MIND4-17 protects retinal pigment epithelium cells and retinal ganglion cells from UV

Abstract

Nrf2 activation would efficiently protect retinal cells from UV radiation (UVR). Recent studies have developed a Nrf2-targeting thiazole-containing compound MIND4-17, which activates Nrf2 through blocking its association with Keap1. In the current study, we demonstrated that pretreatment with MIND4-17 efficiently protected retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs) from UVR. UVR-induced apoptosis in the retinal cells was also largely attenuated by MIND4-17 pretreatment. MIND4-17 presumably separated Nrf2 from Keap1, allowing its stabilization and accumulation in retinal cells, which then translocated to cell nuclei and promoted transcription of ARE-dependent anti-oxidant genes, including HO1, NQO1 and GCLM. Significantly, shRNA-mediated knockdown of Nrf2 almost completely abolished MIND4-17-induced cytoprotection against UVR. Further studies showed that MIND4-17 largely ameliorated UVR-induced ROS production, lipid peroxidation and DNA damages in RPEs and RGCs. Together, MIND4-17 protects retinal cells from UVR by activating Nrf2 signaling.

Keywords: MIND4-17; Nrf2; UV radiation; retinal ganglion cells; retinal pigment epithelium cells.

Figure 1. MIND4-17 protects retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs) from UVR

The molecule structure of MIND4-17 was presented (A). ARPE-19 cells (B–C) or primary cultured human RGCs (D and E) were pretreated for 30 min with applied concentration of MIND4-17, cells were then subjected to UV radiation (UVR, UVA2 + B, 30 mJ/cm²) and were further cultured for 48 hours; Cell viability was tested by the CCK-8 assay (B and D); Cell death was examined by LDH release in the conditional medium (C and E). For each assay, n = 5. “C” stands for untreated control cells. *p < 0.05 vs. “C” cells. ^#p < 0.05 vs. “UVR” only cells. Experiments in this figure were repeated four times to insure consistency of results.

Figure 2. MIND4-17 inhibits UVR-induced apoptosis in RPEs and RGCs

ARPE-19 cells (A–D) or primary cultured human RGCs (E) were pretreated for 30 min with MIND4-17 (5 μM), cells were then subjected to UV radiation (UVR, UVA2 + B, 30 mJ/cm²) and were further cultured for applied time; Expressions of cleaved-PARP (“Clvd-PARP”) and cleaved-caspase-3 (“Clvd-Caspase-3”) were tested (A, GAPDH was shown as the loading control); Cell apoptosis was tested by the assays mentioned in the text (B–E). Annexin V ratio included both early (PI negative) and late (PI positive) apoptotic cells (C). For TUNEL assay, at least 200 cells in five random views (1×100 magnification) of each condition were analyzed to calculate TUNEL ratio (D and E). “C” stands for untreated control cells. *p < 0.05 vs. “C” cells. ^#p < 0.05 vs. “UVR” only cells. Experiments in this figure were repeated three times to insure consistency of results.

Figure 3. MIND4-17 activates Nrf2 signaling in retinal cells

ARPE-19 cells (A–F) or primary cultured human RGCs (G) were treated with applied concentration of MIND4-17, cells were further cultured for 3 hours; The real-time quantitative PCR (RT-qPCR) assay was employed to test mRNA expressions of listed genes (A–D and (G), GAPDH mRNA was tested as the internal control); Listed proteins in total cell lysates (E) and nuclear fraction lysates (F) were also tested by Western blotting assay (GAPDH was tested as the loading control, which was absent in the nuclear fractions). “C” stands for untreated control cells. *p < 0.05 vs. “C” cells. Experiments in this figure were repeated three times to insure consistency of results.

Figure 4. Nrf2 is required for MIND4-17-mediated retinal cytoprotection against UVR

Stable ARPE-19 cells, expressing scramble control shRNA (“sh-sc”) or Nrf2 shRNA (“sh-Nrf2”), were treated with MIND4-17 (5 μM) for 3 hours, Nrf2 mRNA (A) and listed proteins (B) were tested by RT-qPCR assay (GAPDH mRNA was tested as the internal control) and Western blotting assay (GAPDH was tested as the loading control), respectively; Cells were exposed UV radiation (UVR, UVA2 + B, 30 mJ/cm²) and cultured for applied time; Cell survival and apoptosis were tested by CCK-8 assay (C) and ssDNA ELISA assay (D), respectively. “Ctrl” stands for parental ARPE-19 cells (A and B). For each assay, n = 5. *p < 0.05 vs. “sh-sc” cells. Experiments in this figure were repeated three times to insure consistency of results.

Figure 5. MIND4-17 efficiently attenuates UVR-induced oxidative stress in retinal cells

ARPE-19 cells (A–C) or primary cultured human RGCs (D) were pretreated for 30 min with MIND4-17 (5 μM), cells were then subjected to UV radiation (UVR, UVA2 + B, 30 mJ/cm²) and were further cultured for applied time; ROS production was tested by DCFH-DA intensity assay (A and D); DNA damages and lipid peroxidation were tested by γ-H2AX assay (B) and TBAR activity assay (C), respectively. “C” stands for untreated control cells. *p < 0.05 vs. “C” cells. ^#p < 0.05 vs. “UVR” only cells. Experiments in this figure were repeated three times to insure consistency of results.