Role Of Nur77 In Metformin Inhibition Of Vascular Smooth Muscle Cell Proliferation And Migration And Vascular Remodeling And Its Mechanism

Objective:1.To study the effects of metformin on the function of vascular smooth muscle cells.2.Identification of the link between Nur77 and metformin inhibiting VSMCs proliferation and migration and vascular remodeling.3.To explore the signal pathways involved,and provide theoretical basis for the application of metformin in the treatment of cardiovascular diseases.Methods:1.In vitro experiments,rat VSMCs were treated with metformin at different concentrations.CCK-8 and VSMCs scratch experiments were used to observe the migration and proliferation of VSMCs after metformin was added for different times.Western blot(WB)was used.)And Quantitative Real-time PCR(qRT-PCR)were used to detect the relationship between the expression of Nur77,TET2,Calponin and other related genes and the concentration and time of metformin.VSMCs that silence TET2 and Nur77 were constructed,and qRT-PCR and WB were used to detect the expression of Nur77 and MMP-9.2.In vivo experiments,healthy SD rats were randomly divided into 3 groups,including the control group(Contorl,Con),the injury group(Intimal injury,Ⅱ),and the injury+metformin group(Intimal injury+Metformin,ⅡM).The arterial dilation balloon injured the common carotid arteries,and then metformin was administered to the ⅡM group at 300 mg/kg/d,while the group Con and the group Ⅱ were administered only to the stomach without adding metformin.After 21 days,the blood vessels in the injured segment of the common carotid artery were taken from each group(the control group was taken from the common carotid artery at the same site as the injured segment),and hematoxylin-eosin staining(HE)was used to observe under the microscope Vessel morphology,the thickness and area of neointimal-medium and the ratio of neointimal area to median area were calculated to comprehensively evaluate the degree of injury,and immunohistochemical staining was used to detect Nur77,PCNA,Calponin and a-SMA expression.Results:1.In cell experiments,data from the CCK-8 experiment and the VSMCs scratch test indicate that metformin can significantly inhibit the proliferation and migration of VSMCs in a concentration-and time-dependent manner.The results of WB and qRT-PCR showed that metformin can promote the expression of TET2,Nur77 and Calponin.After the VSMCs of TET2 were silenced,the expression of Nur77 also decreased,and the expression of MMP-9 increased significantly when VSMCs of Nur77 were silenced.From these data,metformin can increase the expression of TET2 and Nur77 and regulate the phenotype transition of VSMCs.Thereby inhibiting the proliferation and migration of VSMCs.2.In vivo experiments,healthy SD rats were randomly divided into 3 groups,including the control group(Con),the Intimal injury group(Ⅱ),and the intimal injury+ metformin group(ⅡM).The arterial dilation balloon injured the common carotid arteries,and then metformin was administered to the ⅡM group at 300 mg/kg/d,while the group Con and the group Ⅱ were administered only to the stomach without adding metformin.After 21 days,the blood vessels in the injured segment of the common carotid artery were taken from each group(the control group was taken from the common carotid artery at the same site as the injured segment),and hematoxylin-eosin staining(HE)was used to observe under the microscope Vessel morphology,the thickness and area of neointimal-medium and the ratio of neointimal area to median area were calculated to comprehensively evaluate the degree of injury,and immunohistochemical staining was used to detect Nur77,PCNA,Calponin and a-SMA expression.Conclusion:1.Metformin can regulate the expression of TET2,Nur77,MMP-9,Calponin,a-SMA and PCNA,thereby regulating the phenotype transformation of VSMCs,regulating the proliferation,migration and vascular remodeling of VSMCs.2.TET2 and Nur77 play an important role in the occurrence and development of vascular remodeling.It is speculated that metformin can inhibit the proliferation and migration of VSMCs and vascular remodeling through the metformin-TET2-Nur77 signaling pathway.