Study On The Neuroprotective Effect And Mechanism Of Phenyl Ethyl Caffeate Derivative FA-97

ObjectiveIn this paper,the neuroprotective effect and mechanism of phenylethyl caffeate derivative fa-97 were comprehensively studied in vivo and in vitro by establishing H2O2-induced oxidative damage model of nerve cells,scopolamine induced cognitive impairment mice and APP/PS1 transgenic AD mouse model.MethodsExperiment 1:Protective effect and mechanism of FA-97 on H2O2-induced oxidative stress model of neurons。The oxidative damage model of SH-SY5Y and PC12 cells was established by H202 stimulationo The experiment included normal group,H2O2(500 M)model group,H2O2+FA-97 low dose group(0.25 M),H202+FA-97 medium dose group(0.5 M),and H2O2+FA-97 high dose group(1 M).Cell morphology was observed under an inverted microscope,LDH leakage rate was detected by LDH assay kit,and cell viability was detected by CCK8。The apoptosis rate of cells was detected by the Flow cytometry and the expression of apoptosis-related proteins Bax,Bcl-2,Cytochrome c and caspase—9 were detected by Western-Blot。ROS levels were detected by fluorescence microscopy,and SOD and GSH activities,MDA and protein carbonyl levels were detected by the assay kit。Expression level of Nrf2/HO-1 signaling pathway related proteins and Nrf2 in the nucleus and cytoplasm were detected by Western-Blot.Nuclear translocation of Nrf2 was observed by laser confocal microscopy.Luciferase reporter gene was used to detect the transcriptional activity of Nrf2,and cells were transfected with Nrf2 siRNA to verify the mechanism of Nrf2.Experiment 2:Effect of FA-97 on learning and memory function in scopolamine induced memory impairment mice model and its mechanism。The 18-22g male KM mice were divided into eight groups:the normal group,the scopolamine group(SCOP,3mg/kg),the scopolamine+FA-97(2.5mg/kg)treated group,the scopolamine+FA-97(5mg/kg)treated group,the scopolamine+FA-97(10mg/kg)treated group,the scopolamine+donepezil(DNP,3mg/kg)treated group,and the scopolamine+phenyl caffeinate(CAPE,10mg/kg)treated group。FA-97,CAPE and DNP were administered orally once a day for 30 consecutive days.Mice were intraperitoneal injected with SCOP(3 mg/kg)from the 21th day,while control mice were intraperitoneal injected with normal saline.All mice were given a behavioral test 30 minutes after the SCOP injection。After behavioral tests,mice were sacrificed。Morris water maze experiment and novel objective recognition experiment were used to evaluate the learning and memory ability of mice。Nissl staining was used to observe the damage of neurons in the CA1 and CA3 regions of the hippocampus。Acetylcholine(Ach)level,AchE and ChAT activity in hippocampal and cortex tissues were detectedo MDA content and total antioxidant capacity in hippocampal and cortical tissues were detected.Protein expression level of Nrf2/HO-1 signaling pathway was detected by Western-Blot and immunohistochemistry.Experiment 3:Study on the effect and mechanism of FA-97 on learning and memory function of APP/PS1 mice。In this study,8-month-old APP/PS1 mice were divided into 6 groups with 12 mice in each group.They were divided into normal group(homozygosis APP/PS1 transgenic negative mice),model group(APP/PS1 mice),low dose FA-97 group(2.5mg/kg),medium dose FA-97 group(5mg/kg),high dose FA-97 group(10mg/kg),and DNP group(3mg/kg)。FA-97,CAPE and DNP were administered orally once a day for 60 consecutive days.Morris water maze experiment and novel objective recognition experiment were used to evaluate the learning and memory ability of mice。Levels of Aβ1-40 and Aβ 1-42 in hippocampal and cortex tissues were measured by Elisa.The deposition of Aβ plaque in the brain tissues of mice was observed by Thio S staining.Nissl staining was used to observe the damage of neurons in the CA1 and CA3 regions of the hippocampus.Western-Blot was used to detect the expression of apoptotic proteins(Bax,Bcl-2,Cytochrome c)and synaptic proteins(PSD95,Synapsin,Synaptophysin,and BDNF)in the brain of mice.ResultsExperiment 1:Protective effect and mechanism of FA-97 on H2O2-induced oxidative stress model of neurons。FA-97 inhibited the level of pro-oxidants(MDA and protein carbonyl)and ROS in SH-SY5Y and PC12 cells induced by H202,and significantly increased the activity of several major antioxidant enzymes(SOD and GSH).FA-97 can inhibit the expression of apoptosis-related proteins in H202-induced nerve cells,inhibit the apoptosis rate of nerve cells,and promote the activity of nerve cells.In terms of mechanism research,FA-97 promotes the transcriptional activity of Nrf2 and activates the expression of downstream ho-1 and nqo-1。Experiment 2:Effect of FA-97 on learning and memory function in scopolamine induced memory impairment mice model and its mechanismThe results of the Morris water maze showed that FA-97 could promote the escape latency.The space exploration experiment showed that times of mice in the FA-97 group to traverse the original platform area were significantly increased,and the new object recognition experiment showed that the time of mice in the FA-97 group to explore new objects were increased.FA-97 can promote Ach level and ChAT activity in hippocampus and cortex of scop-treated mice,and inhibit AchE activity.FA-97 down-regulated the expression of apoptosis index Bax/Bcl-2 and cytochrome c in hippocampus and cortex of scop-treated mice.In addition,FA-97 can significantly inhibit malondialdehyde(MDA)content in mice,and significantly improve the total antioxidant capacity of scop-induced mice.In terms of mechanism research,FA-97 can up-regulate the expression of HO-1 and NQO-1,up-regulate the Nrf2 level,and activate the Nrf2/HO-1 signaling pathway.Experiment 3:Study on the effect and mechanism of FA-97 on learning and memory function of APP/PS1 mice.The results of the Morris water maze showed that FA-97 could promote the escape latency of APP/PS1 mice.The space exploration experiment showed that times of the mice in the FA-97 group traversing the original platform area increased significantly.The results of the new object recognition experiment showed that the time to explore new objects increased in the FA-97 group.Fa-97 down-regulated levels of Aβ1-40 and Aβ1-42 in the hippocampal and cortical tissues of APP/PS1 mice.FA-97 can down-regulated the expression levels of pro-apoptotic proteins Bax and cytochrome c and promoted the expression levels of anti-apoptotic proteins Bcl-2.FA-97 can promote the expression levels of Synaptophysin,Synapsinl,PSD95 and BDNF。ConclusionFA-97 has a significant neuroprotective effect,which can reduce the level of H2O2-induced cellular oxidative stress in vitro,inhibit the apoptosis rate of neurons,and alleviate the cognitive impairment induced by scopolamine in vivo.This effect is related to the activation of Nrf2/HO-1 signaling pathway.At the same time,FA-97 can improve the cognitive impairment of the APP/PS1 transgenic AD mice,which may be related to the down-regulation of the level of Aβ in the brain tissue by FA-97 and the protection of synaptic function and the reduction of neuron death.