Insulin-like growth factor-1 activates PI3K/Akt signalling to protect human retinal pigment epithelial cells from amiodarone-induced oxidative injury

Abstract

Background and purpose: Amiodarone is one of the most effective anti-arrhythmic drugs available, but its clinical applications are limited by toxic side effects including optic toxicity. The purpose of this study was to investigate the toxic effect of amiodarone on D407 cells (a human retinal pigmented epithelial (RPE) cell line) and the mechanisms of the protective effect of insulin-like growth factor-1 (IGF-1).

Experimental approach: The involvement of the kinases, Akt and ERK, was analysed by Western blot. Intracellular accumulation of ROS was measured using fluorophotometric quantification. A pharmacological approach with inhibitors was used to investigate the pathways involved in the protective action of IGF-1.

Key results: Amiodarone concentration-dependently augmented the production of ROS, lipid peroxidation and apoptosis in D407 cells. IGF-1 time- and concentration-dependently reversed these effects of amiodarone and protected D407 cells from amiodarone-mediated toxicity. Amiodarone inhibited the pAkt but not pErk, and IGF-1 reversed this inhibitory effect of amiodarone. However, IGF-1 failed to suppress amiodarone-induced cytotoxicity in the presence of PI3K/Akt inhibitor LY294002 suggesting the direct involvement of the PI3K/Akt pathway. Furthermore, in vivo rat flash electroretinogram (FERG) recordings showed that IGF-1 reverses the amiodarone-induced decrease in a- and b-waves. The immunocytochemistry findings confirmed that vitreous IGF-1 injections promote the survival of RPE cells in rat retina treated with amiodarone.

Conclusion and implications: IGF-1 can protect RPE cells from amiodarone-mediated injury via the PI3K/Akt pathway in vivo and in vitro. IGF-1 has potential as a protective drug for the prevention and treatment of amiodarone-induced optic toxicity.

Figure 1

Effect of amiodarone (AM) on the survival and apoptosis of D407 cells. D407 cells were treated with various concentrations of amiodarone; (A) cell viability was determined by MTT; (B, C) the apoptosis was determined by Hoechst DNA colour staining (24 h) and (D) the Annexin V‐FITC/PI assay (16 h). Data are presented as means ± SEM of the results obtained from five to six experiment.*P < 0.05 versus control.

Figure 2

Effects of amiodarone (AM) on the generation of ROS and lipid peroxidation in D407 cells. D407 cells were treated as indicated in the figure with amiodarone and then stained by DCFH‐DA and analysed by fluorometry (A) to detect ROS or followed by the application of MDA detection kit (B) to measure MDA. Data are presented as means ± SEM of the results obtained from five experiments. *P < 0.05 versus control.

Figure 3

IGF‐1 prevents amiodarone‐induced cell death in D407 cells. (A) D407 cells were treated with various concentrations of IGF‐1 and exposed to toxic levels of amiodarone (AM). The viability of the cells was determined by the MTT assay. (B) The apoptosis of D407 cells was determined by Hoechst DNA staining and presented as the quantified data. Data are presented as means ± SEM of the results obtained from five experiments, #P < 0.05 versus control, *P < 0.05 versus amiodarone‐treated group.

Figure 4

Effect of IGF‐1 on the activity of intracellular ROS and MDA in amiodarone (AM)‐treated D407 cells. (A–C) After pretreatment with IGF‐1(100 ng·mL⁻¹) for 1 h, D407 cells were incubated with or without 50 μM amiodarone for another 24 h then stained with DCFH‐DA and analysed by fluorometry to detect ROS, or by the application of MDA detection kit to measure MDA; data are presented as means ± SEM of the results obtained from five experiments. #P < 0.05 versus control, *P < 0.05 versus amiodarone‐treated group.

Figure 5

IGF‐1 attenuated amiodarone (AM)‐induced loss of mitochondrial membrane potential (△ψm) loss and increase in caspase 3/7 activity in D407 cells. (A, B) After pretreatment with IGF‐1(100 ng·mL⁻¹) for 1 h, D407 cells were incubated with or without 50 μM amiodarone for another 6 h. The △ψm was determined by the JC‐1 assay. (C) Quantification of caspase 3/7 activity was determined by caspase 3/7 activity assay. Data are presented as means ± SEM of the results obtained from five to six experiments. #P < 0.05 versus control group; *P < 0.05 versus amiodarone‐treated group.

Figure 6

Effects of amiodarone (AM) on phosphorylation of the Akt and ERK1/2. (A, B) D407 cells were treated with 50 μM amiodarone for various times or with different concentrations of amiodarone for 24 h, and then, the phosphorylation of Akt and MAPK (ERK1/2) in D407 cells was determined as described in Methods. Blots and quantitative data (OD) are shown (C–F). Data are presented as means ± SEM of the results obtained from five experiments. *P < 0.05 versus control.

Figure 7

Effect of LY294002 and PD98059 on the activation of Akt and ERK1/2 and the survival effects of IGF‐1 on amiodarone‐treated D407 cells. D407 cells pretreated with LY294002 to block PI3K/Akt signalling or PD98059 to block the MAPK (ERK1/2) pathway were treated with IGF‐1 and then incubated with (AM) or without (CTL) amiodarone as indicated. Cell viability (A) and caspase 3/7 activity (B) were determined. Data are presented as means ± SEM of the results obtained from five to six experiments. #P < 0.05 versus control, *P < 0.05 versus amiodarone‐treated group.

Figure 8

Effect of LY294002 on the activation of Akt and the IGF‐1 mediated attenuation of the damaging effects of amiodarone (AM) on D407 cells injury. D407 cells, pretreated with LY294002 to block PI3K/Akt signalling or PD98059 to block the MAPK (ERK1/2) pathway, were treated with IGF‐1 and then incubated with (AM) or without (CTL) amiodarone as indicated. Data are presented as means ± SEM of the results obtained from five experiments. #P < 0.05 versus control, *P < 0.05 versus amiodarone‐treated group.

Figure 9

IGF‐1 showed a protective effect and reversal of the effects of amiodarone (AM) on entoretina function detected by FERG morphological injury after vitreous injection with amiodarone (1.5 μM). Representative photographs showing RPE‐65 protein expression in IGF‐1‐treated retina induced by photochemical damage detected by immunofluorescence staining (A) and haematoxylin–eosin staining of retinal sections (B). FERG was tested after 24 h. Rats were vitreous injected with normal saline 5 μL; amiodarone (1.5 μM) 2.5 μL + normal saline 2.5 μL, IGF‐1(100 ng·mL⁻¹) 2.5 μL + AM (1.5 μM) 2.5 μL respectively. FERG studies were performed as described in Methods. Representatives of each group in Max reaction are shown (C, D). Data are presented as means ± SEM of the results obtained from five experiments. #P < 0.05 compared with control, *P < 0.05 compared with amiodarone.

Figure 10

Schematic presentation of the antioxidant and protective effects of IGF‐1 on amiodarone‐treated human retinal pigment epithelial cells. Amiodarone inhibited the activation of Akt, increased ROS/MDA and induced mitochondrial membrane failure and the activation of caspase, causing the apoptosis of D407 cells while IGF‐1 reversed the effects of amiodarone and promoted the survival of D407 cells.