1. Introduction
2. Results
2.1. Endocannabinoid (EC) Levels Are Increased in Plasma of Patients with Chronic Hepatitis C (CHC)


Anandamide | ||||||
Parameter | Unadjusted Coefficients | 95% CI | p-Value | Adjusted Coefficients | 95% CI | p-Value |
Age | −0.00066 | −0.009–0.007 | 0.87 | −0.0036 | 0.013–0.0057 | 0.45 |
Female | 0.075 | −0.08–0.23 | 0.35 | 0.14 | −0.04–0.33 | 0.13 |
BMI | 0.011 | −0.009–0.032 | 0.27 | 0.018 | −0.0045–0.041 | 0.12 |
Diabetes | 0.16 | −0.12–0.43 | 0.26 | 0.14 | −0.16–0.45 | 0.35 |
Alcohol | 0.046 | −0.18–0.27 | 0.69 | 0.056 | −0.21–0.32 | 0.68 |
ALT | 0.0006 | −0.0003–0.002 | 0.20 | 0.001 | −0.0005–0.0026 | 0.18 |
AST | 0.00064 | −0.0007–0.002 | 0.35 | −0.0003 | −0.0025–0.0019 | 0.81 |
Fibrosis grade (0–2 vs. 3/4) | −0.30 | −0.20–0.24 | 0.73 | −0.09 | −0.28–0.10 | 0.36 |
Fibrosis grade cont. | −0.0034 | −0.063–0.056 | 0.91 | |||
Genotype (1 vs. 2–4) | 0.087 | −0.071–0.25 | 0.28 | |||
Activity grade (1 vs. 2, 1 vs. 3) | 0.082 −0.27 | −0.16–0.33 −0.98–0.43 | 0.51 0.45 | |||
Steatosis | −0.092 | −0.33–0.14 | 0.44 | |||
2-AG | 0.012 | −0.069–0.092 | 0.78 | |||
2-AG | ||||||
Parameter | Unadjusted Coefficients | 95% CI | p-Value | Adjusted Coefficients | 95% CI | p-Value |
Age | 0.033 | 0.009–0.058 | 0.007 | 0.043 | 0.014–0.072 | 0.004 |
Female | −0.069 | −0.59–0.45 | 0.80 | −0.14 | −0.72–0.44 | 0.64 |
BMI | 0.018 | −0.049–0.085 | 0.59 | 0.029 | −0.042–0.10 | 0.42 |
Diabetes | −0.15 | −1.05–0.75 | 0.75 | −0.54 | −1.49–0.41 | 0.27 |
Alcohol | −0.33 | −1.06–0.40 | 0.38 | −0.07 | −0.91–0.77 | 0.87 |
ALT | 0.003 | −0.002–0.007 | 0.23 | −0.00008 | −0.005–0.005 | 0.97 |
AST | 0.002 | −0.0009–0.005 | 0.17 | 0.0027 | −0.004–0.01 | 0.44 |
Fibrosis grade (0–2 vs. 3/4) | −0.03 | −0.58–0.51 | 0.90 | −0.25 | −0.85–0.35 | 0.41 |
Fibrosis grade cont. | 0.009 | −0.18–0.20 | 0.93 | |||
Genotype (1 vs. 2–4) | 0.087 | −0.071–0.25 | 0.28 | |||
Activity grade (1 vs. 2, 1 vs. 3) | 0.15 −0.43 | −0.56–0.86 −2.50–1.63 | 0.69 0.68 | |||
Steatosis | −0.52 | −1.21–0.18 | 0.14 |
2.2. Activities of EC Degradation Enzymes Are not Affected in CHC Patients
2.3. 2-AG Has a Major Impact on Expression of Components of the EC System in PBMC


2.4. ECs Suppress Inflammatory Cytokine Production in PBMC


2.5. ECs Reduce Inflammatory Response from HCV-Activated PBMC

2.6. 2-AG Induces Inflammatory Cytokine Expression in Primary Hepatocytes and Fibrogenic Activation of HSC

3. Discussion
4. Material and Methods
4.1. Human Tissues and Patients’ Characteristics
Control | HCV | |
---|---|---|
Number of patients | 25 | 57 |
Female (n, %) | 15 (60) | 22 (38.6) |
Age (mean, SD) | 44.2 (11.4) | 52.7 (10.0) |
Diabetes (n, %) | 0 | 5 (8.8) |
BMI (mean, SD) | NA | 24.7 (3.8) |
ALT (mean, SD) | NA | 115.8 (84.4) |
AST (mean, SD) | NA | 91.1 (58.8) |
