Therapeutic Effect Of Koumine On Intrahippocampal Aβ_1-42 Injection Induced-alzheimer’s Disease And Its Possible Mechanism With NLRP3 Inflammasome

Objective:In order to clear and definite the therapeutic effect of koumine on Alzheimer’s disease（AD）,rats were injected oligomeric Aβ_1-42（amyloid-beta 1-42）into bilateral hippocampus.And then the study reserach the effect of koumine on cognitive performance by behavioral perspectives and impact of hippocampal pathology on rats.And then the study explored the effect of koumine the relationship with astrocyte and microglia anti-AD.Next,we investigated whether koumine could affect the NLRP3inflammasome,and finally we analyzed whether koumine could affect downstream signaling molecules by affecting the NLRP3 inflammasome pathway.The study provid theoretical and experimental evidence for the potential clinical application of koumine,and is helpful to explore the role of the NLRP3 inflammasome pathway in the pathogenesis of AD.Methods:（1）The rats were randomly diveded into sham,model,koumine（0.056,0.28,1.4 mg/kg）and positive drug group（Huperzine A 0.36 mg/kg）.AD animals were established by injecting oligomeric Aβ_1-42（amyloid-beta 1-42）into bilateral hippocampus.The normal saline,corresponding doses of koumine hydrochloride solution and Huperzine A solution were administered intragastrically,once a day for ten days.We observe the effect of koumine on cognitive performance by the step-down test、Morris water maze test and novel object recognition test.（2）After the behavioral experiment,HE staining and Nissl staining were used to observe the effects of koumine on pathological characteristics in AD rats.（3）The expression of GFAP and IBA-1 were observed by western blot and immunofluorescence,which determine whether the effect of koumine on the improvement of AD-like rat was related to the inhibition of glial cell.（4）The protein and m RNA levels of NLRP3,ASC and caspase-1 were analyed by western blot and RT-q RCR.To determine whether the effect of koumine on improving AD-like rat was related to NLRP3 inflammasome,the caspase-1 activity assay kit was used to test the caspase-1 activity.（5）To analyze the downstream signaling molecules of NLRP3 inflammasome,the proteins of Gasdermin D was detected by western blot,and then the level of proinflammatory cytokines IL-1βand IL-18 were detected by western blot and RT-q RCR.Result:（1）The step-down test showed that the escape latency of the model group was decreased and the error times was increased compared with the sham group,the koumine treatment group significantly increased the escape latency and decreased the error times.The MWM test showed that,for 5 consecutive days of directional navigation,compared with the sham group,the escape latency of the model group was prolonged,and the number of platform-site crossovers and the time in target quandrant of the model group was decreased.Compared with the model group,the koumine significantly shortened the escape latency and increased the number of platform-site crossovers and the time in target quandrant.The novel object recognition test showed that the index of exploring new objects in model group was lower than that the sham group,while the preferential index and discrimination index of the koumine group were higher than that of the model group.The above behavioral results showed that the koumine can improve the learning and memory ability of AD rat.（2）The results of HE indicated that,compared with the sham group,cells in the hippocampus of the model group were damaged,cells arrangement were sparse,the number of cells were significantly reduced.Compared with the model group,the koumine group could improve the abnormal cell structure in the hippocampus,the pyramidal cells were closely arranged.Nissl staining showed that nissl body was lost in the model group,more nissl bodies could be observed in the koumine-treat group.The above pathological results showed that the koumine can improve morphology of AD rats.（3）Comparing with the sham group,western blot and immunofluorescence showed that the expression of GFAP and IBA-1 of the AD group was remarkablely increased and the koumine group was remarkablely decreased.The results showed that koumine may reduce the expression of GFAP and IBA-1 to improved AD symptoms.（4）Western blot showed that the expression of NLRP3 protein and cleaved-caspase-1 were increased in the model group,which were decreased in the koumine group,while the expression of ASC and pro-casapse-1 were not significantly different.RT-q PCR results showed that transcription levels of NLRP3 and caspase-1 m RNA were remarkablely increased in the model group,the koumine group could reverse these characteristics.Caspase-1 activity results showed that caspase-1 activity was increased in the model group,which was decreased in the koumine group.The above results suggested that the koumine may inhibit NLRP3 inflammasome activation to cure AD.（5）Western blot results showed that the expression level of Gasdermin D of the key protein of pyroptosis was increased compared with the sham group,and compared with the model group,the expression levels of Gasdermin D was decreased in the koumine group.The levels of pro-inflammatory cytokine IL-1βand IL-18 were increased in the model group.Compared with model group,transcription and expression of proinflammatory cytokines IL-1βand IL-18 were decreased in the koumine group.The above western blot and RT-q PCR results suggested that the therapeutic effect of koumine may reduce the expression of Gasdermin D and proinflammatory cytokine IL-1βand IL-18.Conclusion:（1）Koumine could improve the learning and memory impairment and hippocampal pathology on intrahippocampal Aβ_1-42 injection induced-Alzheimer’s Disease.（2）The therapeutic effect of koumine on AD might be related to inhibit astrocytes and microglia,decrease NLRP3 inflammasome,inhibit the cleaved-caspase-1,and reduce the expression of Gasdermin D and extracellular proinflammatory cytokine IL-1βand IL-18.