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医药导报, 2019, 38(5): 555-559
doi: 10.3870/j.issn.1004-0781.2019.05.004
新型芹菜素纳米脂质体对糖尿病心肌病大鼠心肌细胞凋亡的影响
Effect of Apigenin-loaded Nanoliposomes on Myocardial Cells Apoptosis Induced by Diabetic Cardiomyopathy
章雅丹

摘要:

目的 观察新型芹菜素纳米脂质体对糖尿病心肌病大鼠心肌细胞凋亡水平的影响。方法 采用薄膜水合结合超声分散技术制备新型芹菜素纳米脂质体并进行质量考察。将健康雄性SD大鼠60只,随机分成正常对照组、模型对照组、空白脂质体组及芹菜素脂质体组,每组15只。采用大鼠腹腔注射链脲佐菌素(70 mg·kg-1)建立糖尿病模型,2周后,芹菜素脂质体组尾静脉给予芹菜素纳米脂质体30 μg·kg-1,空白脂质体组尾静脉给予等剂量空白纳米脂质体,模型对照组及正常对照组给予同等剂量0.9%氯化钠溶液。12周之后处死大鼠,取出心肌组织,分别采用TUNEL凋亡染色测定大鼠心肌细胞凋亡指数,Western blotting分析各组大鼠心肌Bax及Bcl-2蛋白表达。结果 TUNEL凋亡染色显示,与正常对照组比较,模型对照组及空白脂质体组大鼠心肌细胞凋亡指数显著升高(P<0.05);芹菜素脂质体组凋亡指数显著下降(P<0.05)。Western blotting分析显示,与正常对照组比较,模型对照组及空白脂质体组大鼠心肌细胞Bax含量显著升高(P<0.01),Bcl-2含量显著下降(P<0.01);与模型对照组及空白纳米脂质体组比较,芹菜素脂质体组心肌细胞Bax含量显著下降(P<0.01),Bcl-2含量显著升高(P<0.01)。结论 芹菜素纳米脂质体能够通过调节细胞凋亡相关蛋白Bcl-2/Bax途径,降低糖尿病心肌病大鼠心肌细胞凋亡水平。

关键词: 芹菜素 ; 纳米脂质体 ; 细胞凋亡 ; 心肌病 ; 糖尿病

Abstract:

Objective To investigate the effect of apigenin-loaded nanoliposomes on myocardial cells apoptosis induced by diabetic cardiomyopathy. Methods The apigenin-loaded nanoliposomes were prepared by film hydration and ultrasonic dispersion technology and their quality inspections were also investigated. Sixty SD rats were randomly divided into normal control group, model control group, blank and apigenin-loaded nano-liposomes group. Rat model of typeⅠdiabetes was induced by single intraperitoneal injection of STZ. After two weeks of STZ injection, the model rats were used for this study. The rats of apigenin loaded nano-liposomes treatment group were treated with apigenin loaded nano-liposomes via caudal vein administration for 12 weeks (three times a week). And the rats of normal control group and model control group were administrated equivalent volume of 0.9% sodium chloride solution. After treatment for 12 weeks, the experimental animals were sacrificed and their hearts were harvested after 0.9% sodium chloride solution perfusion. The myocardial cell apoptosis index was detected by TUNEL staining and the expressions of Bcl-2 and Bax, which were related to the cell apoptosis, were examined by Western blotting analysis. Results TUNEL staining showed the apoptosis index in the rats of diabetes and blank nano-liposomes groups were significant higher than the normal control group (P<0.05). However, there was significant decrease of apoptosis index in the diabetes rats after the treatment of apigenin loaded nano-lipsomes (P<0.05). Moreover, Western blotting analysis confirmed that there was significant lower expression of Bcl-2 and higher expression of Bax in the hearts of model control group and blank nano-liposomes treated rats compared with normal control rats (P<0.01). Compared with the diabetes and blank nano-liposomes group, the apigenin loaded nano-liposomes treated groups showed higher expression of anti-apoptosis protein Bcl-2 (P<0.01) and lower expression of pro-apoptosis protein Bax (P<0.01). Conclusion Apigenin loaded nano-liposomes have reduced the apoptosis of myocardial cells in diabetic cardiomyopathy rats via the Bcl-2/Bax pathway.

