Effects Of Sphingosine Signaling In The MPTP-induced Parkinson’s Disease And The Underlying Mechanism

Backgroud:Parkinson’s disease（PD）is the second largest neurodegenerative disease associated with aging in the world,characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta（SNpc）.To date,there is no ideal treatment for PD.Most of the patients use Dopamine Replacement Therapy to relieve the motion symptoms of PD in clinical practice,but it is unable to delay the pathological process of PD,and continuous application may even cause a series of side effects.So far,the molecular mechanisms underlying the degeneration of dopaminergic neurons in the pathological process of PD have not been fully elucidated,which may include neuronal apoptosis,mitochondrial dysfunction,oxidative stress,and decreased ability to scavenge free radicals,inflammation the central nervous system（Central Neural System,CNS）and other mechanisms.Among these,CNS inflammation plays an important role in the pathogenesis of PD,and inhibition of neuroinflammation protect dopamine neurons.Studies have shown that in the early phase of PD,neuroinflammation reaction in CNS is induced,and a variety of nerve cells are involved in the release of pro-inflammatory factors,such as interleukin 1β（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）.Sphingosine,is an octadecanoamino alcohol containing an unsaturated hydrocarbon chain,and is also one of the components of the cell membrane.Sphingosine is phosphorylated by Sphk1（Sphingosine Kinase 1）and Sphk2 kinases to produce sphingosine-1-phosphate（S1P）.S1P binds to S1P receptors on the cell membrane and exerts different effects,including cell migration,proliferation,survival,and differentiation.FTY720,a structural analog of S1P,is a novel immunosuppressive agent.Phosphorylated FTY720 binds to the S1P1 receptor and exerts its effect.It has been reported that FTY720 has therapeutic effects on animal models of CNS inflammation.However,little is known about the mechanisms of action of sphingosine signaling in PD.PartⅠThe Mechanism of Sphingosine Kinase-1 in MPTP-induced Parkinson’s DiseaseObjective:This part of study is aimed to explore the roles and mechanisms of the sphingosine kinase-1 in MPTP-induced Parkinson’s disease model mice.Methods:8-week-old male C57BL/6J mice and Sphk1^-/-mice were divided into control group and model group.Saline was subcutaneously injected into the control group,and MPTP（20 mg/kg）was subcutaneously injected into the model group for5 consecutive days.Samples were collected 7 days after the last dose of MPTP.Three groups of mice were evaluated for behavior by open field test and rotarod test,the number of apoptptic dopamine neurons were observed by immunohistochemical TH staining,and the monoamine neurotransmitters of dopamine and its metabolites were detected by HPLC.Immunofluorescence was used to observe the number of Iba-1 positive cells.ELISA was used to detect the expression changes of mid-brain TNF-α,IL-6 and IL-1βin four groups of mice.Mice microglial cell line BV2 wwas treated with PF-543 for 30 minutes,and then stimulated with MPP⁺for 12 hours.ELISA was used to detect expression changes of TNF-α,IL-6 and IL-1βin midbrain of four groups of mice.BV2 was treated with PF-543 or equivalent saline for 30minutes and then stimulated with 500μM MPP+for 3 hours or 6 hours.Signal molecules NF-κB,STAT3,and TRAF2 were then detected by Western Blot.BV2was pretreated with PF-543 or equivalent saline for 30 minutes,followed by MPP⁺and S1P for 6 hours.Signal molecules such as NF-κB,p-AKT,and TRAF2 were detected by Western Blot.After replenishing S1P,levels of IL-1β,TNF-α,and IL-6were detected by ELISA.Western Blot was performed to detect the expression of ERK,AKT,NF-κB and STAT3 signal molecules.Results:In vivo experiments,lack of Sphk1 improved the behavioral impairment of MPTP-induced Parkinson’s disease model mice,reduced the number of apoptotic dopamine neurons,increased monoamine neurotransmitter content,inhibited microglia activation and levels of the inflammatory factors TNF-α,IL-6 and IL-1β.In vitro,inhibition of Sphk1 significantly reduced MPP⁺-induced inflammatory cytokines TNF-α,IL-6,and IL-1βsecretion,decreased NF-κB,STAT3phosphorylation,and TRAF2 expression.After supplementation,S1P causes the increase of inflammatory cytokines TNF-α,IL-6 and IL-1β,and increases the phosphorylation of signal molecules such as NF-κB and AKT.Conclusion:Sphk1 mainly induces downstream signaling pathway by generating S1P.In the absence of Sphk1,it can reduce the expression of TRAF2,thus