The Effect Of Probucol On Proliferation And Apoptosis Of Vascular Smooth Muscle Cell Induced By Oxidation

Background:The cardia and cerebrovascular diseases such as atherosclerosis are common and frequently occurring illness.The mechanisms are very complicated and not be illuminated completely at present.Excessive proliferation or apoptosis of vascular smooth muscle cells induced by active oxygen,mediators of inflammation, cytokines may be one of the major mechanisms of the atherosclerosis formation, atheromatous plaque rupture and restenosis after interventional therapy.VSMCs The proliferation and apoptosis of VSMCs are effected and regulated by intracellular redox equilibrium state.Trx system and GSH system are two important reduction systems in cell.The Trx-1 is an important growth promotion helper factor,and has anti-apoptosis activity.The GSH has many physiologic functions incuding anti-apoptosis,regulating proliferation and signal transduction that are sensitive to redox.The intracellular redox environment will be influenced by the changes of concentrations of Trx-1 and GSH/GSSH,and the proliferation and apoptosis will be affected subsequently.Probucol is a hypolipidemic agent synthesized in 1970s.In recent years,the effects of probucol on signal protein,activity of transcription factor,VSMC proliferation and apoptosis,as well as on the atherosclerotic plaque stability and the pathomechanism of restenosis after the interventional theraty have became a hot spot lately,following by recognition of oxidative damage in the artherosclerosis and the investigation of antioxidation mechanism.The present study was designed to investigate the effects of probucol on VSMC proliferation and apoptosis induced by oxidation and the expression of ERK1/2,MKP-1,HO-1 and ASK-1 to reveal its duplicate mechanism.Motheds:Primary cultured VSMCs were used.The concentrations of probucol were 100μmol·L^-1,10μmol·L^-1 and 1μmol·L^-1 respectively.1.The effects of probucol on VSMC proliferation and signal transduction induced by ox-LDL(1) MTT assay and trypan blue staining was used to investigate the effects of ox-LDL on VSMC proliferation with different ox-LDL concentration and incubated time in order to select the best ox-LDL concentration and incubated time.(2) The ratio of GSH/GSSH was measured by the GSH,GSSG kit,and the expression of Trx-1 was determined by western blot assay.(3) The effect of probucol on VSMC viability was assessed by MTT assay.The VSMC cycle was evaluated by flow cytometry.The Cell growth curve was mapped to observe the effect of probucol on the inhibition of VSMC growth; The VSMC migration was evaluated by the scratch assay.(4) The expression of ERK1/2,p-ERK1/2,MKP-1,HO-1 and Trx-1 was determined by Western blot assay.2.The effects of probucol on VSMC apoptosis and signal transduction(1) In order to select the best H₂O₂ concentration that induces apoptosis,the MTT assay and Annexin V/PI staining was used to observe the VSMC apoptosis with different concentrations of H₂O₂.(2) The ratio of GSH/GSSH was measured by the GSH,GSSG kit,and the expression of Trx-1 was determined by western blot assay.(3) The effect of probucol on VSMC viability was assessed by MTT assay.The morphology of apoptotic cells was observed by Hoechest 33258 staining.The apoptosis ratio of VSMC cell was evaluated by flow cytometry.TUNEL assay was used to detect apoptosis in situ to evaluate the apoptotic cell morphous and apoptosis ratio. (4) The expression of ASK-1 and Trx-1 determined by Western blot assay.Results:1.The effects of probucol on VSMC proliferation and signal transduction induced by ox-LDL(1) The proliferation of VSMC induced by 35 mg·L^-1 ox-LDL for 48h was the most significant(P＜0.01).With time,the role of ox-LDL to promote proliferation weakened gradually.Therefore,35 mg·L^-1 ox-LDL for 48h was determined as the best concentration and incubated time.