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低浓度神经生长因子存在下GPI-1046刺激鸡胚神经节突起生长

https://www.doczj.com/doc/ff7027827.html,

Research Paper

Received 2007-05-10 Accepted 2007-09-16

This work was supported by the National Natural Science Foundation of China (No. 30140001) and the National Basic Research Development Program of China (No. 2001CB510206).

**Contribute equally to this work.

*

Corresponding author. Tel: +86-10-66931304; Fax: +86-10-68213039; E-mail: liusj@https://www.doczj.com/doc/ff7027827.html,

GPI-1046 stimulates chicken dorsal root ganglion neurite outgrowth in the presence of nerve growth factor at low concentration in vitro

QUE Hai-Ping 1,**, LI Xin 1,**, LI Song 2, LIU Shao-Jun 1,*

1

Department of Neurobiology, Institute of Basic Medical Sciences; 2Department of Medical Chemistry, Institute of Pharmacology and

Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Abstract: The purpose of this investigation was to re-evaluate the neurotrophic effect of GPI-1046 on neurite outgrowth in vitro . GPI-1046 was synthesized and identified with mass spectrometry, nuclear magnetic resonance and elemental analysis. Chicken dorsal root ganglions (DRGs) were removed and divided into three groups: (1) The DRGs were cultured in DMEM containing different concentra-tions of GPI-1046; (2) The DRGs were cultured in DMEM containing nerve growth factor (NGF) alone at 0.8 and 8 ng/mL, respectively;(3) The DRGs were cultured in DMEM containing both different concentrations of GPI-1046 and NGF at 0.8 ng/mL. The results showed that GPI-1046 alone could not stimulate chicken DRG neurite outgrowth; however, GPI-1046 stimulated DRG neurite outgrowth only in the presence of NGF at low concentration in the culture medium.

Key words: GPI-1046; nerve growth factor; immunophilin; neurite outgrowth; chicken dorsal root ganglia

低浓度神经生长因子存在下GPI-1046刺激鸡胚神经节突起生长

阙海萍1,**,李 欣1,**,李 松2,刘少君1,*

军事医学科学院1基础医学研究所神经生物学研究室;2毒物药物研究所,北京 100850

摘 要:对GPI-1046是否具有神经营养作用目前有两种不同的认识。Steiner 等认为GPI-1046能促进体外培养的感觉神经节神经元突起生长。但Harper 等却没能证明GPI-1046有这样的作用。由于GPI-1046在临床上具有重要应用价值和前景,我们重新评价了GPI-1046对体外培养鸡胚神经节的神经营养作用,发现在低浓度神经生长因子(nerve growth factor, NGF)存在下,GPI-1046能明显促进鸡背根神经节神经突起的生长。

关键词:G P I -1046;神经生长因子;免疫亲和素;神经突起;鸡胚背根节中图分类号:Q 42

There is considerable evidence that immunophilin legends,cyclosporine A (CsA) and FK506 can promote the rege-neration of axons in peripheral nerves, and they can act as neuroprotective agents in the central nervous system (CNS)[1-4].It was reported that in the absence of exogenous nerve growth factors (NGFs), both FK506 and CsA could en-hance the effect of NGF at low concentrations in promot-ing neurite outgrowth in PC12 cells [1], and also could sti-mulate the structural and functional recovery in neurode-generative animal models [2]. FK506 and CsA are shown to increase both the number of neurites emanating from the ganglia and the length of these neurites [2]. Recent studies suggested that the protective properties of FK506 and its non-calcineurin inhibiting derivatives are realized by a fast induction of heat shock proteins [5]. The induction of the heat shock response by immunophilin legends might be an interesting target for neuroregeneration and neuroprotec-tion [5-7]. Although immunosuppressive immunophilin

legends promote neurite outgrowth in vitro, their neu-rotrophic activities are clearly independent of their immu-nosuppressive activity. A novel nonimmunosuppressive im-munophilin ligand, GPI-1046 [3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate], is designed based on the structure of FK506 and could bind to FK506 binding protein-12[2]. GPI-1046 is also evaluated in established experimental systems to promote neuron survival and axonal growth in vitro and in vivo. It was also reported that GPI-1046 could elicit neu-rite outgrowth from cultured sensory neurons with pico-molar potency, and its maximal effects were comparable to that of NGF[3]. Some researchers reported that GPI-1046 might ever have therapeutic potential for neurologi-cal disorders in clinical applications[5,8-10]. However, the re-verse research reports about GPI-1046 were also put for-ward recently. Harper et al.[11], Bocquet et al.[12], and Eber-ling et al.[13] failed to demonstrate GPI-1046 having such a robust effect on neurite outgrowth from chicken dorsal root ganglions (DRGs) in explant cultures under condi-tions where a very robust NGF effect was observed by Steiner et al[2,3]. They also did not obtain evidence support-ing the notion of a general neuroprotective effect of the compound or an effect on morphologic nerve regenera-tion in vitro and in vivo.

