Study On Effects Of Learning Memory In Rats And Molecular Mechanism Of The Apoptosis Of Neurocytes Induced By Benzo[a]Pyrene

【Objective】To study the effects of benzo[a]pyrene on learning and memory in rats in vi-vo, the molecular mechanism of the apoptosis of neurocytes induced by benzo[a]pyrene at cell and molecular levels, to look for the periphery specific effect biomarker, and to provide the imp-ortant and scientific basis for the specific prevention of benzo[a]pyrene intoxication and the imp-rovement of benzo[a]pyrene-exposed workers life.【Methods】40SD rats, adult and healthy, were divided into 4 groups at random, i.e.3 exposed groups and the vehicle control group. The expos-ed groups were treated by lateral cerebral ventricle micro-injection with B[a]P dissolved in olive oil at 126.2, 63.1 and 31.5 g/kg respectively, one time every week, for 3 weeks, the vehicle control group was given olive oil. 1.After exposure, the step-down test, the passive avoidance test and the Morris water maze test were performed to test the learning and memory abilities of rats. 2. After the experiment, slices of the cerebral cortex hippocampus and cerebellum were made and morphological changes were observed by optical microscope. 3. The neural cell apop- tosis of brain tissue in Rats was detected by the technology of situ hybridization. 4. Expression of Bcl-2 and Bax in neural cells in rats' brain tissue were measured with ABC of immunohistoc-hemical method. Results were analyzed by Imaging Analyzing System. 5. Caspase-3, Caspase-9,Cyt-c, Bax and Bcl-2 in brain tissue and cytoplasm were detected with methods of Western-blo-tting ECL, and results were analyzed with Gelatum Analyzing System.【Results】1. learning and memory capability test: 1) In Step-down Test, it was showed that compared with the olive oil group , the latency(LT) was shortened (P<0.05) in the low-dose group; both EN1 and EN2 decreased significantly(P<0.01)and the LT was shortened (P <0.05) in the medium-dose group ; EN1 increased significantly(P<0.01), the EN2 increased (P<0.05) and the LT was shortened obviously(P<0.01) in the high-dose group; 2) In the Passive avoidance Test, it was showed that compared with the olive oil group, the latency two(LT2) was shortened (P<0.05),EN2 decreased (P<0.05)in the low-dose group; and the LT were shortened (P <0.05) , EN1 increased (P<0.05, P<0.01), the EN2 increased significantly (P<0.01) in the medium-dose and high-dose groups; 3 ) In the Morris water maze test,￠ùThe place navigation test, the LT was shortened in rats of all groups (P<0.05).￠úThe spatial probe test, the benzo-[a] pyrene-exposed rats were swimming around the platform quadrant, but the control animals were swimming directly to the platform quadrant. Compared with the olive oil group , the number of times passing over hidden platform decreased in the low-dose group (P > 0.05), the swimming distance through the quadrant of hidden platform and whole distance were shortened(P<0.05); In the medium-dose and high-dose groups, the number of times passing hidden platform decreased significantly (P<0.05, P<0.01),The swimming distance of hidden platform and whole distance were shortened significantly(P<0.01).2. The observation under light microcope showed:arrangement of the neurocytes was orderly in the vehicle control group, but with the increasing of the doses of benzo[a]pyrene exposed, the neurocytes displayed disorderly, the connections among the neurocytes were loosened, the karyon shrinked, some rings around the neurocyte were observed. 3. benzo[a]pyrene can make the apoptosis index increased obviously (P<0.01). Apoptosis indices(AI) of neurocytes in rats' brain tissue in the medium-dose group and high- dose group were significantly higher than that of controls (P<0.01). 4.The analysis of immunohi-stochemistry show benzo[a]pyrene can promote the expression of Bax in rats' brain significantly (P<0.01), it can make the expression of Bcl-2 and Bcl-2/Bax decreased obviously (P<0.01,P<0.05), A negative correlation between AI and the expression ofBcl-2 (r =-0.927, -0.934,P<0.01)and Bcl-2/Bax( r =-0.939, -0.942,P<0.01), and a positive correlation between AI and the expression of Bax (r =0.858, 0.874,P<0.01) were observed. Rrsults were analyzed by Imaging Analyzing System. 5. The analysis of Western- blotting ECL showed benzo[a]pyrene can promote significantly the expression of Caspase-3, Caspase-9, Cyt-c, Bax (P<0.01, P<0.05), it can make the expression of Bcl-2 decreased obviously (P<0.01, P<0.05), A negative correlation between AI and the expression of Bcl-2 (r =-0.631, -0.594 P<0.01), and a positive correlation between AI and the expression of Caspase-3, Caspase-9, Cyt-c and Bax (r=0.872,0.843; 0.869,0.812 ; 0.578, 0.549 ; 0.798,0.731; P<0.01, P<0.05)wereobserved. Results were analyzed by Imaging Analyzing System.【Conclusion】The effects on learning and memory in rats and the molecular mechanism of the apoptosis of neurocytes induced by benzo[a]pyrene are related to the downwards regulation of Bcl-2, the upwards regulation of Bax and the decrease of Bcl-2/Bax, Benzo[a]pyrene may induce neurocytes apoptosis also through the upwards regulation of Caspase-3, Caspase-9 expression, release of Cyt-c.