The Preparation Of Nanoparticle Realgar Powders (NRP) And Its Mechanism Inducing U937 Cell Apoptosis

The arsenical agents(As) such as realgar and AS₂O₃ show an obvious effect on hematological oncology,especially Acute Promyelocytic Leukemia(APL).The previous research of our work found the alleviation rate of the compound realgar natural indigo tablets on APL could be 98.6%.The main component of this tablet is a kind of mineral medicine-realgar,which is indissoluble and hard for absorption. There were references that the physi-chemical properties,efficiency and toxicity of realgar could be changed by nano-technology.For the purpose of promoting the bio-availability,and decreasing the toxicity of it,the preparation of nanoparticle realgar powders(NRP) by nano-technology,and the phamacokinetics in mice, together with the further mechanism of human histocytic lymphoma U937 cell apoptosis were studied.NRP were milled with a plenary mill which was full of nitrogen.And the factors, such as the mass ratio of steel balls to realgar(4:1-16:1),the velocity of revolution (23-3 8Hz),the time of ball milling(4-16h),the temperature time(-20¹0℃) and the volume of H₂O(10-150mL) were checked and analysed under the arrangement of orthogonal design of experiment.The result showed tharthe optimum parameters in the preparation of NRP were as follows:the mass ratio of mill balls to realgar was 16:1,the velocity of revolution was 38Hz,the temperature was -20℃,and the ball milling time was 12h adding 50 mL H₂O as surfactant.NRP were dectected by scanning electron microscopy(SEM),atomic force microscopy(AFM),and laser scattering particle size distribution analyzer(PSD).The result exhibited that the size of about 90%of realgar particles were under 100 nm,and the bigger particles were the polymers of small grains at 5-30 nm and amorphous bodies around them.These methods of AFM,PSD and SEM showed the rapid,simple and accurate characters for the detection size distribution and apparent state.The data obtained from a single oral administration of NRP revealed that the elimination model of As was in accordance with first order kinetics and one compartment model.The pharmacokinetics parameters of NRP were obviously different from these of traditional realgar,the absorption phase of which increased while elimination phase decreased.The previous study showed that NRP could induce NB₄,HL-60,K562,U937 cell apoptosis.We herein research on the mechanism by which nanoparticle realgar powders(NRP) induce human histocytic lymphoma U937 cell apoptosis.The U937 cells were treated with 20,40,60,80 or 100μg/mL NRP for 12,24,36 or 48 h, respectively,and the cell death ratio was detected by MTT assay.The result showed that the NRP inhibited the cell growth in a concentration- and time- dependent manner. We also observed the morphologic changes by acridine orange(AO) staining.U937 cells treated with NRP showed membrane blebbing,condensed nuclear and granular apoptotic bodies under the fluorescence microscopy.The apoptotic and necrotic ratio was measured by LDH activity-based assay.The results indicated that when the cells were cultured with 20-80μg/mL NRP for 24 h,the majority of U937 cells underwent apoptosis and increased in a concentration-dependent manner,and the ratio of necrotic cells were still below 10.8%.However,incubation of U937 cells with 120μg/mL NRP made the necrotic ratio augmented markedly,showing that NRP induced U937 cell death through mediating the balance between apoptosis and necrosis.In order to investigate the effects of PI3-K/Akt signal pathway on the NRP-induced U937 cell death,flow cytometric analysis and western blot analysis were carried out.The results showed that NRP inhibited the activation of the PI3-K/Akt signal pathway,and consequently promoted the cell death.Furthermore, the western blot analysis in combination with MTT assay was applied to examine the roles of SIRT1 and p53 in the NRP-induced U937 cell death,and the results indicated that NRP activated p53,and the SIRT1 inhibitor sirtinol further promoted the activation of p53.In the experiment of western blot analysis,the PI3-K inhibitor wortmannin decreased the expression of SIRT1,and then,made the p53 activated further.Based on all the results,PI3-K/Akt signal pathway was intimately associated with SIRT1/ p53 signal pathway in NRP-induced U937 cell apoptosis.In NRP-treated cells,PI3-K/Akt pathway was restrained,which further inhibited the expression of SIRT1,and played a positive role for p53 activation.Consequently,all these contributed to the NRP-induced U937 cell apoptosis by means of caspase pathway.