In view of the mimetic importance of the small molecu-lar neurotrophic factor in potential clinical application, we re-evaluated the neurotrophic potential of GPI-1046 on neurite outgrowth and neuron survival in DRG neurons to determine whether GPI-1046 has a neurotrophic effect.

1 MATERIALS AND METHODS

1.1 Materials

GPI-1046 was synthesized as described in the litera-ture[3], and was then analyzed using mass spectrometry, nuclear magnetic resonance and elemental analysis. The results showed that it was identical to the published struc-ture originally developed by Guilford Pharmaceuticals (USA). For cultures, it was dissolved first in 20% SMO, then in liquid solution. NGF was isolated and purified from rat angularis glandula. All other reagents were purchased from Sigma.

1.2 Dissecting and explanting chicken DRGs

DRGs were removed from 8- to 10-day incubated chicken embryos as described[14]. After washed in salt solution, the DRGs were explanted in 15 mL culture bottles coated with collagen, at a density of 6-8 DRGs/bottle. One hour later,the DRGs were observed to attach to the wall of the bottle

fast and then DMEM medium (high glucose content, Life Technologies, Paisley, UK) was added at 37 o C in a water vapor-saturated atmosphere containing 5% CO

2

for another 2 h.

1.3 Grouping

The cultured DRGs were then divided into 3 groups: (1) Group 1, the DRGs were cultured in DMEM containing GPI-1046 at different doses (1 pmol/L, 10 pmol/L, 100 pmol/L, and 1 nmol/L) without exogenous growth factors.

(2) Group 2, the DRGs were cultured in DMEM contain-ing NGF alone at 0.8 ng/mL and 8 ng/mL, respectively.

(3) Group 3, the DRGs were cultured in DMEM contain-ing GPI-1046 at different concentrations of 0.5 pmol/L, 1 pmol/L, 10 pmol/L, 100 pmol/L, 500 pmol/L and 1 nmol/L, beside 0.8 ng/mL of NGF. All cultures were incubated at 37 o C in a water vapor-saturated atmosphere containing

5% CO

2

for another 24 h. The DRGs and their neurite outgrowth were observed and recorded under IX70 Olympus microscope.

1.4 Data collecting and statistical analysis

The absolute length of neurites around the body of DRGs was measured and the longest neurite in each DRG was recorded and used as statistical data. The densities of neurites within the range of 100 μm2, at 0.2-mm distance away from the body of DRGs were accounted and re-corded . Statistical significance was determined by ANOV A followed by Newman-Keul’s multiple comparison t-test when appropriate.

2 RESUL TS

2.1 GPI-1046 alone could not stimulate neurite out-growth from DRGs without NGF

GPI-1046 alone showed little and non-significant effect on neurite outgrowth (Fig.1). In Fig.1A-C, no obvious neu-rite outgrowth from DRGs was observed although GPI-1046 at concentrations from 1 to 100 pmol/L was added. Moreover, a little notable dose-dependent manner of GPI-1046 was noted. When the concentration of GPI-1046 was added up to 1 nmol/L, quite short and sparse neurites were observed around the body of DRGs (Fig.1D). But, these short neurite outgrowths did not show any statistical significance. Compared with that in group 1, NGF had significant stimulatory effects on chicken DRG neurite outgrowth in group 2 (Fig.2). Our investigation was iden-tical to previous reports that GPI-1046 did not have stimula-tory effects on neurite outgrowth when it was used

793

QUE Hai-Ping et al : GPI-1046 Stimulates Neurite Outgrowth in the Presence of NGF

Fig. 2. Effects of NGF on chicken dorsal root ganglion (DRG) neurite outgrowth. A : 0.8 ng/mL NGF . B : 8 ng/mL NGF . A significant increase

in neurite outgrowth was observed in DRGs treated with NGF. Scale bar, 50 μm.

Fig. 3. Effects of GPI-1046 on neurite outgrowth from DRG in the presence of exogenous NGF (0.8 ng/mL). A : 0.8 ng/mL NGF and 0.5 pmol/L GPI-1046. B : 0.8 ng/mL NGF and 1 pmol/L GPI-1046. C : 0.8 ng/mL NGF and 10 pmol/L GPI-1046. D : 0.8 ng/mL NGF and 100 pmol/L GPI-1046. E : 0.8 ng/mL NGF and 500 pmol/L GPI-1046. F : 0.8 ng/mL NGF and 1 nmol/L GPI-1046. The neurite outgrowth from DRG increased with the increased concentration of GPI-1046. Scale bar, A -E : 25 μm; F : 40 μm.

