羅 超，李意奇，王俊逸，李葉麗，呂俊遠(yuǎn)，楊丹莉

(1.遵義醫(yī)科大學(xué) 基礎(chǔ)藥理教育部重點實驗室暨特色民族藥教育部國際合作聯(lián)合實驗室，貴州 遵義 563099；2.遵義醫(yī)科大學(xué)珠海校區(qū)，廣東 珠海 519041；3.遵義醫(yī)科大學(xué)附屬醫(yī)院,貴州 遵義 563099)

1 Introduction

Percutaneous transluminal coronary angioplasty is now widely used in the treatment of obstructed atherosclerotic vessels.However,its overall benefits are limited by the fact that it may induce neointimal hyperplasia and restenosis after interventional surgery in many patients who are initially treated successfully[1-2].The evidences that transforming growth factor-beta1(TGF-β1)/Smads plays a key role in the development of neointimal hyperplasia and restenosis have been presented at the cellular,molecular,and genetic levels[3-4].

Icariside II (ICS II) is one of active ingredient of Herba Epimedii,which has a variety of pharmacological effects,such as anti-inflammation,immune regulation,anti-tumor,protecting cardiovascular system and so on[5-7].However,it is not known whether ICSII has effects on neointimal hyperplasia and restenosis.In this study,we sought to determine the effects of ICSII on intimal hyperplasia in rat carotid arteries induced by balloon injury.We additionally explored whether the mechanism is relative to TGF-β1/Smads.

2 Materials and methods

2.1 Materials Icariside (ICS II,whose purity was more than 98% as assayed by HPLC) was purchased from the Nanjing Zelang Medical Technology Co.,Ltd.(Nanjing,China).The 2-F Fogarty arterial embolectomy balloon catheter was obtained from Edwards Lifesciences Co.(USA);Leica optical microscope and photographic system (Leica Microsystems Ltd,Germany);Centrifuge 5415D (Eppendorf,Germany);Nano Drop 2 000 ultramicro spectrophotometer (Thermo,America);Centrifuge 5417R (Eppendorf Germany);I-mark enzyme labeling instrument (BIO-RAD Company),Transforming growth factor-beta (TGF-β,Abcam,Cambridge,MA,USA),p-Smad2 (Phospho-Ser465),Biotechnology,Shanghai,China) and p-Smad3 (Phospho-Ser425),SanYing Biotechnology,Wuhan,China) and Matrix metalloproteinase 2 (MMP-2,Sanying Biotechnology,China),GAPDH (Sanying Biotechnology,China),goat anti-rabbit IgG-HRP (Sanying Biotechnology,China).

2.2 Animal and the rat carotid artery balloon injury model Male SD rats (300～350 g) were obtained from Liaoning Changsheng Biotechnology Co.,Ltd (Liaoning,China),certificate number:SCXK (Liao) 2018-0001.The rats were maintained in accordance with institutional animal care and use committee procedures and guidelines.To establish the common carotid artery intimal denudation model,after fasting 12 hours,the rats were anaesthetized with an intraperitoneal injection of 2% pentobarbital sodium,and the rat carotid artery balloon injury model was made[8].

2.3 Experimental design and arterial harvest Male rats were randomly divided into the sham operation group and the carotid artery balloon-injured groups.Animal carotid artery models were established by balloon injury,and the survival rats were randomly divided into three groups:model,ICSII-L,ICSII-H groups.ICSII-L and ICSII-H groups were administered with ICSII (10,20mg/(kg·d)，qd) by gavage for 14 days.The same volume distilled water was administered in sham group and model group for 14 days.Twenty-four hours after the last dose,the rats were euthanatized with an intraperitoneal injection of pentobarbital sodium.The injured left common carotid artery segments were quickly removed,and the middle portions of the balloon-treated segments were immediately fixed with 4% formaldehyde solution (40% formaldehyde solution was diluted to 4% with PBS solution,and the pH value was about 7.4) for morphological examination.The other portions of the segments were frozen at -80℃ for other tests.

2.4 Haematoxylin and eosin (H.E) staining analysis of the rat carotid arteries For histological analysis,rat arteries were taken from the middle portions of the balloon-treated segments that had been fixed in 10% neutral-buffered formalin solution for 24 h.After embedded in paraffin,the rat arteries were further cut into 5-μm-thick sections,and then analysed by haematoxylin and eosin (H.E) staining.To evaluate the effects of Icariside on balloon injury-induced neointimal hyperplasia,the slices were observed by a Leica light microscope.

2.5 Western blot assay The protein expression levels of TGF-β1,p-Smad2,p-Smad3 and MMP-2 were measured via western blotting and normalized to the protein expression levels of GADPH.The carotid arteries were homogenized with RIPA lysis buffer and centrifuged at 12 000 g for 35 minutes at 4°C to obtain the supernatant.The protein concentration was determined using a Bicinchoninic Acid Protein Assay Kit.Samples containing 10 μg of protein were resolved by 12% SDS-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes,which were subsequently blocked with 5% defatted milk in tris-buffered saline Tween (TBST) for 1 h at room temperature.After the membranes were washed with TBST,they were incubated with TGF-β1 (1∶1 000),p-Smad2 (1∶1 000),p-Smad3 (1∶2 000),MMP-2 (1∶1 000),GAPDH (1∶10 000) antibodies in 1% BSA in TBST overnight at 4°C before being incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h.Finally,the Bio-Rad CCD imaging system was used to obtain images and analyze the gray value of the protein band after ECL chemiluminescence.

