FTO-regulated Glucose And Lipid Metabolism Involved In The Neurotoxicity Induced By Benzo[a]pyrene And The Intervention Effect In Mice

Objective:To explore the intervention effect of metformin（MET）on neurotoxicity in mice induced by benzo[a]pyrene（B[a]P）treatment and the possible mechanism from the aspect of glucose and lipid metabolism.The finding is expected to provide a scientific basis for the study of the mechanism and prevention of B[a]P neurotoxicity.Methods:Forty-five healthy Specific pathogen free（SPF）grade male ICR mice（8 weeks,weighed 29～34g）were randomly assigned into 5 groups（N=9/group）according to their body weights（bw）,including the solvent control group,three B[a]P-treated groups,and a MET intervention group,respectively.The treated groups were gavaged with B[a]P solution（2.5,5,or 10 mg/kg）at 0.1m L/10g bw（at 9:00 am）once every other day,while the solvent control was gavaged with oil.The intervention group was administrated MET（300 mg/kg）by gavage on the day after 10 mg/kg B[a]P treatment.A total of 45 administrations last for 90 days.During the whole administration period,mice were recorded the daily intake of water and food,neurological symptoms and signs,and were weighed once a week.After the last treatment,the anxiety and depression-like behavior of mice were first tested in the Self-grooming test,followed by the elevated plus maze（EPM）.Pole test and beam balance test（BBT）were used to evaluate the motor coordination ability of mice.We evaluated the learning and memory ability of mice using the Morris water maze（MWM）and the new location recognition（NLR）test.After completing the neurobehavioral tests,5 mice out of each group were tested on their fasting glucose level and glucose tolerance.The glucose tolerance for each mouse was evaluated using the area under the curve（AUC）based on the trapezoidal method.Plasma samples were detected the levels of Total Cholesterol（TC）,Triglycerides（TG）,Low-density lipoprotein（LDL-C）,and High-density lipoprotein（HDL-C）using the corresponding biochemical kits. Transmission electron microscopy was used to observe the ultrastructural changes in the synaptic of the hippocampus,quantitative analysis was then performed.The expression of glutamate receptor-associated proteins（NMDAR1,GLUA1,GLUA2）in the cortex of mice,fat mass and obesity-associated protein（FTO）and forkhead protein O6（Fox O6）proteins in the cortex and liver of mice,as well as the N-6 adenylate methylation（m⁶A）modified proteins（Mettle3,Mettle14,ALKBH5）in the cortex of mice were detected using Western blot（WB）.The m⁶A levels in the cerebral cortex of mice were detected by m⁶A RNA methylation quantification kit,the expression of Fox O6 m RNA in the cortex and liver of mice were detected by real-time quantitative PCR（q RT-PCR）,and the co-localization of FTO and Fox O6protein in the cortex of mice was observed using double immunofluorescence（IF）staining.Results:Compared to the solvent controls,following B[a]P treatment,the mice were more prone to mood swings,sloppy appearance,hair drying out and shedding in the mid and high doses B[a]P-treated groups（5 and 10 mg/kg）.The body weights were significantly decreased in the mid and high doses B[a]P-treated groups（5 and 10 mg/kg）compared to the controls（P<0.05）at the beginning of the 12^thweek. Furthermore,after the completion of exposure,the self-grooming test and the EPM showed that compared with the solvent control group,the grooming time of mice in all the B[a]P-treated groups significantly increased（P<0.05）,the entries to the open arm showed a downward trend（P>0.05）,as well as the duration time in the open arm of the EPM were significantly reduced（P<0.05）in the mid and high doses B[a]P-treated groups（5 and 10 mg/kg）.The results of the pole climbing test and BBT showed that compared with the solvent control group,the pole climbing time of mice in all the B[a]P-treated groups significantly increased（P<0.05）,the time required to cross a beam was significantly increased（P<0.05）in the mid and high doses B[a]P-treated groups（5 and 10 mg/kg）.Results from the MWM test indicated that the escape latency of mice in the high dose B[a]P-treated group（10 mg/kg）was significantly higher on day 5 compared to the solvent control,time to the first platform crossing was significantly elevated whereas the number of platform crossing,the percentage of swimming distance and swimming time in the target quadrant were significantly decreased in the high dose group（10 mg/kg）compared with solvent control（P<0.05）.The RI and DI in the NLR test were significantly declined in all three B[a]P-treated groups（2.5,5,and 10 mg/kg）compared to the