| Objective:To observe the effects of vitamin D and pin1 inhibitor Juglone on streptozotocin（STZ）-induced aortic endothelial senescence and expression of pin1,SIRT1 and P66shc in diabetic SD rats through animal experiments;To observated the effect of vit D and Juglone on high glucose-induced umbilical vein endothelial（HUVECs）aging and the possible mechanism of vit D/pin1 axis imbalance to regulate diabetic endothelial aging in vitro.Methods:Animal experiment:the SD rats were established to intraperitoneal injection of STZ.Then divided into:SD rats group,DM group（high fat-STZ group）,VitD group（high fat-STZ-VitD irrigation）,juglone group（high-fat-STZ-jugoquinone intraperitoneal injection group）,Combination medicine group（high-fat-STZ-VitD intragastric+juglone intraperitoneal injection group）;each group were 10 animals.Oxidase method was used to determine fasting blood glucose level;H-E staining was used to observe the structure of aorta,vascular ring was used to detect the vasodilation function of thoracic aorta,and WB was used to detect the protein related to cell aging.Cell experiment:in vitro culture of umbilical vein endothelial cells with collagenase digestion method,morphology,immunocytochemistry method for HUVECs cell identification;use 33mM glucose solution to establish high glucose induced aging model,divided into Control group,high glucose group,high glucose-VitD Group,high-glucose-jugone group;high-glucose-VitD+juglone group;β-galactosidase staining to identify senescent cells;flow cytometry to detect cell cycle;immunoblotting（Western blot）and PCR to detect senescence-related proteins;immunity Fluorescence detection of living cell ROS,JC-1 detection of cell mitochondrial membrane potentialResultes:Animal experiment:1)Compared with the Ctr group,the blood glucose of the DM group was significantly increased.the blood glucose of the VitD group,the juglone group and the combined drug group were significantly reduced comparing with the DM group,,and the VitD group,the juglone group and the combined drug group with No significant difference in blood glucose2)The intima structure of the thoracic aorta in the Ctr group was complete and smooth,the thickness of the intima was uniform,and the arrangement of vascular smooth cells was regular;the aorta intima in the DM group was uneven and the thickness of the intima was uneven.Partially defective;in the VitD group,the juglone group,and the combined medication group have a complete intima structure,a uniform medial structure,and a regular vascular smooth cell structure3)Compared with the Ctr group,the thoracic aorta in the DM group has significantly reduced ACH endothelium-dependent and SNP-independent endothelium-dependent diastolic function.When the ACH concentration is 10-8M,the SNP10-7M diastolic function in the two groups,the endothelium-dependent and endothelium-dependent relaxation function of thoracic aorta in SD rats in VitD group,juglone group and combination medication group were significantly improved compared to DM group.4)Diabetes can increase the expression level of Pin1 protein,mitochondrial oxidative regulation protein P66Shc and senescence-related marker molecule P21protein in thoracic aorta of SD rats,reducing the expression level of mitochondrial oxidative regulation protein SIRT1;compared with Ctr group,thoracic aorta in DM group Arterial P66Shc protein,senescence-related marker molecule P21 protein expression level was significantly increased,SIRT1 protein was significantly reduced;calcitriol and juglone could inhibit the expression of P66Shc,SIRT1 and P21 protein in thoracic aorta of diabetic miceCell experiment:1)Compared with the Ctr group,the staining cells in the high glucose group were significantly increased,the VitD group and the juglone group staining cells were higher than the DM group,In contrast,the combined intervention group had fewer stained cells than the VitD group and the juglone group.2)Compared with the ctr group,the HUVECs in the HG group are significantly blocked,in G1 phase;VitD,juglone and combined drug intervention can significantly inhibit this blocking effect3)Compared with the Ctr group,high glucose（33mM）can significantly enhance the production of ROS in the cell,showing that the fluorescence is significantly enhanced,compared with the high glucose group,VitD group(10-6M),juglone group(10-7M)and joint intervention group can inhibit the fluorescence intensity produced by cells under high glucose conditions,while VitD group(10-6M),juglone group(10-7M)No significant difference4)Compared with the HG group,the VitD group(10-6M)and the juglone group（10-7M）can significantly inhibit the high glucose-induced Mitochondrial membrane potential decreased,and the effect of the juglone group(10-7M)group was stronger(p<0.05 vs VitD group(10-6M)5)High glucose（33mM）can induce the expression levels of pin1,mitochondrial oxidative regulatory protein P66Shc and senescence-related molecule P21 in HUVEC cells,and inhibit the expression level of mitochondrial oxidative regulatory protein SIRT1.Compared with Ctr group,high glucose（33mM）the expression levels of Pin1,P66Shc and P21 in HUVECs cells were significantly increased（p<0.05）.The expression level of SIRT1 was significantly reduced;VitD could reduce the expression levels of Pin1,P66Shc and P21 in HUVECs cells induced by high glucose.Compared with the high glucose group,VitD(10-6M)group and juglone group(10-7M)Pin1,P66Shc and P21 expression levels were significantly reduced;there was no significant difference between Pin1 inhibitor group and VitD groupConclusion:1)Both VitD and juglone can inhibit diabetes-induced thoracic aortic injury and improve vascular function;2)VitD and juglone can inhibit Pin1,SIRT1,P66Shc and P21 protein expression levels of thoracic aorta in diabetic rats;3)Both VitD and Juglone can inhibit HUVECs oxidative stress and cell cycle arrest;4)VitD and juglone may delay the aging of endothelial cells by regulating SIRT1/P66Shc imbalance... |
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