Scopolamine Treatment and Muscarinic Receptor Subtype-3 Gene Ablation Augment Azoxymethane-Induced Murine Liver Injury

Abstract

Previous work suggests that vagus nerve disruption reduces hepatocyte and oval cell expansion after liver injury. The role of postneuronal receptor activation in response to liver injury has not been ascertained. We investigated the actions of scopolamine, a nonselective muscarinic receptor antagonist, and specific genetic ablation of a key cholinergic receptor, muscarinic subtype-3 (Chrm3), on azoxymethane (AOM)-induced liver injury in mice. Animal weights and survival were measured as was liver injury using both gross and microscopic examination. To assess hepatocyte proliferation and apoptosis, ductular hyperplasia, and oval cell expansion, we used morphometric analysis of 5-bromo-2Ј-deoxyuridine-, activated caspase-3-, hematoxylin and eosin-, cytokeratin-19-, and epithelial cell adhesion molecule-stained liver sections. Sirius red staining was used as a measure of collagen deposition and its association with oval cell reaction. In AOM-treated mice, both muscarinic receptor blockade with scopolamine and Chrm3 ablation attenuated hepatocyte proliferation and augmented gross liver nodularity, apoptosis, and fibrosis. Compared with control, scopolamine-treated and Chrm3(Ϫ/Ϫ) AOM-treated mice had augmented oval cell reaction with increased ductular hyperplasia and oval cell expansion. Oval cell reaction correlated robustly with liver fibrosis. No liver injury was observed in scopolamine-treated and Chrm3(Ϫ/Ϫ) mice that were not treated with AOM. Only AOMtreated Chrm3(Ϫ/Ϫ) mice developed ascites and had reduced survival compared with AOM-treated wild-type controls. In AOMinduced liver injury, inhibiting postneuronal cholinergic muscarinic receptor activation with either scopolamine treatment or Chrm3 gene ablation results in prominent oval cell reaction. We conclude that Chrm3 plays a critical role in the liver injury response by modulating hepatocyte proliferation and apoptosis.Acetylcholine activates two classes of cholinergic receptors: nicotinic, which function as cation channels, and muscarinic (CHRM), which mediate G protein-coupled signaling. Five mammalian CHRM subtypes (CHRM1-5) are described; oddnumbered CHRM (CHRM1, 3, and 5) preferentially activate G q/11 proteins, whereas even-numbered CHRM (CHRM2 and 4) activate G i/o proteins. Cell type-specific expression of CHRM subtypes mediates diverse signaling events. In the nervous system, muscarinic receptors play a major role in synaptic transmission and regulate sensory, motor, and autonomic functions. In non-neuronal tissue, muscarinic receptor activation regulates cell function (Shah et al., 2009). For example, muscarinic receptors mediate proliferation of lung and colon cancer cells (Raufman et al., 2008;Shah et al., 2009).In the liver, stimulation of the vagus nerve induces proliferation of oval cells (designated intermediate hepatobiliary Article, publication date, and citation information can be found at

Brown seaweeds protect against azoxymethane-induced hepatic repercussions through up-regulation of peroxisome proliferator-activated receptor gamma and attenuation of oxidative stress

“…These findings are consistent with previous studies demonstrated that AOM induces chronic injury in murine liver (Shan et al 2008;Khurana et al 2010). Further, our findings confirm previous observations (Khurana et al 2010) by showing that AOM administration induces oval cell expansion.…”

“…These findings are consistent with previous studies demonstrated that AOM induces chronic injury in murine liver (Shan et al 2008;Khurana et al 2010). Further, our findings confirm previous observations (Khurana et al 2010) by showing that AOM administration induces oval cell expansion. The recorded oval cell proliferation in the present study has been reported in human chronic liver diseases and animal models of liver injury (Roskams et al 2004;Clouston et al 2005).…”

“…Several studies revealed that AOM is a potent carcinogen for the liver (Chastre et al 2012) and at low doses induce hepatotoxicity with ductular hyperplasia (Shan et al 2008;Khurana et al 2010). Although derived from a rare plant and human exposure to AOM is infrequent, exposure to structurally related hydrazine compounds that are found in herbicides, rocket fuels, tobacco and drugs is common (Gamberini et al 1998).…”

M1 muscarinic receptors modify oxidative stress response to acetaminophen-induced acute liver injury

“…In each section, at least five areas were examined. Nitrotyrosine staining was expressed as the percentage of summed pixels per unit area of liver section (in arbitrary units) determined using Image-Pro Plus software (version 5.0; Media Cybernetics, Silver Spring, MD) as described previously [18]. The stained sections were evaluated independently by two investigators.…”

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

“…5,6 Only during chronic injury are liver stem cells observed to proliferate. 1,[5][6][7][8][9][10][11] These adult, organ specific stem cells are proposed to differentiate into both hepatocytes and cholangiocytes. 1,[5][6][7][8] Interestingly, the vast majority of liver cancer develops on the background of chronic injury, and thus liver stem cells are also hypothesized to be the precursors for a subset of liver cancer.…”