盐酸埃他卡林预防压力超负荷大鼠心血管重构作用及其可能的内皮机制
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摘要
背景:
高血压是最常见的心血管疾病,其主要病理生理学特征表现为压力超负荷(pressure overload)和心血管重构(cardiovascular remodeling),包括血管重构(vascular remodeling)和心脏重构(cardiac remodeling)。大量的临床研究表明,高血压心肌重构不但可导致心室的收缩及舒张功能障碍,还是心肌梗塞、甚至心衰死亡的重要独立危险因素。因此能否逆转高血压所致的心血管重构,从而降低严重心脏事件的发生率和病死率,已成为临床高血压治疗的关键,开发新型的抗高血压、抗心血管重构的新药仍是心血管领域研究的热点之一。
方法:
在体实验:雄性SD大鼠,体重180～220 g。麻醉后于左肋弓下缘纵切口,分离出腹主动脉后用外径为0.7 mm的小圆棒和腹主动脉一起结扎形成腹主动脉缩窄模型,诱导心肌肥大和重构。
离体实验:取新生的1～3d的SD大鼠乳鼠心脏,消化后行心肌细胞原代培养。用异丙肾上腺素(isoproterenol ,ISO)诱导心肌细胞肥大,观察ISO对心肌细胞蛋白质含量和心肌细胞内游离Ca~(2+)的影响。
第一部分盐酸埃他卡林(iptakalim, IPT)对压力超负荷大鼠心血管重构的作用大鼠随机分为6组:⑴假手术对照组;⑵模型对照组;⑶IPT 1 mg/kg组;⑷IPT 3 mg/kg组;⑸IPT 9 mg/kg组;⑹L isenopril 15 mg/kg组。其中假手术对照组大鼠只行手术通路,不结扎腹主动脉,其余手术操作均和手术组相同。其它各组大鼠均做模型处理。各组药物均溶于NS中,于术后第3天灌胃给药,给药容积为0.5 ml/l00 g体重。每天1次,假手术对照组和模型对照组仅给予等体积普通蒸馏水,连续灌胃给药6周。实验结束后八导生理记录仪观察血流动力学和心脏功能的变化,HE染色和Masson’s染色观察心血管组织病理学改变,RT-PCR检测心肌肥大的标志基因——心钠素(atrial natriuretic peptide, ANP)和脑钠素(brain natriuretic peptide, BNP)的表达。
第二部分:IPT逆转心血管重构的内皮机制
(一)内皮素系统在压力超负荷大鼠模型中的作用
大鼠随机分为5组:⑴假手术对照组;⑵模型对照组;⑶IPT 1 mg/kg组;⑷IPT 3 mg/kg组;⑸IPT 9 mg/kg组。手术方案同第一部分。放射免疫法测定血浆中内皮素-1(endothelin-1, ET-1)的含量,RT-PCR观察心肌组织ET-1和内皮素转化酶(endothelin converting enzyme, ECE)的表达,免疫组化法检测心肌组织ETA和ETB的表达。
(二) eNOS-NO信号通路在压力超负荷大鼠模型中的作用
大鼠随机分为5组:⑴假手术对照组;⑵模型对照组;⑶I PT 3 mg/kg组;⑷左旋硝基精氨酸甲酯(N G-nitro-L- arginine methyl ester, L-NAME) 50 mg/kg (L-NAME 50)组;⑸IPT 3 mg/kg + L-NAME 50 mg/kg (IPT 3+L-NAME 50)组。手术方案同第一部分。实验结束后八导生理记录仪观察血流动力学和心脏功能的变化,HE染色和Masson’s染色观察心肌组织病理学改变,RT-PCR心肌组织内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS )mRNA的表达,免疫组化法检测心肌组织eNOS的表达,生化法观察血浆中一氧化氮(nitric oxide, NO)含量的变化。
(三)前列环素(prostacyclin, PGI_2)在压力超负荷大鼠模型中的作用
大鼠随机分为5组:⑴假手术对照组;⑵模型对照组;⑶IPT 3 mg/kg组;⑷i ndomethacin 2 mg/kg (Indo 2)组;⑸IPT 3mg/kg + indomethacin 2 mg/kg (IPT 3+Indo 2)组。手术方案同第一部分。实验结束后八导生理记录仪观察血流动力学和心脏功能的变化,HE染色和Masson’s染色观察心肌组织病理学改变,放射免疫法观察血浆中PGI_2含量的变化。
第三部分:IPT对异丙肾上腺素诱导的培养心肌细胞肥大的影响
1:确定ISO诱导心肌肥大的最适浓度。将培养72 h后的心肌细胞按1×105/孔接种到24孔板中,随机分为6组,每组设4个复孔。⑴Control;⑵ISO 1×10-8 mol/L;⑶1×10~(-7) mol/L;⑷1×10~(-6) mol/L;⑸1×10~(-5) mol/L;⑹1×10~(-4) mol/L。培养48h后,Lowry’s法测定心肌细胞蛋白含量;2:不同浓度IPT对ISO诱导的肥大心肌细胞总蛋白含量的影响。将培养72 h后的心肌细胞按1×105/孔接种到24孔板中,随机分为6组,每组设4个复孔。⑴Control;⑵ISO 1×10~(-5)mol/L;⑶ISO 1×10~(-5)mol/L+IPT 1×10~(-7)mol/L;⑷ISO 1×10~(-5)mol/L+IPT 1×10~(-6)mol/L;⑸ISO 1×10~(-5)mol/L+IPT1×10~(-5)mol/L;⑹ISO 1×10~(-5)mol/L+IPT 1×10~(-4)mol/L。培养48h后,Lowry’s法测定心肌细胞蛋白含量;3:将培养72 h后的心肌细胞按1×105/孔接种到24孔板中,随机分为6组,每组设4个复孔。⑴Control;⑵ISO 1×10~(-5)mol/L;⑶ISO 1×10~(-5)mol/L+IPT 1×10~(-7)mol/L;⑷ISO 1×10~(-5)mol/L+IPT 1×10~(-6)mol/L;⑸ISO 1×10~(-5)mol/L+IPT 1×10~(-5)mol/L;⑹ISO 1×10~(-5)mol/L+IPT 1×10~(-4)mol/L。培养48h后,激光共聚焦显微镜测定每组心肌细胞Ca2+含量,每组取20个细胞。
结果:
第一部分IPT对压力超负荷大鼠心血管重构的作用
血压:整个实验6 wk内,假手术组收缩压(systolic blood pressure, SBP)基本保持稳定,模型组大鼠血压呈时间依赖性增加,各时间点血压均比基础值升高约1.5 kPa左右,IPT 1,3,9 mg/kg/d和Lisenopril 15 mg/kg/d治疗自试验第2周起,可以明显降低大鼠尾动脉SBP。腹主动脉缩窄6 wk后,与假手术组相比,模型组大鼠颈动脉SBP,舒张压(diastolic blood pressure, DBP),平均动脉压(mean blood pressure, MBP)和腹主动脉跨狭窄部位收缩压压力差(Transtenosis gradient of SBP)都明显增高(P<0.01 vs Sham组)。经过IPT 1,3,9 mg/kg/d治疗6 wk后,各组大鼠颈动脉SBP,DBP,MBP,腹主动脉跨狭窄部位收缩压压力差呈剂量相关性降低。
心功能:与假手术组相比,模型组大鼠的左室收缩压(left ventricular systolic pressure, LVSP),左室压力最大上升速率(maximal rate of left ventricular systolic pressure, +dp/dtmax),左室压力最大下降速率(maximal rate of left ventricular diastolic pressure, -dp/dtmax),心肌纤维缩短速度的生理最大值(Vmp),心肌纤维缩短速度的理论最大值(Vmax)均明显增加,表明为了适应腹主动脉缩窄后后负荷的增加,模型组大鼠动用了左心室的心力储备,收缩功能明显增强(P<0.01 vs Sham组),而左心室舒张末期压(left ventricular end diastolic pressure, LVEDP)和假手术组相比明显降低,表明模型组大鼠左心室的舒张功能减弱,心肌的顺应性降低。而经过IPT 1,3,9 mg/kg/d治疗6 wk后,各组大鼠的心室舒缩功能明显改善。
心血管重构:腹主动脉缩窄后6 wk,可引起全心指数(heart weight/body weight, HW/BW)、左心指数(left ventricular weight/body weight, LVW/BW)、右心指数(right ventricular weight/body weight, RVW/BW)和肺指数(lung weight/body weight, LW/BW)都明显增加,表明心脏发生了大体上的重构,模型组大鼠心肌细胞横截面积(cardiomyocyte cross-section area)、心肌间质胶原容积分数(collagen volume fraction, CVF)、心肌血管周围胶原面积(perivscular collagen area, PVCA)心肌的羟脯氨酸含量(hydroxyproline)和对照组相比明显增加(P<0.05,P<0.01 vs Sham组)。与假手术组相比,模型组大鼠主动脉的血管总面积(total artery area, TAA)、管腔总面积(lumen area, LA)、血管管壁面积(cross-section area, CSA)、CSA/TAA、血管平均直径(artery diameter, AD)、血管管壁平均厚度(media)以及M/L等参数均明显增加,表明模型组大鼠的主动脉发生了明显的重构(P<0.01 vs Sham组)。腹主动脉缩窄6 wk后,大鼠心肌组织ANP、BNP mRNA的表达明显上调(P<0.01 vs Sham组)。经IPT 1,3,9 mg/kg/d治疗后,各剂量组均能明显降低心脏指数和肺指数,可剂量相关地抑制心肌细胞横截面积增加,分别为41.54%,43.59%,44.10%(P<0.01 vs Control组);可剂量相关地抑制CVF的升高,分别为35.71%,73.21%(P<0.01 vs Control组),87.98%(P<0.01 vs Control组);可剂量相关地抑制PVCA的升高,分别为34.96%,56.50%(P<0.05 vs Control组),66.26%(P<0.05 vs Control组);可剂量相关地抑制大鼠心肌的羟脯氨酸含量的升高,抑制率分别为56.72%,92.53%(P<0.05 vs Control组)和92.53%(P<0.05 vs Control组),可剂量相关地下调大鼠心肌组织ANP、BNP mRNA的表达(P<0.05, P<0.01 vs Control组)。
