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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (10): 1127-1134.doi: 10.3736/jcim20121009

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Effects of the combination of active component extracts from Astragalus membranaceus and Panax notoginseng on apoptosis, reactive oxygen species and mitochondrial membrane potential of PC12 cells with oxidative injury

Xiao-ping Huang1, Xiao-dan Liu2, Chang-qing Deng2()   

  1. 1. College of Medicine, Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
    2. College of Integrated Traditional Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
  • Received:2012-05-22 Accepted:2012-06-12 Online:2012-10-20 Published:2018-10-15
  • Contact: Chang-qing Deng

Objective: To explore the effects and mechanisms of combining astragaloside Ⅳ (the effective component of Astragalus membranaceus) with notoginsenoside R1, ginsenoside Rb1 and ginsenoside Rg1 (the effective components of Panax notoginseng) against oxidative injury in PC12 cells induced by cobalt chloride (CoCl2). .

Methods: CoCl2 was used to stimulate PC12 cells to induce injury after transdifferentiation with nerve growth factor. Then the PC12 cells were divided into 10 groups and cultured with corresponding drugs. After culture, apoptotic cells were tested by using Hocchst 33258 fluorescent staining, the level of mitochondrial membrane potential (MMP) was analyzed by rhodamine 123 fluorescent staining and the content of reactive oxygen species (ROS) in PC12 cell was measured by dichlorofluorescin diacetate fluorescent staining.

Results: CoCl2 induced apoptosis along with the obvious decrease of MMP as well as overproduction of ROS in PC12 cells. Astragaloside Ⅳ, ginsenosides Rg1, ginsenosides Rb1 and notoginsenoside R1 had inhibition effects in different degree on PC12 cell apoptosis induced by CoCl2, reduced the overproduction of ROS and the decrease of MMP. The effects of the combination were better than those of active component alone.

Conclusion: Active components extracted from Astragalus and Panax notoginseng can inhibit PC12 cell apoptosis induced by oxidative injury, furthermore, the effects were enhanced by combination of these components, which may be associated with jointly antagonizing the generation of ROS and raising MMP.

Key words: plant extracts, compatibility (TCD), apoptosis, reactive oxygen species, mitochondrial membrane potential, PC12 cells

Figure 1

Chemical structures of astragaloside Ⅳ, notoginsenoside R1, ginsenoside Rg1 and ginsenoside Rb1"

Figure 2

Detection of apoptosis in PC12 cells by Hocchst 33258 fluorescent staining (Light microscopy, ×400) A: Blank group; B: Ethanol-PBS group; C: CoCl2 group; D: CoCl2-ethanol-PBS group; E: Astragaloside Ⅳ group; F: Ginsenosides Rg1 group; G: Ginsenosides Rb1 group; H: Notoginsenoside R1 group; I: Combination group; J: Edaravone group. The arrows point to the apoptosis of PC12 cells."

Table 1

Apoptosis rate, ROS content and MMP level of PC12 cells ($\overline{x}$±s)"

Group n Apoptosis rate (%) ROS content (MFI) MMP level (MFI)
Blank 3 1.57±0.26 26.75±0.61 81.43±4.16
Ethanol-PBS 3 30.03±10.33** 39.44±1.54* 79.47±2.89
CoCl2 3 54.17±1.60** 55.54±2.08** 44.56±5.56**
CoCl2-ethanol-PBS 3 89.83±6.26△△▲▲ 73.37±9.06△△▲▲ 30.70±1.92△△▲
Astragaloside Ⅳ 3 37.94±4.37□□■■ 45.10±0.76□□■■ 31.68±0.62■■
Ginsenosides Rg1 3 11.47±1.13▲▲■ 38.29±3.04▲▲■ 45.24±1.53■■
Ginsenosides Rb1 3 16.66±3.13▲▲■■ 35.35±0.95▲▲ 49.57±2.74■■
Notoginsenoside R1 3 13.00±1.52▲▲■■ 32.17±2.91▲▲ 48.47±4.31■■
Combination 3 5.53±0.92□□ 34.05±1.35□□ 56.65±3.36□□
Edaravone 3 16.30±0.65▲▲■■ 28.90±0.47▲▲■■ 47.44±2.13■■

Figure 3

Detection of reactive oxygen species content in PC12 cells by fluorescent staining (Light microscopy, ×400) A: Blank group; B: Ethanol-PBS group; C: CoCl2 group; D: CoCl2-ethanol-PBS group; E: Astragaloside Ⅳ group; F: Ginsenosides Rg1 group; G: Ginsenosides Rb1 group; H: Notoginsenoside R1 group; I: Combination group; J: Edaravone group."

