| Activin A is a member of the transforming growth factorβ(TGF-β) family of growth and differentiation factors and plays important roles in the early development of embryo, neurogenesis, hematopoiesis, apoptosis and hormone secretion from new born to adult. Two types of activin receptors have been identified, type I and type II, which belong to the family of serine/threonine kinase receptors. Activin A also acts as a neurotrophic and neuroprotective for neurons and can maintain neuron survival in the central nervous system (CNS) and protect the cultured neuron from neural poison injury. However, it is still unclear whether activin A can stimulate neurite outgrowth of dorsal root ganglia (DRG) neurons and keep DRG neuronal survival for a long time and inhibit DRG glial growth.In order to evaluate the effect of activin A on the neurite outgrowth of the DRG neurons, we observed the activin A-induced neurite outgrowth by the primary culture method of DRG of chicken embryo, which method is convenient and accurate to evaluate the biological activity of nerve growth and differentiation factor. In this study, ActRII and calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) mRNA, inducible nitricoxide synthase (iNOS) mRNA expressions and neurotransmitter 5-hydroxytryptamine (5-HT), Nitric oxide (NO) secretion level in the supernatant of cultured DRG were analysed by semi-quantitative PCR and liquid chromatography/mass spectrometry (LC/MS), the possible mechanism of activin A effects on neurite outgrowth from DRG neurons was further examined.Our data first time demonstrated that activin A could stimulate neurite outgrowth and maintain DRG neuron survival for a long time and inhibit glial growth of DRG of the chicken. The results support some new trends and lines of thinking in the broad field of neural regeneration and neuron survival of activin A. Meanwhile,the present study suggests the possibility that activin A may be useful to prevent neurodegenerative changes to improve nerve tissue reconstruction through inhibiting glial activation.This study was composed of following parts:Part I: The role of Activin A in neurite outgrowth of DRGIn this study, DRG were dissected from embryonic day 8 (E8) chicken under a dissecting microscope and maintained in primary cultures, and the role of activin A in stimulating neurite outgrowth from DRG analyzed in vitro. The results showed that activin A significantly stimulated neurite outgrowth from DRG neurons in a dose and time-dependent manner in vitro, there's a statistical significance (p<0.01) compared with the culture control. To further confirm whether activin A specifically influence neurite outgrowth from DRG neurons, we used follistatin(FS), an activin-binding protein, to neutralize the effects of activin A. The results showed that FS significantly inhibited the neurite outgrowth of the activin A-treated DRG neurons, but FS could not influence the effect of nerve growth factor (NGF)on inducing neurite outgrowth. These data suggested that activin A could stimulate neurite outgrowth from DRG analyzed in vitro. The previous studies have reported that NGF could also stimulate neurite outgrowth from DRG, so we investigated whether activin A act a synergistic role with NGF in stimulating neurite outgrowth of the DRG. The results showed that NGF and activin A co-inducingd neurite outgrowth was more significant than that of single activin A, it is a synergistic role of NGF and activin A in stimulating neurite outgrowth.Part II: The role of Activin A in DRG neuron survivalEffects of activin A on maintaining ganglion cell survival were examined using monolayer-cultured ganglion cells of DRG. To assay the number of living ganglion cells, the viability of ganglion cells of DRG was detected by Trypan Blue exclusion method and the living ganglion cells were counted for each sample under the high magnification using blood counting chamber. The results showed that activin A could maintain long-time survival of DRG neurons, more than 23% of ganglion cells were living during the time of observation for 10 days, while no living cells were found in the culture control on day 5, there's a statistical significance (p<0.01). To further confirm whether activin A specifically keep DRG neurons survival, we used FS to neutralize the effects of activin A. The results showed that FS restrained DRG neurons survival induced by activin A, but FS could not influence the effect of NGF on maintaining DRG neurons survival. These data suggested that activin A could maintain DRG neuron survival for a long time in vitro. As well as, NGF could also keep DRG neurons survival, we investigated whether