| Background and objective: Parkinson’s disease(PD)is a complex age-related neurodegenerative disorder characterized primarily by the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain,resulting in a significant reduction in dopamine levels in the striatum.The increasing prevalence of PD poses a serious threat to the physical and mental health of the elderly population and places a heavy economic burden on families and society.Treatment for PD includes medication,surgical procedures,physical therapy,psychological counseling,and caregiving.Currently,there is no definitive evidence to suggest that existing treatments can effectively slow the progression of PD.The ongoing in-depth research into cell replacement therapy techniques is making it a promising approach for treating PD and is currently a hot topic of investigation.In recent years,the study of neural stem cells(NSCs)has provided potential applications for cell replacement therapy.Transplanting NSCs not only holds the promise of replacing lost neurons but also has the potential to improve PD symptoms by secreting neurotrophic factors.The precise mechanisms underlying the effects of NSCs replacement therapy for PD are not fully understood,and there are several unresolved issues,such as the low long-term survival rate of transplanted cells and limited therapeutic effects.Therefore,the current research is dedicated to addressing the challenge of enhancing the survival rate of transplanted cells and promoting their differentiation and integration into the host tissue.The survival,differentiation,and maturation of NSCs are greatly influenced by the non-cell autonomous mechanisms within their microenvironment.Studies have revealed the abundant expression of A1-type astrocytes in the brains of PD patients and in the pathological regions of PD animal models.As a critical component of the microenvironment,changes in astrocytes may have a significant impact on the survival of NSCs.Previous studies have suggested that Glucagon-like peptide-1 receptor(GLP1R)agonists may play a neuroprotective role by preventing astrocyte transformation to the A1 neurotoxic phenotype.In light of this,we propose a research approach centered around cell transplantation therapy.Specifically,we intend to treat PD mice by transplanting NSCs in combination with a GLP1R agonist,and we will conduct in vitro cell experiments to investigate the impact of A1-type astrocytes on the survival and differentiation of NSCs.Our goal is to enhance the integration efficiency of transplanted cells by inhibiting A1-type astrocytes,thereby offering a new perspective for PD treatment.Methods: This study was divided into four parts.Part 1: We established a PD mouse model using stereotaxic injection of 6-hydroxydopamine(6-OHDA)and validated the model’s success after 4weeks of injection through an apomorphine(APO)-induced rotational behavior test.The expression of tyrosine hydroxylase(TH)was measured by Western Blot and immunofluorescence staining.C57BL/6J mice were randomly divided into two groups: the control group(Control group)and the PD model group(PD group).After successful modeling,we observed changes in the phenotype of astrocytes in PD mice using WB and immunofluorescence staining techniques.We also examined endogenous neurogenesis through immunofluorescence staining techniques and EDU staining.Part 2: Astrocytes and NSCs were cultured in vitro,and the purity of the cultured cells was determined by immunofluorescence staining.Astrocytes were cultured with interleukin-1α(IL-1α),tumor necrosis factorα(TNFα)and classical complement component C1 q,and whether they could induce A1-reactive astrocytes was verified by Reverse transcriptionquantitative real-time polymerase chain reaction(RT-qPCR).Conditionally co-cultured NSCs using normal astrocyte conditioned medium(Control ACM)and A1 reactive astrocyte conditioned medium(A1 ACM).The impact of A1 reactive astrocytes on NSCs activity,proliferation,and differentiation was assessed through assays including Calcein/PI cell viability and cytotoxicity tests,EDU staining,and immunofluorescence staining.Transcriptomic analysis was conducted to identify differentially expressed genes between A1 reactive astrocytes and normal astrocytes.This was coupled with functional enrichment analysis