The Role And Mechanism Of High Glucose In Phosphate-induced Senescence And Calcification Of Vascular Smooth Muscle Cells

Objective vascular calcification is one of serious complication in diabetes mellitus and chronic kidney disease(CKD)patients,and is closely related to the occurrence of cardiovascular events.The common pathogenesis of VC are aging,inflammation,oxidative stress,uremia,hyperphosphatemia,hyperglycemia and so on.NAD~+dependent deacetylase Sirtuin 1(SIRT1)is a histone deacetylase that regulates cellular senescence and inflammation,and plays an important role in regulating high glucose and high phosphate-related vascular calcification.However,it is unclear whether hyperglycemia can promote hypercalcemia-induced vascular smooth muscle cells(VSMCs)calcification and and its mechanisms for promoting calcification.The purpose of this study is to investigate the role of high glucose in the calcification of VSMCs induced by high phosphate and the molecular mechanism of SIRT1 involved in its regulationMethods:1.Primary mice VSMCs was cultured from thoracic and abdominal aorta of8-week-old C57BL/6 mice,and were identified according to cell morphology and immunofluorescence staining of smooth muscle cell specific markers smooth muscle22 α(SM22 α)and α smooth muscle actin(α-SMA).2.Mice primary VSMCs was used as a cell model,we investigated whether high glucose and high phosphorus promote VSMCs calcification by affecting cellular senescence.(1)The same batch of VSMCs were treated with normal,high phosphate and / or high glucose medium for 3 and 9 days respectively.The alizarin red S staining and intracellular calcium content quantitative test were used to detect the degree of calcification.Western blot(WB)was used to detect the protein expression of osteoblast phenotypic markers bone morphogenetic protein(BMP2),Runt related transcription factor 2(RUNX2),smooth muscle cell phenotypic molecule(α-SMA),and type III sodium dependent phosphate cotransporter-1(Pit1)in order to observe the effect of high glucose on osteoblast transdifferentiation-related calcification.(2)Senescence-associated-β-galactosidas(SA-β-Gal)staining was used to detect the senescence of cells in each group,and WB was used to detect the expression levels of senescence related marker molecules in cells in each group,such as p21 and SIRT1 proteins,so as to explore the relationship between high glucose and senescence of VSMCs3.In order to verify the role of SIRT1 in senescence and calcification,mice VSMCs was treated with high glucose and high phosphorus for 9 days with or without SIRT1 activator SRT1720 and inhibitor Sirtinol.Alizarin Red S staining,determination of intracellular calcium content was used to detect cell calcification,WB to detect the expression of marker molecules related to osteoblast phenotype,to explore the effect of SIRT1 activators and inhibitors on the degree of cells calcification;SA-β-Gal staining and the protein expression of p21 were performed to investigate the effects of SIRT1 activators and inhibitors on cellular senescence.cellular senescence and calcification is closely related to reactive oxygen species(ROS),and the level of ROS was detected by flow cytometry.4.In order to study whether SIRT1 affects high glucose and phosphorus induced aging and calcification of VSMCs by regulating the NF-κB/p53 signaling pathway,the expression levels of signal molecules SIRT1,p65,p-p65,ace-p65 and p53 were detected by WB after treatment with SRT1720 or Sirtinol,and the expressions of SIRT1 and Ace-p65 were also detected by immunofluorescence.Results1.High glucose promotes phosphate-induced calcium deposition associated with VSMC-osteoblast transition.Mice VSMCs exposed to high phosphate and high glucose in vitro showed increased calcification.Transdifferentiation related marker molecules BMP2 and RUNX2,the sodium and phosphate transporter Pit1 also increased significantly after treatment with high phosphorus and high glucose.And the calcification was the strongest under both high glucose and high phosphorus treatment.2.Pharmacological changes the activity of SIRT1 regulates osteoblastic transdifferentiation and calcium deposition of VSMCs by exposure to high glucose and phosphate overload.(1)The expression of SIRT1 was significantly decreased in VSMCs induced by high phosphate and high glucose,and vascular calcification occurred in VSMCs in the treatment group.(2)Compared with the control group,the degree of calcification was further aggravated by the addition of SIRT1 inhibitor Sirtinol in the high phosphate and high glucose group,but significantly inhibited by the treatment with SIRT1 activator SRT1720.3.High glucose promotes phosphate-induced senescence of VSMCs in a SIRT1-dependent manner;(1)compared with the control group,in the group treated with high concentration of phosphate and glucose,the number of senescent cells was increased by SA-β-Gal staining,and the expression of senescence-related marker p21 and the level of reactive oxygen species(ROS)were significantly increased.And the degree of cellular senescence was most severe in both high phosphate and high glucose group.(2)in addition,compared with the non-inhibitor group,the addition of inhibitor Sirtinol inhibited SIRT1 and further accelerated cellular senescence and ROS level.After treatment with SIRT1 activator SRT1720,SIRT1 was activated,ROS and the degree of cell senescence were significantly decreased compared with the untreated group.4.Activation of NF-κB/p53 signal pathway involves in osteoblastic transition and sencence of VSMCs exposed by high glucose and phosphate overload.(1)After VSMCs was treated with high glucose and phosphorus,WB and immunofluorescence results showed that the expression of Ace-p65,p-p65,p53 protein was significantly increased and NF-κB/p53 signal pathway was activated.(2)When SIRT1 was activated by SRT1720,the activation state of Ace-p65,p-p65,p53 was significantly inhibited.On the contrary,when SIRT1 was inhibited by Sirtinol,Ace-p65,p-p65,p53 was further activated,but p65 had no significant change.The results of immunofluorescence SIRT1 and Ace-p65 were consistent with those of WB.Conclusion1.High glucose promotes phosphate-induced senescence and vascular calcification of VSMCs.2.High glucose can inhibit the expression of deacetylase SIRT1 in vascular smooth muscle cells.3.SIRT1 inhibits vascular smooth muscle cell senescence and osteogenic differentiation by reducing p65 acetylation levels,inhibits the activity of NF-κB,thereby inhibiting vascular calcification.