UVA Induced Oxidative Stress Was Inhibited by Paeoniflorin/Nrf2 Signaling or PLIN2

Abstract

Photodamages caused by UVA radiation induced oxidative injuries are closely related to photoaging and skin cancer. Paeoniflorin (PF), extracted from the root of Paeonia lactiflora, has been reported to be an effective antioxidant. PLIN2, known as adipose differentiation-related protein, has been previously involved in the regulation of oxidative stress. In this study, we were sought to investigate the photo-protective property of PF and PLIN2 in UVA-radiated human dermal fibroblasts (HDFs). HDFs were pre-treated with PF (800 μM) followed by UVA radiation (22.5 J/cm2). MTS activity, cell apoptosis, ROS, MDA, and SOD were detected, respectively. The expressions of Nrf2, HO-1, NQ-O1, and PLIN2 were determined using RT-qPCR or western blot. Nrf2 was silenced by siRNA, and PLIN2 was overexpressed via lentiviral transduction. Comparing to the UVA radiation, PF pre-treatment could prominently increase the MTS activity, decrease cell apoptosis, reduce the generations of ROS and MDA, increase the activity of SOD and increase the expression of Nrf2 and its target genes HO-1 and NQ-O1. When Nrf2 was knocked down, PF lost above protective properties. In addition, UVA induced oxidative stress led to upregulation of PLIN2 and the latter could be decreased by PF. Overexpression of PLIN2 improved MTS activity and reduced MDA level in HDFs. The combination of PLIN2 overexpression and PF pre-treatment corporately inhibited UVA-induced injury. Besides, we also found that PF and PLIN2 had a compensatory protection against UVA induced oxidative stress. In conclusion, our study demonstrated that UVA induced photodamages could be inhibited by PF via Nrf2/HO-1/NQ-O1 signaling pathway or by PLIN2, and the combination of PLIN2 overexpression and PF played additive effects against UVA-related oxidative stress.

Keywords: Nrf2; PLIN2; UVA; oxidative stress; paeoniflorin.

Figure 1

PF suppressed UVA-induced injuries in HDFs. (A) MTS assay on the cytotoxicity of PF (0, 200, 400, and 800 μM) in fibroblasts at 24, 48, and 72 h. (B) MTS assay on the cytotoxicity of UVA (0, 20, 22.5, and 25 J/cm²) in fibroblasts at 24, 48, and 72 h. (C) MTS assay detected the cytoprotective function of PF (0, 200, 400, and 800 μM) against UVA (22.5 J/cm²) in fibroblasts. (D) Cellular morphology of fibroblasts with different treatments visualized by inverted microscope. (E) The cells apoptosis was detected by using flow cytometry with annexin-V-FITC and PI staining. The Q3 quadrant contained the vital population; the Q1 quadrant contained the death cells; the Q2 quadrant contained the late apoptotic cells; the Q4 quadrant contained the early apoptotic cells. (F) The data represented the apoptosis percentage containing the Q2 and Q4 quadrants in each group. All data were expressed as the mean ± SD according to at least three independent experiments. ^**p < 0.01, ^***p < 0.001, ^nsp > 0.05.

Figure 2

PF inhibited UVA-induced oxidative stress in HDFs. (A) The levels of ROS were indicated by DCF fluorescence emitting green fluorescent under fluorescence microscope. (B) The DCF fluorescence intensity was quantified by Image-Pro Plus 6.0. and the intensity of DCF fluorescence in each group was normalized to the control group. Detection of the generation of MDA (C) and the activity of SOD (D) in each group. (E) Western blot showing the protein levels of Nrf2, HO-1, and NQ-O1 in fibroblasts treated with PF (0, 200, 400, and 800 μM), the representative result of western blot and the quantitative analyses of three independent experiments were shown. (F) The effects of UVA and PF pre-treatment +UVA on the protein expressions of Nrf2, HO-1, and NQ-O1 at time points of 6, 12, and 24 h, the representative result of western blot and the quantitative analyses of three independent experiments. All data were expressed as the mean ± SD according to at least three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

PF inhibited UVA-induced oxidative stress and cytotoxicity via Nrf2. (A) The mRNA levels of Nrf2 in fibroblasts at 24 and 48 h of transfection. The relative expression level of Nrf2 in each group was normalized to the control group. (B) The protein levels of Nrf2 in fibroblasts at 48 and 72 h of transfection, the representative result of western blot and the quantitative analyses of three independent experiments were shown. (C) Western blot detection of the protein levels of Nrf2, HO-1, and NQ-O1 after UVA and PF-pretreatment in absent of Nrf2, the representative result of western blot and the quantitative analyses of three independent experiments were shown. (D) Detection of MDA level in fibroblasts treated with UVA radiation and PF in absence of Nrf2. (E) The MTS activity of fibroblasts with UVA radiation and PF-pretreatment in absence of Nrf2. All data were expressed as the mean ± standard deviation according to at least three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^nsp > 0.05.

Figure 4

Regulation of PLIN2 by UVA and PF. (A) The effects of UVA and PF pre-treatment +UVA on the mRNA expression of PLIN2 tested by RT-qPCR at time points of 6, 12, and 24 h. (B) The effects of UVA and PF pre-treatment +UVA on the protein expressions of PLIN2 tested by Western blot at time points of 6, 12, and 24 h. (C) The protein expression of PLIN2 after Nrf2 was knocked down in fibroblasts. (D) The protein expression of PLIN2 in fibroblasts with UVA radiation and PF-pretreatment in absence of Nrf2. The representative result of western blot and the quantitative analyses of three independent experiments were shown (B, D). All data were expressed as the mean ± SD according to at least three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^nsp > 0.05.

Figure 5

PLIN2 overexpression had a protective role in HDFs. (A) PLIN2 transfection was confirmed by observing EGFP positive cells under an inverted fluorescent microscope. (B) The transfection efficiency of PLIN2 was evaluated by RT-qPCR. (C) The level of MDA in PLIN2 overexpressed cells. (D) EdU staining was observed under fluorescence microscopy, red florescence indicated EdU-positive (EdU⁺) cells and blue florescence indicated cell nucleus, respectively. (E) The percentage of EdU⁺ cells in PLIN2 overexpressed cells. (F) The MTS activity of PLIN2 overexpressed cells and negative cells at the time points of 24, 48, and 72 h. (G) Detection of the level of MDA in cells treated with UVA+PLIN2 overexpression and/or UVA+PF-pretreatment. (H) The MTS activity of cells treated with UVA+PLIN2 overexpression and/or UVA+PF-pretreatment. All data were expressed as the mean ± SD according to at least three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 6

Schematic illustration of this study showing the regulation of UVA-related oxidative stress by PF via Nrf2/HO-1/NQ-O1 signaling pathway or by PLIN2.