盐酸乐卡地平对高血压伴动脉粥样硬化载脂蛋白E基因敲除小鼠主动脉弹力纤维降解、胶原合成的影响

Effects of lercanidipine hydrochloride on elastic lamina degradation and collagen synthesis in aortic artery of the Apo E gene knock-out hypertensive mice with atherosclerosis

目的观察盐酸乐卡地平对高血压载脂蛋白E基因敲除小鼠主动脉弹力板降解及胶原合成的影响及相关机制。方法雄性载脂蛋白E基因敲除小鼠60只,自6周龄始予以高脂饲料,8周龄时随机分为对照组(n=20)、高血压组(n=20)、盐酸乐卡地平组(n=20),高血压组和盐酸乐卡地平组小鼠8周龄时接受改良主动脉缩窄术制作高血压模型,盐酸乐卡地平组饲料中加入盐酸乐卡地平1.50mg/(kg·d),给药22周。治疗结束测定各组血压、升主动脉弹力纤维分级及胶原含量,ELISA方法测定可溶性细胞间黏附分子1(ICAM-1)水平,放射免疫法测定肾素活性,采用荧光法测定超氧化物阴离子的含量、免疫荧光的方法测定抗鼠单核细胞/巨噬细胞2(MOMA-2)单克隆抗体的表达、实时定量PCR(real-timePCR)法测定核转录因子κB的P65亚基(NFκBP65)、基质金属蛋白酶9(MMP-9)mRNA的表达。结果高血压组主动脉内膜中层厚度[(147.3±22.6)比(104.0±17.2)μm,P<0.01]、弹力纤维Ⅲ级病变[(15.2±3.7)%比(8.1±3.3)%,P<0.01]、Ⅲ型胶原含量[(23±6)%比(11±4)%,P<0.01]、主动脉超氧阴离子(5.9±0.8比3.1±0.3,P<0.01)、MOMA-2(6.1±0.5比4.0±0.3,P<0.01)的表达及NFκBP65(5.2±0.4比1.1±0.1,P<0.01)、MMP-9mRNA的表达(5.1±0.2比1.0±0.1,P<0.01)较对照组均有明显升高。与高血压组比较,盐酸乐卡地平组主动脉内膜中层厚度[(111.8±31.1)比(147.3±22.6)μm,P<0.01]、主动脉弹力纤维Ⅲ级病变[(10.9±4.1)%比(15.2±3.7)%]、Ⅲ型胶原含量[(15±5)%比(23±6)%,P<0.05],主动脉超氧阴离子(3.5±0.4比5.9±0.8,P<0.01)、MOMA-2(4.4±0.5比6.1±0.5,P<0.01)的表达及NF-κBP65(3.9±0.2比5.2±0.4,P<0.01)、MMP-9mRNA(3.8±0.2比5.1±0.2,P<0.01)的表达均降低。结论盐酸乐卡地平能够抑制高血压ApoE基因敲除小鼠弹力板降解及胶原合成,其作用与抑制氧化应激及炎症作用有关。

Objective To study the mechanism and effects of Iercanidipine on elastic lamina degradation and collagen changes in aortic artery of the Apo E gene knock-out hypertensive mice with atherosclerosis. Methods Sixty male Apo E gene knock-out mice were fed with high-fat diet at six weeks old. At 8-week old, they were randomly divid- ed into control group （n=20）, hypertension group （n=20）, and lercanidipine group （n=20）. The mice in hyper- tension group and lercanidipine hydrochloride group underwent the improved operation of aortic coarctation to induce hypertension. Mice in lercanidipine hydrochloride group took lercanidipine hydrochloride 1.50 mg/（kg 〈 d） for 22 weeks. At the end of experiment, the blood pressure,elastic lamina grading and the content of collagen of ascending aorta in each group were measured. The level of soluble intercellular adhesion molecular-1/ICAM-1 ） and rennin activity were determined by enzyme linked immunosorbent assay （ELISA） and radioimmunoassay, respectively. The level of superoxide anion, expression of anti-mice Monocytes /macrophages 2 （MOMA-2）monoclonal antibody and mRNA expression of nuclear factor-kappa B （NF-KB） P65, and matrix metalloproteinases 9 （MMP-9） were determined by fluorescence, immunofluorescence, and real-time quantitative PCR, respectively. Results In the hypertension group, the intima-media thickness, elastic lamina degradation Grade 〈l, content of collgen type ]]I, level superoxide anion, expression of MOMA-2 ,and mRNA expression of NF〈B P65 and MMP-9 mRNA were significantly increased compared with those in the control group[ （ 147.3±22.6 ） vs （ 104.0±17.2 }Um, （ 15.2 ± 3.7 ） vs { 8.1 ± 3.3 }, （23±6）% vs 11±4）, （5.9±0.81 vs （3.1±0.3）, （6.1±0.51 vs 14.0±0.3）, （5.2±0.4） vs （1.1±0.1}, { 5.1 ± 0. 2） vs { 1.0 ± 0.1 ）, respectively; all P〈0.01]. In the lercanidipine group, the intima-media thickness, e- lastic lamina degradation Gradedation of collgen type 111, level of superoxide anion,expression of MOMA-2, and mRNA expression of NFB P65 and MMP-9 were significantly decreased compared with those of the hyperten- sion group [{111.82=31.1） vs （147.3±22.6）vs, P〈0.01; {10.9±4.1） vs （15.2±3.7）, P〈0.01; （15±5）% vs （23±6）〈, P〈0.05; （3.5±0.4） vs （5.9±0.8）, P〈0.01; （4.4〈0.5） vs （6.1±0.5）, P〈0.01; （3.9± 0.2） vs （5.2±0.4）, P〈0.01; （3.8±0.2） vs （5. li0.2）, P〈0.01]. Conclusions Lercanidipine hydrochloride can significantly inhibit elastic lamina degradation and collagen synthesis in aortic artery of the Apo E gene knock-out hypertensive mice with atherosclerosis. The mechanism is related to suppressing oxidative stress and inflammation.