The Effect And Mechanism Of Calcitriol In The Model Of Parkinson's Disease

Part 1 The level of vitamin D and PARP1 in patients of Parkinson's disease and MPP+-induced SH-SY5 Y cellsObjective: To analyze the expression levels of vitamin D and PARP1 in the plasma of PD patients and healthy controls,as well as the expression of vitamin D receptors and PARP1 in the MPP+ cell model,and then verify whether MPP+ induces parthanatos.Methods: The plasma of patients with Parkinson's disease and healthy controls were collected,the level of 25-hydroxyvitamin D in plasma was detected by ELISA analysis and the level of PARP1 and cleaved-PARP1 was detected by western blotting.MPP+ was used to treat SH-SY5 Y cells,CCK-8 assay was conducted to verify the toxicity of MPP+,the expression levels of VDR,PAR,PARP1,and AIF were detected by western blotting.SH-SY5 Y cells were pretreated with different concentrations of Z-VAD-FMK or Nec-1 before MPP+ treatment,then cell viability was assessed by CCK8 assay.Results: The level of 25-hydroxyvitamin D in the plasma of patients with Parkinson's disease was significantly lower than that of the healthy control group.Western blotting showed the expression of PARP1 and cleaved-PARP1(89k D,40 k D,25 k D)in PD patients was increased.As the concentration of MPP+ treatment gradually increased,the cell viability of SH-SY5 Y gradually decreased.Western blotting results showed that the expression level of VDR gradually decreased,PARP1 and PAR were activated,and the nuclear localization of AIF increased,with the most obvious increasing in 100?M of MPP+ treatment.Z-VAD-FMK and Nec-1 pretreatment could not reverse the cell activity reduction caused by MPP+.Conclusions: The plasma vitamin D level of PD patients was reduced and PARP1 was activated compared to healthy controls.In MPP+-induced cell model,the VDR expression was reduced and PARP1 signaling pathway,as well as parthanatos was activated.Part 2 Calcitriol protected MPP+-induced SH-SY5 Y cells from parthanatosObjective: To observe the effect of calcitriol in reducing parthanatos induced by MPP+ in SH-SY5 Y cells.Methods: CCK-8 assay was used to determine the concentration of calcitriol treated in MPP+ cell model.Cells were divided into four groups,the Control group,Calcitriol group,MPP+ group and Calcitriol+ MPP+ group.The intracellular ROS and Ca2+ levels in each group were detected by flow cytometry,and the level of NAD+ in each group was detected by NAD+ kit.The expression of PAR,PARP1,p-H2 A.X and AIF in each group were detected by western blotting.In addition,TUNEL staining and immunofluorescence staining of PAR and AIF were performed.Results: 25,50,75 n M calcitriol pretreatment could partially restore cell viability reduction caused by MPP+,and 50 n M of calcitriol had the most obvious effect and this concentration was also selected as the cell intervention concentration of calcitriol.Flow cytometry showed that the levels of ROS and Ca2+ in MPP+ group were significantly increased,and calcitriol pretreatment alleviated ROS production and Ca2+ level.The level of NAD+ in MPP+ group was significantly decreased,reflecting the excessive consumption of NAD+,calcitriol pretreatment reduced NAD+ consumption.Western blotting showed that the expression of PAR,PARP1,p-H2 A.X in the MPP+ group was increased,and the expression of these proteins in Calcitriol+ MPP+ group were lower than that in MPP+ group.Calcitriol can upregulate the expression of VDR.TUNEL staining showed an increase in DNA fragmentation in MPP+ group,and the number of TUNEL stained positive cells in Calcitriol+ MPP+ group was less than that in MPP+ group.Immunofluorescence staining verified the results of PAR and AIF expression in western blotting,and also showed that the nuclear localization of AIF increased after MPP+ treatment.The nuclear localization of AIF in the Calcitriol+ MPP+ group was significantly reduced.Conclusions: Calcitriol pretreatment improved the cell viability,reduced the increased levels of ROS and Ca2+,and alleviated parthanatos caused by MPP+.Part 3 Calcitriol alleviated parthanatos in the MPTP-subacute modelObjective: To observe the effect of calcitriol in reducing parthanatos in MPTP-subacute model.Methods: C57/BL6 mice were randomly divided into 4 groups as follows: MPTP group,injected with MPTP(30 mg/kg/d,i.p.)for seven days;Calcitriol + MPTP group,injected with