using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,as well as network interaction analysis,to explore the potential mechanisms underlying the influence of A1 astrocytes on NSCs.Part 3: Astrocytes and microglia were cultured in vitro,and the purity of the cultured cells was determined by immunofluorescence staining.Astrocytes were cultured using either normal microglia conditioned medium(MCM)or lipopolysaccharide(LPS)-induced microglia conditioned medium(LPS MCM),along with IL-1α,TNFα,and C1 q,and their ability to induce A1 reactive astrocytes was validated using RT-qPCR.Using the GLP1R agonist semaglutide to intervene in microglia or A1 reactive astrocytes,we aim to investigate how GLP1R agonists regulate the phenotypic transformation of astrocytes.Part 4: The primary NSCs from midbrain tissues were cultured in vitro and the purity of the cultured cells was determined by immunofluorescence staining.The PD mouse model was constructed by stereotaxically injecting6-OHDA into the striatum.C57BL/6J mice were randomly divided into five groups: the control group(Control group),the PD model group(PD group),the group receiving intraperitoneal injection of semaglutide in the PD model is referred to as the "SEG group"(SEG group),the group where only NSCs transplantation is performed in the PD model is referred to as the "NSCs group"(NSCs group),and the group where semaglutide is intraperitoneally injected in combination with NSCs transplantation in the PD model is referred to as the "SEG+NSCs group"(SEG+NSCs group).The ability of spontaneous activity and motor coordination of mice in each group was measured by tasks such as the pole-climbing test,Rotarod test,grip strength test,balance beam test,and gait analysis.In vivo fluorescence imaging techniques were used to trace the NSCs labeled with Di D fluorescent dyes to determine their survival status and distribution in vivo.Immunofluorescence staining was employed to determine the survival,distribution,differentiation of NSCs in vivo,as well as the phenotype transformation of astrocytes.Results:Part 1:(1)Successfully established a mouse model of Parkinson’s disease with unilateral striatal damage.(2)Western Blot analysis showed that the expressions of GFAP and C3 d in PD group were significantly increased compared with those in Control group;the results of immunofluorescence staining showed that the number of GFAP+ cells and GFAP+/C3d+ cells increased in PD group compared with Control group.(3)Four weeks after stereotactic injection of 6-OHDA,there was a significant increase in EDU+ cells,but hardly colocalized with Neu N+ cells and GFAP+cells;the number of EDU+ cells decreased dramatically at 6 weeks after surgery compared to 4 weeks after modeling,and similarly,EDU+ cells were hardly colocalized with Neu N+ cells and GFAP+ cells.Part 2:(1)Immunofluorescence staining showed that the purity of astrocytes cultured in vitro was greater than 95%.In vitro culture of NSCs in midbrain tissue showed that the cells grew in spherical suspension,and the purity was more than 95% identified by immunofluorescence staining.(2)RT-qPCR results showed that IL-1α,TNF-α and C1 q interfered with astrocytes,significantly increasing the expression of A1-related genes(Serping1,Gbp2,Iigp1,H2-T23,Ugt1 a,Psmb8).These results indicated that IL-1α,TNF-α and C1 q could successfully induce A1-reactive astrocytes.(3)The results of Calcein/PI cell viability and cytotoxicity tests indicated that,compared to the Control ACM group,both the number of dead NSCs and the cell death rate were significantly increased in the A1 ACM group.(4)The EDU staining results showed that compared with Control ACM group,the EDU+ cell rate in the A1 ACM group was significantly reduced,suggesting that A1 reactive astrocytes affected the proliferation of NSCs.(5)Immunofluorescence staining results indicated that,compared to the Control ACM group,the differentiation ratio of NSCs into Tu J1+ cells and TH+ cells in the A1 ACM group were significantly reduced,indicating that A1 reactive astrocytes influenced the differentiation of NSCs.