| Alzheimer’s disease(AD)is a neurodegenerative disease with the pathological characteristics of senile plaques(SP)formed by the deposition of amyloid β-protein plaques and Neurofibrillary tangles(NFT)formed by abnormal Tau protein.Additionally,the decreased neurogenesis and neuron loss contribute to the cognitive impairment in AD patients.Thedecreased neurogenesis and neuron loss are also important factors in leading to the cognitive impairment in AD patients.The response element binding protein(CREB)plays a crucial role in regulating gene transcription,changing neuronal excitability and regulating synaptic plasticity,promoting neurogenesis and improving cognitive impairment.It is well known that CREB isin favourof the neurogenesis and CREB phosphorylation compensates for the neuron loss,which can improve the cognitive dysfunction.The neurogenesis-related proteins recombinant doublecortin(DCX),neuron nucleus(NeuN)and neurogenic differentiation factor 1(NeuroD1)are all involved in neurogenesis.It is possible for them to directly reflect the state of neurons and the level of neurogenesis.Meanwhile,the high level DCX,NeuN,and NeuroD1 also contribute to the neuronal differentiation and synaptic growth,and advance to improve the neurogenesis.A variety of signaling pathways can regulate the neurogenesis as well.RasERK-CREB signaling pathway is a relatively classic pathway involved in the neurogenesis regulation.The signaling molecules TrkB,Ras,ERK,and RSK are activated through a series of cascade reactions to activate CREB by phosphorylation.The activated CREB can activate its downstream neurotrophic factors,such as BDNF,NGF,and EGR-1 expressed,which may produce the neurogenesis and memory formation.The usually model of AD was set up based on SPs and NFTs of the characteristic pathological changes of AD.In this study,the AD memory impairment model was established from the perspective of reduction of AD neurons and neuronal damage.In this study,the memory impairment model of AD was established from the perspective of neurogenesis reduction and neuronal damage.It is known that BI-D1870 inhibits the BDNF-ERK-CREB signaling pathway by inhibiting RSK activity.The RSK inhibitor BI-D1870 inhibits the activity of RSK in the BDNF-ERK-CREB signaling pathway,indirectly reduces the phosphorylation of CREB at Ser133 site and its activation,reduces the synthesis of its downstream BDNF,EGR-1 and other nerve growth factors,and reduces neurogenesis and memory formation.Scutellariabarbata flavonoids(SBFs),a flavonoids compounds,were extracted from the aerial part of Scutellariabarbata D.Don,and the main component is scutellarin,which has the function in promoting the brain cell metabolism and protecting neurons.However,whether SBFs affects the neurogenesis decline and cognitive dysfunction caused by BI-D1870 and its mechanism has not been reported.In this study,AD rat model of learning and memory impairment established by BI-D1870 was used as the research subject.The effects of SBFs in promoting neurogenesis and improving cognitive impairment were determined by detecting the learning and memory abilities,the neuron morphology and Nissl bodies,the neurogenesis-related protein DCX,NeuN and NeuroD1 and the Ras-ERK-CREB signaling pathway signaling molecules.Rolipram,a phosphorylated activator of CREB,was used as a positive control drug to further elucidate the effective mechanism of SBFs on neurogenesis reduction and memory impairment in rats induced by BI-D1870.Objective:The aims of the study were to establish a rat model with learning and memory impairment induced by BI-1870 and to investigate the effects and molecular mechanisms of SBFs on neurogenesis reduction and memory impairment in rats induced by BI-D1870.Methods:SPF-grade male SD rats were randomly divided into control group,solvent control group and BI-D1870 group.The BI-D1870 group rats were intraperitoneally injected with 0.35 mg/kg BI-D1870,the solvent control group rats were intraperitoneal injected 10% DMSO + 90% sulfobutyl-β-cyclodextrin solvent and the control group rats were intraperitoneally injected with equal volume of saline.The changes in appearance and behavior of rats were observed,and the weight of rats in the three groups were recorded.Morris water maze was used to screen the memory impairment model of rats.The successful model rats were randomly divided into three groups.The model group,SBFs group and positive control Rolipram group.Morris water maze,Barnes maze,step-through test,shuttle box and step-down test were used to test the learning and memory abilities of rats;HE staining was used to observe the morphology of neurons in hippocampus CA1 region and cerebral cortex;Crystal violet staining was used to observe intracellular Nissan bodies in hippocampus CA1 region and cerebral cortex;The immunohistochemical(IHC)was used to detect the expression of DCX,NeuN and NeuroD1 proteins in the hippocampal gyrus;Immunofluorescence assay(IF)was used to assay the expression of p-CREB-Ser133 and p-CREB-Ser142 proteins;qPCR and Western blotting were used to measure the mRNA and protein expression of the upstream signal molecules in Ras-ERK-CREB signal pathway,BDNF,TrkB,Ras,ERK,RSK,CREB,and NGF and EGR-1 induced by CREB in the hippocampus and cerebral cortex of rats.Results:1.The Effects of BI-D1870 on appearance,behavior and body weight changes in ratsThere are no markedly different in appearance and hair color of rats between control group,solvent control group and BI-D1870 group on the day82 of intraperitoneal injection of BI-D1870.The rats in the BI-D1870 group appeared irritated on the day 10 of BI-D1870 intraperitoneal