| Objective:Exploring the role of glycolysis in B[a]P activation of microglia-induced cognitive impairment in mice from in vivo animal and in vitro cell experiments.To provide a certain scientific basis for the study of the mechanism of B[a]P neurotoxicity.Methods:In vivo experiment: Fifty SPF adult ICR mice were randomly divided into five groups according to body weight,with 10 mice in each group.They are control group(peanut oil),2.5,5,10 mg/kg B[a]P exposure group and 10 mg/kg B[a]P+ 2-DG intervention group.The mice in the exposure group were given B[a]P orally,and the mice in the solvent control group were given the same amount of peanut oil according to the weight of the mice,once every other day,and were exposed continuously for 45 times for a total of 90 days.Mice in the intervention group were given orally 2-DG dissolved in drinking water at a concentration of 0.03% 2-DG.Prepare 2-DG of drinking water daily.After accomplishing the treatment,the cognitive function of the mice was assessed using the hole-board test,T-maze test,pole climbing test,Selffleece experiment,and Morris water maze test.Western blot analysis was used to detect the expression of Iba-1,GLUT1,HK-2,PKM-2,HIF-1α and LDHA proteins in rat cerebral cortex.In vitro experiment: BV2 cells were cultured in complete DMEM medium containing 10% fetal bovine serum.When BV2 cells grew to the logarithmic growth phase,the cells were randomly divided into 5 groups: DMSO control group,0.2,2,20μM B[a]P exposure group,20 μM B[a]P + 80 μM 2-DG Intervention group.The complete DMEM medium was replaced with low serum DMEM(3% fetal bovine serum),and BV2 cells were treated with DMSO or B[a]P.The intervention group received a solution of 2-DG diluted in PBS along with B[a]P exposure.After culturing at 37°C and 5% CO2 for 24 hours,observe morphological changes of cells with optical microscope,and detect the changes in cell viability with CCK-8 kit.The cell glycolysis,glycolytic capacity and glycolytic reserve were tested using Agilent Seahorse XFP.Iba-1,GLUT1,HK-2,PKM-2,HIF-1α and LDHA protein expression were detected using Western blot.Results:In vivo experiments showed that With the increasing of B[a]P doses,The mice in the high-dose B[a]P group reacted slowly,their hair luster decreased,the hair was dry and easy to comb,multiple yellow abscesses appeared on the back,neck and abdominal cavity,and their body weight decreased significantly.Compared with the solvent control group,the 2.5,5,and 10 mg/kg B[a]P groups significantly reduced the frequency and duration of caving in hole-board test(P < 0.05),and the correct rate of the T maze was significantly reduced(P < 0.05),the time of climb from top to bottom significantly was increased(P < 0.05),self-fleece time was significantly increased(P< 0.05);the water maze escape latency,the frequency of crossing the platform and target quadrant dwell time of the 10.0 mg/kg B[a]P exposure group were significantly Increase(P < 0.05).This suggests that B[a]P exposure can impair the cognitive ability of mice.Transmission electron microscopy showed increased doses of B[a]P.In the hippocampus of mice,the nuclei of microglial cells gradually deformed,decreased in size,and produced vacuoles between them and the cytoplasm;the expression of Iba-1in the cerebral cortex was significantly increased(P < 0.05),suggesting that B[a]P can activate microglia,glucose content detection showed that compared with the solvent control group,the glucose level in the cerebral cortex of mice after B[a]P exposure was significantly reduced(P < 0.05),suggesting that B[a]P exposure may cause brain energy crisis in mice.The results of Western blot showed that the protein levels of PKM-2 and LDHA were significantly increased in all B[a]P groups(P<0.05),and the protein levels of GLUT1,HK-2 and HIF-1α were significantly increased in the 10.0mg/kg B[a]P groups(P<0.05).Increased expression of glycolytic proteins indicated a shift in brain glucose metabolism towards reprogramming of glucose metabolism in mice exposed to B[a]P.After 2-DG intervention,the mice in the 10 mg/kg B[a]P + 2-DG group had improved neurobehavioral performance compared with the B[a]Pexposed group.The nuclear membrane of mice hippocampal microglial cells is complete and smooth,the chromatin is evenly distributed,and the mitochondrial structure is complete,the expression of Iba-1 in the mice cerebral cortex is significantly reduced(P < 0.05),and the glucose content in the mice cerebral cortex is significantly increased(P < 0.05).Compared with the solvent control group,the expressions of glycolytic pathway-related proteins including GLUT1,HK-2,PKM-2,HIF-1α and LDHA were significantly reduced in mice(P < 0.05),Glucose levels are significantly elevated in the brains of mice.As the central immune cells in the central nervous system,microglia are like a double-edged sword.Transient activation promotes neuronal growth,but sustained activation under chronic stimulation induces neuroinflammation,which leads to neuronal inflammation.Microglia play a role in the brain no less than neurons.Studying the cause of microglia activation has great scientific significance for the prevention of cognitive dysfunction.The results of in vitro studies showed that B[a]P exposure decreased cell number,increased cell aggregates,increased cell debris,shortened number of prominent branches.Cell morphology changes from normal dumbbell-like to amoebalike.Cell viability was significantly decreased in the B[a]P high-dose group compared with the DMSO group(P<0.05).The results of Western blot showed that the expression of Iba-1 protein in each group exposed to B[a]P was significantly increased compared with the DMSO control group(P < 0.05).It suggests that B[a]P can activate BV2 cells after exposure.The results of glucose content detection showed that compared with the DMSO group,the glucose levels in each B[a]P group were significantly decreased(P < 0.05).The glycolytic stress test showed that compared to the DMSO control group the 20.0 μM B[a]P group had significantly increased the basal glycolytic capacity,the glycolysis capacity and glycolytic reserve(P < 0.05).Western blot results showed that compared with the DMSO group,the expression levels of HK-2,PKM-2,HIF-1α and LDHA proteins in the 20.0 μM B[a]P group increased(P<0.05).The expressions of Iba-1 and GLUT1 proteins in all B[a]P groups were significantly increased(P<0.05),Indicates that B[a]P activates BV2 cells by regulating glycolysis.After 2-DG intervention,the B[a]P-induced amoeba-like morphological transition of BV2 cells was delayed or attenuated.Compared with the20.0 μM B[a]P group,the BV2 cell viability and intracellular glucose content in the intervention group increased significantly(P < 0.05).The results of Seahorse showed that the basic glycolytic ability of cells in the intervention group,the glycolytic capacity and glycolytic reserve were all significantly reduced(P < 0.05).The results of Western blot showed that the expression of Iba-1 protein decreased significantly,the expression of glycolysis-related proteins GLUT1,HK-2,PKM-2,HIF-1α and LDHA decreased significantly.Conclusion:Glycolysis plays an important role in B[a]P-induced cognitive dysfunction and microglia activation in mice,and the potential mechanism may be related to the activation of GLUT1/HK-2/PKM-2/HIF-1α/LDHA glycolysis pathway.Inhibition of glycolysis can significantly improve B[a]P-induced sustained activation of microglia and cognitive dysfunction. |
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