| BackgroundAtherosclerosis(AS) is the most common disease of the cardiovascular system diseases and is also the pathological basis of coronary heart diseases.Fibrosis of the intima of vessels and plaque formation cause the hardening of wall, narrowness of lumen and lead a series of secondary lesions. As an important part of the vascular wall, vascular smooth muscle cells(VSMCs) have a key role in atherosclerosis. Oxidative stress injury is an important mechanism for the formation of atherosclerosis. Proliferation and phenotypic change of VSMCs is one of the main pathological basis of AS, is also an important cause of Stent Restenosis. Therefore anti-inflammation, anti-oxidation and inhibition of smooth muscle cell proliferation are important ways to treat atherosclerosis and inhibit in-stent restenosis.Atorvastatin is the basis drug for the treatment of atherosclerosis. A large amount of new studies manifest that atorvastatin has effects of anti-inflammation, anti-oxidation and inhibition of smooth muscle cell proliferation. These effects are important mechanisms of atorvastatin in treating atherosclerosis. As an immunosuppressant, Rapamycin can inhibit the GO and G1phase of the cell cycle and block G1into the S phase through combining with the the corresponding immune addicted prime RMBP. Rapamycin is commonly used in drug-eluting stents in order to reduce intimal hyperplasia, but there is no clear conclusion whether rapamycin has effect on VSMCs, especially on oxidative stress injury of VSMCs. Therefore, these experiments were conducted to explore the effect of the combination of rapamycin and atorvastatin on oxidative stress injury in the rat vascular smooth muscle cells.ObjectivesPrimary cultured VSMCs separated from rat aorta were chosen as a model to identify the effect of the combination of rapamycin and atorvastatin on oxidative stress injury in the rat vascular smooth muscle cells by preforming modern molecular biology, such as Western blot, RT-PCR, CCK8, WST-1, WST-8, and so on. These result may provide theoretical basis for using rapamycin and atorvastatin for treating atherosclerosis in clinical practice.MethodsRat aortic smooth muscle cells were obtained from the thoracic aorta of male Wistar rats. Isolated VSMCs were cultured in DMEM plus10%FBS at37℃in a humidified atmosphere. The purity and identity of VSMCs were verified using a monoclonal antibody against smooth muscle α-actin. Cell from passages4-6were used in all experiments.Assessment of cell toxicity of t-BHP:VSMCs were divided into six groups:1) control group,2)40μmol/L t-BHP group,3)60μmol/L t-BHP group,4)80μmol/L t-BHP group,5)100μmol/L t-BHP group,6) t-BHP(200μmol/L group). After cells were treated for12h,24h,48h and72h, CCK8was performed to detect the cell viability(%). The purpose is to explore the model of concentration and time of t-BHP-induced oxidative stress injury of aortic smooth muscle cells.60μmol/L t-BHP treated24h was chosen.The effect of the combination of rapamycin and atorvastatin on the proliferation rate of the rat vascular smooth muscle cells with oxidative stress injuryVSMCs were divided into six groups, including control, DMSO, atorvastatin3μmol/L(A3), rapamycin50nmol/L(R50), rapamycin100nmol/L(R100) and atorvastatin combined with rapamycin100nmol/L. After pretreated with corresponding stimulants for2hours, VSMCs were treated with t-BHP to induce oxidative stress. After24hours of incubation, cell proliferation was detected.The effect of the combination of rapamycin and atorvastatin on superoxide dismutase(SOD) of the rat vascular smooth muscle cells with oxidative stress injuryVSMCs were divided into six groups, including control, DMSO, atorvastatin3μmol/L(A3), rapamycin50nmol/L(R50), rapamycin100nmol/L(R100) and atorvastatin combined with rapamycin100nmol/L. After pretreated with corresponding stimulants for2hours, VSMCs were treated with t-BHP to induce oxidative stress. After24hours of incubation, superoxide dismutase(SOD) were detected.The effect of the combination of rapamycin and atorvastatin on malonyldialdehyde(MDA) of the rat vascular smooth muscle cells with oxidative stress injuryVSMCs were divided into six groups, including control, DMSO, atorvastatin3μmol/L(A3), rapamycin50nmol/L(R50), rapamycin100nmol/L(R100) and atorvastatin combined with rapamycin100nmol/L. After pretreated with corresponding stimulants for2hours, VSMCs were treated with t-BHP to induce oxidative stress. After24hours of incubation, malonyldialdehyde(MDA) were detected.The effect of the combination of rapamycin and atorvastatin on senescence associated-β-galactosidase(SA-β-GAL) of the rat vascular smooth muscle cells with oxidative stress injury VSMCs were divided into six groups, including