(6)Transcriptomic analysis revealed that there were 3,140 differentially expressed genes between A1 reactive astrocytes and normal astrocytes(with 1,669 genes upregulated and 1,471 genes downregulated).The Biological Processes of GO functional enrichment analysis showed that the upregulated genes in A1 reactive astrocytes primarily enriched functions related to inflammation/immune response,cellular response to interferon,cytokine-mediated signaling pathways,positive regulation of tumor necrosis factor production,positive regulation of interleukin-1 beta production,NF-kappa B transcription factor activity,and positive regulation of I-kappa B kinase/NF-kappa B signaling.Conversely,the downregulated genes were mainly enriched in functions related to multicellular organism development,mitotic cell cycle,membrane regulation,nervous system development,DNA repair,cell cycle,and cell division.The KEGG pathway enrichment analysis results showed that the upregulated genes primarily enriched in signaling pathways including the TNF signaling pathway,IL-17 signaling pathway,chemokine signaling pathway,NOD-like receptor signaling pathway,NF-kappa B signaling pathway,C-type lectin receptor signaling pathway,cytokinecytokine receptor interaction,and more.On the other hand,the downregulated genes were mainly enriched in signaling pathways such as DNA replication,cell cycle,GABA/glutamatergic synapse,neuroactive ligand-receptor interaction,c AMP signaling pathway,mismatch repair,and axon guidance.Part 3:(1)Immunofluorescence staining identified that the purity of in vitro cultured astrocytes and microglia was greater than 95%.(2)The RTqPCR results indicate that LPS MCM,as well as IL-1α,TNFα,and C1 q interventions,significantly increase the expression of A1-related genes(Serping1,Gbp2,Iigp1,H2-T23,Ugt1 a,Psmb8)in astrocytes,suggesting that both LPS MCM and IL-1α,TNFα,and C1 q can successfully induce A1 reactive astrocytes.(3)Direct intervention with GLP1R agonist semaglutide in IL-1α,TNFα,and C1q-induced astrocytes did not reduce the expression of A1 reactive astrocyte-related genes(Serping1,Gbp2,Iigp1,H2-T23,Ugt1 a,Psmb8).(4)Treatment with GLP1R agonist semaglutide reduced the expression of IL-1α,TNFα,and C1 q in LPSstimulated microglial cells.Compared to the LPS MCM group,the LPS+SEG MCM group significantly reduced the expression of A1 reactive astrocyte-related genes(Serping1,Gbp2,Iigp1,H2-T23,Ugt1 a,Psmb8),suggesting that GLP1R agonist semaglutide may inhibit astrocyte conversion to A1 phenotype by acting on microglia.Part 4:(1)In vitro cultivation of NSCs from midbrain tissue showed that the cells exhibit spherical suspended growth,and immunofluorescence staining confirmed a purity greater than 95%.(2)Behavioral results indicated that GLP1R agonist semaglutide treatment(SEG group)and standalone NSCs transplantation treatment(NSCs group)could partially improve the performance of PD mice in the pole-climbing test,Rotarod test,grip strength test,balance beam test,and gait analysis.The GLP1R agonist semaglutide treatment combined with NSCs transplantation(SEG+NSCs group)further improved the performance of mice in the above experiments.(3)In vivo fluorescence imaging and immunofluorescence staining results revealed that the combination of GLP1R agonist treatment and NSCs transplantation therapy increased the survival of transplanted NSCs,promoted the migration of transplanted NSCs to the surrounding tissue,and increased the number of transplanted cells co-labeled with NeuN+ cells.(4)The combination of GLP1R agonist and NSCs transplantation therapy reduced the expression of A1 reactive astrocytes in PD mice.Conclusion:1.Abundant expression of A1 reactive astrocytes in the striatum of PD mouse models and endogenous neurogenesis disorders(newborn cells failed to differentiate into neurons and astrocytes,and gradually died over time).2.A1 reactive astrocytes severely impair the activity,proliferation,and differentiation capacity of NSCs.3.Classical inflammatory mediators activate microglia,which can transform astrocytes into a neurotoxic A1 phenotype.The action of GLP1R agonist semaglutide in blocking the transformation of astrocytes into the A1 neurotoxic phenotype may be mediated through the inhibition of microglial activation.4.GLP1R agonist semaglutide combined with NSCs transplantation promoted the survival,migration and differentiation of transplanted cells,and improved the effect of NSCs transplantation in the treatment of PD... |
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