Effects of Ketamine on Proliferation and Apoptosis of Pheochromocytoma Cell
|Keywords||ketamine pheochromcytoma cell toxicity schizophrenia cell proliferation cell apotosis|
Objective: To investigate the effects of ketamine-induced PC12 apoptosis.Methods: Rat adrenal pheochromcytoma cells (PC12) were treated with different concentration of ketamine , after various time of ketamine treatment , the cell viability was measured by MTT method . Apoptosis and cell cycle of PC12 cells after 48h of different concentration of ketamine exposure were detected by flow cytometry (PI and AnnexinV-FITC staining ); The morphous of apoptosis was observed by Hoechst staining to ; The ultrastructure of PC12 cells was examined by transmission electron microscopy. mRNA expressions of bax、bcl-2、caspase9、caspase3 were assessed by RT-PCR, and the protein expressions of bax、bcl-2 were assayed by western blot .Results: (1)MTT assay showed that ketamine had a significant anti-proliferation effect on pheochromcytoma cells in a dose-dependant and time-dependant manner. After treated with 0.9 mmo/L ketamine for 12 h, 24 h, 48 h the inhibition rates were 13.36%、40.13% and 42.02%, respectively; After treated with 1.2 mmo/L ketamine for 12 h, 24 h, 48 h ,the inhibition rates were 17.14%、41.8% and 60.72%, respectively ; And After treated with 1.5 mmo/L ketamine for 24 h, 48 h, 72 h the inhibition rates were 36.26%、58.76% and 80.04%, respectively. The IC50 of 12h、24h、48h were1.659 mmol/L、1.261mmol/L and 1.182 mmol/L; respectively. (2) Apoptosis morphological changes about chromatic agglutination and nuclear condensation were detected by Hoechst 33258 fluorochrome staining.(3) Apoptosis rates of pheochromcytoma cells treated with 0, 0.9, 1.2 , 1.5 mmo/L ketamine for 48 hours were 2.52%、6.53%、8.61% and 19.77%, respectively; After trated with ketamine for 72 h , the apoptosis rates were 3.9 %、42.74 % and 66.48 % respectively (P<0.01); Cell cycle arrest was examined by Flow Cytometry (FCM). After treated with 0.9, 1.2 , 1.5 mmo/L ketamine for 48 hours, the S phase rates were 21.59%、25.81% and 29.07%, respectively. It was 17.5% in control group(P< 0.05). (4) AnnexinV-FITC staining detects the apoptosis rates after treated with 0.9, 1.2 , 1.5 mmo/L ketamine for 48h , They were 8.2%、12.5% and 14.5% respectively , the apoptosis rates of control group was 1.2% (P< 0.05) ; (5) After treated with 0.9, 1.2 , 1.5 mmo/L ketamine for 48h , the mitochondria mediated mRNA bax、caspase9、caspase3 were upregulated and bcl-2 was downregulated by RT-PCR . They were also in a dose-dependant(P<0.05).(6)After treated with 0.9, 1.2 , 1.5 mmo/L ketamine for 48h , the protein of bax was upregulated , bcl-2 and An-TH were downregulated by western blot. They were also in a dose-dependant(P<0.05).Conclusion Ketamine can inhibit the proliferation of PC12 cell, inducing PC12 cell to apoptosis remarkably and concentrations-dependent . The mitochondrial signal transduction pathway was involved with its mechanism .