Key words: Apigenin ; Nanoliposomes ; Cells apoptosis ; Cardiomyopathy ; diabetic

糖尿病已经成为危害人类健康的重大疾病之一[1,2]。糖尿病心肌病(diabetic cardiomyopathy,DCM)是糖尿病患者心肌细胞原发性损伤引起的心脏结构以及功能的障碍,已经成为患者致死的主要原因[3]。糖尿病引起的高血糖、高脂血症可以造成心肌细胞凋亡,是DCM的主要病理特征之一,可进一步引起左心室功能紊乱以及心肌细胞纤维化[4]。研究显示,芹菜素能够显著降低糖尿病动物血糖及血脂水平,是潜在的防治DCM的药物[5]。然而,由于其存在溶解性低、在体应用难吸收等问题限制了临床应用[6]。脂质体是一种新型的被动靶向药物递送载体,应用脂质体包载难溶性药物,能够提高药物溶解度,同时促进药物的细胞膜渗透性,增加药物的吸收[7,8],从而实现难溶性药物在体临床应用。笔者制备新型纳米脂质体包载难溶性药物芹菜素,考察其对于DCM大鼠心肌细胞凋亡的预防作用及相关机制。

1 材料与方法
1.1 实验动物

健康雄性SD大鼠60只,体质量为180~220 g,清洁级,由温州医科大学实验动物中心购自上海斯莱克实验动物有限公司。实验动物使用许可证号:SYXK(浙)2015-0001,合格证号:wydw2013-0050,饲养条件符合实验动物管理与使用指南。

1.2 主要试剂

芹菜素(Sigma-Aldrich 公司,含量≥99%,CAS号:520-36-5),注射级蛋黄卵磷脂(上海艾韦特医药科技有限公司,CAS号:93685-90-6),戊巴比妥钠(Sigma-Aldrich 公司,CAS号:57-33-0),链脲佐菌素(Sigma-Aldrich 公司,CAS号 :18883-66-4),TUNEL凋亡试剂盒(Roche Applied Science,批号:11684795910),Bcl-2蛋白(Santa Cruz Biotechnology,批号:sc-71957)以及Bax一抗(Santa Cruz Biotechnology,批号:sc-70407),辣根过氧化酶标记羊抗兔二抗(Santa Cruz Biotechnology,批号:sc-2004),血糖试纸(德国罗氏活力型血糖仪专用试纸,批号:Accu-Chek Performa test strips)。

1.3 芹菜素纳米脂质体的制备及其性质考察

参考JIN等[9]介绍的方法制备新型芹菜素纳米脂质体:芹菜素1 mg,蛋黄卵磷脂10 mg,加入到处方量无水乙醇中完全溶解,将上述溶液在真空条件下旋转蒸发乙醇,形成载药脂质薄膜,然后加入适量纯化水水化12 h,形成胶体溶液。将上述胶体溶液在室温(25 ℃)及高压(73.5 MPa)条件下,超声分散15 s(循环3次),制备芹菜素纳米脂质体。应用上述方法同时制备空白纳米脂质体。

采用扫描电子显微镜考察载药脂质体表观形态,同时应用动态光衍射法测定载药及空白脂质体的粒径和Zeta电位分布。

包封率测定:将上述制备载药脂质体溶液放入透析袋(截留相对分子质量10 000~12 000),室温透析2 h,除去未包封药物及他制备辅料。然后甲醇破坏脂质体,应用高效液相色谱(HPLC)法测定载药脂质体中药物含量。

载体包封率(%)=载药脂质体中药物含量/最开始加入药物×100%。

1.4 糖尿病实验动物模型建立及给药方法

采用随机数字表法将60只大鼠分为正常对照组、模型对照组、空白脂质体组、芹菜素脂质体组,每组15只。饲养1周后,参考ZHAO等[10]介绍的方法建立大鼠1型糖尿病模型,腹腔注射1%链脲佐菌素(70 mg·kg-1),正常对照组腹腔注射同剂量0.9%氯化钠溶液。于注射后第3天、第7天以及第14天尾静脉取血测定血糖。3次测定结果均大于16.7 mmol·L-1且实验动物明显出现多饮、多食、多尿以及体质量下降等现象即说明造模成功。此后芹菜素脂质体组尾静脉给予芹菜素纳米脂质体(30 μg·kg-1),空白脂质体组尾静脉给予等剂量空白纳米脂质体,每周3次,持续给药12周。模型对照组以及正常对照组每周尾静脉给予同剂量0.9%氯化钠溶液。12周之后处死大鼠,迅速开胸,取出心脏,在左心室中部沿横轴切取厚度为0.5 cm的心肌组织,放置在4%多聚甲醛中,剩余心肌细胞-80 ℃保存。

