Protective properties of ginsenoside Rb3 against UV-B radiation-induced oxidative stress in HaCaT keratinocytes

Figures & data

Fig. 1. The chemical structure of ginsenoside Rb3 (Rb3).

Fig. 2. Effect of Rb3 on reactive oxygen species (ROS) elevation in HaCaT keratinocytes under irradiation with 70 mJ/cm² UV-B radiation. HaCaT cells were subjected to fresh medium with the indicated concentrations (0, 5, 12, or 30 μM) of Rb3 for 30 min prior to the irradiation. The intracellular ROS level was quantitated using DCFH-DA in a microplate fluorometer (A) and dihydrorhodamine 123 in confocal microscopic analysis (B).

Notes: In (A), ROS levels are represented as DCF fluorescence arbitrary units expressed as percentage of control. In the lower panel of (B), the ROS-associated fluorescent signals were quantified using Adobe Photoshop software. Each experiment was repeated three times. *p < 0.05; **p < 0.01 vs. the non-treated control (UV-B irradiation alone).

Fig. 3. Effect of Rb3 on cellular viability in HaCaT keratinocytes under irradiation with 70 mJ/cm² UV-B radiation.

Notes: HaCaT cells were subjected to fresh medium with the indicated concentrations (0, 5, 12, or 30 μM) of Rb3 for 30 min prior to the irradiation. The viable cell numbers, represented as the relative percentages, were determined using MTT assay. The experiment was repeated three times.

Fig. 4. Effect of Rb3 on the gelatinolytic activities of proMMP-2 (A) and proMMP-9 (B) in the conditioned media obtained from HaCaT keratinocyte cultures under irradiation with 70 mJ/cm² UV-B radiation.

Notes: HaCaT cells were subjected to fresh medium with the indicated concentrations (0, 5, 12, or 30 μM) of Rb3 for 30 min prior to the irradiation. The gelatinolytic activities of proMMP-2 and proMMP-9 in conditioned medium were detected using gelatin zymography. In (C), the equal loading of conditioned media was shown by the use of silver staining of the identical gel. The relative band strength was determined with densitometry using the ImageJ software. The experiment was repeated three times. *p < 0.05; **p < 0.01 vs. the non-treated control (UV-B irradiation alone).

Fig. 5. Effect of Rb3 on proMMP-2 (A) protein levels in conditioned medium and proMMP-9 protein levels (B) in cellular lysate.

Notes: HaCaT cells were subjected to fresh medium with the indicated concentrations (0, 5, 12, or 30 μM) of Rb3 for 30 min prior to the irradiation. The proMMP-2 and proMMP-9 proteins were determined using Western blotting analysis with anti-MMP-2 and anti-MMP-9 antibodies. In (B), GAPDH was used as an internal loading control. The equal loading of conditioned media was shown in Fig. 4(C). The relative band strength was determined with densitometry using the ImageJ software. Representatives of three independent experiments were shown.

Fig. 6. Effect of Rb3 on total glutathione (GSH, (A)) and superoxide dismutase (SOD, (B)) activity levels in cellular lysates prepared from HaCaT keratinocyte cultures under irradiation with 70 mJ/cm² UV-B radiation.

Notes: HaCaT cells were subjected to fresh medium with the indicated concentrations (0, 5, 12, or 30 μM) of Rb3 for 30 min prior to the irradiation. In (A), total GSH content, expressed as relative percentages, was determined with enzymatic recycling assay using GR. In (B), total SOD activity, expressed as relative percentages, was measured using a spectrophotometric assay. Each experiment was repeated three times. ^##p < 0.01 vs. non-irradiated control; **p < 0.01 vs. the non-treated control (UV-B irradiation alone).