INR (mean, SD) | NA | NA |
Albumin (mean, SD) | NA | NA |
CHILD | ||
A | 14 (24.6) | |
B | 2 (3.5) | |
C | 0 | |
Unknown/NA | 25 (100.0) | 41 (71.9) |
Fibrosis stage | ||
0 | 4 (7.0) | |
1 | 21 (36.8) | |
2 | 11 (19.3) | |
3 | 5 (8.8) | |
4 | 15 (26.3) | |
Unknown/NA | 25 (100.0) | 1 (1.8) |
Activity grade | ||
19 (33.3) | ||
14 (24.6) | ||
1 (1.8) | ||
Unknown/NA | 25 (100.0) | 23 (40.4) |
Steatosis | ||
No | 15 (26.3) | |
Yes | 16 (28.1) | |
Unknown | 25 (100.0) | 26 (45.6) |
Genotype | ||
1 | 2 (3.5) | |
1A | 11 (19.3) | |
1B | 18 (31.6) | |
2 | 8 (14.0) | |
3 | 14 (24.6) | |
4 | 3 (5.3) | |
Negative | 1 (1.8) | |
Unknown/NA | 100 (100.0) | 0 (0.0) |
Viral load (mean/SD) Missing | NA | 2,182,222 (2,705,243) 1 (1.8) |
Control (Bariatric Surgery) | HCV | |
---|---|---|
Number of patients | 6 | 17 |
Female (n, %) | 4 (66) | 6 (35.3) |
Age (mean, SD) | 34 (8.4) | 57.5 (12.7) |
Diabetes (n, %) | 6 (100) | NA |
BMI (mean, SD) | 49.5 (7.3) | NA |
ALT (mean, SD) | NA | NA |
AST (mean, SD) | NA | NA |
INR (mean, SD) | NA | NA |
Albumin (mean, SD) | NA | NA |
CHILD | ||
A | 5 (29.4) | |
B | 0 (0.0) | |
C | 0 (0.0) | |
Unknown/NA | ||
Fibrosis stage | ||
0 | 6 (100.0) | 1 (5.9) |
1 | 0 (0.0) | |
2 | 3 (17.6) | |
3 | 8 (47) | |
4 | 5 (29.4) | |
Unknown/NA | ||
Activity grade | ||
1 | 6 (35.3) | |
2 | 10 (58.8) | |
3 | 1 (5.9) | |
Unknown/NA | ||
Steatosis | ||
No | 6 (100.0) | 5 (29.4) |
Yes | 12 (70.5) | |
Unknown | ||
Genotype | 6 (100.0) | |
1 | ||
1A | 12 (70.5) | |
1B | NA | |
2 | NA | |
3 | 1 (5.9) | |
4 | 2 (11.7) | |
Negative Unknown/NA | 6 (100.0) | 2 (11.7) |
Viral load (mean/SD) | NA | 2,530,938 (3,483,784) |
4.2. Materials
4.3. Analysis of ECs in Human Plasma Samples and Liver Biopsies
4.3.1. FAAH and MAGL Activity Assay
4.3.2. In Vitro Experiments
4.3.3. Quantitative Real-Time PCR
4.3.4. ELISpot
4.4. Statistical Analysis
Supplementary Materials
Acknowledgments
Author Contributions
Abbreviations
AEA | anandamide |
2-AG | 2-arachidonoyl glycerol |
CHC | chronic hepatitis C |
FAAH | fatty acid amid hydrolase |
MAGL | monoacylglycerol lipase |
hHep | human hepatocytes |
hHSC | human hepatic stellate cells |
Conflicts of Interest
References
- Nagarkatti, P.; Pandey, R.; Rieder, S.A.; Hegde, V.L.; Nagarkatti, M. Cannabinoids as novel anti-inflammatory drugs. Future Med. Chem. 2009, 1, 1333–1349. [Google Scholar] [CrossRef] [PubMed]
- Julien, B.; Grenard, P.; Teixeira-Clerc, F.; van Nhieu, J.T.; Li, L.; Karsak, M.; Zimmer, A.; Mallat, A.; Lotersztajn, S. Antifibrogenic role of the cannabinoid receptor CB2 in the liver. Gastroenterology 2005, 128, 742–755. [Google Scholar] [CrossRef] [PubMed]
