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心力衰竭,简称心衰,是由于心脏器质性或功能性异常导致的心室充盈或射血能力受损所引起的复杂临床综合征[1]。随着人口老龄化程度不断加深以及许多患者从急性心脏疾病中存活,慢性心衰的患者数呈现增长态势,尽管过去30年其生存率有了显著提高,但5年病死率仍约为50%[2, 3]。慢性心衰因其高发病率和高病死率,长期以来一直是社会的主要医疗负担[4]。慢性心衰主要表现为射血分数降低的心衰,其药物治疗主要包括血管紧张素转换酶抑制剂或血管紧张素受体脑啡肽酶抑制剂、β受体阻滞剂、盐皮质激素受体拮抗剂、钠-葡萄糖协同转运体2抑制剂组成的四联疗法[5]。患者尤其是长期用药者,由于低血压、电解质紊乱等不良反应的影响,通常用药依从性欠佳。
传统中药在治疗心衰方面有着悠久的历史和独特的理论[6],具有多靶点且成本较低等优势,如黄芪[7]。黄芪注射液是临床上治疗心衰的常用中药注射剂之一,疗效肯定[8]。大量研究已经证实了黄芪治疗心衰的有效性和安全性[9, 10]。黄芪甲苷是黄芪治疗心衰的主要活性成分[11]。但是,由于黄芪甲苷水溶性差,生物利用度极低,限制了黄芪甲苷药物制剂在临床上的转化[12, 13]。因此,黄芪甲苷可作为先导化合物,进行结构优化,既保持其原有的活性,又能增强其水溶性,以便增加生物利用度,充分发挥黄芪甲苷治疗心衰的作用。本研究主要从合成的系列水溶性黄芪甲苷衍生物中,筛选具有抗心衰效用的化合物,并初步探讨其对心肌重塑和心肌纤维化的改善作用,以期为临床提供抗心衰的候选新药。
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通过2.5%异氟烷麻醉小鼠,气管插管接入呼吸机保持麻醉状态。开胸暴露心脏,在左心耳下缘约1 mm处用线缝扎冠状动脉,缝合后,待小鼠恢复自主呼吸,饲养3~4周,通过心超检测慢性心衰模型,从中筛选慢性心衰造模成功的小鼠(EF<50%)。
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首先对黄芪甲苷衍生物进行药效筛选。将LAD结扎造模成功后的小鼠随机分为模型组、卡托普利组、系列黄芪甲苷衍生物(HHQ、HHQ12CS、HHQ18TC)组、假手术组,共6组。造模后4周使用0.5%CMC-Na溶液配制黄芪甲苷衍生物,并按照小鼠每10 g体重0.1 ml进行灌胃给药,黄芪甲苷衍生物组分别给予10 mg/kg的HHQ、HHQ12CS、HHQ18TC,假手术组和模型组分别给予同等剂量的0.5%CMC-Na溶液,卡托普利组给予8.6 mg/kg卡托普利混悬液,每日给药1次,连续28 d。HHQ衍生物药效筛选分组同上,即造模成功的小鼠随机分为模型组、HHQ组、HHQ16组和HHQ19组、假手术组,一共5组。假手术组和模型组处理同上,HHQ衍生物组分别用10 mg/kg的HHQ、HHQ16、HHQ19灌胃,每日给药1次,连续28 d。
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通过吸入2.5%异氟烷气体麻醉小鼠,在检测过程中提供1.0%~1.5%浓度的异氟烷以维持麻醉状态。通过二维及M型超声诊断仪检测小鼠的心功能,筛选造模后的成功模型(EF<50%)以及评价给药后小鼠的心功能。主要检测指标有左室射血分数(LVEF)、左室短轴缩短率(LVFS)等参数。分别于给药18、28 d对各组小鼠进行心脏超声检测。
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对小鼠进行心脏超声检测之后称重,然后取心脏称重。计算心脏重量和体重的比值,即心体比(mg/g)。心脏标本用10%福尔马林固定24 h后,将心脏取出,排列整齐,拍照,观察心脏的大小和形态。取小鼠心脏组织于4%多聚甲醛中固定24 h后,常规石蜡包埋切片处理。用HE染色和Masson染色观察心脏大体形态及胶原形成。
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提取心肌组织总RNA,逆转录成cDNA,实时定量PCR体系包括:SYBR 10 μl,引物(上游引物和下游引物)1 μl,cDNA 3 μl,以及DEPC水7 μl,引物合成序列见表1。
表 1 引物合成序列
名称 上游引物 下游引物 COL1A1 TAAGGGTCCCCAATGGTGAGA GGGTCCCTCGACTCCTACAT COL3A1 ACGTAGATGAATTGGGATGCAG GGGTTGGGGCAGTCTAGTG αSMA GGACGTACAACTGGTATTGTGC TCGGCAGTAGTCACGAAGGA TGF-β1 GAGCCCGAAGCGGACTACTA TGGTTTTCTCATAGATGGCGTTG Gapdh GTATGACTCCACTCACGGCAAA GGTCTCGCTCCTGGAAGATG -
心脏组织于4%多聚甲醛组织固定液中固定。梯度乙醇脱水后将组织浸入热石蜡中,将浸好的石蜡放入包埋机中包埋。将组织进行横切后去石蜡,使用Target Retrieval Solution进行抗原修复操作。用内源性过氧化物酶封闭液消除内源性过氧化物酶的干扰。用1%牛血清白蛋白封闭,使用相应的特异性抗体进行染色,根据种属关系选择对应二抗染色。为了可视化染色,使用DAB显色,并用苏木素进行复染。
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实验处理及统计图表的绘制利用GraphPad Prism软件完成。实验结果均采用均数±标准误(mean ± SEM)的方式呈现。两组样本之间的比较采用两独立样本t检验(t-test);两组以上两两之间比较采用单因素方差分析(one-way ANOVA)或双因素重复方差分析(two-way ANOVA)。以P<0.05为差异有统计学意义。
Efficacy and mechanism of astragaloside Ⅳ derivatives on chronic heart failure in mice
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摘要:
目的 对黄芪甲苷系列衍生物治疗慢性心力衰竭的药效学进行评价,筛选候选化合物,初步探讨候选化合物黄芪甲苷衍生物HHQ16抗心衰的作用机制。 方法 采用左冠状动脉前降支结扎四周诱导C57BL/6小鼠慢性心衰模型,将小鼠分为假手术组、模型组、阳性对照卡托普利组、系列黄芪甲苷衍生物组,共4组。小鼠灌胃给药4周,超声心动检测心功能,筛选出治疗心衰的最优黄芪甲苷衍生物HHQ16。进一步对HHQ16进行初步机制研究,通过大体形态学观察心脏的大小变化;HE染色观察心脏的病理变化;Masson染色观察心肌胶原沉积;免疫组化染色检测心肌纤维化指标Ⅰ型胶原(COL1)、III型胶原(COL3)、α平滑肌肌动蛋白(αSMA)蛋白水平变化;qPCR技术测定心肌纤维化指标COL1、COL3、αSMA和转化生长因子β1(TGF-β1)mRNA水平的变化。 结果 黄芪甲苷衍生物均能显著增加左室射血分数(LVEF)和左室短轴缩短率(LVFS),改善心功能,以HHQ16为最优化合物。与模型组相比,HHQ16组心脏体积显著减小,心肌肥大减轻,心肌组织胶原沉积显著下降,心肌纤维化指标COL1、COL3、αSMA和TGF-β1 mRNA水平以及COL1、COL3、αSMA蛋白水平显著降低。 结论 HHQ16是治疗小鼠心衰的最优黄芪甲苷衍生物,它通过改善心肌重塑,抑制心肌肥大和心肌纤维化,改善心功能。 Abstract:Objective To evaluate the pharmacodynamics of astragaloside Ⅳ derivatives for chronic heart failure, screen the candidate compounds and preliminarily explore the mechanism of the candidate compound HHQ16 against heart failure. Methods Chronic heart failure was induced by left anterior descending artery ligation in C57BL/6 mice for 4 weeks, and the mice were divided into 4 groups, including sham group, model group, positive control captopril group, and astragaloside Ⅳ derivatives group. After continuous intragastric administration for four weeks, the cardiac function was detected by echocardiography, and the optimal astragaloside Ⅳ derivative HHQ16 was selected for the treatment of heart failure. The preliminary mechanism for HHQ16 was further explored. The size of heart was observed by gross morphology; pathological changes were observed by HE staining; collagen deposition in the myocardium was observed by Masson staining; protein levels of myocardial fibrosis indexes COL1, COL3, and αSMA were detected by immunohistochemical staining, and mRNA levels of myocardial fibrosis indexes COL1, COL3, αSMA, and TGF-β1 were determined by qPCR technique. Results All astragaloside Ⅳ derivatives significantly improved cardiac function with increasing LVEF and LVFS, of which HHQ16 was the optimal compound. Compared with the model group, the heart volume of HHQ16 group was significantly reduced; myocardial hypertrophy was reduced; collagen deposition in myocardial tissues was reduced; and myocardial fibrosis indexes, COL1, COL3, αSMA and TGF-β1 mRNA levels, as well as the protein levels of COL1, COL3 and αSMA were significantly reduced. Conclusion HHQ16 is an optimal astragaloside Ⅳ derivatives for the treatment of chronic heart failure in mice, which could improve cardiac function by improving myocardial remodeling, and inhibit myocardial hypertrophy and myocardial fibrosis. -
表 1 引物合成序列
名称 上游引物 下游引物 COL1A1 TAAGGGTCCCCAATGGTGAGA GGGTCCCTCGACTCCTACAT COL3A1 ACGTAGATGAATTGGGATGCAG GGGTTGGGGCAGTCTAGTG αSMA GGACGTACAACTGGTATTGTGC TCGGCAGTAGTCACGAAGGA TGF-β1 GAGCCCGAAGCGGACTACTA TGGTTTTCTCATAGATGGCGTTG Gapdh GTATGACTCCACTCACGGCAAA GGTCTCGCTCCTGGAAGATG -
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