养心康片调节自噬对慢性心力衰竭小鼠心肌纤维化的影响
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摘要
目的:研究养心康片对心衰后小鼠心肌纤维化的影响,探讨其作用机制。方法:采用胸主动脉缩窄法(TAC)建立小鼠慢性心衰模型,建模成功后随机分为假手术组,模型组,3-甲基腺嘌呤(3-MA)组(15 mg·kg~(-1)),养心康片高、中、低剂量组(1 170,585,390 mg·kg~(-1)),假手术组给予等体积蒸馏水灌胃。30 d后行心脏超声检查,收集血流动力学参数;通过心脏石蜡切片,马松(Masson)染色观察心肌细胞形态学变化及心肌纤维化程度;蛋白免疫印迹法(Western blot)检测微管相关蛋白轻链3(LC3),酵母ATG6同源物(Beclin-1),溶酶体膜蛋白(LAMP)心肌自噬蛋白,Ⅰ型胶原酶(CollagenⅠ),Ⅲ型胶原酶(CollagenⅢ),α-平滑肌肌动蛋白(α-SMA)心肌纤维化标志性蛋白表达情况。结果:与假手术组比较,模型组小鼠左心射血分数(LVEF),短轴缩短分数(FS)明显降低(P <0. 05),左心室舒张末期内径(LVDd),左心室收缩末期内径(LVDs)明显增大(P <0. 05),心肌纤维化程度显著加重(P <0. 01),α-SMA,CollagenⅠ,CollagenⅢ心肌纤维化标志性蛋白,LAMP,LC3,Beclin-1自噬蛋白表达均明显升高(P <0. 05);与模型组比较,3-MA组,养心康高、中剂量组心脏超声各项指标均显著改善(P <0. 05),心肌纤维化程度明显改善(P <0. 01),α-SMA,CollagenⅠ,CollagenⅢ,LAMP,LC3,Beclin-1表达明显降低(P <0. 05),低剂量组心功能、心肌纤维化及各项蛋白表达差异不明显。结论:养心康片可通过下调自噬减缓慢性心力衰竭小鼠心肌纤维化,改善心功能。
Objective: To study the effect of Yangxinkang tablets on myocardial fibrosis in mice after heart failure,and to explore its mechanism. Method: The model of chronic heart failure in mice was established by thoracic aorta constriction( TAC). After successful modeling,mice were randomly divided into sham operation group,model group,3-methyladenine( 3-MA,15 mg·kg~(-1)) autophagy inhibitor group,Yangxinkang tablets high,medium,and low dose groups( 1 170,585,390 mg·kg~(-1)). The sham operation group received equal volume of distilled water. After 30 days,cardiac ultrasound was performed to collect hemodynamic parameters.Cardiac paraffin slices were stained with Masson to observe the morphological changes and fibrosis of cardiomyocytes. Western blot was used to detect lysosome-associated membrane protein( LAMP),microtubuleassociated protein light chain 3( LC3),Beclin-1 autophagyportein,α-smooth muscle activin( α-SMA),CollagenⅠ,Collagen Ⅲ protein expression. Result: As compared with normal group,the left ventricular ejection fraction( LVEF) and fractional shortening( FS) were significantly decreased( P < 0. 05),the left ventricular enddiastolic dimension( LVDd) and left ventricular end-systolic dimension( LVDs) were significantly increased( P <0. 05),the degree of myocardial fibrosis was significantly aggravated in model group( P < 0. 01),and the protein expression levels of α-SMA,CollagenⅠ,Collagen Ⅲ,LAMP,LC3,and Beclin-1 were significantly increased in model group( P < 0. 05). As compared with model group,the cardiac ultrasound indexes of the 3-MA group,Yangxinkang high and medium dose groups were significantly improved( P < 0. 05),the degree of myocardial fibrosis was significantly reduced( P < 0. 01),the protein expression levels of α-SMA,CollagenⅠ,CollagenⅢ,LAMP,LC3 and Beclin-1 were decreased in 3-MA group,Yangxinkang high and medium dose groups( P <0. 05),while the protein expression levels between the model group and Yangxinkang low-dose group showed no significant difference. Conclusion: Yangxinkang tablets can reduce myocardial fibrosis and improve cardiac function in mice with heart failure probably by down-regulating autophagy.
