1. Antifibrogenic role of the cannabinoid receptor CB2 in the liver
Boris Julien, Pascale Grenard, Fatima Teixeira-Clerc, Jeanne Tran Van Nhieu, Liying Li, Meliha Karsak, Andreas Zimmer, Ariane Mallat, Sophie Lotersztajn 
Gastroenterology 
Volume 128, Issue 3, Pages (March 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
CB2 receptor protein is expressed during chronic liver diseases. (A and B) Representative distribution of CB2 receptor immunostaining on liver tissue sections obtained from human biopsies of (A) normal liver and (B) active cirrhosis (original magnification, ×200). Arrows indicate representative immunostaining of mesenchymal cells within and at the edge of the fibrotic septa. Inset: Negative control staining on active cirrhosis obtained after preadsorption of the anti-CB2 antibody with the CB2 synthetic peptide (original magnification, ×200). (C) Representative double immunostaining for CB2 receptor (brown) and smooth muscle α-actin (red) in cirrhotic liver (original magnification ×630). Arrows indicate cells immunopositive for both CB2 receptor and smooth muscle α-actin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
CB2 receptors are expressed and functional in cultured hepatic myofibroblasts, and their expression is up-regulated during hepatic stellate cell activation. (A) Expression of CB2 receptor mRNA in human hepatic myofibroblasts. RT-PCR for CB2 receptors was performed as described in the Methods section. A band of 337 bp corresponding to the expected size of the CB2 receptor PCR product was identified in cultured human hepatic myofibroblasts (hMF) and human spleen taken as control. RT, reverse transcriptase. (B) Expression of CB2 receptor protein. CB2 receptor protein was detected by immunofluorescence as described in the Methods section (original magnification ×630). No signal was detected when preadsorbing with the corresponding CB2 blocking peptide. (C) THC and JWH-015 enhance GTPγS binding in human hepatic myofibroblasts (hMF) and CHO-CB2 cells. Membranes from human hepatic myofibroblasts or CHO-CB2 cells were assayed for [35S]GTPγS binding assays as described in the Methods section, with varying concentrations of THC. Results (mean ± SEM, n = 7–9) are expressed as percentage of control. Inset shows stimulation of [35S]GTPγS binding by 300 nmol/L THC or JWH-015 in human hepatic myofibroblasts. P < .05 for agonist treatments. (D) CB2 receptor expression increases during activation of hepatic stellate cells. RT-PCR for CB2 receptors and β2 microglobulin was performed as described in the Methods section on RNA prepared from HSC cultured for either 4 days (D4, smooth muscle α-actin negative) or 12 days (D12, smooth muscle α-actin positive) after isolation. A band of 409 bp corresponding to the expected size of the rat CB2 receptor PCR product was identified in activated hepatic stellate cells and in rat testis taken as control (not shown).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Cannabinoids inhibit DNA synthesis in human hepatic myofibroblasts via a CB2 receptor-dependent pathway. (A) Effects of cannabinoids on DNA synthesis. Cells were stimulated for 30 hours with varying concentrations of THC, JWH-015, and ACEA in the presence of 20 ng/mL of PDGF-BB. [3H]Thymidine incorporation into DNA was measured as described in the Methods section. Results (mean ± SEM, n = 3–6) are expressed as percentage of control. P < .05 for THC and JWH-015 vs. control. Inset: Effects of THC on human hepatic myofibroblast viability. Cells were stimulated for 30 hours with the indicated concentrations of THC and JWH-015 in the presence of 20 ng/mL of PDGF-BB. Cell viability was determined as described in the Methods section. (B) Inhibition of DNA synthesis by THC is blocked by a CB2 receptor antagonist and is unaffected by a CB1 receptor antagonist. Cells were pretreated for 1 hour with either 1 μmol/L of the CB2 receptor antagonist SR , 1 μmol/L of the CB1 receptor antagonist SR A, or vehicle. DNA synthesis was then measured as described in (A). Results represent the mean ± SEM of 9 determinations obtained from 3 experiments. P < .05 for THC vs. vehicle and for THC + SR vs. THC. (C) The endocannabinoids methanandamide and 2-arachidonoylglycerol inhibit DNA synthesis by a CB2-independent mechanism. Cells were pretreated for 1 hour with 1 μmol/L of the CB2 receptor antagonist SR prior to stimulation for 30 hours with varying concentrations of methanandamide (met-AEA) or 2-arachidonoylglycerol (2-AG) in the presence of 20 ng/mL of PDGF-BB. DNA synthesis was then measured as described in panel A. Results represent data obtained from a typical experiment repeated twice and are the mean ± SEM of triplicate determinations. P < .05 for met-AEA or 2-AG vs. vehicle. PDGF-BB induced a 4.3-, 4.0-, and 4.5-fold increase over basal levels in vehicle, SR A-, and SR treated cells, respectively.