Regulation And Mechanism Of FGF7 On The Proliferation And Activation Of Astrocytes In Parkinson's Disease Model Mice

Parkinson's disease(PD)is the second most common neurodegenerative disease.It is characterized by progressive loss of dopaminergic neurons in the substantia nigra of the midbrain.The clinical symptoms include resting tremor,muscle stiffness,and slow movement.And accompanied by non-motor symptoms such as cognitive decline.Since the understanding of the pathogenesis of PD is not yet fully understood,the current clinical treatment is still based on symptomatic treatment,which cannot fundamentally reverse or prevent the progression of the disease.Various hypotheses have been proposed regarding the pathogenesis of PD,including excessive release of oxygen free radicals,damage to mitochondrial function,loss of nutritional support,abnormal kinase activity,destruction of calcium homeostasis,and protein degradation dysfunction.With the deepening of research,these neuron-centered hypotheses cannot completely reveal the molecular mechanism of PD.The role of non-neuronal cells in the occurrence and development of PD has been paid more and more attention by researchers.In the past,it was believed that as interstitial cells in brain tissue,astrocytes mainly function to support tissue structure,repair and fill damaged areas.In recent years,studies have found that astrocytes can guide the development of neurons and regulate synaptic function,regulate the extracellular concentration of ions and neurotransmitters,participate in the immune response and repair process,and regulate the cerebral microcirculation and blood-brain barrier(BBB)permeability,etc.It is currently believed that astrocytes and nerve cells form a complete neuro-glia network,which is a necessary condition for maintaining the basic physiological functions of the brain.In such a network microenvironment,microglia can release the fibroblast growth factor FGF and inflammatory factors act on astrocytes,and at the same time,astrocytes will release FGF and inflammatory factors into the microenvironment,forming a network of interactions between growth factors and inflammatory factors.In the pathological process of PD,astrocytes play an important role in regulating the inflammatory response.Activated astrocytes can accelerate the loss of neurons in the PD model.However,this molecular mechanism is still unclear.The fibroblast growth factor(FGFs)family belongs to one of the most diverse groups of growth factors in vertebrates,and is involved in the early development of the brain and the repair of brain tissue after injury.FGF7 is a paracrine fibroblast growth factor.It is expressed in brain,lung,vas deferens,tongue,skin and other tissues.It is involved in damaged tissue repair,neovascularization,stem cell proliferation and differentiation,nerve regeneration,and calcium and phosphorus metabolism.Studies have shown that FGF/FGFR plays an important role in regulating inflammation.There are few reports on the exact role of FGF7 in the central nervous system.The previous study of our group found that the protein expression level of FGF7 in astrocytes under the MPP~+model was significantly increased,which may regulate the proliferation of astrocytes and cause inflammation The important molecule of the reaction.This topic intends to further study the exact role and mechanism of FGF7 in the proliferation and inflammation of astrocytes in the PD model from the animal,cell and molecular levels.Object:To explore the regulatory effect and molecular mechanism of FGF7 on the proliferation and inflammation of astrocytes in PD mice.Methods:2-3 months old mice of WT were used to prepare mouse MPTP subacute PD models.Immunohistochemistry and Nissl staining were used to detect the loss and survival of Th positive neurons in midbrain.Transcriptome sequencing screened out the differentially expressed gene FGF7.The activation of GFAP and the distribution and localization of FGF7 in astrocytes and neurons were detected by immunofluorescence.The protein expressions of FGF7 and GFAP were detected by Western blot.Primary WT astrocytes were cultured,and MPP~+stimulation was given to detect the protein expressions of FGF7 and GFAP by Western blot.Use lentivirus to knock down FGF7 on primary astrocytes,and Western blot to verify the expression of GFAP protein.In vivo preparation of midbrain FGF7 knockdown MPTP chronic PD Model,behavioral testing of mouse movement coordination ability,Western blot and immunohistochemical testing of GFAP activation and TH loss.Then explore the biological function of FGF7,culture primary astrocytes,stimulate with exogenous FGF7 factors,Western blot Detect the expression of proliferating cell nuclear antigen PCNA,Brdu staining and immunofluorescence to detect the proliferation number and morphological changes of astrocytes;then explore the regulation of FGF7 in the process of astrocyte activation in the regulation of inflammation,and cultivate primary astrocytes Cells were stimulated with LPS,and Western blot was used to verify