Objective: To study the effect of Yangxinkang tablets on myocardial fibrosis in mice after heart failure,and to explore its mechanism. Method: The model of chronic heart failure in mice was established by thoracic aorta constriction( TAC). After successful modeling,mice were randomly divided into sham operation group,model group,3-methyladenine( 3-MA,15 mg·kg~(-1)) autophagy inhibitor group,Yangxinkang tablets high,medium,and low dose groups( 1 170,585,390 mg·kg~(-1)). The sham operation group received equal volume of distilled water. After 30 days,cardiac ultrasound was performed to collect hemodynamic parameters.Cardiac paraffin slices were stained with Masson to observe the morphological changes and fibrosis of cardiomyocytes. Western blot was used to detect lysosome-associated membrane protein( LAMP),microtubuleassociated protein light chain 3( LC3),Beclin-1 autophagyportein,α-smooth muscle activin( α-SMA),CollagenⅠ,Collagen Ⅲ protein expression. Result: As compared with normal group,the left ventricular ejection fraction( LVEF) and fractional shortening( FS) were significantly decreased( P < 0. 05),the left ventricular enddiastolic dimension( LVDd) and left ventricular end-systolic dimension( LVDs) were significantly increased( P <0. 05),the degree of myocardial fibrosis was significantly aggravated in model group( P < 0. 01),and the protein expression levels of α-SMA,CollagenⅠ,Collagen Ⅲ,LAMP,LC3,and Beclin-1 were significantly increased in model group( P < 0. 05). As compared with model group,the cardiac ultrasound indexes of the 3-MA group,Yangxinkang high and medium dose groups were significantly improved( P < 0. 05),the degree of myocardial fibrosis was significantly reduced( P < 0. 01),the protein expression levels of α-SMA,CollagenⅠ,CollagenⅢ,LAMP,LC3 and Beclin-1 were decreased in 3-MA group,Yangxinkang high and medium dose groups( P <0. 05),while the protein expression levels between the model group and Yangxinkang low-dose group showed no significant difference. Conclusion: Yangxinkang tablets can reduce myocardial fibrosis and improve cardiac function in mice with heart failure probably by down-regulating autophagy.
引文
[1] Mozaffarian D,Benjamin E J,Go A S,et al. Heart disease and stroke statistics——2016 update:a report from the American Heart Association[J]. Circulation,2016,133(4):38-360.
[2] Fonseca C,Maggioni A P,Marques F,et,al. A systematic review of in-hospital worsening heart failureas an endpoint in clinical investigations of therapy for acute heart failure[J]. Int J Cardiol,2018,250:215-222.
[3] Schelbert E B,Piehler K M,Zareba K M. et,al.Myocardial fibrosis quantified by extracellular volume is associated with subsequent hospitalization for heart failure,death,or both across the spectrum of ejection fraction and heart failure stage[J]. J Am Heart Assoc,2015,4(12).
[4] QIAO B,HE B X,CAI J H,et al. MicroRNA-27a-3p modulates the Wnt/β-catenin signaling pathway to promote epithelial-mesenchymal transition in oral squamous carcinoma stem cells by targeting SFRP1[J].Sci Rep,2017,7:44688.
[5] ZHOU L C,MA B H,HAN X Z. The role of autophagy in angiotensinⅡ-induced pathological cardiac hypertrophy[J]. Mol Endocrinol,2016,57(4):143-152.
[6] LIU L,WANG C,SUN D,et al. Calhex231 ameliorates cardiac hypertrophy by inhibiting cellular autophagy in vivo and in vitro[J]. Cell Physiol Biochem,2015,36(4):1597-1612.
