| Objective: In order to determine the therapeutic effect of koumine on AD from the behavioral and pathological perspectives,the study observe the effect of koumine on cognitive performance and protein expression of Tau in hippocampus with AD model rats submitted to intrahippocampal-OA injection.The study explored the effect of koumine on the protein expression of the hallmark for astrocytes,microglia and neuron in hippocampus with AD model induced by intrahippocampal OA injection,in order to determine the possible target cells.Then,we observed the level of proinflammatory cytokines,the mRNA expression of NLRP3 and caspase-1,to analyze whether therapeutic effect of koumine on AD is associated with inflammatory protective mechanisms and to analyze whether it is associated with NLRP3 inflammasome.The study provid a theoretical basis for the development and application of koumine,and deepen the understanding of the pathogenesis of AD.Methods:(1)Rats was injected with OA into the right dorsal hippocampus to induced AD models.The experimental rats were randomly divided into control group,sham operation group,model group and koumine treatment group(koumine 0.056,0.28,1.4 mg/kg),positive drug treatment group(Huperzine A 0.36 mg/kg).At the same time as the first injection of OA,normal saline,different doses of koumine hydrochloride solution and Huperzine A solution was administered intragastricly,once a day for ten consecutive days.Assess the effect of koumine on the cognitive performance of rats by Morris water maze test on D 15~20 after the first hippocampus injection of OA.After behavioral study was finished,rat brain was harvested and the hippocampus was dissected.The expression of tau and p-tau in hippocampus of rats was detected by western blot,in order to investigate the effect of koumine on the characteristic pathological biochemical changes of OA-induced AD-like rats.(2)The expression of GFAP ? Iba-1 and NeuN was deteced by western blot and the positive immunoreactivity of hippocampal astrocytes was observed by Immunofluorescence histochemistry to clarify possible role of effector cells of koumine against.(3)And then the level of pro-inflammatory cytokine TNF-? and IL-1? was detected by enzyme-linked immunosorbent assay(ELISA)to elucidate whether therapeutic effect of koumine on AD is associated with inflammatory protective mechanismst.(4)The mRNA levels of NLRP3 and caspase-1 in rat hippocampus was analyed by RT-qPCR,to reveal the relationship between the therapeutic effect of koumine on AD and the NLRP3 inflammatory pathways.Results:(1)The MWM test and western blot showed that compared with the sham operation group,the escape latency of the model group was significantly increased in the place navigation test.And during the spatital probe trial,the number of platform-site crossovers,the distance and the time in target quandrant of the model group was decreased.The phosphorylation level of tau protein in hippocampus increased in model group,suggesting the successful establishment of AD model induced by hippocampus injection of OA.Compared with the model group,the koumine(0.056-1.4 mg/kg)significantly shortened the escape latency and increased the number of platform-site crossovers,the distance and time in target quandrant.And the level of phosphorylation tau was decreased.And the data showed a significant inverse correlation with last behavior,suggesting that koumine can improve the effect of AD model induced by intrahippocampal OA injection.(2)Western blot analysis showed that in the model group,the expression of GFAP and Iba-1 was significantly increased,and the expression of NeuN was significantly decreased.The expressions of GFAP and Iba-1 in the group were significantly lower than those in the model group,and all of them were dose-dependent.The results were moderately negatively correlated with the last behavioral data.The expression of NeuN in the koumine group was increased.However,there was no statistical difference compared with the model group,suggesting that the target cells of koumine anti-hippocampal injection of OA-induced AD may be hippocampal astrocyte and microglia.The results of immunofluorescence histochemistry showed that the number of GFAP immunoreactive cells in the model group increased,and the number of GFAP immunoreactive cells in the koumine group was significantly lower than that in the model group.The result was consistent with the results of western blot described above.(3)The results of enzyme-linked immunosorbent assay showed that the TNF-? and IL-1? levels in the model group were significantly higher than those in the sham operation group.The levels of TNF-? and IL-1? in the koumine group were also significantly lower than those in the model group.The results were slightly negatively correlated with the last behavioral data,and its inhibition of IL-1? was dose-dependent,suggesting that the effect of koumine on hippocampus-induced AD is related to the decrease of TNF-? and IL-1?.(4)RT-qPCR results showed that the transcription levels of NLRP3 and caspase-1 mRNA in the model group were significantly higher than those in the sham operation group.The transcription levels of NLRP3 and caspase-1 mRNA in the koumine group were also significantly lower than those in the model group,and they were all dose-dependent.There was a negative correlation with the last behavioral data,suggesting that the effection of koumine on AD iinduced by intrahippocampal OA injection may be associated with inhibition of NLRP3 inflammasome.Conclusion: 1.Koumine can ameliorate the learning and memory dysfunction and characteristic pathological changes in AD model induced by intrahippocampal OA injection.2.The hippocampal astrocytes and microglia may be the effector cells of the therapeutic effect of koumine on AD induced by OA.3.The therapeutic effect of koumine on AD induced by OA may be related to the protective mechanism of inflammation.The therapeutic effect of koumine on AD might be related the inhibition of the activation of microglia and astroyte in hippocampus,and the reduction of the expression of proinflammatory cytokine TNF-? and IL-1? in hippocampus,and the inhibition NLRP3 inflammasome function. |
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