| Objective: Alzheimer's disease (AD) is the most frequent neurodegenerative disorder. The pathogenesis of AD is unknown. It has been well established that generation and deposition of beta-amyloid (Aβ) peptide is important in AD pathogenesis.Puerarin is an effective natural drug monomer, which is isoflavone extracted from the dry roots of Wild Kudzu Vine, a plant in the genus Pueraria of the family Leguminosae. Puerarin has shown a wide variety of pharmacological action, but its effect on AD is unclear.In the present study, it was performed that the injection of Aβ25～35 into the hippocampus of Wistar rats induced AD. The aims of the study are to try to investigate the effect of puerarin on Aβ-induced learning and memory deficits in rats and its mechanisms of action.Methods and Results:1 The effect of puerarin on Aβ-induced learning and memory deficits in ratsImpairment of learning and memory functions was examined using the Morris Water Maze test. In place navigation test, the average escape latency (AEL) shortening gradually indicated that all rats became more efficient at recognizing and remembering the hidden platform on successive trainings. On days 1-3 of the 5-days training duration, the increased AEL was significantly different for AβGroup compared to Control Group (P<0.05), suggesting that hippocampus injections of Aβ(25–35) resulted in impairment of learning and memory performances; AEL shortened statistically in Puerarin Group when compared to AβGroup (P<0.05), but the result was not statistically different for Puerarin Group vs. Control Group (P>0.05), suggesting that puerarin treatment improved Aβ(25–35)-induced learning and memory deficits in Wistar rats. On the last 2 days of training duration, learning and memory performances of all rats approached to the stable level, therefore, AEL did not differ among groups (P>0.05).In spatial probe test, AβGroup rats always swam along the wall, and spent more time in outer ring of the pool, while Puerarin and Control Group rats spent more time in the platform quadrant. The frequency of crossings over the platform position was significantly decreased in AβGroup compared to Control Group (P<0.05), while the frequency of crossings over the platform position was significantly increased in Puerarin Group compared to AβGroup (P<0.05). It suggested that hippocampus injections of Aβ(25–35) resulted in impairment of spatial memory function in rats, but puerarin treatment can lessen the Aβ-induced learning and memory deficits. This result is in good agreement with the above results in place navigation test.2 The intervention of puerarin to Aβ-induced inflammatory response in hippocampus of ratsOn HE-stained section, the pyramidal cell layer of the hippocampus of Wistar rat was well-defined and integrity structurally, and neurons disposed in regular, integrity and compact multiplayer with well-defined cytoplasm and large, round and clear nuclei and clear nucleoli in Control Group. But there was the phenotype of severe inflammatory response in the AβGroup: structurally destroyed pyramidal cell layer of the hippocampus, decreased and confused number of layer, large amounts of inflammatory cells aggregation in and beside pyramidal cell layer, even"strip breakage"presence (all neurons loss in a segmental pyramidal cell layer and substitution with lots of inflammatory cells and cell debris). Number-decreased neurons were in paramorphia, size disparity and maldistribution, and some neurons were obscure boundary or hyperchromic or hypochromic, even"vacuolizational". There is the phenotype of mild inflammatory response in the Puerarin Group: pyramidal cell layer of the hippocampus was still integrity structurally but less and confused number of layer and a small number of flammatory cells infiltration. Some neurons were paramorph, and"vacuolization"is seldom. On silver-stained section, the neurofibra were clear, close-up and well-arranged in regularity in Control Group, and the neurofibra were sparse, thick or thin, deep or light, and disposed chaotically in AβGroup, and in Puerarin Group, the neurofibra were well-arranged and similar to those in Control Groups.The number of neurons in pyramidal cell layer of the hippocampus was counted on HE-stained section, and statistical comparisons were made. This number significantly decreased in AβGroup compared to Control Group (P<0.05), and this number significantly increased in Puerarin Group compared to AβGroup (P<0.05). These indicate that puerarin can preserve neuron, and prevent from Aβ-induced neuron loss. It could be one of pathological mechanisms of the beneficial effect of puerarin on Aβ-induced learning and memory deficits in ratsOn Nissl-stained section, intracytoplasmic Nissl bodies are decreased, vague, or even disappeared in impaired and parafunctional neurons. The number of neurons containing less, vague or disappeared Nissl bodies in hippocampus was counted and statistical comparisons were performed. This number significantly increased in AβGroup compared to Control Group (P<0.05), and this number significantly decreased in Puerarin Group compared to