[7] Garcia A G,Wilson R M,Heo J,et al. Interferon-γablation exacerbates myocardial hypertrophy in diastolic heart failure[J]. Am J Physiol Heart Circ Physiol,2012,303(5):587-596.
[8] Nakai K,Tsuboi J,Okabayashi H,et al. Development of a signal-averaged vector-projected 187-channel highresolution electrocardiogram for the evaluation of the spatial location of high-frequency and abnormal ventricular repolarization[J]. Int Heart J,2007,48(6):701-713.
[9] WENG L Q,ZHANG W B,YE Y,et al. Aliskiren ameliorates pressure overload-induced heart hypertrophy and fibrosis in mice[J]. Acta Pharmacol Sin,2014,35(8):1005-1014.
[10]任培华,王鹏,廖东江,等.养心康片对心梗后心力衰竭模型兔血管紧张素Ⅱ、醛固酮和心肌病理形态的影响[J].中国中医急症,2018,27(2):205-207,218.
[11]沈凌,王嵩.养心康治疗无症状心力衰竭临床研究[J].新中医,2014,46(9):26-27.
[12] Tavakoli R,Nemska S,Jamshidi P,et al. Technique of minimally invasive transverse aortic constriction in mice for induction of left ventricular hypertrophy[J]. J Vis Exp,2017,dio:10. 3791/56231.
[13] Songstad N T,Johansen D,How O J,et al. Effect of transverse aortic constriction on cardiac structure,function and gene expression in pregnant rats[J]. PLo S One,2014,9(2):e89559.
[14]唐晓霞.高分辨力超声评价小鼠心脏结构和功能的价值[D].南京:南京医科大学,2006.
[15]赵菁,王迎超,张玲.超声心动图评价不同心肌梗死模型小鼠心功能的改变[J].中华急诊医学杂志,2015,24(11):1242.
[16]魏伟,吴希美,李健.药理实验方法学,第四版[M].北京:人民卫生出版社,2010:1698.
[17] YI J,HE G,YANG J,et al. Heat acclimation regulates the autophagy-lysosome function to protect against heat stroke-induced brain injury in mice[J]. Cell Physiol Biochem,2017,41(1):101-114.
[18] Go A S,Mozaffarian D,Roger V L,et al. Executive summary:heart disease and stroke statistics-2014update:a report from the American Heart Association[J]. Circulation,2014,129(3):399-410.
[19] Porter K E,Turner N A. Cardiac fibroblasts:at the heart of myocardial remodeling[J]. Pharmacol Ther,2009,123(2):255-278.
[20] Weber K T, SUN Y,et al. Myofibroblast-mediated mechanisms of pathological re-modelling of the heart[J]. Nat Rev Cardiol,2013,10(1):15-26.
[21] Sw V D B,Diez J,Blankesteijn W M,et al. Myocardial remodeling after infarction:the role of myofibroblasts[J]. Nat Rev Cardiol,2010,7(1):30-37.
[22]陈艳俏,陈少军,曲畅.益气养阴活血法配合西医综合疗法治疗慢性充血性心力衰竭疗效观察[J].辽宁中医杂志,2012,39(7):1307-1308.
[23]郑颖,黄芪,张运.益气养阴活血中药治疗慢性心力衰竭急性加重36例临床疗效观察[J].天津中医药,2014,31(7):412-415.
[24]黄衍寿,冼绍祥,吴辉.养心康治疗充血性心力衰竭临床研究[J].中国中西医结合急救杂志,2000,7(2):71-74.
[25]任培华,冼绍祥,孙敬和,等.养心康对慢性心功能不全兔心室重构的影响[J].中药新药与临床药理,2012,23(1):58-60.
[26] XIAN S X,YANG Z Q,REN P H,et al. Effect of yangxinkang tablets on chronic heart failure:a multicenter randomized double-blind placebo-controlled trial[J]. Chin J Integr Med,2015,21(10):733-742.
[27]徐国良,余日跃,李冰涛,等.中药复方量效关系剂量阈研究方法探讨[J].世界中医药,2014,9(1):11-13.
