Formononetin Attenuated Platelet Aggregation And Vascular Intimal Hyperplasia In Rats With Balloon-Induced Arterial Injury

BackgroundPercutaneous transluminal angioplasty(PTA)treatments for cardiovascular diseases and peripheral arterial disease are commonly used in clinical;however,a frequent complication of restenosis after PTA often limits the long-term beneficial effects of PTA.Caulis spatholobi,as a traditional Chinese medicine,has been used in China for thousands of years in the treatment of various human diseases.Formononetin is one of the main active ingredients of caulis spatholobi,formononetin was determined as a qualitative indicator of caulis spatholobi in Chinese pharmacopoeia(2010).Formononetin has been reported to have multiple pharmacological activity including antitumor,scavenging oxygen free radicals,reducing cell lipid peroxides,reducing cholesterol and so on.Therefore,it will be reasonable to hypothesize that formononetin may inhibit platelet aggregation and thrombosis after PTA.In this study,the effects of formononetin were investigated on rats with balloon-induced arterial injury.We further investigated the effects of formononetin against balloon-induced neointima formation in vivo,inhibition effects on proliferation and migration of VSMCs induced by PDGF-BB in vitro,and the underlying mechanisms of the effects.MethodsPart 1 UPLC-MS/MS assay for simultaneous determination of four compounds in rat plasma:application to pharmacokinetic study after oral administration of Caulis Spatholobi extractsThe water decoction method commonly used clinically is adopted,with dehydration and concentration to prepare freeze-dried suberect spatholobus stem extract.Decoct caulis spatholobi for 3 hours,dehydrate the filtrate to make the freeze-dried CSE.Preparation of standard and quality control sample.Mixing mother solution was concentrated with protocatechuic acid,catechuic acid,gallocatechin of 0.5 mg/ml,and formononetin of 0.8 mg/ml in methanol(1:1:1:1,v/v/v/v).The internal standard(Carnation)mother solution was 0.5 mg/ml in methanol.Preparation of samples.Plasma samples were prepared by liquid extraction with ethyl acetate,and genkwanin was selected as internal standard.UPLC-MS/MS was used to establish the method for the determination of protocatechuic acid,catechuic acid,gallocatechin,and formononetin in plasma samples.Validation process:chromatographic conditions and MS conditions optimization,selectivity and linear range,precision and accuracy experiments,matrix effects and extraction recovery,and stability tests.Wistar rats are selected as experimental subjects.Intragastric administration with suberect spatholobus 0.638g/kg(equivalent to protocatechuic acid 0.64g/kg,catechin acid 6.73g/kg,epigallocatechin 5.17g/kg and formononetin 1.06 g/kg)is conducted.UPLC-MS/MS method is used to determine the contents of four flavonoid compounds including protocatechuic acid,catechin acid,gallocatechin and fonnononetin in the serum of rats.Blood samples are taken from the infraorbital vein of rats at 9 time points before and after the intragastric administration with caulis spatholobi extract and put in the heparin test tube.After centrifugation,the samples are stored at-80?.The DAS 2.1 suite procedure is applied,and non-compartment model is adopted to analyze the plasma concentration VS time data to calculate the pharmacokinetic parameters.Part 2 Formononetin attenuated platelet aggregation and vascular intimal hyperplasia in rats with balloon-induced arterial injuryRats model with balloon-induced arterial injury were established to detect the inhibition effects of formononetin on intimal hyperplasia.90 rats were randomly divided into sham group and model group.After establishment of the balloon-induced arterial injury model,rats were randomly divided into 6 groups,and the treatments were as follows:Rats in sham group were treated with intragastric administration of saline(10 ml/kg);Rats with balloon-induced arterial injury were divided into model group(intragastric administration of saline,10 ml/kg),and formononetin group(intragastric administration of formononetin,25,50,100 mg/kg);Aspirin group((intragastric administration of aspirin,10 ml/kg)).Drug treatment was started on the second day after modeling,once a day and the treatment was lasted for 14 days.Platelet aggregation rate was measured by turbidimetric method of Born.Nitrate reductase method was used to detect serum nitric oxide(NO)level.Levels of serum endothelin-1(ET-1)and thromboxane A2(TXA2)were measured by radioimmunoassay.Levels of platelet derived growth factor(PDGF)and epoprostenol(PGI2)were measured by enzyme-linked immunoSorbent assay(ELISA).Part 3 Formononetin protects against balloon-induced neointima formation via regulating proliferation and migration of vascular smooth muscle cells through TGF-a1/Smad3 signalingCarotid artery-injury rat model was established to examine the effects of formononetin against balloon-induced neointima formation in vivo.Histological observation of the carotid artery tissues was done by hematoxylin and eosin staining.Rat aortic SMCs were isolated,and the effects of formononetin on SMCs proliferation and migration ability induced by PDGF-BB was measured