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Journal of Chinese Integrative Medicine ›› 2010, Vol. 8 ›› Issue (1): 67-73.doi: 10.3736/jcim20100113

• Original Experimental Research • Previous Articles     Next Articles

An in vitro study on neuroprotective effects of serum containing Gengnianchun decoction and its main monomers against amyloid beta protein-induced cellular toxicity

 Wen-jun Wanga, Da-jin Lia, Jun Lia, Wen-jiang Zhoub   

  1. a Department of Integrated Traditional Chinese and Western Medicine,Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
    b Department of Experimental Animal, Fudan University, Shanghai 200023, China
  • Received:2009-08-25 Accepted:2009-10-12 Online:2010-01-20 Published:2010-01-15
  • Contact: Jun Li


To observe the effects of serum containing Gengnianchun (GNC) decoction, a compound traditional Chinese herbal medicine, and its monomers (paeoniflorin, berberine, timosaponin A-Ⅲ and icariin) on neurotoxicity in PC12 cells induced by amyloid beta-protein (Aβ).

Injury of PC12 cells was induced by in incubating with Aβ25-35 in vitro.Ovariectomized rats were intragastrically administered with GNC decoction twice daily for 5 days and then sera were obtained. Different concentrations of serum containing GNC decoction and its main monomers including paeoniflorin, berberine, timosaponin and icariine and the monomer mixtures were cultured with PC12 cells to determine the best concentration of the drugs by methyl thiazolyl tetrazolium (MTT) method. The effective concentration of Aβ25-35 was detemined by culturing PC12 cells with different concentrations of Aβ25-35. Then, the activity of PC12 cells with Aβ25-35-induced injury was observed with MTT method. Cellular morphological change was observed by phase contrast microscopy. Flow cytometry and fluorescence microscopy were employed to observe the Aβ25-35-induced early apoptosis of PC12 cells. 


After Aβ25-35 induction, the PC12 cells were fewer in number, less viable with shrinked cel1 body, many fragments, adhered less and nuclei shrinked. The cell proliferation was inhibited by Aβ25-35concentration- and time-dependently. Aβ25-35 at concentration of 20 μmol/ L was selected to construct the Alzheimer's disease model in vitro. The sera containing GNC decoction could reinforce PC12 cell activity, and concentration at 20% was better than other concentrations after 24-, 48- and 72-hour culture. The 20% serum containing GNC decoction, 0.1 μmol/L berberin and monomer mixture 3 including 1 mg/mL paeoniflorin, 1 μmol/L berberine, 1 μmol/L timosaponin and 1 ng/mL icariine could antagonize neurotoxicity induced by Aβ25-35. Moreover, they could inhibit Aβ25-35-induced early apoptosis of PC12 cells, with the effect of 20% serum containing GNC decoction better than 0.1 μmol/L berberine and monomer mixture 3.

Serum containing GNC decoction at 20% concentration has the potential neuroprotective effect on Aβ-induced cellular impairment. The serum containing GNC decoction was found to be stronger in action than the main monomers.

Key words: Chinese herbal drugs, serum, pharmacology, Gengnianchun Decoction, Chinese herb main monomers, p-amyloid, Alzheimer’s disease, PC12 cells, Apoptosis

Table 1

Effects of different concentrations of serum with GNC Decoction on PC12 cell proliferation at 24, 48 and 72 h ($\bar{x}±s$)"

Group n A value
24 h 48 h 72 h
20% serum containing GNC 5 0.760±0.045 1.109±0.029 1.428±0.039
10% serum containing GNC 5 0.538±0.020** 0.919±0.045** 1.332±0.040**
5% serum containing GNC 5 0.510±0.015** 0.748±0.039** 0.942±0.020**
2.5% serum containing GNC 5 0.404±0.022** 0.554±0.040** 0.721±0.065**

Table 2

Effects of 20% serum with GNC Decoction on PC12 cells proliferation at 24, 48 and 72 h ($\bar{x}±s$)"

