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Volume 39 Issue 3
May  2021
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JIN Xiaoling, CHEN Lan, ZHANG Feng, HUANG Doudou, LIAO Lina, CHEN Wansheng. Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(3): 240-244. doi: 10.12206/j.issn.1006-0111.202006080
Citation: JIN Xiaoling, CHEN Lan, ZHANG Feng, HUANG Doudou, LIAO Lina, CHEN Wansheng. Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(3): 240-244. doi: 10.12206/j.issn.1006-0111.202006080

Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation

doi: 10.12206/j.issn.1006-0111.202006080
  • Received Date: 2020-06-21
  • Rev Recd Date: 2021-01-27
  • Available Online: 2021-05-25
  • Publish Date: 2021-05-25
  •   Objective  To study the protective effect of Shengxian decoction and the single herb decoction against myocardial injury induced by hypoxia/reoxygenation.  Methods  The H9c2 cells were cultured to establish hypoxia/reoxygenation model. Rats were divided into 8 groups: normal control group, hypoxia/reoxygenation group (model group) and treated groups (Shengxian decoction and the single herb decoction). The apoptotic rate of cardiomyocytes, the activity of reactive oxygen species (ROS) and intracellular calcium concentration (Ca2+) were measured.  Results  Compared with hypoxia/reoxygenation group, the apoptosis rate, ROS activity and intracellular Ca2+ concentration were significantly lower in all treated groups (P<0.05). The ROS activity and intracellular Ca2+ concentration was decreased by 41.37% and 15.20% in Shengxian decoction group compared to the model group.  Conclusion  Shengxian decoction and the single herb decoction had protective effect on myocardial injury induced by hypoxia/reoxygenation.
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Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation

doi: 10.12206/j.issn.1006-0111.202006080

Abstract:   Objective  To study the protective effect of Shengxian decoction and the single herb decoction against myocardial injury induced by hypoxia/reoxygenation.  Methods  The H9c2 cells were cultured to establish hypoxia/reoxygenation model. Rats were divided into 8 groups: normal control group, hypoxia/reoxygenation group (model group) and treated groups (Shengxian decoction and the single herb decoction). The apoptotic rate of cardiomyocytes, the activity of reactive oxygen species (ROS) and intracellular calcium concentration (Ca2+) were measured.  Results  Compared with hypoxia/reoxygenation group, the apoptosis rate, ROS activity and intracellular Ca2+ concentration were significantly lower in all treated groups (P<0.05). The ROS activity and intracellular Ca2+ concentration was decreased by 41.37% and 15.20% in Shengxian decoction group compared to the model group.  Conclusion  Shengxian decoction and the single herb decoction had protective effect on myocardial injury induced by hypoxia/reoxygenation.

JIN Xiaoling, CHEN Lan, ZHANG Feng, HUANG Doudou, LIAO Lina, CHEN Wansheng. Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(3): 240-244. doi: 10.12206/j.issn.1006-0111.202006080
Citation: JIN Xiaoling, CHEN Lan, ZHANG Feng, HUANG Doudou, LIAO Lina, CHEN Wansheng. Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(3): 240-244. doi: 10.12206/j.issn.1006-0111.202006080
  • 1960年,Jenning首次提出了心肌再灌注损伤的概念[1]。心肌缺血/再灌注损伤的主要原因包括氧自由基增多、细胞内钙超载及微血管损伤等[2-3],缺血组织细胞恢复灌注后发生的再灌注损伤(MIRI)在恢复血流的过程中常会引起心肌细胞的氧化应激损伤[4-5],往往造成患者预后不佳 [6]

    升陷汤出自张锡纯《医学衷中参西录》,该方由黄芪、柴胡、升麻、桔梗、知母组成,主治大气下陷之证,临床广泛用来防治心肌缺血性疾病[7]、治疗老年慢性充血性心力衰竭[8]、治疗不稳定型心绞痛[9]、治疗青少年病毒性心肌炎[10-11]。课题组前期考察了升陷汤及各单味药对阿霉素致心肌细胞的保护作用[12],尚未见升陷汤对心肌细胞缺氧/复氧损伤模型保护作用的报道。本实验基于经典的心肌细胞缺氧/复氧损伤模型[13],从细胞凋亡的角度初步探讨升陷汤及单味药对心肌缺氧/复氧的作用机制。

  • 纯净水(美国Millipore公司)、Fura-3/AM(碧云天生物技术有限公司)、生理盐水(海军军医大学附属长征医院);0.25%胰蛋白酶溶液(Gibco公司);DMEM低糖培养基、培养液(美国Hyclone公司);MTT工作液(美国sigma公司);二甲基亚砜(德国GmbH公司);PBS缓冲液(南京滴纯生物科技有限公司);胎牛血清(美国Gibco公司);DCFH-DA(碧云天生物有限公司);Annexin V-FITC/PI 凋亡检测试剂盒(美国BD公司);16%甲醛(无甲醇,赛默飞世尔科技有限公司);其他试剂为分析纯。

