Protective Effect And Mechanism Of Physcion On Vascular Injury Induced By Homocysteine

ObjectiveHyperhomocysteinemia?HHcy?is an important and independent risk factor of atherosclerosis and hypertension.It is closely related to the occurrence and development of cardiovascular disease.High plasma levels of Hcy can stimulate vascular endothelial cells,induce vascular damage and disorder,and leading to cardiovascular disease.Physcion?Phys?is one of the main active ingredients of rhubarb and belongs to the anthraquinones compounds.It has many biological effects such as anti-inflammatory,anti-oxidation and anti-apoptosis.However,the protective effect of physcion on vascular injury induced by Hcy has not been reported.In order to explore the potential mechanism of physcion on Hcy-induced vascular injury,in vivo and in vitro experiments were performed.This study will provide a new theoretical basis for the prevention and treatment of vascular injury by Hcy.Methods1.Protective effect of physcion on vascular function in HHcy ratsHHcy was induced in rats by feeding a highmethionine diet,which consisted of a regular diet plus 3%methionine?wt/wt?.Animals were assigned into 4 groups:Control,HHcy,Phys-L+HHcy and Phys-H+HHcy.Rats in Hcy group,Phys-L+HHcy group and Phys-H+HHcy group were fed a high methionine diet,which consisted of a regular diet plus 3%methionine?wt/wt?.Rats in Phys-L+HHcy group were intragastrically administrated Phys by 30 mg/kg/day.Rats in Phys-H+HHcy group were intragastrically administrated Phys by 50 mg/kg/day.The effect of acetylcholine?ACh?on the relaxation effect of phenylephrine pre-contracted rat isolated aortic rings was observed using a vivo vascular ring device and a BL-420E⁺biofunctional experiment system.The plasma levels of Hcy,NO,SOD and MDA in rat were measured with a microplate reader.2.Protective Effect of physcion on Hcy-induced HUVECs damageHuman umbilical vein endothelial cells?HUVECs?were cultured in vitro,and the model of Hcy injury cells was established.HUVECs were divided into control group,Hcy group,and Phys+Hcy group.The following experiments were performed:?1?MTT assay was used to determine cell viability.?2?Hoechst 33342 staining method and Annexin V-FITC Apoptosis Detection Kit were used to determine apoptosis of HUVECs.3.The molecular mechanism of the protective effect of physcion on Hcy-induced HUVECs damageThe following experiments were performed according to the following.?1?ROS production in HUVECs was determined by DCFH-DA as a probe for the presence of ROS.?2?Mitochondrial membrane potential?MMP?was identified with JC-1 staining.?3?After labeling of HUVECs with the fluorescent probe Fluo-3,intracellular level of calcium was detected by fluorescence microscopy.?4?The level of NO in cell supernatant was detected by nitrate reductase method;?5?Western blot was used to observe the expression of Akt,eNOS and apoptotic proteins such as Bcl-2,Bax,Caspase-3,Caspase-9 in HUECs.Results:1.Protective effect of physcion on vascular function in HHcy ratsPlasma levels of Hcy and MDA were significantly higher while the SOD and NO were significantly lower in the HHcy group compared with control group.Treatment with physcion significantly reversed the changes.Acetylcholine?ACh?,a neurotransmitter of vagal nerve,can induce vasorelaxation through the accentuation of the nitric oxide?NO?release in endothelium.Vascular tone results showed that ACh-induced vasorelaxation was significantly attenuated in aortic rings relaxation effect of HHcy rats compared to the control group,and physcion significantly reversed the inhibitory effect.The results showed that Hcy could damage the endothelial cells of blood vessels and attenuate ACh-induced vasorelaxation,while physcion could protect the vascular endothelium from the injury of Hcy.2.Protective Effect of physcion on Hcy-induced damage of HUVECsThe results of MTT assay shown that the survival rate of HUVECs was decreased by Hcy in a dose-dependent manner while pre-treatment with physcion could prevente the inhibition of the survival rate nduced by Hcy.To examine the effect of Phys on Hcy-induced apoptosis,flow cytometry analysis using Annexin V-FITC and PI double staining was used.The result showed that Hcy markedly increased HUVECs apoptosis while pre-treatment with physcion could inhibit Hcy-Induced increase in HUVECs apoptosis in a dose-dependent manner.3.The Molecular mechanism of protective effect of physcion on Hcy-induced HUVECs damageTo investigate whether increased oxidative stress is associated with Hcy-induced apoptosis in HUVECs,flow cytometry analysis using DCFH-DA staining was performed.The result showed that treatment of HUVECS with Hcy markedly caused ROS generation.Hcy induced increase in ROS production was considerably reduced under treatment with physcion in a dose dependent manner.Hcy decreased MMP in HUVECs,while pre-treatment with Phys markedly inhibited the Hcy-induced MMP loss.The levels of calcium,NO,the expression of p-Akt/Akt and p-eNOS/eNOS in HUVECs were significantly lower in the Hcy group than that of the control group.Pre-treatment with Phys restored the changes.In the presence of 2-APB,a novel inhibitor of store-operated Ca²⁺entery?SOCE?,the effect of Phys on the Hcy-induced decrease in the levels of NO in HUVECs was significantly inhibited.Meanwhile,the effect of Phys to the level of NO was inhibited by PI3K/Akt inhibitor WT.Phys could inhibit Hcy-induced decrease in the levels of NO in HUVECs through the PI3K/Akt andCa²⁺-eNOS-NO signaling pathway.Hcy caused a signifcant reduction in the Bcl-2/Bax ratio in HUVECs,pre-treatment with Phys dose dependently attenuated Hcy-induced decrease in the Bcl-2/Bax ratio.The study also detected the protein expression of cleaved caspase-9,procaspase-9,cleaved caspase-3 and procaspase-3,which were the hallmark of apoptotic execution enzymes.Hcy increased the cleaved caspase-9/procaspase-9 ratio and cleaved caspase-3/procaspase-3 ratio in HUVECs,but pre-incubation with Phys inhibited the changes.It was indicated that Phys exerted its protective effect on Hcy-induced HUVECs damage through anti-apoptotic signaling pathway.Conclusions:Phys could inhibits Hcy-induced decrease in the level of NO in HUVECs through PI3K/Akt and Ca²⁺-eNOS-NO signal pathways,and reduces Hcy-induced increase in the level of oxidative stress and apoptosis,thus exerting its protective effect on Hcy-induced vascular injury.