Effects And Molecular Mechanisms Of MRSP On Neointimal Hyperplasia In Balloon Injured Rat Carotid Artery

Part ? MRSP attenuates neointimal hyperplasia after vascular injury by inhibiting cell proliferationObject:Mfn2 is a negative regulatory factor of Ras,which can effectively inhibit proliferation of vascular smooth muscle cells?VSMCs?.We previously found that P21^rasas signature motif is the smallest functional sequence of Mfn2 on inhibition of VSMCs proliferation.We also synthesized a 16-amino acids?aa?Mfn2 Gene Related Peptide?MRSP?based on the sequence of P21^ras signature motif,which can inhibit VSMCs proliferation in vitro.The aim of this study is to explore the effects of MRSP on cell proliferation in vivo in carotid artery of rats after balloon injury.Methods:Male SD rats were divided into four groups randomly:Sham group,Model group,BSA group?2 mg?and MRSP group?2 mg?.We successfully established the carotid artery balloon injury model,and euthanized them at 7 days and 14 days after injury.Neointimal hyperplasia was observed by HE staining.Neointimal cell proliferation rate was evaluated by immunohistochemical staining of PCNA.The protein expressions of P-Raf-1,ERK1/2,P-ERK1/2,cyclin D1,and p21 were evlauated by WB.Results:The results of HE staining showed that there was no statistical difference of intimal area,media area,I/M ratio and lumen area between BSA group and Model group.MRSP reduced neointimal area and I/M ratio?P<0.01?,increased lumen area?P<0.05?,but had no influence on media area.MRSP significantly reduced proportion of PCNA positive cells at 14 days after injury?p<0.01?.Results of WB showed MRSP obviously decreased cyclin D1 protein expression and increased P21protein expression levels?p<0.01?;MRSP significantly inhibited P-raf-1 and P-ERK1/2 expression?p<0.01?.Conclusion:MRSP can significantly inhibit neointimal hyperplasia in carotid artery after balloon injury of rats by inhibiting cell proliferation,and regulate the expression of cell cycle protein cyclinD1 and P21.It is mainly mediated by inhibiting the raf-ERK1/2 signaling pathway.This finding provides experimental basis for the development of drug coating stent.Part ? MRSP attenuates neointimal hyperplasia after vascular injury by promoting cell apoptosisObject: Previous study found MRSP based on the sequence of P21 ras signature motif can promote VSMCs apoptosis.It lacks research in vivo.This research is to explore the effects and mechnisms of MRSP on cell apoptosis in vivo in carotid artery of rats after balloon injury.Methods: Male SD rats were randomly divided into four groups: Sham group,Model group,BSA group?10mg?and MRSP group?10mg?.Rats were euthanized at 14 days after injury.TUNEL test was used to evaluate apoptosis rate of neointimal cells.The protein expressions of cleaved caspase-3,Bax,Bcl-2,cleaved caspase-8,cleaved caspase-9,p-ATK,AKT were detected by WB.Vascular reendothelialization index was evaluated by immunohistochemical staining of CD31.Results: The results of TUNEL staining showed that there was no obvious apoptosis in Model and BSA group at 14 days after balloon injury,while MRSP significantly increased apoptosis rate of neointimal cells?p < 0.01?.WB test results showed that,compared with Model group and BSA group,MRSP obviously increased expression level of cleaved capase-3 and Bax/Bcl-2 ratio?p < 0.01?,which confirmed that MRSP promoted neointimal apoptosis after injury;MRSP significantly increased the expression of cleaved caspase-9?p < 0.01?,but had no influence on the expression of cleaved caspase-8,indicating that MRSP promotes mitochondrial pathway of apoptosis;MRSP significantly inhibited phosphorylation level of AKT protein?p < 0.01?.Results of CD31 immunohistochemical staining showed that MRSP did not affect the expression of CD31 and reendothelialization index?p>0.05?.Conclusion: This finding strongly suggests that MRSP significantly promotes cell apoptosis of neointima in rat carotid artery after balloon injury.It is mediated by inhibiting phosphorylation of Akt,which triggers the mitochondrial apoptosis pathway via activating caspase-9,caspase 3,and elevating Bax/Bcl-2 ratio.What is more,MRSP had no influence on reendothelialization after vascular injury.Part ? Influence of MRSP on carotid neointimal fibrosis in balloon injured rat carotid arteryObject: Neointimal fibrosis caused by collagen deposition is an important reason of restenosis after balloon injury or stents.This part is to learn the influence of MRSP on carotid intimal fibrosis after balloon injury.Methods: SD rats were divided into four groups randomly: Sham group,Model group,BSA group?2 mg?and MRSP group?2 mg?.Rats were euthanized at 21 days after injury.Neointimal hyperplasia was observed by HE staining.Neointimal fibrosis was evaluated by Masson staining.Expression of MMP2 in situ was detected by immunohistochemical staining.Results: The results of HE staining showed that there was obvious neointimal hyperplasia and lumen stenosis in Model group and BSA group.MRSP reduced neointimal area and I/M ratio?P < 0.01?,increased lumen area?P < 0.01?,but had no influence on media area.Results of Masson staining showed that MRSP significantly reduced the area of collagen?p<0.01?.Expression of MMP2 in situ was decreased by MRSP evaluated by immunohistochemical staining of MMP2?p<0.01?.Conclusion: MRSP decreases collagen membrane area and inhibits carotid neointimal fibrosis at 21 days after balloon injury,which may be mediated by inhibiting the expression of MMP2.This finding further proved the superiority of MRSP on inhibiting neointimal hyperplasia.