670nm photobiomodulation modulates bioenergetics and oxidative stress, in rat Müller cells challenged with high glucose

Abstract

Diabetic retinopathy (DR), the most common complication of diabetes mellitus, is associated with oxidative stress, nuclear factor-κB (NFκB) activation, and excess production of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). Muller glial cells, spanning the entirety of the retina, are involved in DR inflammation. Mitigation of DR pathology currently occurs via invasive, frequently ineffective therapies which can cause adverse effects. The application of far-red to near-infrared (NIR) light (630-1000nm) reduces oxidative stress and inflammation in vitro and in vivo. Thus, we hypothesize that 670nm light treatment will diminish oxidative stress preventing downstream inflammatory mechanisms associated with DR initiated by Muller cells. In this study, we used an in vitro model system of rat Müller glial cells grown under normal (5 mM) or high (25 mM) glucose conditions and treated with a 670 nm light emitting diode array (LED) (4.5 J/cm2) or no light (sham) daily. We report that a single 670 nm light treatment diminished reactive oxygen species (ROS) production and preserved mitochondrial integrity in this in vitro model of early DR. Furthermore, treatment for 3 days in culture reduced NFκB activity to levels observed in normal glucose and prevented the subsequent increase in ICAM-1. The ability of 670nm light treatment to prevent early molecular changes in this in vitro high glucose model system suggests light treatment could mitigate early deleterious effects modulating inflammatory signaling and diminishing oxidative stress.

Fig 1. Culture and treatment flow chart.

All cells were cultured for experimental on day 0. Cells were treated at each indicated time point and assayed 1 hour after treatment with 670nm light.

Fig 2. 670 nm light treatment diminishes oxidative stress under high glucose conditions in Muller cells.

Bar graph displaying ROS production in rMC-1 cells. Cells were cultured in either normal glucose (black bars and white bars) or high glucose (gray bars and hashed bars) medium for 24 hours. 670nm or sham control was applied to the cells at 4.5 J/cm². Oxidative stress was analyzed using DCF-DA. One-way ANOVA statistical analysis yielded *** p<0.001, **** p<0.0001. Three separate experiments were performed in triplicate.

Fig 3. 670nm PBM restores mitochondrial function under high glucose conditions.

rMC-1 cells were cultured in either normal glucose (black bars and white bars) or high glucose medium (gray bars and hashed bars) for 24 hours. 670nm (white bars and hashed bars) or sham control (black bars and gray bars) was applied to the cells at 4.5 J/cm². All assays were normalized to the normal glucose sham levels. (A) Mitochondrial membrane potential was analyzed using TMRE, (B) NAPDH-dependent oxidoreductase activity was analyzed using MTT. Both TMRE and MTT assays were performed in 3 separate experiments, each data point was measured in triplicate. One-way ANOVA analysis yielded statistics as follows: * p<0.05, ** p<0.01, **** p<0.0001.

Fig 4. 670nm light treatment diminishes NFκB activity under high glucose conditions in Muller cells.

Bar graph showing Müller cells were cultured in either normal glucose (black bars and white bars) or high glucose medium (gray bars and hashed bars) for 72 hours while transfected with NFκB-luciferase. NFκB activity was analyzed via reporter gene assay, normalized to the transfection control, Renilla, and then treatments were normalized to normal glucose, sham conditions. 670nm (white bars and hashed bars) or sham control (black bars and gray bars) was applied to the cells at 4.5 J/cm² daily. One-way ANOVA statistical analysis yielded *p>0.05, ** p<0.01 Four separate experiments were performed in duplicate.

Fig 5. 670nm PBM suppresses downstream mediators of NFκB signaling.

Bar graph representing rMC-1 cells were cultured in either normal glucose (black bars and white bars) or high glucose (gray bars and hashed bars) medium for 72 hours. 670nm (white bars and hashed bars) or sham control (black bars and gray bars) was applied to the cells at 4.5 J/cm2 daily while in culture. (A) VEGF mRNA was analyzed via qPCR and normalized to actin. mRNA levels were calculated by ΔΔCt and normalized to Normal glucose sham. Five separate experiments were performed in duplicate. One-way ANOVA statistical analysis yielded * p = 0.0147 (B) ICAM was analyzed via western blot and normalized to GAPDH as the loading control via ImageJ. Inset includes a representative western blot image. Six separate experiments were performed, and one-way ANOVA analysis yielded * p<0.05, ** p<0.01, *** p<0.001.