(2) The intracellular GSH/GSSG ratio decreased slightly compared with the control group when the VSMCs were incubated with 35 mg·L^-1 ox-LDL for 48h,and the GSH/GSSG ratio increased in a dose-dependent when the VSMCs were co-incubated with probucol in different concentrations. Meanwhile,the Trx-1 expression increased markedly with probucol treatment compared with the ox-LDL group.(3) The ox-LDL 35 mg·L^-1 could promote the proliferation of VSMCs.The proliferation was inhibited after treated with probucol(P＜0.01);Flow cytometry analysis indicated that ox-LDL stimulation accelerated the cycle of VSMC.However,probucol had an inhibitory effect on VSMC proliferation stimulated by ox-LDL,resulting in cell sycle arrest in the phase G0/G1 and S, and emerged apoptotic peak.Cell growth curve also showed that probucol inhibited the growth of VSMC(P＜0.01).The scratch wound in control group was closed with time.A significant delay in the migration of VSMC into the empty space was observed after treatment with probucol.(4) The expression of p-ERK1/2 was inhibited obviously by probucol,and the expression of MKP-1 was enhanced notably.Moreover Trx-1 expression was significantly enhanced,and HO-1 expression also increased.The significantly differences were found compared with ox-LDL group in a dose-dependent way.2.The effects of probucol on VSMC apoptosis and signal transduction(1) Compared with the control group,the proliferation of VSMCs was inhibited significantly when treated with H₂O₂ 1000μmol·L^-1 for 6h(P＜0.01).Flow cytometry analysis indicated that H₂O₂ induced the cell of VSMCs apoptosis. Therefore,H₂O₂ 1000μmol·L^-1 for 6h was determined as the best concentration and incubated time.(2) The intracellular GSH/GSSG ratio decreased significantly compared with the control group when the VSMCs were incubated with H₂O₂ 1000μmol·L^-1 for 6h,and the GSH/GSSG ratio increased in a dose-dependent when the VSMCs were co-incubated with probucol in different concentrations. Meanwhile,the Trx-1 expression was increased markedly with probucol treatment.(3) The VSMCs appearanced apoptosis with H₂O₂1000μmol·L^-1.After co-incubaion with probucol,the OD values increased markedly(P＜0.01);By Hoechest 33258 staining,the compact fluorescence was observed under fluorescent microscope with H₂O₂ treated group,but cells in the control group showed uniform,dispersed fluorescence.After treated with probucol, the number of hyperchromic cells decreased in a concentration-dependent way.By flow cytometry analysis,the apoptosis rate increased significantly stimulated by H₂O₂,and decreased in a concentration-dependent way when treated with probucol.In TUNEL assay,most of the nucleus in control group were oval or round,and in uniform blue stain,and there were no obvious apoptotic cells.H₂O₂ treatment group showed brown nuclear staining,nuclear shrinkage and chromatin aggregation into mass.The number of nuclear stained brown reduced in probucol treatment groups.(4) The expression of ASK-1 induced by H₂O₂ was enhanced obviously,and the expression of Trx-1 was degraded notably.However,Trx-1 expression was significantly enhanced,and ASK-1 expression was decreased after treated with probucol.Conclusion:The ox-LDL can induce VSMC proliferation and lower down the GSH/GSSG ratio that this effect on proliferation involved in the inhibition of MKP-1 activity and reinforcement of ERK1/2 activity.However,probucol can enhance the expression of MKP-1 and inhibit ERK1/2 activity,thereby exert anti-proliferative role. In addition,through the enhancement of Trx protein expression,probucol enhanced HO-1 activity and induced VSMCs apoptosis,by which probucol exert anti-proliferative effect.H₂O₂(1000μmol·L^-1) induce VSMCs apoptosis and cut down the GSH/GSSG ratio obviously.Its effect on apoptosis involves in the desent of Trx-1 concentrantion and accrescence of ASK-1 expression.Probucol can reduce the depression of Trx-1 concentration by increasing the GSH/GSSG ratio,thereby inhibited ASK-1 activity and exert anti-apoptotic role.