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Cannabinoids trigger apoptosis of human hepatic myofibroblasts. (A) Phase-contrast analysis (original magnification ×100). Cells were incubated for 8 hours with 4 μmol/L THC. (B) DAPI staining of the nuclei (original magnification ×630). Cells were incubated for 8 hours with 4 μmol/L THC or vehicle. (C) Caspase-3-like activity. Caspase-3-like activity was assayed on lysates obtained from cells treated for various periods of time with 4 μmol/L THC or vehicle, as described in the Methods section. (D) DNA ladder formation. Cells were incubated for 16 hours with either 4 μmol/L THC or vehicle. DNA was extracted, and 2 μg per condition were analyzed by electrophoresis on a 1.8% agarose gel stained with SYBR Green I.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Cannabinoids trigger apoptosis via a CB2 receptor-dependent pathway in hepatic myofibroblasts and in activated hepatic stellate cells. (A) Effects of THC and CB1 or CB2 receptor selective agonists on hepatic myofibroblast and activated hepatic stellate cell viability. Human hepatic myofibroblasts were incubated with varying concentrations of THC, JWH-015, and ACEA for 16 hours. Cell viability (mean ± SEM, n = 3–6) was determined as described in the Methods section. P < .05 compared with vehicle. Inset shows the cytotoxic effects of 5 μmol/L THC and JWH-015 on rat activated hepatic stellate cells cultured for 12 days after isolation. (B) Apoptotic effects of THC are blunted by a CB2 receptor antagonist and unaffected by a CB1 receptor antagonist. Cells were preincubated for 1 hour with 1 μmol/L SR , 1 μmol/L SR A, or vehicle and further incubated with varying concentrations of THC for 16 hours. Cell viability (mean ± SEM, n = 6) was determined as described in the Methods section. P < .05 for THC vs. vehicle and for THC + SR vs. THC. SR and SR A alone had no effect on cell viability. (C) The endocannabinoids methanandamide and 2-arachidonoylglycerol are cytotoxic by a CB2-independent mechanism. Cells were pretreated for 1 hour with 1 μmol/L of the CB2 receptor antagonist SR and further incubated with varying concentrations of methanandamide (met-AEA) or 2-arachidonoylglycerol (2-AG). Cell viability (mean ± SEM, n = 3) was determined as described in A. P < .05 for met-AEA or 2-AG vs. vehicle.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Growth inhibitory and apoptotic effects of THC are mediated by 2 distinct signaling pathways. (A) COX-2 mediates inhibition of human hepatic myofibroblast proliferation by THC. Cells were pretreated for 1 hour with 10 μmol/L NS-398 or vehicle, further stimulated with 20 ng/mL PDGF-BB and varying concentrations of THC, and DNA synthesis was measured as in Figure 3. Results (mean ± SEM, n = 3–5) are expressed as percentage of respective control (17,000 ± 6000 cpm for PDGF-BB; 34,000 ± 12,000 cpm for PDGF-BB + NS-398). P < .05 for NS-398 effects. Inset: Cells were pretreated for 1 hour with 5 mmol/L NAC, 25 μmol/L EUK8, or vehicle and further stimulated with 20 ng/mL PDGF-BB and 750 nmol/L THC. (B) THC induces COX-2 and stimulates COX activity in human hepatic myofibroblasts. Western blot analysis of COX-2 protein induction in extracts of cells were treated with varying concentrations of THC for 8 hours (n = 3). A typical blot is shown. Results were normalized relative to β-actin expression. COX activity was assayed after 8-hour incubation with 1000 nmol/L THC. At the end of incubation, 10 μmol/L of arachidonic acid was added for 30 minutes, and PGE2 released in the supernatants was measured as described in the Methods section. Results are the mean of sextuplate determinations obtained from 2 experiments (#P < .05 vs. control). (C) THC-induced apoptosis involves ROS production. Cells were pretreated for 1 hour with 10 μmol/L NS-398, 5 mmol/L NAC, 25 μmol/L EUK8, or vehicle. Caspase 3-like activity was assayed after a 5-hour treatment with 4 μmol/L THC or vehicle. (mean ± SEM, n = 3; #P < .05 vs. THC). Antioxidants added alone had no effect. Maximal increase of caspase-3 activity by THC was 6.3- ± 2-fold (100%). (D) Effect of THC on ROS production. DCF fluorescence was measured in the presence of varying concentrations of THC or vehicle, as described in the Methods sections. Mean ± SEM, n = 6, P < .05 for THC vs. basal.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
Enhanced liver fibrosis in CB2 knockout mice after chronic intoxication with carbon tetrachloride. (A–D) Representative picro-sirius red staining of liver sections (original magnification ×100). (A) Olive oil-treated WT mice; (B) olive oil-treated CB2−/− mice; (C) CCl4-treated WT mice; and (D) CCl4-treated CB2−/− mice. (E) Score of fibrosis; each point represents the mean ± SEM. *P < .05 vs. WT group. (F) Hepatic hydroxyproline content. Each point represents the mean ± SEM of 3 animals in the olive oil-treated WT group or olive oil-treated CB2−/− group and of 5 animals in the CCl4-treated WT group or CCl4-treated CB2−/− group. *P < .05 vs. olive oil group, #P < .05 vs. WT CCl4-treated group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions