RESUMO
This paper aimed to investigate the role of Duhuo Jisheng decotion (DHJSD) in delaying human disc degeneration and its possible molecular mechanism. The intervertebral disc specimens were divided into normal and degenerated groups according to Pfirrmann classfication. The expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in intervertebral disc tissue were detected by Western blot and PCR. Then degenerated human primary NPCs were cultured in vitro, the viability of NPCs treated with stromal cell-derived factor-1 (SDF-1，10 μg·L⁻¹)and various concentrations of DHJSD was assessed by the CCK-8 assay, and the appropriate concentration was screened. The experiment was divided into three groups, control group, SDF-1 group and DHJSD plus SDF-1 group. The levels of TNF-α, IL-1β, Agg, coIⅡ, MMP-3 and MMP-13 were detected. The levels of CXCR4, NF-κB major groups P65 phosphorylation (p-P65) and nuclear translocation, after treated with CXCR4 siRNA and NF-κB inhibitor (BAY11-7082) were measured by Western blot and immunofluorescence. At the same time, the expression of cell inflammatory factors and extracellular matrix were also measured. The expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in the degenerated intervertebral disc tissue were significantly increased. In vitro study, the results of CCK-8 indicated that the viability of NPCs was significantly increased when DHJSD concentration was 300 mg·L⁻¹. After the experiment was divided into three groups, compared with SDF-1 group, the expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in DHJSD group were significantly decreased, but the expressions of Agg, coIⅡ were significantly increased. When CXCR4-siRNA was transfected into NPCs, SDF-1 increased expressions of CXCR4 and p-P65 and inhibited nuclear translocation of P65, whose effect was suppressed by CXCR4-siRNA and DHJSD. In addition, when BAY11-7082 was used to treat NPCs, the expression of TNF-α, IL-1β, MMP-3 and MMP-13 were significantly decreased. DHJSD could inhibit the production of inflammatory factors and promote the synthesis of extracellular matrix. The potential mechanism may be related to the SDF-1/CXCR4/NF-κB signaling pathway.
RESUMO
<p><b>AIM</b>To investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI), an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells.</p><p><b>METHODS</b>HL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit.</p><p><b>RESULTS</b>DMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0.24 micromol x L(-1). The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated, while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60 cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 micromol x L(-1) of DMTCCI in HL-60 cells.</p><p><b>CONCLUSION</b>DMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might play a role in the apoptosis process induced by DMTCCI.</p>