靶向紫杉醇纳米晶-胱天蛋白酶⁃3复合物对肺动脉高压大鼠肺血管重构的影响

Effects of targeted taxol nanocrystal-caspase-3 complex on pulmonary vascular remodeling in rats with pulmonary arterial hypertension

目的观察靶向紫杉醇纳米晶-胱天蛋白酶-3(caspase-3)复合物对肺动脉高压大鼠肺血管重构的治疗效果。方法选择SPF级健康成年雄性SD大鼠56只,8周龄,体重220~250 g。随机分为七组:对照组(N组)、模型组(M组)、模型+紫杉醇组(T组)、模型+caspase-3组(C组)、模型+紫杉醇复合caspase-3组(TC组)、模型+靶向紫杉醇纳米晶组(GN1组)和模型+靶向紫杉醇纳米晶-caspase-3复合物组(GN2组),每组8只。N组不建立模型,其余组大鼠颈部皮下注射野百合碱60 mg/kg建立肺动脉高压模型。建模第21、25、29天,T组、C组、TC组、GN1组和GN2组分别经尾静脉注射紫杉醇、caspase-3、紫杉醇复合caspase-3简单混合溶液、靶向紫杉醇纳米晶、靶向紫杉醇纳米晶-caspase-3复合物(紫杉醇所用剂量为0.4 mg/kg,caspase-3为0.04 mg/kg)。于建模第35天测定大鼠平均肺动脉压力(mPAP),分别称取大鼠右心室(RV)、左心室与室间隔(LV+S)的重量,计算右心室肥厚指数[RV/(LV+S)],Western blot法检测肺组织中转录因子叉头框O1(FoxO1)蛋白含量,肺组织HE染色和α-平滑肌肌动蛋白(α-SMA)免疫组化染色评估肺血管形态,FoxO1和Ki67免疫组化染色分析其在肺血管中膜的阳性细胞百分比。结果与N组比较,M组、T组、C组、TC组、GN1组和GN2组mPAP、右心室肥厚指数、血管中膜厚度百分比和肌化血管百分比均明显升高,FoxO1蛋白含量、FoxO1阳性细胞百分比明显降低(P<0.05)。与M组比较,T组、C组、TC组、GN1组和GN2组mPAP、血管中膜厚度百分比、Ki67阳性细胞百分比明显降低,FoxO1蛋白含量明显升高,GN2组右室肥厚指数明显降低,T组、TC组、GN1组和GN2组肌化血管百分比明显降低,T组、TC组、GN1组和GN2组FoxO1阳性细胞百分比明显升高(P<0.05)。与T组比较,GN2组血管中膜厚度百分比和肌化血管百分比均明显降低;C组、TC组、GN1组和GN2组FoxO1蛋白含量明显升高;GN1组和GN2组Ki67阳性细胞百分比明显降低,FoxO1阳性细胞百分比明显升高(P<0.05)。与C组比较,GN2组血管中膜厚度百分比和肌化血管百分比均明显降低;TC组和GN2组FoxO1蛋白含量明显升高;GN1组和GN2组Ki67阳性细胞百分比明显降低,FoxO1阳性细胞百分比明显升高(P<0.05)。与TC组比较,GN2组血管中膜厚度百分比、肌化血管百分比、Ki67阳性细胞百分比均明显降低;GN1组FoxO1蛋白含量明显降低;GN1组和GN2组FoxO1阳性细胞百分比明显升高(P<0.05)。与GN1组比较,GN2组肺组织FoxO1蛋白含量明显升高,Ki67阳性细胞百分比明显降低(P<0.05)。结论靶向紫杉醇纳米晶-caspase-3复合物通过上调肺血管中膜FoxO1的表达,改善了野百合碱肺动脉高压大鼠的肺血管重构和右心室肥厚,降低肺动脉压力。

Objective To observe the therapeutic effect of targeted taxol nanocrystal-caspase-3 complex on pulmonary vascular remodeling in rats with pulmonary arterial hypertension.Methods Fifty-six healthy adult male SPF level SD rats,aged 8 weeks,weighing 220-250 g,were randomly divided into 7 groups:control group(group N),model group(group M),model+taxol group(group T),model+caspase-3 group(group C),model+taxol combined with caspase-3 group(group TC),model+targeted taxol nanocrystal group(group GN1)and model+targeted taxol nanocrystal-caspase-3 complex group(group GN2),8 rats in each group.Group N did not establish pulmonary arterial hypertension model,rats in other groups were subcutaneously injected with monocrobiline 60 mg/kg in the neck to establish pulmonary arterial hypertension model.On day 21,25 and 29 after modeling,groups T,C,TC,GN1 and GN2 were intravenously injected with taxol,caspase-3,taxol and caspase-3 mixed solution,targeted taxol nanocrystal,targeted taxol nanocrystal-caspase-3 complex(taxol 0.4 mg/kg,caspase-30.04 mg/kg).Mean pulmonary artery pressure(mPAP)of rats were measured on day 35 after modeling;right ventricle(RV)and left ventricle plus ventricular septum(LV+S)were weighed respectively to calculate RV/(LV+S);Western blot was used to detect transcription factor Forkhead box O1(FoxO1)protein content in lung tissue;HE staining and immunohistochemical staining ofα-smooth muscle actin(α-SMA)in lung tissue were used to evaluate pulmonary vascular morphology;immunohistochemical staining of FoxO1 and Ki67 were used to analyze the percentage of positive cells in pulmonary vascular medium membrane.Results Compared with group N,right ventricular hypertrophy index,the percentage of vessel media thickness and the percentage of muscularized vessels were significantly increased in groups M,T,C,TC,GN1 and GN2,FoxO1 protein content,the percentage of FoxO1 positive cells was significantly decreased(P<0.05).Compared with group M,mPAP,the percentage of vessel media thickness and Ki67 positive cells were significantly decreased in groups T,C,TC,GN1 and GN2,FoxO1 protein content was significantly increased;the right ventricular hypertrophy index was significantly decreased in group GN2;the percentage of muscularized vessels was significantly decreased in groups T,TC,GN1 and GN2;the percentage of FoxO1 positive cells was significantly increased in groups T,TC,GN1 and GN2(P<0.05).Compared with group T,the percentage of vessel media thickness and muscularized vessels were significantly decreased in group GN2;FoxO1 protein content was significantly increased in groups C,TC,GN1 and GN2;the percentage of Ki67 positive cells was significantly decreased,and the percentage of FoxO1 positive cells was significantly increased in groups GN1 and GN2(P<0.05).Compared with group C,the percentage of vessel media thickness and muscularized vessels were significantly decreased in group GN2;FoxO1 protein content was significantly increased in groups TC and GN2;the percentage of Ki67 positive cells was significantly decreased,and the percentage of FoxO1 positive cells was significantly increased in groups GN1 and GN2(P<0.05).Compared with group TC,the percentage of vessel media thickness,the percentage of muscularized vessels and the percentage of Ki67 positive cells were significantly decreased in group GN2;FoxO1 protein content in group GN1 was significantly decreased;the percentage of FoxO1 positive cells was significantly increased in groups GN1 and GN2(P<0.05).Compared with group GN1,FoxO1 protein content of lung tissue in group GN2 was significantly increased,the percentage of Ki67 positive cells was significantly decreased(P<0.05).Conclusion Targeted taxol nanocrystal-caspase-3 complex relieved pulmonary vascular remodeling,right ventricular hypertrophy and reduced pulmonary artery pressure by up-regulating the level of FoxO1 in the pulmonary vascular media in monocrotaline pulmonary arterial hypertension rats.