1.5 心肌细胞TUNEL染色

按照罗氏TUNEL凋亡试剂盒说明书,将组织切片脱蜡、乙醇梯度浸洗,Proteinase K工作液透化,在切片上加TUENEL反应液,盖膜,37 ℃孵育60 min,磷酸盐缓冲液(PBS)冲洗3次,DAB室温显色10 min,PBS冲洗3次,每次5 min,乙醇梯度脱水、透明和封片,于光镜下观察,每只大鼠做切片3张,每张切片随机计数无重叠、具有代表性的5个400倍视野,以平均每100个细胞核中含凋亡细胞的数量作为心肌细胞凋亡指数(apoptosis index,AI)。

1.6 Western blotting检测

将组织从-80 ℃冰箱中取出,在裂解缓冲液中匀浆,4 ℃离心,取上清液,高速离心,沉淀用Triton X-100缓冲液1 mL溶解,二喹啉甲酸试剂盒测定蛋白浓度备用。

每组按20 μg蛋白的溶液体积上样,进行聚丙烯酰胺凝胶电泳,电压设为90 V,根据Maker移动情况适时终止电泳。进行转膜,封闭,洗膜,加入1:5000稀释的Bax以及Bcl-2一抗,4 ℃孵育过夜。TBST洗膜3次,加二抗室温孵育1 h。以β-actin为参照,DAB显色后应用凝胶成像分析仪分析。

1.7 统计学方法

采用SPSS18.0版统计软件进行分析。正态分布的计量资料以均数±标准差( x ¯ ±s)表示,多组间均数比较采用单因素方差分析,组间两两比较采用LSD-t检验。以P<0.05为差异有统计学意义。

2 结果
2.1 芹菜素纳米脂质体的性状表征

扫描电镜观察空白及载药纳米脂质体如图1所示,空白及载药纳米脂质体的形态圆整、分散性好、无粘连。动态光衍射法测定载药及空白脂质体的粒径和Zeta电位分布结果显示:空白及载药纳米脂质体的平均粒径分别(98.3±1.3)和(103.4±1.2) nm,说明载药前后纳米脂质体的粒径无明显变化。同时空白及载药纳米脂质体的多分散系数(polydispersity index,PDI)分别为0.102和0.108,均<0.2,说明载药及空白纳米脂质体稳定性好,粘连少。Zeta电位测定结果显示:空白及载药纳米脂质体的Zeta电位分别为(-23.8±1.6)和(19.8±1.3)mV,绝对值均大于15 mV。包封率是评价纳米载体的重要质量指标,本次研究制备的纳米脂质体的包封率测定结果为(93.42±3.41)%,说明新型纳米脂质体能够较好地实现对于芹菜素的包载。

图1 空白及载药纳米脂质体电镜图
A.空白纳米脂质体;B.芹菜素纳米脂质体

Fig.1 Blank and drug-loaded nanoliposomes observed by scanning electron microscope
A.blank nanoliposomes;B.apigenin-loaded nanoliposomes

2.2 心肌细胞TUNEL凋亡染色结果

各组实验大鼠TUNEL染色结果见图2所示:正常心肌细胞核呈蓝色,凋亡细胞核呈棕褐色,箭头所示为凋亡心肌细胞的细胞核。与正常对照组比较,模型对照组大鼠及空白脂质体干预组12周之后心肌细胞凋亡指数显著升高(P<0.05)。与模型对照组及空白脂质体组比较,经过连续12周芹菜素脂质体治疗的实验大鼠心肌细胞凋亡水平显著下降(P<0.05)。

图2 4组大鼠心肌细胞凋亡情况(n=15)
A.正常对照组;B.模型对照组;C.空白脂质体组;D.芹菜素脂质体组。与正常对照组比较,*1P<0.05;与芹菜素脂质体组比较,*2P<0.05

Fig.2 Myocardial apoptosis in four groups of rats(n=15)
A.normal control group;B.model control group;C.blank nanoliposome group;D.apigenin loaded nanoliposome group.Compared with normal control group, *1P<0.05;compared with apigenin-loaded nanoliposome group, *2P<0.05

2.3 心肌细胞Western blotting检测结果

各实验组大鼠心肌细胞凋亡相关蛋白Bcl-2及Bax的Western blotting结果分析如图3所示。与正常对照组比较,糖尿病大鼠及空白纳米脂质体干预大鼠心肌凋亡相关蛋白Bcl-2显著降低(P<0.01),Bax显著升高(P<0.01)。与模型对照组大鼠及空白纳米脂质体组比较,芹菜素纳米脂质体治疗组的心肌Bcl-2含量显著升高(P<0.01),Bax含量显著降低(P<0.01)。

图3 4组大鼠心肌Bcl-2及Bax的表达情况(n=15)
A.正常对照组;B.模型对照组;C.空白脂质体组;D.芹菜素纳米脂质体组。与正常对照组比较,*1P<0.01;与芹菜素脂质体组比较,*2P<0.01