Fig. 1. Effects of different concentrations of GPI-1046 on chicken dorsal root ganglion (DRG) neurite outgrowth. A : 1 pmol/L GPI-1046. B : 10pmol/L GPI-1046. C : 100 pmol/L GPI-1046. D

: 1 nmol/L GPI-1046. No obvious neurite outgrowth from DRG was observed. Scale bar, 15 μm..

Acta Physiologica Sinica , December 25, 2007, 59 (6): 791-795

794alone [12,13].

2.2 GPI-1046 stimulated neurite outgrowth from DRGs in the presence of NGF at low concentration However, in the presence of NGF at low concentration, it was clearly demonstrated that the neurite outgrowth from DRGs treated with GPI-1046 obviously increased. The

Fig. 4. Length comparison of the neurites emanating from each ganglion.means±SEM, n =3. Significance was determined by analysis of variance followed by Newman-Keul’s multiple comparison t -tests.

*

P <0.05, ***P <0.001

vs

0.8 ng/mL NGF group.

Fig. 5. Density comparsion of the outgrowth neurites after treatment with NGF alone (0.8 ng/mL and 8 ng/mL, respectively) for 24 h, and both NGF (0.8 ng/mL) and GPI-1046 at different concentrations.GPI-1046 caused a marked increase in density of neurites. Signifi-cance was determined by analysis of variance followed by Newman-Keul’s multiple comparison t -tests. *P <0.05, **P <0.01 vs 0.8 ng/mL

NGF group.

length of neurites even doubled, trebled or more, com-pared with the effect of NGF alone. The densities of these neurites also significantly increased (Fig.3). The result sug-gested that GPI-1046 did not directly promote neurite outgrowth, but enhanced the effect of NGF. Moreover,the length and density of outgrowth neurites were increased with the increased concentration of GPI-1046 (Fig.3). It is possible that, in the presence of NGF at quite low concentration, GPI-1046 at high concentrations magnified the effect of NGF. Statistical analysis showed a significant dose-dependent manner (Fig.4 and 5). It is indicated that GPI-1046 potently magnified the stimulatory effect of NGF on neurite outgrowth in a dose-dependent manner.

3 DISCUSSION

Some previous results have suggested that GPI-1046 and other FK506 derivatives are extremely potential in promot-ing neurite outgrowth in chick sensory ganglion, and sig-nificant enhancing effect is achieved at concentrations even as low as 1 pmol/L without NGF in the medium. However,as Harper et al .[11], Bocquet et al .[12] and Eberling et al.[13],we failed to repeat this result when chicken DRGs were cultured with medium containing GPI-1046 alone at con-centrations from 0.5 pmol/L to 1 nmol/L without NGF.The results in the present work suggested that GPI-1046alone had little effect on promoting the neurite outgrowth without exogenous growth factors like NGF. On the other hand, the promoting effect of GPI-1046 on the neurites, if present, was not direct at least. The observations in the present experiment, different from the traditional reports,demonstrated that GPI-1046 potently stimulated axon growth in a dose-dependent manner in the presence of NGF at a low concentration. The stimulatory effect was not dependent on the class of sensory axon.

Previous observations showed that GPI-1046 was a potent stimulator of regenerating axons from adult, primary sen-sory neurons [1,3]. It was also reported that FK506 could promote neurite survival and neurite growth in PC12 cells in the presence of NGF. This observation was similar to the reports [1,3]. It is known that NGF has a promoting ef-fect on the growth of neuroprocesses and a protective effect on injured brain cells. However, NGF, due to its high molecular weight, can not pass through the blood-brain barriers, and hence has little action on the CNS in vivo . The small molecular neurotrophic factors analogous to NGF, such as GPI-1046, FK506 and related agents,may have therapeutic application in various neuroregene-ration disorders. Different conclusions on actions of these

795 QUE Hai-Ping et al:GPI-1046 Stimulates Neurite Outgrowth in the Presence of NGF

small molecular agents affect their applicative prospects. In our investigation, GPI-1046 displays excellent bioavai-lability in promoting DRG neurite outgrowth in the pre-sence of NGF at low concentration and has little action without NGF. Our study provides new understanding for these agents in therapeutic application.

* * * ACKNOWLEDGEMENTS: We thank Professor LI Y uan-Min for his careful reading and revising this manuscript.

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