Original article
Activation of Nrf2 by Ginsenoside Rh3 protects retinal pigment epithelium cells and retinal ganglion cells from UV

https://doi.org/10.1016/j.freeradbiomed.2018.02.001Get rights and content

Highlights

  • Ginsenoside Rh3 protects human RPEs and RGCs from UV radiation.

  • Ginsenoside Rh3 activates Nrf2 signaling in retinal cells.

  • Nrf2 activation is required for Ginsenoside Rh3-mediated retinal cell protection.

  • Ginsenoside Rh3 up-regulates microRNA-141 to downregulate Keap1 in retinal cells.

  • Ginsenoside Rh3 protects mouse retina from light-induced damages in vivo.

Abstract

Excessive Ultra-violet (UV) radiation shall induce damages to resident retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs). Here we tested the potential activity of Ginsenoside Rh3 (“Rh3”) against the process. In cultured human RPEs and RGCs, pretreatment with Rh3 inhibited UV-induced reactive oxygen species (ROS) production and following apoptotic/non-apoptotic cell death. Rh3 treatment in retinal cells induced nuclear-factor-E2-related factor 2 (Nrf2) activation, which was evidenced by Nrf2 protein stabilization and its nuclear translocation, along with transcription of antioxidant responsive element (ARE)-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown by targeted-shRNA almost abolished Rh3-induced retinal cell protection against UV. Further studies found that Rh3 induced microRNA-141 (“miR-141”) expression, causing downregulation of its targeted gene Keap1 in RPEs and RGCs. On the other hand, Rh3-induced Nrf2 activation and retinal cell protection were largely attenuated by the miR-141's inhibitor, antagomiR-141. In vivo, intravitreal injection of Rh3 inhibited retinal dysfunction by light damage in mice. Rh3 intravitreal injection also induced miR-141 expression, Keap1 downregulation and Nrf2 activation in mouse retinas. We conclude that Rh3 protects retinal cells from UV via activating Nrf2 signaling.

Introduction

Long-term or excessive Ultra-violet (UV) radiation shall induce cytotoxicity to resident retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs) [1], [2], which is one important cause of retinal degeneration [3], [4], [5]. Existing evidences have demonstrated that UV radiation will induce profound reactive oxygen species (ROS) production and oxidative stress, along with lipid peroxidation, calcium overload and DNA damages, which eventually will lead to death and apoptosis of RPEs and RGCs [1], [2], [6], [7], [8], [9], [10]. On the other hand, our group and others have shown that ROS scavenging could efficiently protect retinal cells from UV damages [1], [2], [6], [7], [8], [9], [10].

Ginseng, a well-known traditional herbal medicine, has been widely-utilized in ancient China [11], [12]. The ginseng's pharmacological properties are mainly attributed by the active component ginsenosides [11], [12]. Ginsenosides might exert different functions in different types of cells [11], [12]. Ginsenoside Rg5 is the main active component of heat-processed ginseng, which has displayed anti-cancer, anti-inflammatory, and ROS-scavenging activities [13], [14], [15], [16]. Ginsenoside Rh3 (“Rh3”) is a bacterial metabolite of Ginsenoside Rg5 [17], [18]. Existing evidences have investigated the potential cytoprotective and anti-inflammatory function of Rh3 [17], [18]. Its potential activity on UV-induced retinal cell damages has not been studied thus far.

Nuclear-factor-E2-related factor 2 (Nrf2) signaling is arguably one of the most important anti-oxidant axis [19], [20], [21]. As a transcription fact, Nrf2 binds to the antioxidant responsive element (ARE), which is essential for the transcription and expression of multiple key anti-oxidative enzymes [19], [20], [21], including heme oxygenase-1 (HO1), NAD(P)H quinone oxidoreductase 1 (NQO1) and γ-glutamyl cysteine ligase catalytic subunit (GCLC) [19]. Activation of Nrf2 could lead to a profound anti-oxidant response, and protects cells from oxidative stresses [19], [20], [21]. Nrf2's activity is tightly controlled by its suppresser protein Keap1, the latter is an adaptor of a Cul3-ubiquitin ligase complex, which dictates cytosol Nrf2 ubiquitination and proteasomal degradation [19], [20], [21]. Activated Nrf2 disassociates with Keap1 in cytoplasm, causing it accumulation and nuclear translocation [19], [20], [21].

