Study On The Neuroprotection And Its Mechanism Of Basic Fibroblast Growth Factor In The Model Of Parkinson’s Disease

Objective: Neuroprotection and mechanism of b FGF was verified in 6-OHDA induced PD model, and then the pharmacokinetics and safety of b FGF was investigated, which will provide a theoretical basis and rational mode of administration for clinical development and application of Parkinson’s disease. Methods:(1) SD rats were injected with 6-OHDA by use of the stereotaxic apparatus, we demonstrated that liposomes carriers-mediated intranasal delivery of basic fibroblast growth factor(b FGF) provided neurprotection in the 6-Hydroxydopamine(6-OHDA) model for PD in vivo.(2) Rotation, open-field and cat-walk test were used to investigate improvement of b FGF in movement disorder rats.(3) DA,DOPAC and HVA in the rat striatum were detected by HPLC-ECD.(4) Immunohistochemisty and western blotting were used to evaluate the expression of TH in rat striatum and SN.(5) Nissl’s body was detected in the rat lesion striatum by Nissl’s staining.(6) Iconography parameter were obtained from the magnetic resonance imaging(7) Western blotting was used to evaluate the level of PD-associated protein in rat lesion SN.(8) b FGF in the rat serum was tested by ELISA.(9) b FGF in the rat olfactory bulb, cortex, striatum and SN was detected by ELISA and western blotting.(10) Hematoxylin-eosin staining was used to observe the pathology change in rat nasal mucosa, olfactory bulb, striatum, SN, liver and lung.(11) Cell viability was detected by MTT.(12) Cell morphous was observed by scanning electron microscope.(13) Western blotting was used to evaluate the level of PD-associated protein inSH-SY5 Y cells. Results:(1) b FGF in the olfactory bulb, cortex, striatum and SN was significantly increased after intranasal administration of Lip-b FGF, b FGF in the olfactory bulb and striatum was also marked increased after intranasal administration of b FGF(p < 0.05). Lip-b FGF treatment group demonstrated a higher efficiency of b FGF delivery, evidenced in significantly elevated levels of b FGF in prefrontal cortex and striatum compared with b FGF(p < 0.05). Result for ELISA was almost correspondent with western blotting.(2) The rotation number in 6-OHDA-induced rats achieved 6r/min, which was considered to be a PD model.(3) Movement disorder induced by 6-OHDA injection, involved raising number of rotation, hypokinesia, gait disorder and so on(p < 0.05). Lip-b FGF significantly relieved the behavioral dysfunction of PD model rats, including reduction of apomorphine-induced rotation, improvement in locomotor functions and gait imbalance in open field and cat walk(p < 0.05), and the Lip-b FGF leads to better rescuing effect compare to b FGF. No differences were found when the rats were treated with liposomes alone(4) The terminal and neuron loss induced by 6-OHDA affected striatum DA and its metabolites levels(91.2%,84.3%,92.3%)(p < 0.05). Lip-b FGF notably increased dopamine(DA) and its metabolites levels in the lesioned striatum, Lip-b FGF increased DA level in a dose-dependently manner. DA and DOPAC concentrations were also elevated in response to intranasal b FGF solution treatment, but not HVA.(5) 6-OHDA intoxication in rats resulted in losses of TH-positive terminals in striatum(18% remained) and TH-positive neurons in SN(22% remained)(p < 0.05). Lip-b FGF(150 and 300 μg/kg) significantly ameliorated 6-OHDA toxicity, evidenced by significantly increased dopaminergic striatum terminal number(15% for 150 μg/kg, 23% for 300 μg/kg)(p < 0.05) and dopaminergic neuron number in SN(20% for 150μg/kg, 25% for 300 μg/kg)(p < 0.05). There were no significant difference in 600 μg/kg Lip-b FGF and 300 μg/kg b FGF compared to model. Therefore, intranasal Lip-b FGF administration demonstrated a better efficacy compared with b FGF solution.(6) Nissl’s staining demonstrated 6-OHDA reduced 80% nissl’s body(p < 0.05), Lip-b FGF(150 and 300 μg/kg), but not b FGF, marked increased the 6-OHDA-induced loss of nissl’s body in the rat striatum, and Lip-b FGF at dose of 300 μg/kg demonstrated a better efficacy compared with the other treatment groups.