Anandamide Influences Interleukin-1β Synthesis and IL-1 System Gene Expressions in the Ovine Hypothalamus during Endo-Toxin-Induced Inflammation

Abstract

This study evaluated the effect of anandamide (AEA) on interleukin (IL)-1β synthesis and gene expression of IL-1β, its type I (IL-1R1) and II (IL-1R2) receptors, and IL-1 receptor antagonist (IL-1RN) in the hypothalamic structures, involved in the central control of reproduction, during inflammation. Animals were intravenously (i.v.) injected with bacterial endotoxin-lipopolysaccharide (LPS) (400 ng/kg) or saline, and two hours after LPS administration., a third group received i.v. injection of AEA (10 μg/kg). Ewes were euthanized one hour later. AEA injection (p < 0.05) suppressed LPS-induced expression of IL-1β protein in the hypothalamus. The gene expression of IL-1β, IL-1RN, and IL-1R2 in the hypothalamic structures was higher (p < 0.05) in animals treated with both LPS and AEA in comparison to other experimental groups. AEA administration did not influence LPS-stimulated IL-1R1 gene expression. Our study shows that AEA suppressed IL-1β synthesis in the hypothalamus, likely affecting posttranscriptional levels of this cytokine synthesis. However, anti-inflammatory effect of AEA might also result from its stimulating action on IL-1RN and IL-1R2 gene expression. These results indicate the potential of endocannabinoids and/or their metabolites in the inhibition of inflammatory process at the level of central nervous system, and therefore their usefulness in the therapy of inflammation-induced neuroendocrine disorders.

Keywords: IL-1β; LPS; anandamide; central nervous system; endocanabinoids; endotoxin; hypothalamus; inflammation.

Figure 1

The effect of intravenous (i.v.) anandamide (AEA) injection (10 µg/kg; i.v.) and lipopolysaccharide (LPS)-induced inflammation (400 ng/mg; i.v.) on the concentration of interleukin (IL)-1β in the hypothalamus. Data are presented as the mean value ± S.E.M. Different letters indicate significant (p < 0.05) differences according to a one-way ANOVA followed by Fisher’s least significance test.

Figure 2

The effect of intravenous (i.v.) anandamide (AEA) injection (10 µg/kg; i.v.) and lipopolysaccharide (LPS)-induced inflammation (400 ng/mg; i.v.) on the relative interleukin (IL)-1β mRNA level in preoptic area (POA), anterior hypothalamus (AHA), and medial basal hypothalamus (MBH) normalized to the mean expression of three reference genes (GAPDH, HDAC1, ACTB—see Table 2.). All data are presented as the mean value ± S.E.M. Different letters indicate significant (p < 0.05) differences according to a one-way ANOVA followed by Fisher’s least significance test.

Figure 3

The effect of intravenous (i.v.) anandamide (AEA) injection (10 µg/kg; i.v.) and lipopolysaccharide (LPS)-induced inflammation (400 ng/mg; i.v.) on the relative interleukin 1 antagonist (IL-1RN) mRNA level in preoptic area (POA), anterior hypothalamus (AHA), and medial basal hypothalamus (MBH) normalized to the mean expression of three reference genes (GAPDH, HDAC1, ACTB—see Table 2). All data are presented as the mean value ± S.E.M. Different letters indicate significant (p < 0.05) differences according to a one-way ANOVA followed by Fisher’s least significance test.

Figure 4

The effect of intravenous (i.v.) anandamide (AEA) injection (10 µg/kg; i.v.) and lipopolysaccharide (LPS)-induced inflammation (400 ng/mg; i.v.) on the relative receptor type I interleukin 1 (IL-1R1) mRNA level in preoptic area (POA), anterior hypothalamus (AHA), and medial basal hypothalamus (MBH) normalized to the mean expression of three reference genes (GAPDH, HDAC1, ACTB—see Table 2). All data are presented as the mean value ± S.E.M. Different letters indicate significant (p < 0.05) differences according to a one-way ANOVA followed by Fisher’s least significance test.

Figure 5

The effect of intravenous (i.v.) anandamide (AEA) injection (10 µg/kg; i.v.) and lipopolysaccharide (LPS)-induced inflammation (400 ng/mg; i.v.) on the relative receptor type II interleukin 1 (IL-1R2) mRNA level in preoptic area (POA), anterior hypothalamus (AHA), and medial basal hypothalamus (MBH) normalized to the mean expression of three reference genes (GAPDH, HDAC1, ACTB—see Table 2). All data are presented as the mean value ± S.E.M. Different letters indicate significant (p < 0.05) differences according to a one-way ANOVA followed by Fisher’s least significance test.