丹皮酚对心力衰竭大鼠心肌损伤的保护作用及其机制

doi:10.13481/j.1671-587x.20200416

摘要/Abstract

摘要： 目的：探讨丹皮酚对心力衰竭大鼠心肌损伤的保护作用，初步阐明其药理学机制。方法：以腹腔注射阿霉素的方法复制心力衰竭大鼠模型，造模成功后大鼠随机分为模型组（n=10）、卡托普利组（n=11）、低剂量丹皮酚组（n=11）和高剂量丹皮酚组（n=11），并以正常大鼠作为对照组（n=10）。大鼠造模成功后开始给药，连续给药5周，记录大鼠体质量。末次给药后麻醉大鼠，观察大鼠心功能指标[平均动脉压（MAP）、心率（HR）、左室舒张末压（LVEDP）、左室收缩压（LVSP）、左室内压最大下降速率（-dp/dt_max）和左室内压最大上升速率（+dp/dt_max）]的变化。取大鼠眼眶外周血分离血清，采用ELISA法检测各组大鼠血清中肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）水平。随机选取5只大鼠麻醉后，在无菌操作下取出心脏，采用Western blotting法检测各组大鼠心肌组织中Bax、B细胞淋巴瘤2（Bcl-2）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）、半胱氨酸天冬氨酸蛋白酶8（Caspase-8）、半胱氨酸天冬氨酸蛋白酶9（Caspase-9）和细胞色素C（Cyt-C）蛋白表达水平。采用HE染色法观察大鼠心肌组织病理形态表现。结果：与对照组比较，模型组大鼠体质量明显降低（P<0.05），MAP和LVEDP明显升高（P<0.05），而HR、LVSP、+dp/dt_max和-dp/dt_max明显降低（P<0.05），血清中TNF-α和IL-6水平明显升高（P<0.05），心肌组织中Bax、Caspase-3、Caspase-8、Caspase-9和Cyto-C蛋白表达水平明显升高（P<0.05），Bcl-2蛋白表达水平明显降低（P<0.05）；与模型组比较，卡托普利组和低、高剂量丹皮酚组大鼠体质量明显升高（P<0.05），心功能指标明显改善（P<0.05），血清中TNF-α和IL-6水平不同程度降低（P<0.05），心肌组织中Bax、Caspase-3、Caspase-8、Caspase-9和Cyt-C蛋白表达水平明显降低（P<0.05），Bcl-2蛋白表达水平明显升高（P<0.05）。HE染色，与对照组比较，模型组大鼠心肌组织受损明显，心肌细胞广泛水肿；与模型组比较，卡托普利组和低、高剂量丹皮酚组大鼠心肌纤维排列和心肌间质水肿等现象明显好转。结论：丹皮酚对心力衰竭大鼠心肌具有保护作用，其机制可能与其改善血流动力学、抗炎、抗凋亡及保护心肌细胞等有关，高剂量丹皮酚在心功能保护及抗凋亡方面效果较好。