Fig.3 Expression of Bcl-2 and Bax in myocardium in four groups of rats(n=15)
A.normal control group;B.model control group;C.blank nanoliposome group;D.apigenin-loaded nanoliposome group.Compared with normal control group, *1P<0.01;compared with apigenin-loaded nanoliposome group, *2P<0.01

3 讨论

糖尿病引起的高血糖能够通过氧化应激损伤及Bax途径等诱导患者心肌细胞凋亡,加重DCM的进程[11,12],最终导致患者死亡。芹菜素是一种天然的抗氧化剂[13],能够改善脂肪肝患者的血脂异常以及肥胖型糖尿病动物的的胰岛素抵抗,降低其血糖水平[14]。同时芹菜素还能够显著降低缺血-再灌注损伤大鼠心肌细胞凋亡水平[15]。然而芹菜素存在溶解度低及在体吸收差等生物药动学参数缺陷,限制了其临床应用。

载体质量考察结果显示,空白及载药纳米脂质体的形态圆整,载药前后形态无明显变化。多分散系数PDI均小于0.2,说明本次实验制备的载药纳米脂质体分散性好,粘连少。粒径分析及Zeta电位测定显示,载药及空白纳米脂质体的粒径接近100 nm,Zeta电位绝对值均大于15,说明载药及空白纳米脂质体的稳定性好[16]。另外包封率测定结果显示:芹菜素纳米脂质体的包封率高,说明采用薄膜水合结合超声分散法能够较好地实现对于芹菜素的包载。

12周之后模型对照组大鼠及空白脂质体干预大鼠心肌细胞的凋亡指数显著高于正常对照组(P<0.05),说明各组大鼠DCM动物模型成功,其心肌细胞出现明显凋亡。而经过芹菜素纳米脂质体持续干预12周之后的糖尿病大鼠心肌细胞凋亡指数显著低于模型对照组大鼠(P<0.05),接近正常水平,说明芹菜素脂质体能够有效降低DCM引起心肌凋亡。

另外,本次实验研究还初步考察了芹菜素纳米脂质体对于降低DCM大鼠心肌细胞凋亡水平的作用机制。研究表明,在目前已知的凋亡调节蛋白中,Bcl-2蛋白家族在各类刺激信号引起的凋亡中起到关键作用。Bcl-2和Bax蛋白水平的高低与凋亡调控直接相关,Bax升高,促进细胞凋亡;Bcl-2增高,抑制细胞凋亡[17]。本次研究应用Western blotting技术检测了各组大鼠心肌细胞Bcl-2及Bax的蛋白含量。结果显示,模型对照组大鼠及空白纳米脂质体组大鼠的心肌细胞凋亡相关蛋白Bax含量显著提高(P<0.01),抗凋亡相关蛋白Bcl-2含量显著降低(P<0.01);与糖尿病及空白纳米脂质体组大鼠比较,经过剂量芹菜素纳米脂质体干预后的糖尿病大鼠心肌细胞Bcl-2含显著升高(P<0.01),而Bax含量显著下降(P<0.01),说明芹菜素纳米脂质体可能通过调节Bcl-2/Bax途径降低DCM大鼠的心肌细胞凋亡水平。

本实验证实了芹菜素纳米脂质体能够有效抑制糖尿病引起的大鼠心肌细胞凋亡,为临床DCM患者的防治提供了一种新的思路。

The authors have declared that no competing interests exist.

