[Epub ahead of print]

Functional relevance of the cannabinoid receptor 2 – heme oxygenase pathway: A novel target for the attenuation of portal hypertension.

Steib CJ, Gmelin L, Pfeiler S, Schewe J, Brand S, Göke B, Gerbes AL.

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Department of Medicine II (Gastroenterology and Hepatology), Liver Center Munich, University of Munich, Grosshadern, Munich, Germany. Electronic address: christian.steib@med.uni-muenchen.de.

Abstract

AIMS:

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of thecannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

MAIN METHODS:

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10mg/kg b.w.), GP 1a (2.5mg/kg b.w.) or ZnPP IX (1μM). Isolated KC were treated with Zymosan (0.5mg/ml) in addition to JWH-133 (5μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western Blot or confocal microscopy.

KEY FINDINGS:

JWH-133 or GP 1a pretreatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

SIGNIFICANCE:

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.
© 2013.

KEYWORDS:

(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran, 2-Chloro-5-nitro-N-phenylbenzamide, BDL, CB(2), GW 9662, Gp 1a, HO, JWH-133, KC, Kupffer cell, LDH, LPS, N-(Piperidin-1-yl)-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide, TAA, TLR, TX, Zy, Zymosan A, b. w, bile duct ligation, bile duct ligation (BDL), body weight, cannabinoid receptor 2, heme oxygenase, heme oxygenase (HO), lactate dehydrogenase, lipopolysaccharide, thioacetamide, thioacetamide (TAA), thromboxane, toll like receptor

PMID:

24007798
[PubMed – as supplied by publisher]

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Research Support, Non-U.S. Gov’t

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Figures and tables from this article:

: Fig. 1.

Treatment with the cannabinoid receptor 2 agonists JWH-133 and GP 1a lower the basal portal pressure and attenuate the LPS-mediated increase in portal pressure in rat liver cirrhosis. Data are expressed as the means ± S.D. Animals underwent BDL for 28 days. (A) Control group (solvent, n = 5) was compared with the treatment group (JWH-133 for 2 h, 10 mg/kg b. w. intraperitoneally, n = 5). JWH-133 pre-treatment lowered the basal portal pressure (p_min) and the maximal portal pressure (p_max) following KC activation with LPS (LPS from E. coli 026:B6, 4 mg/kg b.w., 100 μl/min over 6 min) (*p < 0.05). (B) Pre-treatment with GP 1a (2.5 mg/kg b. w. intraperitoneally for 2 h, n = 5) reduced the basal portal pressure (p_min) and the maximal portal pressure (p_max) following LPS infusion (LPS from E. coli 026:B6, 4 mg/kg b.w., 100 μl/min over 6 min, n = 5) in comparison to control group (n = 5)(*p < 0.05).; Figure options

: Fig. 2.

Treatment with the cannabinoid receptor 2 agonists JWH-133 results in the increased expression of heme oxygenase 1 and concurrently attenuated portal perfusion pressure increase. Data are expressed as the means ± S.D. (A) Western blot analyses showed that in whole liver lysates, the expression of HO-1 increased by pre-treatment with JWH-133 (10 mg/kg b. w. for 2 h intraperitoneal) in BDL-induced liver fibrosis. Shown are representative Western blots. The densitometric data are additionally shown (*p < 0.05). (B) Expression of cannabinoid 2 receptor was not influenced by JWH-133 pre-treatment of the animals as indicated. The densitometric data are additionally shown beside. (C) KC activation with Zymosan A (Zymosan, 150 μg/ml, minute 40 to 46, n = 5) increased the portal perfusion pressure (maximal portal perfusion pressure and at end of isolated liver perfusion) (*p < 0.05) when compared to basal portal perfusion pressure (p_min) in bile duct-ligated animals. This increase was attenuated by pre-treatment with the CB₂receptor agonist JWH-133 (JWH-133 for 2 h, 10 mg/kg b. w. intraperitoneally, n = 5)(**p < 0.05).; Figure options

: Fig. 3.

