Preliminary Study On The Role And Mechanism Of S-equol In Preventing Phenotypic Switching Of Vascular Smooth Muscle

Backgroud Ischemic stroke?IS?is a major threat to human health worldwide].Cerebral artery stenting?CAS?has become an effective option for treating IS.More and more pepole have have been treated with CAS.However,the benefits of CAS treatment were diminished by ISR.Although drug eluting stent?DES?and drug coated balloon?DCB?have reduced the incidence of ISR after stent implantation,there are still problems such as delayed vascular endothelialization and chronic ISR.The reality of using bare metal stent?BMS?in CAS may make the incidence of ISR in CAS higher than that in PCI.The pathological process of ISR was that vascular smooth muscle cells?VSMCs?were stimulated by factors to transform from a contractile phenot ype in a physiological state to a secretory phenotype,then proliferate and migrate to the intima after the dysfunction of endothelial cells?ECs?or the destruction of the integrity of the intima.Phenotypic switching of VSMCs is a key factor of ISR.The phenotypic switching of VSMCs also plays an important role in various pathological processes such as the formation of atherosclerosis?AS?,affecting the stability of AS plaques and stenosis caused by vasculitis.Our previous study has found that there was a sexual difference in the effect of Rosiglitazone,an PPAR? activator,in the treatment of vascular stenosis in ISR mini-pig.The degree of stenosis reduction in female mini-pigs was better than that in male mini-pigs.Thses results suggested that the effects of estrogen or estrogen receptors may invovled in the effect of Rosiglitazone on vascular stenosis.These results suggest that estrogen and estrogen ralted receptors?EER?may have direct or indirect effects on ISR.Considering the importance of phenotypic transformation of VSMCs in ISR,we hypothesized that estrogen or EER may influence the phenotypic transformation of VSMCs.Therefore,experiments were designed to verify this hypothesis.The protective effect of estrogen on the heart and cerebrovascular has been proven by previous study.The results suggest that the incidence of cardiovascular and cerebrovascular events is lower in women than in men,and lower in premenopausal women than in postmenopausal women .In the use of estrogen replacement therapy?HRT?to treat vascular diseases,it was found that some serious side effects such as feminization of men and carcinogenesis can also occur while obtaining therapeutic effects.These side effects limit the use of estrogen.Phytoestrogens are a class of polyphenolic compounds that exist widely and obtained easily from food.It has a structure like estrogen,exerts estrogen-like effects,has fewer side effects and higher safety .Phytoestrogens are great candidates for estrogen replacement.Binding of phytoestrogens to estrogen receptors is weaker than estrogen.Phytoestrogens can specifically bind to a certain estrogen receptor or subtype.Phytoestrogens are known to include daidzein,soy isoflavones,equol,genistein,resveratrol and other polyphenols derived from different plants.The results have shown that the incidence of cardiovascular and cerebrovascular events in people who consume more phytoestrogen-rich foods is lower than in those who consume less phytoestrogen-rich foods.Beans are the main source of phytoestrogens in the body at present.Therefore,we take legume-derived estrogen as the research object that affects the phenotypic transformation of VSMCs.Daidzein and soy isoflavones are the main phytoestrogens in legumes.They also need to be metabolized into equol by the intestinal flora,and then equol was absorbed into the body to play a biologically role.Therefore,the bioactive effects of daidzein and soy isoflavones in the body are mainly equol.The equol has two conformations,namely the left-handed structure?S-equol?and the right-handed structure?R-equol?.All the equol produced in vivo by metabolism was S-equol .S-equol affected vasomotor function,regulated blood pressure,affected the opening of calcium or potassium ion channels,reduced AS plaque formation and regulated angiogenesis .However,the effect and mechanism of S-equol on the proliferation,migration and phenotypic transformation of VSMCs are still uncle ar,and more research is needed.There were two main types of estrogen receptors in VSMCs: estrogen receptor?ER?and G protein-coupled estrogen receptor?GPER?.ER has two sub-types: ER? and ER?.S-equol was considered to be a specific ligand of ER? because its binding rate to ER? was much higher than that to ER?.It is unclear whether the binding of S-equol and ER? affects the phenotypic transformation of VSMCs.GPER,as known as GPR30,was previously thought to be an orphan receptor but was later found to specifically bind to estrogen.NO production was suppressed after S-equol combined with GPER.Other results shown that activating GPER receptors has a protective effect on cardiovascular disease and regulated the cell proliferation of breast cancer and ovarian cancer .The role of GPER in phenotypic switching of VSMCs is unclear,and further research