关键词: 丹皮酚, 阿霉素, 心力衰竭, 心功能, 血流动力学, 疾病模型,动物

Abstract: Objective: To investigate the protective effect of paeonol on myocardial injury in the rats with heart failure, and to elucidate its preliminary pharmacological mechanism. Methods: The rat models of heart failure were replicated by intraperitoneal injection of doxorubicin. After successful modeling, the rats were randomly divided into model group(n=10), captopril group(n=11), low dose of paeonol group(n=11) and high dose of paeonol group(n=11), and the normal rats were used as control group(n=10).After successful modelring, the rats were given the drug continuously for 5 weeks, and the body weights of rats were recorded.After the last administration,the rats were anesthetized,and the changes of mean arterial pressure(MAP),heart rate(HR),left ventricular end diastolic pressure(LVEDP), left ventricular systolic blood pressure (LVSP), maximum rate of decrease of left ventricular blood pressure (-dp/dt_max), and maximum rate of increase of left ventricular blood pressure (+dp/dt_max)]of the rats were observed.The serum of rat orbital peripheral blood was isolated, and the levels of the serum tumor necrosis factor (TNF-β) and interleukin-6 (IL-6) were detected by ELISA. After anesthesia, 5 rats were randomly selected and the heart was obtained under aseptic operation; Western blotting method was used to detect the expression levels of Bax, B cell lymphoma - 2 (Bcl-2), cysteine aspartic acid protease 3 (Caspase-3), cysteine aspartic acid protease 8 (Caspase-8), cysteine aspartic acid protease 9 (Caspase-9) and cytochrome C (Cyt C) proteins in myocardium tissue of the rats in various groups. HE staining was used to observe the pathomorphology of myocardium tissue of the rats. Results: Compared with control group, the body weight of the rats in model group was significantly reduced (P<0.05),and MAP and LVEDP were increased(P<0.05), HR, LVSP, +dp/dt_max and -dp/dt_max were significantly decreased (P<0.05),the serum TNF-α and IL-6 levels were increased (P<0.01), the expression levels of Bax,Caspase-3,Caspase-8,Caspase-9 and Cyto-C proteins were significantly increased (P<0.01),and the expression level of Bcl-2 was significantly decreased (P<0.05). Compared with model group, the body weights of the rats in captopril,low and high doses of paeonol groups were significantly increased (P<0.05),the cardiac function indexes were improved significantly (P<0.05), the TNF-α and IL-6 levels were decreased (P<0.05), the expression levels of Bax, Caspase-3, Caspase-8, Caspase-9 and Cyto-C proteins in myocardium tissue of the rats were significantly decreased (P<0.05), and the Bcl-2 protein expression levels were increased significantly (P<0.05).The HE staining results showed that compared with control group, the myocardium tissue of the rats in model group was damaged obviously, and the myocardial cells were edema extensively. Compared with model group, the myocardial fiber arrangement and myocardial interstitial edema of the rats in captopril group and low and high doses of paeonol groups were significantly improved. Conclusion: Paeonol has a protective effect on the myocardium of the rats with heart failure, and the mechanism may be related to the improvement of hemodynamics, anti-inflammation and anti-apoptosis, and the protection of myocardial cells. High dose of paeonol is more effective in protecting the cardiac function and anti-apoptosis.

Key words: paeonol, adriamycin, heart failure, myocardial function, hemodynamics, disease model,animal

中图分类号:

R285.5

高瑞敏, 康玲玲, 侯防震. 丹皮酚对心力衰竭大鼠心肌损伤的保护作用及其机制[J]. 吉林大学学报(医学版), 2020, 46(04): 765-770.

GAO Ruimin, KANG Lingling, HOU Fangzhen. Protective effect of paeonol on myocardial injury in rats with heart failure and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 765-770.