第二部分:IPT逆转心血管重构的内皮机制
(一)内皮素系统在压力超负荷大鼠模型中的作用
腹主动脉缩窄6 wk后,大鼠血浆ET-1的含量比假手组明显升高(101.97±19.64 pg/ml vs 135.27±36.91 pg/ml,P<0.05);大鼠心肌组织ET-1、ECE mRNA的表达明显上调(P<0.01 vs Sham组);大鼠心脏的内皮素受体A型(endothelin-1 receptor A type, ETA)、内皮素受体B型(endothelin-1 receptor Btype, ETB)的表达明显上调(P<0.01 vs假手术组)。IPT 1、3、9 mg/kg治疗6 wk后,可剂量相关地抑制大鼠血浆ET-1的升高,分别为(120.11±40.06)pg/ml、(95.98±28.27)pg/ml(P<0.05 vs Control组)和(78.01±21.77)pg/ml(P<0.01 vs Control组);可剂量相关地下调大鼠心肌组织ET-1、ECE mRNA的表达(P<0.05,P<0.01 vs模型组);可剂量依赖性性地下调大鼠心脏ETA、ETB的表达(P<0.05,P<0.01 vs模型组)。
(二) eNOS-NO信号通路在压力超负荷大鼠模型中的作用
血压:在6 wk的实验期间,单独应用L-NAME大鼠的SBP随时间逐渐升高,第2 wk、第4 wk、第6 wk的SBP分别为20.6±2.2、20.8±1.7、21.2±1.4 kPa,与同期模型组的SBP 17.9±2.5、18.7±1.2、18.0±1.2 kPa相比差异有显著性(P<0.05,P<0.01)。而IPT 3+L-NAME 50组和单用L-NAME组相比,在第2 wk、第4 wk、第6 wk能分别降低SBP约10.4%、15.1%和10.7%(P<0.0l)。
心功能:经过6 wk的内源性的NOS抑制后,L-NAME 50组大鼠心室的收缩功能和假手术组相比明显增强,而心室的舒张功能和顺应性明显降低。合用IPT,能明显降低ASBP和LVSP(P<0.05)。此外,我们还发现了一个有趣的现象,L-NAME 50mg/kg/d无论是单独应用还是和IPT合用,都导致了比模型组大鼠还要严重的高血流动力学状态,但却未发生大体形态学上的心肌肥大。
心脏重构:腹主动脉缩窄6 wk后,无论是单用L-NAME还是和IPT合用,2组大鼠的心脏从大体上和病理学检查上都未发现明显的肥大(vs Sham组)。单用L-NAME组的心肌纤维化程度均比假手术组明显升高,心肌组织羟脯氨酸含量明显增加(P<0.01 vs Sham组),甚至诱发了比模型组更严重的胶原沉积,合用IPT可以改善单独应用L-NAME导致的心肌纤维化,分别为0.87%(CVF)和4.3%(PVCA),明显抑制心肌组织羟脯氨酸含量明显增加(P<0.01 vs L-NAME 50)。电镜结果显示,假手术组大鼠心肌肌丝排列整齐、连续,线粒体和心肌纤维呈平行排列,线粒体嵴突排列整齐,Z线清晰。IPT 3+L-NAME 50组的心肌纤维平行排列,Z线均匀分隔肌小节,卵圆形的线粒体呈列分布在肌纤维之间。而单独用L-NAME组的心肌细胞可以看到无论使线粒体的数量还是体积都比假手术组和IPT 3+L-NAME 50组明显增加。提示当内源性的NOS被抑制后,心肌细胞发生了亚细胞结构的重构,表现为线粒体数量的增加(hyperplasia)和体积的增加(hypertrophy)。IPT可以改善这种亚细胞结构的重构。
eNOS-NO:腹主动脉缩窄6 wk后,血浆中的NO含量明显低于假手术组(21.56±8.06μmol/L vs 13.89±1.59μmol/L,P<0.05)。IPT1,3,9 mg/kg可以剂量相关地提高血浆中NO含量(P<0.05,P<0.01 vs Control组)。并且血浆中的NO含量和左心室重构指数、心肌胶原含量、SBP(tail-cuff)呈明显的负相关。模型组大鼠心肌组织的eNOS mRNA和eNOS蛋白表达水平和假手术组相比明显下调。经过IPT治疗6 wk后eNOS mRNA和eNOS蛋白表达水平和模型组相比明显增加。单独应用L-NAME 6 wk后,大鼠心肌组织的eNOS mRNA和eNOS蛋白表达被明显抑制,合用IPT后能明显提高被抑制的eNOS mRNA和eNOS蛋白的表达。
(三)PGI_2在压力超负荷大鼠模型中的作用
血压:单用indomethacin组SBP(tail-cuff)呈时间依赖性升高,颈动脉收缩压(right carotid aorta systolic blood pressure, ASBP)和跨狭窄部位收缩压差值也明显增加,合用IPT后只能部分而不能完全改善indomethacin诱导的高血流动力学状态,和模型组相比差异无显著性。
心功能:单用吲哚美辛Indo 2组的心脏收缩功能明显增加,心肌的顺应性明显下降,合用IPT后能明显抑制心肌收缩力的提高,改善心肌的顺应性。心脏重构:腹主动脉缩窄6 wk后,单用吲哚美辛和假手术组相比HW/BW,LVW/BW和RVW/BW比值明显增加,心肌细胞横截面积亦明显增加,合用IPT后只能部分而不能完全改善心肌细胞的肥大。单用Indomethacin后,心肌组织的CVF、PVCA、心肌组织羟脯氨酸含量明显增加(P<0.05,P<0.01 vs Sham组),合用IPT后可明显抑制CVF和PVCA的恶化(P<0.05,P<0.01 vs Indo 2)。明显抑制心肌组织羟脯氨酸含量的增高(P<0.05 vs Indo 2)。
PGI_2:腹主动脉缩窄6 wk后,模型组血浆中PGI_2含量和假手术组相比明显降低(63.87%,P<0.05 vs Sham组),IPT 1,3,9 mg/kg可以剂量相关地提高血浆中PGI_2含量(P<0.05 vs Control组)。Indo 2组血浆的PGI_2含量和假手术组相比明显降低,和模型组相比也降低了19%,合用IPT后能明显提高血浆中PGI_2的含量(P<0.05 vs Indo 2组)。
第三部分:IPT对异丙肾上腺素诱导的培养心肌细胞肥大的影响
1:与对照组相比,ISO1×10~(-5)mol/L组和ISO1×10-4mol/L组的心肌细胞蛋白含量分别增加了24.53%和31.74%(P<0.01),二者之间没有明显差别,说明1×10~(-5)mol/L ISO可以完全激活β-肾上腺素受体(β-adrenergic receptor,β-AR),使心肌细胞蛋白含量显著增加;2:与对照组相比,ISO组心肌细胞蛋白含量增加了39.62%,IPT 1×10~(-7)mol/L组、1×10~(-6)mol/L组、1×10~(-5)mol/L组、1×10-4mol/L组可剂量依赖性地抑制ISO诱导的心肌细胞肥大和蛋白含量的增加,抑制率分别为52.38%、85.71%、95.24%、95.24%。3:与对照组相比,ISO组心肌细胞荧光强度(fluorescence intensity)明显增加(15.18±10.99 vs 125.82±45.56,P<0.01),IPT 1×10~(-7)mol/L组、1×10~(-6)mol/L组、1×10~(-5)mol/L组、1×10-4mol/L组可剂量依赖性地抑制ISO诱导的心肌细胞荧光强度的增加,抑制率分别为26.36%、31.82%、70.91%、90.91%。
结论:
1 IPT 1,3,9 mg/kg组可以明显逆转腹主动脉缩窄/压力超负荷大鼠的心血管重构,并且这种逆转作用呈现一定的剂量依赖性;
2 IPT逆转心血管重构的作用可能与作用于内皮细胞上的KATP通道,恢复内皮细胞的分泌功能密切相关,即增加NO和PGI_2的分泌,抑制ET-1的合成、分泌,抑制内皮素系统的活化;
3在培养新生乳鼠心肌细胞上,IPT可以剂量依赖性地抑制ISO诱导的心肌细胞肥大和蛋白质含量的增加,其作用机制可能与降低细胞膜静息膜电位,抑制Ca~(2+)内流有关。
Background:
Hypertension is a substantial public health problem, the major pathophysiological character is pressure overload and cardiovascular remodeling, including vascular remodeling and cardiac remodeling. A lot of clinical investigations reveal that essential hypertension and consequent left ventricular hypertrophy not only contribute to ventricular systolic and diastolic dysfunction, but also have been recognized as a strong, virtually independent risk factor. So the desirability to prevent and induce regression of LVH by drug treatment is widely understood, and a variety of antihypertensive drugs has been shown to cause regression of LVH in treated hypertensives. However, the advantages and disadvantages of these drugs have been debated and there is an impetus for the development of new chemical entities with a more desirable therapeutic endpoint, decreasing morbidity and mortality of hypertensives.