Group n A value
24 h 48 h 72 h
Control (5% FBS plus 15% HS) 5 0.581±0.065 0.598±0.055 0.818±0.024
Blank serum 5 0.681±0.023 1.017±0.055 1.211±0.017
20% serum containing GNC 5 0.760±0.045△△▲ 1.109±0.029△△▲▲ 1.428±0.039△△▲▲

Table 3

Effects of GNC main monomers on PC12 cells proliferation ($\bar{x}±s$)"

Group n A value
Control 5 0.669±0.072
100 μmol/L berberine 5 0.666±0.046
10 μmol/L berberine 5 0.741±0.008
1 μmol/L berberine 5 0.747±0.056
0.1 μmol/L berberine 5 0.723±0.036
100 μmol/L timosaponin a-Ⅲ 5 0.523±0.010
10 μmol/L timosaponin a-Ⅲ 5 0.692±0.039
1 μmol/L timosaponin a-Ⅲ 5 0.658±0.021
0.1 μmol/L timosaponin a-Ⅲ 5 0.626±0.036
4 mg/mL paeoniflorin 5 0.523±0.010
2 mg/mL paeoniflorin 5 0.585±0.024
1 mg/mL paeoniflorin 5 0.600±0.022
0.5 mg/mL paeoniflorin 5 0.567±0.025
100 ng/mL icariin 5 0.545±0.039
10 ng/mL icariin 5 0.617±0.013
1 ng/mL icariin 5 0.558±0.032
0.1 ng/mL icariin 5 0.533±0.016
Monomers complex 1 5 0.568±0.037
Monomers complex 2 5 0.619±0.016
Monomers complex 3 5 0.721±0.069
Monomers complex 4 5 0.539±0.031

Table 4

Effects of different concentrations of Aβ25-35 on PC12 cell proliferation ($\bar{x}±s$)"

Group n A value
24 h 48 h
Control 3 0.563±0.009 0.686±0.008
Aβ25-35 5 μmol/L 3 0.410±0.007□□ 0.336±0.006□□■■
Aβ25-35 10 μmol/L 3 0.372±0.003□□ 0.319±0.006□□■■
Aβ25-35 20 μmol/L 3 0.356±0.006□□ 0.304±0.006□□■■
Aβ25-35 40 μmol/L 3 0.301±0.010□□ 0.247±0.007□□■■

Figure 1

Cellular morphological change observed by phase contrast microscopy(×200) A: Normal PC12 cells; B: Apoptosis of PC12 cells after 24-hour incubation with Aβ25-35."


Group n A value
Normal 5 0.436±0.006☆☆
25-35 5 0.281±0.008
20% serum containing GNC 5 0.466±0.038☆☆
0.1 μmol/L berberine 5 0.381±0.018△△☆☆
Monomer mixture 3 5 0.413±0.024△☆☆★
NGF 5 0.461±0.028☆☆

Table 6

Effects of serum containing GNC decoction and its main monomers on early period apoptosis of PC12 cells induced by Aβ25-35 observed by flow cytometry and fluorescence microscopy ($\bar{x}±s$, %)"

Group n Rate of early period apoptosis
Normal 5 4.31±0.36**
Aβ25-35 5 28.41±1.65
20% serum containing GNC 5 4.86±0.26**
0.1 μmol/L berberine 5 10.26±1.30**△△
Monomer mixture 3 5 7.79±0.87**△△▲▲
NGF 5 4.82±0.41**

Figure 2

Aapoptosis of PC12 cells observed by flow cytometry A: Normal group; B: Aβ25-35 group; C: 20% serum containing GNC group; D: 0.1 μmol/L berberine group; E: Monomer mixture 3 group; F: NGF group"

