The Molecular Mechanism Of Insulin-like Growth Factor Binding Protein 2(IGFBP2) In The Pathogenesis Of Parkinson’s Disease

Objective: The neuropathologic features of Parkinson’s disease(PD)are abnormal loss of dopaminergic neurons in the substantia nigra,alphasynaptic nucleoprotein aggregation,and tau phosphorylation.At present,the diagnosis mainly depends on the typical clinical symptoms.The existing treatment are mainly drugs and deep brain stimulation,which cannot stop the pathological process of progressive decrease of dopaminergic neurons.The pathogenesis of PD is very complex and still not completely clear.At present,it is believed that the main pathogenesis is the decrease and even apoptosis of dopaminergic neurons caused by age,mitochondrial dysfunction,oxidative stress,excitatory toxin,genetic factors,neuroinflammation,endoplasmic reticulum stress and other pathological factors.Mitochondrial dysfunction and oxidative stress play an important role in the pathological process of PD development.Therefore,effective relief of mitochondrial dysfunction and oxidative stress may effectively prevent this neurodegenerative process,and may effectively prevent the development or even occurrence of the disease.Insulin-like growth factor binding protein 2(IGFBP2)is a member of the family of IGFBPs,can activate multiple signal pathways,play a role in cell proliferation and migration,may participate in the regulation of advanced cognitive function,and play a role in the progression and pathogenesis of AD.In astrocytes with overexpression of α-synuclein,the expression of IGFBP2 changes significantly,but its role in Parkinson’s disease and its molecular regulation mechanism are not clear.Lnc RNAs and mi RNAs belong to noncoding RNA,which is a kind of RNA that can not encode any protein.Noncoding RNA can affect protein synthesis by regulating protein transcription and translation,plays a role in a variety of pathophysiological processes,is closely related to the occurrence and development of neurodegenerative diseases,and has therapeutic potential in the treatment of cancer,nervous system diseases and other major diseases.The bioinformatics prediction results of the database show that there are binding sites between mi R-130b-5p and IGFBP2 3’UTR,and between lnc-TMEM189-UBE2V1-3:4 and mi R-130b-5p,but it is not clear whether there is a targeting relationship.The purpose of this study is to explore the role and molecular regulation mechanism of IGFBP2 in 6-OHDA-induced PD model,in order to provide an effective marker for the diagnosis of Parkinson’s disease,especially for early diagnosis,and to provide a new target for its treatment.Methods: 6-OHDA-induced Parkinson’s rat model and cell model were used in this study.The overexpression model of IGFBP2 was established by slowly injecting recombinant IGFBP2(r IGFBP2)into the right medial forebrain bundle of rats.The cell models of overexpression of IGFBP2,overexpression of IGF-1 and double overexpression of IGFBP2 and IGF-1were constructed by using r IGFBP2 alone or co-treated with r IGF-1.LncTMEM189-UBE2V1-3:4 silencing plasmid was used to infect SH-SY5 Y cells to construct lnc-TMEM189-UBE2V1-3:4 silencing model.Mi R130b-5p overexpression lentivirus was used to infect SH-SY5 Y cells to construct mi R-130b-5p overexpression model.Lnc TMEM189-UBE2V1-3:4overexpression/ silencing plasmid was transfected into SH-SY5 Y cells alone or cotransfected with mi R-130b-5p mimics/inhibitor to construct lncTMEM189-UBE2V1-3:4 overexpression / silencing model and lncTMEM189-UBE2V1-3:4 and mi R-130b-5p double overexpression / double silencing model.The behavioral changes of the model were tested by behavioral test(rotation behavior,catalepsy study,pole test).The expression of tyrosine hydroxylase in substantia nigra pars compacta was detected by immunohistochemistry,and the expression of lnc-TMEM189-UBE2V1-3:4,mi R-130b-5p and IGFBP2 was determined by fluorescence quantitative PCR.Western blot was used to detect the expression of IGFBP2,α-synuclein,IGF-1,IGF-1R,p-IGF-1R(Tyr1131),AKT,p-AKT(Ser473),cytochrome C in cytoplasm and mitochondria,Bax,Bcl-2,cleaved caspase-9,cleaved caspase-3.The cell viability was detected by CCK-8.The apoptosis of substantia nigra was detected by TUNEL.The level of ROS,the content of MDA,the