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Journal of Chinese Integrative Medicine ›› 2009, Vol. 7 ›› Issue (3): 242-248.doi: 10.3736/jcim20090309

• Original Experimental Research • Previous Articles     Next Articles

Protective effect of spleen-yin-nourishing recipe on amyloid β-peptide-induced damage of primarily cultured rat hippocampal neurons and its mechanism

 Li-bin Zhana, Xin-ping Niub, Hua Suic, Xiao-yang Gongd   

  1. a Department of Traditional Chinese Medicine, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
    b Department of Cadres,Dalian Second People’s Hospital,Dalian 116011,Liaoning Province,China;
    c Department of Pathophysiology, Dalian Medical University, Dalian 116044, Liaoning Province, China
    d Department of Traditional Chinese Medicine, the First Affiliated Hospital of Dalian Medical University, Dalian 116021, Liaoning Province, China
  • Received:2008-09-16 Accepted:2008-12-27 Online:2009-03-20 Published:2009-03-15

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Objective

To observe the relationship among amyloid β-peptide (Aβ)-induced neurotoxicity, serum-inducible kinase (SNK)-spine-associated Rap guanosine triphosphatase activating protein (SPAR) pathway and N-methyl-D-aspartate receptor (NMDAR), and to explore the mechanism of the protective effect of spleen-yin nourishing recipe (Zibu Piyin Recipe, ZBPYR) in hippocampal neurons against Aβ-induced neurotoxicity.
Methods

The Aβ1-40 powder was dissolved in 1×PBS and incubated at 37 ℃, and then aggregated fibrillar Aβ1-40 was obtained 72 h later. We used rat primary hippocampal neurons as cell model. ZBPYR-containing serum was gained by the method of serum pharmacology. ZBPYR-containing serum was added to the culture 1 h before Aβ1-40 (5 μmol/L) exposure. Cells were harvested 2 h after Aβ1-40 exposure for total RNA extracting. Then the mRNA expression levels of SNK, SPAR and NMDAR subunits NR1, NR2A and NR2B were detected by reverse transcription-polymerase chain reaction (RT-PCR).
Results

After 2-hour Aβ1-40 exposure, we found that the expression level of SNK mRNA was up-regulated and the expression levels of SPAR, NR1, NR2A and NR2B mRNAs were down-regulated in hippocampal neurons as compared with control group (P<0.01, P<0.05). While with ZBPYR-containing serum pretreatment, the expression level of SNK mRNA was down-regulated and the levels of SPAR, NR1, NR2A and NR2B were up-regulated as compared with Aβ1-40 exposure, and 2% ZBPYR-containing serum showed the best effect (P<0.05).
Conclusion

Aβ-induced neurotoxicity was related to SNK-SPAR pathway and NMDAR; ZBPYR-containing serum can protect neurons from Aβ-induced neurotoxicity, and this protective effect may be performed by regulating the expression of NMDAR and blocking of the SNK-SPAR pathway.

Key words: Alzheimer disease, Zibu Piyin Recipe, Amyloid beta-peptide, Serum-inducible kinase, Spine- associated Rap guanosine triphosphatase activating protein, Receptors, IV-methyl-D-aspartate, Synapses

Table 1

Sequence of the primers"

Denomination Sequence Size of products (bp)
SPAR Sense: 5'-CCG AAT CCG TCA GCG TAG-3' 334
Anti-sense: 5'-TTC CCT TTC CTT GAA TAG C-3'
SNK Sense: 5'-GGA TGA TAG TTA GAG GGA CG-3' 699
Anti-sense: 5'-TGT TCA GGG CGT ATT CCA-3'
NR1 Sense: 5'-AAC CTG CAG AAC CGC AAG-3' 333
Anti-sense: 5'-GCT TGA TGA GCA GGT CTA TGC-3'
NR2A Sense: 5'-TCC ATT CTT CTG TCA TCC TGC-3' 224
Anti-sense: 5'-AAG ACC GTC TCT CAC TCT TGC-3'
NR2B Sense: 5'-TGC ACA ATT ACT CCT CGA CG-3' 222
Anti-sense: 5'-TCC GAT TCT TCT TCT GAG CC-3'
β-actin Sense: 5'-AAC CCT AAG GCC AAC CGT GAA AAG-3' 241
Anti-sense: 5'-TCA TGA GGT AGT CTG TCA G-3'

Figure 1

Morphous of fibrillar Aβ1-40 after 72-hour incubation at 37 ℃ (Transmission electron microscopy, ×80 000) Long fibrils were observed. Scale bar: 100 nm."

Figure 2

Expressions of SNK and SPAR mRNAs in different groups treated by Aβ1-40 and ZBPYR-containing serum analyzed by RT-PCR"

Table 2

Expressions of SNK and SPAR mRNAs in different groups treated by Aβ1-40 and ZBPYR-containing serum analyzed by RT-PCR (x±s)"

Group n SNK/β-actin SPAR/β-actin
Control 4 0.294±0.046 0.518±0.080
Aβ1-40 4 0.696±0.139** 0.280±0.055**
Aβ1-40 plus 2% control serum 4 0.644±0.070** 0.383±0.075
Aβ1-40 plus 1% ZBPYR-containing serum 4 0.514±0.076**△ 0.394±0.076
Aβ1-40 plus 2% ZBPYR-containing serum 4 0.459±0.103*△△ 0.521±0.065△△
Aβ1-40 plus 3% ZBPYR-containing serum 4 0.519±0.104**△ 0.508±0.187△△

Figure 3

Expressions of NR1, NR2A and NR2B mRNAs in different groups treated by Aβ1-40 and ZBPYR-containing serum analyzed by RT-PCR"

Table 3

Expressions of NR1, NR2A and NR2B mRNAs in different groups treated by Aβ1-40 and ZBPYR-containing serum analyzed by RT-PCR (x±s)"

Group n NR1/β-actin NR2A/β-actin NR2B/β-actin
Control 4 0.622±0.070 0.681±0.119 0.574±0.199
Aβ1-40 4 0.359±0.104* 0.379±0.082** 0.299±0.059**
Aβ1-40 plus 2% control serum 4 0.417±0.119 0.414±0.134** 0.329±0.078*
Aβ1-40 plus 1% ZBPYR-containing serum 4 0.541±0.226 0.470±0.099* 0.358±0.043*
Aβ1-40 plus 2% ZBPYR-containing serum 4 0.605±0.175△ 0.568±0.097△ 0.493±0.037△
Aβ1-40 plus 3% ZBPYR-containing serum 4 0.471±0.115 0.441±0.067** 0.446±0.122
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