The Inhibitory Effect And Action Mechanism Of DJS-F, A New Structure Derivative Of Farrerol, On Balloon-Induced Intima Hyperplasia In Rat Common Carotid Artery

Objective:Farrerol, a cough expectorant active ingredient from natural sources, was proven to have inhibitory effects on abnormal proliferation of VSMC. However, the underlying mechanism remains unclear due to its low active and less natural distribution. In our previous study, a series of new compounds were designed and synthesized with farrerol as the lead compound. The 2-(Furan-2-)-5,7-dihydroxy-6,8-dimethyl-2,3-dihydroben Zo-pyran-4-ketone(DJS-F), as a new structure derivative of farrerol, was the main object in this study. The aim of this study was to investigate the mechanism underlying its inhibitory effects on balloon-induced intima hyperplasia in rat common carotid artery via attenuation of abnormal proliferation and phenotype transformation of VSMC, and explore its protein targets in VSMC. Our study will provide scientific basis for the further structural optimization and in-depth research of these classes of compounds.Methods:(1) Tissue adherent method was used to establish the primary rat aortic smooth muscle cells(RASMCs). The immunocytochemistry method was employed to identifyα-SMA in RASMCs. Cells were starved in 0.5% FBS-1640 medium for 48 h to achieve cell culture synchronization and then treated with 10% FBS for 24 h to induce proliferation of RASMC. MTT assay were applied to examine the actions of farrerol and its new structure derivatives(DJS-NO2 and DJS-F) on 10%FBS-induced proliferation of RASMC.(2) Flow cytometry was used to examine cell cycle distribution in RASMC.Western Blotting assay was performed to evaluate the expression of cell cycle-regulatory proteins including p27, Cyclin E1, CDK2, Cyclin D1 and the phenotypic marker protein including SM22-α, α-SMA, and OPN. Furthermore, the effects of DJS-F on MAPK and PI3K-Akt signaling pathways in 10% FBS-stimulated VSMCs were evaluated.(3) The animal model was established by rubbing the endothelia with a balloon catheter in the common carotid artery of male Sprague Dawley rats. Tail vein injection of Evans blue(EB) was applied to the evaluation of endothelial damage. SD rats were divided into 5groups: balloon injury group, positive control group(Rapamycin, 0.5 mg/kg); DJS-F high-dose treatment group(18 mg/kg), DJS-F treatment middle-dose group(9 mg/kg),DJS-F low-dose treatment groups(4.5 mg/kg), 8 rats in each group. DJS-F was mixed with 40% pluronic gel solution and was topically applied over the injured carotid artery evenly. Two weeks after injury, the arteries were harvested and morphometric analysis was done. Morphology of intima hyperplasia was assessed by hematoxylin/eosin staining and Image-Pro Plus(IPP) Analyzer version 5.1 software. Immunohistochemical staining was performed to examine SM22-α 、 α-SMA 、 OPN and PCNA production.(4) The potential targets for the active compound DJS-F was investigated with a drug affinity responsive target stability(DARTS) technique and MALDI-TOF mass spectrometry to investigate the possible action targets in its proliferation inhibition against VSMCs.Results:(1) Immunocytochemistry staining showed that α-SMA was significantly positive expressed in VSMCs. 10% FBS stimulation concentration-dependently caused VSMC proliferation by more than 50% compared with that of untreated cell. No significant decrease in cell vitality was detected in the concentration range of 0-50μmol/L in VSMC treated with farrerol and its derivatives. DJS, along with its derivatives DJS-NO2 and DJS-F, attenuated 10% FBS-induced RASMC proliferation in a concentration-dependent manner with an IC50 of 30.8 μmol/L, 7.4 μmol/L, and 5.2 μmol/L respectively.(2)DJS-F blocked 10%FBS-induced cell cycle progression from the G0/G1 to the S-phaseand resulted in an accumulation of VSMCs at the G0/G1 phase. DJS-F treatment resulted in inhibition of decrease of p27 expression induced by 10%FBS while increase of PCNA,p21, Cyclin E1, CDK2, and Cyclin D1 expression in a concentration dependent manner.DJS-F treatment resulted in inhibition of 10%FBS-induced decrease expression of contractile phenotype marker α-SMA and SM22α, two VSMC contraction phenotype related proteins, while increase of expression of synthetic phenotype marker osteopontin(OPN), a VSMC synthetic phenotype related protein, in a concentration dependent manner. DJS-F treatment inhibited 10%FBS-induced phosphorylation of ERK1/2,p38 MAPK, JNK and AKT in RASMC without affecting expression of the total proteins.Both of specific MEK1/2 inhibitors and PI3 K inhibitor could inhibit 10%FBS-induced decrease of α-SMA and SM22α expression while increase of OPN expression.(3)Perivascular application of DJS-F inhibits rat carotid neointima hyperplasia induced by balloon injury in a dose-dependent manner. Morphometric analysis showed that neointima hyperplasia was well developed at 14 days after balloon injury. By 14 days,neointima accounted for over 50% of the carotid arterial wall thickness. When compared with that of the balloon injured group, carotid arterial wall thickness was significantly decreased in the three DJS-F-treated groups, both I/M ratio and intima area were lower than that observed at the balloon injured group. Moreover, the inhibitory effect of DJS-F on neointima formation was more significant in the moderate- and high-dose DJS-F groups than that in low-dose DJS-F group. These results suggest that DJS-F dose-dependently inhibits neointima formation induced by balloon injury. The intima-to-media(I/M) ratio were significantly reduced in rapamycin-treated group and DJS-F-treated group(p<0.05). Perivascular delivery of DJS-F with pluronic gel attenuated the development of intimal hyperplasia. DJS-F suppresses up-regulation of OPN and down-regulation of SM22α and α-SMA induced by balloon injury.(4) Two proteins, Tm and GRP78, were identified as the potential targets for the active compounds with a drug affinity responsive target stability(DARTS) technique and MALDI-TOF mass spectrometry.Conclusion:(1) DJS-F is more effective than DJS for the inhibition of 10% FBS-stimulated proliferation of RASMC.(2) The inhibitory effects of DJS-F against 10%FBS-induced proliferation of RASMC is correlated with its regulation of cell cycle progression, which might be associated with the inhibition of 10%FBS-induced decrease expression of p27 and increase expression of Cyclin E1, CDK2 and Cyclin D1.(3) DJS-F inhibits10%FBS-induced phenotypic transition of VSMCs, which might be associated with inactivation of PDGFR-ERK1/2 and AKT in VSMCs.(4) Perivascular application of DJS-F can dose-dependently reduce intimal hyperplasia in a rat carotid balloon injury model, which is likely associated with proliferation inhibition and phenotype modulation of VSMCs.(5) The proteins Tm and GRP78 may be act as the potential protein targets for the active compound DJS-F against the abnormal proliferation of VSMC.(6) This study will provide scientific basis for the further structural optimization and in-depth research of DJS-F.