reducing the phosphorylation level of NF-κB,STAT3 and other signal molecules,reducing the release of inflammatory factors.Inhibiting the activation of microglia reduces the inflammatory levels of the central nervous system,reduces neurotoxicity and exerts a neuroprotective effect on dopaminergic neurons,ultimately improving the behavioral impairment in MPTP-induced Parkinson’s disease model mice.PartⅡThe Mechanism of FTY720 inhibits microglia activation and has neuroprotective effect on Parkinson’s diseaseObjective:To investigate whether S1P analog FTY720 can inhibit the activation of microglia and then influence the MPTP-induced Parkinson’s disease and to provide a clinical method for the treatment of Parkinson’s disease.Methods:8-week-old male C57BL/6J mice were randomly divided into control group,FTY720 group,MPTP model group and treatment group.The mice in the model group were injected subcutaneously with MPTP（20 mg/kg）.The treatment group received intragastric administration of FTY720（2 mg/kg/day）30 min before subcutaneous injection of MPTP（20 mg/kg）.The control group was subcutaneously injected with FTY720 and the same dose of saline was intragastrically administered.The above experiment was continued for 5 days,and the anesthetized mice were sampled on day 7 after the last administration.The behaviors of the four groups of mice were evaluated by open-field test and rotarod test.The number of dopamine neurons in the substantia nigra was observed by immunohistochemical TH staining.Dopamine and its metabolite monoamine transmitters in the striatum were detected by HPLC.Immunofluorescence staining of Iba-1 was used to label microglia in the substantia nigra of mice,and activation of microglia was observed.The midbrain was extracted and the expression of TNF-α,IL-6 and IL-1βin the brain was detected by ELISA.Mouse microglia cell line BV2 was cultured in vitro and pretreated with0.5μM FTY720 or equal physiological saline for 30 minutes,followed by 500μM MPP⁺for 12 hours.The inflammatory cytokines TNF-α,IL-6 and IL-1βwere detected by ELISA.The BV2 was stimulated with 500μM MPP⁺for 12 hours with or without FTY720,and then the supernatant was extracted as a conditioned medium（CM）to treat the SH-SY5Y cell line for 12 hours,after that the apoptotic cells were observed by Hoechst fluorescence staining.At the same time,Annexin-V and PI double staining and flow cytometry were used to detect the apoptosis rate.The phosphorylation levels of PI3K/AKT/GSK-3βand STAT3 signaling molecules were detected by Western Blot.The Mit SOX probe was used to detect the ROS content in different groups of BV2.Western blot was used to detect p65/NLRP3/Caspase-1protein levels.Results:FTY720 significantly alleviated the behavioral impairment in mice with Parkinson’s disease caused by MPTP.The course and speed of the mice in the treatment group,and the time and speed of stay on the rods were significantly increased.The mice in the treatment group were compared with the model group.Apoptosis of dopamine neurons in the substantia nigra were also decreased.Results of HPLC showed that FTY720 partially reversed the decrease of dopamine and its metabolites caused after by MPTP treatment.At the same time,the activation of Iba-1 labeled microglia was significantly reduced.The levels of TNF-α,IL-6,and IL-1βalso decreased.In vitro,FTY720 inhibited the increase of inflammatory factor levels in the microglial cell line after MPP⁺stimulation.The media of BV2 was used as the conditioned medium to act on the SH-SY5Y cell line,and Hoschet fluorescence staining showed that the conditioned medium pretreated with FTY720reduced the number of apoptosis of SH-SY5Y cells,and the rate of apoptosis detected by flow cytometry after Annexin-V and PI double staining was also reduced.At the same time,phosphorylation levels of PI3K/AKT/GSK-3βand STAT3signaling molecules were also decreased.Mitochondrial（ROS）levels in cells treated with FTY720 were significantly lower than those in the model group.FTY720 also reduced the phosphorylation of p65 and the activation of NLRP3 inflammasome,thus reducing the production of Caspase-1.Conclusion:FTY720 can improve microglial mitochondrial dysfunction reduce and p65 phosphorylation induced by MPTP（MPP⁺）,and subsequently reduce the expression of inflammatory cytokines in brain by inhibiting the activation of NLRP3inflammasome in microglia,indirectly reducing apoptosis of neurons,and increase in the content of monoamine neurotransmitters of dopamine and its metabolites,thereby improving behavioral disorders in Parkinson’s disease mice.