| Objective: To investigate the effect of ultrasound-targeted microbubble destruction(UTMD)technique combined with inhibition of glycogen synthase kinase-3β(GSK-3β)in the stability of atherosclerosis(AS)plaque.Methods: In vitro,THP-1 cells were treated with 100 ng/ml phorbol-12-myristate-13-acetate(PMA)for 48 h to differentiate THP-1 cells into macrophages,and then were incubated with 80μg/ml oxidized low-density lipoprotein(ox-LDL)for 48 h as foam cells and randomly divided into Control group,si NC group,si GSK-3β+lip2000 group(lip2000 group)and si GSK-3β+UTMD group(UTMD group);Oil Red “O” staining was used to assess the model of foam cells;cell viability was detected by the CCK-8 assay;the cytoskeleton changes of foam cells in each group was observed by the fluorescence immunocytochemistry;q RT-PCR and western blot were used to detect the m RNA level,protein expression of vulnerable plaque markers(MMP-2,MMP-9 and VEGF)and the m RNA level of the inflammatory factors(TNF-α and IL-1β).In vivo,we established AS plaque model in the right carotid artery of New Zealand rabbits by liquid nitrogen injury and high cholesterol diet,and then randomly divided into Blank group,Model group,sh NC group,sh GSK-3β group,sh GSK-3β+Sono Vue group(Sonovue group)and sh GSK-3β+UTMD group(UTMD group).We used shear wave elastography and contrast-enhanced ultrasonography to evaluate the AS plaque stability.q RT-PCR,western blot were used to detect the m RNA level and protein expression of vulnerable plaque markers(MMP-2,MMP-9 and VEGF);ELISA was used to detect the secretion of inflammatory factors in the serum of New Zealand rabbits(hs-CRP,TNF-α and IL-1β).Results: In vitro:(1)Oil Red “O” staining results revealed that the model of foam cells were successfully established and the expression of GSK-3β in foam cells was significantly higher than that in macrophages(P < 0.05);(2)m RNA levels and protein expression of GSK-3β in group UTMD were lower than those in group lip2000(P < 0.05);(3)CCK-8 test results showed no significant difference in cell viability among these 4 groups(P > 0.05);(4)q RT-PCR and western blot results showed that the m RNA levels,protein expression of vulnerable plaque factors(MMP-2,MMP-9 and VEGF)and the m RNA level of inflammatory factors(TNF-α and IL-1β)in group UTMD were significantly lower than those in group lip2000(P < 0.05);(5)the confocal results demonstrated that the fluorescence intensity of cytoskeleton proteins in group UTMD was significantly weaker than that in group lip2000,and the distribution was less concentrated.The morphology of the cytoskeleton in group UTMD changed significantly,which contained that the microtubules and microfilaments were irregularly arranged and formed the small circular dense distribution.In vivo:(1)GSK-3β m RNA level and protein expression in group UTMD were significantly lower than those in group sh GSK-3β and Sono Vue(P < 0.05);(2)as compared with group sh GSK-3β and Sono Vue,shear wave elastography and contrast-enhanced ultrasound showed the higher Young’s modulus(P < 0.05)and the lower peak intensity of AS plaque in group UTMD(P < 0.001);(3)sh GSK-3β combined with UTMD treatment reduced the expression of the vulnerable plaque markers(MMP-2,MMP-9 and VEGF)in AS plaque and the inflammatory factors(hs-CRP,TNF-α and IL-1β)in serum obviously,which got the better efficiency than those in group sh GSK-3β and Sono Vue(P < 0.05).Conclusion: By establishing the model of foam cells in vitro and the AS plaque in vivo,UTMD combined with inhibition of GSK-3β enhances the transfection efficiency and silencing effect of the target gene,which suppressed the expression of vulnerable plaque markers,inflammatory response and intraplaque angiogenesis remarkably.Moreover,the cytoskeleton of foam cells is significantly changed after silencing GSK-3β by UTMD.Above data indicates that GSK-3β may be a potential target for anti-AS and UTMD combined with GSK-3β inhibition therapy can further improve the AS plaque stability. |
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