activity of SOD and GSH-Px were measured by kit.The changes of mitochondrial membrane potential were detected by JC-1.The content of LDH in the supernatant of the cells was detected by kit.Apoptosis was detected by Hoechst 33258 staining.In situ hybridization was used to determine the expression location of lnc-TMEM189-UBE2V1-3:4.The targeting binding relationship between lnc-TMEM189-UBE2V1-3:4 and mi R-130b-5p,mi R-130b-5p and IGFBP2 was detected by luciferase method.Results: 1.In the PD rat model,218 up-regulated genes and 427 downregulated genes were screened.Combined with the ranking of differentially expressed genes,by analyzing the enrichment of differential genes,and critical review of literatures,IGFBP2 was selected as the target for further research.2.In the substantia nigra pars compacta of PD rats,the expression of IGFBP2was significantly decreased.After overexpression of IGFBP2,the number of rotation,the time for the two front claws to move completely from the horizontal rod,the time for the rat head to turn downward and the time to reach the bottom of the cage were significantly increased,the number of TH positive cells decreased significantly,and the expression level of α-synuclein increased significantly,which aggravated the performance of PD.3.In PD rats and cell models,after overexpression of IGFBP2,the levels of ROS and MDA were significantly increased,while the activities of SOD and GSH-Px were significantly decreased.The mitochondrial membrane potential decreased significantly,the protein expression level of cytochrome C in cytoplasm increased significantly,and the protein expression level of Cytochrome C in mitochondria decreased significantly.The number of apoptotic cells increased significantly.The protein expression levels of IGF-1、p-IGF-1R(Tyr1131)/IGF-1R and p-AKT(Ser473)decreased significantly.In PD cell model,double overexpression of IGFBP2+IGF-1 could reverse the above expression caused by overexpression of IGFBP2 alone.4.In PD cell model,the expression of lnc-TMEM189-UBE2V1-3:4 was significantly increased,while the expression of mi R130b-5p was significantly decreased.Lnc-TMEM189-UBE2V1-3:4 negatively regulated mi R-130b-5p and mi R-130b-5p negatively regulated IGFBP2,luciferase.The specific binding of lnc-TMEM189-UBE2V1-3:4 with mi R-130b-5p,mi R-130b-5p and IGFBP2 was verified.5.After overexpression of mi R-130b-5p,the levels of ROS and MDA were significantly decreased,while the activities of SOD and GSH-Px were significantly increased.The mitochondrial membrane potential increased significantly,the protein expression of Cytochrome C in cytoplasm decreased significantly,and the protein expression of Cytochrome C in mitochondria increased significantly.6.After silencing lnc-TMEM189-UBE2V1-3: 4,the levels of ROS and MDA decreased significantly,while the activities of SOD and GSH-Px increased significantly.The mitochondrial membrane potential increased significantly,the protein expression of Cytochrome C in cytoplasm decreased significantly,the protein expression of Cytochrome C in mitochondria increased significantly,and the protein expression levels of IGF-1、p-IGF-1R(Tyr1131)/IGF-1R and p-AKT(Ser473)increased significantly.Lnc-TMEM189-UBE2V13:4+mi R-130b-5p double silencing could reverse the above performance.Conclusion: 1.IGFBP2 is lowly expressed in the pars compacta of substantia nigra of PD rats.Overexpression of IGFBP2 can aggravate the phenotype of PD rats and promote the occurrence and development of PD.2.In PD model,overexpression of IGFBP2 inhibits the phosphorylation of IGF-1R and AKT by down-regulating the expression of IGF-1,aggravates oxidative stress and mitochondrial damage in PD,promotes apoptosis of PD cells and promotes the occurrence and development of PD.3.In PD model,IGFBP2 is the target gene of mi R-130b-5p.Lnc-TMEM189-UBE2V13:4 negatively regulates mi R-130b-5p.Lnc-TMEM189-UBE2V1-3:4was specifically bound to mi R-130b-5p and mi R-130b-5p was specifically bound to IGFBP2.4.In PD model,silencing lnc-TMEM189-UBE2V1-3:4 suppresses the expression of IGFBP2 through up-regulation of mi R-130b-5p,upregulates the level of IGF-1,and promotes the phosphorylation of IGF-1R and AKT.It reduced the oxidative stress response and mitochondrial dam age in PD,and reduced the apoptosis of PD cells.