is needed.The purpose of this study is to demonstrate the effect and mechanism of S-equol on the phenotypic switching of VSMCs.This results of this may provide a new potential therapeutic target for ISR.The main contents of this study include in vitro and in vivo.Obeject In present study,rat aortic VSMCs and carotid arteries of female SD rats with balloon-damaged were used in vitro and in vivo,respectively.The effects of S-equol on VSMCs proliferation,migration,expression of phenotypic markers,phosphorylation of key molecules in related signaling pathways and vascular stenosis after carotid balloon injury in female SD rats were observed.After the ER or GPER was blocked,the effect of S-equol on the above-mentioned effects of VSMCs was also observed.Methods 1.Cell culture Procell Corp.provied rat aortic VSMCs?CP-R076?.VSMCs were cultured in Dulbecco's Modified Eagle's Medium?DMEM,phenol red-free?supplemented contained 10% fetal bovine serum in a CO₂/O₂ incubator at 37 °C.Due to estrogen-like effects,phenol red was free from media.2.Animals All animal handling and experimental procedures were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University?TMMU?,Chongqing,China.The study protocol was carried out in strict accordance with the recommendations stated in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health and was reviewed and approved by the Institutional Animal Care and Use Committee of TMMU.All Sprague Dawley?SD?rats were obtained from the Laboratory Animal Center of TMMU.Animal feed consisted of corn,flour,bran,sesame?instead of cardamom to remove phytoestrogen from food?,fish meal,oil,stone powder,calcium bicarbonate,salt,and multivitamins.Rats were individually housed in same-sex cages in temperature-controlled rooms on a reverse 12-hour day/night cycle?7 am to 7 pm?.Rats were provided with food and water ad libitum.Animals were anesthetized with 5% chloral hydrate,and maximal efforts were made to minimize suffering.3.Tissue processing The carotid arteries were harvested at post-sugury 4 weeks.The part of arteries was immobilized with 4% paraformaldehyde?PFA?for 24 hours.The protein in arteries were extracted with RIPA solution contained PMSF?1:100?and phosphotransferase inhibitor?1:100?.Centrifuge at 12,000 g for 15 minutes to remove impurities from the solution.The protein abundance of the samples was verified by a bicinchoninic acid?BCA?assay.4.VSMCs experiments VSMCs were seeded into 96-well plates and cultured in DMEM supplemented contained 10% FBS in a CO₂/O₂ incubator at 37 °C.VSMCs were treated with PDGF-BB?25 ng/ml??PDGF group?or S-equol?100 ?M??control+equol group?for 24 h or treated with PDGF-BB?25 ng/ml?together with S-equol?100 ?M??PDGF+equol group?for 24 h.For the receptor blocking experiments,all the above groups of cells were co-treated with G-15?10 n M?or ICI 182,780?100 n M?for 24 h.After treatment,VSMCs were harvested and lysed with RIPA solution contained PMSF?1:100?and phosphorylase inhibitors?1:100?.After centrifugation at 12,000 g for 15 min at 4 °C,the supernatants were harvested.The protein concentrations were determined by the BCA assay.All experiments were repeated at least three times.5.Masson trichrome and immunohistochemistry staining Masson trichrome staining was processed with the instructions of the Masson trichrome staining kit.Immunohistochemistry staining?IHC?was was processed the instructions of the IHC kit.The primary antibodies,namely,anti-?SMA?1:500?and anti-OPN?1:200?were purchased from CST Corp and Abcam Corp,respectively.Images of tissue stained with Masson trichrome and IHC were shoted by an image shooting system and analyzed with 3DHISTECH Case viewer 6.0.All experiments were repeated at least three times.6.Transwell assay Seeded VSMCs into transwell chambers,and then 500 ?L DMEM was added to the lower chambers in a 24-well plate.VSMCs were treated with PDGF-BB?25 ng/ml?for 24 h and further treated with different concentrations of S-equol?0 M,10 n M,100 n M,10 ?M,and 100 ?M?for 24 h.Then,the transwell member was fixed with 4% PFA for 30 min and washed with phosphate-buffered saline?PBS?.VSMCs on the upper of the chamber were removed,and VSMCs on the lower were keeped.Cell morphology was shown by HE staining and shoted by a image shooting system.The numbers of VS MCs were counted with Image J software.The results were statistically analyzed with Graph Pad Prism 8.0 software.All experiments were repeated at least three times.7.Cell Counting Kit-8 assay?CCK-8?CCK-8 assays were processed with the instruction of the CCK-8 kit.VSMCs were seeded into 96-well plates and treated with PDGF-BB?25 ng/ml?or cotreated with a series of concentrations of S-equol?0 M,10 n M,100 n M,10 ?M,and 100 ?M?for 24 h.In each well,100 ?L DMEM contained 10 ?L CCK-8 solution were replaced the culture medium and then incubated in a CO₂/O₂ incubator at 37 °C for 1 h.The OD value at 450 nm was tested.The results were statistically analyzed with Graph Pad Prism 6.0 software.All experiments were repeated at least three times.8.Western blot assay The protein sample from VSMCs or the carotid artery of SD rats were