control, DMSO, atorvastatin3μmol/L(A3), rapamycin50nmol/L(R50), rapamycin100nmol/L(R100) and atorvastatin combined with rapamycin100nmol/L. After pretreated with corresponding stimulants for2hours, VSMCs were treated with t-BHP to induce oxidative stress. After24hours of incubation, senescence associated-β-galactosidase(SA-β-GAL) were detected.The effect of the combination of rapamycin and atorvastatin on mRNA expression of eNOS of the rat vascular smooth muscle cells with oxidative stress injuryVSMCs were divided into six groups, including control, DMSO, atorvastatin3μmol/L(A3), rapamycin50nmol/L(R50), rapamycin100nmol/L(R100) and atorvastatin combined with rapamycin100nmol/L. After pretreated with corresponding stimulants for2hours, VSMCs were treated with t-BHP to induce oxidative stress. After24hours of incubation, mRNA expression of eNOS were detected.The effect of the combination of rapamycin and atorvastatin on protein expression of eNOS of the rat vascular smooth muscle cells with oxidative stress injuryVSMCs were divided into six groups, including control, DMSO, atorvastatin3μmol/L(A3), rapamycin50nmol/L(R50), rapamycin100nmol/L(R100) and atorvastatin combined with rapamycin100nmol/L. After pretreated with corresponding stimulants for2hours, VSMCs were treated with t-BHP to induce oxidative stress. After24hours of incubation, protein expression of eNOS were detected.Results1.The effect of the combination of rapamycin and atorvastatin on the proliferation rate of the rat vascular smooth muscle cells with oxidative stress injuryCompared with control group, the cell proliferation rate of DMSO group decreased(P<0.01). Compared with DMSO group,in other four groups,the cell proliferation rates decreased. There was no significant statistical difference between A3group and R50group. Compared with R50or A3groups, proliferation rate decreased(P<0.05) in R100group. In the comination group, cell proliferation rate was lower than any other groups(P<0.01).2.The effect of the combination of rapamycin and atorvastatin on superoxide dismutase(SOD) of the rat vascular smooth muscle cells with oxidative stress injuryCompared with control group, SOD of DMSO group was not changed. In other four groups, SOD increased(P<0.01). There was no significant statistical difference between A3group and R50group. Compared with R50or A3groups, SOD was higher in R100group(P<0.05). In the comination group, SOD was higher than any other groups(P<0.05).3.The effect of the combination of rapamycin and atorvastatin on malonyldialdehyde(MDA) of the rat vascular smooth muscle cells with oxidative stress injuryCompared with control group, MDA of DMSO group was not changed. In other four groups, MDA decreased(P<0.05). There was no significant statistical difference between A3group and R50group. Compared with R50or A3groups, MDA was lower in R100group(P<0.05). In the comination group, MDA was lower than any other groups(P<0.05).4.The effect of the combination of rapamycin and atorvastatin on senescence associated-β-galactosidase(SA-β-GAL) of the rat vascular smooth muscle cells with oxidative stress injuryCompared with control group, SA-β-GAL of DMSO group was not changed. In other four groups, SA-β-GAL senescence rates decreased (P<0.05). There was no significant statistical difference between A3group and R50group. Compared with R50or A3groups, SA-β-GAL rates was lower in R100group(P<0.05). In the comination group, SA-β-GAL rates was lower than any other groups(.P<0.05).5.The mRNA and protein expression of eNOS of the rat vascular smooth muscle cells with oxidative stress injurymRNA and protein of eNOS didn’t exrpess in the rat vascular smooth muscle cells stimulated by t-BHP.Conclusions1) Both of atorvastatin and rapamycin can inhibit vascular smooth muscle cell proliferation induced by oxidative stress, the inhibition effct of combination therapy is stronger than monotherapy.2) Both of atorvastatin and rapamycin can increase SOD of vascular smooth muscle cell induced by oxidative stress, the beneficial effct of combination therapy is stronger than monotherapy.3)Both of atorvastatin and rapamycin can lower MDA of vascular smooth muscle cell proliferation induced by oxidative stress, the beneficial effct of combination therapy is stronger than monotherapy.4) Both of atorvastatin and rapamycin can lower SA-β-GAL rates of vascular smooth muscle cell proliferation induced by oxidative stress, the beneficial effct of combination therapy is stronger than monotherapy.5) eNOS doesn’t express in the VSMC and drug interventions have no effect on eNOS expression, whicih indicates no correlation between attenuated oxidative stress injury of VSMC by both drugs and eNOS. |
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