Protective Effect And Mechanism Of HDL On Cerebral Ischemia-reperfusion Injury By Inhibiting NLRP3

Objective: In this study,rat models of ischemia-reperfusion were established to observe the degree of brain injury at different perfusion times and to treat different doses of high-density lipoprotein.To investigate the protective effect and related mechanism of high density lipoprotein(HDL)on rat brain injury induced by ischemia-reperfusion.Methods:(1)An middle cerebral artery occlusion ischemic-reperfusion model(MCAO)was established by blocking the middle cerebral artery(MCA)of rats.Rats were sacrificed 1.5 h after ischemia,and then reperfusion for 12 h,24 h,and 72 h,respectively.Rats were sacrificed after reperfusion for 12 h,24 h,and 72 h.Brain injury tissue was taken for TTC staining to detect the infarct volume of rats after different reperfusion injury time,neurological impairment in rats was assessed according to the Bederson scoring criteria.Quantitative Real-time PCR(qRT-PCR)was used to detect the expression of NLRP3 mRNA in inflammatory corpuscles after brain injury at different perfusion times.A suitable animal model was established for the subsequent study of the protective effect of HDL on ischemia-reperfusion brain injury.(2)Further,a suitable ischemia-reperfusion model was selected to study the protective effect of HDL on the nerves of ischemia-reperfusion brain injury and its specific mechanism.Animals were divided into sham operation group,MCAO model group,low dose HDL/MCAO treatment group,medium dose HDL/MCAO treatment group,and high dose HDL/MCAO treatment group.In the HDL treatment group,10 mg/kg,25 mg/kg,50 mg/kg HDL were injected into the tail vein 15 min before ischemia.The rats were sacrificed after reperfusion 72 h.TTC staining was performed to detect the improvement of cerebral infarction volume in rats with ischemia-reperfusion by different doses of HDL.Rat neurological ischemia was scored by the Bederson scoring criteria.The expressions of IL-18 and IL-1 β in the ischemic penumbra were detected by enzyme-linked immunosorbent assay(ELISA)and qRT-PCR.Western blotting was used to detect the expression of NLRP3 and caspase1,and the effects of different doses of HDL on the expression of NLRP3 and caspase1 were analyzed.Results:(1)The results of TTC staining showed that compared with the sham operation group,the ischemic side of the ischemic reperfusion group showed that the ischemic side of the brain was white and different degrees of cerebral infarction occurred.With the prolongation of ischemia-reperfusion time,the TSH staining showed that the ischemic reperfusion was 12 h,24h and 72 h.The volume of cerebral infarction was significantly increased(P<0.05).The neurological function score of the MCAO model group were significantly higher than the sham operation group,and the difference was statistically significant(P<0.05).With the prolongation of ischemia-reperfusion time,the neurological dysfunction in rats was more significant,and the difference was statistically significant(P<0.05).The results of qRT-PCR showed that the mRNA expression of NLPR3 was up-regulated in the brain tissue of MCAO 12 h group compared with the sham operation group(p<0.05).The mRNA expression of NLPR3 in the brain tissue of rats with MCAO 24 h and 72 h injury was significantly up-regulated(p<0.05,p<0.01).The brain damage of rats was the most significant after 72 hours of ischemia-reperfusion.(2)The MCAO 72 h model group was selected to further study the protective effect of HDL on ischemia-reperfusion brain injury.Neurological ischemic scoring showed that the neurological scores of the MCAO model group were significantly higher than the sham-operated group(P>0.05).Compared with the model group,10 mg/kg of HDL did not significantly improve neurological impairment in rats(P>0.05).After 25 mg/kg of HDL treatment,the neurological score of rats was significantly decreased(P<0.05),50 mg/ After HDL treatment of kg,the degree of neurological function score was the most significant(P<0.01).The results of ELISA and qRT-PCR showed that the expression levels of IL-18 and IL-1β protein and mRNA in the cerebral ischemic penumbra were significantly different in the MCAO model group compared with the sham operation group.Compared with the model group,the expression of IL-18 and IL-1β protein and mRNA in the cerebral ischemic penumbra were significantly decreased after treatment with 25 mg/kg or 50 mg/kg HDL(P<0.05,P< 0.01).Western blot results showed that the expression levels of NLRP3 and caspase1 were significantly up-regulated in the MCAO model group 72 h after reperfusion compared with the sham-operated group(P<0.05).There was no significant change in the expression of NLRP3 and caspase1 after treatment with 10 mg/kg HDL(P>0.05),25 mg.After treatment with /kg and 50mg/kg HDL,the expression levels of NLRP3 and caspase1 in the cerebral ischemic penumbra were significantly down-regulated(P<0.05,P<0.01).Conclusion:(1)The model of cerebral ischemia-reperfusion injury was successfully established by the method of middle-arterial occlusion.With the prolongation of ischemia-reperfusion time,the degree of infarction in the brain tissue of rats increased,the neurological function of rats was severe,and the inflammation was small.The mRNA expression level of NLRP3 was significantly up-regulated,which was significantly higher than that of the sham operation group(P<0.05).(2)Middle and high doses of HDL treatment have protective effects on the brain of rats induced by ischemia-reperfusion.Medium and high doses of HDL can significantly reduce the neurological function score of rats with ischemia-reperfusion and improve the infarct volume of ischemia-reperfusion rats after 72 hours of ischemia-reperfusion,down-regulate the expression of IL-18 and IL-1β-protein and mRNA induced by cerebral ischemia-reperfusion.HDL protects against ischemia-reperfusion brain injury by inhibiting the expression level of NLRP3 in cerebral ischemic penumbra tissue.