Methods:
In vivo: Adult male Sprague–Dawley rats (weight range 180–220 g), after rats were anesthetized, under sterile conditions, the abdominal aorta was exposed through a abdominal incision and constricted at the suprarenal level by a 4-0 silk suture tied around both the aorta and a blunted 22-gauge needle, which was then pulled out. A similar procedure was performed for Sham group except without the ligature.
In vitro: Sprague–Dawley rats neonatal ventricular myocytes were isolated and cultured. Cardiomyocytes were exposed to isoproterenol (ISO) for 48 h, and the total protein content of the cardiomyocytes was measured. Laser scanning confocal microscope was used to determine the [Ca~(2+)]_i of cardiomyocytes.
PartⅠThe experimental therapeutic effects of iptakalim on cardiovascular protection in pressure-overload rats
Animals were divided into 6 groups:⑴Sham group;⑵C ontrol group;⑶IPT 1 mg/kg group;⑷IPT 3 mg/kg group;⑸IPT 9 mg/kg group;⑹Lisenopril 15 mg/kg group. The drugs were dissolved in distilled water and administered orally via a gastric tube. Drugs were administered from 3 days after operation through 6 weeks after surgery. At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson’s stain. RT-PCR method was used to measure the cardiac hypertrophy marker gene, ANP and BNP mRNA expression.
PartⅡIPT reverse cardiovascular remodeling: Endothelial mechanism
1 Role of endothelin system in pressure-overload rats
Animals were divided into 5 groups:⑴Sham group;⑵C ontrol group;⑶IPT 1 mg/kg group;⑷IPT 3 mg/kg group;⑸I PT 9 mg/kg group. Radioimmunoassay was used to determine the serum ET-1 content, RT-PCR method was used to measure the cardiac ET-1 and ECE mRNA expression, immunochemistry method was used to evaluate the cardiac ETA and ETB expression.
2 Role of eNOS-NO signal pathway in pressure-overload rats
Animals were divided into 5 groups:⑴Sham group;⑵C ontrol group;⑶IPT 3 mg/kg group;⑷L-NAME 50 mg/kg group;⑸L -NAME 50 mg/kg + IPT 3 mg/kg group. At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson’s stain. RT-PCR method was used to measure the cardiac eNOS mRNA expression, immunochemistry method was used to evaluate the cardiac eNOS expression, and biochemical method was used to determine serum nitric oxide content.
3 Role of PGI_2 in pressure-overload rats
Animals were divided into 5 groups:⑴Sham group;⑵C ontrol group;⑶IPT 3 mg/kg group;⑷i ndomethacin 2 mg/kg group (Indo 2);⑸indomethacin 2 mg/kg + IPT 3 mg/kg group(Indo 2+IPT 3). At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson’s stain. Radioimmunoassay was used to determine the serum PGI_2 content.
PartⅢEffect of ISO on cultured cardiomyocytes hypertrophy induced by ISO
1: Ascertain the appropriate concentration of ISO inducing cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plates for 72 h, 1×105/well, and then divided into 6 groups:⑴Control group;⑵ISO 1×10-8 mol/L;⑶ISO 1×10~(-7) mol/L;⑷I SO 1×10~(-6) mol/L;⑸ISO 1×10~(-5) mol/L;⑹I SO 1×10~(-4) mol/L. After 48 h, Lowry’s method was used to determine the cardiomyocytes total protein content. 2: Effect of IPT on cardiomyocytes total protein content in ISO-induced cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plate for 72 h, 1×105/well, then divided into 6 group:⑴Control group;⑵ISO 1×10~(-5) mol/L;⑶ISO 1×10~(-5) mol/L+IPT 1×10~(-7) ;⑷ISO 1×10~(-5) mol/L+IPT 1×10~(-6);⑸ISO 1×10~(-5) mol/L+IPT 1×10~(-5);⑹ISO 1×10~(-5) mol/L+IPT 1×10~(-4). After 48 h, Lowry’s method was used to determine the cardiomyocytes total protein content. 3: Effect of IPT on cardiomyocytes [Ca2+]i in ISO-induced cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plate for 72 h, 1×105/well, then divided into 6 group:⑴Control group;⑵I SO 1×10~(-5) mol/L;⑶I SO 1×10~(-5) mol/L+IPT 1×10~(-7) ;⑷ISO 1×10~(-5) mol/L+IPT 1×10~(-6);⑸ISO 1×10~(-5) mol/L+IPT 1×10~(-5);⑹ISO 1×10~(-5) mol/L+IPT 1×10~(-4). After 48 h, laser scanning confocal microscope was used to determine [Ca_~(2+)]_i of cardiomyocytes, 20 cells were selected in each group.
Results:
PartⅠThe experimental therapeutic effects of iptakalim on cardiovascular protection in pressure-overload rats
Blood pressure: During the time course, tail artery systolic blood pressure(SBP) in Sham group maintain at the baseline level, tail artery SBP in Control group increased significantly with a time-dependent manner, IPT 1,3,9 mg/kg/d and Lisenopril 15 mg/kg/d treatment inhibited the increase of tail artery SBP markedly from 2 week. At the end of 6 week, carotid aorta SBP, DBP, MBP and transtenosis gradient of SBP increased significantly with a time-dependent manner(P<0.01 vs Sham group), IPT 1,3,9 mg/kg/d and Lisenopril 15 mg/kg/d treatment inhibited the increase of carotid aorta SBP, DBP, MBP and transtenosis gradient of SBP markedly.
Heart function: After 42 d of aortic banding, LVSP,±dp/dtmax,Vmp,Vmax increased significantly, while left ventricular LVEDP decreased significantly compared with Sham group (P<0.01). IPT 1, 3, 9 mg/kg/d and Lisenopril 15 mg/kg/d treatment ameliorated left ventricular systolic and diastolic function, augmented cardiac compliance markedly.
