LNC001209 Regulating PI3K/AKT/mTOR Signaling Pathway Through CD36 Is Involved In Aluminum Induced Cognitive Impairment

Objective:In vivo experiments were conducted to explore that aluminum exposure regulates Lnc001209,CD36 and PI3K/AKT/m TOR signaling pathway,causing neuronal cell apoptosis and prominent plasticity damage,and finally leading to cognitive dysfunction.In vitro experiments were conducted to explore that Lnc001209 regulating PI3K/AKT/m TOR signaling pathway through CD36 is involved in aluminum induced cognitive impairment.Methods:1?Sprague Dawley rats were randomly divided into 4 groups according to body weight:normal saline,low-dose(10?mol/kg),medium-dose(20?mol/kg)and high-dose(40?mol/kg)(15 rats in each group).Rats were exposed to intraperitoneal injection for 3months.The basic conditions of rats in each group were observed and the brain weight ratio was calculated.The content of Aluminium in rat brain tissue and plasma was determined by inductively coupled plasma mass spectrometry(ICP-MS).The spatial exploration ability and learning and memory ability of rats were observed through new object recognition and Morris water maze experiment.The excitatory postsynaptic potential(f EPSP)in hippocampal CA1 was recorded by the technique of long-term enhancement detection in vivo animals.The morphology of hippocampal neurons and the number of dendritic spines were observed by Golgi staining.The synaptic and mitochondrial structures of hippocampal neuron apoptosis were observed by transmission electron microscope.The expression level of Lnc001209 in rat hippocampal tissues was detected by reverse transcription-polymerase chain reaction(RT-PCR).Western blot was used to detect the expression of PI3K,AKT,m TOR and corresponding phosphorylated protein in hippocampal tissues of rats.2?The adrenal medulla pheochromocytoma differentiated cells(PC12 cells)were selected as the study subjects,and were divided into four groups according to the exposure dose,namely the control group,the low-dose group(100?mol/L aluminum maltol),the middle-dose group(200?mol/L aluminum maltol),and the high-dose group(400?mol/L aluminum maltol).PC12 cells at logarithmic growth stage were cultured in100?L culture medium of all concentrations of the poison in an incubator for 24h.The morphology of PC12 cells exposed to aluminum was observed by electron microscope.The survival rate of cells exposed to aluminum was determined by CCK-8 method.Apoptosis rate of cells after al exposure was determined by Annexin V-FITC/PI double staining.RT-PCR was used to detect the expression level of Lnc001209 after aluminum exposure.Cells were transfected with Lnc001209 si RNA cells to detect the transfection efficiency.Western blot was used to detect the expression of p-AKT Ser473,p-AKT Thr308,p-p85 Tyr467,p-m TOR Ser2448 and CD36 in cells transfected with si RNA of Lnc001209,and then the apoptosis rate was detected by flow cytometry.After RNA transcription and purification in vitro,RNA pull-down experiment was conducted to detect the binding protein of Lnc001209.The differential proteins after exposure to aluminum were identified by mass spectrometry.3?PC12 cells were selected as the research objects,and the interaction protein of Lnc001209 was confirmed as CD36 by RNA Pull down-MS method.The expression level of CD36 gene was detected by RT-PCR after aluminum exposure.Western blot was used to detect the expression level of CD36 protein after aluminum exposure and to observe the expression level of p-AKT Ser473,p-AKT Thr308,p-p85 Tyr467,p-m TOR Ser2448 after cell intervention with CD36.Apoptosis rate was determined by Annexin V-FITC/PI double staining.Results:1?During the experiment,rats in the control group developed normally,with good hair color and active and hyperactive,while rats in the low-dose group showed no obvious abnormalities.Rats in the medium-high dose group showed poor reaction ability,slightly reduced activity and dark hair color,and no animals died during the experiment.The brain index of the rats in each aluminum maltol exposure group decreased gradually with the increase of exposure dose,and the difference between the groups was statistically significant(p<0.05).ICP-MS detection found that the contents of brain and blood aluminum in rats exposed to maltol aluminum increased gradually with the increase of exposure dose,and the increase was most obvious in the high-dose group,with statistically significant difference between each group(p<0.05).It was found in the new object recognition experiment that The Times of exploring new objects in each maltol aluminum exposure group decreased gradually with the increase of the exposure dose,the residence time of the new object quadrant decreased gradually,and the ability of recognizing new objects decreased gradually.The experimental data of water maze showed that the incubation period of rats in each maltol aluminum exposure group gradually increased with the increase of the exposure dose,the number of crossing the platform gradually decreased,the residence time of the target quadrant gradually decreased,and the learning and memory functions of the rats gradually decreased.LTP results showed that the excitation electrodes in CA1 area of hippocampus of rats exposed to aluminum maltol gradually weakened with the increase of exposure dose,and the differences between groups were statistically significant(p<0.05).Golgi staining showed that,with the increase of exposure dose,the number of dendritic spines decreased gradually,resulting in neuronal cell loss and neuronal fracture.The results of electron microscope showed that neuronal cell apoptosis occurred in rats exposed to medium and high dose of aluminum maltol,especially in rats exposed to high dose.The synaptic structures of nerve cells in the hippocampus CA1 