Figure 4

Detection of mitochondrial membrane potential level of PC12 cells by fluorescent staining (Light microscopy, ×400) A: Blank group; B: Ethanol-PBS group; C: CoCl2 group; D: CoCl2-ethanol-PBS group; E: Astragaloside Ⅳ group; F: Ginsenosides Rg1 group; G: Ginsenosides Rb1 group; H: Notoginsenoside R1 group; I: Combination group; J: Edaravone group."

[1] Huang XP, Tan H, Chen BY , Deng CQ . Influence of astragalosides and Panax notoginseng saponins compatibility on MMP-9 and TIMP-1 after cerebral ischemia-reperfusion in mice. Zhongguo Zhong Yao Za Zhi. 2010; 35(16):2187-2191.
doi: 10.4268/cjcmm20101634
黄小平, 谭华, 陈北阳, 邓常清 . 黄芪总苷和三七总皂苷配伍对脑缺血再灌注后MMP-9和TIMP-1表达的影响[J]. 中国中药杂志, 2010,35(16):2187-2191
doi: 10.4268/cjcmm20101634
[2] Tan H, Huang XP , Deng CQ. Effects of astragalosides and Panax notoginseng saponins combination on oxidative stress of cerebral ischemic reperfusion injury in mice. J Chin Integr Med. 2010; 8(5): 448-452. Chinese with abstract in English.
谭华, 黄小平, 邓常清 . 黄芪总苷和三七总皂苷配伍对小鼠缺血再灌注脑组织氧化应激的影响[J]. 中西医结合学报, 2010,8(5):448-452
[3] Liu XD, Deng CQ . Compatibility study of main effective components in PNS and AST on oxidative damage of PC12[J]. Hunan Zhong Yi Yao Da Xue Xue Bao, 2010,32(1):8-12
doi: 10.3969/j.issn.1674-070X.2012.
刘晓丹, 邓常清 . 黄芪甲苷和三七总皂苷中主要有效成分抗PC12细胞氧化损伤的配伍研究[J]. 湖南中医药大学学报, 2010,32(1):8-12
doi: 10.3969/j.issn.1674-070X.2012.
[4] Jung JY, Mo HC, Yang KH, Jeong YJ, Yoo HG, Choi NK, Oh WM, Oh HK, Kim SH, Lee JH, Kim HJ, Kim WJ . Inhibition by eqigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells[J]. Life Sci, 2007,80(15):1355-1363
doi: 10.1016/j.lfs.2006.11.033 pmid: 17240404
[5] Kim KS, Rajagopal V, Gonsalves C, Johnson C, Kalra VK . A novel role of hypoxia-inducible factor in cobalt chloride- and hypoxia-mediated expression of IL-8 chemokine in human endothelial cells[J]. J Immunol, 2006,177(10):7211-7224
doi: 10.4049/jimmunol.177.10.7211
[6] Schmalz G, Schweikl H, Hiller KA . Release of prostaglandin E2, IL-6 and IL-8 from human oral epithelial culture models after exposure to compounds of dental materials[J]. Eur J Oral Sci, 2000,108(5):442-448
doi: 10.1034/j.1600-0722.2000.108005442.x
[7] Liao XX, Yang CT, Yang ZL, Li JP, Wang LC, Huang X, Guo RX, Chen PX, Feng JQ . Hydrogen sulfide protects H9C2 cells against chemical hypoxia-induced injury and the underlying mechanisms[J]. Zhongguo Yao Li Xue Tong Bao, 2009,25(8):1012-1017
廖新学, 杨春涛, 杨战利, 李建平, 王礼春, 黄雪, 郭瑞鲜, 陈培熹, 冯鉴强 . 硫化氢对抗化学性缺氧引起的心肌细胞损伤及其机制[J]. 中国药理学通报, 2009,25(8):1012-1017
[8] 孟金兰, 兰爱平, 杨春涛, 杨战利, 王立伟, 陈丽新, 朱琳燕, 陈培熹, 冯鉴强 . 热休克蛋白90在硫化氢保护PC12细胞对抗化学性缺氧损伤中的作用[J]. 中国药理学通报, 2010,26(1):103-107
Meng JL, Lan AP, Yang CT, Yang ZL, Wang LW, Chen LX, Zhu LY, Chen PX, Feng JQ . Role of heat shock protein 90 in protective effect of hydrogen sulfide against PC12 cells injuries induced by chemical hypoxia[J]. Zhongguo Yao Li Xue Tong Bao, 2010,26(1):103-107
