The Mechanism Of Hirulog-Like Peptide(HLP) Improving Brain Ischemia And Recanalization In Cerebral Infarction Model

Background and Objective:Hirulog-like-peptide(HLP),a direct thrombin inhibitor,is successfully inhibited vascular neointimal formation or restenosis in rats,rabbits and minipigs undergoing vascular injury,furthermore,HLP can inhibit the expression of inflammatory cytokines in minipigs.Thrombin plays a central role in ischemic or hemorrhagic brain injury,and exerts its neurotoxicity.The injury of nerve cells during cerebral ischemia is mainly through the activation of thrombin receptor-1(protease-activated receptor-1,PAR-1).Therefore,the inhibition of activated thrombin receptor-1 can reduce the nerve cell necrosis,increase cerebral blood flow and improve cerebral ischemia.According to our pre-trial that HLP can suppress the expression of thrombin receptor in local injury of neointima in rats,rabbits and minipigs.It retains unclear whether HLP can reduce the cerebral tissue damage caused by vascular ischemia by prevented the activation of thrombin or PAR-1,thereby improving cerebral ischemia and getting recanalization of occlusions.The present study aims to clarify whether HLP intervention can alleviate cerebral circulation brain damage to nerve,and to further explore its mechanism.We use thromboembolism model and observe the real-time effect of the treatment to explore the recanalization in occluded the artery of HLP through in Shanghai Synchrotron Radiation Facility.Method:Established mice MCAO(Middle Cerebral Artery Occlusion)model,mice were given HLP(4mg/kg/time),low molecular weight heparin calcium(3mg/kg/time)and the control group as the same dose of saline.1 and 3 days after the intervention,all mice were done the rotarod test to measurement for motor function,neurological severity scores(NSS)for observation of the recovery of neural function,and cresyl violet for determination of infarct volume.PAR-1 was observed with immunohistochemical method.Western blotting are for the protein expression of PAR-1,CAT,Casp-3,Bcl-2 and Bax.MDA content was tested with MDA kit.Results:1.HLP amelioratd the cerebral ischemia in the mouse cerebral infarction model.After 3 days intervention,the features of rotarod test,and NSS of HLP group and low molecular weight heparin calcium(LMWH)treatment group were much better than the saline group,and the infarction area was significantly decreased(HLP group110.06±9.27mm~3,LMWH group 117.01±27.20mm~3 and saline group 243.40±35.68mm~3,P<0.05).HLP and LMWH remarkably slowed down the expression of PAR-1,the following data were saline,LMWH and HLP,respectively,(2.03±0.28,1.74±0.46)vs(1.47±0.2,P<0.05).HLP and LMWH can reduced the production of MDA,and increased the level of CAT,but HLP was better than LMWH.Both groups of treatment significantly alleviated the expression of Casp-3 and increased the ratio of Bcl-2/Bax,while the saline had no effect.After 1 day intervention,HLP and LMWH show improvement effect on cerebral ischemia,but there was no statistically significant compared with the saline group.The effect of HLP on recanalization were examind in rat thromboembolic vascular model.We found that the dose of 4mg/kg/h had obvious vascular recanalization and minimum bleeding risk.Compared to it,8mg/kg dose significantly increased bleeding and caused to death,while 2mg/kg/h showed no obvious effect.Conclusion:The findings from two type of animal models of intervention studies,HLP is better than LMWH in reducing ischemia brain lesion volume of cerebral infarction and improving cerebral ischemia and recanalization.These effects may be related to the inhibition of thrombin receptor expression,anti-oxidation and lessening the apoptotic.