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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."

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