The Study Of Repair Of Spinal Cord Injury In Mice Through GDF-11/smads Signaling Pathway And Pyroptosis Signaling Pathway

Objective:Spinal cord injury(SCI),one serious illness with a high mortality and poor prognosis after spinal trauma,has been included primary injury and secondary injury.The molecular mechanism of spinal cord injury including regulation of nerve regeneration,the repair and protection of nerve cells and the repair application of biological materials has been understood.Pyroptosis has been valued as a caspase-1-dependent inflammatory form of programmed cell death in recent years,which has been rarely reported in the studies related to spinal cord injury.Growth differentiation factor-11(GDF-11)/smads pathway have been certified as be closely to cell growth,differentiation and homeostasis.Our study was to explore the role of GDF-11/smads pathway and pyroptosis pathway in spinal cord injury during second injury period and the underlying mechanism.Methods:Mice of spinal cord injured group were established by arteryclamp through clamping the T10 level spinal cord after laminectomy for one minute,and mice in control group were treated with sham-operation(laminectomy was performed without spinal cord injury.Mouse Scale sores was used to assess the recovery of the injured mice and the behavior of mice was observed during the first two weeks after operation.The expressions of IL-6,IL-18,IL-1?,ASC and caspase-1 in the spinal cord mice were detected by Western Blot,real-time PCR and Immunohistochemistry.The mice of spinal cord injured group and control group were treated with normal saline,caspase-1 inhibitor or smad3 inhibitor through local injection.Mouse Scale sores was used to assess the recovery of the injured mice and the behavior of mice was observed during the first two weeks after operation.The expressions of IL-6,IL-18,IL-1?,ASC,caspase-1,smad4,AIM-2 and NLRP-1 were detected by Western Blot,Real-time PCR and Immunohistochemistry at the 14 th day after surgery.The expressions of caspase-1,smad2/3,GDF-11 and AIM2 in mice of each spinal cord injured group were detected by immunohistochemistry and immunofluorescence.NSC-34 cells were cultured in vitro.The cell viability of NSC-34 treated with aldosterone and smad3 inhibitor was detected by CCK8.Caspase-1 expressions of NSC-34 cells in each group were detected by Western Blot.Results: 1.The model of spinal cord injured mice was successfully constructed by arteryclamp through clamping the T10 level spinal cord after laminectomy for one minute.That including hind limb paralysis,no ankle joint movement were observed in all the mice in spinal cord injured mice and the BMS score was 0 within 24 hours after the injury.The survival rate decreased to 77.78% at the first 3 days.On the 3rd day after the operation,some mice showed ankle joint activity,and the BMS score was 0.64�0.44.On the 5th day after surgery,the BMS score was 1.36�1.05.At the7 th day,the survival rate decreased to 55.56%,and the BMS score was2.80�1.54.Within 7-14 days after the operation,the status of spinal cord injury mice was tended to be stable and the hind limb function of the mice recovered obviously,and there was still residual effect after the injury.The BMS score was3.89�1.76.However,the survival rate of the control group was 100% within 2 weeks.After anesthesia,the hind limbs were not paralyzed and the body balance was normal.The BMS score of the control group was all 9.2.Pyroptosis signaling pathway might be involved in spinal cord injury during the recovery period.The markers of pyroptosis signaling pathways was detected both at m RNA and protein level,we found that the expressions of IL-6,IL-18,IL-1?(except the 5th day)in all SCI groups are higher than that in control group.ASC and caspase-1,the markers of pyroptosis signaling pathway,were highly expressed in spinal cords of mice at the 7th day and14 th day after the laminectomy with clamping spinal cord for one minute,and there were rarely present in spinal cord of control group mice In addition,the caspase-1was broken up into p20 to induce inflammation in sci group and rarely in con group at the 7th day and 14 th day.3.The GDF-11/smads signaling pathway may be involved in mice spinal cord injury,and the results suggested that the expressions of the key markers of the GDF-11 /smads signaling pathway was increased after mice spinal cord injured,especially at the 7th and 14 th day after the surgery.4.Spinal cord injury could be alleviated by caspase-1 inhibitors and smad3 inhibitors.The survival rate of mice and limb function recovery can be improved after spinal cord injury by treating with caspase-1 inhibitors and smad3 inhibitors.5.Smad3 inhibitors might play a role in alleviating spinal cord injury via reducing pyroptosis of neurons induced by caspase-1.The expressions of ASC,p20 and mature IL-1? were obviously decreased in spinal cory injured mice treating with smad3 inhibitors compared with Normal saline.Co-immunostaining of smad2/3 with caspase-1 revealed that smad2/3 was enriched in caspase-1 expressing neurons in ventricornu in pyroptosis neurons of spinal cord mice.The expressions of smad2/3 and caspase-1 could be inhibited and the numbers of pyroptosis neurons were decreased by treating with smad3 inhibitor in spinal cord injured mice.6.AIM-2 or NLRP-1 may be not the targets of GDF-11/smads pathway to regulate the pyroptosis of neurons.There were no evidently differences of NLRP-1 and AIM-2 expressions between spinal cord mice treated with smad3 inhibitor and saline injured mice.7.The cell viability of NSC-34 which reduced by aldosterone,can be increased by smad3 inhibitors in vitro and the expressions of caspase-1 and p20 in NSC-34 cells was decreased.Conclusion : Inhibition of the GDF-11/smads pathway can alleviate spinal cord injury by down-regulating caspase-1 and reduce the pyroptosis of nerve cells during the recovery period after spinal cord injury.