The TSPO Ligands 2-Cl-MGV-1, MGV-1, and PK11195 Differentially Suppress the Inflammatory Response of BV-2 Microglial Cell to LPS

Abstract

The 18 kDa Translocator Protein (TSPO) is a marker for microglial activation as its expression is enhanced in activated microglia during neuroinflammation. TSPO ligands can attenuate neuroinflammation and neurotoxicity. In the present study, we examined the efficacy of new TSPO ligands designed by our laboratory, MGV-1 and 2-Cl-MGV-1, in mitigating an in vitro neuroinflammatory process compared to the classic TSPO ligand, PK 11195. We exposed BV-2 microglial cells to lipopolysaccharide (LPS) for 24 h to induce inflammatory response and added the three TSPO ligands: (1) one hour before LPS treatment (pretreatment), (2) simultaneously with LPS (cotreatment), and (3) one hour after LPS exposure (post-treatment). We evaluated the capability of TSPO ligands to reduce the levels of three glial inflammatory markers: cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitric oxide (NO). We compared the effects of the two novel ligands to PK 11195. Both 2-Cl-MGV-1 and MGV-1 reduced the levels of glial COX-2, iNOS, and NO in LPS-treated BV-2 cells more efficiently than PK 11195. Notably, even when added after exposure to LPS, all ligands were able to suppress the inflammatory response. Due to their pronounced anti-inflammatory activity, 2-Cl-MGV-1 and MGV-1 may serve as potential therapeutics in neuroinflammatory and neurodegenerative diseases.

Keywords: TSPO ligands; cyclooxygenase-2 (COX-2); inducible nitric oxide synthase (iNOS); lipopolysaccharide (LPS); microglial activation; neuroinflammation; nitric oxide (NO).

Figure 1

TSPO ligands reduced COX-2 expression levels in LPS-treated BV-2 cells. BV-2 cells were treated with 100 ng/mL LPS for 24 h, with or without TSPO ligands (25 μM) in three different scenarios. A,C,E: Representative Western blots of COX-2 and β-actin expression levels in pretreatment, cotreatment, and post-treatment, respectively, with the three TSPO ligands. B,D,F: Relative COX-2 protein expression levels normalized to β-actin in pretreatment, cotreatment, and post-treatment, respectively, with TSPO ligands. Results were calculated using densitometry and presented as means ± SEM; n = 4 (pretreatment), n = 5 (cotreatment), n = 3 (post-treatment). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Control; ## p < 0.01, ### p < 0.001 compared to LPS; @@@ p < 0.001 compared to LPS + PK 11195; & p < 0.05 compared to LPS + MGV-1.

Figure 2

TSPO ligands reduced iNOS expression levels in LPS-treated BV-2 cells. BV-2 cells were treated with 100 ng/mL LPS for 24 h, with or without TSPO ligands (25 μM) in three different scenarios. A,C,E: Representative Western blots of iNOS and β-actin expression levels in pre-treatment, co-treatment, and post-treatment, respectively, with the three TSPO ligands. B,D,F: Relative iNOS protein expression levels normalized to β-actin in pre-treatment, co-treatment, and post-treatment, respectively, with TSPO ligands. Results were calculated using densitometry and presented as means ± SEM; n = 4 (pre-treatment), n = 5 (co-treatment), n = 3 (post-treatment). * p < 0.05, *** p < 0.001 compared to Control; ### p < 0.001 compared to LPS; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001 compared to LPS + PK 11195.

Figure 3

TSPO ligands reduced nitrite levels in LPS-treated BV-2 cells. BV-2 cells were treated with 100 ng/mL LPS for 24 h, with or without TSPO ligands (25 μM) in three different scenarios. A,B,C: Nitrite concentrations (μM) in pretreatment, cotreatment, and post-treatment, respectively, with TSPO ligands. Nitrite levels are presented as means ± SEM; n = 3 (pretreatment), n = 3 (cotreatment), n = 3 (post-treatment). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Control; ### p < 0.001 compared to LPS; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001 compared to LPS + PK 11195.