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Comparative Study
. 2008 Feb;324(2):475-83.
doi: 10.1124/jpet.107.131896. Epub 2007 Nov 20.

Regression of fibrosis after chronic stimulation of cannabinoid CB2 receptor in cirrhotic rats

Javier Muñoz-Luque  1 , Josefa Ros, Guillermo Fernández-Varo, Sònia Tugues, Manuel Morales-Ruiz, Carlos E Alvarez, Scott L Friedman, Vicente Arroyo, Wladimiro Jiménez
Affiliations
Comparative Study

Regression of fibrosis after chronic stimulation of cannabinoid CB2 receptor in cirrhotic rats

Javier Muñoz-Luque et al. J Pharmacol Exp Ther. 2008 Feb.

Abstract

Two cannabinoid (CB) receptor subtypes, CB1 and CB2, have been cloned and characterized. Among other activities, receptor activation by cannabinoid ligands may result in pro- or antifibrogenic effects depending on their interaction with CB1 or CB2, respectively. In the current study, we investigated whether selective activation of hepatic CB2 modifies collagen abundance in cirrhotic rats with ascites. mRNA and protein expression of CB receptors in the liver of control and cirrhotic rats was assessed by reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry. The effect of chronically activating the CB2 receptor was investigated in cirrhotic rats with ascites treated daily (9 days) with the CB2 receptor-selective agonist 3-(1,1-dimethylbutyl)-1-deoxy-Delta(8)-tetrahydrocannabinol (JWH-133). At the end of treatment, mean arterial pressure and portal pressure were measured, and liver samples were obtained to evaluate infiltrate of mononuclear cells, hepatic apoptosis, alpha-smooth muscle actin (SMA) expression, collagen content, and matrix metalloproteinase (MMP)-2 abundance in all animals. JWH-133 improved arterial pressure, decreased the inflammatory infiltrate, reduced the number of activated stellate cells, increased apoptosis in nonparenchymal cells located in the margin of the septa, and decreased fibrosis compared with cirrhotic rats treated with vehicle. This was associated with decreased alpha-SMA and collagen I and increased MMP-2 in the hepatic tissue of cirrhotic rats treated with the CB2 agonist compared with untreated cirrhotic animals. Therefore, selective activation of hepatic CB2 receptors significantly reduces hepatic collagen content in rats with pre-existing cirrhosis, thus raising the possibility of using selective CB2 agonists for the treatment of hepatic fibrosis in human cirrhosis.

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Figures

Fig. 1
Fig. 1
RNA expression of cannabinoid receptors in the liver of cirrhotic rats. Reverse transcription-polymerase chain reaction of mRNA for CB1 and CB2 receptors in hepatic tissue of three control and six cirrhotic rats. A, HPRT was amplified as a housekeeping gene. B, densitometric analysis of RT-PCR is shown at the bottom.
Fig. 2
Fig. 2
Protein expression of cannabinoid receptors in the liver of cirrhotic rat. A, Western blot for CB1 (top) and CB2 receptor (bottom) in liver tissue of three control rats and six cirrhotic rats. Eighty micrograms of protein extracts was loaded per lane. B, CB2-positive cells (arrows) was visualized in cirrhotic rats by immunostaining of paraffin liver sections with anti-CB2 antibody (original magnification, 200×).
Fig. 3
Fig. 3
Effect of CB2 receptor activation on infiltrating cells. A, CD68 staining of a representative liver section obtained from a cirrhotic rat with ascites treated with vehicle or receiving JWH-133 (1 mg/kg b.wt. for 9 days). Positive cells were determined by counting the number of CD68-positively stained cells in 16 independent fields per animal. Original magnification, 400×. B, quantitative measurement in all animals (seven nontreated and eight treated cirrhotic rats).
Fig. 4
Fig. 4
Effect of CB2 receptor activation on fibrogenic cells. A, α-SMA staining in hepatic tissue of cirrhotic rats with ascites treated with vehicle or receiving JWH-133 (1 mg/kg b.wt. for 9 days). Original magnification, 100×. Quantification of relative α-SMA-positive area was assessed in 16 fields per animal. B, quantitative measurement in all animals (seven nontreated and eight treated cirrhotic rats).
Fig. 5
Fig. 5
Effect of CB2 receptor activation on apoptosis. A, representative TUNEL assay in hepatic tissue of cirrhotic rats with ascites treated with vehicle or receiving JWH-133 (1 mg/kg b.wt. for 9 days). The number of positive cells was determined by counting the number of positively stained cells in eight independent fields per animal (original magnification, 200×). B, Western blot for activate caspase-3 in liver tissue of cirrhotic rats treated with vehicle or JWH-133 (1 mg/kg b.wt. for 9 days). Eighty micrograms of protein extracts was loaded per lane. Numbers below the panels indicate relative levels based on densitometry.
Fig. 6
Fig. 6
Immunofluorescent localization of activated caspase-3, α-SMA, and CD68 in livers of cirrhotic rats treated with JWH-133. Cell nuclei (blue), activated caspase-3 (green), α-SMA (red), and CD68 (red) fluorescent staining in cirrhotic livers treated with JWH-133 (1 mg/kg b.wt. for 9 days) was performed using 4,6-diamidino-2-phenylindole staining (DAPI) and specific antibodies. Colocalizations of activated caspase-3 with α-SMA (top) or CD68 (bottom) markers are shown in the merge panels (yellow; arrowheads) (n = 3). Original magnification, 400×.
Fig. 7
Fig. 7
Effect of CB2 receptor activation on liver fibrosis. A, Sirius Red staining of a representative liver section obtained from a cirrhotic rats with ascites treated with vehicle (n = 7) or receiving JWH-133 (1 mg/kg b.wt. for 9 days; n = 8). Original magnification, 200×. B, quantification of relative fibrosis area was assessed in 36 fields per animal.
Fig. 8
Fig. 8
Effect of CB2 receptor activation on protein expression of α-SMA, collagen I, and MMP-2. Left, representative Western blot for α-SMA, collagen I, and MMP-2 in the liver tissue of cirrhotic rats with ascites receiving vehicle or chronically treated with JWH-133 (1 mg/kg b.wt. for 9 days). Protein extract (120, 120, and 80 μg) was loaded per lane, respectively. Protein extracts from rat thoracic aorta, skin, and thoracic aorta were used as positive control for α-SMA, collagen I, and MMP-2, respectively. Right, densitometric analysis of all samples (seven nontreated and eight treated cirrhotic rats).
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