The Study On The Mechanism Of Learning And Memory Impairment Induced By Bisphenol A In Developing Mice And The Intervention Of α-lipoic Acid

Objective:Bisphenol A（BPA）is widely used in the production of food or beverage containers,food packaging bags.BPA is an environmental endocrine disruptor that can be widely exposed to human body.BPA can cross the blood-brain barrier,which makes the brain an important target of BPA.Learning and memory ability is the basis of all cognitive activities.Embryo period,infancy and adolescence are considered to be the key periods for the formation and consolidation of learning and memory ability.Studies found that BPA exposure during these critical periods may have adverse effects on brain structure,cognitive behavior and learning and memory ability through destroying synaptic plasticity or interfering with neurotransmitter homeostasis.However,there are still few reports on the effect of BPA on learning and memory ability,and the specific mechanism is not clear.The structure of BPA is similar to diethylstilbestrol（DES）,which can simulate or antagonize the biological function of estrogen.17β-estradiol（E2）is an important estrogen in the body,which is an important neuromodulator that can regulate the process of learning and memory.After binding to estrogen receptorα（ERα）,E2 can quickly activate protein kinase C（PKC）,and then activate extracellular signal regulated kinase（ERK）and its downstream transcription factor c AMP response element binding protein（CREB）,so as to regulate learning and memory ability.BPA can be used as an agonist or antagonist of ERαto interfere with the biological effects of E2.Therefore,researching the effects of BPA on E2,ERα,and its downstream PKC/ERK/CREB signaling pathway,is of great significance to explore the mechanism of BPA on learning and memory.However,the toxic mechanism of BPA is not single.In addition to interfering with hormones’function,BPA can also have adverse effects on the body by inducing oxidative stress.Synapse is the physiological basis of learning and memory formation.When oxidative stress occurs at synapses,the accumulation of reactive oxygen species（ROS）and reactive nitrogen species（RNS）can destroy the membrane structure of synapses and affect various proteins on synapses,such as estrogen receptors,neurotransmitter receptors N-methyl-D-aspartate receptor（NMDAR）andα-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor（AMPAR）,and synaptic structure marker protein synapsin I and postsynaptic density protein-95（PSD-95）.These receptors and structural proteins play an important role in maintaining the structural and functional integrity of synapses.In addition,the activation of NMDAR can make Ca²⁺flow rapidly,activate PKC,and then quickly activate PKC/ERK/CREB signaling pathway to regulate learning and memory.Therefore,the study of the changes of synaptic associated proteins is of great significance to explore the role and mechanism of BPA induced synaptic oxidative damage.At present,the specific mechanism of oxidative stress induced by BPA in hippocampal synapses is not clear.One of the causes of oxidative stress is the increase of the sources of ROS and RNS.Mitochondria are the main places for the production of ROS.The mitochondrial respiratory chain complex I and complex III are the main sites for the production of O₂^·-.The continuous production and accumulation of ROS in mitochondria will lead to mitochondrial dysfunction and more ROS production,resulting in oxidative damage in the whole cell.In addition,neuronal NO synthase（nNOS）can catalyze L-arginine to produce nitric oxide（NO）after NMDAR activation.NO is not only the key signal molecule of synaptic transmission and plasticity,but also a kind of RNS.Another important reason for oxidative stress is the reduction of antioxidant capacity.Kelch-like ECH-associated protein（keap1）/Nuclear factor erythroid 2-related factor 2（Nrf2）signaling pathway can regulate the gene and protein expression of downstream detoxification enzymes（such as heme oxygenase）and antioxidant enzymes（such as superoxide dismutase and catalase）,so as to play an antioxidant role.Studies have reported that the health hazards of BPA exposure are related to oxidative stress.Therefore,to explore the effects of BPA exposure on mitochondrial function,nNOS and keap1/Nrf2 signaling pathway is of great significance to reveal the cause and specific mechanism of synaptic damage caused by BPA exposure.At present,the antioxidants that antagonize the toxicity of BPA mainly include melatonin,vitamin C and E,and the related research is very few.