injection,and gradual increase in the number of irritated rats along BI-D1870 given till the day 82.Meanwhile,the weight gain of the BI-D1870 group was higher than those of the control group and the solvent control group(P<0.05).2.The successful model of memory impairment screened Morris water mazeThe successful model of memory impairment was screened by Morris water maze.It was found that,the latency of rats was gradually shortened in each group to find the platform along with the Morris water maze training.Compared with the solvent control group,the latency of rats in the BI-D1870 group was significantly increased.According to the latency to find the platform on the 25th,55th and 85th day of rats,the successful rate of rat model was50.00%,62.00% and 82.00%,respectively.The follow-up experiment was carried out in the model rats successfully injected with BI-D1870 on the 85 th day.3.The effects of SBFs on learning and memory in rats3.1 The effects of SBFs on learning and memory impairment in rats induced by BI-D1870The results showed the positioning navigation trial in Morris water maze test.Compared with the solvent control group,the latency of model group to find the platform was significantly prolonged(P<0.01).This prolonged latency was significantly shortened by treatment of SBFs and Rolipram(P<0.01).A significant reduction in swimming time was observed in the target quadrant for the model group,as compared with the solvent control group in the probe trial(P<0.01).Both the SBFs and Rolipram significantly prolonged rats’ swimming time in the target quadrant,as compared to the model group(P<0.05,P<0.01).According to the opposite trial,the model group showed significantly longer latency to find the platform than that of the solvent control group(P<0.05).There was a significant reduction in latency to find the platform in the SBF’s and Rolipram’s groups,as compared to the model group(P<0.05).There was no significant difference in the latency to find the platform and the swimming time in the target quadrant between the control group and the solvent control group,as well as between the SBFs group and the Rolipram group.3.2 The effects of SBFs on spatial memory in ratsAccording to the Barnes maze test results,latency and number of errors in the model group were higher than those of the solvent control group(P<0.05).Compared with the model group,the latency and error times of SBFs group and the Rolipram group finding the safe hole were significantly reduced(P<0.01).There were no significant differences between the control group and the solvent control group,as well as between the SBFs group and the Rolipram group in the latency for finding the safe hole and the number of errors.3.3 The effects of SBFs and Rolipram on short-term memory in ratsIn the step-through test,rats in the model group showed a decrease in latency(P<0.01)and increased number of errors,as compared to the solvent control group(P<0.05).SBFs and Rolipram groups exhibited higher latency and the decreased errors than those of the model group(P<0.01,P<0.05).There were no significant differences between the control group and the solvent control group,as well as the SBFs group and the Rolipram group in the latency and errors number in the step-through test.3.4 The effects of SBFs and Rolipram on active avoidance response in ratsIn the shuttle box test,the rats in the model group had shorter latency and more errors than those of the solvent control group(P<0.05).Rats in the SBFs and Rolipram groups showed a significant increased latency and a significant decreased error,as compared to the model group(P<0.05).There were no significant differences between the control group and the solvent control group,as well as the SBFs group and the Rolipram group in the latency and errors number in the shuttle box test.3.5 The effects of SBFs and Rolipram on passive avoidance response in ratsThe step-downexperimental results showed that the passive avoidance latency of rats in the model group was significantly reduced and the number of errors was significantly increased,compared with the solvent control group,(P<0.01).Compared with the model group,the passive avoidance latency in SBFs and Rolipram groups increased and the number of errors decreased(P<0.01).There were no significant differences between the control group and the solvent control group,as well as the SBFs group and the Rolipram group in the latency and errors number in the step-down test.4.Effects of SBFs on hippocampal CA1 region and cortical cell morphology in rats with BI-D1870 memory impairmentHE staining results showed that the pyramidal cells were with neat and compact arrangement,complete cell body and membrane,large and round nucleus,and uniform staining in CA1 region of hippocampus in control group and solvent control group.In the model group,the number of cells in the hippocampal CA1 region decreased,the arrangement was sparse,the number of glial cells increased,the nucleus was hyperchromatic and the edge shifted,and the neurophagic phenomena appeared.Compared with the model group,the number of pyramidal cells in thehippocampal CA1 area and cortex of the SBFs group and Rolipram group increased,the arrangement is more compact,the cell body is basically complete,and the nuclear concentration,edge shift and neurophagic phenomena are less than that of the model group.There were no obvious differences between the control group and the solvent control group,as well as the SBFs group and the Rolipram group in the neural cell morphology.5.The effects of SBFs on changes in Nissl bodies in the hippocampal CA1 region and cortical cortex cells of ratsThe results of crystal