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Journal of Chinese Integrative Medicine ›› 2008, Vol. 6 ›› Issue (1): 68-72.doi: 10.3736/jcim20080113

• Original Experimental Research • Previous Articles     Next Articles

Preliminary assay of p-amyloid binding elements in heart-beneficial recipe

Min Cheng, Qiong Feng, Shu-wen Qian, Hui Gao, Cui-qing Zhu()   

  1. Key State Laboratory of Medical Neurobiology, Shanghai Medical School, Fudan University, Shanghai 200032, China
  • Received:2007-04-05 Online:2008-01-20 Published:2008-01-15
  • Contact: ZHU Cui-qing E-mail:cqzhu@shmu.edu.cn

Objective: To explore whether there are β-amyloid protein (Aβ) binding elements in heart-beneficial recipe (HBR, a compound traditional Chinese herbal medicine), which can ameliorate the cytotoxicity of Aβ.

Methods:The extract of HBR and Aβ1-40 were co-precipitated, and the Aβ1-40 in pellets was detected by immunoblotting. Affi-gel-Aβ1-40 was constructed, and Affi-gel-Aβ1-40 affinity elements from the extract of HBR were analyzed by high-performance liquid chromatography (HPLC). The assay of lactic dehydrogenase (LDH) release from the primary cultured rat cortex neurons was used to evaluate the cytotoxicity of Aβ1-42, and the protection effects of the HBR serum and the Affi-gel-Aβ1-40 treated HBR serum.

Results:Immunoblotting examination showed Aβ1-40 could be co-precipitated with components of HBR following co-incubation, and the amount of Aβ1-40 within pellets decreased when the HBR extract was diluted. Aβ1-40 affinity elements from the extract of HBR, eluted from Affi-gel-Aβ1-40 by glycine solution (pH=2.5), could be detected by HPLC-fluorescent detector system. The analysis of LDH release showed that exposure of neurons to 5 μmol/L Aβ1-42 for 48 h caused a significant increase of LDH release in either a serum free or 10% serum contained culture condition (P<0.01). The rat HBR serum was able to suppress Aβ1-42 induced LDH release (P<0.05), whereas Affi-gel-Aβ1-40 treated HBR serum still maintained the ability to attenuate Aβ1-42 induced LDH release although the effect was somewhat decreased compared with Affi-gel treated HBR serum.

Conclusion:There are Aβ affinity components in HBR, which could not increase the Aβ cytotoxicity, but might be able to inhibit the cytotoxicity of Aβ. The results implied that the exploration of Aβ affinity elements from Chinese medicinal recipe which is effective for Alzheimer disease, might be an important direction in Alzheimer disease therapeutic research area.

Key words: β-amyloid protein, heart-beneficial recipe, lactic dehydrogenase, Alzheimer disease

CLC Number: 

  • R749.16

Figure 1

Co-precipitation of Aβ1-40 with the extracts from heart-beneficial recipe Lane 1 is Aβ1-40 positive marker. The samples of lane 2-4 are pellets precipitated from the solutions containing extract of heart-beneficial recipe (HBR) and Aβ1-40 following incubation. The HBR extract was diluted with PBS by 1∶1 (lane 2), 1∶4 (lane 3) and 1∶8 (lane 4), and co-incubated with Aβ1-40. The Aβ1-40 in centrifuging precipitates was detected by immunoblotting. This experiment was repeated by three times."

Figure 2

HPLC detection of the elements in the heart-beneficial recipe extract absorbed by Affi-gel-Aβ1-40After Affi-gel-Aβ1-40 beads were co-incubated with the extract of HBR, the beads were washed with PBS, and the putative Aβ affinity elements were eluted by glycine solution (pH 2.5). The fluorescent detector in HPLC system could not detect positive signal in the eluents from Affi-gel-Aβ1-40 without OPA derivation (A). After OPA derivation, the positive signals were detected in the eluents from Affi-gel-Aβ1-40 followed by HBR incubation (B, D). However, in the eluents from Affi-gel, as a control sample, only non-specific absorbed elements were detected (C, E)."

Figure 3

Effect of Affi-gel-Aβ1-40 treated heart-beneficial recipe serum on Aβ caused neuronal injuryIn non-serum system (A), the LDH release from primary cultured neurons was significantly increased following 48 h Aβ1-42(1 and 5 μmol/L) treatment. The Sham represented the neurons not treated with Aβ1-42. *P<0.05 and **P<0.01 vs Sham. In a culture system containing 10% serum (B), neurons exposed to Aβ1-42 also caused an increase of LDH release. The serum from HBR administrated rats significantly inhibited Aβ1-42 caused LDH release. And Affi-gel-Aβ1-40 treated HBR serum is still able to inhibit Aβ1-42 cytotoxicity, however it was somewhat less effective than Affi-gel treated HBR serum. *P<0.05 and **P<0.01 vs Aβ1-42 treated normal serum. Each value represents the mean±SD (n=6)."

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