α-Lipoic acid（ALA）is a kind of biological mercaptan,which can freely pass through the biofilm.ALA can play its antioxidant role by scavenging ROS and RNS,and regenerating other endogenous antioxidants.Al A is also an important cofactor in mitochondrial metabolism and mitochondrial redox regulation.It is found that ALA can improve the spatial learning and memory ability of elderly rats.Therefore,exploring the protective effect and mechanism of ALA on learning and memory impairment induced by BPA is of great significance to explore effective antioxidants that can antagonize the neurotoxic effects caused by BPA exposure.In conclusion,the first part of this study will explore the effect of BPA exposure on learning and memory ability in developing mice and the protective effect of ALA.The second part is to explore the mechanism of hippocampal synaptic injury caused by BPA exposure and the protective mechanism of ALA through detecting the changes of hippocampal ERα,synaptic associated protein and PKC/ERK/CREB signal pathway.The third part discusses the specific mechanism of hippocampal synaptic injury caused by BPA exposure and the protective mechanism of ALA from the perspective of oxidative stress through detecting the changes of mitochondrial function,nNOS and keap1/Nrf2 signaling pathway.Through the above three parts,this study will provide a new theoretical basis for further clarifying the neurotoxic effect and mechanism of BPA exposure,and also provide a reference basis for exploring effective antioxidants to antagonize the neurotoxic effect caused by BPA exposure.Methods:1.Animal experiment:Fifty 4-week-old C57BL/6J female mice were randomly divided into 5 groups after one week of adaptive feeding:control group,0.1μg/ml BPA（0.1 BPA）group,0.2μg/ml BPA（0.2 BPA）group,0.6 mg/ml ALA（ALA）group,and 0.2μg/ml BPA+0.6 mg/ml ALA（0.2 BPA+ALA）group.Exposure to BPA and ALA by drinking water for 8 weeks.The body weight,food intake and water consumption of mice were recorded.Morris water maze experiment,object placement（OP）and object recognition（OR）test,and shuttle box experiment were used to detect the learning and memory ability of mice.The levels of Ach and GABA in hippocampus,E2 in serum,and 8-OHd G in serum and hippocampus were detected by ELISA.The levels of MDA,and the activity of CAT and SOD in serum and hippocampus of mice were detected by corresponding kits.H&E staining was used to detect the histopathological changes of mouse hippocampus.The protein expression levels of ERα,NR2B,Glu A1,PSD-95,synapsin I and Nrf2 in hippocampus was detected by immunohistochemistry.The protein expression levels of ERα,synapse related proteins,PKC/ERK/CREB signaling pathway,nNOS and keap1/Nrf2 signaling pathway related proteins in hippocampus were detected by Western blot.2.Cell experiment:Immortalized mouse hippocampal neuron cell line HT-22 cells were used in this experiment.At the same time of BPA and ALA exposure,Nrf2inhibitor was added to further study whether the oxidative stress caused by BPA exposure and the protective effect of ALA were related to Nrf2.According to different experimental purposes,the cells were divided into five groups and six groups.（1）Five groups:control group,50μM BPA group,100μM BPA group,150μM ALA group,and 100μM BPA+ALA group,the exposure time was 24 hours.（2）Six groups:control group,100μM BPA group,150μM ALA group,BPA+ALA group,50 n M Brusatol（BT,inhibitor of Nrf2）group,and BPA+ALA+BT group,the exposure time was 24 hours.CCK-8 was used to detect cell viability and determine the exposure doses of BPA,ALA and BT.The level of ROS was detected by flow cytometry.The activities of mitochondrial complex I and III and the ATP level were detected by corresponding kits.The expression levels of ERα,synapse related proteins,PKC/ERK/CREB signaling pathway,nNOS and keap1/Nrf2 signaling pathway related proteins in HT-22 cells were detected by Western blot.Results:1.The escape distance and escape latency to find the platform were increased in BPA exposed mice on the 3-5th day,and the frequency,latency and cumulative time of mice entering the target quadrant were decreased on the 6th day compared with the control group.The above indexes were improved in 0.2 BPA+ALA group compared with 0.2 BPA group.The recognition index of new placement and new