| Objective:To study the protective effect and mechanism of Rhizoma Drynariae extract on learning and memory ability of APP/PS1 double-transgenic AD model mice,and to investigate the protective effect and mechanism of the Neoeriocitrin from the main phytoestrogens of Rhizoma Drynariae extract on PC 12 cells injured by Aβ25-35.It provides a experimental basis for the prevention and treatment of Alzheimer’s disease with Rhizoma Drynariae extract in kidney-dried Chinese herbs.Methods:1.There are sixty APP/PS1 mice with 3 months old were randomly divided into four groups:model group,western medicine positive control group(donepezil hydrochloride 0.65 mg/Kg·d),traditional Chinese medicine positive control group(kang nao shuai capsules 585 mg/Kg·d)and Rhizoma Drynariae extract group(97.5 mg/Kg·d),and there are fifteen C57BL/6 mice of the same age which were used as control group,control group and model group were given the double distilled water once a day for three months consecutively.New object recognition,water maze,passive avoidance experiment were used to observe the memory and judgment ability of mice.HE staining and electron microscopy were used to observe the pathological changes in the hippocampus and hypothalamus of mice.Immunohistochemistry was used to detect the expression of ERα,ERβ,FSHR and LHR in the hippocampus and hypothalamus of mice.Western Blot was used to detect expression of ERα,ERβ,P-P38/P38,relevant markers of protein expression in Aβ metabolism(APP,BACE1),Tau protein phosphorylation(p-Tau,CDK5)and apoptosis index(Bax,Bcl-2,Caspase-3).The kit method was used to detect acetylcholine system(Ach content and the activity of ChAT and AchE)in the hippocampus.Elisa kit method was used to detect the levels of Aβ,inflammatory factors(IL-1β,IL-6,TNF-α)in the hippocampus and the levels of GnRH,β-EP,and SP in the hypothalamus.2.2×10-5 mol/L Aβ25-35 injured PC 12 cells were used to establish AD cell model.The experiment was divided into control group,model group,estradiol group,and Neoeriocitrin group.The safe concentration and effective concentration of Aβ25-35 injured PC 12 cells were detected by MTT assay.Apoptosis rate was determined by flow cytometry.The kit method was used to detect intracellular Ach content,ChAT,AchE activity.Western Blot method was used to determine the contents of ERβ and P-P38/P38 in cells.Results:1.The results of the new object recognition experiment showed that,the Rhizoma Drynariae extract significantly increased the new object recognition index of APP/PS1 mice(P<0.01).The results of water maze experiments showed that the Rhizoma Drynariae extract significantly reduced the escape latency of APP/PS1 mice(P<0.01),increased the number of platform crossings and the residence time of the target quadrant(P<0.01).The passive avoidance test results showed that Rhizoma Drynariae extract can significantly increase the passive avoidance latency of APP/PS1 mice(P<0.01).2.The results of HE staining showed that,compared with the control group,the hippocampal and hypothalamus cells of the model group were tightly arranged,but the number of neurons became smaller,the cells became smaller,the nucleus was pyknosis,and glial cell proliferation was observed.Neurons in the hippocampus and hypothalamus of the Rhizoma Drynariae extract group were arranged more closely,with more numbers and normal morphology,and there was no obvious phenomenon of nuclear condensation.3.The results of electron microscopy showed that,compared with the control group,the mice in the model group had decreased free ribosome in the hippocampus,mitochondrial flocculation,rough endoplasmic reticulum dilation and degranulation,Nissl body disappeared,and the structure of the synaptic membrane was not clear and the number of vesicles reduced.The number of synaptic vesicles in the nerve carpet area of the Rhizoma Drynariae extract group increased significantly.The cell nuclear membrane was clear and the nucleolus was apparent.The chromatin in the nucleus was patchy,with rich organelles and mitochondria developed.4.The results of immunohistochemistry showed that the expression of ERβ,ERα,FSHR,and LHR in the hippocampus and hypothalamus decreased significantly in the model group compared with the control group(P<0.01),and Rhizoma Drynaria extract could significantly increase the expression of ERβ,ERα,FSHR and LHR in the hippocampus and hypothalamus of APP/PS1 mice(P<0.01).5.Western Blot