Morphine promotes microglial activation by upregulating the EGFR/ERK signaling pathway

Abstract

Although they represent the cornerstone of analgesic therapy, opioids, such as morphine, are limited in efficacy by drug tolerance, hyperalgesia and other side effects. Activation of microglia and the consequent production of proinflammatory cytokines play a key pathogenic role in morphine tolerance, but the exact mechanisms are not well understood. This study aimed to investigate the regulatory mechanism of epidermal growth factor receptor (EGFR) on microglial activation induced by morphine in mouse microglial BV-2 cells. In this research, BV-2 cells were stimulated with morphine or pretreated with AG1478 (an inhibitor of EGFR). Expression levels of cluster of differentiation molecule 11b (CD11b), EGFR, and phospho-EGFR were detected by immunofluorescence staining. Cell signaling was assayed by Western blot. The migration ability of BV-2 cells was tested by Transwell assay. The production of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in the cell supernatant was determined by ELISA. We observed that the expression of CD11b induced by morphine was increased in a dose- and time- dependent manner in BV-2 cells. Phosphorylation levels of EGFR and ERK1/2, migration of BV-2 cells, and production of IL-1β and TNFα were markedly enhanced by morphine treatment. The activation, migration, and production of proinflammatory cytokines in BV-2 cells were inhibited by blocking the EGFR signaling pathway with AG1478. The present study demonstrated that the EGFR/ERK signaling pathway may represent a novel pharmacological strategy to suppress morphine tolerance through attenuation of microglial activation.

Fig 1. Morphine upregulates CD11b in a dose- and time-dependent manner in microglial BV-2 cells.

BV-2 cells were treated with 0, 25, 50, 100, 200, and 400 μM morphine for 2, 4, 6, and 8 h and tested by immunocytochemistry staining. (a) Time- and dose-response effect of morphine on the expression of CD11b. Scale bar: 20 μm. (b) The time- and dose-response curve of BV-2 cells activated by morphine. Data were fit to a sigmoidal dose-response curve and EC50 values calculated using GraphPad Prism software. The mean optical densities of six randomly selected fields of cells from each group were analyzed by ImageJ software for quantitative analysis. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 were considered to be statistically significant compared to the control groups.

Fig 2. Dose- and time-dependent changes in the expression of CD11b proteins with morphine treatment were analyzed by Western blotting.

BV-2 cells were treated with 0, 25, 50, 100, and 200 μM morphine for 24 h (a) or with 200 μm morphine for 0, 2, 4, 6 and 8 h (b). GAPDH was used as a loading control. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 compared to the control group.

Fig 3. The effect of morphine and LPS on microglial activation by immunocytochemical analysis.

(a) BV-2 cells were treated with PBS, DMSO, LPS (1 μg/mL), morphine (200 μM), LPS (1 μg/mL) + morphine (200 μM), morphine (200 μM) + AG1478 (10 μM), and LPS (1 μg/mL) + morphine (200 μM) + AG1478 (10 μM) for 6 h. Cells were stained for the activation marker CD11b. Scale bar: 20 μm. (b) The mean optical densities of six randomly selected fields of cells from each group were analyzed by ImageJ software for quantitative analysis. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 compared to the control groups. ^# p < 0.05 and ^## p < 0.01 morphine vs. morphine + AG1478. ^& p < 0.05 and ^&& p < 0.01 LPS + morphine vs. LPS + morphine + AG1478.

Fig 4. The effect of morphine and LPS on expression of EGFR and p-EGFR.

The expression of EGFR (a) and p-EGFR (b) in BV-2 cells was detected by immunocytochemical analysis. Scale bar: 20 μm. (c), (d) The mean optical densities of six randomly selected fields of cells from each group were analyzed by ImageJ software for quantitative analysis. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 compared to the control groups. ^# p < 0.05 and ^## p < 0.01 morphine vs. morphine + AG1478. ^& p < 0.05 and ^&& p < 0.01 LPS + morphine vs. LPS + morphine + AG1478.

Fig 5. The effect of EGFR inhibition on morphine-induced protein expression.

BV-2 cells were incubated with DMSO, AG1478 (2.5, 5, and 10 μM), morphine (200 μM), and AG1478 (2.5, 5, and 10 μM) + morphine (200 μM) for 24 h. After treatment, cell lysates were examined by Western blot for expression levels of CD11b (a), EGFR and p-EGFR (b) and ERK1/2 and p-ERK1/2 (c). GAPDH was used as a loading control. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 were considered to be statistically significant.

Fig 6. The effect of EGFR inhibition on migration of BV-2 cells induced by morphine.

(a) BV-2 cells were treated with morphine or AG1478 for 48 h, and the number of migrated cells was evaluated by the Transwell assay. Scale bar: 20 μm. (b) Migration of BV-2 cells in six randomly selected fields from each group was quantified. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 compared to DMSO groups. ^## p < 0.01 morphine vs. morphine + AG1478.

Fig 7

The effect of EGFR inhibition on morphine- and LPS- induced IL-1β (a) and TNF-α (b) production in BV-2 microglial cells. The supernatants of BV-2 cells were collected after centrifugation and measured by ELISA. The absorbance of each well was measured at 450 nm using a microplate leader. Data are presented as the means ± SEMs of three independent experiments. * p < 0.05 and ** p < 0.01 compared to PBS groups. ^# p < 0.05 and ^## p < 0.01 morphine vs. morphine + AG1478. ^& p < 0.05 and ^&& p < 0.01 LPS + morphine vs. LPS + morphine + AG1478.