Study On The Effect And Mechanism Of Moxibustion On Vascular Endothelial Function In ApoE^-/- Mice Based On P2Y2 Receptor Regulation Of SIRT1/FOXO3a Signaling Pathwa

Objective:To observe the effect of moxibustion on lipid metabolism levels,pathological morphology,ultrastructural,inflammatory factors,oxidative stress,and endothelial function in Apo E^-/-mice,and to explore the possible mechanisms of moxibustion against atherosclerosis from the P2Y2 receptor-mediated SIRT1/FOXO3a pathway as a starting point.Methods:For the first two parts of the experiment,8-week male Apo E^-/-mice were randomly divided into the model group,simvastatin group,and moxibustion group,with 10 mice in each group,all of them fed with a high-fat diet.C57BL/6J mice were used as the control group and fed with normal diet.The mice in the control group and the model group were grasped for fixation,the simvastatin group were given by gavage（2.5 mg/kg）,and the moxibustion group was treated with moxibustion at Danzhong（CV17）,Shenque（CV8）,Neiguan（PC6,bilateral）,and Xuehai（SP10,bilateral）for 30 minutes,and each group received treatment once daily for12 weeks,five times a week.The general condition and body mass of mice in each group were observed.Serum TG,TC,LDL-C,and HDL-C were detected by automatic biochemical analyzer,serum ox-LDL,NO,ET-1,MDA,ICAM-1,VCAM-1,TNF-αconcentrations,and SOD activity were detected by ELISA,ROS levels were detected by flow cytometry,pathological structural changes of the aorta were observed by HE staining and transmission electron microscopy,and Endomucin expression expression in the aortic was observed by immunofluorescence.For the third part of the experiment,Apo E^-/-mice were divided into five groups:model group,moxibustion group,suramin group,moxibustion+EX527 group,and suramin+EX527 group.Mice in the suramin group were received an intraperitoneal injection of suramin,an inhibitor of the P2Y2 receptor,at a concentration of 5 mg/kg.Those in the moxibustion+EX527 group were received an intraperitoneal injection of EX527,a selective inhibitor of SIRT1,at a concentration of 10 mg/kg 30 minutes before moxibustion.The suramin+EX527 group received both suramin and EX527 injections.Each group received treatment once daily for 12 weeks,five times a week.The other operations were consistent with the first two parts.ELISA was used to detect inflammatory factors,oxidative stress indicators,and SIRT1 deacetylase activity.PCR and Western blotting were used to detect m RNA and protein expression of aortic P2Y2 receptors,SIRT1,and FOXO3a,and CO-immunoprecipitation was used to detect the expression of acetylated FOXO3a protein.Results:1.Body mass:During the 12-week intervention,the body mass of all groups significantly increased（P<0.01）.At weeks 8 and 12,compared with the control group,body mass was significantly higher in the model group（P<0.05）.Compared with the model group,the body mass was significantly reduced in the simvastatin group and moxibustion group（P<0.01）.2.Lipid metabolism:Compared with the control group,the contents of serum TG,TC,LDL-C,and ox-LDL levels were significantly higher（P<0.01）,and HDL-C levels were significantly lower in the model group（P<0.05）.Compared with the model group,the TG,TC,LDL-C,and ox-LDL levels were significantly lower in the simvastatin group and moxibustion group（P<0.01）,and HDL-C levels were significantly higher in the simvastatin group（P<0.05）.3.Pathological observation of thoracic aorta:In the model group,the HE staining results showed that the aortic intima was thickened,the mesothelial layer was necrotic,the elastic membrane was thin or even broken,and the endothelial cells were degenerated,swollen or even detached.The histomorphology of the simvastatin group and moxibustion group were more intact,with thicker vessel walls,more regular lumen,a small amount of endothelial cells degeneration and swelling,and no obvious pathological changes.The results of transmission electron microscopy showed that the aortic vascular endothelial cells in the model group had suffered from necrosis,cell membrane rupture,loss of cytoplasmic contents,incomplete cell structure,widened perinuclear gaps,and swelling