| Objective:To investigate the effects of benzo[a]pyrene exposure on learning and memory,fear memory and other behaviors in mice,as well as the effects of adult neurogenesis on the dentate gyrus of the hippocampus,and further explore the Wnt/?-catenin signaling pathway whether to participate in the regulation of related changesMethods:198 healthy 8-week-old C57BL/6 male mice were randomly divided into 3 groups:the exposed group,the vehicle control group and the saline control group.Each group was intraperitoneally injected with 0.04%benzo[a]pyrene solution(2mg/kg),equal volume of olive oil(5mL/kg)and equal volume of normal saline(5mL/kg)for 10 consecutive days respectively,and then intraperitoneally injected 1%BrdU solution(100mg/kg)for 5 consecutive days.On the day of 1 week post injection and 3 weeks post injection,8 mice of each group were tested in Novel Object Recognition,Morris Water Maze,Fear Conditioning Test and Open Field Test to detect the effects of benzo[a]pyrene exposure on discriminating memory,visual spatial memory and fear memory function in mice Also 6 mice of each group were carried out with perfusions and frozen sections Immunofluorescent staining was used to observe and count the number of double positive neurons of DCX/NeuN and BrdU in the dentate gyrus of hippocampus,and to explore the effect of benzo[a]pyrene exposure on adult neurogenesis in mice Furthermore,on the day of 1 week post injection,6 mice in each group were prepared for Western Blot to investigate the effect of benzo[a]pyrene exposure on the expression of key proteins ?-catenin and Axin-2 in the Wnt/?-catenin signaling pathway and further to explore whether Wnt/?-catenin signaling pathway was involved in the regulation of adult neurogenesis and the above changes.Data were processed for mapping and statistical analysis using GraphPad Prism 7 and SPSS 21.0.Experimental data are expressed as mean ± standard error(Mean ± SEM).One-way ANOVA was used after normality and variance homogeneity test among multiple groups,and Tukey's multiple comparison was used for post-test;repeated measurement data satisfy normality,homogeneity of variance and "spherical symmetry" test using Repeated measurement ANOVA;Two paired sample data were used to satisfy the normality and variance homogeneity test using Two-tailed paired t-test.Set P<0.05 as the statistically significantResults:1.Behavioral results(1)The results of the Novel Object Recognition showed that compared with the control group,the benzo[a]pyrene exposure group were significantly reduced in the number of recognition of novelty(1wpi:F=26.330,P<0.0001;3wpi:F=20.850,P<0.0001),the number of crossing the area where the novelty is located(1wpi:F=37.920,P<0.0001;3wpi:F=16.350,P<0.0001)and the discrimination ratio(1wpi:F=73.440,P<0.0001;3wpi:F=51.160,P<0.0001),and also the difference was statistically significant(P<0.05)(2)The results of Morris Water Maze showed that compared with the control group,the benzo[a]pyrene exposure group significantly increased the latency of finding the platform during the navigational phase(Fbetween group=13.100,P<0.0001;Fwithin group=37.640,P<0.0001).Also the number of crossing the platform in the space exploration phase was significantly reduced(1wpi:F=11.070,P=0.0011;3wpi:F=8.532,P=0.0034),and the difference was statistically significant(P<0.05)(3)The results of Fear Conditioning Test and Open Field Test showed that the level of fear memory in the benzo[a]pyrene exposed group was significantly inhibited compared with the control group(Fbetween group=12.880,P<0.0001;Fwithingroup=363.70,P<0.0001),and thus reduced fear associated mood-anxiety,and the difference was statistically significant(P<0.05).2.Immunohistochemistry resultsAfter coronal frozen section of hippocampus,immunofluorescence double-staining was performed to observe the double labeling of DCX and BrdU and the double labeling of NeuN and BrdU.Using Image J for counting analysis,it was found that compared with the control group,the number of generating adult newborn neuron decreased significantly in the benzo[a]pyrene exposed group(lwpi:F=13.270,P=0.0005;3wpi:F=17.420,P=0.0001),and the difference was statistically significant(P<0.05).3.Western Blot resultsThe results of Western Blot showed that the expression of ?-catenin(F=21.260,P<0.0001)and Axin-2(F=42.170,P<0.0001)was significantly lower in benzo[a]pyrene exposed group compared with the control group.It indicated that benzo[a]pyrene exposure changed the adult neurogenesis in the dentate gyrus of hippocampus by affecting Wnt/?-catenin signaling pathway,and the difference was statistically significant(P<0.05).Conclusion:1.Benzo[a]pyrene exposure could impair learning and memory functions and decrease the level of fear memory in mice;2.Benzo[a]pyrene exposure significantly reduced adult neurogenesis in the dentate gyrus of hippocampus in mice;3.Benzo[a]pyrene exposure could significantly affect the function of the Wnt/?-catenin signaling pathway in mice and might cause these changes. |
PDF Full Text Request