Involvement of PDICAkt pathway in apoptosis induced by semaphorin 3A in cultured rat cortical neurons

Objective To investigate the effect of exogenous semaphorin 3A （Sema3A） on apoptosis in primary cultured rat cortical neurons and the roles of phosphoinositide 3-kinase （PI3K）/serine-threonine kinase （Akt） pathway in apoptosis induced by Sema3A. Methods Newborn Spmgue-Dawley rat cortical neurons were cultured in vitro and they were identified by microtubule associated protein-2 （MAP-2） staining The cultured cortical neurons were treated with various concentrations of Sema3A （0, 500, 1 000, and 2 000 μg/ml） for 48 hours. Neuronal survival rate was detected with CCK8 assay. Neuronal apoptosis was detected with Hoechst33342 staining and TUNEL staining. The expressions of P-Akt, Akt and Bcl-2 in cortical neurons were determined with Western blotting Results The purity of cortical neurons culture was more than 95%. CCK8 assay showed that the survival rates of cortical neurons in the oups of 500, 1 000and 2 000 μg/ml Sema3A were 80. 9% ±5. 3%, 67. 5% ±3.9% and 50. 2% ±4. 4% of the control ffoup, respectively （F= 165. 042,P =0. 000）. Hoechst33342 staining showed that the apoptosis rate in the normal control ffoup and the goups ofS00, 1 000and2 000 μg/ml Sema3A were22. 4% ± 1.2%,34.0% ±1.2%,39.3% ±1.4% and47. 3% ± 2. 3%, respectively （F = 103.237 ,P =0. 000）. TUNEL staining showed that the apoptosis rate in the normal control goup and the ffoups of 500, 1 000and 2 000 μg/ml Sema3A were 23.9% ± 1.1%, 31.9% ± 1.0%, 40. 1% ± 1.5% and 51.4% ± 3.4%, respectively （F = 103. 118 ,P = 0. 000）. Western blotting showed that the expressions of P-Akt （F = 15. 959, P =0. 001） and Bcl-2 （F = 18. 776,P =0. 001） decreased gaduaUy, while the expression of Akt had no sifffificant changes （F = 0. 590, P = 0. 639）. Conclusions Sema3A can decrease the survival rate of the cultured cortical neurons, mainly by inducing apoptosis, and the mechanism of which might be related to the down-realation of expressions of P-Akt and Bcl-2. Key words: Neurons;  Apoptosis;  Semaphorin-3A;  Phosphatidylinositol 3-Kinases;  Proto-OncogeneProteins c-akt;  Proto-Onco~me Proteins c-bcl-2;  Cells, Cultured;  Rats