Effects Of Kaempferol-3-O-rutinoside On The Proliferation,Migration And Activation Of TGFBR1 Signaling Pathway In Vascular Smooth Muscle Cells

BackgroundVascular smooth muscle cells(VSMC)are located in the media layer of the artery,which is mainly constituted with VSMC.Studies show that proliferation and migration of VSMC are the most important pathogenesis of atherosclerosis.The proliferation and migration of VSMC promote the formation and development of atherosclerotic plaque.The stability of the fibrous cap is closely related to the proliferation of VSMC.Inhibition of proliferation and migration of VSMC delays atherosclerosis.Transforming growth factor beta receptor 1(TGFBR1)is a specific receptor for transforming growth factor beta(TGF-?)and participates in the physiopathological process of TGF-?.TGF-P is a multi-functional cytokine that regulates cell proliferation and migration,matrix synthesis,calcification and immune response via the TGFBR1 signaling pathway and participates in the development of atherosclerosis.TGFBR1 expression is upregulated in patients with atherosclerosis and some researchers have proposed TGFBR1 as a biomarker for the early diagnosis of atherosclerosis.Kaempferol-3-O-rutinoside(KR)is a component of safflower.Safflower has the effect of promoting blood circulation and removing blood stasis and is generally used for cardiovascular diseases.Some studies have found that safflower and its components have anti-atherosclerotic effects,including safflower yellow,safflower glycosides.Other studies have found that KR has the effect of reducing blood pressure,slowing heart rate,and inhibiting fat synthesis.However,there is no report on the effect and mechanism of KR on atherosclerosis.ObjectiveTo investigate the effects of kaempferol-3-O-rutinoside(KR)on the proliferation,migration of vascular smooth muscle cells(VSMC)and activation of TGFBR1 signaling pathway in the cells.Method1.The culture of A7R5A7R5 was cultured in DMEM medium containing 10%fetal bovine serum,50U/mL penicillin,50U/mL streptomycin and in 37? 5%CO2 incubator.Medium changed once every 2 days,until the cells growed to 60%-70%of the petri dishes.2.The cell viability of A7R5 was detected by MTT assayA7R5 in the logarithmic phase was plated in 96-well plates at 5,000 cells per well.Five replicate wells were set up in each group.After stimulated with different concentrations of KR for 24 hours,20?L of 5mg·mL-1 MTT solution was in addition to each well to incubate for 4 hours.The supernatant was discarded and 200?L of DMSO was added to each well.The optical density was obserbed by a microplate reader at 570nm.3.The cell proliferation of A7R5 was detected by EdU stainingA7R5 in logarithmic phase was plated in 96-well plates at 5,000 cells per well and incubated with different concentrations of KR for 24 hours.The cells were fixed with 50?L 4%paraformaldehyde for 30min after the supernatant was discarded.Cells were stained with 100?L1×Apollo for 30 min.The supernatant was discarded and 100?L of 1×Hoechst 33342 reaction solution was in addition to each well to incubate for 30min,protected from light.Each well was washed with 100?L PBS for 1-3 times.Fluorescence microscope was utilized to select three fields randomly.The proportion of EDU positive cells was calculated.4.Transwell cell migration assayA7R5 in logarithmic phase was placed in the upper chamber of Transwell at 105 cells per well and incubated with different concentrations of KR in the lower chamber for 24 hours.A7R5 in the upper surface of the filter membrane was wiped out with cotton balls.The filter membrane was fixed with 4%paraformaldehyde and stained with crystal violet.A7R5 migrated to the lower surface of the filter membrane was counted under the microscope.5.Molecule dockingThe spatial structure of TGFBR1(PDB ID:1PY5)was available from the PDB protein structure database.The 3D molecular structure of KR(CID:5318767)was obtained from the PubChem Compound database.Autodock VINA software was used to generate molecular docking.Software YASARA was used for energy minimization.Water molecules and ligands from the original space structure were removed before molecular docking.The known binding sites for TGFBR1 inhibitors were used to predict the relationship between TGFBR1 and KR.The molecular docking of TGFBR1 and KR was performed,and the binding capacity was used to evaluate the binding capacity.Pymol 2.7 and Chimera 1.8.1 software were used for 3D drawing.6.Western BlotA7R5 was incubated with different concentrations of KR for 24h.Proteins was extracted by protein lysates containing protease inhibitors and phosphatase inhibitors.After centrifugation,proteins were separated by 10%SDS-PAGE gel electrophoresis and moved to a nitrocellulose membrane.The membranes were blocked with 5%BSA at room temperature for 2h and then incubated overnight with primary antibody at 4?.Incubation with the secondary antibody at room temperature for 1 hour on the next day.The membranes were washed 3 times in TBST(5min for each wash)before exposure.The result was calculated by KODAK Image Station 4000MM exposure imaging system and quantified using Image J software.7.Statistical methodsAll data were expressed as Mean±SD,and differences among groups were analyzed by one-way ANOVA followed by LSD method.P<0.05 was examined statistically significant.All the analyses were carried out using SPSS 19.0.Result1.KR dose-dependently and time-dependently inhibited the proliferation of VSMCA7R5 was incubated with different concentrations of KR(10,20,40?M)for 24h,and MTT assay was utilized to observe the effect of KR on the proliferation of A7R5.The results showed that KR dose-dependently inhibited the viability of A7R5 compared with the Control group(P<0.05).Meanwhile,KR(40?M)was incubated with A7R5 for 24,48,and 72h.The results showed that KR inhibited the increase of A7R5 in a time-dependent manner compared with the Control group(P<0.05).Results of EdU staining showed that KR dose-dependently reduced the ratio of EdU-positive cells compared with the Control group(P<0.05).2.KR dose-dependently inhibited A7R5 migrationA7R5 was incubated with different concentrations of KR(10,20,40?M)for 24h,Transwell migration assay was used to observe the effect of KR on the migration of A7R5.The results showed that KR dose-dependently decreased the number of A7R5 migration compared with the Control group(P<0.05).3.KR inhibited migration-associated protein levelsA7R5 was incubated with different concentrations of KR(10,20,40?M)for 24h.The protein levels of MMP2 and MMP9 related to A7R5 migration was observed by Western Blot.The results showed that KR dose-dependently reduced the ptotein levels of MMP2,and KR to 20?M and 40?M significantly decreased MMP9 protein levels compared with the Control group(P<0.05).4.Molecular docking simulation of KR and TGFBR1Autodock vina molecular docking method was used to explore the relationship between TGFBRl and KR.The binding capacity of TGFBR1 to KR is-9.804 kcal/mol.Surface modeling of TGFBR1 binding to KR showed that KR was located in the TGFBR1 inhibitor binding site.The binding model of KR to TGFBR1 showed that KR formed 1 hydrogen bond with the amino acid residues SER-280,ARG-215,LYS-335 with the length of hydrogen bond 2.7,2.6,2.5 A and 3 hydrogen bonds with ASP-290 with the hydrogen bond length of 2.3,2.4,2.6 ?.5.KR inhibited the activation of TGFBR1 Signaling PathwayA7R5 was incubated with different concentrations of KR(10,20,40?M)for 24h.The activation of TGFBR1 and its downstream proteins Smad2 and Smad3 was detected by Western Blot.The results showed that KR dose-dependently suppressed the activation of TGFBR1,Smad2,and Smad3 compared with the Control group(P<0.05).ConclusionKR inhibited VSMC proliferation and migration,possibly via blocking TGFBR1 signaling pathway.