of most mitochondria.In the simvastatin group and moxibustion group,the morphological structure of aortic endothelial cells was more intact,with only some mitochondria showing mild swelling and a few cristae being broken.The rough endoplasmic reticulum was not abnormal in these groups.4.Aortic Endomucin fluorescence intensity:Compared with the control group,the fluorescence intensity was significantly lower in the model group（P<0.01）.Compared with the model group,the fluorescence intensity was significantly higher in the simvastatin group and moxibustion group（P<0.01,P<0.05）.5.Endothelial function,inflammatory factors,oxidative stress indicators:Compared with the model group,the NO content and SOD activity were significantly lower（P<0.01）,ET-1,ROS,MDA,TNF-α,ICAM-1,VCAM-1 levels were significantly higher in the model group（P<0.01）.Compared with the model group,the NO content and SOD activity were significantly higher（P<0.01）,and the ET-1,MDA,TNF-α,ICAM-1,VCAM-1,and ROS levels were significantly decreased in the simvastatin group and moxibustion group（P<0.01）,the MDA,TNF-α,ICAM-1,VCAM-1,and ROS levels were significantly lower（P<0.01）,and SOD activity was significantly higher in the Suramin group（P<0.01）.Compared with the moxibustion group,the MDA,TNF-α,ICAM-1,VCAM-1,and ROS levels were significantly higher（P<0.05,P<0.01）,and the SOD activity was significantly lower in the moxibustion+EX527 group and Suramin+EX527 group（P<0.01）.Compared with the Suramin group,the MDA,TNF-α,ICAM-1,VCAM-1,and ROS levels were significantly higher（P<0.05,P<0.01）,and the SOD activity was significantly lower in the moxibustion+EX527 and Suramin+EX527 group（P<0.05,P<0.01）.6.P2Y2 receptors expression level:Compared with the control group,the m RNA and protein expression of P2Y2 receptors were significantly higher in the model group（P<0.01）.Compared with the model group,P2Y2 receptors m RNA and protein expression were significantly lower in the moxibustion group,suramin group,moxibustion+EX527 group,and suramin+EX527 group（P<0.01）.7.SIRT1 and FOXO3a expression levels:Compared with the control group,the m RNA and protein expression of SIRT1 were significantly lower in the model group（P<0.01）.Compared with the model group,the m RNA and protein expression of SIRT1 were significantly higher in the moxibustion group and suramin group（P<0.05,P<0.01）.Compared with the moxibustion group and suramin group,the m RNA and protein expression of SIRT1were significantly lower in the moxibustion+EX527 group and suramin+EX527 group（P<0.05 or P<0.01）.There were no significant differences between groups in m RNA and protein expression of FOXO3a（P>0.05）.Compared with the control group,the aortic SIRT1 activity was significantly reduced（P<0.01）,and the acetylated FOXO3a protein expression was significantly increased in the model group（P<0.01）.Compared with the model group,the SIRT1 activity was significantly increased（P<0.05,P<0.01）,and the acetylated FOXO3a protein expression level was significantly reduced in the moxibustion group and suramin group（P<0.01）.Compared with the moxibustion group and suramin group,the SIRT1 activity was significantly lower（P<0.05or P<0.01）,and the acetylated FOXO3a protein expression level was significantly higher in the moxibustion+EX527 group and suramin+EX527 group（P<0.01）.Conclusion:1.Moxibustion reduces body mass in Apo E^-/-mice,regulated lipid metabolism,and improved pathological structura in the aorta.2.Moxibustion can decrease TNF-α,ICAM-1,VCAM-1,ET-1,and MDA levels,while increasing NO levels and SOD activity,inhibiting inflammatory responses,improving oxidative stress,reducing endothelial damage,and protecting aortic endothelial function,thus delaying the development of atherosclerosis.3.Moxibustion down-regulated the m RNA and protein expression of the P2Y2 receptor,up-regulated the m RNA,protein expression levels,and deacetylation activity of SIRT1,and decrease acetylated FOXO3a protein expression.4.Moxibustion may prevent atherosclerosis by inhibiting the expression of the P2Y2receptor,which in turn regulates the SIRT1/FOXO3a pathway,inhibits the inflammatory response,improves oxidative stress,and reduces endothelial damage.