Cell experiment:

SP-1 keratinocytes are seeded in 96 well plates (2×104 cells/well). After 24 h, the media is replaced with media containing various concentrations of (A) SKRG, or (B) Ginsenoside Rh3 (0.01, 0.1, 1 and 10 μM). Control cells are treated with DMSO at a final concentration of 0.1%. After 24 h, the media containing the compounds or DMSO is replaced with media containing 10% EZ-Cytox. The cells are then incubated at 37℃ for 1 h, and the absorbance is measured using a microplate reader at a wavelength of 450 nm. All assays are performed in triplicate[2].

Animal experiment:

Mice[1]The BALB/c mice (Male, 5-6 week old, 17-18 g weight) are used. The pupillary dilation is performed before exposure to 5000 lx of white fluorescent light. Thirty min before light exposure, Ginsenoside Rh3 (at 5 mg/kg body weight) are injected intravitreally to the right eye. ERG recording after light exposure is also reported early. The b-wave amplitude is measured from the trough of the a-wave to the peak of the b-wave, and the amplitude of the a-wave is measured from the initial baseline.

Ginsenoside Rh3 is a bacterial metabolite of Ginsenoside Rg5. Ginsenoside Rh3 treatment in human retinal cells induces Nrf2 activation.

Ginsenoside Rh3 inhibits UV-induced oxidative damages in retinal cells via activating nuclear-factor-E2-related factor 2 (Nrf2) signaling. Ginsenoside Rh3 treatment in retinal cells induces Nrf2 activation. The potential activity of Ginsenoside Rh3 is tested on Nrf2 signaling in the retinal pigment epithelium cells (RPEs). The qRT-PCR assay results demonstrate that treatment with Ginsenoside Rh3 dose-dependently increases mRNA transcription and expression of key Nrf2-regulated genes, including HO1, NQO1 and GCLC. Consequently, protein expressions of these Nrf2-dependent genes (HO1, NQO1 and GCLC) are also significantly increased in Ginsenoside Rh3 (3-10 μM)-treated RPEs. Notably, although Nrf2 mRNA level is unchanged after Ginsenoside Rh3 treatment, its protein level is significantly increased by Rh3[1]. EZ-Cytox assay is used to assess the effect of ginsenoside-Rh3 on SP 1-keratinocytes viability. Ginsenoside Rh3 (0.01, 0.1, 1 and 10 μM) shows no cytotoxic effect at all concentrations[2].

The potential effect of Ginsenoside Rh3 is examined on mouse retina, using the light-induced retinal damage model. Ginsenoside Rh3 intravitreal injection (5 mg/kg body weight, 30 min pre-treatment) significantly attenuates light-induced decrease of both a- and b-wave amplitude. The electroretinography (ERG)'s a-wave decreases to 46.03±1.62% % of control level after light exposure, which is back to 71.84±7.51% with Ginsenoside Rh3 administration. The b-wave is 40.19±3.34% of control level by light exposure, and Rh3 intravitreal injection brings back to 80.01±2.37% of control level[1].

References:
[1]. Tang CZ, et al. Activation of Nrf2 by Ginsenoside Rh3 protects retinal pigment epithelium cells and retinal ganglion cells from UV. Free Radic Biol Med. 2018 Mar;117:238-246.
[2]. Chung I, et al. Inhibitory mechanism of Korean Red Ginseng on GM-CSF expression in UVB-irradiated keratinocytes. J Ginseng Res. 2015 Oct;39(4):322-30.