Our studies and others have shown that activation of Nrf2 is a fine strategy to protect retinal cells from UV radiation and oxidative stress [8], [22], [23], [24]. For example, we showed that Salvianolic acid A protected RPEs from hydrogen peroxide (H2O2) via activating Nrf2-HO1 signaling [22]. Similarly, activation of Akt-Nrf2 signaling axis by 3H-1,2-dithiole-3-thione (D3T) also exerted cytoprotection in RPEs against UV [8]. A novel Akt activator, SC79-mediated RPE protection against UV also required Akt-Nrf2 signaling [23]. Our recent study found that microRNA-141 (miRNA-141)-mediated silence of Keap1 activated Nrf2 signaling and protected RPEs and RGCs from UV [24]. In the present study, we show that Rh3 inhibits UV-induced oxidative damages in retinal cells via activating Nrf2 signaling.

Section snippets

Reagents, chemicals and antibodies

The analytical standard ginsenoside Rh3 (“Rh3″, CAS no. 105558–26-7) [18], [25], [26], with the formula [3β, 12β,20Z)− 12-Hydroxydammara-20(22),24-dien-3-yl-β-D-glucopyranoside or C36H60O7 (MW: 604.86), was purchased from MCE China (HY-N0606, Shanghai, China). Quality control of Rh3 by HPLC (> 99%) was performed via the supplier. The following antibodies, HO-1 (#70081, 28kD), NQO1 (#3187, 29kD), Nrf2 (#12721, 98kD), Keap1 (#8047, 60kD), α-Tubulin (#2125, 52kD) and Lamin B1 (#13435, 55kD), were

Ginsenoside Rh3 protects human RPEs and RGCs from UV radiation

Here, we studied the potential effect of Ginsenoside Rh3 (“Rh3″) on UV-treated retinal cells. ARPE-19 is a well-established human RPE cell line [8], [23], [36]. In line with our previous findings [8], [23], UV irradiation (UVB/A2, 30 mJ/cm2) in APRE-19 cells (“RPEs”) resulted in significant cell viability reduction (MTT OD decrease, Fig. 1A) and cell death (Trypan blue ratio increase, Fig. 1B). Notably, UV-induced cytotoxicity in RPEs was attenuated with pre-treatment (30 min before UV) of Rh3 (

Discussion

The transcription factor Nrf2 plays a vital role in ARE-mediated expression of phase II detoxifying and antioxidant enzymes, as well as in the prevention of cell damages caused by oxidative stress [38]. In the current study, we provided evidences to support that Rh3 could be an efficient Nrf2 activator in retinal cells. in both human RPEs and RGCs, pretreatment with Rh3 inhibited UV-induced ROS production and following apoptotic/non-apoptotic cell death. Rh3 treatment in human retinal cells

Conclusion

In summary, Rh3 protected RPEs and RGCs from UV radiation through activation of Nrf2 signaling. Expression of miR-141 could be the key mechanism of Rh3-induced Nrf2 activation and retinal cell protection. The in vivo studies demonstrated that Rh3 intravitreal injection protected mouse retinas from UV. Nrf2 activation and miR-141 upregulation were also noticed in Rh3-treated mouse retinas. Our results imply that Rh3 might have therapeutic value for UV or oxidative stress-associated retinal

Acknowledgements

This work was generously supported by the grants from the National Natural Science Foundation of China (Grant No. 81371055, 81570859, 81670878, 81302195, 31371139, 81571282, 81771457 and 81700859), grants from the Medical Science and Technology Development Project Fund of Nanjing (YKK16271, YKK14193, YKK15241, YKK16269, YKK16270), Grants from Natural Science Foundation of Jiangsu Province (BK2016022104, BK20171065, BK20170060).

Competing interests

The authors declare that they have no competing interests.

Author contributions

All authors carried out the experiments, participated in the design of the study and performed the statistical analysis, participated in its design and coordination and helped to draft the manuscript.

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