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Myocardial cell death is a key element in the pathogenesis and progression of various etiological cardiomyopathies such as ischemia-reperfusion, toxic exposure, and various chronic diseases including myocardial infarction, atherosclerosis, and endothelial dysfunction. Myocardial cell death is also observed in the hearts of diabetic patients and antimal models; however, its importance in the development of diabetic cardiomyopathy is not completely understood. The goal of this review is to summarize our current understanding of the characteristics of diabetes-induced myocardial cell death. In the search of themechanisms by which diabetes induces myocardial cell death, multiple cell death pathways have been proposed. Reactive oxygen and nitrogen species accumulation plays a critical role in the cell death by antioxidants or inhibitors for apoptosis-specific signaling pathways results in a significant prevention of diabetic cardiotoxicity, suggesting that cell death in diabetic subjects plays an important role in the development of diabetic cardiomyopathy.
DOI:10.1385/CT:3:3:219      PMID:14555788      URL    
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[5] 刘俊法. 芹菜素对糖尿病大鼠降血糖、调节血脂和抗氧化能力的影响[J].中药药理与临床,2014,5(1):44-47.
目的:研究芹菜素对糖尿病大鼠降血糖、调节血脂和抗氧化能力的影响。方法:将大鼠随机分为正常对照组、糖尿病模型对照组、芹菜素(10、20、40和80 mg/kg)治疗组,连续4周腹腔注射给药治疗,分别测定各组大鼠血糖(GLU)、血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、丙二醛(MDA)含量以及总抗氧化能力(T-AOC);测定肝脏组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)的活性和丙二醛(MDA)的含量;HE染色观察肝脏组织病理学改变。结果:与模型对照组相比,芹菜素(40和80mg/kg)治疗组大鼠血糖水平显著降低;芹菜素(40和80mg/kg)治疗组血清中TC、TG、LDL-C含量水平显著降低、T-AOC显著升高、MDA含量显著降低;芹菜素(80mg/kg)治疗组HDL-C含量显著升高;芹菜素(20、40和80mg/kg)治疗组血清ALT和AST含量显著降低,肝脏组织中SOD、GSH-Px、CAT活性显著升高,MDA含量显著降低;肝组织形态学改变和脂肪病变均明显减轻。结论:芹菜素能够降低实验性糖尿病大鼠血糖水平,增强机体抗氧化能力、降低自由基损伤、改善肝功能、降低血脂,提示芹菜素对实验性糖尿病大鼠具有保护作用,对缓解糖尿病并发症的发生具有重要意义。
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[6] ZHANG J,LIU D,HUANG Y,et al.Biopharmaceutics class-ification and intestinal absorption study of apigenin[J].Int J Pharmac,2012,436(1/2):311-317.
The aim of the study was to characterize the biopharmaceutics classification system (BCS) category of apigenin (AP) using intrinsic dissolution rate (IDR) and rat intestinal permeability, and to investigate the intestinal absorption mechanism of AP in rats. In the present investigation, equilibrium solubility and intrinsic dissolution rate (IDR) of AP were estimated in phosphate buffers. Effective intestinal permeability (Peff) of AP was determined using single-pass intestinal perfusion (SPIP) technique in four segments (duodenum, jejunum, ileum and colon) of rat intestine at three concentrations (10, 50 and 100μg/ml). The aqueous solubility of AP in tested phosphate buffers was very poor with maximum solubility of 2.16μg/ml at pH 7.5. The IDR of AP was very low with a value of 0.006mg/min/cm2. The minimum and maximum Peffs determined by SPIP were 0.198×10614 and 0.713×10614cm/s at jejunum and duodenum site, respectively. In addition, the concentration-dependent permeability behavior was observed in the duodenum and jejunum, which suggested that AP was transported by both passive and active carrier-mediated saturable mechanism in these two intestinal segments. However, the observed concentration-independent permeability behavior in ileum and colon indicated primarily passive transport mechanism of absorption of AP in the last two intestinal segments. AP was classified as class II drug of the BCS due to its low solubility and high intestinal permeability. AP could be well absorbed in the whole intestine with the main absorption site at duodenum. The absorption of AP in four intestinal segments exhibited different transport mechanisms.
DOI:10.1016/j.ijpharm.2012.07.002      PMID:22796171      URL    
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[7] CASTOLDI A,HERR C,NIEDERSTRASSER J,et al.Calcifediol-loaded liposomes for local treatment of pulmonary bacterial infections[J].Eur J Pharmac Biopharmaceutics,2017,118(1):62-67.
DOI:10.1016/j.ejpb.2016.11.026      URL    
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[8] JORAHOLMEN M W,BASNET P,ACHARYA G,et al.PEGylated liposomes for topical vaginal therapy improve delivery of interferon alpha[J].Eur J Pharmac Biopharmaceutics,2017,113(1):132-139.
Recent studies regarding mucosal drug delivery indicate that nanosystems with surface-available polyethylene glycol (PEG) are able to penetrate mucus barrier, assure closer contact with the epithelium, and improve drug delivery to vagina. In the present work, we developed the mucus-penetrating PEGylated liposomes containing interferon alpha-2b (IFN α-2b), destined to provide localized therapy for human papilloma virus (HPV) vaginal infections. The PEGylated liposomes were of a mean size of 18102±02802nm, bearing a negative zeta potential of – 1302mV and an entrapment efficiency of 8102±0210%. In vitro release experiments on model membrane showed a nearly non-existent IFN α-2b release from both the control and liposomally-associated IFN α-2b. However, the ex vivo penetration studies performed on the vaginal tissue obtained from pregnant sheep, showed the clear elevated IFN α-2b penetration from PEGylated liposomes as compared to the control. Furthermore, mucin studies confirmed the absence of interaction between the PEG-modified liposomes and mucin, confirming their ability to penetrate mucus and reach the deeper epithelium. The system holds a promise in improving topical delivery of IFN α-2b through enhanced efficacy of local anti-viral therapy.
