| Spinal cord injury (SCI) involves primary degeneration of the directly injured neurons, but also initiates a self-destructive process that leads to secondary degeneration of neighboring neurons that escaped the initial insult. The traditional belief is that immune activity in the damaged SCI is harmful and must therefore be eliminated or suppressed. Therefore,anti-inflammatory compounds, such as methylprednisolone and dexamethasone have been used in restricting the spread of damage at an early post-traumatic stage.However, accumulating evidence points to one view that endogenous autoimmunity in central nervous system(CNS) is beneficial and a properly controled immune response prodvides a pivotal protective effect in diseased conditions of CNS. Studies have been shown that T cells directed to specific CNS antigens, such as myelin basic protein(MBP)-T cell, play a key role in the physiological processes of CNS protection and repair. Based on the experimental findings in rodents, the concept of"protective autoimmunity"in neurodegenerative conditions was established by Schwartz and colleagues. Boosting of such a T cell response specific for CNS antigens could serve as a therapeutic approach to ameliorate CNS disease including SCI.Dendritic cells (DCs) are professional antigen-presenting cells that play a crucial role as initiators and modulators of adaptive immune responses. DC-based vaccines have received considerable interest in recent years. They have been used successfully to generate cytolytic T cell activity against tumor antigens, induce immune tolerance and bring about changes in T-cell polarization. When DCs used as a vaccine in animals with injured spinal cords, it could evoke protective autoimmune response. Antibodies to major myelin proteins, such as MBP, might also contribute to the efficacy of the therapy. Previously, we have reported that local or systematic injection of DCs pulsed with homogenate protein of spinal cord (hpDC) acquired significant functional recovery from SCI in mice. Compared with mbpDC, hpDC demonstrated better tissue preservation in SCI mice. Therefore, vaccation with hpDC is more potent than mbpDC in improving functional recovery from SCI.Evodiamine(EVO), an alkaloidal component extracted from the fruit of Evodiae fructus, exhibits various biological effects, such as anti-tumor growth and metastasis, vasodilatory effect and anti-inflammatory actions. Recently, experiments in our labarory found that mature DC, after stimulated by EVO in vitro, could enhance the capacity to induce antigen-specific T cell responses. So, wether hpDCevo is better than hpDC in stimulating proliferation of CNS-specific T cell and improving the functional recovery from SCI mice is unclear.Materials and methods:In this study, by using BMS score, histopathological examination, to assess the neuroprotective effect of hpDCevo on injuried spinal cord. To further discussed the neuroprotective mechinisim of hpDCevo, in addition, by using ELISA, MTT and immunofluorescence assay, we examined the ability of T cell proliferation,the expression of cytokines(IFN-γand IL-4) , growth factors production (BDNF and NT-3) , nestin protien and NF200 protein.Results:1. BMS score declined to 0 after SCI, then recovered slowly, and reached a highest plateau (6.29±0.25) on 28dpi in hpDCevo group. Difference was significant between hpDCevo group and hpDC groups (p<0.05). This results suggest that Injected hpDCevo promoted significant functional recovery from SCI in mice2. The ares of injured region(1.93±0.16mm2) and cyst(0.22±0.01mm2) of hpDCevo group were siginificant decreased compared with the ares of injured region(2.18±0.12mm2) and cyst(0.25±0.02mm2) of hpDC group (p<0.05) . These results suggest that injected hpDCevo decreased the ares of injured region and cyst from SCI in mice.3. In vitro, we observed that T lymphocyte proliferation in the stimulation culture with ovaDCevo or ovaDC was more significant than in the culture with EVO, DCevo and DC(p<0.05), MTT assays analysis also revealed that T cells proliferative responses in culture with ovaDCevo(0.99±0.04) were greater when compared with ovaDC(0.62±0.03 )(p<0.05). These results suggest that EVO-stimulated DC enhances T-cell proliferation in vitro. 4. In vitro, we found that the proliferation ability of T lymphocyte in hpDCevo group(0.33±0.03) was greater than hpDC group(0.25±0.04)(p<0.05). In vivo, we found that significantly more T cells in the vicinity of the lesion site were seen in hpDCevo(673.33±167.17/mm2) group than hpDC(500±146.42/mm2) treated group(p<0.05). These results suggest that hpDCevo can evoke a pronounced homogenous T-cell immune response.5. We observed that the amount of IFN-γobtained from T cells supernatant was significantly higher in hpDCevo group(2792.54±162.99 pg/ml) than hpDC group (932.94±60.24pg/ml) (p<0.05). The amount of IFN-γobtained from lesion site, moreover, was also significantly higher in hpDCevo(1882.85±50.67ng/g) group than hpDC group(970.56±19.9 4ng/g)(p<0.05). However, no obvious change of IL-4 expression could be detected in any treated group.6. The expression of BDNF(393.80±48.99 pg/ml) and NT-3(32.17±2.48 pg/ml) obtained from T cells supernatant was significantly higher in hpDCevo group than the expression of BDNF(300.10±14.34 pg/ml) and NT-3(7.95±4.08 pg/ml) in hpDC group, respectively(p < 0.05). Moreover, the amount of BDNF(27.71±0.35ng/g) and NT-3(1.47±0.11ng/g) obtained from lesion site was also significantly higher in hpDCevogroup than the amount of BDNF(25.39±0.55 ng/g) and NT-3(0.98±0.16 ng/g) in hpDC, respectively (p<0.05).7. We also found that the number of Nestin+ cell(187.5±18.73/mm2) and NF200+ cell(88±12.9/mm2) around the areas of injury in hpDCevo group were more than the number of Nestin+ cell(101.75±15.44/mm2) and NF200+ cell(61.75±20.12/mm2) in hpDC group, respectively (p<0.01).Conclusion:1. It is the first finding that immune of hpDCevo can improve functional recovery from SCI in mouse, while pure hpDC have less effect.This suggest that EVO can enhance the neuroprotective effect of hpDC.2. EVO-stimulated DCs enhances T-cell proliferation in vitro.3. The vaccination with hpDCevo in this work evoked a T cell response with a potential T helper 1 (Th1/Th0) bias.4. The vaccination with hpDCevo in this work up-regulation the expression of BDNF and NT-3, the number of Nestin+ cell and NF200+ cell in vivo. These results suggest that hpDCevo could induce the differentiation of endogenous neural stem cell and promote the secretion of neurotrophic factor derived from T cell. So as to promote functional recovery from spinal cord injury in mice.
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