Protective Effect And Mechanism Of Knocking Down PTBP1 On Peripheral Nerve Injury

ObjectivePolypyrimidine tract-binding protein 1(PTBP1)is a known post-transcriptional gene expression regulator that controls splicing,translation,stability and localization of messenger RNA(m RNA),has many molecular functions related to RNA metabolism and is a major repressor of selective splicing.PTBP1 has been shown to play a role in regulating splice site selection and to function as a selective splicing regulator in mammalian cells.Previously,PTBP1 has been shown to be neuroprotective in both Parkinson’s Disease(PD)and optic nerve injury(ONI)models in mice.It is important to note that researchers have successfully reversed the disease phenotypes of mice in both disease models by knocking down this molecule.Nevertheless,studies based on this molecule are still focused on the central nervous system,especially in the brain,while PTBP1-centered therapeutic strategies are rarely mentioned in the field of therapeutic research for peripheral nerve injury(PNS),undermining the potential therapeutic value of this novel target.This study aims to explore the specific role of PTBP1 in peripheral nerve injury,and then search for the potential role of PTBP1 in peripheral nerve injury repair and its molecular mechanism,and provide a theoretical basis for the development of effective neuroprotective drugs,which has important scientific significance and potential clinical application.Methods1.In vivo,male C57BL/6 mice(20 g-30 g),7-8 weeks were randomly divided into 3groups,control group(sham-operated group),Sciatic nerve injury(SNI)group,and SNI+AAV-GFAP-Cas Rx-Ptbp1 group,and the peripheral nerve injury was established by intermittent clamping method of clamping the sciatic nerve in mice.model.The spinal cord and dorsal root ganglion(DRG)tissues of 14 d mice were first taken,and the knockdown level of PTBP1 by AAV adeno-associated virus was verified by immunohistochemistry(IHC)technique.2.In vitro,primary astrocytes were isolated from fetal mice and grown in 12-well plates after 6-7 days of in vitro incubation and trypsin digestion,and astrocytes were transfected with small interfering RNA to reduce PTBP1 expression.Subsequently,a cell knockdown model was constructed.The expression levels of neuron-related molecules and astrocyte polarization marker were detected by q-PCR,Western Blot and immunofluorescence at the m RNA and protein levels respectively.The key molecules and signaling pathways of PTBP1 in peripheral nerve injury repair were screened by transcriptome sequencing.Results1、Immunofluorescence co-staining of PTBP1 with GFAP on mouse spinal cord and DRG tissues showed that PTBP1 molecules were mainly localized in astrocytes in the spinal cord and DRG.2、The spinal cord and DRG tissues of mice at 0d,3d,7d and 14 d after SNI were taken for PTBP1 immunofluorescence and WB assay,and the results demonstrated that PTBP1 showed a trend of increasing in the acute phase and then gradually decreasing after nerve injury.3 、 After administration of AAV-GFAP-Cas Rx-Ptbp1 by intrathecal injection,immunohistochemical and WB results showed reduced expression of PTBP1,confirming the knockdown efficiency of AAV.4、The sciatic nerve tissues of SNI+AAV-GFAP-Cas Rx-Ptbp1 mice were subjected to HE staining and immunofluorescence of tublin-β3 and tunel to detect nerve fiber opening,axon regeneration and local apoptosis,and the results showed that compared with the SNI group,the sciatic nerve fiber opening and axon regeneration of SNI+AAV-GFAP-Cas RxPtbp1 mice were significantly improved,which promoted the repair of sciatic nerve after injury.5、The control group,SNI group and SNI+AAV-GFAP-Cas Rx-Ptbp1 group mice were used to determine the mechanical and thermal pain thresholds by Von Frey and Hot Plate Analgesia test method,and the basal threshold of thermal pain and mechanical pain was derived from the control group mice,and the results showed that compared with the SNI group,the SNI+AAV-GFAP-Cas Rx-Ptbp1 group mice had a shorter time to regenerate to the basal threshold of thermal pain.Ptbp1 group mice showed a shorter decrease in thermal pain threshold to the basal threshold,demonstrating that the SNI+AAV-GFAP-Cas Rx-Ptbp1 group mice had accelerated axon regeneration and promoted nerve repair after injury.6、SI-RNA transfected astrocytes were sampled 48 h later for transcriptome sequencing,and the results indicated that the neuronal cytosol and axon-related components were increased,and the expression of DCX,S100a10,Clcf1 were up-regulated,while the expression of Fkbp5,Fbln5,Serpiing,an A1 astrocyte marker,were down-regulated.In addition,The expression of Glial cell derived neurotrophic factor(GDNF)was also found to be up-regulated.7、SI-RNA transfected astrocytes were sampled for WB after 72 h and showed increased expression of neuronal markers DCX,VGLUT1,MAP2,and A2 astrocyte polarization marker S100a10;q-PCR results showed increased expression of A2 astrocyte markers S100a10,Clcf1 and GDNF,and decreased expression of the A1 astrocyte markers Fkbp5,Fbln5,and Serpiing The immunofluorescence results showed that neurons were present in astrocytes and expressed DCX,MAP2,Neun,β-tublin and other neuronal markers,while the expression of A2 astrocyte polarization marker S100a10 was increased.8、After administration of AAV-GFAP-Cas Rx-Ptbp1 gland by intrathecal injection,immunofluorescence showed increased expression of A2 astrocyte polarization marker S100a10 and decreased expression of A1 astrocyte marker C3 in the spinal cord of mice.ConclusionPTBP1 promotes axonal regeneration and repair after peripheral nerve injury by regulating the transdifferentiation of astrocytes into early neurons and their ability to develop into functional mature neurons,while PTBP1 further promotes the repair of peripheral nerve injury by increasing the secretion of glial cell-derived neurotrophic factor to nourish neurons and axons by promoting the polarization of A2 astrocytes.