DOI:10.1016/j.ejpb.2016.12.029      PMID:28087379      URL    
[本文引用:1]
[9] JIN X,YANG Q,ZHANG Y.Synergistic apoptotic effects of apigenin TPGS liposomes and tyroservatide:implications for effective treatment of lung cancer[J].Int J Nanomedicine,2017,12(5):109-118.
To develop an alternative treatment for lung cancer, a combination of two potent chemotherapeutic agents liposomal apigenin and tyroservatide was developed. The therapeutic potential of this combination was investigated using A549 cells. Apigenin and tocopherol derivative-containing D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) liposomes might improve the delivery of apigenin to tumor cells, both in vitro and in vivo. Importantly, compared to either agent alone, the combination of apigenin TPGS liposomes and tyroservatide exhibited superior cytotoxicity, induced stronger G2 arrest, and suppressed A549 cancer cell invasion at a lower dose. The proapoptotic synergistic effects were also observed in A549 cells using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, flow cytometry, and Western blot analysis. More importantly, in vivo results showed that the combination of apigenin TPGS liposomes and tyroservatide exhibited tumor-growth inhibitory effects in A549 cell-bearing mice. In conclusion, our study showed that this combination therapy could serve as a promising synergistic therapeutic approach to improve outcomes in patients with lung cancer.
DOI:10.2147/IJN.S140096      PMID:5522679      URL    
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[10] ZHAO Y Z,ZHANG M,WONG H L,et al.Prevent diabetic cardiomyopathy in diabetic rats by combined therapy of aFGF-loaded nanoparticles and ultrasound-targeted microbubble destruction technique[J].J Control Rel Soc,2016,223(1):11-21.
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DOI:10.1016/j.jconrel.2015.12.030      PMID:26712588      URL    
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[11] CAI L,LI W,WANG G,et al.Hyperglycemia-induced apoptosis in mouse myocardium:mitochondrial cytochrome C-mediated caspase-3 activation pathway[J].Diabetes,2002,51(6):1938-1948.
Abstract Diabetic cardiomyopathy is related directly to hyperglycemia. Cell death such as apoptosis plays a critical role in cardiac pathogenesis. Whether hyperglycemia induces myocardial apoptosis, leading to diabetic cardiomyopathy, remains unclear. We tested the hypothesis that apoptotic cell death occurs in the diabetic myocardium through mitochondrial cytochrome c-mediated caspase-3 activation pathway. Diabetic mice produced by streptozotocin and H9c2 cardiac myoblast cells exposed to high levels of glucose were used. In the hearts of diabetic mice, apoptotic cell death occurred as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Correspondingly, caspase-3 activation as determined by enzymatic assay and mitochondrial cytochrome c release detected by Western blotting analysis were observed. Supplementation of insulin inhibited diabetes-induced myocardial apoptosis as well as suppressed hyperglycemia. To explore whether apoptosis in diabetic hearts is related directly to hyperglycemia, we exposed cardiac myoblast H9c2 cells to high levels of glucose (22 and 33 mmol/l) in cultures. Apoptotic cell death was detected by TUNEL assay and DAPI nuclear staining. Caspase-3 activation with a concomitant mitochondrial cytochrome c release was also observed. Apoptosis or activation of caspase-3 was not observed in the cultures exposed to the same concentrations of mannitol. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-cmk, suppressed apoptosis induced by high levels of glucose. In addition, reactive oxygen species (ROS) generation was detected in the cells exposed to high levels of glucose. These results suggest that hyperglycemia directly induces apoptotic cell death in the myocardium in vivo. Hyperglycemia-induced myocardial apoptosis is mediated, at least in part, by activation of the cytochrome c-activated caspase-3 pathway, which may be triggered by ROS derived from high levels of glucose.
DOI:10.2337/diabetes.51.6.1938      PMID:12031984      URL    
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[12] ZOU M H,XIE Z.Regulation of interplay between auto-phagy and apoptosis in the diabetic heart:new role of AMPK[J].Autophagy,2013,9(4):624-625.
