The Molecular Mechanism Of Apoptosis In NB4 Cells Induced By Puerariae Radix Flavones In Vitro

Xue Fu Kang, a compound preparation which composed of Qingdai, Ezhu and Gegen, named after its effort of disintoxicating and blood flow promoting, is manufactured by Department of Hematology, Jiangsu traditional Chinese medical hospital. As a classic Chinese herb, Puerariae Radix has a wide range of clinical applications. The ancient books had recorded that it had great clinical effects, such as relieving muscles, letting out skin eruptions, disintoxicating, slaking thirst and so on. And the present writings also summarize its experiences in anti-infection and the protection of cardiocerebral vessels. Constituting the largest and most important group of active compounds in puerariae radix, Puerariae radix flavones (PRF) is used to perform in our experiments. The datas manifest that PRF could inhibit the proliferation of the four sub-types of AML cell lines-HL-60, Kasumi-1, NB4 and U937 respectively. The cell inhibitory rate of NB4 cells is much higher than others.Consequently, the NB4 cells are selected as the experimental object in further study. Treated with PRF in 12.5-200μg/mL, the proliferation of NB4 cells are inhibited in time-dose dependent manner(n=6, p<0.01), the NB4 cells display distinct apoptostic character by Hoechst 33258 and Wright's staining. Cell cycle detection shows that NB4 cells are arrested in synthesis period. FITC-AnnexinV/PI double staining indicates that PRF could induce NB4 cells apoptosis, which presents time-dose dependent manner(n=6,p<0.01).The results of real-time PCR suggest that PRF can degradate the expression level of leukemia genesis PML/RARa fusion gene and down-regulate anti-apoptotic Bcl-2 and survivin gene, With Western blotting assay, the pro-caspase3 protein expression decreases and the active p17 subunit increases which indicates that caspase3 is activated. Simultaneously FasL, caspase8 and PARP are up-regulated, while P38 is down-regulated. In a word, all these effects are triggered by the activation of JNK, which can increase the expression of FasL, recruit and activate Caspase8,finally activate effector of Caspase3 and PARP following the delivery of induce-apoptotic factor and down-regulation of anti-apoptotic factors expression such as p38MAPK, survivin and Bcl-2.Simultaneously, RPF also can degradate the expression of PML/RARa fusion gene to a certain extent.