A study on OPG/RANK/RANKL axis in osteoporotic bile duct-ligated rats and the involvement of nitrergic and opioidergic systems

Amir Hossein Doustimotlagh, Ahmad Reza Dehpour, Shahroo Etemad-Moghadam, Mojgan Alaeddini, Sattar Ostadhadi, Abolfazl Golestani


Chronic liver disease (CLD) affects millions of people and its impact on bone loss has become a subject of interest. Nitric oxide and endogenous opioids are suggested to increase during cholestasis/cirrhosis and may impact bone resorption by different mechanisms. The receptor activator of nuclear factor-κB (RANK)/RANK-ligand (RANKL)/osteoprotegerin (OPG) signaling pathway regulates bone resorption, but its role in metabolic bone disease subsequent to CLD is unknown. We aimed to investigate the involvement of nitrergic and opioidergic systems in bone loss relative to the RANK/RANKL/OPG pathway, in bile duct-ligated (BDL) rats. Eighty BDL/sham-operated (SO) rats received injections of 3 mg/kg/day Nω-Nitro-L-arginine methyl ester ± naltrexone (10 mg/kg/day) or saline for 28 days. Plasma bone turnover markers, OPG, RANK, and RANKL along with mRNA expression levels of the latter three were assessed. Plasma bone turnover markers and OPG level increased, but RANKL decreased in the BDL group compared with their SO controls (both: P ≤ 0.05). Administration of naltrexone reduced bone turnover markers and OPG level while increased RANKL content in comparison to BDL rats (P ≤ 0.05). As compared to untreated BDL rats, nitric oxide inhibition showed no effect on bone turnover marker i.e. OPG, RANK, and RANKL levels. BDL significantly increased RANK mRNA, but had no significant effect on RANKL and OPG mRNA expression. The lack of association between plasma levels and quantitative gene expression of RANKL and OPG suggests an indirect function of these markers in BDL rats. Considering that opioid receptor blockage by naltrexone in BDL animals caused a significant decrease in OPG and an increase in RANKL plasma contents, it could be postulated that the opioidergic system may have a regulatory effect on these bone markers.


Cirrhosis; Bone loss; Nitrergic system; Opioidergic system; BDL rats; RANK/RANKL/OPG axis.

Full Text:



Leslie WD, Bernstein CN, Leboff MS. AGA technical review on osteoporosis in hepatic disorders. Gastroenterology. 2003;125(3):941-966.

Gatta A, Verardo A, Di Pascoli M, Giannini S, Bolognesi M. Hepatic osteodystrophy. Clin Cases Miner Bone Metab. 2014;11(3):185-191.

Szalay F, Hegedus D, Lakatos PL, Tornai I, Bajnok E, Dunkel K, et al. High serum osteoprotegerin and low RANKL in primary biliary cirrhosis. J Hepatol. 2003;38(4):395-400.

Moschen AR, Kaser A, Stadlmann S, Millonig G, Kaser S, Mühllechner P, et al. The RANKL/OPG system and bone mineral density in patients with chronic liver disease. J Hepatol. 2005;43(6):973-83.

Gasser RW. Cholestasis and metabolic bone disease–a clinical review. Wien Med Wochenschr. 2008;158(19-20):553-557.

Ho TY, Santora K, Chen JC, Frankshun AL, Bagnell CA. Effects of relaxin and estrogens on bone remodeling markers, receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG), in rat adjuvant-induced arthritis. Bone. 2011;48(6): 1346-1353.

Tudpor K, van der Eerden BC, Jongwattanapisan P, Roelofs JJ, van Leeuwen JP, Bindels RJ, et al. Thrombin receptor deficiency leads to a high bone mass phenotype by decreasing the RANKL/OPG ratio. Bone. 2015;72:14-22.

Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337-342.

Vega D, Maalouf NM, Sakhaee K. The role of receptor activator of nuclear factor-κB (RANK)/RANK ligand/osteoprotegerin: clinical implications. J Clin Endocrinol Metab. 2007;92(12):4514-4521.

Mahmoud MF, Zakaria S, Fahmy A. Can Chronic Nitric Oxide Inhibition Improve Liver and Renal Dysfunction in Bile Duct Ligated Rats? Adv Pharmacol Sci. 2015;2015:298792.

Lee SK, Huang H, Lee SW, Kim KH, Kim KK, Kim HM, et al. Involvement of iNOS-dependent NO production in the stimulation of osteoclast survival by TNF-α. Exp Cell Res. 2004;298(2):359-368.

Fan X, Roy E, Zhu L, Murphy TC, Ackert-Bicknell C, Hart CM, et al. Nitric oxide regulates receptor activator of nuclear factor-κB ligand and osteoprotegerin expression in bone marrow stromal cells. Endocrinology. 2004;145(2):751-759.

Ebrahimkhani MR, Kiani S, Oakley F, Kendall T, Shariftabrizi A, Tavangar SM, et al. Naltrexone, an opioid receptor antagonist, attenuates liver fibrosis in bile duct ligated rats. Gut. 2006;55(11): 1606-1616.

Queiroz-Junior CM, Maltos KL, Pacheco DF, Silva TA, Albergaria JD, Pacheco CM. Endogenous opioids regulate alveolar bone loss in a periodontal disease model. Life Sci. 2013;93(12):471-477.