Cardiovascular remodeling:After 42 d of aortic banding, heart weight/body weight(HW/BW), left ventricular weight/body weight(LVW/BW), right ventricular weight/body weight(RVW/BW) , lung weight/body weight(LW/BW) total area of total aorta(TAA), area of lumen(LA), cross-section area(CSA), CSA/TAA, aorta radium(AR), media(M) and M/L increased significantly, indicating a morphological remodeling of heart and aortic remodeling. Cardiomyocytes cross-section area, CVF, PVCA and cardiac tissue hydroxyproline increased significantly compared with Sham group(P<0.05,P<0.01). ANP and BNP mRNA expression increased significantly in Control group rats compared with that in Sham group rats(P<0.01). After treatment with IPT 1,3,9 mg/kg/d, HW/BW, LVW/BW, RVW/BW, LW/BW, cardiomyocyte cross-section area(41.54%,43.59%,44.10%, P<0.01 vs Control group),CVF(35.71%,73.21%,P<0.01 vs Control group), PVCA(34.96%,56.50%,P<0.05 vs Control group),cardiac tissue Hydroxyproline content(56.72%,92.53%,P<0.05 vs Control group) were reversed ,respectively. Cardiac ANP and BNP mRNA expression were downregulated significantly(P<0.05, P<0.01 vs Control group).
PartⅡIPT reverse cardiovascular remodeling: Endothelial mechanism
1 Role of endothelin system in pressure-overload rats
After 42 d of aortic banding, plasma ET-1 content was increased (101.97±19.64 pg/ml vs 135.27±36.91 pg/ml,P<0.05 vs Sham group), cardiac tissue ET-1、ECE mRNA and ETA、ETB protein expression were upregulated in Control group(P<0.01). After treatment with IPT 1,3,9 mg/kg/d, plasma ET-1 content was decreased significantly(P<0.01 vs Control group), the upregulation of cardiac tissue ET-1、ECE mRNA and ETA、ETB protein expression were inhibited markedly(P<0.05,P<0.01 vs Control group).
2 Role of eNOS-NO signal pathway in pressure-overload rats
Blood pressure: Chronic L-NAME administration caused persistent severe hypertension. The SBP (of the 2nd, 4th, 6th weeks) were 20.6±2.2, 20.8±1.7 and 21.2±1.4 kPa versus 17.9±2.5, 18.7±1.2 and 18.0±1.2 kPa in Control group rats, respectively (P<0.05, P<0.0l). SBP of L-NAME 50 + iptakalim 3 group in the 2nd, 4th, 6th weeks had 10.4%, 15.1%, 10.7% lower than that of rats given L-NAME alone, respectively(P<0.0l).
Heart function: After 42 days of NO synthase inhibition, ASBP,LVSP,±dp/dtmax were significantly increased(P<0.05,P<0.01 vs Sham group), while LVEDP decreased markedly (P<0.01 vs Sham group), indicating an enhanced contractility but a reduced diastolic compliance were taken place, and iptakalim could statistically or partially ameliorate the LV function(P<0.05 versus L-NAME 50 group). Interestingly, in L-NAME 50 mg/kg/d rats (whether iptakalim was given or not), the LVW/BW and RVW/BW of rats were not statistically different from those of Sham group, despite of the significant increase in blood pressure.
Cardiac remodeling: Histological examination demonstrated that both CVF, PVCA and cardiac tissue hydroxyproline revealed significant difference in both L-NAME 50 mg·kg-1·d-1 and IPT 3 mg·kg-1·d-1+L-NAME 50 mg·kg-1·d-1 compared to that of Sham group, and combined with IPT 3 ameliorated cardiac tissue hydroxyproline compared with that of L-NAME 50 alone. However, the extent of myocyte hypertrophy of rats given IPT 3 mg/kg/d +L-NAME 50 mg/kg/d or given L-NAME 50 mg/kg/d alone were not statistically different from those of Sham group. Ultrastructural examination revealed well-organized myofibrils with mitochondria grouped along the periphery of longitudinally oriented fibers were found in Sham group rats, mitochondria tightly packed, intact horizontally oriented cristae in Sham group rats, well-preserved intercalated disk with smooth contours in Sham group rats. The cardiomyocytes in iptakalim 3+L-NAME 50 group showed almost uniform parallel myofibril arrangement. Z-lines dividing the sarcomeres were linear and perpendicular to the myofilaments. Myofibrils alternated with few rows of ovoid mitochondria. However, the number of mithochondria in the latter group seems to be larger. Ultrastructure of the cardiomyocytes from L-NAME-treated rats presented mitochondrial changes, characterized by diffuse mild to moderate increase in size (hypertrophy) and number (hyperplasia), focally forming several rows of enlarged mitochondria separating the myofibrils. Many mitochondria had the appearance of elongated giant mitochondria. Longitudinally oriented cristae, either parallel to the long axis of the mitochondrion or irregular and concentric, were observed in many areas.
eNOS-NO: After 42 d of aortic banding, serum NO content was decreased (21.56±8.06μmol/L vs 13.89±1.59μmol/L,P<0.05), treatment with IPT 1,3,9 mg/kg increase serum NO content significantly with a dose-dependent manner, significant negative linear correlations were found between LVW/BW, cardiac tissue hydroxyproline , SBP and serum NO. There was a tendency for decreased immunoreactivity to eNOS and eNOS mRNA expression in Control group rats compared with that in Sham group rats. Immunoreactivity for eNOS and eNOS mRNA expression was obviously enhanced in cardiomyocytes by long-term iptakalim treatment. However, long-term L-NAME administration reduced immunoreactivity for eNOS and eNOS mRNA expression which can be completely improved by iptakalim 3 +L-NAME 50.
3 Role of PGI_2 in pressure-overload rats
Blood pressure: Chronic indomethacin administration caused time-dependent severe hypertension, increased ASBP and transtenosis blood pressure. Combined with IPT 3 ameliorated indomethacin induced severe hemodynamics partially. There were no statistically difference between these two groups and Control group.
Heart function: Prolonged administration indomethacin enhanced cardiac contractility and reduced cardiac diastolic compliance, combined with IPT 3 inhibited the increase of cardiac contractility, ameliorated cardiac compliance.
Cardiac remodeling: After 42 d of aortic banding, HW/BW, LVW/BW, RVW/BW and cardiomyocytes cross-section increased significantly in Indo 2 group. Combined with IPT 3 ameliorated cardiac hypertrophy partially but not completely. Long-term treatment with indomethacin cardiac CVF, PVCA and hydroxyproline increased significantly compared with Sham group(P<0.05,P<0.01), combined with IPT 3 inhibited the deterioration of CVF and PVCA(P<0.05,P<0.01 vs Indo 2) ,inhibited the increase of cardiac hydroxyproline(P<0.05 vs Indo 2).
PGI_2: After 42 d of aortic banding, plasma PGI_2 content decreased markedly compared with Sham group(63.87%,P<0.05).Long-term treatment with IPT 1, 3, 9 mg/kg increase plasma PGI_2 significantly with a dose-dependent manner. Chronic administration indomethacin decrease plasma PGI_2 content significantly, combined with IPT 3 inhibited the decrease of plasma PGI_2 content markedly(P<0.05 vs Indo 2). PartⅢEffect of ISO on cultured cardiomyocytes hypertrophy induced by ISO 1: The total protein of cardiomyocytes was increased by 24.53% and 31.74% in ISO 1×10~(-5)mol/L group and ISO 1×10~(-4)mol/L group, respectively(P<0.01 vs Control group), and there is no statistically difference between these two group, indicating 1×10~(-5)mol/L ISO can activatedβ-AR completely; 2: The total protein of cardiomyocytes was increased by 39.62% in ISO 1×10~(-5)mol/L group(P<0.01 vs Control group), in IPT 1×10~(-7)mol/L, 1×10~(-6)mol/L, 1×10~(-5)mol/L, 1×10~(-4)mol/L group, the total protein of cardiomyocytes was markedly decreased with a dose-dependent manner and the inhibition rate was 52.38%, 85.71%, 95.24% and 95.24%, respectively; 3: Compared with values in Control group, the fluorescence intensity increased significantly in ISO 1×10~(-5)mol/L group(15.18±10.99 vs 125.82±45.56,P <0.01)which could be inhibited completely in IPT-treated group, and the inhibition rate was 26.36%, 31.82%, 70.91% and 90.91%, respectively.
Conclusions
1 iptakalim 1, 3, 9 mg/kg can significantly reverse abdominal aorta binding/pressure- overload induced cardiovascular remodeling with a dose-dependent manner;
2 iptakalim might possess cardioreparative properties, its mechanism may contribute to binding KATP channel in endothelial cells, ameliorating endothelium cells function, augmenting NO and PGI_2 secretion, inhibiting ET-1 biosynthesis, secretion, inhibiting ET-1 system activation;
3 In cultured cardiomyocytes in vitro, iptakalim inhibites cardiomyocytes hypertrophy and total protein enhancement, this may contribute to decreasing cell resting membrane potential and inhibiting [Ca~(2+)]_i.
