The Study On Targeting Thrombin Modulating Astrocyte To Repair Spinal Cord Injury

【Objective】Astrogliosis and inflammation play important roles in the pathological process of spinal cord injury(SCI).It is essential to explore novel strategies that target activated astrocytes and inhibit the underlying harmful molecular mechanisms to promote a protective environment after SCI.Protease-activated receptor 1(PAR1)is known to mediate neuroinflammation in the central nervous system(CNS);however,PAR1 and its endogenous activators in astrocytes are poorly characterized in SCI.The purpose of this study is to explore the role of thrombin and PAR1 in SCI,and to evaluate the treatment effect of recombinant hirudin on SCI.【Method】In vitro1.Primary culture cells,including neurons,astrocytes,and microglia were extracted and cultured.We used thrombin to mimic the thrombin high expression environment after the SCI.2.Primary culture astrocytes were treated by lipopolysaccharide(LPS)or thrombin, detected by immunofluorescence staining and western blot to observe the expression of PAR1.3.The astrocytes were treated by thrombin,recombinant hirudin,PAR1 agonist-TFLLR-NH2.Western blot was conducted to determine the expression of PAR1 and evaluated the intervention effect and mechanism of recombinant hirudin.4.We evaluated whether CM from PAR1~+astrocytes pretreated with thrombin and TFLLR-NH2 and examined the treatment effect of r-hirudin.Neurons were cultured in CM from proinflammation astrocytes and evaluated by PI staining.In vivo1.We established a standardized minimally invasive SCI model of Wistar rats and evaluated its advantages.2.The treatment effect of recombinant hirudin,an FDA-approved thrombin inhibitor,was demonstrated in a rat contusion SCI model.The potential functions and mechanisms of thrombin and PAR1 were explored by immunohistochemistry, western blotting,and m RNA sequencing(m RNA-seq).3.Histological evaluation of recombinant hirudin on SCI:Immunofluorescence,HE and Luxol Fast Blue(LFB)staining were conducted to observe the improvement of neurohistology and bladder pathology by recombinant hirudin.4.The effect of r-hirudin on functional and neural recovery after SCI was examined using Basso–Beattie–Bresnahan scores,inclined plane test,Cat Walk and electrophysiological monitoring.【result】In vitro1.The immunofluorescence staining results show that PAR1 and GFAP have achieved co-localization,that is,astrocytes can express PAR1 after thrombin treatment.Neuron PAR1 is negative,that is,the neuron does not express PAR1.2.Astrocytes can express PAR1 through thrombin in vitro,LPS cannot activate astrocytes to express PAR1.3.Recombinant hirudin reduces the expression of PAR1 in astrocytes by inhibiting thrombin activity.4.Recombinant hirudin reverses the toxic effect of PAR1~+astrocytes on neurons.In vitro1.Temporal and spatial expression of thrombin in SCI rats.After SCI,thrombin was abundantly distributed in the injury epicentre of the lesion site.2.The thrombin level began to increase at 12 h after the injury,reached a peak at day three,and then continued to decrease until day 14.3.Recombinant hirudin treatment decreases the expression of thrombin and its receptor-PAR1,and the STAT3 signaling pathway participates in the repair process of recombinant hirudin.4.Recombinant hirudin improves the nerve and behavioral functional recovery after SCI.【Conclusion】This study demonstrate that thrombin and its receptor are involved in the recovery of SCI.Thrombin,as well as PAR1,play a critical role in the regulation of astrocyte reactivation,which mediates neuroinflammatory response and astrogliosis.We,for the first time,suggest the promising treatment effect of recombinant hirudin on spinal cord injury.