(7) Magnetic resonance imaging demonstrated that 6-OHDA reduced MD, T2 and CBF(p < 0.05), increased FA and R2(p < 0.05), but was useless for ADC, MK, RK and KA. Lip-b FGF at dose of 300 μg/kg significantly increased T2 and reduced R2 in the lesion striatum compare to the dose of 150 μg/kg(p < 0.05); but for the CBF, Lip-b FGF(150 μg/kg) demonstrated a marked up-regulation in the ROI than 600 μg/kg.(8) Lip-b FGF significantly increased the level of doublecortin(DCX) and β-tubulin protein in the rat lesion SN(p < 0.05), but was useless for PINK1, DJ-1, Parkin and synapsin protein. Phosphorylation of α-syn at Ser129 was significantly elevated after 6-OHDA brain injection(p < 0.05), which was down-regulated by injection of Lip-b FGF in SN. The expression of CK-1 was increased by 6-OHDA(p < 0.05) and inhibited by Lip-b FGF administration. Tau protein phosphorylation at Ser 396 in rat SN improved by 6-OHDA(p < 0.05), which was released by Lip-b FGF at dose of 150, 300, 600μg/kg and b FGF at 300μg/kg. 6-OHDA-induced significantly reduced the phosphorylation level of GSK-3(Ser21/9), AKT(Ser473) and PI3K(Tyr458) in rat SN, Lip-b FGF improved the phosphorylation level of GSK-3, AKT and PI3K(p < 0.05).(9) The concentration-time fit two-compartment open model after intranasal administration of Lip-b FGF(150, 300 and 600 μg/kg) and b FGF(300 μg/kg) in rat, and the weight was 1. Half life time were respectively 185.7±28.6,172.9±72.2,140.8±22.3 and 26.4±2.5 min for distribution phase, 174.5±29.2 min,207.4±73.6min,260.6±41.6 min and 60.7±16.9 min for elimination phase, and the AUC were respectively 672.6±86.6,1324.9±175.3,2191.8±256.3 and 700.1±122.3μg/L*min.(10) No mouse died in the acute toxicity experiment, and all of the mouse was in good condition. Weight of male mouse was sustainable growth in the entire procedure(2 weeks), correspondence with control group. Nevertheless, weight of female mouse was almost stable in the first week, their weight back to normal after the second week.(11) There was no significant pathology difference between control and b FGF treatment groups form the HE staining.(12) Cell viability determined by MTT assay in the present study showed that 50 and 100μM 6-OHDA had no effect on cells, 150, 200, 250, 300 μM 6-OHDA reduced SH-SY5 Y cell viability respectively about 16%, 40%, 52% and 61%. b FGF at concentration of 100, 500 and 1000 ng/ml significantly increased the amount of cell viability.(13) Morphological examination was confirmed by analysis at an ultrastructural level, most cells were injured after treatment with 200μM 6-OHDA, including severely deformed(not fusiform), even broken, synaptic became shorter, and cytoplasm effluence, accumulation on the cell surface. The impaired cells were significantly ameliorated after treatment with different concentration of b FGF, cell morphous and integrity were also maintained by b FGF.(14) 6-OHDA significantly reduced the level of synapsin and tubulin, increased the level of CK-1 and PLK-2, as well as the phosphorylation tau(Ser 396) and α-syn(Ser 129) in SH-SY5 Y cells. b FGF up-regulated the expression of synapsin and tubulin in SH-SY5 Y cells, and down-regulated CK-1. b FGF protect the SH-SY5 Y cells against 6-OHDA-induced tau and α-syn hyperphosphorylation, meanwhile, up-regulation the phosphorylation level of GSK-3(Ser21/9), AKT(Ser473) and PI3K(Tyr458) in SH-SY5 Y cells. Conclusion:(1) Neuroprotection for b FGF was observed in 6-OHDA induced PD model in vivoand in vitro.(2) b FGF reduced 6-OHDA induced neurotoxicity by decreasing phosphorylation of α-syn at Ser 129 and phosphorrylation tau at Ser 396, as well, b FGF promoted the nerve regeneration.(3) b FGF regulated phosphorylation of α-syn at Ser 129 by decreasing CK-1, and b FGF regulated phosphorylation of tau at Ser 396 via activating PI3K/AKT/GSK-3 pathway.(4) b FGF, loaded by liposomes, could be delivered in to striatum and cerebral cortex. Lip-b FGF enhanced penetration of drug, prolong the half-time and AUC, and provided better absorption into the brain, compare to b FGF alone.(5) TMD of b FGF and Lip-b FGF in the mouse exceed 45mg/kg, which were safe for mouse. There was no pathology alteration in nasal mucosa, olfactory bulb, striatum, SN, liver and lung after intranasal administration of b FGF and Lip-b FGF for 30 days.