Effects And Mechanisms Of Capsaicin On The Senescence And Calcification Of Vascular Smooth Muscle In Diabetes Mellitus

Background and objectives:Vascular calcification(VC),a characteristic change in diabetic vasculopathy,is characterized by the senescence and osteogenic differentiation of vascular smooth muscle cells(VSMCs).In our previous dietary survey of clinical type 2 diabetic patients,we found that patients with a preference for spicy foods had lower pulse wave velocity and pulse pressure compared to non-spicy patients,suggesting that a spicy diet can help improve vascular elasticity and reduce vascular stiffness.Capsaicin,the main pungent and active component of chili peppers,acts by activating Transient receptor potential vanilloid 1(TRPV1).Recent studies have found that longevity proteins Sirtuins(SIRTs)not only regulated cellular senescence but also inhibited vascular calcification.Our previous study found that capsaicin inhibited high glucose-induced vascular endothelial cell senescence through activation of TRPV1 and upregulation of SIRT1 levels.To explore the vascular protective mechanism of a spicy diet in diabetic patients,and investigate whether TRPV1 can be a promising target for preventing vascular calcification,we observed the effects of capsaicin on senescence and calcification of VSMCs in cultured VSMCs and type 2diabetic rat models and used lentiviral transfection of TRPV1 sh RNA to further determine the role of TRPV1,as well as clarify whether its mechanism is related to the regulation of SIRTs levels.Methods:1.Cell level: SD rat thoracic aorta was isolated,and primary VSMCs were cultured by VSMCs were cultured by tissue explants adherent method.Smooth muscle cell-specific marker α-SMA was detected by cell immunofluorescence,and Passages 3-6 of cells were taken for subsequent experiments.(1)VSMCs were treated with high glucose and high phosphorus(30 m M glucose,10 m M sodium β-phosphoglycerate)for 14 days and given different concentrations of capsaicin(1 μM,3 μM,10 μM)to observe its effects on VSMCs senescence and calcification induced by high glucose and high phosphorus.VSMCs senescence-related assays: Western blotting detected the expression levels of senescence-related proteins P21 and P16,β-galactosidase staining detected the number of senescent cells,fluorescence microscopy observed intracellular ROS levels,Elisa detected the levels of senescence-related secretory phenotypes IL-6 and IL-1β in cell supernatants;VSMCs calcification-related assays: Western blotting to detect the expression levels of contractile marker protein α-SMA,osteogenic transcription factor RUNX2 and osteogenic marker proteins OPN,BGLAP,and COLⅠ,and alkaline phosphatase staining and alizarin red S staining to detect intracellular osteogenic differentiation and calcification;Western blotting to detect the expression levels of SIRT1 and SIRT6.(2)To investigate the effect of TRPV1-sh RNA on the senescence and calcification of VSMCs.TRPV1 was silenced by lentiviral transfection of sh RNA,expression of SIRT1 and SIRT6 and expression of osteogenic differentiation-related proteins(RUNX2,OPN,and BGLAP)were observed by Western blotting,and the number of decaying cells was detected by β-galactosidase staining.2.Overall animal level: SD rats were divided into three groups: normal group(Con),diabetic group(DM),and diabetic + dietary capsaicin group(DM+Cap).High-sugar,high-fat diet-fed male SD rats combined with an intraperitoneal injection of streptozotocin(STZ)was used to construct a type 2 diabetic rat model,and the DM+Cap group was given a high-sugar,high-fat diet supplemented with 0.01%capsaicin for 10 weeks.Thoracic aortic tissue and serum were extracted from rats for subsequent experiments.(1)Effect of dietary capsaicin on diabetes-induced vascular smooth muscle aging:HE staining to observe the vascular wall structure of thoracic aorta in each group,immunohistochemistry,and Western blotting to detect the expression levels of P21 and P16 in vascular smooth muscle,Elisa to detect the levels of insulin and aging-related secretory phenotypes IL-6,MMP-9 and TNF-α in serum,and serum to detect the four lipids(cholesterol,triglyceride,low-density lipoprotein,and high-density lipoprotein).(2)Effect of dietary capsaicin on diabetic-induced vascular smooth muscle calcification: Western blotting detected the levels of osteogenic differentiation-related proteins(RUNX2,OPN,BGLAP,and COLⅠ)and longevity proteins(SIRT1 and SIRT6)in three groups of thoracic aortic tissues.Results:1.Capsaicin significantly inhibited the senescence of VSMCs induced by high glucose and phosphorus,as shown by reducing the number of VSMCs senescence induced by high glucose and phosphorus,down-regulating the expression of senescence-related proteins P21 and P16,reducing ROS production induced by high glucose and phosphorus,and inhibiting the secretion of IL-6 and IL-1β.2.Capsaicin significantly inhibited osteogenic differentiation and calcification induced by high glucose and phosphorus: Western blotting results showed that capsaicin dose-dependently restored the level of contractile marker protein α-SMA in VSMCs and inhibited the expression of osteogenic-related marker proteins RUNX2,OPN,BGLAP,and COLⅠ;Alkaline phosphatase staining and alizarin red S staining showed High glucose and high phosphorus promoted VSMCs calcification,and capsaicin significantly reduced intracellular alkaline phosphatase activity and calcium salt deposition.3.High glucose and phosphorus reduced the level of SIRT6 in VSMCs,while capsaicin restored the protein expression of SIRT6.4.TRPV1-sh RNA significantly downregulated TRPV1 protein levels in VSMCs,as well as the protein expression levels of SIRT1 and SIRT6.Compared with the negative control(Ctrl-sh RNA),TRPV1-sh RNA promoted osteogenic differentiation and senescence in VSMCs,as shown by upregulation of the levels of osteogenic marker molecules RUNX2,OPN,and BGLAP and the increased number ofβ-galactosidase staining-positive senescent cells.5.Dietary addition of 0.01% capsaicin did not significantly affect fasting glucose and lipid levels in diabetic rats,but inhibited diabetes-induced vascular aging and calcification,as evidenced by the reduced levels of vascular wall senescence-associated proteins P21 and P16,decreased IL-6 secretion,and the reduced expression of the osteogenic transcription factor RUNX2 and osteogenic marker proteins OPN,BGLAP,and COLⅠ.In addition,the level of SIRT6 in the vascular wall of diabetic rats was significantly decreased,and dietary capsaicin could partially restore the level of SIRT6.Conclusion:1.The TRPV1 agonist capsaicin upregulates the level of longevity protein SIRT6 and inhibits high glucose and high phosphorus-induced senescence and calcification in VSMCs,whereas TRPV1 sh RNA downregulates the level of SIRTs and promotes the senescence and calcification in VSMCs.2.Dietary capsaicin upregulates the level of SIRT6 protein and inhibits vascular aging and calcification in diabetic rats.