    倒置荧光显微镜(重庆奥特光学仪器);流式细胞仪(美国BD公司);全自动酶标仪(美国Multiskan MK3公司);CO2细胞培养箱(德国Heraeus公司);低温高速离心机(德国Heraeus公司);台式高速离心机(德国Eppendorf公司);荧光显微镜(日本OLYMPUS公司);二氧化碳培养箱(美国Forma公司);79-1型磁力搅拌器(江苏周庄科研仪器厂);YJ-II型超声波细胞粉碎机(上海新芝生物技术研究所);海尔低温冰箱。

  • 升陷汤中五味药材饮片由长征医院药材科提供。将5 L水浸泡升陷汤全方提取物或各单味药材24 h后,再煎煮,重复3次,将浓缩水煎煮液合并至650 ml,干燥得提取物浸膏。其中,升陷汤组方饮片(简称药物SXT)包括黄芪600 g,知母300 g,柴胡150 g,桔梗150 g和升麻100 g。SXT、黄芪、知母、柴胡、桔梗和升麻水提液蒸干后的浸膏重量分别为431.2、187.5、89.5、45.2、43.5、28.1 g。将所有提取物分别研细,于−20°C冰箱中保存备用。

  • H9C2细胞培养在含10% FBS的高糖DMEM培养液、37 ℃、5% CO2、95% O2饱和湿度的恒温细胞培养箱,待细胞汇合度达到80%~90%进行传代培养。传代时,弃去培养液,用PBS缓冲液洗2~3次后用含0.25%胰蛋白酶消化液消化细胞,待细胞变圆时立刻停止消化,以1000 r/min离心5 min。弃上清液,用含10% FBS 的DMEM培养基重悬后,并按1∶3分瓶,隔天更换培养液。取对数生长期细胞进行试验。

  • 将心肌细胞随机分为以下8组:对照组:心肌细胞正常培养基培养;模型组:心肌细胞用低糖无血清培养基预孵育1 h后,在含95% N2和5%CO2的培养箱中缺氧6 h,再在5% CO2和95%空气饱和的培养箱复氧1 h造成心肌缺血损伤模型组;药物干预组:心肌细胞用低糖无血清培养基稀释的药物预孵育1 h后,经缺氧6 h-复氧1 h处理后即为药物干预组。其中,干预药物包括SXT、黄芪、知母、柴胡、桔梗和升麻水提液(共6组)。

  • 培养完成后,将对照组和模型组组以及药物干预组中取对数期生长的细胞接种于96孔板,调整细胞的浓度使每孔有5×105个细胞,然后向每孔中加入20 μl MTT(5 mg/ml)工作液,孵育一段时间,后向每孔中加入150 μl DMSO,室温条件先低速震荡让二者充分融合,然后参照MTT比色法试剂盒说明,在490 nm测定各孔下的吸光值,分别记录结果。模型组、各药物干预组与对照组的比值为细胞活力相对值。

  • 培养完成后,分别收集所有组中悬浮细胞,在各组细胞中加入150 μl DCFH-DA溶液,在37 ℃细胞培养箱内避光孵育30 min。每隔5 min震荡1次,使探针和细胞充分接触。用无血清细胞培养液将细胞洗几遍,最后用PBS重悬。采用多功能酶标仪检测细胞内ROS活性(激发波长488 nm,检测波长525 nm)。

  • 培养结束后,用0.25%胰酶消化离心,分别将所有组中细胞用缓冲液PBS冲洗,再与10 μmol/L钙离子荧光探针Fura-3/AM(10)共同孵育60 min,然后用缓冲液冲洗2次,最后用PBS重悬。将制成的标本置于倒置荧光显微镜下,用340 nm紫外光激发获得荧光图像,经过计算机图像处理后根据标准曲线算出细胞内钙浓度。

  • 培养完成的细胞用不含 EDTA 的胰酶消化,离心收集悬浮细胞,2 000 r/min离心5 min,弃培养基;用预冷的PBS洗涤细胞;加入100 μl结合缓冲液悬浮细胞,浓度约为 1 × 106/ml;然后在细胞悬浮液中加入5 μl Annexin V-FITC,轻轻混匀后于 4 ℃ 避光条件下孵育 15 min;再加入 5 μl PI 后轻轻混匀于 4 ℃ 避光条件下孵育5 min;添加PBS至500 μl并轻轻震摇均匀,于60 min内用流式细胞仪检测细胞凋亡率。

  • 使用GraphPad Prism v5.0统计软件进行统计学分析,实验数据以($\bar x \pm s$)表示,组内两两比较采用t-test,当P < 0.05时判定差异具有统计学意义。