高血压是最常见的心血管疾病,其主要病理生理学特征表现为压力超负荷(pressure overload)和心血管重构(cardiovascular remodeling),包括血管重构(vascular remodeling)和心脏重构(cardiac remodeling)。大量的临床研究表明,高血压心肌重构不但可导致心室的收缩及舒张功能障碍,还是心肌梗塞、甚至心衰死亡的重要独立危险因素。因此能否逆转高血压所致的心血管重构,从而降低严重心脏事件的发生率和病死率,已成为临床高血压治疗的关键,开发新型的抗高血压、抗心血管重构的新药仍是心血管领域研究的热点之一。
方法:
在体实验:雄性SD大鼠,体重180～220 g。麻醉后于左肋弓下缘纵切口,分离出腹主动脉后用外径为0.7 mm的小圆棒和腹主动脉一起结扎形成腹主动脉缩窄模型,诱导心肌肥大和重构。
离体实验:取新生的1～3d的SD大鼠乳鼠心脏,消化后行心肌细胞原代培养。用异丙肾上腺素(isoproterenol ,ISO)诱导心肌细胞肥大,观察ISO对心肌细胞蛋白质含量和心肌细胞内游离Ca~(2+)的影响。
第一部分盐酸埃他卡林(iptakalim, IPT)对压力超负荷大鼠心血管重构的作用大鼠随机分为6组:⑴假手术对照组;⑵模型对照组;⑶IPT 1 mg/kg组;⑷IPT 3 mg/kg组;⑸IPT 9 mg/kg组;⑹L isenopril 15 mg/kg组。其中假手术对照组大鼠只行手术通路,不结扎腹主动脉,其余手术操作均和手术组相同。其它各组大鼠均做模型处理。各组药物均溶于NS中,于术后第3天灌胃给药,给药容积为0.5 ml/l00 g体重。每天1次,假手术对照组和模型对照组仅给予等体积普通蒸馏水,连续灌胃给药6周。实验结束后八导生理记录仪观察血流动力学和心脏功能的变化,HE染色和Masson’s染色观察心血管组织病理学改变,RT-PCR检测心肌肥大的标志基因——心钠素(atrial natriuretic peptide, ANP)和脑钠素(brain natriuretic peptide, BNP)的表达。
第二部分:IPT逆转心血管重构的内皮机制
(一)内皮素系统在压力超负荷大鼠模型中的作用
大鼠随机分为5组:⑴假手术对照组;⑵模型对照组;⑶IPT 1 mg/kg组;⑷IPT 3 mg/kg组;⑸IPT 9 mg/kg组。手术方案同第一部分。放射免疫法测定血浆中内皮素-1(endothelin-1, ET-1)的含量,RT-PCR观察心肌组织ET-1和内皮素转化酶(endothelin converting enzyme, ECE)的表达,免疫组化法检测心肌组织ETA和ETB的表达。
(二) eNOS-NO信号通路在压力超负荷大鼠模型中的作用
大鼠随机分为5组:⑴假手术对照组;⑵模型对照组;⑶I PT 3 mg/kg组;⑷左旋硝基精氨酸甲酯(N G-nitro-L- arginine methyl ester, L-NAME) 50 mg/kg (L-NAME 50)组;⑸IPT 3 mg/kg + L-NAME 50 mg/kg (IPT 3+L-NAME 50)组。手术方案同第一部分。实验结束后八导生理记录仪观察血流动力学和心脏功能的变化,HE染色和Masson’s染色观察心肌组织病理学改变,RT-PCR心肌组织内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS )mRNA的表达,免疫组化法检测心肌组织eNOS的表达,生化法观察血浆中一氧化氮(nitric oxide, NO)含量的变化。
(三)前列环素(prostacyclin, PGI_2)在压力超负荷大鼠模型中的作用
大鼠随机分为5组:⑴假手术对照组;⑵模型对照组;⑶IPT 3 mg/kg组;⑷i ndomethacin 2 mg/kg (Indo 2)组;⑸IPT 3mg/kg + indomethacin 2 mg/kg (IPT 3+Indo 2)组。手术方案同第一部分。实验结束后八导生理记录仪观察血流动力学和心脏功能的变化,HE染色和Masson’s染色观察心肌组织病理学改变,放射免疫法观察血浆中PGI_2含量的变化。
第三部分:IPT对异丙肾上腺素诱导的培养心肌细胞肥大的影响
1:确定ISO诱导心肌肥大的最适浓度。将培养72 h后的心肌细胞按1×105/孔接种到24孔板中,随机分为6组,每组设4个复孔。⑴Control;⑵ISO 1×10-8 mol/L;⑶1×10~(-7) mol/L;⑷1×10~(-6) mol/L;⑸1×10~(-5) mol/L;⑹1×10~(-4) mol/L。培养48h后,Lowry’s法测定心肌细胞蛋白含量;2:不同浓度IPT对ISO诱导的肥大心肌细胞总蛋白含量的影响。将培养72 h后的心肌细胞按1×105/孔接种到24孔板中,随机分为6组,每组设4个复孔。⑴Control;⑵ISO 1×10~(-5)mol/L;⑶ISO 1×10~(-5)mol/L+IPT 1×10~(-7)mol/L;⑷ISO 1×10~(-5)mol/L+IPT 1×10~(-6)mol/L;⑸ISO 1×10~(-5)mol/L+IPT1×10~(-5)mol/L;⑹ISO 1×10~(-5)mol/L+IPT 1×10~(-4)mol/L。培养48h后,Lowry’s法测定心肌细胞蛋白含量;3:将培养72 h后的心肌细胞按1×105/孔接种到24孔板中,随机分为6组,每组设4个复孔。⑴Control;⑵ISO 1×10~(-5)mol/L;⑶ISO 1×10~(-5)mol/L+IPT 1×10~(-7)mol/L;⑷ISO 1×10~(-5)mol/L+IPT 1×10~(-6)mol/L;⑸ISO 1×10~(-5)mol/L+IPT 1×10~(-5)mol/L;⑹ISO 1×10~(-5)mol/L+IPT 1×10~(-4)mol/L。培养48h后,激光共聚焦显微镜测定每组心肌细胞Ca2+含量,每组取20个细胞。
结果:
第一部分IPT对压力超负荷大鼠心血管重构的作用
血压:整个实验6 wk内,假手术组收缩压(systolic blood pressure, SBP)基本保持稳定,模型组大鼠血压呈时间依赖性增加,各时间点血压均比基础值升高约1.5 kPa左右,IPT 1,3,9 mg/kg/d和Lisenopril 15 mg/kg/d治疗自试验第2周起,可以明显降低大鼠尾动脉SBP。腹主动脉缩窄6 wk后,与假手术组相比,模型组大鼠颈动脉SBP,舒张压(diastolic blood pressure, DBP),平均动脉压(mean blood pressure, MBP)和腹主动脉跨狭窄部位收缩压压力差(Transtenosis gradient of SBP)都明显增高(P<0.01 vs Sham组)。经过IPT 1,3,9 mg/kg/d治疗6 wk后,各组大鼠颈动脉SBP,DBP,MBP,腹主动脉跨狭窄部位收缩压压力差呈剂量相关性降低。
心功能:与假手术组相比,模型组大鼠的左室收缩压(left ventricular systolic pressure, LVSP),左室压力最大上升速率(maximal rate of left ventricular systolic pressure, +dp/dtmax),左室压力最大下降速率(maximal rate of left ventricular diastolic pressure, -dp/dtmax),心肌纤维缩短速度的生理最大值(Vmp),心肌纤维缩短速度的理论最大值(Vmax)均明显增加,表明为了适应腹主动脉缩窄后后负荷的增加,模型组大鼠动用了左心室的心力储备,收缩功能明显增强(P<0.01 vs Sham组),而左心室舒张末期压(left ventricular end diastolic pressure, LVEDP)和假手术组相比明显降低,表明模型组大鼠左心室的舒张功能减弱,心肌的顺应性降低。而经过IPT 1,3,9 mg/kg/d治疗6 wk后,各组大鼠的心室舒缩功能明显改善。
心血管重构:腹主动脉缩窄后6 wk,可引起全心指数(heart weight/body weight, HW/BW)、左心指数(left ventricular weight/body weight, LVW/BW)、右心指数(right ventricular weight/body weight, RVW/BW)和肺指数(lung weight/body weight, LW/BW)都明显增加,表明心脏发生了大体上的重构,模型组大鼠心肌细胞横截面积(cardiomyocyte cross-section area)、心肌间质胶原容积分数(collagen volume fraction, CVF)、心肌血管周围胶原面积(perivscular collagen area, PVCA)心肌的羟脯氨酸含量(hydroxyproline)和对照组相比明显增加(P<0.05,P<0.01 vs Sham组)。与假手术组相比,模型组大鼠主动脉的血管总面积(total artery area, TAA)、管腔总面积(lumen area, LA)、血管管壁面积(cross-section area, CSA)、CSA/TAA、血管平均直径(artery diameter, AD)、血管管壁平均厚度(media)以及M/L等参数均明显增加,表明模型组大鼠的主动脉发生了明显的重构(P<0.01 vs Sham组)。腹主动脉缩窄6 wk后,大鼠心肌组织ANP、BNP mRNA的表达明显上调(P<0.01 vs Sham组)。经IPT 1,3,9 mg/kg/d治疗后,各剂量组均能明显降低心脏指数和肺指数,可剂量相关地抑制心肌细胞横截面积增加,分别为41.54%,43.59%,44.10%(P<0.01 vs Control组);可剂量相关地抑制CVF的升高,分别为35.71%,73.21%(P<0.01 vs Control组),87.98%(P<0.01 vs Control组);可剂量相关地抑制PVCA的升高,分别为34.96%,56.50%(P<0.05 vs Control组),66.26%(P<0.05 vs Control组);可剂量相关地抑制大鼠心肌的羟脯氨酸含量的升高,抑制率分别为56.72%,92.53%(P<0.05 vs Control组)和92.53%(P<0.05 vs Control组),可剂量相关地下调大鼠心肌组织ANP、BNP mRNA的表达(P<0.05, P<0.01 vs Control组)。