experimental results showed that compared with control group,the expression of ERα,ERβ and Bcl-2 in hippocampus of model group mice were significantly reduced(P<0.01),the expression of P-P38/P38,APP,BACE1,p-Tau/Tau,CDK5,Bax,and Caspase-3 were significantly increased(P<0.01),and Rhizoma Drynariae extract group significantly increased the expression of ERα,ERβ,and Bcl-2 in the hippocampus(P<0.01),and decreased the expression of P-P38/P38,APP,BACE1,p-Tau,CDK5,Bax and Caspase-3(P<0.01).6.The kit test results showed that the Ach content and ChAT activity in hippocampus of model group were significantly lower than control group,and the AchE activity was significantly increased(P<0.01).The extract of Rhizoma Drynariae could significantly increase Ach content and ChAT activity,and decrease AchE activity in the APP/PS1 mice hippocampus(P<0.01).7.The results of the Elisa kit showed that the levels of Aβ,IL-1β,IL-6 and TNF-α in the hippocampus of the model group were significantly higher than those in the control group(P<0.01),and the contents of GnRH,β-EP and SP were significantly decreased in the hypothalamic(P<0.01).The extract of Rhizoma Drynaria could significantly reduce the contents of Aβ,AchE,IL-1β,IL-6 and TNF-α in the APP/PS1 mice hippocampus(P<0.01),and increase the content of GnRH,β-EP,SP in the hypothalamus.8.6×10-2mol/L of Neoeriocitrin could significantly protect PC 12 cells from Aβ injury and reduce the apoptosis rate of Aβ-injured PC 12 cells(P<0.01).It can also significantly increase the expression of ERβ(P<0.01),the content of Ach and the activity of ChAT and reduce the expression of P-P38/P38(P<0.01),and the activity of AchE.Conclusion:1.The results of behavioral experiments showed that Rhizoma Drynariae extract can effectively prevent the learning and memory impairment of the double-transgenic APP/PS1 model of AD mice.2.The Rhizoma Drynariae extract can decrease the neurotoxicity of neurons by regulating the expression of APP and BACE1 protein in the hippocampus of APP/PS1 mice,reducing the deposition of amyloid Aβ in the hippocampus,so as to exert the neuroprotective effect.3.The Rhizoma Drynariae extract can reduce the expression of CDK5 in the hippocampus of APP/PS1 mice,and inhibit the over-phosphorylation of Tau,the formation of neurofibrillary tangles in the hippocampus,so as to exert the neuroprotective effect.4.The Rhizoma Drynariae extract can reduce the neuronal apoptosis by regulating the expression of neuronal apoptotic factors Bax,Bcl-2 and Caspase-3 in the hippocampus of APP/PS1 mice and exert neuroprotective effect.5.The Rhizoma Drynariae extract can increase the activity of cholinergic neurons by regulating the content of Ach in the hippocampus of APP/PS1 mice and the activity of ChAT and AchE,so as to exert its neuroprotective effect.6.The Rhizoma Drynariae extract can improve the inflammatory state by regulating the contents of IL-1β,IL-6 and TNF-α in hippocampus of APP/PS1 mice,so as to exert neuroprotective effects.7.The Rhizoma Drynariae extract can increase the expression of ERβ、ERα、FSHR、LHR in the hippocampus of APP/PS1 mice,enhencing the ration of ERβ/ERα expression.It is suggested that the hippocampal sex hormone receptor,especially ERβ,mediated/involved in the neuroprotective effect of Rhizoma Drynariae extract on APP/PS1 mice.8.The Rhizoma Drynariae extract can increase the expression of ERβ,ERα,FSHR and LHR in the hypothalamus of APP/PS 1 mice,and increase the content of GnRH,β-EP and SP in hypothalamus,suggesting that Rhizoma Drynariae extract can activate the hypothalamus-pituitary-Gonad axis,exert anti-aging effect.9.The major component of Rhizoma Drynariae extract,Neoeriocitrin,can reduce the apoptosis rate of Aβ-injured PC 12 cells,increase the activity of ChAT and decrease the activity of AchE,increase the content of Ach,so as to exert the protective effect on AD model cells.These effects are likely to be mediated through the ERβ/P38 cell signal transduction pathway.10.The Rhizoma Drynariae extract contains a large number of phytoestrogen components,presumably these components can act directly on the ER in the hippocampus,play a neuroprotective role through the ERβ/P38 signaling pathway;which can also initiate the hypothalamic-pituitary-gonadal axis and exert overall anti-aging effects.However,the exact active ingredient of Rhizoma Drynariae against AD has not yet been determined. |
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