[an error occurred while processing this directive]

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 E-mail:wjwwang@yahoo.com.cn

RichHTML

1

PDF (PC)

69

Like

Obiective

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).
Methods

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. 

Results

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.
Conclusion

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☆☆
Aβ25-35 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
[1] Na Wu , Lu Song , Xin-xin Yang, Jiang-lei Wei , Zhen-guo Liu. Effects of Chinese herbal medicine Tianqi Pingchan Granule on G protein-coupled receptor kinase 6 involved in the prevention of levodopa-induced dyskinesia in rats with Parkinson disease. Journal of Chinese Integrative Medicine, 2012, 10(9): 1018-1024.
[2] Avijit Paul, Kausik Bishayee, Samrat Ghosh, Avinaba Mukherjee, Sourav Sikdar, Debrup Chakraborty, Naoual Boujedaini, Anisur Rahman Khuda-Bukhsh. Chelidonine isolated from ethanolic extract of Chelidonium majus promotes apoptosis in HeLa cells through p38-p53 and PI3K/AKT signalling pathways. Journal of Chinese Integrative Medicine, 2012, 10(9): 1025-1038.
[3] Xin-min Li, Hui Chen, Yan-yan Ren, Yan-li Lu. Effects of Chinese herbal medicine Xifeng Capsule on multidrug resistance-associated protein 1 expression in hippocampus and cortex of rats with lithium-pilocarpine-induced epilepsy. Journal of Chinese Integrative Medicine, 2012, 10(8): 911-917.
[4] Yong Zhang, Xiao-wen Sun, Jian-hua Xu, Hai Lu, Zhong-ze Fan, Jue Sun, Xiao-xiao Zhang. Effects of medicated serum prepared with Chinese herbal medicine Changweiqing on pharmacokinetics of oxaliplatin in colon cancer cells. Journal of Chinese Integrative Medicine, 2012, 10(8): 901-910.
[5] Wei-hua Xu, Ze-huai Wen, Wei-xiong Liang, Qi Wang. A review of research on the development of instruments for therapeutic efficacy evaluation of traditional Chinese medicine. Journal of Chinese Integrative Medicine, 2012, 10(7): 726-737.
[6] Lin Wang, Guan-liang Wang, Jia-han Liu, Di Li, De-zeng Zhu, Liang-neng Wu . Effects of ursolic acid in ameliorating insulin resistance in liver of KKAy mice via peroxisome proliferator-activated receptors α and γ. Journal of Chinese Integrative Medicine, 2012, 10(7): 793-799.
[7] Lu Yu, Shui-miao Lin, Ru-qian Zhou, Wei-jun Tang, Pin-xian Huang, Ying Dong, Jian Wang, Zhi-hua Yu, Jiu-lin Chen, Li Wei, San-li Xing, Hui-juan Cao, Hong-bin Zhao. Chinese herbal medicine for patients with mild to moderate Alzheimer disease based on syndrome differentiation: A randomized controlled trial. Journal of Chinese Integrative Medicine, 2012, 10(7): 766-776.
[8] Yogesh Bendale, Vineeta Bendale, Saili Paul, Soumya Sundar Bhattacharyya. Green synthesis, characterization and anticancer potential of platinum nanoparticles Bioplatin. Journal of Chinese Integrative Medicine, 2012, 10(6): 681-689.
[9] Ying-chun Miao , Jin-zhou Tian , Jing Shi , Min Mao. Effects of Chinese medicine for tonifying the kidney and resolving phlegm and blood stasis in treating patients with amnestic mild cognitive impairment: A randomized, double-blind and parallel-controlled trial. Journal of Chinese Integrative Medicine, 2012, 10(4): 390-397.
[10] Min Zhu , Mi Zhou , Ying Shi , Wen-wei Li. Effects of echinacoside on MPP+-induced mitochondrial fragmentation, mitophagy and cell apoptosis in SH-SY5Y cells. Journal of Chinese Integrative Medicine, 2012, 10(12): 1427-1432.
[11] Yu-ting Yin, Xiao-wan Li , Yang Dong , Jian-rong Shi. Research progress in biological basis of cold and heat essence of Chinese medicine. Journal of Chinese Integrative Medicine, 2012, 10(12): 1328-1335.
[12] Rui Jin , Bing Zhang . A complexity analysis of Chinese herbal property theory: The multiple expressions of herbal property (Part 2). Journal of Chinese Integrative Medicine, 2012, 10(12): 1321-1327.
[13] Mukherjee Avinaba, Sikdar Sourav, Bishayee Kausik, Paul Avijit, Ghosh Samrat, Boujedaini Naoual, Rahman Khuda-Bukhsh Anisur. Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro. Journal of Chinese Integrative Medicine, 2012, 10(12): 1451-1459.
[14] Samadder Asmita, Das Jayeeta, Das Sreemanti, Das Durba, De Arnab, Bhadra Kakali, Rahman Khuda-Bukhsh Anisur. Dihydroxy-isosteviol methyl ester of Pulsatilla nigricans extract reduces arsenic-induced DNA damage in testis cells of male mice: Its toxicity, drug-DNA interaction and signaling cascades. Journal of Chinese Integrative Medicine, 2012, 10(12): 1433-1442.
[15] Rui Jin, Bing Zhang. A complexity analysis of Chinese herbal property theory: the multiple formations of herbal property (Part 1). Journal of Chinese Integrative Medicine, 2012, 10(11): 1198-1205.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] Wei-xiong Liang. Problems-solving strategies in clinical treatment guideline for traditional Chinese medicine and integrative medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 1-4
[2] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[3] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[4] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50
[5] . Uniform requirements for manuscripts submitted to biomedical journals: Writing and editing for biomedical publication (Chinese version, part two). Journal of Chinese Integrative Medicine, 2010, 8(11): 1001-1005
[6] Daniel Weber, Janelle M Wheat, Geoffrey M Currie. Inflammation and cancer: Tumor initiation, progression and metastasis,and Chinese botanical medicines. Journal of Chinese Integrative Medicine, 2010, 8(11): 1006-1013
[7] Hong Liu , Guo-liang Zhang, Li Shen , Zhen Zeng, Bao-luo Zhou, Cheng-hai Liu, Guang Nie . Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5Nl avian influenza virus. Journal of Chinese Integrative Medicine, 2010, 8(11): 1036-1040
[8] Zhao-guo Li . A discussion of English translation of 1995 and 1997 Chinese National Standards of Traditional Chinese Medical Terminologies for Clinical Diagnosis and Treatment. Journal of Chinese Integrative Medicine, 2010, 8(11): 1090-1096
[9] Rui Jin, Bing Zhang. A complexity analysis of Chinese herbal property theory: the multiple formations of herbal property (Part 1). Journal of Chinese Integrative Medicine, 2012, 10(11): 1198-1205
[10] Hui-min Liu, Xian-bo Wang, Yu-juan Chang, Li-li Gu. Systematic review and meta-analysis of randomized controlled trials of integrative medicine therapy for treatment of chronic severe hepatitis. Journal of Chinese Integrative Medicine, 2012, 10(11): 1211-1228
[an error occurred while processing this directive]