- Pacher, P.; Gao, B. Endocannabinoids and liver disease. III. Endocannabinoid effects on immune cells: Implications for inflammatory liver diseases. Am. J. Physiol. Gastrointest. Liver Physiol. 2008, 294, G850–G854. [Google Scholar]
- Guillot, A.; Hamdaoui, N.; Bizy, A.; Zoltani, K.; Souktani, R.; Zafrani, E.S.; Mallat, A.; Lotersztajn, S.; Lafdil, F. Cannabinoid receptor 2 counteracts interleukin-17-induced immune and fibrogenic responses in mouse liver. Hepatology 2014, 59, 296–306. [Google Scholar] [CrossRef] [PubMed]
- Teixeira-Clerc, F.; Julien, B.; Grenard, P.; Tran Van Nhieu, J.; Deveaux, V.; Li, L.; Serriere-Lanneau, V.; Ledent, C.; Mallat, A.; Lotersztajn, S. CB1 cannabinoid receptor antagonism: A new strategy for the treatment of liver fibrosis. Nat. Med. 2006, 12, 671–676. [Google Scholar] [CrossRef] [PubMed]
- Mallat, A.; Teixeira-Clerc, F.; Deveaux, V.; Manin, S.; Lotersztajn, S. The endocannabinoid system as a key mediator during liver diseases: New insights and therapeutic openings. Br. J. Pharmacol. 2011, 163, 1432–1440. [Google Scholar] [CrossRef] [PubMed]
- Caraceni, P.; Viola, A.; Piscitelli, F.; Giannone, F.; Berzigotti, A.; Cescon, M.; Domenicali, M.; Petrosino, S.; Giampalma, E.; Riili, A.; et al. Circulating and hepatic endocannabinoids and endocannabinoid-related molecules in patients with cirrhosis. Liver Int. 2009, 30, 816–825. [Google Scholar]
- Biswas, K.K.; Sarker, K.P.; Abeyama, K.; Kawahara, K.; Iino, S.; Otsubo, Y.; Saigo, K.; Izumi, H.; Hashiguchi, T.; Yamakuchi, M.; et al. Membrane cholesterol but not putative receptors mediates anandamide-induced hepatocyte apoptosis. Hepatology 2003, 38, 1167–1177. [Google Scholar] [PubMed]
- Osei-Hyiaman, D.; DePetrillo, M.; Pacher, P.; Liu, J.; Radaeva, S.; Batkai, S.; Harvey-White, J.; Mackie, K.; Offertaler, L.; Wang, L.; et al. Endocannabinoid activation at hepatic cb1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity. J. Clin. Investig. 2005, 115, 1298–1305. [Google Scholar]
- Siegmund, S.V.; Seki, E.; Osawa, Y.; Uchinami, H.; Cravatt, B.F.; Schwabe, R.F. Fatty acid amide hydrolase determines anandamide-induced cell death in the liver. J. Biol. Chem. 2006, 281, 10431–10438. [Google Scholar] [CrossRef] [PubMed]
- Baldassarre, M.; Giannone, F.A.; Napoli, L.; Tovoli, A.; Ricci, C.S.; Tufoni, M.; Caraceni, P. The endocannabinoid system in advanced liver cirrhosis: Pathophysiological implication and future perspectives. Liver Int. 2013, 33, 1298–1308. [Google Scholar] [CrossRef] [PubMed]
- Cencioni, M.T.; Chiurchiu, V.; Catanzaro, G.; Borsellino, G.; Bernardi, G.; Battistini, L.; Maccarrone, M. Anandamide suppresses proliferation and cytokine release from primary human t-lymphocytes mainly via CB2 receptors. PLoS ONE 2010, 5, e8688. [Google Scholar] [CrossRef] [PubMed]
