抑制磷酸二酯酶5的短发夹RNA重组腺病毒载体对阿霉素所致心肌病小鼠的保护作用

Protective effects of phosphodiesterase 5 shRNA on doxorubicin-induced cardiomyopathy in mice

目的研究靶向磷酸二酯酶5(PDE5)抑制剂短发夹RNA(shRNA)[PDE5shRNA]重组腺病毒载体对阿霉素所致心肌病的保护作用。方法用一次性腹腔内注射阿霉素15 mg·kg^(-1)诱导小鼠阿霉素心肌病模型。按照体重将小鼠随机分为3组:模型组(n=16);实验组(阿霉素+PDE5shRNA,n=16)在造模后,一次性心肌多部位注射PDE5shRNA重组腺病毒载体(1×1010粒子/鼠);正常组(n=10):一次性腹腔注射等量0.9%NaCl。给药2周后,对存活小鼠进行心脏超声检查,检测左心室收缩末期内径(LVESD)、左心室舒张末期内径(LVEDD)、左心室射血分数(LVEF)和短轴缩短率(%FS)。以苏木精-伊红(HE)染色法测量心肌细胞直径;以Masson氏染色法测量心肌纤维化面积;以酶联免疫吸附法检测各组小鼠心肌组织中环磷酸鸟苷(c GMP)和蛋白激酶(PKG)含量,以免疫蛋白印迹法检测各组小鼠心肌组织中肌凝蛋白重链(MHC)、肌钙蛋白I(Tropnin1)以及肌间蛋白(Desmin)的表达情况。结果给药后2周后,实验组与模型组的LVESD分别是(2.13±0.10),(2.75±0.12)mm,这2组的LVEDD分别是(2.98±0.10),(3.42±0.12)mm,这2组的LVEF分别是(58.74±1.40)%,(48.53±1.50)%,这2组的%FS分别为(28.52±2.10)%,(19.59±1.67)%,2组比较差异均有统计学意义(均P<0.05)。这2组的心肌细胞直径分别是(14.68±0.42),(13.75±0.38)μm,差异有统计学意义(P<0.05)。实验组与模型组的心肌纤维化面积比率分别为(2.28±0.20)%,(5.10±0.35)%,差异有统计学意义(P<0.05)。这2组的c GMP含量分别是(0.23±0.02),(0.06±0.01)pg·mg^(-1),这2组的PKG含量分别是(0.21±0.02),(0.10±0.01)pg·mg^(-1),2组比较差异均有统计学意义(均P<0.05)。实验组与模型组的MHC表达分别是1.55±0.16,1.15±0.22,这2组的Tropnin1表达分别是1.32±0.08,0.88±0.08,这2组的Desmin表达分别是1.33±0.51,1.10±0.04,差异均有统计学意义(均P<0.05)。结论 PDE5shRNA可能通过激活c GMP/PKG通路减轻阿霉素所致心肌细胞萎缩和心肌纤维化,明显改善左心室功能障碍。

Objective To study the protective effects of phosphodies- terase 5 shRNA [ PDE5shRNA ] on doxorubicin （ DOX ） - induced cardiomyopathy in mice. Methods A single intraperitoneal injection of doxorubicin 15 mg · kg^-1 to induce cardiomyopathy to establish model. Male 10 -week -old C57BL/6J mice were randomly assigned to three groups ： model group （ n = 16） ; experimental group （ DOX ± PDE5 shRNA,n = 16）： a single intraperitoneal injection of same dose of doxorubicin and being simultaneously treated with a single myocardial injection of PDE5shRNA 1 × 10^10 particles;Normal group （ n = 10） ：a single intraperitoneal injection of same volume of saline. After two weeks of administration, left ventricular end - systolic diameter （ LVESD）, left ventricular end - distolic diameter （ LVEDD）, left ventricular ejection fraction （LVEF） and percent fractional shortening （ % FS） were evaluated by echocardiography. Cardiac specimens were then subjected for HE （ Hematoxyum Eosin）, Masson staining, computing for each group of cardiac cell size and fibrosis area. Levels of cyclic gnanosine monophosphate （cGMP） and protein kinase G（ PKG ） in the myocardium were assayed by ELISA. The expression of myosin heavy chain （MHC）, Troponin I and Desmin were evaluated by Western blot. Results Two weeks later of administration, indicators in experimental group and model group were as follows： LVESD were（2. 13 ±0. 10）, （2.75 ±0. 12）ram; LVEDD were（2. 98 ± 0. 10）, （3.42 2 0. 12） mm; LVEF were （58.74 ± 1.40）%, （48.53 ± 1.50）% ;% FS were （28.52 ± 2.10）%, （19.59 2 1.67）%; the transverse diameter of cardiomyocytes were （14.68 ± 0.42）, （13.75 20. 38） p.m; cardiac fibrosis were （2. 28 20. 20）%, （5.10 20. 35）% ;the levels of cGMP expression were （0.23±0.02）,（0.06±0.01）pg· mg^-1; the levels of PKG were （ 0. 21 20.02） ,（0.10±0.01）pg · mg^-1; the myocardial expression of MHC were 1.55 ± 0. 16, 1.15 2 0. 22; the expression of troponin I were 1.32 2 0. 08, 0. 88 ±0.08; the expression of Desmin were 1.327 2 0.512, 1. 103 2 0.038; all the above data were significantly different compared between two groups （all P 〈 0. 05 ）. Conclusion The protective effects of PDE 5shRNA on doxorubicin - induced cardiomyopathy, mitigatting doxorubicin - induced impairment of cardiac function in mice, significantly attenuatting doxorubicin - induced atrophic degeneration of cardiomyocyte and myocardial fibrosis through possible activating cGMP/PKG signaling pathway.