第二部分:IPT逆转心血管重构的内皮机制
(一)内皮素系统在压力超负荷大鼠模型中的作用
腹主动脉缩窄6 wk后,大鼠血浆ET-1的含量比假手组明显升高(101.97±19.64 pg/ml vs 135.27±36.91 pg/ml,P<0.05);大鼠心肌组织ET-1、ECE mRNA的表达明显上调(P<0.01 vs Sham组);大鼠心脏的内皮素受体A型(endothelin-1 receptor A type, ETA)、内皮素受体B型(endothelin-1 receptor Btype, ETB)的表达明显上调(P<0.01 vs假手术组)。IPT 1、3、9 mg/kg治疗6 wk后,可剂量相关地抑制大鼠血浆ET-1的升高,分别为(120.11±40.06)pg/ml、(95.98±28.27)pg/ml(P<0.05 vs Control组)和(78.01±21.77)pg/ml(P<0.01 vs Control组);可剂量相关地下调大鼠心肌组织ET-1、ECE mRNA的表达(P<0.05,P<0.01 vs模型组);可剂量依赖性性地下调大鼠心脏ETA、ETB的表达(P<0.05,P<0.01 vs模型组)。
(二) eNOS-NO信号通路在压力超负荷大鼠模型中的作用
血压:在6 wk的实验期间,单独应用L-NAME大鼠的SBP随时间逐渐升高,第2 wk、第4 wk、第6 wk的SBP分别为20.6±2.2、20.8±1.7、21.2±1.4 kPa,与同期模型组的SBP 17.9±2.5、18.7±1.2、18.0±1.2 kPa相比差异有显著性(P<0.05,P<0.01)。而IPT 3+L-NAME 50组和单用L-NAME组相比,在第2 wk、第4 wk、第6 wk能分别降低SBP约10.4%、15.1%和10.7%(P<0.0l)。
心功能:经过6 wk的内源性的NOS抑制后,L-NAME 50组大鼠心室的收缩功能和假手术组相比明显增强,而心室的舒张功能和顺应性明显降低。合用IPT,能明显降低ASBP和LVSP(P<0.05)。此外,我们还发现了一个有趣的现象,L-NAME 50mg/kg/d无论是单独应用还是和IPT合用,都导致了比模型组大鼠还要严重的高血流动力学状态,但却未发生大体形态学上的心肌肥大。
心脏重构:腹主动脉缩窄6 wk后,无论是单用L-NAME还是和IPT合用,2组大鼠的心脏从大体上和病理学检查上都未发现明显的肥大(vs Sham组)。单用L-NAME组的心肌纤维化程度均比假手术组明显升高,心肌组织羟脯氨酸含量明显增加(P<0.01 vs Sham组),甚至诱发了比模型组更严重的胶原沉积,合用IPT可以改善单独应用L-NAME导致的心肌纤维化,分别为0.87%(CVF)和4.3%(PVCA),明显抑制心肌组织羟脯氨酸含量明显增加(P<0.01 vs L-NAME 50)。电镜结果显示,假手术组大鼠心肌肌丝排列整齐、连续,线粒体和心肌纤维呈平行排列,线粒体嵴突排列整齐,Z线清晰。IPT 3+L-NAME 50组的心肌纤维平行排列,Z线均匀分隔肌小节,卵圆形的线粒体呈列分布在肌纤维之间。而单独用L-NAME组的心肌细胞可以看到无论使线粒体的数量还是体积都比假手术组和IPT 3+L-NAME 50组明显增加。提示当内源性的NOS被抑制后,心肌细胞发生了亚细胞结构的重构,表现为线粒体数量的增加(hyperplasia)和体积的增加(hypertrophy)。IPT可以改善这种亚细胞结构的重构。
eNOS-NO:腹主动脉缩窄6 wk后,血浆中的NO含量明显低于假手术组(21.56±8.06μmol/L vs 13.89±1.59μmol/L,P<0.05)。IPT1,3,9 mg/kg可以剂量相关地提高血浆中NO含量(P<0.05,P<0.01 vs Control组)。并且血浆中的NO含量和左心室重构指数、心肌胶原含量、SBP(tail-cuff)呈明显的负相关。模型组大鼠心肌组织的eNOS mRNA和eNOS蛋白表达水平和假手术组相比明显下调。经过IPT治疗6 wk后eNOS mRNA和eNOS蛋白表达水平和模型组相比明显增加。单独应用L-NAME 6 wk后,大鼠心肌组织的eNOS mRNA和eNOS蛋白表达被明显抑制,合用IPT后能明显提高被抑制的eNOS mRNA和eNOS蛋白的表达。
(三)PGI_2在压力超负荷大鼠模型中的作用
血压:单用indomethacin组SBP(tail-cuff)呈时间依赖性升高,颈动脉收缩压(right carotid aorta systolic blood pressure, ASBP)和跨狭窄部位收缩压差值也明显增加,合用IPT后只能部分而不能完全改善indomethacin诱导的高血流动力学状态,和模型组相比差异无显著性。
心功能:单用吲哚美辛Indo 2组的心脏收缩功能明显增加,心肌的顺应性明显下降,合用IPT后能明显抑制心肌收缩力的提高,改善心肌的顺应性。心脏重构:腹主动脉缩窄6 wk后,单用吲哚美辛和假手术组相比HW/BW,LVW/BW和RVW/BW比值明显增加,心肌细胞横截面积亦明显增加,合用IPT后只能部分而不能完全改善心肌细胞的肥大。单用Indomethacin后,心肌组织的CVF、PVCA、心肌组织羟脯氨酸含量明显增加(P<0.05,P<0.01 vs Sham组),合用IPT后可明显抑制CVF和PVCA的恶化(P<0.05,P<0.01 vs Indo 2)。明显抑制心肌组织羟脯氨酸含量的增高(P<0.05 vs Indo 2)。
PGI_2:腹主动脉缩窄6 wk后,模型组血浆中PGI_2含量和假手术组相比明显降低(63.87%,P<0.05 vs Sham组),IPT 1,3,9 mg/kg可以剂量相关地提高血浆中PGI_2含量(P<0.05 vs Control组)。Indo 2组血浆的PGI_2含量和假手术组相比明显降低,和模型组相比也降低了19%,合用IPT后能明显提高血浆中PGI_2的含量(P<0.05 vs Indo 2组)。
第三部分:IPT对异丙肾上腺素诱导的培养心肌细胞肥大的影响
1:与对照组相比,ISO1×10~(-5)mol/L组和ISO1×10-4mol/L组的心肌细胞蛋白含量分别增加了24.53%和31.74%(P<0.01),二者之间没有明显差别,说明1×10~(-5)mol/L ISO可以完全激活β-肾上腺素受体(β-adrenergic receptor,β-AR),使心肌细胞蛋白含量显著增加;2:与对照组相比,ISO组心肌细胞蛋白含量增加了39.62%,IPT 1×10~(-7)mol/L组、1×10~(-6)mol/L组、1×10~(-5)mol/L组、1×10-4mol/L组可剂量依赖性地抑制ISO诱导的心肌细胞肥大和蛋白含量的增加,抑制率分别为52.38%、85.71%、95.24%、95.24%。3:与对照组相比,ISO组心肌细胞荧光强度(fluorescence intensity)明显增加(15.18±10.99 vs 125.82±45.56,P<0.01),IPT 1×10~(-7)mol/L组、1×10~(-6)mol/L组、1×10~(-5)mol/L组、1×10-4mol/L组可剂量依赖性地抑制ISO诱导的心肌细胞荧光强度的增加,抑制率分别为26.36%、31.82%、70.91%、90.91%。
结论:
1 IPT 1,3,9 mg/kg组可以明显逆转腹主动脉缩窄/压力超负荷大鼠的心血管重构,并且这种逆转作用呈现一定的剂量依赖性;
2 IPT逆转心血管重构的作用可能与作用于内皮细胞上的KATP通道,恢复内皮细胞的分泌功能密切相关,即增加NO和PGI_2的分泌,抑制ET-1的合成、分泌,抑制内皮素系统的活化;
3在培养新生乳鼠心肌细胞上,IPT可以剂量依赖性地抑制ISO诱导的心肌细胞肥大和蛋白质含量的增加,其作用机制可能与降低细胞膜静息膜电位,抑制Ca~(2+)内流有关。
Background:
Hypertension is a substantial public health problem, the major pathophysiological character is pressure overload and cardiovascular remodeling, including vascular remodeling and cardiac remodeling. A lot of clinical investigations reveal that essential hypertension and consequent left ventricular hypertrophy not only contribute to ventricular systolic and diastolic dysfunction, but also have been recognized as a strong, virtually independent risk factor. So the desirability to prevent and induce regression of LVH by drug treatment is widely understood, and a variety of antihypertensive drugs has been shown to cause regression of LVH in treated hypertensives. However, the advantages and disadvantages of these drugs have been debated and there is an impetus for the development of new chemical entities with a more desirable therapeutic endpoint, decreasing morbidity and mortality of hypertensives.
