| ObjectiveThe aim was to study the effect of nimodipine (NMDP) on Fas/FasL and Caspase signal pathway of HL-60cell apoptosis induced by cytarabine (Ara-c).MethodsNMDP and Ara-c with the different concentration were administered to HL-60leukemia cells. After24h, The ATP chemiluminescence method was detected to the cell proliferation inhibition rate.When the concentration of NMDP was0.050g/l and that of Ara-c was0.010g/l,the optimal concentration was selected.Then divided into four after the experimental group:the control group (not add any drug HL-60cell group),NMDP group (concentration for0.050g/L), Ara-group C (0.010g/L concentration),NMDP (0.050g/L)+Ara-C (0.010g/L) group..After acridine orange/ethidium bromide (AO/EB) mixed dye staining for24h, the cell morphology was observed and apoptotic cells were counted under fluorescence microscope.Then Caspase3and Caspase8activity was assayed by Detection kit and Fas/FasL protein expression was assayed by Western blotting method.Results1. Cell proliferation:①Compared with the control group, HL-60Cell inactivation rate induced by NMDP (0.001～0.100g/L) and Ara-c (0.005～0.500g/L) of different concentrations was significantly enhanced (FMNDPp=12231.563, FAra-c=6404.210, p=0.000).②When the concentration of NMDP was0.050g/l and that of Ara-c was0.010g/l, HL-60Cell inactivation rates were the highest (F=916.836,p=0.000).2. Cell apoptosis:①AO/EB dyeing:Cell morpholology observed under fluorescence microscopy showed that most of cells in the control group appeared normal, green and integrated. The number of apoptotic cells in NMDP, Ara-c and NMDP+Ara-c groups significantly increased, showing orange and pyknotic nuclear chromatin, and NMDP+Ara-c group was the most significant.②Cell apoptosis rate:The cell apoptosis rate in the control group was (5.021±0.391)%, while that in NMDP, Ara-c and NMDP+Ara-c group was (14.203±1.204)%,(27.604±3.192)%,(54.929±11.598)%. The cell apoptosis rates in the three groups were statistically significant (F=148.020,p=0.000), and NMDP+Ara-c group was the highest.3. Fas protein expression:Gray scale scan ratio of Fas protein expression in the control group was (0.642±0.008), while that in NMDP, Ara-c and NMDP+Ara-c group was (0.677±0.003),(0.716±0.005),(0.939±0.003). Compared with the control group, the protein expression of Fas in NMDP, Ara-c and NMDP+Ara-c group was significantly enhanced (F=1890.569, p=0.000), among which NMDP+Ara-c group was the highest.4. FasL protein expression:Gray scale scan ratio of FasL protein expression in the control group was (0.493±0.001), while that in NMDP, Ara-c and NMDP+Ara-c group was (0.554±0.007),(0.605±0.001),(0.807±0.003), which is more than control group (F=16065.218,p=0.000). NMDP+Ara-c group was the highest among them.5.Caspase3activity:Caspase3activity in the control group was (116699.000±72834.647) RLU, while that in NMDP, Ara-c and NMDP+Ara-c group was (234964.333±32328.981)RLU,(614644.33±15958.4)RLU,(815261.000±91330.007) RLU, which is more than control group (F=85.045, p=0.000). NMDP+Ara-c group was the highest among them.6. Caspase8activity:Caspase8activity in the control group was (5890.500±1523.108) RLU, while that in NMDP, Ara-c and NMDP+Ara-c group was (16135.000±140.714) RLU,(39399.000±1786.859) RLU,(66113.000±823.779) RLU. Compared with the control group,the differences among groups were statistically significant (F=923.843,p=0.000).Caspase8activity in NMDP, Ara-c group and NMDP+Ara-c group was enhanced, among which NMDP+Ara-c group was the highest.7. Fas/FasL protein expression and cell apoptosis:①Fas protein expression and cell apoptosis:Pearson correlation coefficient was0.893(p=0.000), showing a strong positive linear correlation.②FasL protein expression and cell poptosis:Pearson correlation coefficient was0.939(p=0.000), showing a strong positive linear correlation.③Fas and FasL:Pearson correlation coefficient was0.992(p=0.000), showing a strong positive linear correlation.8. Caspases activity and cell apoptosis:①Caspase3and cell apoptosis:Pearson correlation coefficient was0.967(p=0.000), showing a strong positive linear correlation.②Caspase8and cell apoptosis:Pearson correlation coefficient was0.981(p=0.000), showing a strong positive linear correlation.③Caspase3and Caspase8:Pearson correlation coefficient was0.960(p=0.000), showing a strong positive linear correlation. 9. Fas/FasL protein expression and Caspases activity:①Fas and Caspase3:Pearson correlation coefficient was0.865(p=0.000), showing a strong positive linear correlation.②Fas and Caspase8:Pearson correlation coefficient was0.942(p=0.000), showing a strong positive linear correlation.③FasL and Caspase3:Pearson correlation coefficient was0.905(p=0.000), showing a strong positive linear correlation.④FasL and Caspase8:Pearson correlation coefficient was0.971(p=0.000), showing a strong positive linear correlation.Conclusions1.Nimodipine can promote HL-60cell proliferation inhibited by cytarabine, the inactivation rate of HL-60Cell by NMDP (0.001-0.050g/L) and Ara-c (0.005～0.01Og/L) of different concentrations was inhibited remarkably,showing a concentration dependence.When the concentration of NMDP was0.050g/l and that of Ara-c was0.010g/l, HL-60Cell inactivation rates were the highest.2. Nimodipine can increase Fas/FasL protein expression of HL-60cell apoptosis induced by cytarabine, showing a strong positive linear correlation. It's indicated that enhanced Fas/FasL protein expression is involved into cell apoptosis induced by NMDP and Ara-c.3. Nimodipine can increase Caspase3, Caspase8activity of HL-60cell apoptosis induced by cytarabine, showing a strong positive linear correlation. It's indicated that enhanced caspases activity is involved into cell apoptosis induced by NMDP and Ara-c.4. Fas/FasL protein expression, caspase3and caspase8activity also have a strong positive linear correlation, indicating that Fas/FasL protein was involved in cytarabine-induced HL-60cell apoptosis enhanced by nimodipine through the caspase pathway. |
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