Apoptosis Of MRSP On VSMC And Mechanisms Of MRSP On Neointimal Hyperplasia After Balloon Injury

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MRSP promotes apoptosis of vascular smooth muscle cells through the PI3K-Akt pathwayAim:Previous studies have shown that Mfn2 gene can significantly inhibit cell proliferation and promote apoptosis.The effect of Mfn2 gene related synthesized polypeptide(MRSP)based on the sequence of p21ras signature motif of Mfn2 is poorly understood.The aim of this study was to investigate the proapoptotic roles of MRSP and explore the relevant molecular mechanisms in VSMC.Methods:VSMCs were interfered with MRSP at different concentrations(10 ?M,25?M,50?M),and Cell Counting,Cell Death ELISA and TUNEL fluorescence staining were used to observe the effects of MRSP on VSMCs apoptosis.Western blot analysis was performed to determine the activation of apoptosis-related protease of caspase8,caspase9 and caspase3,and the expression level of apoptotic proteins Bax and Bcl2,the mitochondrial release of cytochrome c in each group to identify the apoptotic pathway.And we also detected the phosphorylation of PI3K and Akt to confirm that MRSP induced apoptosis through this pathway,as evidenced by the effects of 740 Y-P,the PI3K agonist and LY294002,the specific inhibitor of PI3K on VSMC apoptosis.Results:Cell counting and Cell Death ELISA results showed that MRSP obviously increased the degree of apoptosis in the range of 10-50?M with time-and dose-dependent.TUNEL results described that MRSP had a markedly apoptotic effect at 25?M concentration,and some cell nucleus disappeared at a concentration of 100?M at 12h;the apoptosis was more obvious at 24h with nucleus disappeared at 50?M.The data of western blot showed that caspase9 activation was significant and dose-dependent with no alteration of caspase8 activation,and MRSP treatment induced the elevation of cleaved caspase3,Bax/Bcl2 ratio and the release of cytochrome c from mitochondria to the cytoplasm.MRSP inhibits the action of Akt due to its phosphorylation at Ser473 in a concentration-dependent manner.Then we found that the pre-intervention with 740 Y-P partly removed MRSP-mediated inhibition of Akt phosphorylation and prevent caspase3 activation,further suppress apoptosis of VSMC.After early intervention with LY294002,MRSP-regulated inhibition of Akt phosphorylation had obvious difference with LY294002 only.Conclusion:The experimental results in this section clarify the proapoptotic effect of MRSP on VSMCs and the related mechanisms of apoptosis was further studied.The data suggested that the apoptosis role of MRSP depends on the activation of mitochondrial apoptotic signal cascade through suppression of PI3K-Akt pathway.Part ? Effect and mechanism of MRSP on restenosis of carotid artery balloon injury in ratsAim:Investigating the roles of MRSP in carotid balloon injury in rat models and verifying the mechanism of its apoptotic signaling pathway.Methods:Male SD rats(n=36;weight,300-320g)were randomly allocated into three groups:Sham group,Model group and MRSP group(10mg).The carotid artery balloon injury model was established successfully and samples were collected 7 days and 14 days after the operation.TUNEL fluorescence staining was used to detected the apoptosis of neointima after carotid balloon injury.The expression of Bax and Blc2,and caspase3 activation in carotid artery after balloon injury were detected by immunohistochemistry staining.In addition,the level of Ras,the phosphorylation of PI3K and Akt in carotid artery after vascular injury were detected by western blot.Results:The carotid intimal thickening of the model group was obvious at 7 days after balloon injury according to HE staining.MRSP treatment reduced the intimal area,lumen area and I/M ratio were significantly decreased,but exerts no effects on media area.TUNEL staining indicated that MRSP intervention increased TUNEL-positive cells and augmented the apoptotic index of vascular tissues.The results of immunohistochemistry staining revealed that the balloon injury-induced weaken in vessels MOD of Bax was markedly increased by MRSP,which has a opposite trend of reduced Bcl2.MRSP raised the expression of cleaved caspase3.WB results indicated that MRSP significantly inhibited the PI3K and Akt phosphorylation.Conclusion:MRSP promotes intimal cell apoptosis after carotid balloon injury through activating mitochondrial apoptotic signaling cascade regulated by PI3K-Akt pathway,and thus slowing the process of neointimal hyperplasia and further reducing the RS rate.Part ? Effects of MRSP on vascular inflammatory reactions after carotid balloon injuryAim:To analyze the roles of MRSP intervention on oxidative stress and vascular wall inflammation after carotid injury.Methods:Male SD rats(n=36;weight,300-320g)were randomly allocated into three groups:Sham group,Model group and MRSP group(10mg).The carotid artery samples were collected 14 days after the operation.DHE fluorescence staining is used to detected the level of superoxide anion in the artery after carotid balloon injury.Then carotid levels of MDA,SOD and GSH were determined using biochemical assays.The carotid contents of IL6,TNF?,IL-1? were assayed by ELISA kits.Western blot was performed to examine the proteins associated with inflammation response in carotid artery tissues.Results:When compared with the model group,there was decreased level in superoxide anion of MRSP treatment according to DHE fluorescence staining.Furthermore,the oxidative injury-related marker MDA was reduced and the concentration of anti-oxidative indicators including SOD and GSH were heightened after MRSP intervention.In addition,MRSP treatment decreased the expression of proinflammatory factors including IL6,TNF?,IL-1? in the carotid artery tissues and the activation of NF-?B.Conclusion:MRSP treatment not only significantly increased the ability to promote ROS clearance in the carotid arteries of rat,but also attenuated the vascular inflammatory response,indicating that the anti-RS effect of MRSP was related to amelioration of oxidative stress and reduction of inflammation.