Systemic inflammation and ammonia (hyperammonemia) act synergistically in the pathogenesis of hepatic encephalopathy (HE), the neurobehavioral sequelae of advanced liver disease. In cirrhotic patients, we have recently observed elevated levels of circulating neuronal tight junction (TJ) protein, zonula occludens 1 (ZO-1), reflective of a change to blood–brain barrier (BBB) integrity. Moreover, ZO-1 levels positively correlated with hyperammonemia, although any potential relationship remains unclear. Using a carbon tetrachloride (CCl4)–induced mouse model of cirrhosis, we primarily looked to explore the relationship between neuronal TJ protein expression and hyperammonemia. Secondarily, we assessed the potential role of a natural antioxidant, resveratrol, on neuronal TJ protein expression and hyperammonemia. Over 12 weeks, male Swiss mice were randomized (n = 8/group) to either naïve controls or induced cirrhosis, using two doses of intraperitoneal CCl4 (0.5 ml/kg/week). After 12 weeks, naïve and cirrhotic mice were randomized to receive either 2 weeks of par-oral resveratrol (10 mg/kg). Plasma samples were analyzed for ammonia, liver biochemistry (ALT, AST, albumin, and bilirubin), and pro-inflammatory cytokines (TNF-α and IL-1β), and brain tissue for brain water content, TJ protein expression (e.g., ZO-1, claudin 5, and occludin), and tissue oxidative stress and inflammatory markers (NF-κB and iNOS) using western blotting. Compared to naïve mice, cirrhosis significantly increased circulating ammonia, brain water, ALT, AST, TNF-α, IL-1β, 4HNE, NF-κB, and iNOS levels, with a concomitant reduction in all TJ proteins (P < 0.05, respectively). In cirrhotic mice, resveratrol treatment ameliorated these changes significantly (P < 0.05, respectively). Our findings provide evidence for a causal association between hyperammonemia and inflammation in cirrhosis linked to TJ protein alterations, BBB disruption, and HE predilection. Moreover, this is the first report of a potential role for resveratrol as a novel therapeutic approach to managing neurological sequelae complicating cirrhosis.
This is a preview of subscription content, log in to check access.
This work was supported by the 5-year Ramalingaswami Re-entry Fellowship grant (102/IFD/SAN/22/2013-14) awarded to V.B. from the Department of Biotechnology (DBT), Government of India.
V.B. designed the study; V.B. and M.S. conducted the study; V.B. and M.S. analyzed the data statistically; V.B. wrote and critically reviewed the manuscript; B.H.S. interpreted the histology and immunohistochemical findings.
Compliance with Ethical Standards
Conflict of Interest Statement
All the authors declare that there is no conflict of interest.
The manuscript underwent proof read and plagiarism check prior submission to the Journal.
Institutional Review Board Statement
The study was reviewed and approved by the JIPMER Scientific Advisory Committee and Institutional Animal Ethics Committee.
Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1994) Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127(6 Pt 1):1617–1626CrossRefGoogle Scholar
Cording J, Berg J, Kading N, Bellmann C, Tscheik C, Westphal JK, Milatz S, Gunzel D et al (2013) In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization. J Cell Sci 126(Pt 2):554–564. https://doi.org/10.1242/jcs.114306CrossRefPubMedGoogle Scholar
Hu J, Han H, Cao P, Yu W, Yang C, Gao Y, Yuan W (2017) Resveratrol improves neuron protection and functional recovery through enhancement of autophagy after spinal cord injury in mice. Am J Transl Res 9(10):4607–4616PubMedPubMedCentralGoogle Scholar
Lin YL, Chang HC, Chen TL, Chang JH, Chiu WT, Lin JW, Chen RM (2010) Resveratrol protects against oxidized LDL-induced breakage of the blood–brain barrier by lessening disruption of tight junctions and apoptotic insults to mouse cerebrovascular endothelial cells. J Nutr 140(12):2187–2192. https://doi.org/10.3945/jn.110.123505CrossRefGoogle Scholar
Balasubramaniyan V, Wright G, Sharma V, Davies NA, Sharifi Y, Habtesion A, Mookerjee RP, Jalan R (2012) Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats. Am J Physiol Gastrointest Liver Physiol 302(1):G145–G152. https://doi.org/10.1152/ajpgi.00097.2011CrossRefPubMedGoogle Scholar
Dhanda S, Sandhir R (2017) Blood–brain barrier permeability is exacerbated in experimental model of hepatic encephalopathy via MMP-9 activation and downregulation of tight junction proteins. Mol Neurobiol. https://doi.org/10.1007/s12035-017-0521-7
Liebner S, Fischmann A, Rascher G, Duffner F, Grote EH, Kalbacher H, Wolburg H (2000) Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol 100(3):323–331CrossRefGoogle Scholar
Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123(6 Pt 2):1777–1788CrossRefGoogle Scholar
Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, Takano H, Noda T et al (1998) Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol 141(2):397–408CrossRefGoogle Scholar
Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T, Tsukita S (2000) Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell 11(12):4131–4142CrossRefGoogle Scholar
Rochfort KD, Collins LE, McLoughlin A, Cummins PM (2016) Tumour necrosis factor-alpha-mediated disruption of cerebrovascular endothelial barrier integrity in vitro involves the production of proinflammatory interleukin-6. J Neurochem 136(3):564–572. https://doi.org/10.1111/jnc.13408CrossRefPubMedGoogle Scholar
Didier N, Romero IA, Creminon C, Wijkhuisen A, Grassi J, Mabondzo A (2003) Secretion of interleukin-1beta by astrocytes mediates endothelin-1 and tumour necrosis factor-alpha effects on human brain microvascular endothelial cell permeability. J Neurochem 86(1):246–254CrossRefGoogle Scholar
Gu X, Cai Z, Cai M, Liu K, Liu D, Zhang Q, Tan J, Ma Q (2018) AMPK/SIRT1/p38 MAPK signaling pathway regulates alcohol-induced neurodegeneration by resveratrol. Mol Med Rep. https://doi.org/10.3892/mmr.2018.8482