Δ8-Tetrahydrocannabivarin prevents hepatic ischaemia/reperfusion injury by decreasing oxidative stress and inflammatory responses through cannabinoid CB2 receptors

Abstract

Background and purpose: Activation of cannabinoid CB(2) receptors protects against various forms of ischaemia-reperfusion (I/R) injury. Δ(8) -Tetrahydrocannabivarin (Δ(8) -THCV) is a synthetic analogue of the plant cannabinoid Δ(9) -tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB(2) receptors. Here, we assessed effects of Δ(8) -THCV and its metabolite 11-OH-Δ(8) -THCV on CB(2) receptors and against hepatic I/R injury.

Experimental approach: Effects in vitro were measured with human CB(2) receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo.

Key results: Displacement of [(3) H]CP55940 by Δ(8) -THCV or 11-OH-Δ(8) -THCV from specific binding sites in CHO cell membranes transfected with human CB(2) receptors (hCB(2) ) yielded K(i) values of 68.4 and 59.95 nM respectively. Δ(8) -THCV or 11-OH-Δ(8) -THCV inhibited forskolin-stimulated cAMP production by hCB(2) CHO cells (EC(50) = 12.95 and 14.3 nM respectively). Δ(8) -THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-α, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of Δ(8) -THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of Δ(8) -THCV, while a CB(1) antagonist tended to enhance it.

Conclusions and implications: Δ(8) -THCV activated CB(2) receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB(2) receptor activation.

Linked articles: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Figure 1

Synthesis and in vitro effects of Δ⁸-THCV on CB₂ receptors. (A) Scheme of the synthesis of Δ⁸-THCV from 5-propylbenzene-1,3-diol (1) and p-menth-2-ene-1,8-diol (2). (B) Left-hand panels: displacement of [³H]CP55940 from specific binding sites in CHO hCB₂ cell membranes. Each symbol represents the mean percent displacement ± SEM (n= 6). Right-hand panels: inhibition of forskolin-induced stimulation of cyclic AMP production in CHO hCB₂ cells. Each symbol represents the mean percent inhibition ± SEM (n= 7). The derived K_i, EC₅₀ and E_max values are given in the Results.

Figure 2

Δ⁸-THCV pretreatment decreases liver I/R injury. (A and B) Serum transaminase ALT (A) and AST (B) levels in sham-operated mice treated with vehicle (veh) or Δ⁸-THCV (n= 5 per group) or in mice exposed to 1 h of hepatic ischaemia followed by 2h (I + 2h R) or 6 h (I + 6h R) of reperfusion pretreated with vehicle, Δ⁸-THCV (3 or 10 mg·kg⁻¹) or SR144528 (SR2, 3 mg·kg⁻¹), SR141716A (SR1, 3 and 10 mg·kg⁻¹) and 10 mg·kg⁻¹Δ⁸-THCV in combination with SR1 or SR2 (n= 6–10 per group). *P < 0.05 versus vehicle; #P < 0.05 versus corresponding mice exposed to I/R; $P < 0.05 versus corresponding mice exposed to I/R and pretreated with 10 mg·kg⁻¹Δ⁸-THCV.

Figure 3

Δ⁸-THCV treatment at the moment of reperfusion decreases liver I/R injury. (A and B) Serum transaminase ALT (A) and AST (B) levels in mice exposed to 1 h of hepatic ischaemia followed by 6 h of reperfusion (I+6h R) treated with vehicle or Δ⁸-THCV (10 mg·kg⁻¹, n= 7–8 per group) at the start of reperfusion. #P < 0.05 versus corresponding mice exposed to I/R.

Figure 4

Δ⁸-THCV decreases histological damage at 24 h following ischaemia. HE staining of representative liver sections of sham mice treated with vehicle (sham) or Δ⁸-THCV (THCV), and mice exposed to 1 h of ischaemia followed by 24 h of reperfusion treated with vehicle (I + 24 h R) or Δ⁸-THCV (THCV + I + 24 h R). Upper row of images depicts 200× magnification, while the lower one 400× magnification. A similar histological profile was seen throughout each group (n = 3–5).

Figure 5

Δ⁸-THCV decreases neutrophil infiltration. MPO staining (brown) of representative liver sections of sham mice treated with vehicle (sham) or Δ⁸-THCV (THCV), and mice exposed to 1 h of ischaemia followed by 24 h of reperfusion treated with vehicle (I + 24 h R) or Δ⁸-THCV (THCV + I + 24 h R). Slides were counterstained by nuclear fast red. Upper row of images depicts 200× magnification, while the lower one 400× magnification. A similar histological profile was seen throughout each group (n = 3–5).

Figure 6

Δ⁸-THCV attenuates the I/R-induced acute pro-inflammatory response in the liver. Real-time PCR shows significant increase of mRNA for the chemokine CCL3 (A), the chemokine CXCL2 (B), the pro-inflammatory cytokine TNF-α (C) and adhesion molecule ICAM-1 (D) at 2 h of reperfusion (I + 2 hR), and a gradual decrease at 6 h (I + 6 hR). Pretreatment with Δ⁸-THCV at 10 mg·kg⁻¹ significantly attenuates the I/R-induced increased levels of these inflammatory markers at the time points of the reperfusion studied (2 and 6 h). Results are mean ± SEM; n= 6–12. *P < 0.05 versus vehicle; #P < 0.05 versus corresponding I/R mice.

Figure 7

Δ⁸-THCV decreases I/R-induced increased oxidative stress. (A) HNE adducts, a marker for lipid peroxidation/oxidative stress, increase with time following I/R injury. 10 mg·kg⁻¹Δ⁸-THCV pretreatment attenuates these increases at the time points of reperfusion studied, 6h (I + 6h R) and 24 h (I+24h R). (B) Oxidative modification of proteins, measured by carbonyl adducts, increases with time following I/R injury. 10 mg·kg⁻¹Δ⁸-THCV pretreatment attenuates these increases at the time points of reperfusion studied, 6h (I + 6h R) and 24 h (I+24h R). Results are mean ± SEM for both panels and n= 8 per group. *P < 0.05 versus vehicle; #P < 0.05 versus corresponding I/R mice.

Figure 8

Δ⁸-THCV attenuates the I/R-induced enhanced hepatic cell death. (A) Changes in hepatic caspase3/7 activity following I/R, and attenuation of the observed increases by pretreatment with 10 mg·kg⁻¹Δ⁸-THCV. Results are mean ± SEM of n= 8 per group. *P < 0.05 versus vehicle; #P < 0.05 versus corresponding I/R mice. (B) Increase of hepatic DNA fragmentation demonstrated following I/R injury and attenuation by pretreatment with 10 mg·kg⁻¹Δ⁸-THCV. Results are mean ± SEM of n= 8 per group. *P < 0.05 versus vehicle; #P < 0.05 versus corresponding I/R mice.