Rosen H, Bar‐Shavit Z. Dual role of osteoblastic proenkephalin derived peptides in skeletal tissues. J cell Biochem. 1994;55(3):334-339.

Liashev I. [Effect of opioid peptides on the repair regeneration of the bone tissue]. Arkh Patol. 2001;64(1):6-8.

Elhassan AM, Lindgren J, Hultenby K, Bergstrom J, Adem A. Methionine‐enkephalin in bone and joint tissues. J Bone Miner Res. 1998;13(1):88-95.

Dresner–Pollak R, Gabet Y, Steimatzky A, Hamdani G, Bab I, Ackerman Z, et al. Human parathyroid hormone 1–34 prevents bone loss in experimental biliary cirrhosis in rats. Gastroenterology. 2008;134(1):259-267.

Namiranian K, Samini M, Mehr SE, Gaskari SA, Rastegar H, Homayoun H, et al. Mesenteric vascular bed responsiveness in bile duct-ligated rats: roles of opioid and nitric oxide systems. Eur J Pharmacol. 2001;423(2):185-193.

Kholari FS, Dehpour AR, Nourbakhsh M, Doustimotlagh AH, Bagherieh M, Golestani A. Erythrocytes membrane alterations reflecting liver damage in CCl₄-induced cirrhotic rats: the ameliorative effect of naltrexone. Acta Med Iranica. 2016;54(10):632-640.

Doustimotlagh AH, Dehpour AR, Nourbakhsh M, Golestani A. Alteration in membrane protein, antioxidant status and hexokinase activity in erythrocytes of CCl4-Induced cirrhotic rats. Acta Med Iranica. 2014;52(11):795-803.

Minkin C. Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int. 1982;34(1):285-290.

Carter LE, Kilroy G, Gimble JM, Floyd ZE. An improved method for isolation of RNA from bone. BMC Biotechnol. 2012;12(1):5-9.

Hay JE. Bone disease in cholestatic liver disease. Gastroenterology. 1995;108(1):276-283.

Rouillard S, Lane NE. Hepatic osteodystrophy. Hepatology. 2001;33(1):301-307.

Hernandez-Vaquero D, Garcia-Sandoval MA, Fernandez-Carreira JM, Suarez-Vázquez A, Perez-Hernández D. Measurement of bone mineral density is possible with standard radiographs: a study involving total knee replacement. Acta Orthop. 2005;76(6):791-795.

Doustimotlagh AH, Dehpour AR, Etemad-Moghadam S, Alaeddini M, Kheirandish Y, Golestani A, et al. Nitrergic and opioidergic systems affect radiographic density‎ and histomorphometric indices in bile-duct-ligated cirrhotic rats. Histol Histopathol. 2017. DOI: 10.14670/HH-11-836.

Guañabens N, Parés A, Alvarez L, Osaba D, Martínez MJ, Monegal A, et al. Collagen‐related markers of bone turnover reflect the severity of liver fibrosis in patients with primary biliary cirrhosis. J Bone Miner Res. 1998;13(4):731-738.

Van De Wijngaert FP, Burger EH. Demonstration of tartrate-resistant acid phosphatase in un-decalcified, glycolmethacrylate-embedded mouse bone: a possible marker for (pre) osteoclast identification. J Histochem Cytochem. 1986;34(10):1317-1323.

Guañabens N, Enjuanes A, Alvarez L, Peris P, Caballería L, De Osaba MJM, et al. High osteoprotegerin serum levels in primary biliary cirrhosis are associated with disease severity but not with the mRNA gene expression in liver tissue. J Bone Miner Metab. 2009;27(3):347-354.

Fábrega E, Orive A, García‐Suarez C, García‐Unzueta M, Antonio Amado J, Pons‐Romero F. Osteoprotegerin and RANKL in alcoholic liver cirrhosis. Liver Int. 2005;25(2):305-310.

Monegal A, Navasa M, Peris P, Alvarez L, Pons F, Rodés J, et al. Serum osteoprotegerin and its ligand in cirrhotic patients referred for orthotopic liver transplantation: relationship with metabolic bone disease. Liver Int. 2007;27(4):492-497.

GarcÍA-Valdecasas-Campelo E, GonzÁLez-Reimers E, Santolaria-FernÁNdez F, De La Vega-Prieto MJ, Milena-Abril A, SÁNchez-PÉRez MJ, et al. Serum osteoprotegerin and RANKL levels in chronic alcoholic liver disease. Alcohol Alcohol. 2006;41(3):261-266.

Lacey D, Timms E, Tan H-L, Kelley M, Dunstan C, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998;93(2):165-176.

Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, et al. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/ osteoprotegerin. Biochem Biophys Res Commun. 1998;247(3):610-615.

Colucci S, Brunetti G, Rizzi R, Zonno A, Mori G, Colaianni G, et al. T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction. Blood. 2004;104(12):3722-3730.

Browner WS, Lui L-Y, Cummings SR. Associations of serum osteoprotegerin levels with diabetes, stroke, bone density, fractures, and mortality in elderly women. J Clin Endocrinol Metab. 2001;86(2):631-637.

Simonet W, Lacey D, Dunstan C, Kelley M, Chang M-S, Lüthy R, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997;89(2):309-319.


  • There are currently no refbacks.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.