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Objective To explore the different concentrations of paeoniflorin (paeoniflorin, PF) on human melanoma cell line A375 cell proliferation inhibition rate, the rate of apoptosis, and the cell lines of p16INK4a p14INK/ARF nuclear factor-κB (NF-κB) subunit p65 mRNA levels, provide a basis for its clinical application and further study. A375 cells, set the blank control group and 5 corresponding experimental group paeoniflorin 0,0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml culture medium processing (Purity of Paeoniflorin ≥ 92%). (1) inverted microscope to observe changes in cell density and morphology; ② The MTT colorimetric assay in the intervention of various concentrations of drugs in the 24, 48 and 72 hours after the proliferation inhibition rate of A375 cells; (3) Annexin V / PI kit staining, various concentrations of drugs in the 24 hours after the early apoptotic rate of A375 cells by flow cytometry in the intervention; the ④ SYBR Green-PCR detection in the intervention groups of 24, 48 and 72 hours after the A375 cells of p16INK4a p14INK/ARF, NF- κB subunit p65 mRNA levels. 1, paeoniflorin inhibited the growth of A375 cells with the concentration increasing its density decreases, the cells gradually presented stubby dendritic cell body, rather than the normal elongated like perinuclear body and double inverted microscope pole dendritic-like cell body. MTT results showed that paeoniflorin can inhibit the proliferation of A375 cells, and its inhibition in a concentration-dependent manner, without time-dependent manner. Annexin V / PI staining - flow cytometry results show each group of A375 cells in early apoptosis rate was no significant difference (P gt; 0.05). 4, SYBR Green-PCR results show: ① paeoniflorin promote the A375 cells p16INK4a mRNA transcription level, the differences were statistically significant (P lt; 0.05) compared with the control group, and its role in a concentration-dependent manner, and No time-dependent; ② paeoniflorin inhibits NF-κB subunit p65 mRNA transcription in A375 cells, compared with the control group, the differences were statistically significant (P lt; 0.05) in a concentration-dependent manner, and its inhibition without time-dependent; ③ paeoniflorin of A375 cells p14INK/ARF the mRNA levels of no significant impact (P gt; 0.05). Conclusion 1, paeoniflorin can inhibit the proliferation of A375 and dose-dependent manner without time-dependent; but had no significant effect on apoptosis of A375 cells, may be associated with melanoma induce tolerance or tumor from different sources. The paeoniflorin promote A375 p16INK4a expression, inhibition of NF-κB subunit p65, and this effect was concentration-dependent but not time-dependent; but had no significant effect on p14INK/ARF transcription. Inferred paeoniflorin may inhibit the proliferation of A375 through the promotion of p16INK4a inhibition of NF-κB subunits p65 expression.
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