Effect And Mechanism Of Diallyl Trisulfide On The Activity Of NADPH Oxidase In HL-60 Cells

Objective To investigate the effect and mechanism of diallyl trisulfide on the activity of NADPH oxidase, and to search for the target of ROS production induced by diallyl trisulfide in HL-60 cells.Methods The activity of NADPH oxidase was measured by the reduction of the yellow dye nitroblue tetrazolium (NBT) to insoluble blue-black formazan. The mRNA expression of NADPH oxidase subunits, including gp91^phox, p47^phox, p22^phox, Rac2 and Rac1, was detected by RT-PCR. The protein expression of p67^phox,gp91 ^phox and Rac2 was analyzed by Western blot.Results DATS had significant effects on NBT reduction ability of HL-60 cell in a concentration-dependent manner. The NBT reduction ability of HL-60 cell treated by DATS for 1h and 12h had no significant difference compared with control group(P>0.05). But the NBT reduction ablility of HL-60 cell treated by DATS could be seen apparently at 3h and 6h and exhibited a concentration -dependent manner, expressing peak of induction ROS at 150μM(P<0.05). As a positive control,As2O3 had little effect on NBT reduction ablility of HL-60 cells compared with control group(P>0.05)DATS increases expression of NADPH oxidase. mRNA expression of NADPH oxidase subunits(p47^phox,gp91^phox,p22 ^phox , Rac2 and Rac1) and protein levels(Rac2 and gp91^phox)in HL-60 cell treated with DATS increased in a concentration-dependent manner and in a time-dependent manner. HL-60 cells were treated with 150μM DATS for 0h¹2h. RT-PCR analysis showed an increase in mRNA (p47^phox,gp91 ^phox,p22^phox,Rac2 and Rac1)levels within 1h after treatment with DATS. Levels of mRNA were maximally increased at 3 h. Western blot experiments were performed to analyze protein levels of NADPH oxidase subunits (Rac2,gp91^phox and p67^phox). Expression of Rac2 and gp91^phox was steadily increased and got to the peak followed treatement with DATS at 3h in HL-60 cells,but protein level of p67^phox had no distinct difference compared with control group.Then HL-60 cells were treated with DATS of different concentration for 3h.RT-PCR analysis showed an increase in mRNA (p47^phox,gp91^phox,p22^phox,Rac2 and Rac1)levels and got to the peak at 150μM . Western blot analysis showed expression of Rac2 and gp91^phox was steadily increased in a concentration-dependent manner. protein level of p67^phox had no distinct difference compared with control group.Treatment with DATS results in translocation of NADPH oxidase. Activation of NADPH oxidase involves translocation of the cytosolic Rac2 and p67^phox subunits into the plasma membrane . The levels of Rac2 and p67^phox were reduced in the cytosolic fraction and increased in the membrane fraction from HL-60 exposed to DATS in a concentration-dependent manner and in a time-dependent manner., HL-60 cells were treated with150μM DATS for 0h¹2h .Western blot analysis showed translocation of the cytosolic Rac2 and p67^phox subunits into the plasma membrane got to the peak at 3h.HL-60 cells followed treatement with DATS of different concentration.Western blot analysis showed that translocation of Rac2 and p67^phox increased in a concentration-dependent manner.Conclusion DATS induce the activation of NADPH oxidase in HL-60 cells. DATS up-regulate the mRNA expression of p47^phox, gp91^phox, p22^phox, Rac2 and Rac1 subunit and the protein expression of gp91^phox and Rac2 subunit in HL-60 cells. DATS have little effect on protein expression of gp67^phox. DATS induce the plasma membrane translocation of both Rac2 and p67^phox subunit from the cytosol, so our results suggest that DATS induce the activation of NADPH oxidase by both up-regulating the expression of NADPH oxidase subunit and translocating the cytosolic Rac2 and p67^phox subunit to the plasma membrane in HL-60 cells.