| Objective1. The neuroprotective effect of the optimized DNA vaccine pVAX1-IL-4/SYN-B was approached when dopaminergic neurons were subjected to acute neurotoxin damage after prophylactic inoculation.2. The expression effects of cyclooxygenase-2 and inducible nitric oxide synthase were probed when dopaminergic neurons were subjected to acute neurotoxin damage after prophylactic inoculation of optimized DNA vaccine pVAX1-IL-4/SYN-B.MethodsLots of DNA vaccine and empty recombinant plasmid were extracted with endotoxin-free plasmid kit. DNA vaccine and empty recombinant plasmid were got.α-syn DNA vaccine(pVAX1-IL-4/SYN-B plasmid group, empty recombinant plasmid (pVAX1 plasmid group) and PBS(PBS group) were injected into anterior tibial muscle of C57BL mices'bilateral hind limbs at the same site and deep (50μl/times, 3times). Mice were injected with intraperitoneal injection four times (2-h intervals over 1day) with 20mg/kg MPTP two weeks later after the last prophylactic immunization. Ethological variation was observed in the process of immunization and model preparation, and then, success of model preparation or not was evaluated. After the last MPTP injection, the mice were killed at the preset time (1st day, 3rd day, 7th day, n=6). Ethological variation was observed in the process of immunization and model preparation, and then, success of model preparation or not was evaluated. After the last MPTP injection, the mice were killed at the preset time (1st day, 3rd day, 7th day, n=6). The change of dopaminergic neuron, COX-2, CD11b, and iNOS positive neurons in the substantia nigra was detected dynamically with DAB immunohistochemisty and HE staining. Cyclooxygenase-2 and inducible nitric oxide synthase mRNA expressions in different times were detected with RT-PCR.Results1. Behavioral observations: there were no marked changes during the process of immunifaction in the mice of pVAX1-IL-4/SYN-B group, pVAX1 group and PBS group. During the model preparation, the tremor, less movement, pilo-erection, rolling up, and increasing secretion were presented in the all mice in the pVAX1-IL-4/SYN-B group with the first MPTP intraperitoneal injection 10-30 minutes later. The symptoms were increased and irritability in the second injection. The symptoms were increased further in the third. The similar PD symptoms were appeared 10 minutes later, such as circle bulging tails, stiff, micro-tremble, slow movement, bad moter coordination of anterior-posterior limbs and so on. The same symptoms were observed in the pVAX1 group and PBS group. The ethology of mice in each group was in line with the standards of acute PD mouse model.2. Morphological examination results: compared to the pVAX1 group and PBS group, the relic neurons in the substantia nigra were more in the pVAX1-IL-4/SYN-B group, and the activation of microglia was lighter.3.SP immunohistochemistry results: compared to the pVAX1 group and PBS group, the MOD of TH positive neurons in the substantia nigra were more in the pVAX1-IL-4/SYN-B group, the variability was significant (p<0.05). While the number and MODof COX-2, CD11b, and iNOS positive neurons in the substantia nigra was less. There was statistical significance (p<0.05). While the significant devation was not resided in the pVAX1 plasmid group and PBS group(P>0.05).4. RT-PCR results presented that: cyclooxygenase-2 mRNA was expressed in every group at different times. It was weak in the first day, reached the peak time in the third day, and reduced in the seventh day. At the same point. Cyclooxygenase-2 mRNA expression in the pVAX1-IL-4/SYN-B plasmid group (1, 3, 7d was 0.28±0.07, 0.59±0.07, 0.45±0.05 respectively) was lower significantly than pVAX1 plasmid group(1, 3, 7d was 0.43±0.05, 0.74±0.15, 0.61±0.10 respectively) and PBS group(1, 3, 7d was 0.41±0.02, 0.73±0.13, 0.60±0.01 respectively). There was statistical significance in the difference (P<0.01).The iNOS mRNA was also expressed in all groups. In the first day, it was at the peak time, and then, reduced by turns. Compared to the pVAX1 plasmid group and PBS group, it was low in the pVAX1-IL-4/SYN-B group, there was significant difference between them (p<0.01). No statistical significance resided in the last groups (P>0.05).Conclusions1. The optimized hα-synuclein DNA vaccine pVAX1-IL-4/SYN-B had better immunogenicity, and against neurotoxin of MPTP to neurons. The prophylactic immunization effect in the mice protected dopaminergic neurons.2. The Optimized hα-synuclein DNA vaccine pVAX1-IL-4/SYN-B could be against the expression of inflammatory factors and showed a protection to dopaminergic neurons to be free of its impairment.
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