Research Of Nafamostat Repair Spinal Cord Injury Through Thrombin-PAR1-STAT3 Pathway

[Objective]Spinal Cord Injury(SCI)is a serious central nervous system disease characterized by high disability,mortality and incidence of serious complications.In terms of medical treatment,the use of methylprednisolone has been controversial because it can lead to a variety of serious complications.A preliminary study found that Nafamostat(NM)inhibited inflammatory response,reduced glial scar hyperplasia,and promoted motor function recovery after spinal cord injury.Thrombin is a serine protease that plays an important role in the inflammatory response,and can be used as an important target for regulating the imbalance of microenvironment of spinal cord injury.This study is based on the results of the previous study on the effectiveness of Nafamostat in repairing spinal cord injury,further clarifying the time window of Nafamostat,assessing the safety of medication,and exploring its mechanism in the repair of spinal cord injury.[Methods]1.Motor function and nerve conduction function testEight-week-old female Wistar rats(220 g±10 g)were randomly divided into three groups:Sham group,Injury group and NM group.The sham group only removed the T10 lamina;the T10 spinal cord contusion model was prepared for the Injury group and the NM group.The NM component was divided into 7 subgroups,and intervention was started at 7 different time points(0 h,2 h,8 h,12 h,24 h,48 h,5 d).The recovery of motor function was evaluated by BBB score.The neurological recovery was evaluated by neurophysiological detection of Sensory Evoked Potential(SEP)and Motor Evoked Potential(MEP).2.Coagulation function testEight-week-old female Wistar rats(220 g±10 g)were randomly divided into three groups:Sham group,Injury group and NM group.The Sham group only removed the T10 lamina;the spinal cord contusion model was prepared for the Injury group and NM group.NM components were divided into 5 subgroups,and intervention was started at 5 different time points(0 h,2 h,8 h,12 h,24 h).Spinal cord tissue and venous blood were taken 24 h after injury,and the safety of Nafamostat in repairing spinal cord injury was evaluated by Western blot and coagulation function tests.3.Mechanism of Nafamostat repairing spinal cord injuryEight-week-old female Wistar rats(220 g±10 g)were randomly divided into three groups:Sham group,Injury group and NM group.The Sham group only removed the T10 lamina;the spinal cord contusion model was prepared for Injury group and NM group.The expression of Thrombin was detected by immunofluorescence staining;expression of Thrombin,PARI,STAT3 and p-STAT3 was detected by Western blot;The expression of astrocyte precursor marker Vimentin was detected by Western blot;and the activation of astrocytes was detected by immunofluorescence staining;The expression of inflammatory factors was detected by inflammatory microarray technique.[Results]1.The time window of Nafamostat for spinal cord injury repair is 8-12 hThe recovery of motor function was assessed using the BBB score before and after spinal cord injury.The recovery of hindlimb motor function in NM group was better than other NM groups at 8 h and 12 h after injury(P<0.05).Neurophysiological examination was performed 5 weeks after spinal cord injury.The results showed that the latency of MEP and SEP in the simple injury group and the NM group were prolonged in different degrees compared with Sham group.The latency of the NM groups was shorter than other NM groups(P<0.05).The amplitudes of MEP and SEP in Injury group and NM group were lower than Sham group.The amplitude of the 8h and 12h NM groups was smaller than other NM groups,and the difference was statistically significant(P<0.05).2.Nafamostat intervention at 8 h after spinal cord injury had no significant effect on coagulationThe results of coagulation function(PT,PT activity,PT-INR,etc.)showed that in each NM group,the coagulation function administered at 8 h after spinal cord injury was basically the same as that of the simple injury group,and the difference was not statistically significant.3.Nafamostat mediates Thrombin-PAR1-STAT3 pathway to repair spinal cord injuryThe results of Western blot showed that the expression of thrombin in the spinal cord of NM group was significantly lower than Injury group.The immune-fluorescence results showed that the number of Thrombin+ neurons in the injured spinal cord tissue was significantly higher than NM group.The difference was statistically significant(P<0.05);Western blot results showed that the expression of Thrombin,PARI,STAT3,p-STAT3 and Vimentin in NM group was lower than that in Injury group(P<0.05);The number of GFAP+ astrocytes in the spinal cord of NM group was significantly lower than Injury group(P<0.05).The results of inflammatory microarray showed that Nafamostat intervention can reduce the expression of various pro-inflammatory factors after spinal cord injury.[Conclusions]1.The time window of application of Nafamostat to repair spinal cord injury in rats 1s 8 h-12 h after injury,and the time window 1s wider than that of methylprednisolone,which is more clinically meaningful.2.Nafamostat intervention at 8 h after spinal cord injury had no significant effect on the body's coagulation function,and the safety of medication was better,which further proved the rationality of the time window of medication;3.Nafamostat reduced the expression and activity of Thrombin after spinal cord injury,inhibited activation of PARI,interfered with the downstream STAT3 signaling pathway to repair spinal cord injury.