controls（P<0.05）.The above results indicated that mice in all B[a]P-treated groups have increased anxiety and depression like behavior,impaired motor coordination,and impaired cognitive function.Following B[a]P treatment,FBG showed no significant difference among all groups（P>0.05）.The AUC values were significantly increased in the mid and high doses B[a]P-treated groups（5 and 10 mg/kg）（P<0.05）,and the concentrations of TC and LDL-C in plasma were significantly increased in the high dose B[a]P treatment group（10 mg/kg）compared to the solvent controls（P<0.05）,the glucolipid metabolism was disrupted in mice following B[a]P treatment.Under transmission electron microscopy,the pre-and post-synaptic membranes and cleft of hippocampal neurons were relatively clear,the synaptic vesicles in the pre-synaptic membranes were abundant and evenly distributed in the solvent control,whereas the synaptic of hippocampal neurons were more swollen,the synaptic vesicles in the membrane decreased,and the synaptic cleft widened in the mice treated with B[a]P.Compared with the solvent control group,the number of synapses,the curvature of the synaptic interface,the postsynaptic density,and the length of the postsynaptic membrane in the hippocampal neurons of mice have significantly decreased in the high dose B[a]P treatment group（10 mg/kg）（P<0.05）.Following B[a]P treatment,the cortical NMDAR1 protein levels were significantly elevated,while the expression of GLUA1 and GLUA2 decreased（P<0.05）.The FTO and Fox O6 protein levels were both significantly elevated in the cortex and liver of mice（P<0.05）.The IF results showed that the localization of FTO and Fox O6 in the cortex of mice was consistent.Compared with the solvent control group,the fluorescence intensity of FTO and Fox O6 in the cortex of mice were increased in a dose-dependent manner in the B[a]P-treated groups.Quantitative detection of m⁶A RNA methylation showed that the level of m⁶A methylation in the cerebral cortex of mice in the mid and high doses（5 and 10 mg/kg）of B[a]P-treated groups were lower than that in the solvent control group（P>0.05）.The Mettl3 and Mettl14 protein levels were decreased（P<0.05）,while the expression of demethylase protein ALKBH5 was increased（P<0.05）.The results of q RT-PCR showed that compared with the solvent control group,the expression of Fox O6 m RNA in the cerebral cortex and liver of mice were significantly decreased in the mid and high doses（5 and 10 mg/kg）of B[a]P-treated groups（P<0.05）. Compared to the B[a]P alone treatment group（10 mg/kg B[a]P）,MET intervention had significantly restrained the declines in water and food intakes,and had greatly improved the mood swings,sloppy appearance,and hair shedding.MET effectively reversed changes in indicators related to mood,motor coordination,and cognitive function（P<0.05）,meanwhile,MET effectively reversed the disorder of glucose and lipid metabolism caused by B[a]P through significantly reduced the AUC values and bring down the LDL-C levels（P<0.05）.In addition,under transmission electron microscopy,MET intervention alleviated the degree of synaptic swelling,and increased the number of synaptic vesicles in the pre-synaptic membranes,the number of synapses,postsynaptic density,and length of postsynaptic membrane（P<0.05）. MET intervention reversed the overexpression of NMDAR1 and the low expression of GLUA1 and GLUA2 proteins in the cerebral cortex of mice,reversed the high expression of FTO and Fox O6 proteins in the cortex and liver of mice,significantly increased the expression of Mettl3 protein and decreased the expression of ALKBH5protein（P<0.05）,and showed an upward trend in Mettl14 protein expression and m⁶A methylation modification levels（P>0.05）.The IF results showed that the fluorescence intensity of FTO and Fox O6 in the cortex of mice in the MET intervention group was significantly reduced.The results of q RT-PCR showed that MET intervention reversed the low expression of Fox O6 m RNA in the cerebral cortex and liver of mice（P<0.05）.Conclusion:B[a]P treatment can induce increased anxiety and depression-like behavior,decreased motor coordination,cognitive dysfunction,abnormal neuronal synaptic plasticity,and disorder of glucose and lipid metabolism in mice,accompanied by the activation of FTO/Fox O6 signaling pathway,decreased expression of m⁶A methyltransferase proteins,and increased expression of m⁶A demethyltransferase proteins.MET intervention effectively alleviated the neurotoxic induced by B[a]P treatment,mainly by regulating glucolipid metabolism via inhibition of FTO/Fox O6signaling and reversing the abnormal expression of m⁶A modified proteins.