Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway

Argatroban is a synthetic thrombin inhibitor approved by U.S.Food and Drug Administration for the treatment of thrombosis.However,whether it plays a role in the repair of spinal cord injury is unknown.In this study,we established a rat model of T10 moderate spinal cord injury using an NYU Impactor ModerⅢand performed intraperitoneal injection of argatroban for 3 consecutive days.Our results showed that argatroban effectively promoted neurological function recovery after spinal cord injury and decreased thrombin expression and activity in the local injured spinal cord.RNA sequencing transcriptomic analysis revealed that the differentially expressed genes in the argatroban-treated group were enriched in the JAK2/STAT3 pathway,which is involved in astrogliosis and glial scar formation.Western blotting and immunofluorescence results showed that argatroban downregulated the expression of the thrombin receptor PAR1 in the injured spinal cord and the JAK2/STAT3 signal pathway.Argatroban also inhibited the activation and proliferation of astrocytes and reduced glial scar formation in the spinal cord.Taken together,these findings suggest that argatroban may inhibit astrogliosis by inhibiting the thrombin-mediated PAR1/JAK2/STAT3 signal pathway,thereby promoting the recovery of neurological function after spinal cord injury.

Baicalin Attenuates Focal Cerebral Ischemic Reperfusion Injury by Inhibition of Protease-Activated Receptor-1 and Apoptosis

Objective： To investigate the neuro-protective effects of baicaiin in Wistar rats with focal cerebral ischemic reperfusion injury. Methods： Ninety adult male Wistar rats weighing 320-350 g were randomly divided into the following groups （n=5）： （a） sham control group; （b） vehicle group, subjected to middle cerebral artery occlusion and received vehicle intraperitoneally; （c-e） baicalin groups, which were subjected to the middle cerebral artery occlusion and treated with baicalin 25, 50 and 100 mg/kg, respectively. The neurological scores were determined at postoperative 1, 3 and 7 d after the treatment. The expression of protease-activated receptor-1 （PAR-1）, PAR-1 mRNA and Caspase-3 were determined using Western blot, reverse transcription polymerase chain reaction （RT- PCR） analysis and immunohistochemistry, respectively. Results： Significant decrease was noted in the neurological score in the baicalin group compared with that of the vehicle group （P〈0.01）. Additionally, down-regulation of PAR-1 mRNA, PAR-1 and Caspase-3 was observed in the baicalin groups compared with those obtained from the vehicle group （P〈0.01）. Compared with the low-dose baicalin group （25 mg/kg）, remarkable decrease was noted in neurological score, and the expression of PAR-1 mRNA, PAR-1 as well as Caspase-3 in the high-dose group （P〈0.05）. Conclusion： Baicalin showed neuro-protective effects in focal cerebral ischemic reperfusion injury through inhibiting the expression of PAR-1 and apoptosis.

Effect of Thrombin on the Apoptosis of Hippocampal Neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activatingpeptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. Thenumbers of apoptotic cell and apoptotic rate of hippocampal neurons treated bydifferentconcentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyltransferase (TdT) mediated dUTP-biotin nick end-labeling (TUNED method and Flow Cytometry. When theconcentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neuronsreached peak value, were 27. 3 +- 4. 0 and (29. 333 +- 4. 633 ) % , respectively.Immunocytochemistry assay show that Bcl-2 protein expression was down- regulated and Bax proteinexpression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effectof thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombininduced hippocampal neuron apoptosis in a dose-dependent manner through activatingprotease-acti-vated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related withthe effect of high concentration thrombin induced apoptosis on hippocampal neurons.