AβGroup (P<0.05). These indicate that puerarin do not only prevent from Aβ-induced neuron loss, but also preserve neuronal normal structure and functions, and diminish neuronal damage induced by Aβ.Neurofibrillary tangle (NFT) is the characteristic pathological appearance in AD. NFT can be observed on HE-stained and silver-stained sections. On silver-stained section, the number of neurons bearing NFT in hippocampus was counted and statistical comparisons were performed. Neurons bearing NFT found in hippocampus were seldom in Control Group, but the number significantly increased, and it is easy to find in AβGroup compared to Control Group (P<0.05), however, this number significantly decreased in Puerarin Group compared to AβGroup (P<0.05).Ultrastructural changes of neurons in hippocampus of rats were observed under electron microscope, and the analysis of neuronal inflammatory damage score was performed using a scoring standard. The inflammatory damage in neurons in hippocampus of rats was slight in Control Group, but it aggravated significantly in AβGroup (P<0.05), however, puerarin treatment improved it significantly (P<0.05).The results suggested that the intensive inflammatory response induced by hippocampus injections of Aβ25-35 could be the pathological basis of Aβ-induced learning and memory deficits, and that suppression of Aβ-induced inflammatory response in hippocampus by puerarin treatment could be the pathological mechanisms of the beneficial effect of puerarin on Aβ-induced learning and memory deficits.3 The molecular mechanisms of puerarin suppressing Aβ-induced inflammatory response in hippocampusThe transcription factor nuclear factor-κB (NF-κB) exerts an important role in inflammatory response in AD brains. The level of expression of intranuclear NF-κB p65 subunit can demonstrate the activity of NF-κB. Immunohistochemistry staining was performed to show that the p65 subunit immunoreactive positive particles were located in the cytoplasm and the nucleus. Few p65 immunoreactive positive cells and mild p65 immunostaining was observed in the Control Group. Significantly increased p65 positive cells and deeper immunostaining was seen in AβGroup, but significantly less p65 positive cells and lighter immunostaining was found in Puerarin Group. Western blot analysis was performed to visualize the level of p65 subunit expression in the hippocampus. Control Group p65 protein bands were light and thin, and AβGroup p65 protein bands were obviously deeper and thicker than that in Control Group, but Puerarin Group p65 protein bands were lighter and thinner than that in AβGroup. All bands were quantified by Bio ID digital imaging analysis system and presented as relative density ratios. This result showed that there were very low levels of p65 subunit protein expression in Control Group, and sharp increase of p65 subunit protein expression in AβGroup (P<0.01), but this increase was inhibited in Puerarin Group (P<0.05), indicating activation of NF-κB signaling pathway in the process of hippocampus injections of Aβ25-35 resulting in impairment of learning and memory, and suppression of NF-κB activation by puerarin.NF-κB activation could mediate lethal damage to neurons via up-regulation of COX-2 and iNOS. Immunohistochemistry staining was used to show the locations of COX-2 and iNOS in neurons in hippocampus. Few COX-2 immunoreactive positive cells were observed in the Control Group. Significantly increased COX-2 positive cells and deeper immunostaining was found in AβGroup, and the positive particles in the cells increased and were located in the cytoplasm. But less COX-2 positive cells and lighter immunostaining was found in Puerarin Group than those in AβGroup. Western blot analysis was performed for more precise quantitative assay. The results showed that there was very low level of COX-2 protein expression in Control Group and a sharp increase of COX-2 expression in AβGroup (P < 0.01), but COX-2 expression decreased significantly in Puerarin Group compared to AβGroup (P < 0.05). These data demonstrated that puerarin can inhibit COX-2 expression.The iNOS immunoreactive positive particles were located in the cytoplasm. In Control Group, there were hardly iNOS immunoreactive positive cells. Lots of iNOS positive neurons were seen in hippocampus in AβGroup. Less iNOS positive neurons were found in Puerarin Group compared to AβGroup. It is showed by Western blot analysis that there was low level of iNOS protein expression in Control Group and a notable increase of iNOS expression in AβGroup compared to Control Group (P < 0.01), but iNOS expression decreased in Puerarin Group compared to AβGroup (P < 0.05). It suggested that puerarin can inhibit Aβ-induced increase of iNOS expression.4 The intervention of puerarin to Aβ-induced neuronal apoptosis in hippocampus of rats and its mechanisms of actionHE-staining can detect neuronal apoptosis in the pyramidal cell layer of the hippocampus of rat. The apoptotic neurons displayed polygon, triangle, sliver or fusiform in small volume and lumping or scattering. Both nuclei and cytoplasm were pycnotic and