- Pandey, R.; Hegde, V.L.; Singh, N.P.; Hofseth, L.; Singh, U.; Ray, S.; Nagarkatti, M.; Nagarkatti, P.S. Use of cannabinoids as a novel therapeutic modality against autoimmune hepatitis. Vitam. Horm. 2009, 81, 487–504. [Google Scholar] [PubMed]
- Hegde, V.L.; Hegde, S.; Cravatt, B.F.; Hofseth, L.J.; Nagarkatti, M.; Nagarkatti, P.S. Attenuation of experimental autoimmune hepatitis by exogenous and endogenous cannabinoids: Involvement of regulatory t cells. Mol. Pharmacol. 2008, 74, 20–33. [Google Scholar] [CrossRef] [PubMed]
- Klein, T.W.; Newton, C.; Larsen, K.; Chou, J.; Perkins, I.; Lu, L.; Nong, L.; Friedman, H. Cannabinoid receptors and T helper cells. J. Neuroimmunol. 2004, 147, 91–94. [Google Scholar] [CrossRef] [PubMed]
- Klein, T.W.; Newton, C.; Larsen, K.; Lu, L.; Perkins, I.; Nong, L.; Friedman, H. The cannabinoid system and immune modulation. J. Leukoc. Biol. 2003, 74, 486–496. [Google Scholar] [CrossRef] [PubMed]
- Rockwell, C.E.; Snider, N.T.; Thompson, J.T.; Vanden Heuvel, J.P.; Kaminski, N.E. Interleukin-2 suppression by 2-arachidonyl glycerol is mediated through peroxisome proliferator-activated receptor γ independently of cannabinoid receptors 1 and 2. Mol. Pharmacol. 2006, 70, 101–111. [Google Scholar] [PubMed]
- Louvet, A.; Teixeira-Clerc, F.; Chobert, M.N.; Deveaux, V.; Pavoine, C.; Zimmer, A.; Pecker, F.; Mallat, A.; Lotersztajn, S. Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating kupffer cell polarization in mice. Hepatology 2011, 54, 1217–1226. [Google Scholar] [CrossRef] [PubMed]
- Zhou, L.; Ding, L.; Yin, P.; Lu, X.; Wang, X.; Niu, J.; Gao, P.; Xu, G. Serum metabolic profiling study of hepatocellular carcinoma infected with hepatitis B or hepatitis C virus by using liquid chromatography-mass spectrometry. J. Proteome Res. 2012, 11, 5433–5442. [Google Scholar] [CrossRef] [PubMed]
- Mallat, A.; Teixeira-Clerc, F.; Lotersztajn, S. Cannabinoid signaling and liver therapeutics. J. Hepatol. 2013, 59, 891–896. [Google Scholar] [CrossRef] [PubMed]
- Ishida, J.H.; Peters, M.G.; Jin, C.; Louie, K.; Tan, V.; Bacchetti, P.; Terrault, N.A. Influence of cannabis use on severity of hepatitis c disease. Clin. Gastroenterol. Hepatol. 2008, 6, 69–75. [Google Scholar] [CrossRef] [PubMed]
- Hezode, C.; Roudot-Thoraval, F.; Nguyen, S.; Grenard, P.; Julien, B.; Zafrani, E.S.; Pawlotsky, J.M.; Dhumeaux, D.; Lotersztajn, S.; Mallat, A. Daily cannabis smoking as a risk factor for progression of fibrosis in chronic hepatitis c. Hepatology 2005, 42, 63–71. [Google Scholar] [CrossRef] [PubMed]
- Hezode, C.; Zafrani, E.S.; Roudot-Thoraval, F.; Costentin, C.; Hessami, A.; Bouvier-Alias, M.; Medkour, F.; Pawlostky, J.M.; Lotersztajn, S.; Mallat, A. Daily cannabis use: A novel risk factor of steatosis severity in patients with chronic hepatitis c. Gastroenterology 2008, 134, 432–439. [Google Scholar] [CrossRef] [PubMed]
