Lithium is able to minimize olanzapine oxidative-inflammatory induction on macrophage cells

Abstract

Background: Olanzapine (OLZ) is a second-generation antipsychotic drug used for treatment of schizophrenia, bipolar disorder, and other neuropsychiatric conditions. Undesirable side effects of OLZ include metabolic alterations associated with chronic oxidative-inflammation events. It is possible that lithium (Li), a mood modulator that exhibits anti-inflammatory properties may attenuate OLZ-induced oxi-inflammatory effects.

Methodology: To test this hypothesis we activated RAW 264.7 immortalized macrophages with OLZ and evaluated oxidation and inflammation at the gene and protein levels. Li and OLZ concentrations were determined using estimated plasma therapeutic concentrations.

Results: OLZ triggered a significant increase in macrophage proliferation at 72 h. Higher levels of oxidative markers and proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, with a concomitant reduction in IL-10, were observed in OLZ-exposed macrophages. Lithium (Li) exposure triggered a short and attenuated inflammatory response demonstrated by elevation of superoxide anion (SA), reactive oxygen species (ROS), IL-1β, and cellular proliferation followed by elevation of anti-inflammatory IL-10 levels. Li treatment of OLZ-supplemented macrophages was able to reverse elevation of oxidative and inflammatory markers and increase IL-10 levels.

Conclusions: Despite methodological limitations related to in vitro protocols, results suggested that Li may attenuate OLZ-induced oxidative and inflammatory responses that result from metabolic side effects associated with OLZ.

Fig 1. Effect on RAW macrophages cellular proliferation in 72 h cultures e cellular of OLZ (OLZ) at different concentrations.

C = non-treated cells; PHA = cells activated by phytohemaglutin antigen exposure. Treatments were compared using one-way analysis of variance (ANOVA), followed by the Tukey post hoc test. Different letters (i.e., A, B, C, D, E, F) indicated significant statistical differences among treatments at p < 0.05. Treatments with A letter were considered with values similar to C group.

Fig 2. Comparison of cell cycle determined by flow cytometry analysis among RAW macrophages 72 h cultures exposed OLZ (OLZ) at 0.03 μg/mL concentration and to phytohaemagglutinin (PHA), an natural antigen that trigger inflammatory macrophages activation.

(A) Representative graphics with cell phases: (G0/G1 = gap 1; S = synthesis; G2/M = gap2 and mitosis). (B) % of cells the control in at each stage (G0/G1 = gap 1; S = synthesis; G2/M = gap2 and mitosis) of the cell cycle compared by One-way analysis of variance (ANOVA), followed by the Tukey post hoc test. The different letters (i.e., A, B, C, D, E, F) indicate statistical differences in each treatment at p < 0.05. (C) Monolayer culture pattern of RAW macrophages microscopic optic analysis (×40, scale bar = 20 μm) in control (C) cells and cells exposed to OLZ and PHA. No-activated C-cells presented higher frequency of spheric cells typical of monocytes. Cells PHA and OLZ exposed presenting a macrophage spreading pattern that is observed in inflammatory-activated cells.

Fig 3

Interaction between OLZ (0.03 μg/mL) and Li (0.7 mEq/L) on proliferation (A) and modulation of oxidative markers superoxide anion (SA), reactive oxygen species (ROS), and nitric oxide (NO) (B). Treatments were compared by one-way analysis of variance (ANOVA), followed by the Tukey post hoc test. The different letters (i.e., A, B, C, D) indicate statistical differences in each treatment at p < 0.05. Treatments identified with A-letter were statistically s imilar to untreated-cells group (C).

Fig 4. Modulation of protein and gene expression cytokines (IL-1β, IL-6, TNFα, IL-10) involved with inflammatory response of RAW macrophages exposed to OLZ (OLZ, 0.03 μg/mL) and Li (Li, and 0.7 mEq/L).

Treatments were compared by one-way analysis of variance (ANOVA), followed by the Tukey post hoc test. The different letters (i.e., A, B, C, D) indicate statistical differences in each treatment at p < 0.05. Treatments with A letter were considered with values similar to C untreated-cells group. Gene expression of each cytokine in each treatment is represented by colored squares (black = gene expression similar to C-group; green square = gene overexpression ≥ 1.2 < 10 times than C-group; yellow square = gene overexpression ≥ 10 < 50 times than C-group; red square = gene overexpression ≥ 50 times than C-group. The beta-actin housekeeping was used as internal control to normalized gene expression analysis among treatments.