Beneficial paracrine effects of cannabinoid receptor 2 on liver injury and regeneration

Abstract

The cannabinoid receptor 2 (CB2) plays a pleiotropic role in innate immunity and is a crucial mediator of liver disease. In this study, we investigated the impact of CB2 receptors on the regenerative process associated with liver injury. Following acute hepatitis induced by carbon tetrachloride (CCl(4)), CB2 was induced in the nonparenchymal cell fraction and remained undetectable in hepatocytes. Administration of CCl(4) to CB2(-/-) mice accelerated liver injury, as shown by increased alanine/aspartate aminotransferase levels and hepatocyte apoptosis, and delayed liver regeneration, as reflected by a retarded induction of hepatocyte proliferating cell nuclear antigen expression; proliferating cell nuclear antigen induction was also delayed in CB2(-/-) mice undergoing partial hepatectomy. Conversely, following treatment with the CB2 agonist JWH-133, CCl(4)-treated WT mice displayed reduced liver injury and accelerated liver regeneration. The CCl(4)-treated CB2(-/-) mice showed a decrease in inducible nitric oxide synthase and tumor necrosis factor-alpha expression, and administration of the nitric oxide donor moldomine (SIN-1) to these animals reduced hepatocyte apoptosis, without affecting liver regeneration. Impaired liver regeneration was consecutive to an interleukin-6 (IL-6)-mediated decrease in matrix metalloproteinase 2 (MMP-2) activity. Indeed, CCl(4)-treated CB2(-/-) mice displayed lower levels of hepatic IL-6 messenger RNA and increased MMP-2 activity. Administration of IL-6 to these mice decreased MMP-2 activity and improved liver regeneration, without affecting hepatocyte apoptosis. Accordingly, administration of the MMP inhibitor CTTHWGFTLC to CCl(4)-treated CB2(-/-) mice improved liver regeneration. Finally, in vitro studies demonstrated that incubation of hepatic myofibroblasts with JWH-133 increased tumor necrosis factor-alpha and IL-6 and decreased MMP-2 expressions.

Conclusion: CB2 receptors reduce liver injury and promote liver regeneration following acute insult, via distinct paracrine mechanisms involving hepatic myofibroblasts. These results suggest that CB2 agonists display potent hepatoprotective properties, in addition to their antifibrogenic effects.

Figure 1. Expression of CB2 receptor mRNA following CCl_4-induced liver injury

(A) RT-PCR analysis of hepatic CB2 mRNA expression. (B) CB2 receptor mRNA expression in the total liver, parenchymal and non parenchymal liver cell fractions at 24 h following CCl₄ or MO administration. (C) RT-PCR analysis of hepatic F4/80, CCR2, MPO and α-SMA mRNA expression following CCl₄ administration. #P<0.05 for CCl₄ vs MO.

Figure 2. Hepatoprotective properties of CB2 receptors

(A) Representative TUNEL staining and kinetics of mean TUNEL staining (*P<0.05 for CB2^−/− vs WT mice; # P<0.05 for CCl₄ vs MO). (B) ALT and AST levels. (C) Representative TUNEL staining of WT mice treated with JWH-133 or vehicle and sacrified 24 h after CCl₄ administration.*P<0.05 for JWH-133 vs vehicle. (D) ALT and AST levels. (E) RT-PCR analysis of hepatic F4/80 and MPO expressions. #P<0.05 for CCl₄ vs MO. Representative F4/80 and MPO immunostaining of CCl₄-treated CB2^−/− and WT liver tissue sections.

Figure 3. Beneficial effects of CB2 receptor on liver regeneration

Representative western blot of (A) hepatic cyclin D1 expression and (B) hepatic PCNA expression after CCl₄ injection. Immunohistochemical detection of PCNA in hepatocyte nuclei from WT and CB2^−/− mice 48 h after CCl₄ injection, and mean PCNA-positive cells. *P<0.05 for CB2^−/− vs WT mice; # P<0.05 for CCl₄ vs MO; (C) Representative western blot of hepatic PCNA expression in mice treated with JWH-133 or vehicle.*P<0.05 for JWH-133 vs vehicle; # P<0.05 for CCl₄ vs MO. (D) RT-PCR analysis of hepatic CB2 mRNA expression following partial hepatectomy; #P<0.05 for hepatectomized vs control mice. (E) Representative western blot of hepatic PCNA expression after partial hepatectomy. Immunohistochemical detection of PCNA in nuclei of hepatocytes 48 h after partial hepatectomy and mean PCNA-positive cells. *P<0.05 for WT vs CB2^−/− mice.

Figure 4. Comparison of the expression profile of survival and mitogenic factors between CCl₄-treated CB2^−/− mice and WT counterparts

(A) RT-PCR analysis of iNOS TNF-α and IL-6 mRNA (B) MCP-1, IL-10, TLR4 mRNA following CCl₄ administration. *P<0.05 for CB2^−/− vs WT mice; # P<0.05 for CCl₄ vs MO.

Figure 5. Effect of the NO donor SIN-1 on TUNEL staining and cyclin D1 expression in CCl₄–treated CB2^−/− and WT mice

Mice were injected with SIN-1 or vehicle and sacrified 24 h after CCl₄ injection. (A) Representative TUNEL staining and mean TUNEL-positive area. (B) Representative immunoblot and mean densitometric analysis of cyclin D1 expression. *P<0.05 for SIN-1 vs vehicle.

Figure 6. Effect of IL-6 administration to CCl₄–treated CB2^−/− and WT mice on TUNEL staining and PCNA expression

(A) Representative TUNEL staining and mean TUNEL-positive area 24 h after CCl₄ treatment *P<0.05 vs vehicle-injected WT mice administered CCl₄. (B) Representative immunoblot and mean densitometric analysis of PCNA expression 48 h after CCl₄ exposure. *P<0.05 for IL-6 vs vehicle.

Figure 7. CB2 receptor inactivation delays liver regeneration by a mechanism involving enhanced MMP-2 activity

Gelatin zymography showing (A) MMP-9 and MMP-2 activities and (B) MMP-2 activities of liver homogenates from WT and CB2^−/− mice injected with IL-6 or vehicle and sacrified 24 h after CCl₄ administration. (C) Representative western blot of hepatic cyclin D1 expression 24 h after CCl₄ injection, following CTT or vehicle treatment. *P<0.05 for CTT vs vehicle.

Figure 8. Consequences of CB2 receptor activation in cultured macrophages and hepatic myofibroblasts

(A) RT-PCR analysis of TNF-α, IL-6 and MMP-2 mRNA on human hepatic myofibroblasts incubated with 5 μM JWH-133 or vehicle for 6 or 24 h. *P<0.05 for JWH-133 vs vehicle. (B) RT-PCR analysis of TNF-α and IL-6 mRNA on bone marrow-derived macrophages incubated with 5 μM JWH-133 or vehicle for 7 h. *P<0.05 for JWH-133 vs vehicle.