抑制磷酸二酯酶5的短发夹RNA重组腺病毒载体可改善小鼠心肌梗死后心力衰竭

Adenoviral short hairpin RNA targeting phosphodiesterase 5 attenuates cardiac remodeling and cardiac dysfunction following myocardial infarction in mice

目的探讨抑制磷酸二酯酶5（PDE5）的短发夹RNA重组腺病毒载体（PDE5shRNA）对小鼠心肌梗死后心室重构和心力衰竭的影响。方法雄性10周龄C57BL／6J小鼠，随机分为假手术组（仅穿线不结扎左冠状动脉前降支，n=6）、PDE5shRNA组（结扎左冠状动脉前降支＋心肌注射PDE5shRNA，n=12）、普通腺病毒组（结扎左冠状动脉前降支＋心肌注射普通腺病毒，n=15）和DMEM组（结扎左冠状动脉前降支＋心肌注射DMEM，n=8）。4周后，统计各组小鼠存活情况，通过心脏超声检查和组织学染色的方法分别检测各组小鼠左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）、左心室舒张末期内径（LVEDD）、左心室收缩末期内径（LVESD）、心肌梗死面积和心肌纤维化面积，以评价PDE5shRNA对小鼠心功能及心室重构的影响。同时采用免疫组织化学、ELISA、Westernblot等方法，检测血管密度以及PDE5、环磷酸鸟苷（cGMP）和蛋白激酶G（PKG）等蛋白的表达情况。结果（1）小鼠存活情况：4周后，假手术组小鼠全部存活（100％），DMEM组死亡3只（37％），PDE5shRNA组小鼠死亡1只（8％）与普通腺病毒组死亡5只（33％）比较差异无统计学意义（P=0．11）。（2）心功能和心室重构指标的检测结果：PDE5shRNA组小鼠心肌梗死面积显著小于普通腺病毒组和DMEM组[（25．44-2．9）％比（42．0±3．2）％和（43．4±2．6）％，P均〈0．05]，LVFS显著高于普通腺病毒组和DMEM组[（21．1l±3．72）％比（14．22±2．91）％和（14．22±2．91）％，P均〈0．05]，低于假手术组（41．3±1．8）％（P〈0．05），LVEF显著高于普通腺病毒组和DMEM组[（48．23±7．06）％比（34．59±6．23）％和（38．1±2．8）％，P均〈0．05]，低于假手术组（77．7±3．1）％（P〈0．05），LVEDD显著小于普通腺病毒组和DMEM组[（4．88±0．36）mm比（5．72±0．62）mm和（5．49±0．37）mm，P均〈0．05]，大于假手术组（3．26±0．29）mm（P〈0．05），LVESD显著小于普通腺病毒组和DMEM组[（3．87±0．45）mm比（4．91±0．62）mm和（4．63±0．37）mm，P均〈0．05]，大于假手术组（1．93±0．25）mm（P〈0．05）。（3）血管密度检测结果：PDE5shRNA组小鼠血管密度显著高于普通腺病毒组，心肌梗死区为（14．3±2．0）个比（6．6±1．2）个（P〈0．05）、梗死周边为（23．6±2．1）个比（13．7±2．4）个（P〈0．05）。（4）PDE5蛋白表达水平检测结果：PDE5shRNA组显著低于普通腺病毒组，0．448-4-0．105比0．993±0．057（P〈0．05）。（5）eGMP和PKG蛋白表达的检测结果：PDE5shRNA组两种蛋白的表达水平均显著高于对照组（P均〈0．05）。结论PDE5shRNA可能通过上调cGMP和PKG表达减少小鼠心肌梗死面积和抑制心肌纤维化，从而抑制心室重构、改善心功能。

Objective To observe the function following myocardial infarction in mice. impact of PDE5shRNA on cardiac remodeling and heart Methods Myocardial infarction （MI） was induced in mice by left coronary artery ligation. Mice were randomly assigned to sham group （n = 6）, PDE5shRNA group （ n = 12） , common adenovirus group （ n = 15） and DMEM group （ n = 8 ）. Four weeks post-MI, the survival rate was evaluated. Cardiac function was examined by echocardiography. HE staining and Masson staining were used to evaluate the myocardial infarction size and fibrosis. The number of blood vessels was evaluated by immunohistochemistry, PDE5 protein expression in the left ventricular was detected using Western blot, level of cGMP or PKG activity in the left ventricle was evaluated with ELISA. Results Four weeks post-MI, all mice survived in the sham group, 3 （37%） mice died in the DMEM group, 1 （8%） died in the PDE5shRNA group and 5 died in the common adenovirus group （ 33% ）. Infarct size was significantly reduced in PDE5shRNA group compared with the common adenovirus group and DMEM group [ （ 25.4 ± 2. 9 ） % vs. （42. 0 ± 3.2 ） % and （ 43.4 ± 2. 6 ） %, P 〈 0. 051. Cardiac function was significantly improved in PDE5shRNA group compared to common adenovirus group and DMEM group [ LVFS： （21.1 ± 3.7）% vs. （14.2±2.9）% and （14.22±2.91）%,allP〈0.05; LVEF： （48.2±7.1）% vs. （34.6± 6.2）% and （38.1 ±2.8）%, allP〈0.05; LVESD： （3.87 -±0.45） mm vs. （4.91 ±0.62） mm and （4. 63 ± 0. 37 ） ram, all P 〈 0. 05 ] . The blood vessel density was also higher in PDE5shRNA group compared with common adenovirus group （ infarct area ： 14. 3 ± 2. 0 vs. 6. 6 ± 1.2, P 〈 0. 05 ; periinfarct area： 23.6 ±2. 1 vs. 13.7 ±2.4, P 〈0. 05）. Compared with common adenovirus group, level of PDE5 was significantly downregulated and level of eGMP or PKG was significantly upregulated in PDE5shRNA group （ all P 〈 0. 05 ） . Conclusions Present study suggests PDE5shRNA improves cardiac function and attenuates cardiac remodeling through reducing infarction size and cardiac fibrosis and these beneficial effects are possibly mediated by activating cGMP/PKG signaling pathway.