Methods:
In vivo: Adult male Sprague–Dawley rats (weight range 180–220 g), after rats were anesthetized, under sterile conditions, the abdominal aorta was exposed through a abdominal incision and constricted at the suprarenal level by a 4-0 silk suture tied around both the aorta and a blunted 22-gauge needle, which was then pulled out. A similar procedure was performed for Sham group except without the ligature.
In vitro: Sprague–Dawley rats neonatal ventricular myocytes were isolated and cultured. Cardiomyocytes were exposed to isoproterenol (ISO) for 48 h, and the total protein content of the cardiomyocytes was measured. Laser scanning confocal microscope was used to determine the [Ca~(2+)]_i of cardiomyocytes.
PartⅠThe experimental therapeutic effects of iptakalim on cardiovascular protection in pressure-overload rats
Animals were divided into 6 groups:⑴Sham group;⑵C ontrol group;⑶IPT 1 mg/kg group;⑷IPT 3 mg/kg group;⑸IPT 9 mg/kg group;⑹Lisenopril 15 mg/kg group. The drugs were dissolved in distilled water and administered orally via a gastric tube. Drugs were administered from 3 days after operation through 6 weeks after surgery. At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson’s stain. RT-PCR method was used to measure the cardiac hypertrophy marker gene, ANP and BNP mRNA expression.
PartⅡIPT reverse cardiovascular remodeling: Endothelial mechanism
1 Role of endothelin system in pressure-overload rats
Animals were divided into 5 groups:⑴Sham group;⑵C ontrol group;⑶IPT 1 mg/kg group;⑷IPT 3 mg/kg group;⑸I PT 9 mg/kg group. Radioimmunoassay was used to determine the serum ET-1 content, RT-PCR method was used to measure the cardiac ET-1 and ECE mRNA expression, immunochemistry method was used to evaluate the cardiac ETA and ETB expression.
2 Role of eNOS-NO signal pathway in pressure-overload rats
Animals were divided into 5 groups:⑴Sham group;⑵C ontrol group;⑶IPT 3 mg/kg group;⑷L-NAME 50 mg/kg group;⑸L -NAME 50 mg/kg + IPT 3 mg/kg group. At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson’s stain. RT-PCR method was used to measure the cardiac eNOS mRNA expression, immunochemistry method was used to evaluate the cardiac eNOS expression, and biochemical method was used to determine serum nitric oxide content.
3 Role of PGI_2 in pressure-overload rats
Animals were divided into 5 groups:⑴Sham group;⑵C ontrol group;⑶IPT 3 mg/kg group;⑷i ndomethacin 2 mg/kg group (Indo 2);⑸indomethacin 2 mg/kg + IPT 3 mg/kg group(Indo 2+IPT 3). At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson’s stain. Radioimmunoassay was used to determine the serum PGI_2 content.
PartⅢEffect of ISO on cultured cardiomyocytes hypertrophy induced by ISO
1: Ascertain the appropriate concentration of ISO inducing cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plates for 72 h, 1×105/well, and then divided into 6 groups:⑴Control group;⑵ISO 1×10-8 mol/L;⑶ISO 1×10~(-7) mol/L;⑷I SO 1×10~(-6) mol/L;⑸ISO 1×10~(-5) mol/L;⑹I SO 1×10~(-4) mol/L. After 48 h, Lowry’s method was used to determine the cardiomyocytes total protein content. 2: Effect of IPT on cardiomyocytes total protein content in ISO-induced cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plate for 72 h, 1×105/well, then divided into 6 group:⑴Control group;⑵ISO 1×10~(-5) mol/L;⑶ISO 1×10~(-5) mol/L+IPT 1×10~(-7) ;⑷ISO 1×10~(-5) mol/L+IPT 1×10~(-6);⑸ISO 1×10~(-5) mol/L+IPT 1×10~(-5);⑹ISO 1×10~(-5) mol/L+IPT 1×10~(-4). After 48 h, Lowry’s method was used to determine the cardiomyocytes total protein content. 3: Effect of IPT on cardiomyocytes [Ca2+]i in ISO-induced cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plate for 72 h, 1×105/well, then divided into 6 group:⑴Control group;⑵I SO 1×10~(-5) mol/L;⑶I SO 1×10~(-5) mol/L+IPT 1×10~(-7) ;⑷ISO 1×10~(-5) mol/L+IPT 1×10~(-6);⑸ISO 1×10~(-5) mol/L+IPT 1×10~(-5);⑹ISO 1×10~(-5) mol/L+IPT 1×10~(-4). After 48 h, laser scanning confocal microscope was used to determine [Ca_~(2+)]_i of cardiomyocytes, 20 cells were selected in each group.
Results:
PartⅠThe experimental therapeutic effects of iptakalim on cardiovascular protection in pressure-overload rats
Blood pressure: During the time course, tail artery systolic blood pressure(SBP) in Sham group maintain at the baseline level, tail artery SBP in Control group increased significantly with a time-dependent manner, IPT 1,3,9 mg/kg/d and Lisenopril 15 mg/kg/d treatment inhibited the increase of tail artery SBP markedly from 2 week. At the end of 6 week, carotid aorta SBP, DBP, MBP and transtenosis gradient of SBP increased significantly with a time-dependent manner(P<0.01 vs Sham group), IPT 1,3,9 mg/kg/d and Lisenopril 15 mg/kg/d treatment inhibited the increase of carotid aorta SBP, DBP, MBP and transtenosis gradient of SBP markedly.
Heart function: After 42 d of aortic banding, LVSP,±dp/dtmax,Vmp,Vmax increased significantly, while left ventricular LVEDP decreased significantly compared with Sham group (P<0.01). IPT 1, 3, 9 mg/kg/d and Lisenopril 15 mg/kg/d treatment ameliorated left ventricular systolic and diastolic function, augmented cardiac compliance markedly.
