Effect Of SHARPIN Overexpression On Phenotypic Transformation Of Astrocytes And Glial Scar Formation After Spinal Cord Injury In Rats

Background During tissue repair following SCI,astrocytes play a crucial role.In the acute stage of injury,RA play a neuroprotective role in limiting inflammation diffusion.In the chronic stage of injury,SA form glial scars and block axon regeneration.Promoting the neuroprotective effect of RA and inhibiting the adverse effect of SA on tissue repair may be one of the therapeutic strategies for spinal cord injury.Studies have expressed that the transformation of RA into SA after spinal cord injury is regulated by the type I collagen-Integrin β1-N-Cadherin signaling pathway,and glial scar can be prevented by inhibiting integrin β1.According to numerous research,SHARPIN,as an endogenous inhibitor of integrin β1,can directly inhibit Talin-mediated integrin activation.In this study,lentivirus was used to construct a rat model of SHARPIN overexpression in order to investigate whether SHARPIN overexpression in astrocytes can inhibit the formation of glial scar and thus promote tissue repair.Methods In vitro experiments:1.The surface marker GFAP of astrocytes was detected by cellular immunofluorescence.2.The SHARPIN gene fragment was amplified by PCR and then integrated into the lentivirus vector to obtain the lentivirus containing the SHARPIN gene fragment and the control lentivirus.Astrocytes were infected with LV-SHARPIN and LV-NC,and cell models overexpressing SHARPIN were established,which were divided into LVSHARPIN infection group AC(LV-SHARPIN)and LV-NC infection group AC(LVNC).3.Cell immunofluorescence and western blotting were used to detect SHARPIN expression levels in the two groups of astrocytes,in order to verify the successful establishment of the overexpressed SHARPIN astrocyte model.4.The migration ability of astrocytes in the two groups was detected by scratch test.In vivo experiments:The spinal cord contusion model of thoracic vertebra(T10)was used in SD rats.Lentivirus was injected in situ immediately after spinal cord injury.Thirty healthy female SD rats were randomly divided into LV-SHARPIN in situ injection group(LVSHARPIN group),LV-NC in situ injection group(LV-NC group)and no lentivirus in situ injection group(Control group).5.Tissue immunofluorescence and Western blot were used to detect the activation of collagen I-integrin β1-N type adhesin signaling pathway and the expression level of SHARPIN in rats in the Control group after spinal cord injury.6.Tissue immunofluorescence was used to detect the temporal and spatial distribution of RA(Nestin+)and SA(N-Cadherin+)at 7,14,and 28 dpi in rats in LV-SHARPIN and LV-NC groups.7.Tissue immunofluorescence was used to detect the distribution of inflammatory cells(CD68+)and fibrous scar(PDGFRβ+)in the injured core of rats in both groups at 28 days after spinal cord injury.8.Tissue immunofluorescence technique was used to quantitatively analyze the number of Neu N+ neurons and NF+ axons in the two groups 28 days after spinal cord injury.9.BMS score and footprint analysis were used to evaluate the recovery of motor function 28 days after spinal cord injury in the two groups.Result In vitro experiment results showed that:1.Astrocytes were typically star-shaped,with large amounts of GFAP expressed on the cell surface,so it was confirmed that the used cells were astrocytes.2.According to the results of cell immunofluorescence and Western blot,the SHARPIN expression level in LV-SHARPIN group was significantly higher than that in LV-NC group(P<0.05).3.According to scratch test results,there was no significant difference in cell migration rate between the LV-SHARPIN group and the LV-NC group.In vivo results showed that:4.According to the results of cell immunofluorescence and Western blot,the expressions of type I collagen,integrin β1,N-type cadherin and SHARPIN were significantly increased after SCI(P < 0.0001).This suggests that the type I collagenintegrin-β1-N cadherin signaling pathway is activated and SHARPIPN expression is upregulated after spinal cord injury.5.Tissue immunofluorescence results of rats in LV-SHARPIN group and LV-NC group showed that,compared with the LV-NC group,the expression levels of SHARPIN and Nestin in LV-SHARPIN group were significantly increased(P < 0.0001),while the expression levels of N-type calcin were significantly decreased(P < 0.0001).This suggests that SHARPIN can inhibit RA to SA transformation after spinal cord injury.6.The immunofluorescence results of CD68,PDGFRβ,Neu N and NF in the two groups showed that compared with the LV-NC group,the expression of CD68 in the LVSHARPIN group was significantly increased(P < 0.0001),and the injury area was significantly increased(P < 0.001).The expression of PDGFRβ was significantly decreased(P < 0.0001),and the number of Neu N+ neurons and NF+ axons was significantly decreased(P < 0.001).7.The BMS score and footprint analysis of the two groups showed that the motor function of the rats in the LV-NC group recovered better than that in the LV-SHARPIN group(P < 0.001).Conclusion SHARPIN can inhibit RA’s transformation into SA by inhibiting integrinβ1,which leads to the obstruction of glial scar formation and the impairment of tissue repair and functional improvement.