Effect Of PGC-1? Overexpression On The Mitochondria Of PD Cell Model And The Dopaminergic Neurons Of PD Mouse Model

Parkinson's disease(PD)is a neurodegenerative disease characterized by degrading death of dopamine(DA)neurons,which the average onset age is 60 years.Clinical manifestations are muscle rigidity,resting tremor,gait and posture instability and other movement disorders,as well as insomnia,depression and other non-motor symptoms,the health of the elderly are seriously affected by the symptoms.Factors that cause the disease include inheritance,environment,drugs,response caused by these factors such as,mitochondrial dysfunction,oxidative stress,protein aggregation,inflammatory are the main mechanism.Mitochondrial dysfunction may be one of the important mechanisms.Mitochondria are the energy supply centers of cells and are also the main sites generating oxygen free radicals(ROS).These factors may cause the mitochondrial electron transport chain generate ROS and lead to dysfunction.ROS can damage the DA neurons by oxidative stress and make the neurons degeneration.Some studies showed that autophagy-lysosomal system was associated with the pathogenesis of PD,especially closely related to mitochondrial autophagy.In recent years,the peroxisome proliferator activated receptor ? coactivator-1?(PGC-1?)was found highly expressed in the muscle after strenuous exercising and is closely related to mitochondrial biogenesis,autophagy/mitochondrial autophagy.So it may play an important role in the remodeling of skeletal muscle cells.The suggested that PGC-1? may have a protective effect on cell damage by regulating the autophagy and biogenesis of mitochondria.So,if improve the expression of PGC-1?,can the MPP+ damaged DA neurons be protected? Does it affect autophagy/mitochondrial autophagy? Is there a protective effect on the damage of DA neurons in vivo? Does it affect the inflammatory responses? These are worthy of doing further studies.The clarification of these problems will help to determine whether PGC-1? can provide the necessary experimental evidence as a drug target for the prevention and treatment of PD,which has certain theoretical and practical significance.In this study,SH-SY5 Y cells and C57BL/6 mice were used to study the cell PD model of SH-SY5 Y induced by MPP+,and the animal PD model C57BL/6 mice was injected by intraperitoneal injection of MPTP.First,MTS assay,Hoechst 33342 staining and FJC staining were used to explore the best damage condition of MPP+ on SH-SY5 Y cells.The experiment was divided into CON group,MPP+(MPTP)group,NCOE+MPP+(MPTP)group and OE+MPP+(MPTP)group.SH-SY5 Y cells and mice were transfected with PGC-1? overexpressing lentivirus to overexpression.The survival rate of SH-SY5 Y cells was detected by MTT assay.Real-time PCR was used to investigate the changes of mitochondrial-related gene and autophagy-related gene mRNA level after PGC-l? overexpression.Immunofluorescence staining was used to observe the expression changes of dopaminergic neurons in astrocytes and microglia,so as to find the answers to the above questions.The experimental results are as follows:1.When the concentration of MPP+ was 1 mmol/L and treat the cell for 24 h,the cell survival rate was close to 50%.Therefore,MPP+ concentration was 1 mmol/L for 24 h,which was the optimal condition for MPP+ injury.2.The apoptosis of SH-SY5 Y cells was detected by FJC staining and Hoechst 33342 staining.The apoptotic rate of MPP+ increased with the increase of MPP+ incubation time,which indicated that MPP+ injury was positively correlated with incubation time.From the above two experimental results to consider the damage time of MPP+ was 24 h and the concentration was 1 mmol/L.3.PCR was used to obtain the target gene and then ligated with the linear vector.The positive transformants were identified by PCR.After sequencing,the sequence of the recombinant plasmid was consistent with the target gene.The expression vector was obtained.4.The titer of the virus was determined by ELISA.NCOE is 1×109 TU/ml and OE is 2×108 TU/ml.5.The cell viability after overexpression of PGC-1? was determined by MTT assay.The cell viability was significantly increased after overexpression of PGC-1? gene.NCOE group had no significant difference compared with the normal group.The survival rate of OE group was significantly higher than that of MPP+ group and NCOE group(P<0.01),and there was significant difference compared with CON group(P<0.05).6.Real-time PCR was used to detect the expression of mitochondrial related genes(NRF1,Drp1,Mfn2)and autophagy-related genes(LC3B,p62).The expression of NRF1 mRNA was also significantly increased after overexpression of PGC-1?,and there was a significant difference between MPP+ and NCOE groups(P<0.01).The mRNA expression of mitochondrial fission and fusion genes of the Drp1 and Mfn2 were also significantly increased.Drp1 OE group had significant difference compared with MPP+ group(P<0.01)and had no significant difference between compared with NCOE group.The expression of Mfn2 mRNA was significantly higher than that of MPP+ and NCOE groups(P<0.01).The expression of LC3 B mRNA was significantly decreased in MPP+ group and NCOE group(P <0.01).The expression of p62 mRNA was significantly higher than that of MPP+ and NCOE groups(P<0.01).7.The results showed that the number of dopaminergic neurons in the substantia nigra of mice was not significantly improved,and the expression of TH-positive neurons has no statistical difference between groups.The expression of PGC-1? in OE group was significantly higher than that in MPTP group and NCOE group,and there was a significant difference(P<0.01).Especially in the substantia nigra of the substantia nigra,the number of PGC-1? positive cells increased significantly.Although the expression of PGC-1? was increased in the number of cells,the immunoprecipitation pictures showed that PGC-1? was more abnormal and the cells became irregular.In addition,immunofluorescence statistics also show that microglia and astrocytes appear over-proliferated.We can draw the conclusions through analyzing the above results:1.This study was used to 1 mmol/L MPP+ at 24 h for SH-SY5 Y cells.The MPP+ induced PD in vitro model was successfully replicated.2.In the PD cell model,overexpression of PGC-l? can resist MPP+-induced cytotoxicity,inhibit the occurrence of autophagy.It also can increase the expression of NRF1 after MPP+ injury,increase the expression of mitochondrial fusion gene Mfn2,improve the mitochondrial function,and maintain the balance of mitochondrial fusion and fission,thereby reducing cell apoptosis,and thus produce a protective effect on cells.3.In the PD mice model,overexpression of PGC-1? triggers the proliferation of astrocytes and microglia in substantia nigra and lead to the secondary damage to animal body.Thus,overexpression of PGC-1? may be a damaging effect on dopaminergic neurons.The results of this study will provide a basis for the further study of PGC-1? as a drug target for the prevention and treatment of PD.