| Benzo [a] pyrene (B[a]P) is a major component of environmental pollutants of polycyclic aromatic hydrocarbons, because of good liposolubility, it can cross the blood-brain barrier, studies also showed it can enter into the central nervous system from the olfactory bulb, and accumulate in the cerebellum and other parts. Autonomic nerve function disorder and short-term memory damage were observed among workers who exposed to coke oven emissions. A large number of studies on neurotoxicity of B[a]P were launched between domestic and foreign researchers. Results showed that B[a]P can impaired short-term memory and spatial related memory both in human and rats, and the impairment were correlated with the concentration of both B[a]P and metabolite of B[a]P. Studies suggest that B[a]P caused neuronal apoptosis may be related to learning and memory decline related, but the exact mechanism is unclear.This study aims to analysis the mechanism which B[a]P induced neuronal apoptosis and the effects of B[a]P on LTP signaling pathway, and explore the molecular mechanisms of B[a]P impaired learning and memory abilities in rats.ObjectiveTo investigate the apoptosis correlated gene and protein expression, proteion antivity, still the gene and protein expression and phosphorylation level of PKA/ERK/CREB pathway. To explore the molecular mechanisms of B[a]P induced learning and memory impairment.Method Learning and memory ability of50SD rats were measured before contamination and then they were randomly divided into blank control group, olive control group,1.0,2.5and6.25mg/kg B[a]P group according to the results. After intra-peritoneal injected with B[a]P for90days, learning and memory ability of rats were assessed again by using Morris water maze. Hippocampus of rats were obtained after learning and memory ability was test, then extract total mRNA and tissue protein. Use flow cytometry to test apoptosis ratio of neuron in rat hippocampus. mRNA expression were mensurated by fluorescent quantitation PCR. Utilize western blotting to detect the expression protein and phosphorylation level. And test the activity of Caspase3, Caspase6and Caspase9protein using spectrophotometry.Results1. Swimming speed for each group showed no statistacally significant in the first five days, and average swimming speed also showed no significantlly change(P>0.05). Escape latency of6.25mg/kg B[a]P group were higher than that of blank control and olive control group and1.0mg/kg B[a]P group at the1,2and5th day(P<0.05). Escape latency of1.0and2.5mg/kg B[a]P group for lth day,2.5mg/kg B[a]P group for3th day,2.5and6.25mg/kg B[a]P group for5th day were higher than blank control group(P<0.05).Average escape latency of B[a]P exposure groups were significantlly higher than blank control group and olive control group (P<0.05).Then the platform was moved away, frequance of platform crossing of2.5and6.25mg/kg B[a]P group were significantly lower than that of blank control group, olive control group and1.0mg/kg B[a]P group(P<0.05). platform quadrant retention time of6.25mg/Kg B[a]P group was significantly less than the blank control, solvent control,1.0and2.5mg/Kg in B[a]P group, the differences were statistically significant (P<0.05); Percentage of platform quadrant retention time of6.25mg/Kg B[a]P group was significantly lower than that of blank control, solvent control,1.0mg/kg B[aP group(P<0.05) and2.5mg/kg B[a]P group significantly lower than blank control group.2. Results came from flow cytometry showed that early apoptosis ratio were increased as the dose of B[a]P ascend(Ptrena<0.05) and early apoptosis ratio of1.0,2.5and6.25mg/Kg B[a]P group were significantly higher than blank and Olive control group. BAX protein was up-regulated at6.25mg/Kg B[a]P group while BCL-2protein and BCL-2/BAX down-regulated.Caspase3,6protein was significantly rised at2.50and6.25mg/Kg B[a]P group compared to blank, Olive control group and1.0mg/Kg B[a]P group (P<0.05), but we didn’t observed modification of Caspase9protein (P>0.05).Activity of Caspase3,Caspase6and Caspase9were increased at2.5and6.25mg/Kg B[a]P group, and significantly more than the tow control groups and1.0mg/Kg B[a]P group(P<0.05). And there exist a positive correlation between Caspase3, Caspase6, Caspase9activity and early apoptosis ratio of neuron in rat hippocampus(r=0.793,p=0.019; r=0.886,p=0.006; r=0.773, p=0.025). Expression of apoptosis correlated gene in hippocampus present no significantly modification (P>0.05).3. It was present that PKA mRNA expression in2.5and6.25mg/kg group were less than that of blank control group and olive control group, Both ERK1mRNA in1.0,2.5,6.25mg/kg group and ERK2mRNA in1.0mg/kg group were less than that of blank control group; The expression of CREB in6.25mg/kg group was less than that of blank control group, olive control group1.0and2.5mg/kg group. Both C-FOS mRNA in1.0,2.5,6.25mg/kg group and C-JUN mRNA in2.5,6.25mg/kg group were more than that of blank control group. All the differences above were statistically significant (P<0.05). Protein expression of PKA in2.5and6.25mg/kg group were lower than that of blank control group and olive control group, and that, PKA level in6.25mg/kg group was less than2.5and6.25mg/kg group, all differences were statistically significant (P<0.05). Interestingly, in spite of the expression of ERK1/2, CREB, BDNF, C-FOS and C-JUN protein were didn’t significantly changed (P>0.05), we observed that the phosphorylation level of PKA, ERK1/2and CREB protein in2.5and6.25mg/kg were significantly lower than that in blank control group and olive control group (P<0.05).Conclusions1.Sub-chronically exposed to B[a]P can impair learning and memory ability of rats.2. Subchronic B[a]P expose can induce apoptosis of neuron in hippocampus; Overexpress of BAX,caspase3,caspase6and up-regulate of caspase3, caspase6and caspase9activity, coupled with deregulate of BCL-2and BCL-2/BAX would be one of the apoptosis mechanism induced by B[a]P.3. The mechanism that Benzo(a)pyrene subchronic exposure impair learning and memory ability might correlated with depressing PKA, ERK1/2, CREB protein phosphorylation, B[a]P induced down regulation of PKA mRNA and protein levels may contribute to the down regulation of PKA,ERK1/2and CREB phosphorylation. |
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