Acetylcholine induces fibrogenic effects via M2/M3 acetylcholine receptors in non-alcoholic steatohepatitis and in primary human hepatic stellate cells.
BACKGROUND
The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear.


OBJECTIVE
We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH).


METHODS
Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis.


RESULTS
We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers.


CONCLUSIONS
We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.
Maelle L
ML
Morgan
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.
Barbara
B
Sigala
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.
Junpei
J
Soeda
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.
Paul
P
Cordero
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.
Vi
V
Nguyen
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.
Chad
C
McKee
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.
Angelina
A
Mouraliderane
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.|Gastroenterology Unit, Department of Medical Sciences, Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy.|Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, UK.
Manlio
M
Vinciguerra
University College London, Institute for Liver and Digestive Health, Royal Free Hospital, London, UK.|Guy's and St Thomas' National Health Service Foundation Trust, London, UK.
Jude A
JA
Oben
D019870:1-Phosphatidylinositol 4-Kinase / Q000502:physiology; D000109:Acetylcholine / Q000009:adverse effects* / Q000096:biosynthesis / Q000502:physiology*; D000110:Acetylcholinesterase / Q000096:biosynthesis; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D018450:Disease Progression; D005355:Fibrosis; D015870:Gene Expression; D055166:Hepatic Stellate Cells / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D008103:Liver Cirrhosis / Q000209:etiology* / Q000473:pathology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D065626:Non-alcoholic Fatty Liver Disease / Q000209:etiology* / Q000473:pathology; D010275:Parasympathetic Nervous System / Q000502:physiology; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D015854:Up-Regulation
D011976:Receptors, Muscarinic; D002795:Choline O-Acetyltransferase; D019870:1-Phosphatidylinositol 4-Kinase; D020929:Mitogen-Activated Protein Kinase Kinases; D000110:Acetylcholinesterase; D000109:Acetylcholine
fibrosis; liver disease; parasympathetic nervous system