violet staining showed that there were abundant Nissl bodies(tiger spots)in the hippocampal CA1 area and cortex cytoplasm,and the staining was deeper and more numerous in the control group and solvent control group.the purple staining of intracellular Nissl bodies in the hippocampal CA1 area and cortex of the model group became less,the number of Nissl bodies decreased,and some neurons disappeared.Compared with the model group,the staining of intracellular Nissl bodies in hippocampal CA1 area and cortex of rats in the SBFs group and Rolipram group was significantly deepened and the number increased.There were no marked differences between the control group and the solvent control group,as well as the SBFs group and the Rolipram group in Nissl bodies.6.The effects of SBFs on the protein expression of DCX,NeuN and NeuroD1 in the hippocampal gyrus of ratsThe results of IHC showed that the positive expression cells number of DCX,NeuN and NeuroD1 protein in the hippocampal gyrus of the model group were decreased,as compared with the solvent control group(P<0.05).Compared with the model group,the decreased positive expression cells number of DCX,NeuN and NeuroD1 in the hippocampal gyrus were reversed by treatment of the SBFs group and the Rolipram(P<0.05).There were no significant differences between the control group and the solvent control group,as well as the SBFs group and the Rolipram group in positive expression cells of DCX,NeuN and NeuroD1.7.The effects of SBFs on the protein’s expression of p-CREB-Ser133 and pCREB-Ser142 in the hippocampal CA1 region and cerebral cortex of ratsIF detection results showed that p-CREB-Ser133 and p-CREB-Ser142 were positively expressed by red fluorescence in the cytoplasm,and the nuclear DAPI staining showed blue fluorescence.The expression of p-CREB-Ser133 in hippocampal CA1 region and cortex of control and solvent control group was higher,and the fluorescence intensity was heavier.Compared with the solvent control group,the expression of p-CREB-Ser133 was decreased and the fluorescence intensity was weak in the model group.Compared with the model group,the expression of p-CREB-Ser133 was increased by SBFs and Rolipram treated,and the fluorescence intensity were stronger.However,the expression of p-CREB-Ser142 was lower in control and solvent control group,and the fluorescence intensity was weak.Compared with the solvent control group,the expression of p-CREB-Ser142 was increased and the fluorescence intensity was enhanced in the model group.This increase in protein expression was significantly reduced and the fluorescence intensity was weakened by SBFs and Rolipram treated.There were no significant differences between the control group and the solvent control group,as well as the SBFs group and the Rolipram group in p-CREB-Ser133 and p-CREB-Ser142 protein expression.8.Effects of SBFs on the mRNA expression of BDNF,TrkB,Ras,ERK,RSK,CREB,NGF and EGR-1 in the hippocampus and cortex of ratsqPCR results showed that compared with solvent control group,mRNA expressions of BDNF,TrkB,ERK,RSK,NGF and EGR-1 in hippocampus of model group were significantly increased(P<0.01),Ras and CREB mRNA had no significant changes.The mRNA expressions of TrkB,Ras,ERK,RSK,CREB,NGF and EGR-1 were significantly decreased in the cortex(P<0.01)and increased the expression of BDNF mRNA(P<0.05).Compared with the model group,SBFs and Rolipram could regulate the abnormal expression of BDNF,TrkB,Ras,ERK,RSK,CREB,NGF and EGR-1 mRNA in the hippocampus and cortex of rats induced by BI-D1870 to varying degrees(P<0.01).The mRNA expressions of BDNF,TrkB,RSK,CREB,NGF and EGR-1 were significantly different between the control group and the solvent control group(P<0.05);There were significant differences in the expression of BDNF,RSK,NGF and EGR-1 mRNA between the SBFs and Rolipram groups(P<0.05).9.Effects of SBFs on the expression of BDNF,TrkB,Ras,ERK,RSK,CREB,NGF and EGR-1 proteins in the hippocampus and cortex of ratsWB results showed that compared with the solvent control group,BDNF,TrkB,Ras,ERK,RSK,CREB,NGF and EGR-1 proteins in the hippocampus and cortex of the model group were decreased(P<0.05).However,compared with the model group,SBFs and Rolipram significantly reversed the abnormal expression of BDNF,TrkB,Ras,ERK,p-ERK,p90 RSK,CREB,NGF and EGR-1 in the hippocampus and cortex of rats induced by BI-D1870(P<0.05).There was no significant difference in protein expression between the control group and the solvent control group;The expression of p-ERK,NGF and EGR-1 protein in SBFs was significantly different from that in Rolipram group(P<0.05).Conclusion:1.Rats were intraperitoneally injected with 0.35 mg/kg BI-D1870 to establish a memory impairment model,which can be used to study AD from neurogenesis in the laboratory.2.BI-D1870 can produce the learning and memory impairment,decrease neurogenesis and induce the neuropathological changes in rats,and also result in the abnormal expression of signal molecules in Ras-ERK-CREB signaling pathway in rat brain.3.SBFs can improve the rats’ neuropathological changes,promote neurogenesis and improve cognitive impairment induced by BI-D1870,and also positively regulates these molecules expression of Ras-ERK-CREB signaling pathway in rat brain.4.The effects of SBFs are parallel to the positive drug Rolipram.Both can improve learning and memory disorders,promote the neurogenesis and increase CREB phosphorylation at Ser133 site.These results suggested that the effective mechanism of SBFs in improving the memory and neurogenesis disorders,is primary from promoting CREB phosphorylation. |
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