Rhein Protects Against Endothelial Function And Atherosclerosis By Targeting SIRT1 Signaling Pathway

ObjectiveTo investigate Rhein protects against endothelial dysfunction and atherosclerosis by targeting SI[RTI signaling pathway and its mechanism.MethodsSixty 6-week-old male ApoE-/-mice were randomly divided into normal diet group(STD),high fat and high cholesterol diet group(HFHC)and high fat and high cholesterol + Rhein group(Rhein),20 in each group.In Rhein group,Rhein was given intragastrically(120 mg?kg,suspended in 1%sodium cellulose solution)once a day for 8 weeks.STD group and HFHC group were given the same volume of 1%cellulose sodium intragastrically.The changes of body weight of mice were dynamically monitored every week.After 8 weeks of intervention,the following expernments were earried out:1)Six mice in each group were randomly selected.The plaque size of brachmocephalic artery at the beginning of aortic arch branch and carotid pulse wave conduction velocity(PWV)in ApoE-/-mice were measured by ultrasound biomicroscopy(UBM)in vivo;2)The serum samples of mice were taken to detect the levels of blood glucose and blood lipid;3)After killed the mice,six mice in each group were randomly selected,the thoracic aorta was separated and the paraventricular adipose tissue was removed.The endothelium-dependent vasodilation function of mouse aorta was evaluated in vitro by vascular tension tester(Wire Myograph);4)Four mice in each group were randomly selected to separate the whole aorta(thoracic aorta and abdominal aorta).Moreover,five mice were randomly selected to prepare firozen sections of cardiac aortic valve.Oil red O staining was used to evaluate the degree of lipid deposition in the whole aortic wall and aortic root of mice;5)The content of ROS in aortic valve was measured by DHE staining;6)The whole aorta of the remaining mice in each group were isolated and removed paraventricular adipose tissue.The mRNA expression levels of inflammatory factors MCP-1,ICAM-1,VCAM,TNF-and SIRT1 in aorta of mice in each group were detected by RT-PCR method;the expression of SIRT1 in aorta was detected by Western blot methodResults1)Comparison of general date of mice in each group:The body weight of ApoE-/-mice fed with high fat and high cholesterol diet was significantly higher than that of normal diet group,but there was no significant difference between Rhein group and HFHC group.After 8 weeks of feeding,there was no significant difference in fasting blood glucose among the three groups,but the serum levels of TC and TG in HFHC group and Rhein group were significantly higher than those in STD group,but there was no significant difference between Rhein group and HFHC group;2)Comparison of endothelium-dependent vasodilation in mice in each group:In STD group,the maximum relaxation amplitude of thoracic aorta induced by acetylcholine(Ach)in ApoE-/-mice was 88.33±11.38%,and the logarithmic value(logEC50)of acetylcholine half-effective dose was-7.62±0.32 mol/L.The maximum relaxation amplitude of thoracic aorta in HFHC group was 72.50±3.78%,which was significantly lower than in STD group,and log EC50 was-6.64±0.43 mol/L,which was significantly higher than in STD group.After 8 weeks of Rhein intervention,the maximum relaxation amplitude of aorta in ApoE-/-mice was significantly increased(89.80±6.07%)and the half-effective dose of acetylcholine was decreased(logEC50:-7.37±0.3 lmol/L,P<0.05).These results suggest that Rhein can improve endothelium-dependent vasodilation dysfimction in ApoE-/-mice induced by high fat;3)Comparison of carotid PWV in mice in each group:Compared with STD mice,PWV in HFHC group increased significantly(3.65×0.43vs5.7?1.19mm/ms),while Rhein intervention significantly decreased carotid PWV(4.11±0.95mm/ms),suggesting that Rhein can reduce vascular stiffness and improve vascular elasticity;4)Comparison of the size and stability of aortic plaques in each group:?The results of ultrasound biomicroscopy showed that there was no plaque formation at the origin of brachiocephalic artery in STD group,but obvious plaque deposition in HFHC group.After 8 weeks of Rhein intervention,the plaque area in brachiocephalic artery was significantly reduced.?The results of oil red O staining showed that the whole aortic wall of STD group was smoother,there was no obvious plaque deposition in oil red staining,and there was a little amount of lipid in aortic root.The plaque deposition in the aortic wall of ApoE-/-mice fed with high fat diet was obvious,the lumen was narrow,and a large amount of lipid accumulated in the aortic valve of the heart.However,Rhein intervention could significantly reduce the size of plaque?plaque deposition and lumen stenosis in the inner wall of aorta;5)Comparison of the expression of inflammatory factors mRNA in aorta of mice in each group:compared with STD group,the mRNA of MCP-1,ICAM-1 and VCAM-1 in HFHC group was significantly increased,and Rhen intervention could significantly inhibit the expression of these inflammatory factors mRNA.It is suggested that Rhein can reduce the level of aortic inflammation;6)Comparison of ROS in aortic valve of mice in each group:compared with STD group,the level of ROS in aorta of HFHC group was significantly increased,while the level of ROS in Rhein group was significantly lower than in HFHC group,suggesting that Rhein can reduce the production of ROS;7)Comparnson the expression of SIRT1 protein and mRNA in aorta of mice in each group:compared with STD group,the expression of SIRT1 and mRNA in aorta of HFHC group decreased significantly,while the expression of SIRT1 and mRNA increased significantly after Rhein intervention.ConclusionsRhein can significantly improve endothelial dysfunction and atherosclerosis in ApoE-/-mice induced by hyperlipidemia.The mechanism may be related to the activation of SIRT1 expression in aorta to reduce oxidative stress and inflammation.