[28] FENG Y,HE D,YAO Z,et al. The machinery of macroautophagy[J]. Cell Res,2014,24(1):24-41.
[29] LI B,SUN Y,WANG J P. Antioxidant N-acetylcysteine inhibits maladaptive myocyte autophagy in pressure overload induced cardiac remodeling in rats[J]. Eur J Pharmacol,2018,839:47-56.
[30] Mari1o G,Pietrocola F,KONG Y,et al. Dimethylα-ketoglutarate inhibits maladaptive autophagy in pressure overload-induced cardiomyopathy[J]. Autophagy,2014,10(5):930-932.
[31] ZHU H X,Tannous P,Johnstone J L,et al. Cardiac autophagy is a maladaptive response to hemodynamic stress[J]. Clin Invest,2007,117(7):1782-1793.
[32] WANG H B,YANG X,YANG Q Q,et al. Parp-1inhibition attenuates cardiac fibrosis induced by myocardial infarction through regulating autophagy[J].Biochem Biophys Res Commun,2018,503(3):1625-1632.
[33] Sw V D B,Isobe S,Verjans J W,et al. Molecular imaging of interstitial alterations in remodeling myocardium after myocardial infarction[J]. J Am Coll Cardiol,2008,52(24):2017-2028.
[2] Fonseca C,Maggioni A P,Marques F,et,al. A systematic review of in-hospital worsening heart failureas an endpoint in clinical investigations of therapy for acute heart failure[J]. Int J Cardiol,2018,250:215-222.
[3] Schelbert E B,Piehler K M,Zareba K M. et,al.Myocardial fibrosis quantified by extracellular volume is associated with subsequent hospitalization for heart failure,death,or both across the spectrum of ejection fraction and heart failure stage[J]. J Am Heart Assoc,2015,4(12).
[4] QIAO B,HE B X,CAI J H,et al. MicroRNA-27a-3p modulates the Wnt/β-catenin signaling pathway to promote epithelial-mesenchymal transition in oral squamous carcinoma stem cells by targeting SFRP1[J].Sci Rep,2017,7:44688.
[5] ZHOU L C,MA B H,HAN X Z. The role of autophagy in angiotensinⅡ-induced pathological cardiac hypertrophy[J]. Mol Endocrinol,2016,57(4):143-152.
[6] LIU L,WANG C,SUN D,et al. Calhex231 ameliorates cardiac hypertrophy by inhibiting cellular autophagy in vivo and in vitro[J]. Cell Physiol Biochem,2015,36(4):1597-1612.
[7] Garcia A G,Wilson R M,Heo J,et al. Interferon-γablation exacerbates myocardial hypertrophy in diastolic heart failure[J]. Am J Physiol Heart Circ Physiol,2012,303(5):587-596.
[8] Nakai K,Tsuboi J,Okabayashi H,et al. Development of a signal-averaged vector-projected 187-channel highresolution electrocardiogram for the evaluation of the spatial location of high-frequency and abnormal ventricular repolarization[J]. Int Heart J,2007,48(6):701-713.
[9] WENG L Q,ZHANG W B,YE Y,et al. Aliskiren ameliorates pressure overload-induced heart hypertrophy and fibrosis in mice[J]. Acta Pharmacol Sin,2014,35(8):1005-1014.
[10]任培华,王鹏,廖东江,等.养心康片对心梗后心力衰竭模型兔血管紧张素Ⅱ、醛固酮和心肌病理形态的影响[J].中国中医急症,2018,27(2):205-207,218.
[11]沈凌,王嵩.养心康治疗无症状心力衰竭临床研究[J].新中医,2014,46(9):26-27.
[12] Tavakoli R,Nemska S,Jamshidi P,et al. Technique of minimally invasive transverse aortic constriction in mice for induction of left ventricular hypertrophy[J]. J Vis Exp,2017,dio:10. 3791/56231.
