The Molecular Mechanism Of Scutellaria Baicalensis Georgi Stems And Leaves Flavonoids In Promoting Neuroregeneration And Improving Memory Impairment Mediated By PI3K-AKT-CREB And CAMP-PKA-CREB Signaling Pathway In Rats

Alzheimer disease（AD）is an age-related chronic neurodegenerative disease,which is mainly manifested by impairment in memory and cognitive function.Its characteristic pathological changes include extracellular senile plaques（SP）,which are composed of aggregated beta-amyloid（Aβ）,and intracellular neurofibrillary tangles（NFT_S）,which consist of aggregates of hyperphosphorylated tau protein.The deposition of SP and NFT can result in the brain parenchymal lesions,which are positively correlated with neuron loss and learning,memory,and cognitive dysfunction in AD patients.It is reported that neuroregeneration is the process of generating new neurons,which can compensate for the damaged neurons,and improve synaptic plasticity and memory impairment,and advance to ameliroate the clinical symptoms for AD patients.Many studies have found that neuroregeneration is regulated by some signal pathways,among which PI3K-AKT-CREB related molecules tropomyosin related kinase B（TRKB）,phosphatidylinositol3-kinase（PI3K）,protein kinase B（AKT）,and cAMP-PKA-CREB related molecules Gs proteinα（Gαs）,adenylate cyclase 1（AC1）,cyclic adenosine monophosphate（cAMP）and protein kinase A（PKA）are involved in synaptic plasticity,memory formation and the neuron servive.SSF,flavonoids from stems and leaves of Scutellaria baicalensis Georgi,isolated from aerial parts of Scutellaria baicalensis Georgi,has showed the functions in anti-inflammation,antioxidation,scavenging free radicals,and improving memory impairment.It has been proved that SSF can improve memory impairment and promote neuroregeneration by regulating Ca²⁺-Ca MK-CREB signaling pathway.However,whether SSF can promote nerve regeneration and improve memory impairment by regulating PI3K-AKT-CREB and cAMP-PKA-CREB signaling pathways have not been reported.In the present study,the model of AD-like memory impairment in rats was established by intracerebroventricular injection of Aβ_25-35 combined with Al Cl₃ and RHTGF-β1.By detecting the effects of SSF on the conditional and spatial learning and memory ability,an indicator of neuronal neuroregeneration BrdU and the expression of PI3K-AKT-CREB and cAMP-PKA-CREB signal pathway related molecules,Which elucidate the molecular mechanism of SSF promoting neuroregeneration and improving memory impairment by regulating PI3K-AKT-CREB and cAMP-PKA-CREB signaling pathways.Objective:The aim of this study was to investigate the effect and the molecular mechanism of SSF in promoting neuroregeneration and improving memory impairment mediated by the PI3K-AKT-CREB and cAMP-PKA-CREB signaling pathway.Methods:Sixty healthy Wistar male rats were adaptively feeded in the SPF-degreed laboratory environment for one week,and randomly divided into the operated group（50 rats）and sham group（10 rats）.Rats in the operated group were intracerebroventricularly injected Aβ_25-35 combined with Al Cl₃ and RHTGF-β1for establishing the model of AD-like.Rats in the sham group were intracerebroventricularly injected the equal volume of normal saline.The Morris water maze was used to screen the successful model by memory impairment in rats.The successful model rats were randomly divided into a model group and three doses of drug.The rats in the drug group were treated with 35,70,and 140 mg/kg of SSF for 30 days.The shuttle box was used to measure the conditioned learning and memory ability by the active avoidance response rate（AARR）,the general avoidance response rate（GARR）,the active avoidance response latency（AARL）and the passive avoidance response latency（PARL）of rats.The Eight-arm maze was assessed to detect the spatial learning and memory abilities of the rats,including the working memory errors（WME）and the reference memory errors（RME）.The immunohistochemistry was assessed to assay the protein expression of BrdU in the hippocampal gyrus and cerebral cortex of rats.The content of cAMP was detected by Elisa method.qPCR and Western blotting methods were used to determine the mRNA and protein expressions of the PI3-KAKT-CREB and cAMP-PKA-CREB signal pathway,Gαs,AC1,and PKA,as well as CREB downstream factors IGF2 and VEGF in the hippocampus and cerebral cortex of rats.All data were analyzed by SPSS 21.0 software and expressed as mean±SD.The data of the shuttle box and the eight-arm maze were analyzed by two-way ANOVA,other data were analyzed by one-way ANOVA,and P<0.05 was considered as statistically significant.Results:1 The memory impairment model of rats screenedThe rats training for 4 days in Morris water maze is to screened the memory impairment model.Along with the increase of training days,all rats gradually took shorted latency for finding the hidden platform.According to the SR≥0.2 of each operated rat on the