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway following Kupffer cell activation. Data are expressed as the means ± S.D. (A) KC activation with Zymosan A (Zymosan, 150 μg/ml, minute 40 to 46, n = 5) increased the portal perfusion pressure (maximal portal perfusion pressure and at end of isolated liver perfusion) compared to basal portal perfusion pressure (pmin) (*p < 0.05) 28 days after bile duct ligation. KC activation with Zymosan A (Zymosan, 150 μg/ml, minute 40 to 46, n = 5) was combined with inhibition of heme oxygenase 1 (HO-1) by ZnPP IX (1 μM, minute 30 to 60, n = 4). Hereby portal perfusion pressure increase was further enhanced (*p < 0.05) when compared to KC activation by Zymosan (Zymosan, 150 μg/ml, minute 40 to 46, n = 5) in bile duct-ligated animals. (B) Absolute increase in portal perfusion pressure was also enhanced (*p < 0.05) following additional treatment with ZnPP (1 μM, minute 30 to 60, n = 4) compared to KC activation by Zymosan A alone (Zymosan, 150 μg/ml, minute 40 to 46, n = 5). (C) The increase in portal perfusion pressure with Zymosan A (Zymosan, 150 μg/ml, minute 40 to 46, n = 5) either in combination with ZnPP IX (1 μM, minute 30 to 60, n = 4) or alone, was paralleled by the efflux of TXB₂, as determined by ELISA. The TXB₂ efflux increased with KC activation by Zymosan A compared to basal values (*p < 0.05). The TXB₂ efflux was further increased by additional inhibition of HO-1 (**p < 0.05), indicating the functional relevance of HO-1.; Figure options

: Fig. 4.

A potential link between CB₂ receptor and HO-1: PPARγ. Data are expressed as the means ± S.D. (A) KC activation with Zymosan A (Zymosan, 150 μg/ml, minute 40 to 46, n = 5) increased the portal perfusion pressure (maximal portal perfusion pressure and at end of isolated liver perfusion) (*p < 0.05) when compared to basal portal perfusion pressure (p_min) in bile duct-ligated animals. JWH-133 (JWH-133 for 2 h, 10 mg/kg b. w. intraperitoneally, n = 5) pre-treatment attenuated this portal perfusion pressure increase (**p < 0.05). Additional pre-tretament with GW 9662 (5 mg/kg b.w., intraperitoneally for 2 h) counteracted the JWH-induced decrease of maximal portal perfusion pressure and at end of perfusion concomitant treatment of JWH-133 plus GW 9662 increased portal perfusion pressure (#p < 0.05) compared to pre-treatment with JWH-133 alone. (B) Western blot analyses of whole liver homogenates showed that additional pre-treatment with the PPARγ inhibitor GW 9662 (5 mg/kg b.w.) counteracted increase of HO-1 expression following JWH-133 pre-treatment (10 mg/kg b.w.)(compare to Fig. 2A). The densitometric data are additionally shown.; Figure options


: Fig. 5.

Involved pathways in isolated Kupffer cells: TXB₂ production influenced by the CB₂ receptor agonist and HO-1. Data are expressed as the means ± S.D. (A) KC were isolated as indicated. Confocal microscopy showed only slight expression of the CB₂ receptor in control cells. Expression of the CB₂ receptor increased with TLR stimulation by LPS (3 mg/ml) or Zymosan A (0.5 mg/ml). In addition, stimulation by the CB₂ receptor agonist JWH-133 (5 μM) resulted in the enhanced expression of the CB₂ receptor. Co-stimulation by JWH-133 (5 μM) and LPS (3 mg/ml) or Zymosan A (0.5 mg/ml) did not further enhance CB₂ receptor expression. (B) Confocal microcopy of isolated KC showed only slight expression of HO-1 in control cells. TLR stimulation by LPS (3 mg/ml) or Zymosan A (0.5 mg/ml) increased HO-1 expression. As observed in whole liver lysates, HO-1 expression was also enhanced by JWH-133 (5 μM) treatment for CB₂ receptor agonism in isolated KC cells. Co-stimulation with JWH-133 (5 μM) and LPS (3 mg/ml) or Zymosan A (0.5 mg/ml) did not further enhance the expression of HO-1. (C) Isolated KC from normal livers (pooled from n = 4 animals) were treated with JWH-133 (5 μM) for 2 h, resulting in an increase in the expression of HO-1 in KC lysates. Representative Western blots are shown and additionally the densitometric data (*p < 0.05). (D) The activation of KC with Zymosan A for 1 h (0.5 mg/ml) resulted in a significant increase (*p < 0.05) in the TXB₂ efflux into the media, compared to unstimulated cells. Additional pre-incubation with JWH-133 (5 μM, 2 h) reduced the TXB₂ efflux into the media (**p < 0.05).; Figure options

: Corresponding author at: Department of Medicine II, Liver Center Munich (LCM), Hospital of the Ludwig-Maximilians University, Campus Grosshadern, Marchioninistrasse 15, 81377 Munich, Germany. Tel.: + 49 89 7095 3176; fax: + 49 89 7095 2392.

1: These authors contributed equally to this manuscript.

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Functional relevance of the cannabinoid receptor 2 – heme oxygenase pathway: A novel target for the attenuation of portal hypertension.

Functional relevance of the cannabinoid receptor 2 – heme oxygenase pathway: A novel target for the attenuation of portal hypertension.

Source

Abstract

AIMS:

MAIN METHODS:

KEY FINDINGS:

SIGNIFICANCE:

KEYWORDS:

Publication Types, MeSH Terms, Substances

Publication Types

MeSH Terms

Substances

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