Ras-ERK-CREB Signal Pathway Mediates The Molecular Mechanism Of SSF In Promoting Neurogenesis And Improving Memory Impairment In Rats

Alzheimer’s disease（AD）the clinical manifestations of these patient are impair their independent living ability.Then the neuropathology of patients with AD is characterized by senile plaque and neurofibrillary tangles formed on neurons,loss of neurons,apotosis and other neuronal pathological changes.The loss of neurons is one of the direct causes for clinical manifestations such as behavioral abnormalities and intellectual impairment of AD.However,the multiple signal pathways participate in the regulation of neurogenesis.The neurogenesis process can produce new functional neurons to compensate for the loss of neurons,improve the neural structure and cognitive impairment,then which plays important role in against-AD.Studies have shown that the occurrence and development of AD is related to a number of abnormal signal pathways,in which Ras-ERK-CREB signaling pathway has been proved to be a important pathway in regulating of neurons,and also in ameliorating the learning and memory impairment.Scutellaria baicalensis Georgi stems and leaves flavonoids（SSF）has been confirmed that showed the antiviral,and improvement in learning memory enhancement.However,there is no reported whether the AD memory impairment rat model established by intracerebroventricular microinjection of amyloid-beta protein 25-35(Aβ_25-35)in combination with aluminum trichloride（AlCl₃）and recombinant human transforming growth factor-β1（RHTGF-β₁）（composited Aβ）promotes neurogenesis through Ras-ERK-CREB signal transduction pathway.In the present study,this rats AD model was used to detect the effects of SSF in learning and memory function,neurogenesis associated protein Ki67 and the molecules changes in Ras-ERK-CREB signal pathway.The aim of this study is to evaluate the molecular mechanism of SSF in improving learning and memory impairment and promoting neurogenesis mediated by the Ras-ERK-CREB signal pathway.Objective:The models of AD memory impairment and nerve injury were established by intracerebroventricular microinjection of composited Aβin rats.The effects of Ras-ERK-CREB signal pathway mediated Scutellaria baicalensis Georgi stems and leaves flavonoids（SSF）on memory impairment and neurogenesis and its regulatory mechanism were observed.Methods:70 SPF male Wistar rats,weighing 280-300g,were placed in a barrier environment and fed adaptively for 7 days before operation.60 rats were intracerebrovenentricularly injected composited Aβto build AD mode.On the first day of the operation,1μL of（10 ng）RHTGF-β₁ was microinjected intracerebroventricular.On the second day of operation,1μL Aβ25-35（4μg）was microinjected into the intracerebroventricular on every morning for 14consecutive days,and 3μL of AlCl₃（1%）was microinjected every afternoon for 5 consecutive days.In addition,10 rats underwent the same operation during the same period,and an equal volume of 0.9%NaCl was injected into the intracerebroventricular as a sham-operated group.On day 45 after the operation,the Morris water maze was used to screen successful rats with memory impairment.The swimming performance of rats in sham-operated group and intracerebroventricular microinjection composited Aβgroup on day 4 of water maze was as the screening rate（SR）.The rats with successful model were randomly divided into model group the SSF three-dose groups.The SSF group were daily intragastrically administered for 41 days.By Morris water maze and step down,the learning and memory ability of rats was tested.All rats were decapitated on the day 89 after operation.The expression of neurogenesis-related molecule Ki67 protein in the rat hippocampcus and cerebral cortex was detected by immunohistochemical method（IHC）.The expression levels of m RNA,and protein of molecules,BDNF,Grb2,SOS1,Ras,c-Raf,P-MEKs,P-ERK1/2 and Rsk in the Ras-ERK-CREB signaling pathway in hippocampus and cerebral cortex were assayed by the Quantitative real-time PCR（qPCR）and Western blotting methods,respectively.Results:1.Successful rat model of AD was screened using Morris water mazeThe latency for all rats to find a hidden platform in the water maze was gradually shortened in 1-4 days.And the latency of finding hidden platform in sham-operated group was significantly shorter than that of intracerebroventricular injection of composited Aβgroup.The success rate of the rat model with memory impairment in this experiment was 83.30%.2.The effect of SSF on spatial learning and memory in rats with composited Aβby Morris water maze testOn day 1-2 positioning navigation trial,and on day 4-6 opposited platform trial of the Morris water maze,the latency of finding the hidden platform in the model group was significantly longer than that of sham-operated group（P<0.01）.The latency of finding the hidden platform in the SSF three-dose groups were significantly shorter than that of model group（P<0.01）.On day 3 of the Morris water maze test,the swimming time and platform crossing times of rats in the model group in the target quadrant（quadrant IV）in the target quadrant were significantly less than of the sham-operated group（P<0.01）,the swimming time and the times of crossing the platform in the target quadrant of rats in the SSF three-dose groups were increased in different degrees（P<0.01）.3.The effect of SSF on passive avoidance learning and memory impairment in rats with composited Aβtreated using step down testIn the passive avoidance experiment conducted 1-2 days test using step down box.Compared with the sham-operated group,the errors times in the model group was significantly increased（P<0.01）,but the latency was significantly shortened（P<0.01）.Compared with the model group,the errors times in the SSF three-dose groups was significantly decreased（P<0.01）,but the latency significantly prolonged（P<0.01）.4.The effect of SSF on protein expressions of Ki67 in the hippocampal gyrus of rats with composited AβtreatedCompared with the sham-operated group,the positive expression of Ki67 protein in hippocampal gyrus was significantly inhibited（P<0.01）.Compared with the model group,the three doses SSF group could reverse the decrease of Ki67 protein induced by composited Aβin hippocampal gyrus（P<0.01）,the color of brown granules gradually deepened.5.The effects of SSF on the expressions of BDNF,Grb2,SOS1,Ras,c-Raf,P-MEKs,P-ERK1/2 and RSK m RNA and protein in hippocampus and cerebral cortex of rats with composited AβtreatedThe m RNA and protein levels of BDNF,Grb2,SOS1,Ras,c-Raf,P-MEKs,P-ERK1/2 and Rsk in the rat brain of the model group were higher than those of sham-operated group（P<0.01）.However,SSF significantly regulated these abnormal expression in the brain of rats induced by composited Aβ,that 35,70 and 140 mg/kg SSF differently lowered the m RNA and protein expression of BDNF,Grb2,SOS1,Ras,c-Raf,P-MEKs,P-ERK1/2 and Rsk in hippocampus and cerebral cortex（P<0.01）.Conclusion:Intracerebroventricular microinjection of Aβ25-35 combination with AlCl₃ and RHTGF-β₁ can produce the learning and memory impairment,decrease neurogenesis and cause the abnormal expression of factors in molecules in the Ras-ERK-CREB signal pathway in the brain.After intragastric administration of SSF for 41 days,the impaired cognition,the decreased neurogenesis and abnormal changes of molecules in Ras-ERK-CREB signaling pathway induced by composited Aβwere ameliorated.These results suggested that the effects SSF in improving the learning and memory impairment and promoting the neurogenesis maybe related to the regulation of SSF in these molecules in Ras-ERK-CREB signal pathway,which clarified the molecular mechanism of SSF in improving memory impairment and promoting neurogenesis mediated by Ras-ERK-CREB signaling pathway.