Experimental Research About Influence Of PRE To The Autophagy On The NB4 Cell

[Background and Objective]Puerariae radix flavones (PRF) is the main effective constituent of Chinese medicine Puerariae radix. In recent five years, our research group found that the PRF in the concentration of 0-50μg/mL could induce the NB4 cells to apoptosis in a time and concentration-dependent manner and retard the cell in S-phase. It has also been proved that when PRF induces NB4 cell to apoptosis, the JNK protein is activated via up-regulation of phosphorylation level, the expression of the cleaved caspase3 and PARP is up-regulated, while p38 down-regulated. By Flow cytometry, we confirmed that early cell apoptosis rate increase concentration dependent. The mitochondrial membrane potential of the NB4 cell was decreased confirmed by flow cytometry. According to these results, we inferred PRF induce NB4 cells to apoptosis by the activation of JNK. PRF induced NB4 cells apoptosis via mitochondrial pathway, with triggering the caspase cascade. We found PRF couldn’t completely inhibit NB4 cells proliferation when pretreating with JNK and caspase inhibitors. Pretreating with JNK inhibitors for 2 hours, the cellular proliferation inhibition rate of 10μg/ml PRF group increased obviously. We speculated there may be other mechanism of death induced by PRF. NB4 cells incubated by 30μg/ml PRF, pretreating with SB203580 for 2 hours, the expression level of JNK, Bax, p53 and ERK was increased, while the difference of cell proliferation inhibition rate was not statistically significant. Weather the p38 is a protective signal to NB4 cells interfered by PRF, or mediate anti-apoptosis or autophagy. In this study, we will observe the impact on the autophagy of NB4 cells incubated by PRF via electron microscope and western blot, and validate the anti-apoptosis of p38 on NB4 cells by blocking p38 signal pathway.[Method]1.0,10,30,50μg/ml PRF interfere NB4 cells for 48 hours, detect the expression of LC3-Ⅰ, LC3-Ⅱ, Bcl-2, p-Bcl-2, Beclin1, Atg3, Atg5, Atg7, Atg12, Atg16 by Western blot.2.0,10,30,50μg/ml PRF interfere NB4 cells for 48 hours, detect autophagosome with electron microscope.3. 10μg/ml PRF interfere the NB4 cell from 0 to 48 hours, detect the the expression of LC3-Ⅰ, LC3-Ⅱ, Beclinl, Atg3, Atg5, Atg7, Atg12, Atg16 by Western blot.4. Detecting the the expression of pro-apoptotic protein, such as PARP, p-JNK, JNK, ERK, Bax by Western blot, with blank control versus 10μg/ml PRF interfering NB4 cells for 48 hours.5.0,10,30,50μg/ml PRF±10μmol/L SB203580 interfere the NB4 cell for 24,48 and 72 hours, detect the NBA cell cellular proliferation inhibition rate by MTT assay. [Results]1. Comparing to the control, NB4 cells exposed to 10μg/mL PRF for 48 hours, the expression of Atg7, Atg16, Atg5, Beclin-1, Bcl-2 and p-Bcl-2 were raised. Others interfered by 30 and 50μg/mL PRF, the expression of Atg5, Beclin-1, Bcl-2, p-Bcl-2 were up-regulated, and the expression of Atg7, Atg16 were down-regulated. LC3-Ⅱ/LC3-1 gradually rised in 0,10,30 μg/ml PRF, and reach the peak in 30 μg/ml.2.0,10,30,50μg/ml PRF interfered NB4 cells for 48 hours, detecting more autophagosome in 10,30μg/ml groups by electron microscope, while there is no obvious change in 50μg/ml group.3. As the time of NB4 cell incubated by 10μg/ml PRF prolonged to 12 hours, the expression of Beclin-1, Atg5 were up-regulated in a time-dependent manner, and reached the peak at 12 hours, then declined gradually, but the expression of Beclin-1 was down-regulated at 36 hours compared with 48 hours, while the expression of Atg5 was down-regulated at 48 hours compared with 36 hours. The expression of Atg16, LC3-Ⅱ/LC3-1 were up-regulated in 1-24 hours,24 hours to spike, then gradually decreased, but the expression of Atg16, LC3-Ⅱ/LC3-I were up-regulated at 48 hours compared with 36 hours. The expression of Atg3, Atg7 were up-regulated in 1-8 hours,8 hours to spike, then gradually decreased, but the expression of Atg7 at 48 hours was 36 hours increased. The expression of Atg12 was up-regulated in 4-36 hours,36 hours to peak, and then gradually reduced.4. NB4 cells exposed to SB203580 in comparison with blank control, the expression of cleaved PARP and JNK, ERK were increase.5. The SB203580 doesn’t affect the proliferation inhibition rate of NB4 cells. There is no difference.[Conclusion]Combined with previous studies, we found PRF can induce NB4 cells to apoptosis, and increase the level of autophagy. 10μg/ml PRF could enhance the level of autophagy in NB4 cells, but the influence between autophagy and apoptosis needs further research. Meanwhile, according to the changes of apoptosis related protein by blocking p38 signal pathway, we hypothesize that p38 pathway may prevent apoptosis in the NB4 cell, and it may play a same role in the apoptosis of NB4 cells induced by PRF.