参考文献

[1] RONCO C, CICOIRA M, MCCULLOUGH P A. Cardiorenal syndrome type 1:pathophysiological crosstalk leading to combined heart and kidney dysfunction in the setting of acutely decompensated heart failure[J]. J Am Coll Cardiol, 2012,60(12):1031-1042.
[2] DAMMAN K, TESTANI J M. The kidney in heart failure:an update[J]. Eur Heart J, 2015, 36(23):1437-1444.
[3] DAMMAN K, TANG W H, TESTANI J M, et al. Terminology and definition of changes renal function in heart failure[J]. Eur Heart J, 2014, 35(48):3413-3416.
[4] BAGSHAW S M, CRUZ D N, ASPROMONTE N, et al. Epidemiology of cardio-renal syndromes:workgroup statements from the 7th ADQI Consensus Conference[J]. Nephrol Dial Transplant,2010,25(5):1406-1416.
[5] 叶婷, 张宇, 张梦, 等. 中医药治疗慢性心力衰竭药理机制研究进展[J]. 中西医结合心脑血管病杂志, 2016, 14(8):841-843.
[6] 苏志远, 叶小汉, 吴锦波. 心康方治疗慢性心力衰竭临床观察[J]. 新中医, 2016, 48(3):12-14.
[7] 孙慧萍, 曹军平, 徐丽, 等. 丹皮酚对动脉粥样硬化大鼠炎症因子的影响[J]. 中华中医药学刊, 2016, 34(1):14-16.
[8] LU L, QIN Y T, CHEN C,et al. Beneficial effects exerted by paeonol in the management of atherosclerosis[J].Oxid Med Cell Longev, 2018, 2018:1098617.
[9] 张竞之, 陈小忆, 金伟孝, 等. 丹皮酚对高血压病血瘀证患者血清损伤的HUVEC-C形态学、活性的影响[J]. 辽宁中医药大学学报, 2012, 14(6):29-31.
[10] 陆建洪, 翟昌林, 陈捷. 丹皮酚对大鼠缺血再灌注损伤心肌细胞凋亡及其Bcl-2和Bax表达的影响[J]. 中国中医药科技, 2013, 20(2):151-152.
[11] SUEMATSU Y, JING W H, NUNES A,et al. LCZ696(sacubitril/valsartan), an angiotensin-receptor neprilysin inhibitor, attenuates cardiac hypertrophy, fibrosis, and vasculopathy in a rat model of chronic kidney disease[J]. J Card Fail, 2018,24(4):266-275.
[12] HAO G P, ZHAI J, JIANG H M, et al. Acetylshikonin induces apoptosis of human leukemia cell line K562 by inducing S phase cell cycle arrest, modulating ROS accumulation, depleting Bcr-Abl and blocking NF-κB signaling[J].Biomed Pharmacother, 2019, 122:109677.
[13] LIU X Y, CHEN J Y, LI W, et al. Inhibition of casein kinase Ⅱ by CX-4945, but not yes-associated protein (YAP) by verteporfin, enhances the antitumor efficacy of temozolomide in glioblastoma[J].Transl Oncol, 2020, 13(1):70-78.
[14] BELOUSOV V V, FRADKOV A F, LUKYANOV K A, et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide[J]. Nat Methods, 2006, 3(4):281-286.
[15] BERTERO E, MAACK C. Metabolic remodelling in heart failure[J]. Nat Rev Cardiol, 2018,15(8):457-470.
[16] VON LUEDER T G, WANG B H, KOMPA A R, et al. Angiotensin receptor neprilysin inhibitor LCZ696 attenuates cardiac remodeling and dysfunction after myocardial infarction by reducing cardiac fibrosis and hypertrophy[J]. Circ Heart Fail, 2015, 8(1):71-78.
[17] BOEHM O, ZUR B, KOCH A, et al. Clinical chemistry reference database for Wistar rats and C57/BL6 mice[J]. Biol Chem, 2007,388(5):547-554.
[18] BRAISSANT O, MCLIN V A, CUDALBU C. Ammonia toxicity to the brain[J].J Inherited Metab Dis, 2013,36:595-612.
[19] ZHANG X X,ZHAI Y H,YUAN J H, et al. New insights into Paeoniaceae used as medicinal plants in China[J].Sci Rep, 2019, 9(1):18469.
[20] CHEN G, JIA P, YIN Z Y, et al. Paeonol ameliorates monosodium urate-induced arthritis in rats through inhibiting nuclear factor-κB-mediated proinflammatory cytokine production[J].Phytother Res, 2019, 33:2971-2978.
[21] BRAUNWALD E. Heart failure[J]. JACC, 2013,1:1-20.
[22] STEINHORN B, SORRENTINO A, BADOLE S, et al. Chemogenetic generation of hydrogen peroxide in the heart induces severe cardiac dysfunction[J]. Nat Commun, 2018, 9(1):4044.