Study On The Effect Of Simvastatin On Angiogenesis Under Immune Regulation

Background:The physiological processes of tissue repair,wound healing,placental development,and astumor development are all closely linked to angiogenesis,as well as pathologica processes such as tumor development,myocardial infarction and atherosclerosis.Macr ophages play a major role in the process of angiogenesis and vascular remodeling.The pro-angiogenic activity of macrophages depends to a large extent on whether they are ac tivated and their polarized phenotypes.Therefore,it is feasible to improve angiogenesis by regulating macrophage subtypes.Studies have found that simvastatin can promote an giogenesis through PI3 k / Akt and VEGF pathways,and can also regulate macrophage p olarity.However,whether it can promote angiogenesis by regulating macrophage polari ty has not been explored.Therefore,we propose a scientific hypothesis that simvastatin promotes angiogenesis under immune regulation.Objective:1.To study the effect of different concentrations of simvastatin on HUVEC angiogenesis.2.The immune microenvironment was simulated by macrophages to detect the effect of different concentrations of simvastatin in regulating the polarization of M1 macrophages.3.Detect the effect of different concentrations of simvastatin on vascularization under immune regulation.4.To provide experimental basis for our subsequent preparation of simvastatinloaded gelatin-chitosan bilayer guided bone regeneration membrane.Methods:(一)Simvastatin acts on human umbilical vein endothelial cells HUVEC cultured in vitroHUVEC cells were randomly divided into 7 groups(blank control group: DMSO group;Experimental group-Simvastatin group with different concentrations: 0.1u M group;0.5u M group;1u M group;2u M group;5u M group;10u M group)and inoculated into the orifice plate according to the group.The cells were inoculated into the well plate according to the groups,and the proliferation of the cells was detected by CCK8 kit at12 h,24h,48 h and 72 h,and the appropriate concentration was selected for follow-up experiments.Tubule-forming ability was observed by tubule-forming assay,cell migration rate was observed by scratch assay,positive expression intensity of CD31 and VEGF 、 HIF-1α was observed by immunocytochemical staining,and expression of CD31,VEGF and HIF-1α genes at m RNA level was detected by RT-q PCR to evaluate the effect of simvastatin on angiogenesis ability.(二)Simvastatin acts on human umbilical vein endothelial cells HUVEC by mediating macrophage polarizationRAW264.7 cells were divided into control group and experimental group,each group was added with the same volume of DMSO,the experimental group was treated with various concentrations(0.1u M;0.5 u M;1u M;2u M;5u M;10u M)of simvastatin to act on cells.The cells were added to the well plate,and the proliferation of the cells at12 h,24h,48 h and 72 h was detected by CCK8,and the appropriate concentration of simvastatin was selected.After that,RAW264.7 cells were divided into control group,positive control group and experimental group.The same volume of DMSO was added to each group.The positive control group was treated with LPS for 24 hours.The experimental group was treated with different concentrations of simvastatin for 12 hours.Macrophages were observed under light microscopy,pyrecyclic peptide staining,and immunofluorescence staining,and then photographed and semi-quantitatively analyzed to observe the effect of simvastatin on M1 macrophage polarization.RAW264.7 cells were inoculated in the upper compartment of transwell plate.After 12 hours of simvastatin treatment and 24 hours of LPS stimulation,the upper compartment of RAW264.7 cells were co-cultured with HUVEC inoculated in the lower compartment.Angiogenesis assay was used to observe the ability of tube formation,wound healing assay was used to observe the rate of cell migration,immunocytochemical staining was used to observe the positive expression of CD31 and VEGF,RT-q PCR was used to detect the expression of CD31,VEGF and HIF-1αm RNA to evaluate the effect of simvastatin on angiogenesis under immune regulation.Results:(1)CCK8 experiment showed that high concentration of simvastatin inhibited the proliferation of HUVEC cells,while low concentration of simvastatin promoted the proliferation of HUVEC cells.(1)Therefore,simvastatin less than or equal to 2u M was selected for the follow-up experiment.The scratch experiment,tubule formation experiment,cellular immunofluorescence experiment and PCR experiment showed that simvastatin could promote the formation of blood vessels,and the optimal concentration for the formation of blood vessels was 0.1u M.(2)CCK8 assay showed that high concentration of simvastatin had a significant inhibitory effect on cell proliferation in RAW264.7 cells,and low concentration of simvastatin had a slight effect on cell proliferation.The results of light microscopy,podacryptin staining and immunofluorescence staining showed that simvastatin could inhibit macrophage differentiation to M1 type.Besides,Different concentrations of simvastatin acted on RAW264.7 cells and co-cultured with HUVEC in transwell.Scratch test,tubule formation experiment,PCR experiment and cell immunofluorescence experiment showed that simvastatin could promote angiogenesis under immune regulation,and the optimal concentration for angiogenesis was 0.5 u M.