基于PI3K/AKT/eNOS通路研究芪丹通脉片对ApoE^(-/-)小鼠动脉粥样硬化易损斑块的影响

Effect of Qidan Tongmai Tablet on Atherosclerotic Vulnerable Plaque in ApoE^(-/-) Mice Based on PI3K/AKT/eNOS Pathway

目的:基于磷脂酰肌醇-3-激酶(phosphatidylinositol-3-hydroxykinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)通路探索芪丹通脉片对ApoE^(-/-)小鼠动脉粥样硬化易损斑块的影响。方法:从72只SPF级雄性ApoE^(-/-)小鼠中随机选取12只作为对照组,其余小鼠采用颈总动脉外置管术联合高脂饲料建立小鼠易损斑块模型。将造模成功的60只小鼠随机分为模型组、黄芪甲苷组(40 mg·kg^(-1))、丹参酮ⅡA组(90 mg·kg^(-1))、黄芪甲苷+丹参酮ⅡA组、芪丹通脉片组(700 mg·kg^(-1)),每组12只,给予相应药物灌胃12周,每日1次,对照组和模型组给予等体积生理盐水。末次灌胃后,处死小鼠并分离右颈总动脉,TUNEL染色观察细胞凋亡情况,免疫荧光染色检测CD68、α-actin蛋白表达水平,RT-PCR检测eNOS mRNA表达水平,Western Blot检测p-PI3K/PI3K、p-AKT/AKT、eNOS、基质金属蛋白酶(matrix metalloprotein-9,MMP-9)蛋白表达水平。结果:TUNEL结果显示:对照组平滑肌细胞几乎没有凋亡,模型组平滑肌细胞凋亡最多;与模型组比较,各给药组平滑肌细胞凋亡明显减少;黄芪甲苷+丹参酮ⅡA组、芪丹通脉片组平滑肌细胞凋亡较黄芪甲苷组、丹参酮ⅡA组有所减少。CD68、α-actin免疫荧光结果显示:对照组几乎没有巨噬细胞,而平滑肌细胞数量最多;与对照组比较,模型组巨噬细胞数量明显增多,而平滑肌细胞数量显著减少;与模型组比较,各给药组巨噬细胞数量明显减少,而平滑肌细胞数量明显增多;与黄芪甲苷组、丹参酮ⅡA组比较,黄芪甲苷+丹参酮ⅡA组、芪丹通脉片组巨噬细胞数量明显减少,而平滑肌细胞数量明显增多。RT-PCR结果显示:与对照组比较,模型组eNOS mRNA表达水平显著降低(P<0.05);与模型组比较,各给药组eNOS mRNA表达水平显著升高(P<0.05);与黄芪甲苷组、丹参酮ⅡA组比较,黄芪甲苷+丹参酮ⅡA组、芪丹通脉片组eNOS mRNA表达水平显著升高(P<0.05)。Western Blot结果显示:与对照组比较,模型组p-PI3K/PI3K、p-AKT/AKT、eNOS蛋白表达水平显著降低,MMP-9蛋白表达水平显著升高(P<0.05);与模型组比较,各给药组p-PI3K/PI3K、p-AKT/AKT、eNOS蛋白表达水平显著升高,MMP-9蛋白表达水平显著降低(P<0.05);与黄芪甲苷组、丹参酮ⅡA组比较,黄芪甲苷+丹参酮ⅡA组、芪丹通脉片组p-PI3K/PI3K、p-AKT/AKT、eNOS蛋白表达水平显著升高,MMP-9蛋白表达水平显著降低(P<0.05)。结论:芪丹通脉片能够稳定动脉粥样硬化易损斑块,其机制可能与激活PI3K/AKT/eNOS信号通路有关。

Objective:To explore the effect of Qidantong Maipian on vulnerable atherosclerotic plaques in ApoE^(-/-)mice based on the pathway of phosphatidylinositol-3-hydroxykinase(PI3K)/protein kinase B(AKT)/endothelial nitric oxide synthase(eNOS).Method:Twelve male ApoE^(-/-)mice of SPF grade were randomly selected as the control group(Group A),while the remaining mice were subjected to common carotid artery catheterization combined with high-fat diet to establish a mouse vulnerable plaque model.Sixty successfully modeled mice were randomly divided into a model group(Group B),an astragalosideⅣgroup(Group C,40 mg·kg^(-1)),a tanshinoneⅡA group(Group D,90 mg·kg^(-1)),an astragalosideⅣ+tanshinoneⅡA group(Group E),and a Qidan Tongmai tablet group(Group F,700 mg·kg^(-1)),with 12 mice in each group.The corresponding drugs were administered orally for 12 weeks,once a day.The control group and model group were given equal volumes of physiological saline.After the last gavage,the mice were euthanized and the right common carotid artery was separated.TUNEL staining was used to observe cell apoptosis,and immunofluorescence staining was used to detect the expression level of CD68 andα-actin,RT-PCR detection of eNOS mRNA expression,Western Blot detection of p-PI3K,p-AKT,eNOS,and matrix metalloproteinase-9(MMP-9)protein expression levels.Results:TUNEL results showed that group A had almost no apoptosis in smooth muscle cells,while group B had the highest apoptosis in smooth muscle cells;Compared with group B,the apoptosis of smooth muscle cells in the C-F group was significantly reduced;E.The apoptosis of smooth muscle cells in group F was reduced compared to groups C and D.CD68 andα-actin immunofluorescence results showed that Group A had almost no macrophages,while the number of smooth muscle cells was the highest;Compared with group A,the number of macrophages in group B significantly increased,while the number of smooth muscle cells significantly decreased;Compared with group B,the C-F group showed a significant decrease in macrophages and a significant increase in smooth muscle cells;Compared with C and D groups,E and F groups showed a significant decrease in macrophages and a significant increase in smooth muscle cells.The RT-PCR results showed that compared with group A,the expression of eNOS mRNA in group B was significantly reduced(P<0.05);Compared with Group B,the expression of eNOS mRNA in Group C-F was significantly increased(P<0.05);Compared with groups C and D,the expression of eNOS mRNA in groups E and F was significantly increased(P<0.05).Western blot results showed that compared with group A,the expression of p-PI3K,p-AKT,and eNOS proteins in group B was significantly reduced,while the expression of MMP-9 protein was significantly increased(P<0.05);Compared with Group B,the expression of p-PI3K,p-AKT,and eNOS proteins in Group C-F was significantly increased,while the expression of MMP-9 protein was significantly reduced(P<0.05);Compared with groups C and D,the expression of p-PI3K,p-AKT,and eNOS proteins in groups E and F was significantly increased,while the expression of MMP-9 protein was significantly reduced(P<0.05).Conclusion:Qidan Tongmai Tablet can stabilize vulnerable atherosclerotic plaque,and its mechanism may be related to the activation of PI3K/AKT/eNOS signaling pathway.