Study On The Change Of Mitochondria Membrance Potential And Reactive Oxygen Species Of HL-60 Cells Induced By Cytarabine And Nimodipine

Objective Study the changes of mitochondria membrane potential of HL-60 cells andobserve the change of reactive oxygen species in HL-60 induced by cytarabine andnimodipine(NMDP). Study the correlation between mitochondria membrane potential andcell apoptosis of HL-60 cells and correlation between cell apoptosis of HL-60 cells andthe change of reactive oxygen species; To further study the mechanism of HL-60 cellsapoptosis inducing by Cytarabine and nimodipine.Methods HL-60 cells were stained with Rhodamine123, change of mitochondriamembrane potential of HL-60 cell were analyzed by flow cytometry. AO/EB stain, flowcytometry were used to examine the apoptosis of HL-60cells. The change of reactiveoxygen species were detected by fluorescence microscope and reactive oxygen speciesassay kit.Results①The levels of HL-60 cells△Ψm in Cytarabine groups were decreased sincethey were cultured for 6 hours. In Ara-c 0.05mg/ml group, the Rhodamine123fluorescence intensity in mitochondrion of HL-60 cell at 6h, 12h, 24h respectively were117.9±7.6,100.9±7.7,87.6±10.7, there was significant difference among the differenttime culture (P＜0.05); In Ara-c 0.1mg/ml group, the Rhodamine123 fluorescenceintensity in mitochondrion of HL-60 cell at 6h,12h,24h respectively were 111.9±10.1,86.6±9.2,68.4±12.2, there was significant difference among the different time culture(P＜0.05); and there was significant difference between the two groups at 12h, 24h(P＜0.05). In Ara-c 0.05mg/ml group, the apoptosis rate of HL-60 cells at 6h,12h,24hrespectively were (41.2±3.0)%, (53.7±5.1)%,(65.8±2.6)%, there was significantdifference among the different time culture (P＜0.01); In Ara-c 0.1mg/ml group, the.apoptosis rate of HL-60 cells at 6h,12h,24h respectively were (45.7±4.1)%,(58.2±4.3)%,(70.1±2.3)%, there was significant difference among the different time culture(P＜0.01); The apoptosis rate of HL-60 cells was no significant difference between thetwo groups at same time. The changes of mitochondria membrane potential and theapoptosis rate of HL-60 cells were significant negatively correlated. In Ara-c 0.05mg/mlgroup, r=-0.89, P＜0.01.In Ara-c 0.1mg/ml group, r=-0.76, P＜0.01.②InAra-c+NMDPgroup(Ara-c 0.05mg/ml, NMDP0.01mg/ml), the Rhodamine123fluorescence intensity in mitochondrion of HL-60 cell at 6h, 12h, 24h respectively were99.8±9.8,85.3±8.0,67.2±7.6, there was significant difference among the different timeculture (P＜0.05); and there was significant difference between Ara-c+NMDPgroup and Ara-c0.05mg/ml group (P＜0.05). The apoptosis rate of HL-60 cells at 6h,12h,24h inAra-c+NMDPgroup respectively were (51.4±3.2)%,(63.9±2.4)%,(83.1±4.8)%, therewas significant difference among the different time culture (P＜0.01); and there wassignificant difference between Ara-c+NMDPgroup and Ara-c0.05mg/ml group (P＜0.05).The changes of mitochondria membrane potential and the apoptosis rate of HL-60 cellswere significant negatively correlated. In Ara-c+NMDP group, r=-0; 73, P＜0.01.③Thefluorescence intensity in single Ara-c group and control group were very low and theexpression of reactive oxygen species was negative. In Ara-c+NMDP group, theexpression of reactive oxygen species was all positive. In single NMDPgroup, theexpression of reactive oxygen species was weakly positive.Conclusions 1,The△Ψm of HL-60 cells was time-dependently andconcentration-dependently decreased when Ara-c induce HL-60 cells apoptosis andNMDP can strengthen the above function of Ara-c. The changes of mitochondriamembrane potential and the apoptosis rate of HL-60 cells were significant negativelycorrelated. The decrease of mitochondria membrane potential is may be one of theimportant mechanisms in HL-60 apoptosis induced by Ara-c. 2, Ara-c can not induceproduction of reactive oxygen species. NMDP can induce the production of reactiveoxygen species and this is may be one of the mechanism of nimodipine strengthening theHL-60 apoptosis rate induced by Ara-c.