulate the levels of LDH,MDA and ROS to alleviate the cell damage induced by t-BHP and protected endothelial cells,with a dose-dependent manner.(2)The six main active ingredients showed different protective effects on vascular endothelial cells injured by t-BHP.And it also had a certain inhibitory effect on the release of LDH.The effective concentration 50 of salvianolic acid B(EC50=0.52 ?mol/L)was better than that of salvianic acid A sodium(EC50=49.79 ?mol/L),caffeic acid(EC50=8.28 ?mol/L),protocatechualdehyde(EC50=6.49 ?mol/L),rosmarinic acid(EC50=6.48 ?mol/L),ferulic acid(EC50 cannot figure out).And 400 ?mol/L salvianolic acid B had the most significant expression of HO-1 mRNA(compare with the control group,P<0.01).The above results indicated that salvianolic acid B had the strongest antioxidant activity among the six ingredients of Guanxinning injection.(3)Guanxinning injection increased the mRNA and protein expression of HO-1,NQO1,and Nrf2 with a dose-dependent manner when Guanxinning was treated alone.After t-BHP injured,the expression of HO-1 and NQO1 protein were significantly down-regulated,and the expression of Keap1 protein was significantly up-regulated(compare with the control group,P<0.05).After pretreated with Guanxinning injection and salvianolic acid B,the protein expression of HO-1 and NQO1 were up-regulated,and the expression of Keap1 protein was down-regulated(compare with the model group,P<0.01).Immunofluorescence results showed that after pretreated with Guanxinning injection and salvianolic acid B,the expression of Nrf2 protein was increased into the nucleus(compare with the model group,P<0.01).Guanxinning injection decreased the expression of the key proteins HO-1 and NQO1 in the downstream after Nrf2 inhibitor treated.It is suggested that Guanxinning injection down-regulated the expression of Keap1,increased the nuclear expression of the Nrf2 uncoupled to Keap1,and sequentially up-regulated the expression of HO-1 and NQO1 to play the protective role of vascular endothelial cells.Conclusion:This study shows that Guanxinning injection can protect the damaged vascular endothelial cells caused by oxidative stress and play a role in reducing lipid peroxidation and enhancing antioxidant capacity by increasing the activity of SOD and decreasing the content of LDH,MDA and ROS.The six main phenolic acids in Guanxinning injection(ferulic acid,caffeic acid,rosmarinic acid,protocatechualdehyde,salvianic acid A sodium and salvianolic acid B)have different effects on endothelial cells injury.Salvianolic acid B is the most active ingredient in Guanxinning injection.Guanxinning injection and salvianolic acid B can play a protective role on damaged vascular endothelial cells by activating the Keap1-Nrf2-ARE signaling pathway. |
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