object were decreased in BPA exposed group mice compared with the control group.The recognition indexes in 0.2 BPA+ALA group were higher than that in 0.2 BPA group.The frequency and time of active avoidance response were decreased,and the frequency and time of escape response were increased in BPA exposed groups compared with the control group.The above indexes of mice were improved in 0.2BPA+ALA group compared with 0.2 BPA group.There was no significant difference in body weight,brain weight,hippocampal weight and brain tissue coefficient among groups.The hippocampal tissue coefficient of 0.2 BPA group mice was lower than that of other groups.There was a significant cell layer decrease,cell shape change,indistinct boundary,nuclear pyknosis,and number decrease in CA1 region,and a significant nuclear pyknosis in DG region in BPA exposed mice compared with the control group.Those pathological changes of hippocampus were improved in 0.2 BPA+ALA group compared with 0.2 BPA group.2.The levels of Ach and GABA in hippocampus,E2 levels in serum were decreased in BPA exposed groups compared with the control group,and were increased in 0.2BPA+ALA group compared with 0.2 BPA group.The protein levels of ERα,p-NR2B,NR2B,p-Glu A1,Glu A1,PSD-95 and synapsin I decreased significantly in hippocampus of mice and HT-22 cells exposed to BPA compared with the control group.Al A could significantly alleviate the decrease of the above proteins.The protein levels of PKC,PKCα,PKCε,p-ERK,p-CREB and CREB were decreased in hippocampus of mice exposed to BPA compared with the control group.The protein levels of PKCα,PKCε,p-ERK,ERK,p-CREB and CREB were decreased in HT-22cells exposed to BPA compared with the control group.Al A can significantly alleviate the decrease of the above protein expression levels in hippocampus of mice and HT-22cells induced by BPA expousre.3.In animal experiment and cell experiment（five groups）,the levels of MDA and8-OHd G in serum and hippocampus were increased,and the enzyme activities of CAT and various types of SOD in serum and hippocampus were decreased in BPA exposed groups compared with the control group,and were improved in 0.2 BPA+ALA group compared with 0.2 BPA group.The level of ROS was increased,the enzyme activities of mitochondrial complexes I and III were decreased,and the level of intracellular ATP were decreased in BPA exposed HT-22 cells compared with the control group.The above indexes were improved in 100μM BPA+ALA group compared with the100μM BPA group in HT-22 cells.Compared with the control group,the protein expression level of nNOS was increased in hippocampus of mice and HT-22 cells exposed to BPA,and the protein expression levels of Nrf2,HO-1 and NQO1 were decreased in hippocampus of mice exposed to BPA,and the protein expression levels of keap1,Nrf2,HO-1 and NQO1 were decreased in HT-22 cells exposed to BPA.Al A could significantly improve the level changes of these proteins in hippocampus and HT-22 cells induced by BPA exposure.4.In the cell experiment（six groups）,the level of ROS was increased,and the protein expression levels of keap1,Nrf2,HO-1,NQO1,ERα,p-NR2B,NR2B,p-Glu A1,Glu A1,PSD-95,synapsin I,PKCα,PKCε,p-ERK,ERK,p-CREB and CREB were decreased in BPA exposed HT-22 cells compared with the control group.The level of ROS was decreased,and the expression level of the above proteins were increased in BPA+ALA group compared with 100μM BPA group.The level of ROS was increased,and the expression level of the above proteins were decreased in BPA+ALA+BT group increased compared with BPA+ALA group.Conclusion:1.BPA exposure can damage the learning and memory ability of mice,and its mechanism is related to the reduce of E2 level,down regulation of the ERαand synapse related proteins,and then inhibiting the PKC/ERK/CREB signaling pathway.2.BPA exposure can reduce mitochondrial function,up regulate nNOS expression,inhibit keap1/Nrf2 signaling pathway and then lead to oxidative damage in hippocampus,which may be the important pathological mechanisms that BPA damaging synaptic structure and function through reducing levels of ERαand synapse related proteins.3.ALA can antagonize the impairment of learning and memory ability caused by BPA exposure.Its mechanism may be that ALA activates keap1/Nrf2 signaling pathway,increases antioxidant enzyme activity,reduces ROS levels,and then inhibits the effect of oxidative stress on hippocampal ERαand synapse related proteins.