Diabetes induces cardiomyocyte apoptosis and suppresses cardiac autophagy, indicating that the interplay between autophagy and apoptotic cell death pathways is important in the pathogenesis of diabetic cardiomyopathy. The potential mechanism, however, remains unknown. We recently reported that diabetes depresses AMP-activated protein kinase (AMPK) activity, inhibits MAPK8/JNK1-BCL2 signaling, and promotes the interaction between BECN1 and BCL2. Concomitantly, diabetes induces cardiomyocyte apoptosis and suppresses cardiac autophagy. Activation of AMPK directly phosphorylates MAPK8, which mediates BCL2 phosphorylation and subsequent BECN1-BCL2 dissociation, leading to restoration of cardiac autophagy, protection against cardiac apoptosis, and ultimately improvement in cardiac structure and function. We conclude that dissociation of BCL2 from BECN1 through activation of MAPK8-BCL2 signaling may be an important mechanism by which AMPK activation restores autophagy, protects against cardiac apoptosis, and prevents diabetic cardiomyopathy.
DOI:10.4161/auto.23577      PMID:23380689      URL    
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[13] LEFORT E C,BLAY J.Apigenin and its impact on gastro-intestinal cancers[J].Molecular Nutrit Food Res,2013,57(1):126-144.
Apigenin (4 ,5,7-trihydroxyflavone, 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a flavonoid found in many fruits, vegetables, and herbs, the most abundant sources being the leafy herb parsley and dried flowers of chamomile. Present in dietary sources as a glycoside, it is cleaved in the gastrointestinal lumen to be absorbed and distributed as apigenin itself. For this reason, the epithelium of the gastrointestinal tract is exposed to higher concentrations of apigenin than tissues at other locations. This would also be true for epithelial cancers of the gastrointestinal tract. We consider the evidence for actions of apigenin that might hinder the ability of gastrointestinal cancers to progress and spread. Apigenin has been shown to inhibit cell growth, sensitize cancer cells to elimination by apoptosis, and hinder the development of blood vessels to serve the growing tumor. It also has actions that alter the relationship of the cancer cells with their microenvironment. Apigenin is able to reduce cancer cell glucose uptake, inhibit remodeling of the extracellular matrix, inhibit cell adhesion molecules that participate in cancer progression, and oppose chemokine signaling pathways that direct the course of metastasis into other locations. As such, apigenin may provide some additional benefit beyond existing drugs in slowing the emergence of metastatic disease.
DOI:10.1002/mnfr.201200424      PMID:23197449      URL    
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[14] JUNG U J,CHO Y Y,CHOI M S.Apigenin ameliorates dyslipidemia,hepatic steatosis and insulin resistance by modulating metabolic and transcriptional profiles in the liver of high-fat diet-induced obese mice[J].Nutrients,2016,8(5):E305.
Severalin vitroandin vivostudies have reported the anti-inflammatory, anti-diabetic and anti-obesity effects of the flavonoid apigenin. However, the long-term supplementary effects of low-dose apigenin on obesity are unclear. Therefore, we investigated the protective effects of apigenin against obesity and related metabolic disturbances by exploring the metabolic and transcriptional responses in high-fat diet (HFD)-induced obese mice. C57BL/6J mice were fed an HFD or apigenin (0.005%,w/w)-supplemented HFD for 16 weeks. In HFD-fed mice, apigenin lowered plasma levels of free fatty acid, total cholesterol, apolipoprotein B and hepatic dysfunction markers and ameliorated hepatic steatosis and hepatomegaly, without altering food intake and adiposity. These effects were partly attributed to upregulated expression of genes regulating fatty acid oxidation, tricarboxylic acid cycle, oxidative phosphorylation, electron transport chain and cholesterol homeostasis, downregulated expression of lipolytic and lipogenic genes and decreased activities of enzymes responsible for triglyceride and cholesterol ester synthesis in the liver. Moreover, apigenin lowered plasma levels of pro-inflammatory mediators and fasting blood glucose. The anti-hyperglycemic effect of apigenin appeared to be related to decreased insulin resistance, hyperinsulinemia and hepatic gluconeogenic enzymes activities. Thus, apigenin can ameliorate HFD-induced comorbidities via metabolic and transcriptional modulations in the liver.
DOI:10.3390/nu8050305      PMID:4882717      URL    
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[15] HU J,LI Z,XU L T,et al.Protective effect of apigenin on ischemia/reperfusion injury of the isolated rat heart[J].Cardiovas Toxicol,2015,15(3):241-249.