hyperchromic. They were often found in the CA1 and CA3 regions of the hippocampus. Control Group apoptotic neurons were rare and scattering, but apoptotic neurons were frequent and lumping, even occupying a segment of hippocampal pyramidal cell layer in AβGroup, and the situation in Puerarin Group was between the two groups. On TUNEL-stained section, most of neurons with above-mentioned morphous were TUNEL positive neurons, so confirmed to be apoptotic neurons. The TUNEL positive neurons were counted and analyzed. Control Group TUNEL positive neurons were rare. The number increased obviously for AβGroup than Control Group (P<0.05), but the number decreased obviously for Puerarin Group than AβGroup (P<0.05). The ratios of apoptotic neurons in hippocampus were detected by flow cytometry. The apoptotic ratio was low in Control Group, and there was a notable increase of the ratio in AβGroup compared to Control Group (P<0.05), but the ratio was obviously lower in Puerarin Group compared to AβGroup (P<0.05). It suggested that hippocampus injections of Aβ25-35 can induce neuronal apoptosis in hippocampus of rats, and puerarin can inhibit it.It was showed by immunohistochemistry staining that the BAX protein immunostaining positive particles were located in the cytoplasm. Few BAX immunoreactive positive neurons were observed in the Control Group. Significantly increased BAX positive neurons and deeper immunostaining was found in AβGroup than those in Control Group, and less BAX positive neurons and lighter immunostaining was found in Puerarin Group than those in AβGroup. Western blot analysis was performed for quantitative assay of BAX expression in hippocampus of rat. BAX expression increased significantly in AβGroup compared to Control Group (P<0.05), but BAX expression decreased significantly in Puerarin Group compared to AβGroup (P<0.05). Immunohistochemistry staining was performed to show the expression of bcl-2 in neurons in hippocampus. There was basal level of BCL-2 protein expression in Control Group, and the immunostaining positive particles were located in the cytoplasm. Less BCL-2 positive cells and lighter immunostaining was found obviously in AβGroup than those in Control Group. Significantly increased BCL-2 positive cells were found in Puerarin Group compared to AβGroup. Western blot analysis was performed for quantitative assay of bcl-2 expression in hippocampus of rat. BCL-2 expression decreased significantly in AβGroup compared to Control Group (P<0.05), BCL-2 expression increased significantly in Puerarin Group compared to AβGroup (P<0.05). These data demonstrated that hippocampus injections of Aβ25-35 induced proapoptotic protein BAX expression increase and antiapoptotic protein BCL-2 expression decrease, but puerarin treatment can inhibit it.Casepase-3 is one of the most important protease in execution of apoptosis. Western blot analysis and immunohistochemistry were performed to detect the expressions of casepase-3 in hippocampus. It was showed by immunohistochemistry staining that casepase-3 protein expression was located in the neuronal cytoplasm. In Control Group, casepase-3 immunoreactive positive cells with light staining were seldom found. Significantly increased and deeper-staining casepase-3 positive neurons was found in AβGroup, and the situation in Puerarin Group was between the two groups. It was showed by Western blot analysis that casepase-3 expression was low in Control Group, and casepase-3 expression increased significantly in AβGroup compared to Control Group (P<0.05), but casepase-3 expression decreased significantly in Puerarin Group compared to AβGroup (P<0.05). These data demonstrated that hippocampus injections of Aβ25-35 induced apoptosis-related protease casepase-3 expression increase, which is a signal of increase of neuronal apoptosis, and puerarin treatment can inhibit it, which could be the molecular mechanisms of puerarin suppressing Aβ-induced neuronal apoptosis.Conclusions:1 The AD model was successfully duplicated by hippocampus injections of Aβ25-35 in rats, and evidences were provided for neuronal damage ethologically and morphologically. 2 Aβ2535 can induce inflammatory respond in hippocampus via activation of NF-κB signal pathway and overexpression of proinflammatory genes COX-2 and iNOS.3 Aβ2535 can promote neuronal apoptosis in hippocampus of rat via induction of proapoptotic protein BAX and apoptosis-related protease casepase-3 expression, and inhibition of antiapoptotic protein BCL-2 expression.4 High dose of puerarin can improve impairment of learning and memory induced by Aβ25-35 in rat. Morphological analysis confirmed that puerarin can prevent from Aβ-induced neuron loss in the pyramidal cell layers of the hippocampus, and maintain normal ultrastructure of neuron.5 Puerarin can inhibit Aβ-induced activation of NF-κB signal pathway and overexpression of proinflammatory genes COX-2 and iNOS.6 Puerarin can inhibit Aβ-induced neuronal apoptosis in hippocampus of rat via suppression of BAX and casepase-3 expression, and increase in BCL-2 expression.
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