by CCK-8 and Transwell/wound healing assay.Immunohistochemistry staining and immunocytochemical assay was applied to measure the TGF-a expression in carotid artery tissues and SMCs respectively.Smad3/p-Smad3 expression was examined by western blotting.ResultsPart 1 UPLC-MS/MS assay for simultaneous determination of four compounds in rat plasma:application to pharmacokinetic study after oral administration of Caulis Spatholobi extractsIn optimized chromatographic and mass spectrometric analyzes,both positive and negative ionization modes were optimized simultaneously.The analyte and internal standard ionization,relative to the negative ion mode,gave a more intense reaction than the cation mode.By optimization,0.05%formic acid enhanced the effect of ionization for all compounds tested,obtaining more than pure distilled water.To obtain satisfactory sensitivity and good peak morphology,we selected 0.05%formic acid and methanol/water(35:65,v/v)as isocratic eluent.The retention times of protocatechuic acid,catechuic acid,gallocatechin,formononetin and internal standard were 1.1,1.1,1.1,2.2,and 4 min,respectively.Liquid-liquid extraction with ethyl acetate was finalized and simple,reproducible and reproducible.A linear range of protocatechuic acid,catechuic acid,gallocatechin was 0.5-200 ng/ml,and that of formononetin was observed in the range of 0.8-320 ng/ml.The coefficient(r)of the calibration curve was over 0.99 for all calibration curves.The lowest limit of quantification(LLOQ)was defined as a signal-to-noise ratio surpass 10,with accuracy and accuracy within±20%.The pharmacokinetic study shows that the AUC0-t values are 67.52±9.47 and 63.64±19.37?g h/1 for catechin acid and gallocatechin,respectively.Catechin acid and gallocatechin(6.73 mg/kg catecholic acid and 5.17 mg/kg gallocatechin)show similar oral absorbance at the same dose level,as a result of their similar chemical structure.At the same time,it is observed that the AUC0-t values in the serum concentration-time curve are also similar when the oral concentration of gallocatechin is 5 times higher than that of formononetin,indicating that the absorption of formononetin by rats is greater than that of gallocatechin.The results of the research are of great significance for studying the mechanism of action of the main constituents of caulis spatholobi extract,and have also provided an effective tool for the study pm pharmacokinetics of traditional Chinese medicine.Part 2 Formononetin attenuated platelet aggregation and vascular intimal hyperplasia in rats with balloon-induced arterial injuryTreatment with formononetin significantly improved histopathological structure of neonatal endometrium.Compared with model group,treatment with formononetin significantly inhibited platelet aggregation,reduced PAGmax and IR,increased levels of NO and PG12,and decreased the level of ET-1,TAX2 and PDGF.Formononetin could improve histopathological structure of vascular intimal hyperplasia and attenuate platelet aggregation in rats through inhibiting ADP,collagen or AA induced platelet aggregation,increasing NO level,reducing ET-1 and PDGF levels,and keeping TXA2/PGI2 balance in serum.Part 3 Formononetin protects against balloon-induced neointima formation via regulating proliferation and migration of vascular smooth muscle cells through TGF-a1/Smad3 signalingFormononetin treatment significantly improved the abnormal proliferation of smooth muscle cells in neonatal endometrium,and the vascular structure changes were attenuated than model group.Pretreatment of formononetin could effectively inhibit the proliferation of SMCs stimulated by PDGF-BB(p<0.05).Formononetin notably reduced cell numbers migrated to the lower surface of the transwell chamber and decrease the wound healing percentage(p<0.05).TGF-a level was decreased by formononetin treatment both in vivo and in vitro,and the Smad3 expression was also profoundly inhibited.Conclusions1.The UPLC-MS/MS method for the simultaneous determination of the four flavonoids(protocatechuic acid,catechuic acid,gallocatechin and formononetin)of caulis spatholobi in rat serum was established,which had the advantages of high speed,accuracy,good linearity,high precision,and high recovery rate.It could be used for the determination of active components in caulis spatholobi and caulis spatholobi mixture,and could be used as the method to control the quality of caulis spatholobi.The determination of protocatechuic acid,catechuic acid and gallocatechin could be used as an evaluation method for the quality control of caulis spatholobi,which provided a new reference method for pharmacodynamics and pharmacokinetic study of caulis spatholobi.2.Formononetin might be the basic active ingredient of the caulis spatholobi in inhibiting platelet aggregation and intimal hyperplasia.Formononetin can significantly increase NO level,reduce levels of ET-1 and PDG and maintain TXA2/PGI2 ba ance.3.Formononetin significantly protected against balloon-induced neointima formation in a rat model,and a possible mechanism might be related with the regulation of SMCs proliferation and migration through adjusting TGF-a1/Smad3 signaling,thus attenuating balloon-induced neointima formation in the carotid artery.