Isoorientin Attenuates Cisplatin-Induced Nephrotoxicity Through the Inhibition of Oxidative Stress and Apoptosis via Activating the SIRT1/SIRT6/Nrf-2 Pathway

Figures

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  FIGURE 1. Effects of Iso on CDDP-induced cell viability and apoptosis in mTECs. mTECs were administered various concentrations of Iso (5, 10, and 20 μM) for 1 h, and the cells received CDDP (20 μM) for 24 h. (A,C) Cellular morphology and the structure of mTECs was assessed by bright field (40×magnification) microscopy, and cell viability was analyzed by the CCK-8 assay. (B,D) Staining with Hoechst/PI was performed for 15 min. The fluorescence was immediately detected by fluorescence microscopy, and the number of PI-positive cells was calculated. The results show the average of three independent experiments. **p < 0.01 versus the control group; ##p < 0.01 versus the CDDP group; and p < 0.05 versus the Iso (20 μM) plus CDDP group.

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  FIGURE 2. Effects of Iso on CDDP-induced ROS levels and antioxidant pathway in mTECs. (A–D) mTECs were treated with Iso at the indicated concentration and for the indicated time, and Western blot analysis was used to determine the protein levels. (E) The enzymatic activity of SIRT1 in mTECs. (F) Effects of Iso on SIRT1, SIRT6, Nrf2, and HO1 genes expression. Total RNA was extracted from mTECs and genes expression was quantified using real-time PCR. (G) mTECs were treated with Iso (5, 10, and 20 μM) for 18 h, treated with CDDP (20 μM) for 10 min, and then stained with 50 μM DCFH-DA for 40 min. The fluorescence was immediately detected by a fluorescence microscope and a multidetection reader. (H) The effects of Iso on mitochondrial membrane potential were tested using the JC-1 method and were determined by flow cytometry and a fluorescent microscope. **p < 0.01 versus the control group; #p < 0.05 and ##p < 0.01 versus the CDDP group; and p < 0.05 versus the Iso (20 μM) plus CDDP group.

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  FIGURE 3. Effects of Iso on CDDP-induced oxidative stress, inflammation and the apoptosis pathway in mTECs. (A,B) mTECs were treated with various concentrations of Iso (5, 10, and 20 μM) for 1 h, and the cells were administered CDDP (20 μM) for 24 h. Whole-cell lysates were collected for Western blot to determine the protein levels. Representative Western blots and the statistical results are shown. **p < 0.01 versus the control group; ##p < 0.01 versus the CDDP group.

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  FIGURE 4. Effects of SIRT1 and SIRT6 on CDDP-induced cytotoxicity and the regulation of Nrf2 in mTECs. (A,B) The effects of Iso on the protein levels of SIRT1, SIRT6, and Nrf2 in mTECs after treatment with SIRT1 and SIRT6 inhibitors. (C) The effects of Iso on the expression levels of Nrf2, HO1, and NQO1 in mTECs after treatment with a Nrf2 inhibitor. (D) SIRT1, SIRT6, Nrf2, HO1, and NQO1 signal intensities were quantified. (E) The effects of Iso on the in vitro viability of mTECs treated with or without SIRT1, SIRT6, and a Nrf2 inhibitor. **p < 0.01 versus the control group; NS, not significant.

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  FIGURE 5. Effect of Iso on CDDP-induced renal dysfunction. (A) Experimental study design and time chart for CDDP alone and combined with Iso treatments. After fasting for 12 h, mice were treated with 50 mg/kg Iso and administered 20 mg/kg CDDP after 1 h via ip injection. Iso was subsequently administrated twice at 24 and 48 h post-injection, respectively. Mice were sacrificed at 24 h after the last Iso administration. (B) Body weight changes were calculated by subtracting body weights at 72 h with body weights at –1 h. (C) Kidney indexes were expressed as kidney weights divided by body weights at 72 h. (D,E) BUN and serum creatinine were measured to estimate kidney dysfunction caused by CDDP. (F) Representative macroscopic mouse kidney. (G) Kidney specimens were stained with H&E. (H) Tubular injury was scored using the quantitative evaluation method as described in the section “Materials and Methods” **p < 0.01.

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  FIGURE 6. Effects of Iso on oxidative stress in CDDP-induced acute kidney injury. (A–D) Effects of Iso on MPO, MDA, SOD, and GSH levels in renal tissues from mice. **p < 0.01 versus the control group; #p < 0.05 and ##p < 0.01 versus the CDDP group.

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  FIGURE 7. Effects of Iso on the protein expression of oxidative stress, inflammation and apoptosis in CDDP-induced acute kidney injury. (A) Representative Western Blots and statistical results show the expression levels of SIRT1, SIRT6, total Nrf2, nuclear Nrf2, cytoplasmic Nrf2, HO-1, NQO1, and NOX4 in renal tissues from mice. (B) The enzymatic activity of SIRT1 in the kidney. (C) Representative Western Blots and statistical results show the expression levels of phosphorylated JNK, p38, ERK, and HMGB1, NF-κB in renal tissues from mice. (D) Representative Western Blots show the expression levels of Ac-p53, p53, p-p53, BCL2, BAX, and cleaved Caspase-3 in the kidneys. *p < 0.05 and **p < 0.01 versus the control group; #p < 0.05 and ##p < 0.01 versus the CDDP group.

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  FIGURE 8. Effects of Iso-mediated regulation of Nrf2 on CDDP-induced acute kidney injury in mice. After fasting for 12 h, WT and Nrf2–/– mice were intraperitoneally injected with 50 mg/kg Iso and 20 mg/kg CDDP after 1 h. Iso was subsequently administrated twice at 24 and 48 h post-injection, respectively. Mice were sacrificed at 24 h after the last Iso administration. (A) Body weight changes of WT and Nrf2–/– mice were calculated by subtracting body weights at 72 h with body weights at –1 h. (B) Kidney indexes of WT and Nrf2–/– mice were expressed as kidney weights divided by body weights at 72 h. (C,D) Creatinine and BUN of WT and Nrf2–/– mice were measured to estimate kidney dysfunction caused by CDDP. (E) Representative macroscopic kidney in WT and Nrf2–/– mice. (F) Kidney specimens of WT and Nrf2–/– mice were stained with H&E. (G) Effects of Iso on the protein expression of WT and Nrf2–/– mice. All data represent the mean ± SEM (n = 5 per group). *p < 0.05 and **p < 0.01 versus the CDDP group; ##p < 0.01 versus the control group; NS, not significant.