day 4 of training,the successful model rate in this present study was 83.3%.2 Effect of SSF on the memory ability of composited AβratsThe shuttle box results showed that SSF can affect the conditioned memory ability of composited Aβrats.Compared with the sham group,the AARR and GARR were remarkable decreased（P<0.01）,the AARL and PARL were significantly prolonged（P<0.01）in model group.Compared with the model group,three doses of SSF increased AARR and GARR（P<0.01）,shortened AARL and PARL（P<0.05,P<0.01）,which indicated that three doses of SSF can improve the conditioned learning and memory impairment induced by composited Aβ.The Eight-arm maz results showed that SSF can also affect the spatial memory ability of composited Aβrats.Compared with the sham group,the number of WME and RME was significantly increased（P<0.01）in the model group of rats,while three doses of SSF could reduce the number of WME and RME（P<0.01）induced by composited Aβ.These results indicated that SSF could improve the spatial memory ability of rats in composited Aβ.3 Effect of SSF on the protein expression of BrdU in the hippocampal gyrus and cerebral cortex of ratsThe immunohistochemical results showed that the intracerebroventricular injection of composited Aβcould decrease the protein expression of BrdU in hippocampal gyrus and cerebral cortex of rats.Compared with the sham group,the expression of BrdU protein in the hippocampal gyrus and the cerebral cortex was significantly reduced（P<0.01）,and the color of brown granules became lighter in the model group.Compared with the model group,the three doses SSF group can differently increase the protein expression of BrdU in the hippocampal gyrus and cerebral cortex in composited Aβgroup（P<0.01）,the color of brown granules gradually deepened.The positive expression of BrdU protein in the three dose of SSF showed an inverted U shape.4 Effect of SSF on the content of cAMP in the hippocampus and cerebral cortex of ratsThe content of cAMP in the hippocampus and cerebral cortex of rats was detected by the Elisa method.The results showed that the content of cAMP significantly lowered（P<0.01）in the model group,as compared with the sham group.However,three doses of SSF can enhance the content of cAMP in composited Aβrats by varying degrees（P<0.01）.Three doses of SSF showed an inverted U shape of cAMP content in the cerebral cortex and U shape of cAMP content in the hippocampus.5 Effect of SSF on the mRNA expression of TRKB,PI3K,AKT,Gαs,AC1,PKA,IGF2 and VEGF in the hippocampus and cerebral cortex of rats.In the hippocampus,the mRNA expression of TRKB,PI3K,Gαs and IGF2 was significantly lowered(P<0.01,P<0.05 and mRNA expression VEGF increased（P<0.01）,and the mRNA of expression in AKT and AC1has no significant change in model group,as compared with sham group.In the cerebral cortex,the mRNA expression of TRKB,AKT and AC1 decreased（P<0.01,P<0.05）,PI3K and VEGF increased（P<0.01,P<0.05）,IGF2 and Gαs have no significant change in model group,as compared with sham group.Compared with the model group,three doses of SSF could regulate the abnormal changes of TRKB,PI3K,AKT,AC1,PKA,IGF2 and VEGF mRNA expression in the hippocampus and cerebral cortex induced by composited Aβ（P<0.01）.6 Effect of SSF on the protein expression of TRKB,PI3K,AKT,P-AKT,Gαs,AC1,PKA,IGF2 and VEGF in hippocampus and cerebral cortex of rats.Compared with the sham group,the protein expression of TRKB,PI3K,P-AKT/AKT,Gαs,AC1,PKA and IGF2 were significantly lowered（P<0.01,P<0.05）,VEGF protein were increased（P<0.01）in hippocampus and cerebral cortex in the model group.Compared with the model group,three doses of SSF could increase the protein expression of TRKB,PI3K,P-AKT/AKT,Gαs,AC1,PKA,IGF2 and VEGF in the hippocampus and cerebral cortex of rats（P<0.05,P<0.01）.Conclusion:1 Intracerebroventricular injection of composited Aβcould disturb the ability of conditioned and spatial learning and memory and reduce neuroregeneration in the hippocampus and cerebral cortex of rats.2 Intracerebroventricular injection of Aβcan affect the expression of PI3KAKT-CREB and cAMP-PKA-CREB signal pathway related molecules TRKB,PI3K,AKT,P-AKT,Gαs,AC1,and PKA,as well as CREB downstream factors IGF2,VEGF mRNA and protein in the hippocampus and cerebral cortex of rats.3 SSF can improve the impairment of conditioned learning and spatial memory in rats induced by composited Aβand promote neuroregeneration in hippocampus and cerebral cortex.4 SSF can promote neruroregeneration by regulating the mRNA and protein expressions of PI3K-AKT-CREB and cAMP-PKA-CREB signal pathway related molecules of TRKB,PI3K,AKT,P-AKT,Gαs,AC1,and PKA,as well as CREB downstream factors IGF2,VEGF in the hippocampus and cerebral cortex in composited Aβrats,and thus improve memory impairment.5 The effect of SSF in promoting neuroregeneration and improving memory impairment is completed by regulating PI3K-AKT-CREB and cAMP-PKA-CREB signaling pathways.