[13] Songstad N T,Johansen D,How O J,et al. Effect of transverse aortic constriction on cardiac structure,function and gene expression in pregnant rats[J]. PLo S One,2014,9(2):e89559.
[14]唐晓霞.高分辨力超声评价小鼠心脏结构和功能的价值[D].南京:南京医科大学,2006.
[15]赵菁,王迎超,张玲.超声心动图评价不同心肌梗死模型小鼠心功能的改变[J].中华急诊医学杂志,2015,24(11):1242.
[16]魏伟,吴希美,李健.药理实验方法学,第四版[M].北京:人民卫生出版社,2010:1698.
[17] YI J,HE G,YANG J,et al. Heat acclimation regulates the autophagy-lysosome function to protect against heat stroke-induced brain injury in mice[J]. Cell Physiol Biochem,2017,41(1):101-114.
[18] Go A S,Mozaffarian D,Roger V L,et al. Executive summary:heart disease and stroke statistics-2014update:a report from the American Heart Association[J]. Circulation,2014,129(3):399-410.
[19] Porter K E,Turner N A. Cardiac fibroblasts:at the heart of myocardial remodeling[J]. Pharmacol Ther,2009,123(2):255-278.
[20] Weber K T, SUN Y,et al. Myofibroblast-mediated mechanisms of pathological re-modelling of the heart[J]. Nat Rev Cardiol,2013,10(1):15-26.
[21] Sw V D B,Diez J,Blankesteijn W M,et al. Myocardial remodeling after infarction:the role of myofibroblasts[J]. Nat Rev Cardiol,2010,7(1):30-37.
[22]陈艳俏,陈少军,曲畅.益气养阴活血法配合西医综合疗法治疗慢性充血性心力衰竭疗效观察[J].辽宁中医杂志,2012,39(7):1307-1308.
[23]郑颖,黄芪,张运.益气养阴活血中药治疗慢性心力衰竭急性加重36例临床疗效观察[J].天津中医药,2014,31(7):412-415.
[24]黄衍寿,冼绍祥,吴辉.养心康治疗充血性心力衰竭临床研究[J].中国中西医结合急救杂志,2000,7(2):71-74.
[25]任培华,冼绍祥,孙敬和,等.养心康对慢性心功能不全兔心室重构的影响[J].中药新药与临床药理,2012,23(1):58-60.
[26] XIAN S X,YANG Z Q,REN P H,et al. Effect of yangxinkang tablets on chronic heart failure:a multicenter randomized double-blind placebo-controlled trial[J]. Chin J Integr Med,2015,21(10):733-742.
[27]徐国良,余日跃,李冰涛,等.中药复方量效关系剂量阈研究方法探讨[J].世界中医药,2014,9(1):11-13.
[28] FENG Y,HE D,YAO Z,et al. The machinery of macroautophagy[J]. Cell Res,2014,24(1):24-41.
[29] LI B,SUN Y,WANG J P. Antioxidant N-acetylcysteine inhibits maladaptive myocyte autophagy in pressure overload induced cardiac remodeling in rats[J]. Eur J Pharmacol,2018,839:47-56.
[30] Mari1o G,Pietrocola F,KONG Y,et al. Dimethylα-ketoglutarate inhibits maladaptive autophagy in pressure overload-induced cardiomyopathy[J]. Autophagy,2014,10(5):930-932.
[31] ZHU H X,Tannous P,Johnstone J L,et al. Cardiac autophagy is a maladaptive response to hemodynamic stress[J]. Clin Invest,2007,117(7):1782-1793.
[32] WANG H B,YANG X,YANG Q Q,et al. Parp-1inhibition attenuates cardiac fibrosis induced by myocardial infarction through regulating autophagy[J].Biochem Biophys Res Commun,2018,503(3):1625-1632.
[33] Sw V D B,Isobe S,Verjans J W,et al. Molecular imaging of interstitial alterations in remodeling myocardium after myocardial infarction[J]. J Am Coll Cardiol,2008,52(24):2017-2028.