- Van der Poorten, D.; Shahidi, M.; Tay, E.; Sesha, J.; Tran, K.; McLeod, D.; Milliken, J.S.; Ho, V.; Hebbard, L.W.; Douglas, M.W.; et al. Hepatitis C virus induces the cannabinoid receptor 1. PLoS ONE 2010, 5, e12841. [Google Scholar]
- Parfieniuk, A.; Flisiak, R. Role of cannabinoids in chronic liver diseases. World J. Gastroenterol. 2008, 14, 6109–6114. [Google Scholar] [CrossRef] [PubMed]
- Siegmund, S.V.; Schwabe, R.F. Endocannabinoids and liver disease. II. Endocannabinoids in the pathogenesis and treatment of liver fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 2008, 294, G357–G362. [Google Scholar] [CrossRef]
- Clugston, R.D.; Jiang, H.; Lee, M.X.; Piantedosi, R.; Yuen, J.J.; Ramakrishnan, R.; Lewis, M.J.; Gottesman, M.E.; Huang, L.S.; Goldberg, I.J.; et al. Altered hepatic lipid metabolism in c57bl/6 mice fed alcohol: A targeted lipidomic and gene expression study. J. Lipid Res. 2011, 52, 2021–2031. [Google Scholar]
- Balvers, M.G.; Verhoeckx, K.C.; Meijerink, J.; Bijlsma, S.; Rubingh, C.M.; Wortelboer, H.M.; Witkamp, R.F. Time-dependent effect of in vivo inflammation on eicosanoid and endocannabinoid levels in plasma, liver, ileum and adipose tissue in c57bl/6 mice fed a fish-oil diet. Int. Immunopharmacol. 2012, 13, 204–214. [Google Scholar] [CrossRef] [PubMed]
- Di Marzo, V.; Petrosino, S. Endocannabinoids and the regulation of their levels in health and disease. Curr. Opin. Lipidol. 2007, 18, 129–140. [Google Scholar] [CrossRef] [PubMed]
- Hauer, D.; Schelling, G.; Gola, H.; Campolongo, P.; Morath, J.; Roozendaal, B.; Hamuni, G.; Karabatsiakis, A.; Atsak, P.; Vogeser, M.; et al. Plasma concentrations of endocannabinoids and related primary fatty acid amides in patients with post-traumatic stress disorder. PLoS ONE 2013, 8, e62741. [Google Scholar]
- Sun, L.J.; Yu, J.W.; Wan, L.; Zhang, X.Y.; Shi, Y.G.; Chen, M.Y. Endocannabinoid system activation contributes to glucose metabolism disorders of hepatocytes and promotes hepatitis C virus replication. Int. J. Infect. Dis. 2014, 23, 75–81. [Google Scholar] [CrossRef] [PubMed]
- Shahidi, M.; Tay, E.S.; Read, S.A.; Ramezani-Moghadam, M.; Chayama, K.; George, J.; Douglas, M.W. Endocannabinoid CB1 antagonists inhibit hepatitis C virus production, providing a novel class of antiviral host targeting agents. J. Gen. Virol. 2014, 95, 2468–2479. [Google Scholar] [CrossRef] [PubMed]
- Patsenker, E.; Stoll, M.; Millonig, G.; Agaimy, A.; Wissniowski, T.; Schneider, V.; Mueller, S.; Brenneisen, R.; Seitz, H.K.; Ocker, M.; et al. Cannabinoid receptor type I modulates alcohol-induced liver fibrosis. Mol. Med. 2011, 17, 1285–1294. [Google Scholar]
