| [Objective] To study the involvement of tau hyperphosphorylation in memory impairment after espousing to B[a]P in SD rats, and the role of P25/CDK5-related signal pathway in B[a]P-induced tau hyperphosphorylation.[Methods]1. Male SD rats were randomly assigned to five groups:the blank control,0,1.0,2.5,6.25mg/kg.bwB[a]P. The rats were received intraperitoneal injection for1,2and3months respectively.2. Morris water maze was employed to observe the learning and memory impairment, the spatial learning was observed by place navigation test, and spatial memory was tested by space probe test.3. BPDE-DNA adducts in rat brain tissues were detected by a BPDE-DNA adducts ELISA kit. MDA content in rat brain tissue were measured by TBA assay, and SOD activity was detected by WST-1method.4. The expression of P35/P25, CDK5, Tau, Tau[pS199], Tau[pS396], Tau[pT181]and Tau[pT231] in the rat brain were detected by western blot.[Results]1. Learning and memory abilities test:After exposure to B[a]P for1,2and3months respectively, escape latency of each B[a]P treated group (0,1.0,2.5,6.25mg/kg.bw) decreased in a testing day-dependent manner and increased in a exposure time-dependent manner. B[a]P-administered rats for different exposure duration (1,2and3months) showed prolonging escape latencies in the MWM compared with controls(P<0.01). The higher-dose group (2.5,6.25mg/kg.bw) exposed for2and/or3months had longer escape latencies.Then, a three-factor ANOVA with repeat measure was performed to analyze the interactions among related factors (day, dose and time) on escape latency in hidden platform test. The results suggest there would exist interactive effects among day, dose and time on the escape latency. The probe trial displayed that B[a]P exposure can decrease the time spent in the target quadrant and the number of crossing the platform, prolong the first time arriving at the target space in a time-dependent and dose-dependent manner. But in rats exposed for1month, the differences of above-mentioned indexes between groups were not statistically significant (F=0.027, P=0.998; F=0.555, P=0.697; F=1.940, P=0.126). Furthermore, the rats exposed to2.5and6.25mg/kg.bw B[a]P in different exposure duration groups spent less time in the target area, had prolonged first time arriving at the target space and less number crossing the platform than the controls. Probe trial suggests that B[a]P exposure could cause deleterious effects on spatial memory.2. The BPDE-DNA adducts in the rat brain was an upward trend with increasing exposure dose and time. Compared with the blank control group, and the DNA adducts increased by9.3%,27.3%and45.9%for rats exposed to6.25mg/kg B[a]P for1,2and3months respectively, and the different between each dose groups was statistically significant (P<0.01); as for rats exposed2.5mg/kg.bw B[a]P for3months, DNA adducts were increased by20.3%compared with the control, the difference was statistically significant difference (P<0.05). MDA contents were increased as exposure dose and time increased. For rats exposed2,3months,2.5,6.25mg/kg.bwB[a]P groups were increased approximately48.6%and98.6%than the control group. And the difference was statistically significant (P<0.05, P<0.01). SOD activity wad decreased with increasing exposure dose and duration, when rats in6.25mg/kg.bw B[a]P group for2and3months, SOD activity decreased by25.4%and37.5%compared with the blank control groups, and the different between each dose groups was statistically significant (P<0.01). And the SOD activity in2.5mg/kg.bw B[a]P group for3months reduced14.6%compared with the blank control group (P<0.05).3. The relative intensities of Tau, Ser199, Thr181and Thr231increased significantly in a dose-and time-dependent manner compared with the control groups. B[a]P did not alter the tau phosphorylation at Ser396, recognized by pS396antibody, of rats exposed for1,2and3months (F=1.266, P=0.287; F=0.713, P=0.584; F=1.392, P=0.241). The interactions of dose and time on the indexes of Ser199, Thrl81and Thr231were statistically significant (F=4.385, P<0.001; F=8.789, P<0.001; F=2.236, P=0.025) with the increase of exposure time and doses.4. The expression level of P25, P35and CDK5increased as the exposure time and dose increased.Compared with the blank control group, P35protein expression in6.25mg/kg.bwB[a]P group when exposed for3months increased by8.25%,28.93%and63.96%respectively. P25protein had no expression in rats brain for1month, since exposure to2months P25expression began to extend an upward trend with the increase in the dose and time of exposure,6.25mg/kg.bw B[a]P group when exposed for2and3months, the expression of P25had an increase of104.67%and197.64%compared with the control. And the dose and duration of interaction of above indexes were statistically significant (P<0.01, P<0.01). CDK5protein expression in rat brain tissue increased as exposure doses of the time increased, CDK5protein expression in rats brains of1.0,2.5and6.25mg/kg.bwB[a]P group were significantly increased compared with the control group (P<0.01), and CDK5expression in6.25mg/kg.bw B[a]P group had an increase of64.99%,80.06%and128.3%respectively.[Conclusion]1. B[a]P can induce learning and memory impairment in SD rats, and with the increase of the exposure dose and time, the damage is more serious.2. B[a]P enables to cause hyperphosphorylation of the SD rats brain tau protein at Ser199,Thr181and Thr231.3. The possible mechanism of tau phosphorylation and learning and memory impairment may be relevant with the P25/CDK5related signal pathways:B[a]P exposure causes oxidative stress and DNA damage in rat brain tissue, which can upgrade the expression of P25protein and activation of CDK5, leading to tau protein hyperphosphorylation. |
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