Mechanisms Underlying Neuroprotective Effects Of P7C3 In MPTP- And LPS-induced Mouse Models Of Parkinson’s Disease

Part1 Mechanisms underlying neuroprotective effects of P7C3 in MPTP-induced mouse model of Parkinson’s diseaseAim:To detect the neuroprotective efficacy of P7C3 in the MPTP-induced mouse model ofParkinson’s disease and the mechanisms underlying its neuroprotective efficacy.Methods:MES23.5 cells damaged by MPP~+were used as the model of Parkinson’s disease(PD)in vitro,while a dose of 30 mg/kg/d of MPTP for consecutive 5 days through intraperitoneal injection was carried out to establish the model of PD in vitro.In the cell model of PD,various concentrations of P7C3(1μM,5μM,10μM)were used to treat the cells before MPP~+administration,while the mice were delivered with P7C3 with a dose of30 mg/kg/d for a sequential 21-days intraperitoneal injection before MPTP treatment in the mouse model of PD.We detected the cell death of MES23.5 with an Annexin V-FITC Apoptosis Detection Kit.The mitochondrial membrane potential was measured using TMRM,healthy mitochondria can be marked by TMRM in red,while damaged mitochondria lost the red fluorescence rapidly.Mitochondrial separation experiment was applied to detect the levels cytochrome c in cytosol and mitochondria.Western blot was also performed to evaluate the activation of caspase-3,the expression of Bax,the activation of p53 and GSK3βin P7C3-or MPP~+-treated MES23.5 cells.Besides,GSK3βinhibitor was used to compared with the effects of P7C3.Finally,in the mouse model of PD,Wesren blot and immunohistochemistry were administrated to observe the activation of p53,GSK3βand TH-positive dopaminergic(DA)cells in the middle brain of different treated mice.Results:Annexin V-FITC detection assay showed that pretreatment of P7C3markedly attenuated the MPP~+-induced cytotoxicity to MES23.5 cells with a concentration dependent manner.Western blot indicated that P7C3 inhibited MPP~+-mediated activation of caspase-3,the up-regulation of Bax protein level,activation of p53 and GSK3β,the analogous effects was also found in the pretreatment of GSK3βinhibitor.In the MPTP-induced mouse model of PD,Western blot combining with immunohistochemistry demonstrated that P7C3 suppressed the GSK3βand p53 activation along with the TH-positive DA cells loss caused byMPTP.Conclusion:P7C3 inhibits GSK3βactivation to protect DAneurons against neurotoxin-induced cell death in vitro and in vivo.Part2 Mechanisms underlying neuroprotective effects of P7C3 in LPS-mediated mouse model of Parkinson’s diseaseAim: To exlpore neuroprotective efficacy of P7C3 in the LPS-mediated mouse model of Parkinson’s disease and the potential mechanisms underlying its effects.Methods: Vitro LPS-induced model of Parkinson’s disease was established through the way of culturing the MES23.5 with the conditioned medium from LPS-treated BV2 microglia,while using bilateral stereotaxic injection into the mice midbrain nigra area with a dose of 1 mg/m L LPS served as the LPS-induced mouse model of Parkinson’s disease.In the cell model of PD,various concentrations of P7C3(0.1 μM,1 μM,10 μM)were used to treat the cells before LPS administration,while the mice were conducted with P7C3 with a dose of 30 mg/kg/d for a sequential 21-days intraperitoneal injection before LPS treatment in the mouse model of PD.Western blot and real-time quantitative PCR(q RT-PCR)were applied to detect the protein and m RNA levels of i NOS,COX-2,IL-6 and TNF-α in LPS-treated BV2 with or without the pretreatment of P7C3.In addition,Western blot was also used to assess the activation inflammation related proteins and kinases invoved in the regulation of inflammatory response,including MAPKs kinases mainly in charge of AP-1 pathway and IKK kinases and IκBα responsible for NF-κB pathway.Besides,we performedsubcellular fractionation assay and immunofluorescence to detect the nuclear distribution of NF-κB subunit p65.Also,Luciferase reporter gene assay was carried out to test the transcriptional activity of NF-κB in different treated BV2.Finally,in the LPS-mediated mouse model of Parkinson’s disease,we carried outthe immunohistochemistry to observe the expression IBA1,GFAP and TH expression to detect the activation of microglia,astrocytes and the loss of TH-postive DA cells in the mice midbrain nigra area.Results: Western blot and qRT-PCRshowed that PC73 remarkably inhibited the LPS-induced inflammatory factors.Moreover,Western blot,subcellular fractionation assay and Luciferase reporter gene assay indicated that P7C3 suppressed the LPS-mediated activation of NF-κB,without influencing the AP-1 activation caused by LPS administration.In LPS-mediated mouse model of Parkinson’s disease,immunohistochemistry showed that pretreatment of P7C3 significantly restrained microglia and astrocytes activation combining with the remitting the TH-positive loss in mice midbrain nigra area.Conclusion: P7C3 inhibits NF-KB activation to protect the dopaminergic neurons against LPS-induced inflammatory damage in vitro and in vivo.