The Effect Of Benzo[a]pyrene On Hsp70 Expression And Nerve Cell Damage In Cultured Nerve Cell In Rat

Objective:To study the effect of benzo[a]pyrene (B[a]P)on Hsp70 expression and nerve cell damage in cultured nerve cell in rat, and investigate the function of Hsp70 in the neurotoxicity of B[a]P, providing intervening measure for neurotoxicity of B[a]P.Methods:The cerebral cortex were isolated from the newborn 1 to 3 days of SD rats under sterile conditions and digested with 0.25% trypsin, then seeded with 1×106 density. The nerve cells were exposed with B[a]P and B[a]P+S9, respetively, at the concentration 0,0.1,0.5,1,5, 10μmol/L for 24h to investigate them on Hsp70 expression in dose-response relationship. The nerve cells were also exposed with B[a]P+S9 at the concentration 0,0.5,5μmol/L for 24h,48h and 72h to explore it on Hsp70 expression in time-response relationship. Western blot was used to measues the expression of Hsp70 and immunofluorescence staining was used to observe the expression and location of Hsp70. The morphological changes of nerve cells were observed by invert microscope. The cell viability was determined by MTT assay. The content of LDH, MDA was detected by special kit. The flow cytometry was used to detect the cell apoptosis. Single cell gel electrophoresis was used to detect cell DNA damage. The correlation between Hsp70 and cell injury was analyzed by the curve fitting.Results:The Hsp70 expression in nerve cells appeared firstly increased with the dose and then decreased after exposed with B[a]P and B[a]P+S9, Hsp70 arrived to the peak when B[a]P was in 50μmol/L and when in 100μmol/L Hsp70 was significantly decreased; and when exposed to 0.1μμmol/L B[a]P+S9 only firstly slightly increase and then decrease significantly with the increase of the dose, while 10μmol/L group nerve cellular Hsp70 expression values (0.10±0.05) was ignificantly lower than 0μ/L group (0.28±0.05, P<0.05),0.5,1,5, 10μmol/L group Hsp70 expression values (0.23±0.05,0.21±0.15,0.16±0.10 and 0.10±0.05) were significantly lower than 0.1μmol/L group (0.41±0.11,P＜0.05); time-response experiments indicated that 0.5, 5μmol/L B[a]P+S9 exposed to nerve cells, along with the exposure time, Hsp70 expression values of a downward trend,72h of nerve cell groups Hsp70 expression values (0.28±0.15 and 0.17±0.03) were significantly lower than the 24h(0.36±0.03 and 0.25±0.02, P<0.05); show B[a]P metabolites on nerve cells can inhibit the expression of Hsp70. Immunohistochemistry showed that the control group only in the cytoplasm of Hsp70 expression,0.1μmol/L group of fluorescence expression, in both cytoplasm and nuclear expression. As the dose increased expression of nucleus gradually increased, but the expression intensity of fluorescence decreased.The nerve cell injury test showed that as the B[a]P+S9 dose increased, the number of axons and dendrites of nerve cells were decresed and the length of synapses was shortened. Some cells showed vacuolization, chromatin condensation into a block. Cell viability decreased with increasing dose,1,5, 10μmol/L group MTT OD value (0.50±0.45,0.49±0.28 and 0.47±0.25) was significantly lower than the control group (0.65±0.81,P<0.05). Cell culture medium LDH activity and MDA content, cell apoptosis, comet tail length and tail moment increased as the dose rose.1,5, 10μmol/L group LDH activity and MDA content (520.2±11.6,540.5±40.6, 588.4±63.7u/L and 1.61±0.08,1.65±0.06,1.67±0.13nmol/ml) was significantly higher (321.5±36.8u/L and 1.38±0.06nmol/ml, P＜0.05).0.1,0.5,1,5, 10μmol/L group of early apoptosis rate and total apoptosis (3.36±0.55,4.73±0.50,6.5±0.71,8.2±0.75,10.43±0.73 and 3.47±0.56,4.83±0.39,6.65±0.63,8.44±1.09,10.57±0.83) were significantly higher than control group (2.13±0.25 and 2.18±0.23, P<0.05), while 0.5,1,5, 10μmol/L group comet tail length and tail moment (4.85±3.19,14.79±5.62,36.23±4.97,62.82±8.85μm and 1.8±2.95,6.23±4.58, 12.78±8.89,24.72±17.41) significantly higher than control group(2.97±1.02μm and 0.81±0.37, P<0.05).Curve fitting analyzed the relationship between the nerve cells Hsp70 expression and cell injury show that a linear correlation exist in Hsp70 expression and LDH and MDA value, and correlation coefficient r was 0.857 (P＜0.05) and 0.828 (P<0.05), while a quadratic curve exist in Hsp70 expression and apoptosis rate and the value of comet tail moment, and curve correlation coefficient R2 was 0.917 (P＜0.05) and 0.987, (P<0.01).Conclusion:Large doses of B[a]P metabolites may inhibit the expression of Hsp70 in nerve cells by a dose and time-dependent relationship. B[a]P metabolites can cause nerve cell membrane injury, increase apoptosis and DNA damage, which may be associated with B[a]P metabolite-induced peroxidation. In the higher dose range, Hsp70 expression in nerve cells was negatively correlated with cell injury. B[a]P metabolites on nerve cells inhibit the expression of Hsp70 that may lead to loss of nerve cell protection and Hsp70 participate in the process of cell injury.