region of rats exposed to maltol aluminum were decreased,and the synaptic vesicles of the presynaptic membrane were decreased,and the post-synaptic density of the presynaptic membrane was thinner,which was most obvious in the high-dose group.The mitochondrial cristal fractures of hippocampal nerve cells of rats exposed to aluminum maltol were observed in the high-dose group.RT-PCR results showed that the expression level of hippocampal Lnc001209 in each maltol aluminum exposure group decreased gradually with the increase of exposure dose,and the difference between each group was statistically significant(p<0.05).Western blot results showed that the expression of CD36 protein in the hippocampus of rats exposed to aluminum maltol gradually increased with the increase of exposure dose,and the difference between the groups was statistically significant(p<0.05).The expression of PI3K,AKT,m TOR protein gradually decreased with the increase of exposure dose.But p-AKT Ser473,p-AKT Thr308,p-p85 Tyr467,p-m TOR Ser2448 expression quantity increase with exposure dose increased,the activity of proteins in the pathways,the activity of p-AKT strong or continuous enhance will promote nerve cell apoptosis,so maltol aluminum exposure can by activating PI3K-AKT-m TOR pathways caused apoptosis of rats,lead to injury of synaptic plasticity,show the function of learning and memory is reduced.2?When the morphology of PC12 cells was observed under a microscope,the number of cells in each group exposed to maltol aluminum was significantly lower,and the morphology of the cells changed,the cell body became round,the axial mutation was short,and the intercellular connections gradually decreased,especially in the high dose group.Cell viability was detected by CCK-8,and the cell viability of each maltol aluminum exposure group decreased gradually with the increase of exposure dose,and the difference between groups was statistically significant(p<0.05).Flow cytometry analysis of PC12 cell apoptosis showed that the apoptosis rate of PC12 cells in the medium and high dose groups was increased,especially in the high dose group,and the difference between the groups was statistically significant(p<0.05).RT-PCR results showed that the expression level of PC12 cells Lnc001209 in each maltol aluminum exposure group decreased gradually with the increase of exposure dose,and the difference between groups was statistically significant(p<0.05).In maltol aluminum exposed PC12 cells transfection Lnc001209 si RNA,found that p-AKT Ser473,p-AKT Thr308,p-p85 Tyr467,p-m TOR Ser244 protein expression levels,maltol aluminum exposed histone phosphorylation level also rises,maltol aluminum exposure than inhibit Lnc001209 is strong,the function of phosphorylated proteins exposed aluminum+inhibit Lnc001209 group of protein expression levels,the most obvious and the difference between groups was statistically significant(p<0.05).Lnc001209 si RNA was transfected into PC12 cells and the expression level of CD36 protein was increased in the aluminum exposure group as well.Compared with the blank control group and the negative transfection control group,the apoptosis rate of cells in the three groups Lnc001209si RNA,200?mol/L Al(mal)₃and the Lnc001209si RNA+200?mol/L Al(mal)₃was increased,and the difference between the groups was statistically significant(p<0.05).3?RNA transcription and purification in vitro,and agarose gel electrophoresis results showed that the observed bands were consistent with the expected position of Lnc001209,enabling RNA pull-down experiment.There were no obvious bands in the magnetic bead samples of Lnc001209 RNA pull-down experiment,indicating that the proteins had been eluted for further mass spectrometry analysis.The 78 different proteins in the experimental group after the control group were subtracts for bioinformatics analysis,and CD36 expression was significantly different in all the different proteins(p=0.00075),but further verification was needed.RNA pull-down MS experiment found that CD36 could be detected in the eluent of the justice strand probe group,while CD36protein was not detected in the eluent of the antisense strand probe group,indicating that Lnc001209 had a direct interaction with CD36 protein.RT-PCR showed that the expression of CD36 gene in PC12 cells exposed to maltol aluminum was increased.Western blot results showed that CD36 protein expression in PC12 cells of each maltol aluminum exposure group was increased,and the difference between the groups was statistically significant(p<0.05).CD36 intervention showed that the expression levels of p-AKT Ser473,p-AKT Thr308,p-p85 Tyr467,p-m TOR Ser244 increased after overexpression of CD36,and the phosphorylation level of AKT was the most obvious,and the pathway was activated.Flow cytometry detection of PC12 cell apoptosis showed that,compared with the blank control group and the negative control group,the apoptosis rate of the CD36 overexpressed aluminum exposure group and the CD36 overexpressed aluminum exposure group were increased,and the difference between the groups was statistically significant(p<0.05),in which the apoptosis rate of PC12 cells in the CD36overexpressed aluminum exposure group was the most significant.Conclusion:1?Aluminum exposure caused differences in the expression of Lnc001209,CD36,PI3K/AKT/m TOR signaling pathway in rats,suggesting that aluminum exposure may influence the pathway to induce neuronal apoptosis,leading to cognitive dysfunction in rats.2?Aluminum exposure inhibited apoptosis of cells caused by Lnc001209 via PI3K/AKT/m TOR signaling pathway,suggesting that aluminum exposure may induce apoptosis by affecting this pathway.3?The regulation of CD36 by Lnc001209 through PI3K/AKT/m TOR signaling pathway may be involved in the aluminium-induced neuronal cell apoptosis.