2.6 Statistical analysis All data were expressed as the mean ±S.D.and processed with SPSS 18.0 statistical software (IBM,CA,USA).A single factor analysis of variance was used for comparisons among groups.The level of α=0.05,P< 0.05 was considered statistically significant.

3 Results

3.1 Effects of Icariside II on neointimal hyperplasia in rat common carotid arteries after balloon injury HE staining showed that the carotid artery,which was not subjected to balloon angioplasty in the sham group,exhibited normal vessel thickness (Fig 1).The carotid artery in the model group displayed clear neointimal hyperplasia compared with the sham group on day 14 after balloon injury.However,Icariside II administration (10,20 mg/(kg·d),qd) was effective in preventing neointimal hyperplasia induced by the balloon-treated in carotid artery (Fig 1).

The images were taken at 100× magnification from the sham,model,ICSII-L(10 mg/(kg·d))and ICSII-H(20 mg/(kg·d))groups(bar=200 μm).Fig 1 Effects of Icariside II on neointimal hyperplasia in rat common carotid arteries after balloon injury

3.2 Effects of ICSII on TGF-β1,MMP-2 protein expression and Smad2/3 phosphorylation in the rat carotid arterie as determined by western blotting after 14 days It was showed that compared with sham group (Fig 2),the TGF-β1,MMP-2 protein expression in the rat carotid arteries of the model group was increased significantly (P<0.05).The activation of Smad2/3 was increased in the Model group compared with sham group (P<0.05).However,the protein expression of TGF-β1,MMP-2 and the phosphorylation of Smad2/3 in carotid arteries was decreased evidently by ICSII administration at doses of 10 mg/(kg·d)and 20 mg/(kg·d) (P<0.05) suggesting that ICSII reduced the protein expression of TGF-β1,MMP-2 and the phosphorylation of Smad2/3 in carotid artery injury after 14 days.

A:The representative strip of TGF-β1,MMP-2 expression protein and Smad 2/3 phosphorylation levels；B:Relative optical density of TGF-β1,MMP-2 protein expression and Smad 2/3 phosphorylation levels was normalized to that of GAPDH.Data are presented as the mean±S.D;n = 5.(*：P<0.05 versus sham;and #：P<0.05 versus model). Fig 2 Effects of ICSII on TGF-β1,MMP-2 protein expression in the rat carotid arteries as determined by western blotting after 14 days

4 Discussion

It is known that when arteries are injured by mechanical stimuli,the vascular smooth muscle cell (VSMCs) are critical target cells that responded by proliferating and then forming a neointimal lesion[9-10].In the present study,the model of vasclar neointimal hyperplasia was induced by balloon injury to simulate the restenosis after the percutaneous coronary angioplasty.It was showed that ICS II had an in vivo antiproliferation activity to block the neointimal hyperplasia in the rat carotid arteries model at day 14 after balloon-injury.

In the early stages of acute vascular injury,inflammatory cytokines and thrombosis can cause the release of transforming growth factor-beta (TGF-β)[10-12].The role of TGF-β in the pathogenesis of intimal hyperplasia is very important[11-12].TGF-β includes the TGF-β1-5 isoforms.TGF-β1 can stimulate the proliferation and differentiation of VSMCs,as well as the deposition of extracellular matrix,leading to angiostenosis[8,12].It is known that TGF-β1 levels increase evidently during the acute phase and the elevations of TGF-β1 levels can persist for up to 2 weeks after balloon-injury[8].In the present study,TGF-β1 protein expression in the rat carotid arteries was increased after balloon injury,which was consistent with the past studies.Interestingly,it was showed that ICS II could reduce the protein expression level of TGF-β1 in the rat carotid arteries at day 14 after balloon-injury in the present study.

In the meantime,TGF-β1 can induce the rapid activation of the Smad.When activated,Smad2 and Smad3 are phosphorylated and subsequently combine with Smad4 to initiate the transcription of their target genes[10,12-13].In the present study,it was found that p-Smad2，p-Smad3 protein expression were increased after the arterial injury,which was consistent with the up-regulation expression in TGF-β1 protein.The present findings again demonstrated that neointimal hyperplasia was closely related to TGF-β1/Smad2 signalling pathway,which was in accordance with the past study[8,12-16].However,ICSII administration not only could block the neointimal hyperplasia,but also inhibit the up-regulation of TGF-β1 protein in the rat carotid arteries.Meanwhile Smad2/3 phosphorylation were inhibited.

Matrix metalloproteinase 2 (MMP-2),one of gelatinase,is expressed and secreted by vascular parietal cells[13].The expression and activity of MMP is increased evidently in restenosis vascular tissue after percutaneous coronary angioplasty,which can promote the deposition of extracellular matrix[7,15,17].In our study,the MMP-2 protein expression was increased,which was consistent with the up-regulation of protein expression levels of TGF-β1,p-Smad2/3 after balloon-injury.However,ICSII administration could inhibit the increased protein expression of MMP-2 in the rat carotid arteries.

In conclusion,ICSII significantly suppresses intimal hyperplasia in rat carotid arteries induced by balloon injury,which might be at least partially mediated through the suppression of TGF-β1/Smads signaling pathways.