the expression of GFAP;FGF7 was pre-incubated and then stimulated with LPS.ELISA and QPCR were used to detect the secretion of TNF-?,IL-6 and IL-1?in the supernatant and cells,respectively;Western blot to detect NF-?B activation indicators,and simultaneously detect PI3K/AKT,JAK/STAT3,RAS/MAPK,PKC/GSK3?signaling pathway related indicators;give GSK3?inhibitors,Western blot verification related indicators.Results:1.Proliferation and activation of astrocytes in PD mice and cell models.In the MPTP subacute PD model,MPTP can induce the loss of TH~+neurons,and at the same time cause the proliferation and activation of astrocytes in the SNc region of the mouse midbrain.MPP~+stimulates the proliferation and activation of primary astrocytes.2.RNA-Seq found that MPP~+induced an increase in the level of Fgf7 m RNA in astrocytes.3.Up-regulation of FGF7 protein expression in PD mice and cell models.In the MPTP subacute PD model,mouse midbrain FGF7 protein levels are highly expressed.In the mouse midbrain SNc area FGF7 can be co-labeled with the astrocyte marker GFAP and neuron marker Neu N,suggesting that FGF7 is in astrocytes It is expressed in neurons and in neurons,and MPTP can induce a further increase and up-regulation of FGF7 expression.WT primary astrocytes were stimulated by MPP~+,and the protein levels of GFAP and FGF7 increased.4.FGF7 knockdown inhibits MPP~+-induced astrocyte activation.In the MPP~+cell model,transfection of Fgf7 sh RNA-LV can reduce the proliferation of astrocytes.5.Loss of FGF7 reduces the proliferation and activation of astrocytes in the SNc region of the midbrain of MPTP/p mice.MPTP/p can induce the activation of GFAP~+glia in the SNc region of NC mice and FGF7 knockdown mice,and FGF7knockdown alleviates the activation and proliferation of GFAP induced by MPTP/p to a certain extent.6.Loss of FGF7 alleviates the loss of TH~+neurons in the midbrain SNc area of MPTP/p model mice.MPTP/p induces the loss of TH~+neurons in the SNc area of mice,while knocking down FGF7 improves the loss of TH~+neurons in the SNc area of mice.7.Loss of FGF7 improves the abnormal behavior of MPTP/p model mice.In the open field experiment,MPTP/p stimulation caused the mice to shorten the running distance and reduce the speed of movement within a certain period of time.After knocking down FGF7,the running distance of MPTP/p model mice was longer than that of the corresponding control group.Knockdown of FGF7 has a certain effect on the shortening of the falling time of the rotating rod and the extension of the climbing time caused by MPTP/p.8.FGF7 factors stimulate the proliferation of astrocytes.Exogenous FGF7cytokines stimulated the primary astrocytes of mice,and the astrocytes proliferated,which was manifested by increased expression and number of proliferating nuclear antigen PCNA,increased GFAP expression,morphologically manifested as astrocyte proliferation and fibrosis.9.FGF7 factor stimulation promotes the inflammatory response of astrocytes.FGF7 cytokine(20 ng/m L)stimulated mouse primary astrocytes,causing the levels of inflammatory factors TNF-?and IL-6 to increase.The cells were pretreated with FGF7for 12 h,and then stimulated with LPS for 12 h,and the cell supernatant and RNA were collected.QPCR results showed that FGF7 further promoted the increase of TNF-?,IL-6,IL-1?m RNA levels induced by LPS;ELISA results also showed that FGF7aggravated the increase of TNF-?and IL-6 expression levels induced by LPS stimulation.10.Stimulation of FGF7 factors intensifies the activation of NF-?B signaling pathway in astrocytes induced by LPS.FGF7(20 ng/m L)stimulates astrocytes to activate the NF-?B pathway.LPS stimulation leads to an increase in the phosphorylation levels of IKK and P65.FGF7 pretreatment can furtherly aggravate the phosphorylation of IKK and P65 induced by LPS.11.FGF7 significantly up-regulates the phosphorylation level of GSK3?.Both FGF7 and LPS stimulation can activate GSK3-?,and FGF7 pretreatment can further promote the increase of LPS-induced GSK3-?phosphorylation.12.Inhibition of GSK3?alleviates FGF7-induced astrocyte proliferation and activation.13.Silencing GSK3?relieves FGF7-induced astrocyte proliferation.Conclusions:1.The increase of FGF7 level is an important factor causing the proliferation and activation of astrocytes in the brain of PD mice.2.FGF7 activates GSK3?and NF-?B pathways to promote the proliferation and activation of astrocytes and the release of inflammatory factors,which aggravate PD nerve damage.The major contributions of the present study lie in:FGF7 mediates the abnormal proliferation and activation of astrocytes and increases the level of inflammatory factors in the process of PD,which aggravates the nerve damage of PD.It explains the mechanism of astrocyte hyperproliferation and activation in PD model from a new perspective,broadens the understanding of the important functions of FGF7 in the glial network,and enriches the scientific hypotheses about the interaction of growth factors-inflammatory factors in the development of neurodegenerative diseases.