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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 E-mail:wjwwang@yahoo.com.cn

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☆☆
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"

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