Azithromycin Protects Retinal Glia Against Oxidative Stress-Induced Morphological Changes, Inflammation, and Cell Death

Abstract

The reactivity of retinal glia in response to oxidative stress has a significant effect on retinal pathobiology. The reactive glia change their morphology and secret cytokines and neurotoxic factors in response to oxidative stress associated with retinal neurovascular degeneration. Therefore, pharmacological intervention to protect glial health against oxidative stress is crucial for maintaining homeostasis and the normal function of the retina. In this study, we explored the effect of azithromycin, a macrolide antibiotic with antioxidant, immunomodulatory, anti-inflammatory, and neuroprotective properties against oxidative stress-induced morphological changes, inflammation, and cell death in retinal microglia and Müller glia. Oxidative stress was induced by H₂O₂, and the intracellular oxidative stress was measured by DCFDA and DHE staining. The change in morphological characteristics such as the surface area, perimeter, and circularity was calculated using ImageJ software. Inflammation was measured by enzyme-linked immunosorbent assays for TNF-α, IL-1β, and IL-6. Reactive gliosis was characterized by anti-GFAP immunostaining. Cell death was measured by MTT assay, acridine orange/propidium iodide, and trypan blue staining. Pretreatment of azithromycin inhibits H₂O₂-induced oxidative stress in microglial (BV-2) and Müller glial (MIO-M1) cells. We observed that azithromycin inhibits oxidative stress-induced morphological changes, including the cell surface area, circularity, and perimeter in BV-2 and MIO-M1 cells. It also inhibits inflammation and cell death in both the glial cells. Azithromycin could be used as a pharmacological intervention on maintaining retinal glial health during oxidative stress.

Figure 1

AZM inhibits H₂O₂-induced intracellular ROS production in BV-2 microglial and MIO-M1 Müller glial cells in vitro. (A) Representative images of DCFDA staining for BV2 cells, (B) spectrofluorometric measurement of DCFDA biochemical assay for BV2 cells, (C) representative images of DCFDA staining for MIO-M1 cells, (D) spectrofluorometric measurement of DCFDA biochemical assay for MIO-M1 cells. Red bars indicate fluorescence quantified in control cells, faint red bars indicate AZM-treated cells, green bars indicate H₂O₂-treated cells, and faint green bars indicate AZM-pretreated H₂O₂-treated cells. DMEM was taken as 100% and it was compared with the rest of the groups. All the results were presented as mean ± SD, n = 6, and one-way ANOVA was performed with Tukey’s multiple comparisons, ****p < 0.0001, black stars are differences as compared to DMEM, and blue stars are differences as compared to the H₂O₂.

Figure 2

Morphological changes in control and H₂O₂-treated BV-2 microglial and MIO-M1 Müller glial cells in vitro in the presence and absence of AZM evaluated by phase-contrast microscopy. (A) Phase-contrast image of BV-2 cells, (B) surface area of the BV-2 cells, (C) circularity of the BV-2 cells, (D) perimeter of the BV-2 cells, (E) phase contrast image of MIO-M1 cells, (F) surface area of the MIO-M1 cells, (G) circularity of the MIO-M1 cells, and (H) perimeter of the MIO-M1 cells. All the results were presented as mean ± SD, n = 6, and one-way ANOVA was performed with Tukey’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns represents p > 0.05, black stars as compared to DMEM, and blue stars are as compared to the H₂O₂.

Figure 3

Confirmation of the anti-inflammatory effect of AZM on BV-2 microglial cells by ELISA. (A) TNF-α, (B) IL-1β, and (C) IL-6 secreted by BV-2 cells. Red bars indicate fluorescence quantification in control cells, faint red bars indicate AZM-treated cells, green bars indicate H₂O₂-treated cells, and faint green bars indicate AZM-pretreated H₂O₂-treated cells. (D) Representative images of the anti-GFAP antibody staining in Müller glia. (E) Quantification of fluorescence intensity from GFAP immunostaining images. All the results were presented as mean ± SD, n = 6, and one-way ANOVA was performed with Tukey’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns represents p > 0.05, black stars are as compared to DMEM and blue stars are as compared to the H₂O₂. AZM inhibits oxidative stress-induced cell death in retinal glia.

Figure 4

Visualization of live and dead cells by acridine orange/propidium iodide (AO/PI) staining. AO gives green fluorescence for live cells, and PI gives red fluorescence for necrotic cells and yellow for apoptotic cells. (A) Representative images of AO/PI staining for BV-2 cells, (B) quantification of the percentage PI-positive cells compared to the total number of microglial cells (n = 8), (C) representative images of AO/PI staining for MIO-M1 cells, (D) quantification of the percentage of PI-positive cells compared to the total number of MIO-M1 cells (n = 8). All the results were presented as mean ± SD, and one-way ANOVA was performed with Tukey’s multiple comparisons, **p < 0.01, ***p < 0.001, ****p < 0.0001, black stars as compared to DMEM, and blue stars are as compared to the H₂O₂.

Figure 5

MTT assay measurement of the cell viability. (A) MTT assay for BV-2 microglial cells and (B) MTT assay for MIO-M1 cells. All the results were presented as mean ± SD, n = 6, and one-way ANOVA was performed with Tukey’s multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns represents p > 0.05; black stars as compared to DMEM, and blue stars are as compared to the H₂O₂.