Sodium Danshensu Improves Angiotensin Ⅱ-induced Endothelial Dysfunction Through Activation Of AMPK

Background and PurposeHypertension is one of the most common chronic diseases,which cause high morbidity and death rate of cardiovascular and cerebrovascular danger.Vascular environment under hypertension is a vital risk factor for endothelial dysfunction.Endothelial dysfunction refers to the metabolic changes of available nitric oxide or the imbalance of several endothelium-derived diastolic and contractile factors,which will aggravate the progression of other cardiovascular diseases.Improving endothelial function is one of the effective means to prevent and manage cardiovascular diseases.As a key regulator of cell and body metabolism,AMPK is a heterotrimeric serine /threonine kinase and regulates a variety of enzymes and substrates.It is a therapeutic target that plays a significant role in cardiovascular protection.In endothelium,activated AMPK promotes the production of NO by phosphorylating e NOS and improves endothelial injury by reducing the adhesion and migration of inflammatory cells.Traditional Chinese medicine Salvia miltiorrhiza has a valuable effect in the prevention and treatment of cardiovascular diseases,which is widely used in clinic.Sodium danshensu is one of the main active components of Salvia miltiorrhiza,exerting therapeutic functions including relaxation of coronary artery,anti-oxidation,anti-inflammation,protection of myocardial ischemia/reperfusion injury,but there are few studies to explore the potential mechanism of it in improving endothelial dysfunction.In this study,we will observe the effect of Sodium danshensu on endothelial cell relaxation and adhesion,especially pay attention to the role of AMPK and the underlying molecular mechanism,which provide more sufficient experimental basis for Sodium danshensu in the prevention and treatment of cardiovascular diseases.Methods1.Human umbilical vein endothelial cells((HUVEC))were treated with Sodium danshensu(12.5 μM,25 μM,50 μM,100 μM,200 μM,400 μM)for 24 hours.MTS was used to detect the effect of Sodium danshensu on cell viability.2.After HUVEC was pretreated with Sodium danshensu(50 μM and 100 μM)for 12 h,the endothelial injury model was induced with Ang II(10 μM for 12 h.Nitric oxide(NO)content in the supernatant was measured by nitric acid reduction method,endothelin-1(ET-1)content was detected by ELISA,and vasodilation-related proteins were detected by Western Blot to explore the effect of Sodium danshensu on endothelial dilation dysfunction.3.The model of endothelial injury was established,and the vascular adhesion-related proteins in HUVEC were detected by monocyte-endothelial cell adhesion test and Western Blot to explore the effect of Sodium danshensu on vascular endothelial adhesion.4.The vascular rings of mouse thoracic aorta were prepared,and the effects of Sodium danshensu on vasodilation function and vascular adhesion molecules of mouse thoracic aorta were detected by vascular ring test and Western Blot method.5.Use molecular docking and molecular dynamics technology to observe the relationship between Sodium danshensu and AMPK.6.Western Blot was used to detect the relationship between Sodium danshensu and AMPK in both HUVEC and mouse thoracic aorta.7.Taking HUVEC as the research object,after si-AMPKα1/α2 was used to interfere with AMPK,NO content was tested and Western Blot was used to detect the effect of interfering AMPK on vascular endothelial relaxation induced by Sodium danshensu.8.Taking HUVEC as the research object,after si-AMPKα1/α2 was used to interfere with AMPK,monocyte-endothelial cell adhesion test and Western Blot was used to detect the effect of interfering AMPK on the improvement of vascular endothelial adhesion by Sodium danshensu.9.The thoracic aorta of mice was used as the research object.AMPK inhibitor was used to interfere with the phosphorylation of AMPK.The tension test of isolated vascular rings and Western Blot were used to observe the effect of inhibition of AMPK on the vasodilation effect of Sodium danshensu.10.The thoracic aorta of mice was used as the research object.AMPK inhibitor was used to interfere with the phosphorylation of AMPK.Western Blot was used to detect VCAM-1 and ICAM-1,to observe the effect of inhibition of AMPK on vascular adhesion improved by Sodium danshensu.Results1.After HUVEC was treated with Sodium danshensu(12.5 μM,25 μM,50 μM,100μM,200 μM,400 μM)for 24 h,compared with the control group,Sodium danshensu promoted the increment of HUVEC,the difference was statistically significant(P < 0.05).2.After the establishment of endothelial injury model,pretreatment with Sodium danshensu(50 μM and 100 μM)could increase the secretion of NO,reduce the release of ET-1 and promote the phosphorylation level of e NOS.The difference was statistically significant(P < 0.05).3.After the establishment of endothelial injury model,compared with the model group,Sodium danshensu could inhibit the adhesion of monocytes to HUVEC and phosphorylation of adhesion molecules VCAM-1,ICAM-1 as well as nuclear transcription factor p-p65,the difference was statistically significant(P < 0.05).4.In the test of vascular ring tension in mice,compared with the model group,the treatment of Sodium danshensu could increase the vasodilation rate of blood vessels.Western Blot showed that Sodium danshensu could promote the phosphorylation of e NOS and inhibit the protein expression of VCAM-1,ICAM-1 and p-p65 in the mouse thoracic aorta,the difference was statistically significant(P < 0.05).5.Molecular docking showed that AMPK had a binding ability with Sodium danshensu,and the binding energy was-6.30 kcal/mol.Molecular dynamics results showed that the bingding between AMPK and Sodium danshensu was stable.6.Western Blot showed that Sodium danshensu could activate AMPK both in HUVEC and mouse thoracic aorta(P < 0.05).7.After RNA interfered with AMPKα1/α2,the improvement effect of Sodium danshensu on HUVEC diastolic function was partially blocked,suggesting that the improving effect of Sodium danshensu on endothelial diastolic function may be carried out through AMPK.8.After RNA interfered with AMPKα1/α2,the inhibitory effect of Sodium danshensu on monocyte-endothelial cell adhesion was partially blocked,suggesting that the inhibitory effect of Sodium danshensu on adhesion may be carried out through AMPK.9.Under the interference of AMPK inhibitor,the relaxing effect and anti-adhesion effect of Sodium danshensu in blood vessels of mice were partially blocked,suggesting that the effect of Sodium danshensu on improving relaxation and inhibiting adhesion in vivo may be carried out through AMPK.Conclusion1.Sodium danshensu improves Ang II-induced endothelial dilation dysfunction and inhibits monocyte-endothelial adhesion.2.Sodium danshensu protects endothelial function and inhibits vascular endothelial adhesion by AMPK activation.