calcitriol(2.5 ?g/kg/d,i.p.)commencing seven days prior to MPTP administration and lasting for 21 days;Calcitriol group,administered calcitriol(2.5 ?g/kg/d,i.p.)for 21 days;and Control group,administered solvent control(n=10/group).Behavioral rotarod and pole tests were carried out to detect the movement function of mice.Proteins in the substantia nigra and striatum of the mouse were extracted to detect the expression of TH by western blotting,and the expression of TH was further verified by immunohistochemistry and immunofluorescence staining performed on the section of the substantia nigra and striatum.Western blotting was performed on proteins of substantia nigra and striatum to detect the levels of PAR,PARP1,cleaved-PARP1,p-H2 A.X,and VDR.TUNEL staining was conducted on sections of substantia nigra and striatum.Immunohistochemical staining of cleaved-PARP1 and immunofluorescence staining of VDR,PAR and AIF were conducted on sections of substantia nigra.Results: The MPTP group had a decrease in latency time to falling from the rotarod and the total time taken to climb down the pole was significantly extended compared to the Control group,and calcitriol treatment reversed this effect.Western blotting showed that the level of TH protein in the MPTP group was significantly decreased compared to that in the Control group and Calcitriol+ MPTP group.The immunofluorescence and immunohistochemical staining of TH demonstrated consistent results with western blotting results in the substantia nigra and striatum.Western blotting of the substantia nigra and striatal proteins showed that MPTP group had a significantly lower expression of VDR and increasing expression of PAR,PARP1,cleaved-PARP1 and p-H2 A.X compared to Calcitriol+ MPTP group and Control group.Compared with the Calcitriol+ MPTP group and the Control group,the number of TUNEL positive cells in the substantia nigra and striatum of the MPTP group increased significantly.Compared with the Calcitriol+ MPTP group and the Control group,the cleaved-PARP1 staining and PAR staining results of MPTP group in substantia nigra were consistent with western blotting results.AIF staining of MPTP group mice showed a significant increase in nuclear localization,and calcitriol reduced the nuclear accumulation of AIF.Conclusions: Calcitriol alleviated behavioral impair,dopaminergic neuron damage and parthanatos in a MPTP model of PD.Part 4 The mechanism of calcitriol inhibits PARP1Objective: To study the mechanism of calcitriol inhibits PARP1.Methods: Directly upregulated the expression of VDR in SH-SY5 Y cells to verify whether the protective effect of calcitriol was achieved by VDR.VDR was upregulated and downregulated at gene level,and changes in PARP1 expression were observed.The expression of VDR was knocked down by si RNA-VDR transfected,and the control group was transfected with negative control si RNA.Real-time PCR and western blotting were used to detect the m RNA and protein expression levels of VDR and PARP1.VDR was overexpressed by transfection of VDR plasmid,and the control group was transfected with negative control plasmid.Real-time PCR and western blotting were used to detect the m RNA and protein expression levels of VDR and PARP1.Co-immunoprecipitation was used to confirm whether VDR and PARP1 had directly combination.Immunofluorescence staining of VDR and PARP1 was conducted in sections of substantia nigra to observe whether VDR and PARP1 were co-localized.Results: Directly upregulated VDR at gene level could alleviate the activation of PARP1 caused by MPP+,showing that the effect of calcitriol on PARP1 was achieved through VDR.The m RNA level of VDR in the si RNA-VDR group was significantly reduced which verified the knockdown efficiency of si RNA.Meanwhile,VDR knockdown increased the mRNA expression of PARP1,western blotting also showed consistent results.Plasmid that overexpressed VDR downregulated the m RNA expression of PARP1.Western blotting showed that VDR level of the VDR overexpression plasmid group was significantly higher than the control group,and the expression of PARP1 decreased.Co-immunoprecipitation results showed that VDR and PARP1 had a direct binding,and immunofluorescence staining showed that VDR and PARP1 were co-localized.Conclusions: The protective effect of calcitriol was achieved by VDR directly binding to PARP1 and regulating the expression of PARP1.