activin A act a synergistic role with NGF on maintaining DRG neurons survival. The results showed that NGF and activin A co-increased living ganglion cells was more significant than that of single activin A, more than 47% of ganglion cells were living during the time of observation for 10 days, it is a synergistic role of NGF and activin A on maintaining DRG neurons survival. To detect the percentage of DRG neurons and cell type in living ganglion cells, neurons in the ganglion cells cultured by activin A and NGF could be found by staining Nissl bodies with toluidine blue. Dual-label Immunohistochemistry staining for MAP2 and GFAP further showed that MAP2-immunoreactive cells (dark) represented neurons and GFAP-immunoreactive cells (yellow) were morphologically more similar to Schwann cells than astroctyes. In cell composition of the surviving ganglion cells, more than 60% of ganglion cells were neurons, about 30% non-neurons including Schwann cells in activin A group and positive NGF group. These data demonstrated that activin A might prolong the survival of DRG neurons in vitro.Part III: The role of Activin A in DRG glial growthTo determine if activin A suppresses glial activation in vitro, the effects of activin were evaluated in DRG activated with L-Arginine (L-Arg)treatment. A significant neurite outgrowth from DRG neurons and no glial growth was observed in the activin A + L-Arg group after 5 days, an obvious neurite outgrowth from DRG neurons and a large number of glial growth were observed in the NGF+ L-Arg group. The results showed that the inhibitory actions for glial activation by activin A may account for the accumulating findings that activin A possesses neuroprotective properties. NGF could also stimulate neurite outgrowth from DRG, however,it could not inhibit glial activation. A bulk of neurons cultured by activin A and L-Arg could be found by staining Nissl bodies with toluidine blue, and L-Arg could induce a large number of glial growth, and a part of glial growth with NGF treatment .The present study also demonstrated that activin A significantly inhibited L-Arg induced production of DRG glial in vitro.PartⅣ: Assay mechanism of Activin A in DRG neuronIn order to further investigate the possible mechanism of activin A effects, by using semi-quantitative RT-PCR, expressions of ActRIIA and ActRIIB, CGRP, VIP and iNOS mRNA were examined. The results showed that activin A significantly induced the expressions of the ActRIIA and CGRP mRNA, inhibited the expressions of iNOS mRNA, but not influenced ActRIIB and VIP mRNA expressions. Neurotransmitter 5-HT secretion level in the supernatant of cultured DRG were analysed by LC/MS, the results showed that 5-HT secretion levels of the supernatant of cultured DRG increased significantly in activin A groups, and had significant difference compared with control groups (P<0.05), it suggested that activin A could stimulate 5-HT secretion. The secretion of NO in the supernatant of cultured DRG was examined by NO assay kit, the results showed that NO secretion levels of the supernatant of cultured DRG decreased significantly in activin A groups, and had significant difference compared with control groups (P<0.05). Previous studies had demonstrated that CGRP has a neuroprotective action, 5-HT could stimulate neurite outgrowth, and beyond NO secretion could induce nerve cell apoptosis. These data suggested that activin A regulate NO, 5-HT secretion and CGRP expression through Activin A-ActRIIA pathway might mediate the effects of activin A on stimulating neurite outgrowth and maintaining neurons survival of embryonic DRG of the chicken.In this study, a synergistic effect of activin A and NGF were investigated. We found that NGF and activin A co-increased ActRIIA and CGRP expression, 5-HT secretion was more significant increased than that of single activin A and NO secretion was more significant decreased than that of single activin A,and had significant difference compared with control groups (P<0.05). It is a synergistic role of activin A and NGF in stimulating neurite outgrowth and maintaining DRG neurons survival. These data suggested that there are two cell mass that is sensitive to activin A and NGF in nerve cell, neither activin A nor NGF could ensure all nerve cells survival, it is more effective that NGF and activin A co-apply in curing nerve injury and related disease. These findings reveal that activin A and NGF has the potential to play a synergistic role in the DRG neuron development, and provide the theory and experiment basis for discovering drug treatment of nervous system-relatated disease.
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