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. 2021 May;14(5):45.
doi: 10.3892/br.2021.1421. Epub 2021 Mar 16.

Cocaine potentiates an inflammatory response in C6 astroglia-like cells

Maryam Agharahimi  1 , Ramesh B Badisa  1 , Elizabeth Mazzio  1 , Karam F Soliman  1 , Carl B Goodman  1
Affiliations

Cocaine potentiates an inflammatory response in C6 astroglia-like cells

Maryam Agharahimi et al. Biomed Rep. 2021 May.

Abstract

Cocaine is a highly addictive drug that mediates its effect through altering dopamine metabolism in the central nervous system (CNS), resulting in a feeling of euphoria. Owing to its high lipophilicity, cocaine easily crosses the blood brain barrier of the CNS and reaches various domains of the brain, where it can trigger cellular damage. Cocaine-induced CNS damage may arise due to increased levels of free radicals and nitric oxide (NO) in immunecompetent astroglial cells. In the present study, the potential ability of cocaine to exacerbate the production of inflammatory products, primarily superoxide free radicals (O2 -), hydrogen peroxide (H2O2) and NO/nitrite (NO2 -) was examined in rat C6 astroglia-like cells challenged with lipopolysaccharide (LPS), a bacterial endotoxin, and interferon gamma (IFNγ), a pro-inflammatory cytokine. Furthermore, the role of cocaine in increasing the expression of hypoxia inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) in cells was also determined. First, the viability of the cells was assessed when treated with cocaine (0.5-7 mM) for 24 and 48 h. The results showed that cocaine toxicity was both time and dose-dependent. In subsequent studies, cells were challenged with or without LPS and IFNγ, followed by co-treatment with cocaine (1-4 mM) for 24 h. Cocaine treatment did not increase O2 - or H2O2 production in the challenged or unchallenged cells. Similarly, cocaine treatment did not increase NO/NO2 - production in the unchallenged cells; however, NO/NO2 - levels in the challenged cells was increased 40-50-fold upon cocaine treatment compared with the corresponding unchallenged group. The HIF-1α and VEGF levels were significantly increased in the challenged cells at higher cocaine doses compared with the unchallenged cells. Since high concentrations of NO are associated with inflammation, the high levels of NO production observed in the present study suggested that cocaine may have potentiated the inflammatory response in the challenged C6 astroglia-like cells.

Keywords: C6 astroglia-like cells; HIF-1α; VEGF; cocaine; nitric oxide/nitrite.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Effect of cocaine on the viability of cells. Cells were treated with various concentrations of cocaine for (A) 24 or (B) 48 h in 96-well culture plates. n=8. **P<0.01, ***P<0.001, ****P<0.0001 vs. respective control (0 mM).
Figure 2
Figure 2
Effect of cocaine on the formation of inflammatory products. (A) O2- free radicals or (B) H2O2 were measured in cells cultured in the media lacking phenol red and treated with various concentrations of cocaine in the absence or presence of both LPS (0.2 µg/ml) and IFNγ (6 µg/ml) for 24 h. n=4, not significant compared to control (0 mM). LPS, lipopolysaccharide; IFNγ, interferon-γ.
Figure 3
Figure 3
Standardization of LPS and IFNγ. Cells cultured in the media lacking phenol red were treated with (A) various concentrations of IFNγ and 2.5 µg/ml LPS, or (B) various concentrations of LPS and 2.5 µg/ml IFNγ for 24 h. n=4. ***P<0.001 vs. respective control (0 µg/ml). LPS, lipopolysaccharide; IFNγ, interferon-γ.
Figure 4
Figure 4
NO/NO2- production in cells. Cells in the media lacking phenol red were treated with various concentrations of cocaine in the absence or presence of LPS (0.2 µg/ml) alone or IFNγ (6 µg/ml) alone, or both together for 24 h in 96-well culture plates. n=4. @P<0.01 vs. respective 0 mM cocaine with LPS alone. *P<0.01 vs. respective 0 mM cocaine with LPS and IFNγ. #P<0.001 vs. stimulated cocaine treated cells with un-stimulated cocaine treated cells. LPS, lipopolysaccharide; IFNγ, interferon-γ.
Figure 5
Figure 5
Detection of HIF-1α using ELISA. Cells in the media lacking phenol red were treated with various concentrations of cocaine in the absence or presence of LPS (0.2 µg/ml) and IFNγ (6 µg/ml) together for 24 h in 96-well culture plates. n=3. *P<0.01 vs. respective 0 mM cocaine with LPS and IFNγ. #P<0.001 stimulated cells treated with 4 mM cocaine vs. un-stimulated cells treated with 4 mM cocaine. LPS, lipopolysaccharide; IFNγ, interferon-γ.
Figure 6
Figure 6
Detection of VEGF using ELISA. Cells in the media lacking phenol red were treated with various concentrations of cocaine in the absence or presence of LPS (0.2 µg/ml) and IFNγ (6 µg/ml) together for 24 h in 96-well culture plates. n=3. *P<0.01 vs. respective 0 mM cocaine with LPS and IFNγ. #P<0.001 stimulated cells treated with 3 or 4 mM cocaine vs. un-stimulated cells treated with 3 or 4 mM cocaine. LPS, lipopolysaccharide; IFNγ, interferon-γ.
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