Opposite effects of methanandamide on lipopolysaccharide-induced prostaglandin E2 and F2α synthesis in uterine explants from pregnant mice

Abstract

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE(2) and PGF(2α) biosynthesis, by inhibiting PGE(2) production and increasing PGF(2α) levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.

Figure 1. LPS-induced PGE₂ and PGF_2α production is mediated by endocannabinoids.

A) PGE₂ production for uterine explants incubated with LPS (1 ug/ml) and AM251 (10 nM) or SR144528 (10 nM) for 24 h. ANOVA test. a, p<0.001 vs control or AM251 or SR144528; b, p<0.001 vs control or AM251; c, p<0.001 vs LPS. n = 6. B) PGF_2α production for uterine explants incubated with LPS (1 ug/ml) and AM251 (10 nM) or SR144528 (100 nM) for 24 h. ANOVA test. a, p<0.001 vs control or AM251 or SR144528; b, p<0.01 vs control or AM251; c, p<0.001 vs control or SR144528; d, p<0.001 vs LPS; e, p<0.01 vs LPS. n = 5.

Figure 2. AEA mediates LPS-induced PG production.

A) PGE₂ production for uterine explants incubated with LPS (1 ug/ml), m-AEA (100 nM), URB597 (1 uM), LPS plus m-AEA (100 nM) or LPS plus URB597 (1 uM) for 24 h. ANOVA test. a, p<0.001 vs control; b, p<0.01 vs LPS; c, p<0.001 vs LPS. n = 7. B) PGF_2α production for uterine explants incubated with LPS (1 ug/ml), m-AEA (100 nM), URB597 (1 uM), LPS plus m-AEA (100 nM) or LPS plus URB597 (1 uM) for 24 h. ANOVA test. a, p<0.001 vs control; b, p<0.01 vs LPS; c, p<0.05 vs control; d, p<0.01 vs control. n = 7.

Figure 3. Effect of LPS on CB1 and CB2 levels.

Densitometric analysis and expression of CB1 and CB2. Uterine explants were incubated without/with LPS (1 ug/ml) for 24 h and where subjected to RT-PCR and western blot analysis. Student t Test. a, p<0.05 vs control. Each gel was repeated three times with different samples. One representative gel/blot is shown.

Figure 4. LPS-induced COX-2 expression is mediated by endocannabinoids.

Densitometric analysis of A) mRNA and B) protein expression of COXs. Uterine explants were incubated with LPS alone (1 ug/ml), LPS plus AM251 (10 nM) or LPS plus SR144528 (10 nM) for 24 h. A) ANOVA test. a, p<0.001 vs control; b, p<0.001 vs LPS; c, p<0.01 vs control; d, p<0.05 vs control. n = 5. B) ANOVA test. a, p<0.001 vs control; b, p<0.001 vs LPS; n = 4. One representative gel/blot is shown.

Figure 5. LPS-induced COX-2 activity is mediated by endocannabinoids.

Uterine explants were incubated with LPS alone (1 ug/ml), LPS plus AM251 (10 nM) or LPS plus SR144528 (10 nM) for 24 h and COX-2 enzyme activity was determined by measuring the disappearance of the radiolabelled substrate [¹⁴C]-AA. Enzyme activity is reported as nmol of disappeared [¹⁴C]-AA/mg protein/h. ANOVA test. a, p<0.001 vs control; b, p<0.01 vs control; c, p<0.001 vs LPS; n = 4.

Figure 6. Effect of AEA on

COX-2 expression. Densitometric analysis of A) mRNA and B) protein expression of COXs. Uterine explants were incubated without/with m-AEA (100 nM) for 24 h. Student t test. a, p<0.05 vs control; b, p<0.001 vs control. n = 4. One representative gel/blot is shown.

Figure 7. AEA increases COX-2 activity.

Uterine explants were incubated with m-AEA (100 nM) or URB597 (1 uM) for 24 h and COX-2 enzyme activity was determined by measuring the disappearance of the radiolabelled substrate [¹⁴C]-AA. Enzyme activity is reported as nmol of disappeared [¹⁴C]-AA/mg protein/h. ANOVA test. a, p<0.05 vs control; b, p<0.01 vs control; n = 4.

Figure 8. Endocannabinoids modulate mPGES-1 mRNA levels.

Densitometric analysis and mRNA expression of PGES isoforms. Uterine explants were incubated with LPS alone (1 ug/ml), LPS plus AM251 (10 nM) or LPS plus SR144528 (10 nM) for 24 h. A) One representative gel is shown. B) Densitometric analysis. ANOVA test. a, p<0.001 vs control; b, p<0.01 vs LPS; c, p<0.001 vs LPS. n = 5.

Figure 9. Endocannabinoids modulate mPGES-1 protein levels.

Densitometric analysis and protein expression of mPGES-1. Uterine explants were incubated with LPS alone (1 ug/ml), LPS plus AM251 (10 nM) or LPS plus SR144528 (10 nM) for 24 h. ANOVA test. a, p<0.001 vs control; b, p<0.001 vs LPS; c, p<0.01 vs LPS. n = 4.

Figure 10. AEA decreases mPGES-1 mRNA and protein levels.

Densitometric analysis of A) mRNA and B) protein expression of mPGES-1. Uterine explants were incubated without/with m-AEA (100 nM) for 24 h. Student t test. a, p<0.05 vs control; b, p<0.001 vs control. n = 5. One representative gel/blot is shown.

Figure 11. Quantitative real-time PCR analysis of mPGES-1 expression.

Uterine explants were incubated without/with m-AEA (100 nM) for 3 h and 6 h and then subjected to qPCR analysis. Data was normalized against β-actin and mPGES-1 mRNA levels under control conditions (no m-AEA) were set to 1 (dotted line). Experiments were independently run three times. In each experiment, cDNA samples were performed in triplicate. Student t Test. a, p<0.05 vs control. n = 9.

Figure 12. Effect of AEA on PGE synthase activity.

Uterine explants were incubated with m-AEA (100 nM), INDO (1 µM) or m-AEA plus INDO for 24 h. Results were expressed as pg PGE2/mg protein/h. ANOVA Test. a, p<0.001 vs control or INDO. n = 4. PGE₂ levels were assessed by RIA.

Figure 13. AEA augments PGE₂ metabolite levels.

Uterine explants were incubated without/with m-AEA (100 nM) for 24 h. A) PGE₂ production assessed by RIA; B) PGEM quantification assessed by PGE metabolite EIA kit. Student t Test. a, p<0.001 vs control; b, p<0.01 vs control. n = 5. Both metabolites were assessed in the same sample. PGEM, Prostaglandin E₂ metabolite.