| Objective:The middle cerebral artery occlusion(MCAO)rats were used as animal models,To explore the effects of cognitive impairment after cerebral ischemic stroke in different time windows with electroacupuncture based on NLRP3/Caspase-1 pathway.Methods:Sixty SPF grade male SD rats weighing(260±20)g were randomly divided into sham group,model group and electroacupuncture group.The electroacupuncture group was divided into three subgroups: 1-hour electroacupuncture group,6-hour electroacupuncture group and24-hour electroacupuncture group,with 12 rats in each group.The MCAO model was prepared in the model group and the electroacupuncture group with reference to the Longa modified suture method.In the sham group,the blood vessels were sutured after blunt separation,and the remaining procedures were the same as in the model group.The electroacupuncture group received electroacupuncture at Shenting and Baihui points for intervention.The 1-hour electroacupuncture group received the frist electroacupuncture1-hour after operation(before removing the embolus),the 6-hour electroacupuncture group received the frist electroacupuncture 6-hour after operation,and the 24-hour electroacupuncture group received the frist electroacupuncture 24-hour after operation,once a day,for 20 minutes,7 consecutive days.The model group and the sham group were subjected to the same conditions as the electroacupuncture group for grasping and binding,and then returned to the cage,without treatment.The neurobehavioral score and body weight were recorded on the 1st,3rd and 7thday after operation;Watr maze test was performed 3-7 days after operation;At the end of the electroacupuncture intervention on the 7th day after the operation,3 rats in each group were randomly selected to observe the cerebral ischemia status with 2,3,5-triphenyl tetrazolium chlo-ride(TTC)staining;Three rats in each group were randomly selected to observe the pathological changes of hippocampal cells by HE(hematoxylin-eosin)staining;Real-time quantitative polymerase chain reaction(RT-q PCR)detection of NLRP3、Caspase-1、 IL-1β 、IL-18 in hippocampus,Western blot(WB)was used to detect the expression of NLRP3、Caspase-1、IL-1β and IL-18 in the hippocampus.Results:1.Nerve function defect score: It can be seen that there was no neurological deficit in the sham group,and the neurological deficit scores in the electroacupuncture group and the model group were significantly higher than those in the sham group on the 1st,3rd,7th day after operation(P < 0.05);On the 1st and 3rd day after operation,there was no statistically significant difference in the scores between each electroacupuncture group and the model group.On the 7th day after operation,the scores of each electroacupuncture group were significantly improved compared to the model group(P<0.05);Compared with the first day,the body mass of the model group decreased(P<0.05),while the body mass of the 1-hour and6-hour electroacupuncture groups increased(P<0.05).2.Body mass: On the 1st day after operation,the body mass of the model group and the24-hour electroacupuncture group was significantly lower than that of the sham group(P<0.05);On the 3rd day after operation,the body mass of the model group and each electroacupuncture group was significantly lower than the sham group(P<0.05),The body mass of the 1-hour and 6-hour electroacupuncture group was significantly higher than the model group(P<0.05),Compared with the 1st day,the body mass of the model group and the24-hour group decreased significantly(P<0.05);On the 7th day after operation,the body mass of the model group and the 24-hour electroacupuncture group was lower than that of the sham group(P<0.05),and the body mass of each electroacupuncture group was higher than that of the model group(P<0.05).3.Rat escape latency: On the 3rd day after operation,the escape latency in the model group and the 24-hour electroacupuncture group was significantly higher than sham group(P<0.05);On the 4th day after operation,the escape latency in the model group was higher than sham group(P<0.05);On the 5th day after operation,the escape latency in the model group was higher than sham group(P<0.05),and the escape latency in each electroacupuncture group was significantly lower than model group(P<0.05);On the 6th day after