- Mueller, M.; Spangenberg, H.C.; Kersting, N.; Altay, T.; Blum, H.E.; Klenerman, P.; Thimme, R.; Semmo, N. Virus-specific cd4+ T cell responses in chronic hcv infection in blood and liver identified by antigen-specific upregulation of cd154. J. Hepatol. 2010, 52, 800–811. [Google Scholar] [CrossRef] [PubMed]
- Glassner, A.; Eisenhardt, M.; Kokordelis, P.; Kramer, B.; Wolter, F.; Nischalke, H.D.; Boesecke, C.; Sauerbruch, T.; Rockstroh, J.K.; Spengler, U.; et al. Impaired CD4+ T cell stimulation of nk cell anti-fibrotic activity may contribute to accelerated liver fibrosis progression in HIV/HCV patients. J. Hepatol. 2013, 59, 427–433. [Google Scholar]
- Schwabe, R.F.; Siegmund, S.V. Potential role of CB2 receptors in cannabis smokers with chronic hepatitis C. Hepatology 2005, 42, 975–976; author reply 976–977. [Google Scholar] [CrossRef] [PubMed]
- Di Marzo, V. Targeting the endocannabinoid system: To enhance or reduce? Nat. Rev. Drug Discov. 2008, 7, 438–455. [Google Scholar] [CrossRef] [PubMed]
- Cao, Z.; Mulvihill, M.M.; Mukhopadhyay, P.; Xu, H.; Erdelyi, K.; Hao, E.; Holovac, E.; Hasko, G.; Cravatt, B.F.; Nomura, D.K.; et al. Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice. Gastroenterology 2013, 144, 808–817. [Google Scholar]
- Von Schonfels, W.; Patsenker, E.; Fahrner, R.; Itzel, T.; Hinrichsen, H.; Brosch, M.; Erhart, W.; Gruodyte, A.; Vollnberg, B.; Richter, K.; et al. Metabolomic tissue signature in human non-alcoholic fatty liver disease identifies protective candidate metabolites. Liver Int. 2014, 35, 207–214. [Google Scholar]
- Semmo, N.; Krashias, G.; Willberg, C.; Klenerman, P. Analysis of the relationship between cytokine secretion and proliferative capacity in hepatitis C virus infection. J. Viral Hepat. 2007, 14, 492–502. [Google Scholar] [CrossRef] [PubMed]
- Chicca, A.; Marazzi, J.; Nicolussi, S.; Gertsch, J. Evidence for bidirectional endocannabinoid transport across cell membranes. J. Biol. Chem. 2012, 287, 34660–34682. [Google Scholar] [CrossRef] [PubMed]
- Patsenker, E.; Popov, Y.; Stickel, F.; Jonczyk, A.; Goodman, S.L.; Schuppan, D. Inhibition of integrin αvβ6 on cholangiocytes blocks transforming growth factor-β activation and retards biliary fibrosis progression. Gastroenterology 2008, 135, 660–670. [Google Scholar] [CrossRef] [PubMed]
- Semmo, N.; Barnes, E.; Taylor, C.; Kurtz, J.; Harcourt, G.; Smith, N.; Klenerman, P. T-cell responses and previous exposure to hepatitis c virus in indeterminate blood donors. Lancet 2005, 365, 327–329. [Google Scholar] [CrossRef] [PubMed]
- Semmo, N.; Day, C.L.; Ward, S.M.; Lucas, M.; Harcourt, G.; Loughry, A.; Klenerman, P. Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection. Hepatology 2005, 41, 1019–1028. [Google Scholar] [CrossRef] [PubMed]
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).