[9] Correa F, Soto V, Zazueta C . Mitochondrial permeability transition relevance for apoptotic triggering in the post-ischemic heart[J]. Int J Biochem Cell Biol, 2007,39(4):787-798
doi: 10.1016/j.biocel.2007.01.013
[10] Fujimura M, Morita-Fujimura Y, Narasimhan P, Copin JC, Kawase M, Chan PH . Copper-zinc superoxide dismutase prevents the early decrease of apurinic/apyrimidinic endonuclease and subsequent DNA fragmentation after transient focal cerebral ischemia in mice[J]. Stroke, 1999,30(11):2408-2415
doi: 10.1161/01.STR.30.11.2408
[11] Chan PH . Reactive oxygen radicals in signaling and damage in the ischemic brain[J]. J Cereb Blood Flow Metab, 2001,21(1):2-14
doi: 10.1097/00004647-200101000-00002 pmid: 11149664
[12] Liu Z, Li WH, Zhang J, Jing P . The study on cytotoxicity of rotenone to human SH-SY5Y cells in vitro[J]. Huan Jing Yu Zhi Ye Yi Xue, 2003,20(2):73-74, 77
[13] Haeberlein SL . Mitochondrial function in apoptotic neuronal cell death[J]. Neurochem Res, 2004,29(3):521-530
刘祯, 李卫红, 张杰, 景鹏 . 鱼藤酮对神经瘤细胞线粒体膜电位的影响[J]. 环境与职业医学, 2003,20(2):73-74, 77
[14] Yoshida H, Yanai H, Namiki Y, Fukatsu-Sasaki K, Furutani N, Tada N . Neuroprotective effects of edaravone: a novel free radical scavenger in cerebrovascular injury[J]. CNS Drug Rev, 2006,12(1):9-20
doi: 10.1111/cns.2006.12.issue-1
[15] Rajesh KG, Sasaguri S, Suzuki R, Maeda H . Antioxidant MCI-186 inhibits mitochondrial permeability transition pore and upregulates Bcl-2 expression[J]. Am J Physiol Heart Circ Physiol, 2003,285(5):H2171-H2178
doi: 10.1152/ajpheart.00143.2003
[16] Luo Y, Qin Z, Hong Z, Zhang X, Ding D, Fu JH, Zhang WD, Chen J . Astragaloside Ⅳ protects against ischemic brain injury in a murine model of transient focal ischemia[J]. Neurosci Lett, 2004,363(3):218-223
doi: 10.1016/j.neulet.2004.03.036
[17] Qu YZ, Li M, Zhao YL, Zhao ZW, Wei XY, Liu JP, Gao L, Gao GD . Astragaloside Ⅳ attenuates cerebral ischemia-reperfusion-induced increase in permeability of the blood-brain barrier in rats[J]. Eur J Pharmacol, 2009,606(1-3):137-141
doi: 10.1016/j.ejphar.2009.01.022
[18] Liu Q, Kou JP, Yu BY . Ginsenoside Rg1 protects against hydrogen peroxide-induced cell death in PC12 cells via inhibiting NF-κB activation[J]. Neurochem Int, 2011,58(1):119-125
doi: 10.1016/j.neuint.2010.11.004
[19] Zhang YF, Fan XJ, Li X, Peng LL, Wang GH, Ke KF, Jiang ZL . Ginsenoside Rg1 protects neurons from hypoxic-ischemic injury possibly by inhibiting Ca2+ influx through NMDA receptors and L-type voltage-dependent Ca2+ channels[J]. Eur J Pharmacol, 2008,586(1-3):90-99
doi: 10.1016/j.ejphar.2007.12.037
[20] Yuan QL, Yang CX, Xu P, Gao XQ, Deng L, Chen P, Sun ZL, Chen QY . Neuroprotective effects of ginsenoside Rb1 on transient cerebral ischemia in rats[J]. Brain Res, 2007, 11671-12
doi: 10.1016/j.brainres.2007.06.024 pmid: 17663984
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