Effects And Mechanism Of SSF On Memory Impairment In AD-like Rats Mediated By Ca²⁺-CaMK-CREB Signal Pathway

Alzheimer’s disease（AD）is a neurodegenerative disease characterized by memory and cognitive impairment.Extracellular Aβdeposits to form senile plaques and intracellular tau hyperphosphorylation to form fibrous tangles are its main pathological states.Studies have found that neuron loss,synaptic dysfunction,and the decrease of neurogenesis are also associated with the occurrence and development of AD.The loss of nerve cells can disrupt the neural networks and disturb the learning and memory ability.New neural connections and neural networks can be established to compensate and repair functional damage by promoting the neurogenesis.The neurogenesis is regulated by multiple pathways,which one is of Ca²⁺-Ca MK-CREB signaling pathway.The reduced levels of Ca M、Ca MKⅡ、Ca MKⅣ、CREB and downstream factors FGF2、Egr-1、Hu B、Hu C、Hu D and Frettin in this pathway could inhibit neurogenesis.Then,the positive regulation of Ca²⁺-Ca MK-CREB signaling pathway,as well as the promotion of FGF2,Egr-1,Hu B,Hu C,Hu D and Frettin expression can increase neurogenesis and improve learning and memory impairment in AD.Flavonoids from stems and leaves of Scutellaria baicalensis（SSF）are extracted from the aerial parts,which have been proved to have antibacterial,antioxidant,anti-hypoxia and improving memory impairment effects.However,the effect of SSF on the reduction of neurogenesis in AD rats caused by intraventricular injection of Aβ_25-35 in combination with Al Cl₃ and RHTGF-β1（composited Aβ）and whether the regulation is by Ca²⁺-Ca MK-CREB signaling pathway have not been reported.In this study,a model of AD-like memory disorder was established in rats to investigate the effect of Ca²⁺-Ca MK-CREB signaling pathway mediated SSF on memory disorder in AD rats and its mechanism,and for further elucidating the mechanism of SSF in treatment of AD.Objective:To investigate the effects and mechanism of SSF on memory impairment in AD-like rats mediated by Ca²⁺-Ca MK-CREB signaling pathway.Method:Sixty healthy adult SD male rats were selected and 50 of them were intraventricular injected with composite Aβ(d1:RHTGF-β1,1μL;d2-d15:Aβ_25-35,4μL;d2-d6:1%Al Cl_3,3μL)to establish a AD-like model.10 rats were conducted the same operation and injected with normal saline as the sham-operated group.The Morris water maze was used to screen the memory impairment model at day of 45 after the operation.The successful model rats were randomly divided into the model group and three-dose of SSF drug group.Rats in the drug group were orally administrated daily with SSF at dose of 35 mg/kg,70 mg/kg and 140 mg/kg for 30 d,and the rats in the model group and sham group were orally administrated daily with equal volume of saline.The positioning navigation trial was used to evaluate memory acquisition on days 1 and 2,the probe trial was used to evaluate memory retention on day 3 of the Morris water maze test and The reversal trial was used to evaluate re-learning for three consecutive days on days 4,5,and 6using Morris water maze test.Immunohistochemistry detected the expression of Neu N protein in the hippocampus of rats.Real-time fluorescence quantitative（q PCR）and western blotting methods were used to measure m RNA and protein expressions of Ca M,Ca MKⅡ,Ca MKⅣ,p-CRE-Ser133,FGF2,Egr-1,Hu B/D,Hu D and Frettin in hippocampus and cerebral cortex of rats.All data were expressed as Mean±SD,and SPSS 19.0 Statistical Software was used to perform statistical analysis.One-way ANOVA and two-way ANOVA were used for Water maze data,and one-way ANOVA was used for other data.P<0.05 was considered statistically significant.Result:1 Screening the memory impairment model of AD-like ratsOver the 4 days of screening model rats in the Morris water maze,the time for finding the hidden platform（latency）progressively declined in all animals.When the screening ratio（SR）,which was based on the latency to find the hidden platform on day 4 for composited Aβand sham rats,was more than 0.2,this animal was considered as a successful model rat.The percentage of successful model rats in this study was 83.3%.2 Effects of SSF on memory acquisition,retention and reproduction ability to AD-like rats.The results showed that,in the positioning navigation trial（1-2 d）,compared with the sham group,the time to find the hidden platform of the model group was significantly increased（P<0.01）.The time to find the platform in the AD-like model rats was shortened by three doses SSF,which indicats that the three-dose