operation,the escape latency in the model group was still higher than that in the sham group(P<0.05),and the escape latency in each electroacupuncture group was significantly lower than model group(P<0.05),the escapelatency in the 1-hour electroacupuncture group was significantly lower than that in the 24-hour electroacupuncture group(P<0.05);Except for the model group,the escape latency in the sham group and each electroacupuncture group was significantly reduced compared to the 1st day(P<0.05).4.Cerebral infarction area: Brain slices in the sham group showed bright red,while large areas of gray white ischemic foci could be observed on the affected side of brain slices in the model group.Small areas of ischemic foci could be found on the affected side of each electroacupuncture group,and this area was smaller than that in the model group.It can be observed in the figure that the ischemic area of each electroacupuncture group was 1-hour electroacupuncture group < 6-hours electroacupuncture group < 24-hours electroacupuncture group.5.Pathological damage of hippocampus: the morphology and structure of neuronsin the sham group were normal,the division of nucleus and cytoplasm was clear,the nucleolus was obvious,and there was no inflammation;In the model group,the cerebralcortex of the affected side was extensively infarcted,the tissue staining became hallow,a large number of neurons were necrotic,the nuclear dissolution,the eosinophilicity of the cytoplasm was enhanced,accompanied by a large number of new blood vessels,and a large number of lymphocytes and macrophages were infiltrated,multiple edema was seen in the meninges,and the arrangement of connective tissue was loose,suggesting that the brain injury was serious;Compared with the model group,the pathological damage of rats in the electroacupuncture group hasimproved to varying degrees,thenumber of nerve cells has increased,the vacuolar degeneration has decreased,the nucleus is clear,and the staining is more uniform.A small number of neurons in the electroacupuncture group still have pyknotic nuclei,unclear cytoplasmic boundaries,and enhanced basophilia.6.The expression level of NLRP3、Caspase-1、IL-1β、IL-18 m RNA in affected hippocampus:Compared with the sham group,the expression of NLRP3、Caspase-1、IL-1β、IL-18 m RNA in the model group was significantly increased(P<0.05);Compared with the model group,the expression of NLRP3、Caspase-1、IL-1β、IL-18 in the 1-hour electroacupuncture and 6-hour electroacupuncture group decreased significantly(P<0.05),The expression of caspase-1 、IL-1β、IL-18 in the 24-hour electroacupuncture group significantly decreased(P<0.05);The relative expression of NLRP3 in the 24-hour electroacupuncture group was significantly higher than sham group and 1h、6h electroacupuncture groups(P<0.05).7.Relative expression of NLRP3、Caspase-1、IL-1β、IL-18 protein in affected hippocampus:Compared with sham group,the protein expression of NLRP3、Caspase-1、IL-1β、IL-18 in model group increased(P<0.05);Compared with the model group,the protein expression of NLRP3、Caspase-1、IL-1β、IL-18 in each electroacupuncture group decreased(P<0.05);The protein expression of NLRP3、Caspase-1、IL-1β、IL-18 in the 24-hour electroacupuncture group was significantly higher in the sham group than in the sham group(P<0.05),and significantly lower than the model group(P<0.05);Compared with the 1-hour electroacupuncture group,the expression of Caspase-1 protein in the 24-hour electroacupuncture group significantly decreased(P<0.05);The expression of Caspase-1protein in the 24-hour electroacupuncture group was significantly lower than 6-hour electroacupuncture group(P<0.05).Conclusions:1.Electroacupuncture at Shenting and Baihui points can effectively improve neurological and cognitive dysfunction in rats with cerebral ischemia and reperfusion.2.The effect of interventional electroacupuncture treatment at 1 hour and 6 hour after the onset of CIS is better than that at 24 hours.3.The mechanism of electroacupuncture improving cognitive dysfunction after CIS may be related to the NLRP3/Caspase-1 pathway.Electroacupuncture inhibits NLRP3、Caspase-1、IL-1β、IL-18 on the NLRP3/Caspase-1 pathway.protect brain neuron cells,reduce cell death,and improve cognitive function in CIS. |
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