[1] Jing SY, Wei MJ . Clinical study on relationship between memory quotient, estrogen and Chinese nouri-shing kidney herbs in perimenopausal women[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 2002,22(7):494-495
doi: 10.7661/CJIM.2002.7.494
景苏玉, 魏美娟 . 围绝经期妇女的记忆商与雌激素及补肾中药之间关系的临床研究[J]. 中国中西医结合杂志, 2002,22(7):494-495
doi: 10.7661/CJIM.2002.7.494
[2] Yu J, Li CJ . The clinical and experimental study of herbal formula gengnianchung in treating perimeno-pausal syndrome: regulation of neuroendocrine and immune network[J]. Sheng Zhi Yi Xue Za Zhi, 2000,9(5):266-271
doi: 10.3969/j.issn.1004-3845.2000.05.003
俞瑾, 李超荆 . 更年春治疗更年期综合征的临床和药理研究——对神经生殖内分泌免疫网络的调节[J]. 生殖医学杂志, 2000,9(5):266-271
doi: 10.3969/j.issn.1004-3845.2000.05.003
[3] Zhao FG, Wang WJ, Zhou WJ, Li DJ . Effect of Gengnianchun Recipe on learning memory function and hippocampal cholinergic system in ovariectomized rats[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 2008,28:234-237
doi: 10.7661/CJIM.2008.3.233
赵凡桂, 王文君, 周文江, 李大金 . 更年春方对切除卵巢大鼠学习记忆能力和海马胆碱能系统的影响[J]. 中国中西医结合杂志, 2008,28(3):234-237
doi: 10.7661/CJIM.2008.3.233
[4] Xu SY, Bian RL, Chen X. Pharmacology experiment methodology(3rd ed)[M]. Beijing: People’s Medical Publishing House, 2002,( 3rd ed):202-203
徐叔云, 卞如濂, 陈修 . 药理实验方法学(第3版)[M]. 北京: 人民卫生出版, 2002: 202-203
[5] Luo M, Xie RM . Apoptosis of PC12 cell induced by amyloid beta-peptide fragment 25-35[J]. Fudan Xue Bao Yi Xue Ban, 2004,31(6):618-621
罗蔓, 谢瑞满 . β-淀粉样蛋白25-35片段诱导PC12细胞凋亡[J]. 复旦学报(医学版), 2004,31(6):618-621
[6] Hosoda T, Nakajima H, Honjo H . Estrogen protects neuronal cells from amyloid beta-induced apoptotic cell death[J]. Neuroreport, 2001,12(9):1965-1970
doi: 10.1097/00001756-200107030-00038 pmid: 11435931
[7] Hardy JA, Higgins GA . Alzheimer's disease: the amyloid cascade hypojournal[J]. Science, 1992,256(5054):184-185
doi: 10.1126/science.1566067 pmid: 1566067
[8] Zhou LJ, Zhu XZ . Reactive oxygen species-induced apoptosis in PC12 cells and protective effect of bilobalide[J]. J Pharmacol Exp Ther, 2000,293(3):982-988
pmid: 10869401
[9] Chu J, Li L . The dementia models induced by intracerebroventricular infusion of β-amyloid peptide in mice[J]. Zhongguo Yao Li Xue Tong Bao, 2004,20(7):827-829
doi: 10.3321/j.issn:1001-1978.2004.07.028
楚晋, 李林 . 脑室灌注β-淀粉样肽致痴呆动物模型[J]. 中国药理学通报, 2004,20(7):827-829
doi: 10.3321/j.issn:1001-1978.2004.07.028
[10] Greene LA, Tischler AS . Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A[J]., 1976,73(7):2424-2428
[11] Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J . Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta[J]. Nature, 2000,403(6765):98-103
doi: 10.1038/47513 pmid: 10638761
[12] Anderson AJ, Su JH, Cotman CW . DNA damage and apoptosis in Alzheimer’s disease: colocalization with c-Jun immunoreactivity, relationship to brain area, and effect of postmortem delay[J]. J Neurosci, 1996,16(5):1710-1719
doi: 10.1523/JNEUROSCI.16-05-01710.1996
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