L-3-N-Butylphthalide Alleviates Cobalt Chloride Induced Oxygen Deficit In Human Neuroblastoma Cells

Objective:To study the protective effect and mechanism of action of L-3-n-butylphthalide which alleviates cobalt chloride induced oxygen deficit in human neuroblastoma SK-N-SH cells.Methods:Human neuroblastoma SK-N-SH cells are divided into four groups, including a group of L-3-n-butylphthalide pretreatment group (Cells were pretreated by L-3-n-butylphthalide at dose of 1μmol/L, 10μumol/L 4 hours before, then added cobalt chloride at dose of 250μmol/L for 6 hours), a group of cobalt chloride (Cells were treated by cobalt chloride at dose of 250μmol/L for 6 hours) and a group of control(Cells did not make any processing).Cells were cμltured in incubator of 5%　CO2 in 37℃, Cells of logarithmic phase were used for experiment. The cell morphology changes of SK-N-SH were observed by phase-contrast microscope. Then, the cell viability was detected by using MTT assay. Meanwhile, western blot was used to detect the expression of HIF-la, Bax, Bcl-2. Hoechst were used to measure the cells of each group.Results:l.The microscopic observation between different groups of SK-N-SH cell phenotype results show that the group of cobalt chloride has fewer shorter nerve synapse and fewer floating dead cells by phase-contrast microscopy observation (compared with the control group). At the same time, the morphological of SK-N-SH cells have changed with the increase of the dose of L-3-n-butylphthalide, the quantity of floating dead cells is decreased.2.MTT assay showed that the cell viability of drug intervention groups are significantly decreased compared with the control group (P<0.001) (compared with the control group).The cell viability of L-3-n-butylphthalide group at low dose (1μumol/L) is significantly increased (P<0.01) (compared with the group of cobalt chloride) and the cell viability of L-3-n-butylphthalide group at high dose (10μmol/L) is significantly increased (P< 0.001) (compared with the group of cobalt chloride). The cell viability of L-3-n-butylphthalide group at high dose (10μmol/L) is significantly increased compared with the group of L-3-n-butylphthalide group at low dose (1μmol/L) (P<0.001)3.The result of hoechst shows that cells nucleus of the group of cobalt chloride pyknosised,strong blue fluorescence presented at the microscopic and apoptosis propotion rase (compared with the control group).With the increasing dose of the L-3-n-butylphthalide,the number of strong blue fluorescence decreased,the apoptosis propotion decreased.4.Western blot showed that the expression of Bax and HIF-1αin the drug intervention group are significantly increased (P<0.001),while the expression of Bcl-2 is significantly decreased (P<0.01) (compared with the control group); the expression of Bax in L-3-n-butylphthalide group are significantly decreased (P< 0.001) (compared with the control group), while the expression of Bcl-2 and HIF-1α is significantly increased (P<0.001) (compared with the group of cobalt chloride);the expression of Bax in L-3-n-butylphthalide group at high dose (10μmol/L) are significantly decreased (P<0.001), while the expression of Bcl-2 and HIF-1α is significantly increased (P<0.001) (compared with the group of L-3-n-butylphthalide group at low dose).Conclusion:1.L-3-n-butylphthalide markedly increased the cell viability of SK-N-SH caused by hypoxia and protect it.2. L-3-n-butylphthalide makes SK-N-SH cell apoptosis rate decline induced by cobalt chloride through activating HIF-1α way which increases Bcl-2,reduces Bax and block apoptosis of SK-N-SH cells induced by hypoxia.