| Parkinson’s disease(PD)is a common degenerative disease of the central nervous system.The pathological feature of PD is the progressive loss of dopamine(DA)neurons in the substantia nigra pars compacta(SNpc)and the formation of Lewy bodies,leading to the reduction of DA content in the striatum(Str).Although the etiology and pathogenesis of PD have not yet been elucidated,many factors such as oxidative stress,inflammatory response,apoptosis,loss of mitochondrial function,and dysfunction of the ubiquitin-proteasome system are considered to be involved in the pathogenesis of PD.Alpha-synuclein is the main component of the pathological inclusion body(Lewy body)of the PD.Increasing evidence supports that the abnormal expression and aggregation of α-synuclein are critical contributors to the pathogenesis of PD.Therefore,clearance of abnormally aggregatedα-synuclein is of great significance for the treatment of PD.Ndfip1 is a transmembrane protein containing 221 amino acid residues and structurally containing two PPx Y motifs that bind to the WW domain of the Nedd4 ubiquitin ligase to mediate ubiquitin-dependent protein degradation.Studies have shown that Ndfip1 acts as an early sensory protein for the ubiquitination of certain proteins,and acts together with the E3 ligase to eliminate harmful misfolded proteins in nerve cells,thereby promoting the survival of nerve cells and thus exerting its function of anti-apoptosis.Studies have shown that Ndfip1 is found to be highly expressed in neurons after cerebral ischemia and traumatic brain injury,accompanied by an increase in the expression level of its binding protein Nedd4.This suggests that the high expression and interaction of these two proteins may contribute to promote the survival of damaged nerve cells.However,the neuroprotective effect and mechanisms of Ndfip1 in PD have not been fully elucidated.Therefore,in this study,real-time quantitative PCR,western blotting,adenovirus and immunofluorescence were performed on human neuroblastoma SH-SY5 Y cells to study the neuroprotective effects and possible mechanisms of Ndfip1 on the mitochondrial complex I inhibitor rotenone-induced neurotoxicity,and further explore the effect of Ndfip1 on α-synuclein protein levels in rotenone-induced PD cell model.The experimental results provide a new theoretical basisand therapeutic target for revealing the pathogenesis of PD and developing a new generation of therapeutic drugs.The results are as follows:1.The m RNA level of α-synuclein was measured after SH-SY5 Y cells were treated with300 nmol/L rotenone for 3hrs,6 hrs,9 hrs,12 hrs and 24 hrs.After rotenone treatment for 12 hrs,24 hrs,the results showed the m RNA level of synuclein in SH-SY5 Y cells increased by164.3%(P<0.01),219.5%(P<0.001)compared with the control group.2.After SH-SY5 Y cells were treated with 300 nmol/L rotenone for 3 hrs,6 hrs,9 hrs,12 hrs,24 hrs,the protein level of α-synuclein was detected.Results showed that the protein level ofα-synuclein began to increase at 6 hrs of rotenone treatment compared with the control group.The expression levels of α-synuclein were upregulated by 106.6%(P<0.05),188.9%(P<0.01),213.6%(P<0.01),and 311.6%(P<0.001)respectively at 6 hrs,9 hrs,12 hrs and 24 hrs of rotenone treatment.3.After SH-SY5 Y cells were treated with 300 nmol/L rotenone for 3 hrs,6 hrs,9 hrs,12 hrs,and 24 hrs,the m RNA level of Ndfip1 was detected.Results showed that the m RNA level of Ndfip1 was upregulated by 83.7%(P<0.05)at 6 hrs and 69.6%(P<0.05)at 9 hrs of rotenone treatment.4.After SH-SY5 Y cells were treated with 300 nmol/L rotenone for 3 hrs,6 hrs,9 hrs,12 hrs,and 24 hrs,the expression level of Ndfip1 protein was detected.Results showed that the protein levels of Ndfip1 were up-regulated by 34.2%(P<0.01),62.9%(P<0.001),and 27.4%(P<0.01)respectively after SH-SY5 Y cells were treated with rotenone for 3 hrs,6 hrs and 9hrs.The expression level of Ndfip1 protein decreased by 22.5%(P<0.05)at 24 hrs of rotenone treatment compared with the control group.5.After the SH-SY5 Y cells were infected with recombinant adenovirus for 24 hrs and incubated with 300 nmol/L rotenone for 24 hrs,study demonstrated that TH protein expression levels in the Rotenone group and the Ad.GFP/Rotenone group were down-regulated by 37.5%(P<0.001)and 44.2%(P<0.001)respectively compared with the control group.The expression level of TH protein in the Ad.Ndfip1/Rotenone group cells was up-regulated by 37.7%(P<0.01)compared with the Rotenone group.6.After the SH-SY5 Y cells were infected with recombinant adenovirus for 24 hrs and incubated with 300 nmol/L rotenone for 24 hrs,the expression level of caspase-3 protein in Rotenone group was up-regulated by 60.3%(P<0.05)compared with the control group.The expression of caspase-3 protein in Ad.GFP/Rotenone group cells was up-regulated by 75.4%(P<0.01)compared with the control group.The expression level of Ndfip1 protein in Ad.Ndfip1/Rotenone group was down-regulated by 29.0%(P<0.05)compared with Rotenone group.7.SH-SY5 Y cells wereinfected by Ad.Ndfip1 for 24 hrs and then incubated with 300nmol/L rotenone for 24 hrs.The expression of Ndfip1 protein in the Rotenone group and Ad.GFP/Rotenone group was decreased by 35.5%(P<0.05),49.5%(P<0.01),compared with the control group;the expression level of Ndfip1 protein in Ad.Ndfip1/Rotenone treated cells was up-regulated by 130.2%(P<0.001)and 194.5%(P<0.001)compared with the Rotenone group and Ad.GFP/Rotenone group.8.After the recombinant adenovirus Ad.Ndfip1 infected SH-SY5 Y cells for 24 hrs,cells were co-incubated with 300 nmol/L rotenone for further 24 hrs.The expression levels ofα-synuclein in the Rotenone group and the Ad.GFP/Rotenone group were up-regulated by86.8%(P<0.01)and 88.7%(P<0.01)respectively compared with the control group;The expression level of Ndfip1 protein in Ad.Ndfip1/Rotenone group was decreased by 31.9%(P<0.05)and 37.4%(P<0.05)compared with Rotenone group and Ad.GFP/Rotenone group;The fluorescence results showed that the expression of α-synuclein was significantly increased after rotenone treatment,which could be inhibited by Ad.Ndfip1 infection in SH-SY5 Y cells.The above experimental results show that the decrease of Ndfip1 in rotenone-induced PD cell model is accompanied by the increased expression of α-synuclein.Further studies showed that high expression of Ndfip1 could inhibit rotenone-induced morphological changes,up-regulation of α-synuclein and caspase-3 expression,and down-regulation of TH.In summary,it is suggested that Ndfip1 has a protective effect on rotenone-induced PD cell model.Ndfip1 might be involved in rotenone-induced up-regulation of α-synuclein expression and high levels of Ndfip1 were able to significantly inhibit rotenone-induced increase in α-synuclein levels.The experimental study provides further insights and new experimental basis into the neurotoxicity caused by the abnormal accumulation ofα-synuclein in PD and provide new experimental evidence for the treatment of PD. |
PDF Full Text Request