of SSF can improve the memory acquisition disorder induced by composite Aβ.In the probe trial,compared with the sham group,the swimming time in the target quadrant of rats in the model group was significantly reduced（P<0.01）.However,the three-dose of SSF significantly increased the swimming time in the target quadrant（P<0.01）,as compared with model group,which indicates that the three-dose SSF can ameliorate the memory retention disturbance induced by composite Aβ.In the reversal trial（4-6 d）,compared with the sham group,the time to find the hidden platform in the model group was significantly increased（P<0.01）,while the time to find the platform in the AD-like model rats was shortened by the three-dose SSF.This indicates that the three-dose SSF can improve the memory reproduction ability of the rats with composite Aβtreated.3 Effect of SSF on Neu N protein expression in hippocampus of AD-like ratsImmunohistochemical results showed that the positive expression of Neu N protein in the hippocampus gyrus of rats in the sham group was significantly higher,with brown-yellow granules and the staining most in the nucleus and a few in the cytoplasm.Compared with the sham group,Neu N positive expression in hippocampal gyrus in the model group was significantly decreased,and the staining in the cytoplasm and few in the nucleus.However,three doses of SSF can markedly raise the protein expression level of Neu N in hippocampus gyrus of AD-like rats by varying degrees.Compared with the model group,the positive expression of Neu N in the hippocampus gyrus of the35,70 and 140 mg/kg SSF groups were successively increased and most of the staining was in the cytoplasm and nucleus.The staining color increased,which showed that the effect of SSF on Neu N protein expression is at dose-dependent manner.4 Effects of SSF on m RNA expression of Ca M,Ca MKⅡ,Ca MKⅣ,p-CREB-Ser133,FGF2,Egr-1,Hu B,Hu C,Hu D and Frettin of hippocampus and cerebral cortex in AD-like rats.Compared with sham group,the m RNA expression of Ca MKⅡ,Ca MKⅣand Egr-1 in hippocampus and cerebral cortex of rats in model group were significantly decreased（P<0.05,P<0.01）,and the m RNA expression of FGF2,Hu B,Hu C and Hu D increased significantly in the hippocampus（P<0.05,P<0.01）,while there were no statistical significant in FGF2,Hu B,Hu C and Hu D in the cerebral cortex.However,three doses of SSF can reversed the expression of Ca MKⅡ,Ca MKⅣ,FGF2 and Egr-1 in hippocampus and cortex of AD-like rats induced by composite Aβ（p<0.05,p<0.01）.5 Effects of SSF on protein expressions of Ca M,Ca MKⅡ,Ca MKⅣ,p-CREB-Ser133,FGF2,Egr-1,Hu B/D,Hu D+Hu C and Frettin in the hippocampus and cortex of AD-like rats.Western blotting method to detect the protein expression levels of Ca M、Ca MKⅡ,Ca MKⅣ、p-CREB-Ser133、FGF2、Egr-1、Hu B/D、Hu D+Hu C an Frettin in the rat hippocampus and cerebral cortex.Compared with the sham group,the protein expression of Ca M,Ca MKⅡ,Ca MKⅣ,p-CREB-Ser133,FGF2,Egr-1,Hu D+Hu C,Hu B/D and Frettin in hippocampus and cerebral cortex of rats in model group were dramatically decreased（p<0.05,p<0.01）.It is interested that the three doses SSF can reverse the protein expression of Ca M,Ca MKⅡ,Ca MKⅣ,p-CREB-Ser133,FGF2,Egr-1,Hu B/D,Hu D+HUC and Frettin in hippocampus and cerebral cortex of rats（P<0.05,P<0.01）.Conclusion:1 Intraventricular injection of Aβ_25-35 combined with Al Cl₃and RHTGF-β1can reduce the number of mature neurons in rats’hippocampus gyrus,reduce neurogenesis and cause memory impairment in AD-like rats.2 Intraventricular injection of Aβ_25-35 combined with Al Cl₃ and RHTGF-β1can disturb Ca²⁺-Ca MK-CREB signal pathway in rat brain,Change the expression of Ca M,FGF2,Hu B,Hu C,Hu D and Frettin m RNA and protein in hippocampus and cortex of rats.3 SSF can regulate the m RNA expression levels of Ca M,CAMK II,CAMK IV,p-CREB-Ser133,FGF2,Egr-1,Hu B,Hu C,Hu D in AD-like rats to varying degrees.Reverses the decrease of expression of Aβ_25-35combined with Al Cl₃ and RHTGF-β1 in hippo Ca Mpus cortex cam,Ca MKⅡ,Ca MKⅣ,p-CREB-Ser133,FGF2,Egr-1,Hu C+Hu D and Frettin proteins,thus increasing mature nerve cells of AD-like rats,promoting nerve regeneration and improving memory impairment,and its effect is stronger in hippocampus than in cortex.4 The improvement of SSF on memory impairment in AD-like rats results from its promotion of neurogenesis.The enhancement of neurogenesis is primary from the up-regulation of Ca²⁺-Ca MK-CREB signal pathway by SSF.These results suggested that the amelioration of SSF in AD-like rats’memory impairment is mediated by Ca²⁺-Ca MK-CREB signaling pathway.