Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice

Abstract

Background & aims: The endocannabinoid and eicosanoid lipid signaling pathways have important roles in inflammatory syndromes. Monoacylglycerol lipase (MAGL) links these pathways, hydrolyzing the endocannabinoid 2-arachidonoylglycerol to generate the arachidonic acid precursor pool for prostaglandin production. We investigated whether blocking MAGL protects against inflammation and damage from hepatic ischemia/reperfusion (I/R) and other insults.

Methods: We analyzed the effects of hepatic I/R in mice given the selective MAGL inhibitor JZL184, in Mgll(-/-) mice, fatty acid amide hydrolase(-/-) mice, and in cannabinoid receptor type 1(-/-) (CB1-/-) and cannabinoid receptor type 2(-/-) (CB2-/-). Liver tissues were collected and analyzed, along with cultured hepatocytes and Kupffer cells. We measured endocannabinoids, eicosanoids, and markers of inflammation, oxidative stress, and cell death using molecular biology, biochemistry, and mass spectrometry analyses.

Results: Wild-type mice given JZL184 and Mgll(-/-) mice were protected from hepatic I/R injury by a mechanism that involved increased endocannabinoid signaling via CB2 and reduced production of eicosanoids in the liver. JZL184 suppressed the inflammation and oxidative stress that mediate hepatic I/R injury. Hepatocytes were the major source of hepatic MAGL activity and endocannabinoid and eicosanoid production. JZL184 also protected from induction of liver injury by D-(+)-galactosamine and lipopolysaccharides or CCl4.

Conclusions: MAGL modulates hepatic injury via endocannabinoid and eicosanoid signaling; blockade of this pathway protects mice from liver injury. MAGL inhibitors might be developed to treat conditions that expose the liver to oxidative stress and inflammatory damage.

Figure 1. MAGL inactivation exerts bidirection al control over endocannabinoid and eicosanoid levels during hepatic I/R

(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.

Figure 2. MAGL inactivation attenuates hepatic I/R-induced tissue injury

(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).

Figure 3. MAGL inactivation attenuates hepatic I/R-induced inflammation and oxidative stress

(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.

Figure 4. Hepatoprotective effects conferred by MAGL are partially due to CB2, but not CB1 signaling

(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.

Figure 5. MAGL inhibition exerts hepatoprotection even when given after reperfusion

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.

Figure 6. MAGL inactivation by JZL184 attenuates liver damage in acute murine liver injury models induced by GalN/LPS or CCl4

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.