4'-O-methylhonokiol increases levels of 2-arachidonoyl glycerol in mouse brain via selective inhibition of its COX-2-mediated oxygenation

Abstract

Background and purpose: 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time.

Methods: CB2 receptor modulation ([35S]GTPγS, cAMP, and β-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS.

Results: MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and β-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM).

Conclusions: LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.

Figure 1

Chemical structures of 4′-O-methylhonokiol (-4′-methoxy-5,3′-di-(2-propenyl)-biphenyl-2-ol), 27a (2-methoxy-5,3′-di-(2-propenyl)- biphenyl-4′-ol), 32 (5,5′-di-sec-butyl- biphenyl-2,2′-diol) and 34a (2-butyloxy-5,3′-di-(2-propenyl)- biphenyl-4′-ol).

Figure 2

Modulation of CB₂ receptor activity by MH. (A) [³⁵S]GTPγS binding assay was performed in stably hCB₂ receptor overexpressing CHO-K1 membranes, in the presence of different concentrations of MH and 2-AG alone or (B) in combination. The same experiments were performed in endogenously CB₂ expressing (C) HL60 cells and (D) mouse spleen membranes. (E) Inhibition of forskolin-induced cAMP formation by MH in CHO-hCB₂ cells transfected with pGloSensorTM 22-F plasmid. The experiments were performed in the presence (circles) and in the absence (triangles) of constitutive activity. (F) β-arrestin recruitment induced by increasing concentrations of MH, CP55,940, and AM630, measured in PathHunter® β-arrestin cells (CHO-K1-HOMSA-CNR2). ^** P < 0.01 MH vs. 2-AG (Figure 2A); ^** P < 0.01 MH (1 μM) plus 2-AG vs. 2-AG, ⁺⁺ P < 0.01, ⁺ P < 0.05 MH (0.3 μM) plus 2-AG vs. 2-AG (Figure 2B). ^* P < 0.05 MH vs. 2-AG and CP55,940 (Figures 2C, D).^** P < 0.01 MH vs. CP55,940 (Figure 2F).

Figure 3

Dose-dependent COX-2 inhibition induced by MH in different biological matrices. (A) MH-mediated inhibition of hCOX-2 using 10 μM of AA (black), AEA (blue), and 2-AG (red) as substrates. MH-mediated inhibition of PGE₂ and PGE₂-GE formation in (B) RAW264.7 cell homogenate and (C) intact cells after 12 h of incubation with LPS (1 mg mL⁻¹). Homogenates and intact cells were incubated for 30 min with 10 μM of 2-AG after the pre-treatment (30 min) with different concentrations of MH or vehicle. ^* P < 0.05, ^** P < 0.01 PGE₂ vs. PGE₂-GE.

Figure 4

MS chromatograms of PGE₂ and PGE₂-GE measured in different biological matrices. Quantification of PGE₂ and PGE₂-GE formation measured after incubation of (A) RAW264.7 cell homogenate and (B) intact cells with 10 μM of AA or 2-AG in the presence of different concentrations of MH or vehicle. Homogenates and cells were treated for 12 h with LPS (1 mg mL⁻¹) followed by 30-min incubation with 10 μM of 2-AG after the pre-treatment (30 min) with different concentrations of MH or vehicle.

Figure 5

MH-mediated inhibition of PGE₂ and PGE₂-GE formation in RAW264.7 cells without external addition of 2-AG. Concentration-dependent inhibition of (A) PGE₂ and (B) PGE₂-GE formation (picogram per million of cell) in RAW264.7 intact cells previously stimulated with LPS (1 mg mL⁻¹) ATP (1 mM) and thapsigargin (2 μM) for 8 h. (C) PGE₂ and PGE₂-GE formation inhibition upon different concentrations of MH (expressed as % of vehicle-treated cells). ^* P < 0.05, ^** P < 0.01 MH treated samples vs. vehicle for panels (A) and (B); ^* P < 0.05, ^** P < 0.01 PGE₂-GE vs. PGE₂ for panel (C).

Figure 6

LC-MS/MS quantification of different analytes in mouse brain. (A) AEA, (B) 2-AG, (C) AA, (D) PGE₂, and (E) corticosterone were quantified in the brains from mice (6 to 14 animals per group) challenged for 6 h with LPS (i.p., 2.5 mg kg⁻¹, or saline), after 1 h of pre-treatment with MH (i.p., 3, 10, and 20 mg kg⁻¹) or vehicle. (F) MH quantification in the brain of LPS-challenged mice. ^* P < 0.05, ^** P < 0.01 treated vs. not-treated animals (Figure 6A-E) and ^* P < 0.05 MH 10 mg kg⁻¹ vs. 20 mg kg⁻¹ (Figure 6F).

Figure 7

COX-2 inhibition by biphenyl neolignans in different biological matrices. (A-D) 27a, (E-H) 32, and (I-L) 34a inhibition of 2-AG, AEA, and AA oxygenation in hCOX-2 (A, E, I), RAW264.7 cell homogenates (B, F, J), and intact cells (C, G, K) incubated with 10 μM of substrate (2-AG, AEA, or AA depending on the assay). (D, H, L) Inhibition of PGE₂ and PGE₂-GE formation in RAW264.7 cells upon LPS (1 mg mL⁻¹) ATP (1 mM) and thapsigargin (2 μM) treatment without externally adding 2-AG. ^* P < 0.05, ^** P < 0.01 PGE₂-GE vs. PGE₂.

Figure 8

Modulation of CB₂ receptor activity by 27a and 34a. (A) [³⁵S]GTPγS binding assay performed in CHO-K1 membranes containing hCB₂ receptors, in the presence of different concentrations of 27a and 34a. The CB₂ inverse agonist AM630 is reported as positive control (red line). Experiments were performed in the presence (dotted line) and in the absence (solid line) of constitutive activity of the receptors. (B) Inhibition of forskolin-induced cAMP formation by 27a and 34a in CHO-hCB₂ cells transfected with pGloSensorTM 22-F plasmid. The experiments were performed in the presence (dotted line) and in the absence (solid line) of constitutive activity. (C) β-arrestin recruitment induced by increasing concentrations of 27a and 34a, measured in PathHunter® β-arrestin cells (CHO-K1-HOMSA-CNR2). ^* P < 0.05, ^** P < 0.01 27a vs. 2-AG (Figure 8A); ^* P < 0.05, ^** P < 0.01 27a and 34a vs. CP55, 940 (Figure 8C).