PMC

Coagulation necrosis (A), neutrophil infiltration (B), and liver damage markers ALT and AST (C) are reduced in hepatic I/R even when JZL184 is administered at 3 h of reperfusion. All parameters were measured at 24 h of reperfusion (24h I/R). Data represent mean±SEM of n=6 mice/group. *p<0.05 versus vehicle-treated I/R group.

Liver damage/necrosis markers ALT and AST are significantly elevated in mouse serum upon treatment with GalN/LPS for 7 h (A, lower panels) or CCl4 for 24 h (B, lower panels), and JZL184 (20 mg/kg, i.p.) pretreatment substantially reduces these elevated enzyme levels. Liver histology (H&E staining, original magnification 40×) showing representative images of liver tissue damage induced by GalN/LPS (A, upper panels) or CCl4 (B, upper panels), which is significantly attenuated upon MAGL inhibition by JZL184. *p<0.05 versus vehicle-treated control groups.

(A, B) Pharmacological (A) and genetic (B) inactivation of MAGL enhances 2-AG levels but does not affect the levels of the other endocannabinoid anandamide in the liver. MAGL blockade also lowers AA and eicosanoids in the I/R livers (A, B), measured after 6 h of reperfusion (peak injury). The potent and selective MAGL inhibitor JZL184 (40 mg/kg, i.p.) was given 1 h prior to inducing ischemia in the liver and endocannabinoid and eicosanoid levels were measured after 6 h of reperfusion after ischemia by SRM-based LC-MS/MS analysis. Data represent mean±sem of n=4–5 mice/group. Significance is represented as *p<0.05 between all groups versus sham vehicle-treated groups or sham Mgll^+/+ groups; #p<0.05 between JZL184-treated or Mgll^−/− I/R groups and the I/R vehicle-treated or Mgll^+/+ groups.

(A) Both pharmacological and genetic blockade of MAGL causes massive delayed infiltration of neutrophils as assessed by MPO staining (brown staining) of livers after 24 h of reperfusion following induction of 1 h hepatic ischemia (I/R 24h). Representative images are shown. This neutrophil infiltration is significantly attenuated upon JZL184-treatment (40 mg/kg, i.p.) prior to ischemia or in Mgll^−/− mice. (B) The I/R-induced early elevations in pro-inflammatory cytokines, chemokines TNF-α, IL-1β, MIP1-α, and MIP-2, and adhesion molecule ICAM-1 assessed after 2 h of reperfusion (I/R 2h) as well as the late oxidative stress markers NOX2 and HNE assessed after 24 h of reperfusion (I/R 24h), are significantly attenuated upon JZL184 treatment (40 mg/kg, i.p., prior to induction of ischemia) as well as in Mgll^−/− I/R groups (C). Data represent mean±SEM of n=6–12 mice/group. Significance is represented as *p<0.05 between the indicated groups and the sham surgery vehicle-treated groups or sham Mgll^+/+ groups; #p<0.05 versus the corresponding I/R vehicle-treated groups or Mgll^+/+ groups.

(A) Liver damage/necrosis markers ALT and AST are significantly elevated in mouse plasma upon I/R induction 2, 6, and 24 h after reperfusion, and both pharmacological (JZL184, 40 mg/kg, i.p.) and genetic (Mgll^−/−) inactivation of MAGL substantially reduces these elevated levels. (B) Liver histology (H&E staining) of mice subjected to sham surgery or 1 h ischemia followed by 24 h of reperfusion (I/R 24h) in vehicle versus JZL184-treated (40 mg/kg, i.p., given prior to induction of ischemia) or in Mgll^+/+ versus Mgll^−/− mice, showing representative images of coagulation necrosis (lighter areas) subjected to I/R that is significantly attenuated upon pharmacological or genetic ablation of MAGL. (C) MAGL inhibition attenuates I/R-induced delayed cell death markers (caspase 3/7 activity, DNA fragmentation, PARP activity) after 24 h of reperfusion (I/R 24h). Data represent mean±sem of n=6–9 mice/group for (A) and 5–11 for (C). Significance is represented as *p<0.05 between vehicle-treated I/R groups and sham vehicle-treated groups, and #p<0.05 between JZL184-treated or Mgll^−/− I/R groups and the corresponding I/R vehicle-treated or Mgll^+/+ groups in (A); *p<0.05 between vehicle-treated I/R group and the sham groups, and #p<0.05 between JZL184 treated I/R groups and vehicle-treated I/R groups in (C).

(A, B) The effect of the CB1 and CB2 receptor antagonists SR1 (A) and SR2 (B) on ALT and AST levels after 6h of reperfusion (I/R 6h), respectively. SR1 (3 mg/kg, i.p.) reduces ALT and AST levels in I/R mice and this effect is additive when given in combination with JZL184 mice. SR2 (3 mg/kg, i.p.) partially reverses the JZL184-induced reductions in ALT and AST levels. (C, D) Pharmacological blockade of MAGL (JZL184, 40 mg/kg, i.p.) after 6 h of reperfusion (I/R 6h) exerts further decreases or attenuation of ALT and ASTlevels in Cnr1^−/− (C) and Cnr2^−/−(D) mice, respectively. (E) Liver histology (H&E staining) of Cnr2^+/+or Cnr2^−/− mice subjected to 1 h ischemia followed by 6 h of reperfusion (I/R 6h) or sham surgery in vehicle versus JZL184-treated (40 mg/kg, i.p., given prior to induction of ischemia) mice, showing an attenuated hepatoprotective response to JZL184 in Cnr2^−/− I/R mice compared to JZL184-treated Cnr2^+/+ mice. Data represent mean±sem of n=6–12 mice/group. Significance is represented as *p<0.05 between the indicated groups and vehicle-treated I/R group (A and B) or vehicle-treated Cnr1^+/+or Cnr2^+/+ I/R groups (C and D), and #p<0.05 between SR1 or SR2-treated JZL184-treated I/R groups (A and B) or JZL184-treated Cnr1^−/−or Cnr2^−/− I/R groups (C and D) versus the corresponding JZL184-treated I/R groups or vehicle-treated Cnr1^−/−or Cnr2^−/− I/R groups, respectively.