Cardiovascular remodeling:After 42 d of aortic banding, heart weight/body weight(HW/BW), left ventricular weight/body weight(LVW/BW), right ventricular weight/body weight(RVW/BW) , lung weight/body weight(LW/BW) total area of total aorta(TAA), area of lumen(LA), cross-section area(CSA), CSA/TAA, aorta radium(AR), media(M) and M/L increased significantly, indicating a morphological remodeling of heart and aortic remodeling. Cardiomyocytes cross-section area, CVF, PVCA and cardiac tissue hydroxyproline increased significantly compared with Sham group(P<0.05,P<0.01). ANP and BNP mRNA expression increased significantly in Control group rats compared with that in Sham group rats(P<0.01). After treatment with IPT 1,3,9 mg/kg/d, HW/BW, LVW/BW, RVW/BW, LW/BW, cardiomyocyte cross-section area(41.54%,43.59%,44.10%, P<0.01 vs Control group),CVF(35.71%,73.21%,P<0.01 vs Control group), PVCA(34.96%,56.50%,P<0.05 vs Control group),cardiac tissue Hydroxyproline content(56.72%,92.53%,P<0.05 vs Control group) were reversed ,respectively. Cardiac ANP and BNP mRNA expression were downregulated significantly(P<0.05, P<0.01 vs Control group).
PartⅡIPT reverse cardiovascular remodeling: Endothelial mechanism
1 Role of endothelin system in pressure-overload rats
After 42 d of aortic banding, plasma ET-1 content was increased (101.97±19.64 pg/ml vs 135.27±36.91 pg/ml,P<0.05 vs Sham group), cardiac tissue ET-1、ECE mRNA and ETA、ETB protein expression were upregulated in Control group(P<0.01). After treatment with IPT 1,3,9 mg/kg/d, plasma ET-1 content was decreased significantly(P<0.01 vs Control group), the upregulation of cardiac tissue ET-1、ECE mRNA and ETA、ETB protein expression were inhibited markedly(P<0.05,P<0.01 vs Control group).
2 Role of eNOS-NO signal pathway in pressure-overload rats
Blood pressure: Chronic L-NAME administration caused persistent severe hypertension. The SBP (of the 2nd, 4th, 6th weeks) were 20.6±2.2, 20.8±1.7 and 21.2±1.4 kPa versus 17.9±2.5, 18.7±1.2 and 18.0±1.2 kPa in Control group rats, respectively (P<0.05, P<0.0l). SBP of L-NAME 50 + iptakalim 3 group in the 2nd, 4th, 6th weeks had 10.4%, 15.1%, 10.7% lower than that of rats given L-NAME alone, respectively(P<0.0l).
Heart function: After 42 days of NO synthase inhibition, ASBP,LVSP,±dp/dtmax were significantly increased(P<0.05,P<0.01 vs Sham group), while LVEDP decreased markedly (P<0.01 vs Sham group), indicating an enhanced contractility but a reduced diastolic compliance were taken place, and iptakalim could statistically or partially ameliorate the LV function(P<0.05 versus L-NAME 50 group). Interestingly, in L-NAME 50 mg/kg/d rats (whether iptakalim was given or not), the LVW/BW and RVW/BW of rats were not statistically different from those of Sham group, despite of the significant increase in blood pressure.
Cardiac remodeling: Histological examination demonstrated that both CVF, PVCA and cardiac tissue hydroxyproline revealed significant difference in both L-NAME 50 mg·kg-1·d-1 and IPT 3 mg·kg-1·d-1+L-NAME 50 mg·kg-1·d-1 compared to that of Sham group, and combined with IPT 3 ameliorated cardiac tissue hydroxyproline compared with that of L-NAME 50 alone. However, the extent of myocyte hypertrophy of rats given IPT 3 mg/kg/d +L-NAME 50 mg/kg/d or given L-NAME 50 mg/kg/d alone were not statistically different from those of Sham group. Ultrastructural examination revealed well-organized myofibrils with mitochondria grouped along the periphery of longitudinally oriented fibers were found in Sham group rats, mitochondria tightly packed, intact horizontally oriented cristae in Sham group rats, well-preserved intercalated disk with smooth contours in Sham group rats. The cardiomyocytes in iptakalim 3+L-NAME 50 group showed almost uniform parallel myofibril arrangement. Z-lines dividing the sarcomeres were linear and perpendicular to the myofilaments. Myofibrils alternated with few rows of ovoid mitochondria. However, the number of mithochondria in the latter group seems to be larger. Ultrastructure of the cardiomyocytes from L-NAME-treated rats presented mitochondrial changes, characterized by diffuse mild to moderate increase in size (hypertrophy) and number (hyperplasia), focally forming several rows of enlarged mitochondria separating the myofibrils. Many mitochondria had the appearance of elongated giant mitochondria. Longitudinally oriented cristae, either parallel to the long axis of the mitochondrion or irregular and concentric, were observed in many areas.
eNOS-NO: After 42 d of aortic banding, serum NO content was decreased (21.56±8.06μmol/L vs 13.89±1.59μmol/L,P<0.05), treatment with IPT 1,3,9 mg/kg increase serum NO content significantly with a dose-dependent manner, significant negative linear correlations were found between LVW/BW, cardiac tissue hydroxyproline , SBP and serum NO. There was a tendency for decreased immunoreactivity to eNOS and eNOS mRNA expression in Control group rats compared with that in Sham group rats. Immunoreactivity for eNOS and eNOS mRNA expression was obviously enhanced in cardiomyocytes by long-term iptakalim treatment. However, long-term L-NAME administration reduced immunoreactivity for eNOS and eNOS mRNA expression which can be completely improved by iptakalim 3 +L-NAME 50.
3 Role of PGI_2 in pressure-overload rats
Blood pressure: Chronic indomethacin administration caused time-dependent severe hypertension, increased ASBP and transtenosis blood pressure. Combined with IPT 3 ameliorated indomethacin induced severe hemodynamics partially. There were no statistically difference between these two groups and Control group.
Heart function: Prolonged administration indomethacin enhanced cardiac contractility and reduced cardiac diastolic compliance, combined with IPT 3 inhibited the increase of cardiac contractility, ameliorated cardiac compliance.
Cardiac remodeling: After 42 d of aortic banding, HW/BW, LVW/BW, RVW/BW and cardiomyocytes cross-section increased significantly in Indo 2 group. Combined with IPT 3 ameliorated cardiac hypertrophy partially but not completely. Long-term treatment with indomethacin cardiac CVF, PVCA and hydroxyproline increased significantly compared with Sham group(P<0.05,P<0.01), combined with IPT 3 inhibited the deterioration of CVF and PVCA(P<0.05,P<0.01 vs Indo 2) ,inhibited the increase of cardiac hydroxyproline(P<0.05 vs Indo 2).
PGI_2: After 42 d of aortic banding, plasma PGI_2 content decreased markedly compared with Sham group(63.87%,P<0.05).Long-term treatment with IPT 1, 3, 9 mg/kg increase plasma PGI_2 significantly with a dose-dependent manner. Chronic administration indomethacin decrease plasma PGI_2 content significantly, combined with IPT 3 inhibited the decrease of plasma PGI_2 content markedly(P<0.05 vs Indo 2). PartⅢEffect of ISO on cultured cardiomyocytes hypertrophy induced by ISO 1: The total protein of cardiomyocytes was increased by 24.53% and 31.74% in ISO 1×10~(-5)mol/L group and ISO 1×10~(-4)mol/L group, respectively(P<0.01 vs Control group), and there is no statistically difference between these two group, indicating 1×10~(-5)mol/L ISO can activatedβ-AR completely; 2: The total protein of cardiomyocytes was increased by 39.62% in ISO 1×10~(-5)mol/L group(P<0.01 vs Control group), in IPT 1×10~(-7)mol/L, 1×10~(-6)mol/L, 1×10~(-5)mol/L, 1×10~(-4)mol/L group, the total protein of cardiomyocytes was markedly decreased with a dose-dependent manner and the inhibition rate was 52.38%, 85.71%, 95.24% and 95.24%, respectively; 3: Compared with values in Control group, the fluorescence intensity increased significantly in ISO 1×10~(-5)mol/L group(15.18±10.99 vs 125.82±45.56,P <0.01)which could be inhibited completely in IPT-treated group, and the inhibition rate was 26.36%, 31.82%, 70.91% and 90.91%, respectively.
Conclusions
1 iptakalim 1, 3, 9 mg/kg can significantly reverse abdominal aorta binding/pressure- overload induced cardiovascular remodeling with a dose-dependent manner;
2 iptakalim might possess cardioreparative properties, its mechanism may contribute to binding KATP channel in endothelial cells, ameliorating endothelium cells function, augmenting NO and PGI_2 secretion, inhibiting ET-1 biosynthesis, secretion, inhibiting ET-1 system activation;
3 In cultured cardiomyocytes in vitro, iptakalim inhibites cardiomyocytes hypertrophy and total protein enhancement, this may contribute to decreasing cell resting membrane potential and inhibiting [Ca~(2+)]_i.
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