Division of Biological Science, Institute of Natural Sciences, Wonkwang University, Iksan, Chonbuk 570-749, South Korea.
Naunyn Schmiedebergs Arch Pharmacol. 2010 Jul;382(1):89-101. doi: 10.1007/s00210-010-0522-9. Epub 2010 May 28.
Our previous study demonstrated the increase in the repair of UVB damage by mRg2, a mixture of ginsenosides containing 60% Rg2 in NIH3T3 cells. In the present study, the effects of purified Rg2 on the repair and apoptosis in ultraviolet B (UVB)-exposed HaCaT cells were investigated on gene expression levels. When cells were exposed to UVB and post-incubated in normal medium for 24 h, the cell viability decreased to about 50% of that in nontreated control. When Rg2 was post-incubated, however, the UVB-induced cytotoxicity was significantly prevented in an Rg2 concentration- and time-dependent manner. The apoptotic nuclear fragmentation resulting from UVB exposure was also significantly protected by the Rg2 post-incubation. Microarray analysis showed that the genes stimulated by the Rg2-alone treatment include those involved in p53 signaling pathway such as GADD45alpha, GADD45beta, and cell communication genes. RT-PCR analysis showed that the Rg2-alone treatment slightly upregulated the p53 and GADD45 transcript and protein levels by about 1.5-fold as compared with the nontreated control. The mRNA levels of p53 and GADD45 in cells exposed to UVB and post-incubated with Rg2 for 24 h decreased in an Rg2 concentration-dependent manner as compared with that post-incubated in normal medium. However, the mRNA level of the UVB-exposed cells post-incubated with 5 microM retinol was essentially the same as that post-incubated in normal medium. Time course experiment showed that the mRNA levels of p53 and GADD45 in UVB-exposed cells were upregulated by post-incubation with 50 microM Rg2 until 6 and 9 h, respectively, and then gradually decreased until 24 h. By Western blot analysis, it was also revealed that the Rg2 post-incubation decreases the expression of p53, phospho-p53, GADD45, and ATM in UVB-exposed cells. Time course analysis also indicated that these decreased expressions were due to the earlier upregulation of p53 and GADD45 proteins. When UVB-exposed cells were post-incubated with Rg2 for 24 h after UVB exposure, the level of remaining cyclobutane pyrimidine dimers decreased in both Rg2 concentration- and time-dependent manner. All these results suggest that Rg2 protects cells against UVB-induced genotoxicity by increasing DNA repair, in possible association with modulation of protein levels involved in p53 signaling pathway.
我们之前的研究表明，mRg2（一种含有 60% Rg2 的人参皂苷混合物）可增加 NIH3T3 细胞中 UVB 损伤的修复。在本研究中，我们研究了纯化的 Rg2 对 UVB 照射 HaCaT 细胞修复和凋亡的影响，观察其在基因表达水平上的作用。当细胞暴露于 UVB 并在正常培养基中孵育 24 小时后，细胞活力下降至未处理对照组的约 50%。然而，当用 Rg2 孵育时，UVB 诱导的细胞毒性呈 Rg2 浓度和时间依赖性显著降低。由 UVB 暴露引起的核碎片凋亡也得到了 Rg2 孵育的显著保护。微阵列分析表明，Rg2 单独处理刺激的基因包括参与 p53 信号通路的基因，如 GADD45alpha、GADD45beta 和细胞通讯基因。RT-PCR 分析表明，与未处理对照组相比，Rg2 单独处理可将 p53 和 GADD45 的转录物和蛋白水平轻微上调约 1.5 倍。用 Rg2 孵育 24 小时后，暴露于 UVB 并孵育 Rg2 的细胞中的 p53 和 GADD45mRNA 水平呈 Rg2 浓度依赖性降低，而在正常培养基中孵育的细胞中的 p53 和 GADD45mRNA 水平基本相同。时程实验表明，用 5 μM 视黄醇孵育 UVB 暴露细胞可使 p53 和 GADD45mRNA 水平在 6 和 9 小时时分别上调，然后逐渐下降直至 24 小时。通过 Western blot 分析，还发现 Rg2 孵育可降低 UVB 暴露细胞中 p53、磷酸化 p53、GADD45 和 ATM 的表达。时程分析还表明，这些表达的降低是由于 p53 和 GADD45 蛋白的早期上调。当 UVB 暴露细胞在 UVB 暴露后用 Rg2 孵育 24 小时时，环丁烷嘧啶二聚体的残留水平呈 Rg2 浓度和时间依赖性降低。所有这些结果表明，Rg2 通过增加 DNA 修复来保护细胞免受 UVB 诱导的遗传毒性，这可能与调节参与 p53 信号通路的蛋白质水平有关。
