| Objective1.To observe the relationship between brain injury and NLRP3(The nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)inflammasome in rats after cardiopulmonary bypass(CPB).2.To investigate the effects of alpha-7 nicotinic acetylcholine receptor(α7 nAChR)agonist on NLRP3 inflammasome and brain injury after CPB in rats.MethodsForty clean and healthy adult male SD rats,weighed 350-450g and aged 10-12 weeks,were randomly divided into four groups:sham-operated group(group S)、CPB group(group C)、α7nAChR agonist PHA568487+CPB group(group CP)、and α7nAChR antagonist Methyllycaconitine(MLA)+PHA568487+CPB group(group CPM),with 10 rats in each group.Rats received 2%Sodium pentobarbital 50 mg/kg intraperitoneal injection for anesthesia,then tracheal intubation and mechanical ventilation with small animal ventilator.After skin preparation and disinfection,left saphenous artery of rats were punctured and cathetered for monitoring blood pressure;left great saphenous vein were punctured and cathetered for infusion,before heparin 400 IU//kg intravenous injection.Then right saphenous artery combined with right internal jugular vein were punctured and cathetered on group C for CPB 1 h;group S also punctured and cathetered,but with no CPB;group CP received 0.8mg/kg PHA568487 intraperitoneal injection 30 minutes before CPB,then the same with group C;group CPM received 6mg/kg MLA intraperitoneal injection 45 minutes before CPB,then the same with group CP.Morris water maze navigation training was performed twice a time on every morning and afternoon for five days in all rats before operation.On the third day after operation,Morris water maze was used again to test the escape latency and times of 120s crossing the original platform of rats in each group.After 2 hours,rats were received anesthesia and perfused with normal saline at 4℃before blood drawn from vena cava.Then rats were decapitated immediately,and one side of hippocampus was extracted and frozen at-80℃.Apoptotic index(AI)was calculated by TUNEL staining on the other side brain.The frozen hippocampus was ground and centrifuged.The concentrations of IL-1β and IL-18 in plasma and hippocampus were detected by ELISA,caspase-1 activity in hippocampus was detected by caspase-1 kit,expression of NLRP3,ASC and pro-caspase-1 protein in hippocampus was detected by Western blot,and mRNA expression of NLRP3,ASC and caspase-1 in hippocampus was detected by RT-PCR.SPSS 19.0 software was used to analyze the data.The measurement data were expressed as mean± standard deviation(x±s).One-way ANOVA was used to compare differences of groups,and LSD-t test was used to compare two groups.The test level α=0.05,P<0.05 was considered to have statistical significance.Results1.Results of the escape latency time and times of traversing original platform in 120s in rats three days after CPB.Compared with group S,the escape latency time was prolonged and times of traversing original platform in 120s was decreased in goup C、CP and CMP;compared with group C,the escape latency time was shorted,and times of traversing original platform in 120s was increased in goup CP;compared with group CP,the escape latency time was prolonged and times of traversing original platform in 120s was decreased in goup CMP(P<0.05).2.Results of TUNEL staining in rats three days after CPB.Compared with group S,the AI increased in group C、CP and CMP;compared with group C,the AI decreased in group CP;compared with group CP,the AI increased on group CPM(P<0.05).3.Results of IL-1β and IL-18 levels in plasma and hippocampus detected by ELISA in rats three days after CPB.Compared with group S,the levels of IL-1β and IL-18 in plasma and hippocampus increased in group C、CP and CPM;compared with group S,the levels IL-1β and IL-18 decreased in group CP;and compared with group CP,the levels of IL-1β and IL-18 increased in group CPM(P<0.05).4.Results of caspase-1 enzyme activity test in rats three days after CPB.Compared with group S,the level of activity of Caspase-1 increased in group C、CP and CPM;compared with group C,the level of activity of caspase-1 decreased in group CP;compared with group CP,the level of activity of caspase-1 increased in group CPM(P<0.05).5.Results of protein expression of NLRP3、ASC and pro-caspase-1 detected by Western blot in rats three days after CPB.Using GAPDH as internal reference,the relative protein expression of NLRP3、ASC and pro-caspase-1 in hippocampus of rats showed that:compared with group S,the protein expression of NLRP3、ASC and pro-caspase-1 increased in group C、CP and CPM;compared with group C,the protein expression of NLRP3、ASC and pro-caspase-1 decreased in group CP;and compared with group CP,the protein expression of NLRP3、ASC and pro-caspase-1 increased in group CPM(P<0.05).6.Results of mRNA expression of NLRP3、ASC and caspase-1 detected by RT-PCR in rats three days after CPB.Relative quantitative method 2-△△Ct was used to calculate the expression of mRNA in each group relatively to group S.The results showed that:compared with group S,the mRNA expression of NLRP3、ASC and caspase-1 increased in group C、CP and CPM;compared with group C,the mRNA expression of NLRP3、ASC and caspase-1 decreased in group CP;and compared with group CP,the mRNA expression of NLRP3、ASC and caspase-1 increased in group CPM(P<0.05).Conclusions1.CPB can induce inflammatory brain injury in rats,and NLRP3 inflammasome play an important role in this process.2.α7nAChR agonist PHA568487 can play a protective role by inhibiting NLRP3 inflammasome and alleviating brain injury after CPB in rats. |
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