  • 本实验根据临床给药剂量及预实验结果,在药物试验浓度分别为2.5、5.0、10.0、20.0、40.0、80.0、160.0和320.0 μg/ml的药物浸膏时,研究全方及各单味药对心肌细胞缺氧/复氧后细胞活力的影响。MTT结果显示:升陷汤、黄芪、知母、桔梗、升麻在20 μg/ml时,细胞活力最佳,因此确定上述提取物浓度组均为20 μg/ml;由于20 μg/ml柴胡提取物对心肌细胞有一定损伤,经试验最终确定药物浓度为0.3 μg/ml时对缺氧/复氧后细胞活力无明显影响(表1)。

    组别终浓度(μg/ml)MTT增殖率(%)
    全方2018.745
    黄芪2014.300
    知母2011.199
    桔梗20 9.244
    升麻2010.977
    柴胡0.326.512
  • 实验结果表明:心肌细胞在缺氧/复氧条件下,细胞内活性氧含量明显增加,升高至对照组的2.49倍(P<0.01,图1),表明缺氧/复氧引起细胞内自由基损伤;给药后,除柴胡外,升陷汤全方及各单味药均能明显降低心肌缺氧/复氧致心肌损伤模型的细胞内ROS的荧光强度,升陷汤全方、黄芪、知母、桔梗和升麻处理组分别为对照组的1.46、1.40、1.79、1.52和1.83倍(P<0.01,图1)。其中,升陷汤与黄芪作用最强,两者无明显统计学差异。

  • 实验结果表明:心肌细胞在常氧下(对照组),Ca2+荧光强度较低,缺氧/复氧后,Ca2+荧光强度增加(P<0.01,图2),表明心肌细胞低氧/复氧时存在Ca2+超载。升陷汤全方和黄芪、知母药物干预后,各组细胞内荧光强度与模型组相比,分别降低了15.20%、23.98%和15.79%(P<0.05,图2),表明其对低氧/复氧时心肌细胞内Ca2+ 超载有抑制作用。桔梗、升麻药物处理后,上述值虽然有降低,但差异不明显。

  • 采用Annexin V-FITC/Pl双染流式细胞术检测药物对缺氧/复氧损伤后心肌细胞凋亡情况。从检测结果发现,心肌细胞在常氧下(对照组),细胞凋亡较低;缺氧/复氧后,细胞凋亡增加;给药组中升陷汤全方、黄芪、桔梗可以降低细胞的凋亡率(P<0.05,图3)。

  • 心肌细胞缺氧/复氧损伤是模拟MIRI的病理生理过程的经典模型,心肌细胞短时间内缺血再灌注造成的组织细胞功能代谢发生障碍及结构功能破坏加重,甚至发生不可逆性损伤的现象[14]。Ca2+超载一直被认为是心肌缺血再灌注的主要机制。缺血缺氧时,心肌细胞内Ca2+超载,线粒体膜的通透性转换孔开放;再灌注恢复使得心肌细胞重新摄取O2,产生大量的线粒体内活性氧,进一步增加线粒体胞质内的Ca2+浓度,线粒体的氧化磷酸化进而受阻促使心肌细胞死亡[15-16]。另外,线粒体内活性氧触发氧化应激反应并加重心肌细胞的凋亡和坏死[17-19]。由此可见,细胞内ROS活性和Ca2+浓度是MIRI重要的检测指标[20-21]

    本课题组前期采用大鼠冠状动脉结扎急性心肌缺血致慢性心力衰竭模型,心肌组织病理学切片证实了升陷汤全方对心肌细胞损伤和炎症发展起到有效的控制作用,通过药理指标测定结合代谢组学研究证实了升陷汤对心衰具有显著的治疗作用。升陷汤通过改善心肌细胞损伤,减少炎症反应,增强左室射血功能,调节机体磷脂代谢和脂肪酸生物合成来发挥其保护心肌作用,从而治疗慢性心力衰竭[22-23]。本课题组通过体外细胞实验从缺氧/复氧致心肌细胞损伤角度,基于心肌细胞活力角度考察并证实了升陷汤的保护作用,可通过抑制细胞凋亡、降低细胞内ROS以及Ca2+的浓度实现。同时,发现各单味药也具有一定的保护作用,但弱于全方的保护功效,进一步证实了全方治疗“大气下陷”的合理性。升陷汤全方是以黄芪补气升陷为主药,知母凉润制主药之温燥,柴胡、升麻助黄芪升陷之力,桔梗载药力上达胸中,共奏升补大气之效[24]。药材柴胡提取液在本实验中尚未发现具有保护缺氧/复氧损伤心肌细胞的作用,在一定程度上证实了其他药味配伍的合理有效性。

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