The Effects Of ABPP On Nerve Growth And Relative Mechanism In Vitro And In Vivo

ObjectiveTo determine the effects of ABPP(Achyranthes bidentata Blum Polypeptides) on neurite growth in cultured hippocampal neurons of rats.To study the potential ability of ABPP to induce the neuronal differentiation of PC12 cells and to preliminary investigate the activation of ERK1/2 cascade induced by ABPP.To study the repair effects of ABPP on the crushed sciatic rat nerve in mice.Methods1.The cytotoxicity of ABPP was tested with MTT.After 24 h of incubation by different concentrations(0.1μg/ml,0.5μg/ml,1.0μg/ml) of ABPP,the hippocampal neurons were photographed by TCS SP2 confocal microscope with fluorescent immunocytochemistry,and the neurite length was analyzed using the Image-Pro Express software.Real-time quantitative RT-PCR was performed to examine the mRNA levels of GAP-43 and NF-H after 6 h with different concentrations of ABPP.Western blotting were performed to further examine the protein levels of GAP-43 and NF-H in cultured hippocampal neurons after 24 h incubation with different concentrations of ABPP,and detected the activities of ERK1/2 in hippocampal neurons co-culture with ABPP and the MEK1/2 inhibitor,PD98059.2.By cell culture in the low-concentration of serum,the morphological changes of PC12 cells treated with of ABPP at different concentration(0.25μg/ml,0.5μg/ml,1.0μg/ml) was detected.Fluorescent immunocytochemistry was performed to examine the expression of NF-H,a well-known neuronal marker,in differentiated PC12 cells induced by 1.0μg/ml ABPP.In order to study the signaling pathways involved in the differentiation induced by ABPP,Western blotting was performed to detect the acitvities of ERK1/2 in PC12 cells activated by 1.0μg/ml ABPP after different periods(0h,6h,12h,1d,2d,3d and 7d) using the activated(Diphosphorylated ERK1&2) antibody and mitogen activated protein kinase(MAP Kinase,MAPK,ERK-1 & ERK-2) antibody when PC12 cells were cultured in low-concentration of serum medium.3.Fifty mice were performed sciatic nerve crush injury operation.After that the animals were randomly divided into 5 groups of 10 animals each and treated tail vein administration as follows:ABPP groups(low,middle and high dose,1,4,16 mg/kg, respectively),Methycobal group(served as positive control) at 65μg/kg and saline group (served as negative control).The treatment lasted for 21d.At different time after nerve crush,a combination of experiments,including walking track analysis, electrophysiological assessments,immunohistochemistry and electron microscopy to the regenerated sciatic nerve,masson trichome staining to the gastrocnemius muscle,as well as morphometric analyses,was carried out to evaluate the regenerative outcomes of ABPP administration.Results1.ABPP promoted the neurite growth of cultured hippocampal neurons.Real-time quantitative RT-PCR showed that the mRNA level of GAP-43 and NF-H in cultured hippocampal neurons was up-regulated by ABPP in a dose-dependent manner.Meanwhile, fluorescent immunocytochemistry and Western blotting showed that the protein level of GAP-43 and NF-H was up-regulated by ABPP.ABPP-induced phosphrylation of ERK1/2 was blocked by PD98059.2.After 3 days treated with ABPP(0.25μg/ml,0.5μg/ml,1.0μg/ml),neuron morphology was observed in PC12 cells:compaction of cell bodies,the outgrowth of neurites,unproliferation.As the time went on,the number of the differentiated PC12 cells increased.At day 7 the differentiated PC12 cells could produced the neurite network and at day 14 the phenomena became mo(?)e obvious.It has been shown that ABPP could promote the differentiation of PC12 by dose-effect relation.ABPP activated ERK1/2 in PC12 cells by dose-effect relation and time-dependent character,with the best concentration of 1.0μg/ml after being co-cultured with PC12 cells for 2 days.And PD98059 significantly inhibited ABPP-induced phosphorylation of ERK1/2.3.The results indicated that treatment with ABPP at a dose range(1-16 mg/kg) promoted histological regeneration and functional recovery of the injured sciatic nerve and its target muscle,yielding a desired efficacy greater than that by vehicle treatment and close to or even greater at some parameters than that by methylcobalamin,a positive control. Conclusion1.ABPP could promote the neurite growth and modulate the expression of GAP-43 and NF-H in cultured hippocampal neurons,suggesting neurotrophic effects of ABPP.2.ABPP could induce the neuronal differentiation of PC12 cells.The differentiation of PC12 cells induced by ABPP may be mediated through ERK1/2 phosphorylation cascade potentially.3.Plant polypeptides,ABPP,may be a potential agent in ameliorating the effects of neuropathy caused by sciatic nerve crash.