applied to 4-20% Bis-Tris-PAGE and immunoblot analysis.The transferred membrane was incubated with an anti-?SMA rabbit primary antibody?1:2000?,anti-OPN rabbit primary antibody?1:2000?,anti-MAPK^p38 rabbit primary antibody?1:2000,8690 S,CST,USA?,anti-p-MAPK^p38 rabbit primary antibody?1:2000,4511 S,CST,USA?,anti-NF-?B^p65 rabbit primary antibody?1:2000?,or anti-p-NF-?B^p65 rabbit primary antibody?1:2000?.Anti-GAPDH rabbit primary antibody?1:2000?was used as the loading control.HRP-labeled goat anti-rabbit Ig G was used as the secondary antibody?1:5000?.The results were visualized with Chemi Doc Touch Imaging System and analyzed using Image Lab 6.0 software.All experiments were repeated at least three times.9.Enzyme-linked immunosorbent assay?ELISA?The experimen was processed with the instruction of ELISA kit.A blank control well was setted on the pre-coated plate.Two holes are set at each standard point.The standard sample was added into each standard point hole at 50 ?L / well.The plasma samples were added into the hole?three replicates in each group?at 50 ?L / well,repectively.HRP enzyme-labeled antigen was add into each well at 50 ?L,excepted the blank control wells.The plate was incubated at 37 ° C for 1 h in dark.The plate was turned over,and then the liquid in the plate was shaked off.Washing solution?350 ?L / well?add into each hole.The liquid in the plate was shaked off again after 10 min.The operation was repeat 3 times.Substrate solutions?100 ?L?was added into the hole.The plate was incubated at 37 ° C for 12 min in dark.The incubation was terminated when the visible differences appeared.The OD valuce at 450 nm were recored by a microplate reader.Results 1.S-equol prevented the phenotypic switching of VSMCs in vitro 1.1 S-equol inhibited the proliferation and migration of VSMCs at high doses The results of CCK-8 show that a dose-response relationship exists in the effect of S-equol on inhibiting the proliferation of VSMCs.When the S-equol concentration was 100 ?M,the effect of inhibition of VSMCs proliferation was the strongest.The increase of S-equol dose made the migration ability of VSMCs weaker.When the S-equol concentration was 100 ?M,the migration ability of VSMCs was significantly inhibited.1.2 S-equol prevented the expression of phenotypic marker Compared with the control group,when the dose of S-equol was 100 ?M,the expression of ?-SMA and OPN of VSMCs which detected by western blot asssy was not significant.When the dose of S-equol was low?1 ?M,10 ?M?,the expression of ?-SMA or OPN was significantly different from that of the control group.2.S-equol prevented the phenotypic switching of VSMCs in vivo 2.1 S-equol reduceed stenosis at the injury site of the common carotid artery in female SD rats Compared with the control group,the stenosis of the common carotid artery in operation group was obious at 4 weeks post-surgery.However,after S-equol treatment,the vascular stenosis at the injury site was significantly reduced,which was not signif icantly different from that of the control group.2.2 S-equol prevented the phenotypic switching of VSMCs in ISR rats Compared with normal female SD rats?control group?,the expression of ?-SMA in the common carotid artery of the rats after surgery was decreased,while the expression of OPN was increased in the detection of IHC and western blot.However,the treatment of S-equol reversed this trend.3.The mechanism of the effect of S-equol on VSMCs both in vitro and in vivo 3.1 GPER-MAPK^p38-NF-?B^p65 signaling pathway was invovled the effect of S-equol on VSMCs in vitro Compared with the control group,the expression of ?-SMA decreased and of OPN increased,and the phosphorylation levels of MAPK^p38 and NF-?B^p65 increased after VSMCs induced by PDGF-BB for 24 hours.S-equol?100 ?M?eliminates the effect of PDGF-BB on VSMCs.Blocked the ER did not eliminate the effect of S-equol on VSMCs,but blocked GPER receptor did.3.2 GPER-MAPK^p38-NF-?B^p65 signaling pathway was invovled the effect of S-equol on VSMCs in vivo Compared with the control group,the expression of ?-SMA was decreased and of OPN was increased in VSMCs at common carotid artery injury site in SD rats.The treatment of S-equol could reversed this result.Bloced ER with ICI 182,780,the effect of S-equol on VSMCs was not eliminated.However.blocked GPER couled eliminated the effect of S-equol on VSMCs.MAPK^p38 and NF-?B^p65 phosphorylation were higher in VSMCs of common carotid artery injured lesions in the operation group than in the control group.The treatement of S-equol reduced the phosphorylation levels of MAPK^p38 and NF-?B^p65.Blocked the ER did not eliminate these effects of S-equol on VSMCs,but blocked GPER receptor did.4.S-equol affects BP of female SD rats S-equol reduced the BP of ISR rat model.Conclusions Our results demonstrated that S-equol can prevent the phenotype of VSMCs switch from Contractile phenotype to Secreted phenotype.It was also confirmed that the mechanism of S-equolaffecting phenotype switching of VSMCs may be related to the inhibition of phosphorylation of MAPK^p38-NFkB^p65 signaling pathway after S-equol bind to GPER.