Abstract Apigenin (Api), a mainly bioactive component of Apium graveolens L. var. dulce DC. (a traditional Chinese medicinal herb), possesses a wide range of biological activities, including antioxidant effects. It also has been shown to associate with lower prevalence of cardiovascular diseases, but its mechanisms of action remain unclear. The aim of the present study is to investigate the role of Api in isolated rat heart model of ischemia/reperfusion (I/R). Langendorff-perfused isolated rat hearts were used in our study. Api was added to the perfusate before ischemia and during reperfusion in the isolated pulsed rat heart exposed to 30-min ischemia followed by 50-min reperfusion. The treatment with Api conferred a cardioprotective effect, and the treated hearts demonstrated an improved ischemic cardiac functional recovery, a decreased myocardial infarct size, a reduced activities of creatine kinase isoenzyme and lactate dehydrogenase in the coronary flow, a reduced number of apoptotic cardiomyocytes, a reduced activity of caspase-3, up-regulation of the anti-apoptotic protein Bcl-2 and down-regulation of the pro-apoptotic protein Bax. In addition, Api inhibited the phosphorylation of p38 MAPKS during I/R. In conclusion, these observations provide preliminary evidence that Api can protect cardiomyocytes from I-/R-induced injury, at least partially, through the inhibition of p38 MAPKS signaling pathway.
DOI:10.1007/s12012-014-9290-y      PMID:25377428      URL    
[本文引用:1]
[16] ZHAO Y Z,ZHANG M,TIAN X Q,et al.Using basic fibroblast growth factor nanoliposome combined with ultrasound-introduced technology to early intervene the diabetic cardiomyopathy[J].Int J Nanomedic,2016,11(4):675-686.
Basic fibroblast growth factor (bFGF)-loaded liposome (bFGF-lip) combined with ultrasound-targeted microbubble destruction (UTMD) technique was investigated to prevent diabetic cardiomyopathy (DCM). Cardiac function and myocardial ultrastructure were assessed. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, immunohistochemistry staining, and Western blot assay were used to investigate the signal pathway underlying the expression of bFGF in DCM treatment. From Mason staining and TUNEL staining, bFGF-lip + UTMD group showed significant differences from the diabetes group and other groups treated with bFGF or bFGF-lip. The diabetes group showed similar results (myocardial capillary density, collagen volume fraction, and cardiac myocyte apoptosis index) to other bFGF treatment groups. Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis, and metabolic disturbances could be suppressed by the different extents of twice-weekly bFGF treatments for 12 consecutive weeks (free bFGF or bFGF-lip +/- UTMD), with the strongest improvements observed in the bFGF-lip + UTMD group. The group combining bFGF-lip with UTMD demonstrated the highest level of bFGF expression among all the groups. The bFGF activated the PI3K/AKT signal pathway, causing the reduction of myocardial cell apoptosis and increase of microvascular density. This strategy using bFGF-lip and UTMD is a potential strategy in early intervention of DCM in diabetes.
DOI:10.2147/IJN.S99376      PMID:26937188      URL    
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[17] XIE Z,KOYAMA T,SUZUKI J,et al.Coronary reperfusion following ischemia:different expression of bcl-2 and bax proteins,and cardiomyocyte apoptosis[J].Japan Heart J,2001,42(6):759-770.
The aim of this work was to examine factors that could be involved in the occurrence of apoptosis in rat hearts subjected to coronary occlusion followed by reperfusion. To this end, we studied the expression of the pro- and anti-apoptotic factors, bax and bcl-2, respectively, in reperfused ischemic hearts and in hearts injected with bFGF or saline. In anesthetized rats the left coronary artery was occluded for 45 min, the anesthesia withdrawn and the occlusion removed to allow reperfusion; in sham-operated rats the occlusion was omitted. After 4 hours the rats were decapitated and the heart excised. Sections from the left ventricle were stained with anti-bcl-2-antibody and anti-bax-antibody using the TUNEL method which detects apoptosis. Fragmentation of DNA isolated from reperfused ventricles was examined by agarose electrophoresis. In reperfused hearts no bcl-2 staining was observed in the discrete area in which many cardiomyocyte nuclei were stained by the TUNEL method; outside this area staining for bcl-2 was more marked than in sham-operated rats. Sections from reperfused hearts were stained for bax protein over a wide area including the apoptotic region; sham-operated hearts showed little reaction. Staining for bcl-2 was demonstrable in some nuclei in hearts from saline-injected rats; the numbers were unaffected by i. v. bFGF. Ischemia/reperfusion increases the overall expression of both bcl-2 and bax proteins, but bcl-2 is lost from the reperfused area as indicated by TUNEL staining. Accordingly, the ratio of bcl-2 to bax was reduced in the reperfused area, indicating a pro-apoptotic trend. The marked increase in bcl-2 outside the reperfused area could be a mechanism with which to salvage surviving cardiomyocytes.
DOI:10.1536/jhj.42.759      PMID:11933925      URL    
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关键词(key words)
芹菜素
纳米脂质体
细胞凋亡
心肌病
糖尿病

Apigenin
Nanoliposomes
Cells apoptosis
Cardiomyopathy
diabetic

作者
章雅丹

ZHANG Yadan