Butyrylcholinesterase alleviates hepatic stellate cell activation via paracrine muscarinic signaling [2W-CCL4-DOX-HSC]

BuChE expression and HSC activation were assessed in liver biopsies from patients with liver fibrosis using qPCR and immunostaining. To investigate the functional role of BuChE in fibrogenesis, we employed BucheΔHep, knock-in (BucheKI/KI-Hep) mice, as well as pharmacological inhibition. The inhibitory effect of BuChE on HSC activation was further confirmed using conditioned medium from cells expressing mutant BuChE, in hepatocyte-HSC co-culture experiments, and analyses of recombinant BuChE protein.

The release of ACh was measured with patch-clamp and the expression and functionality of its receptor were detected via intracellular Ca2+ influx measurements. The role of the muscarinic acetylcholine receptor 3 (ChRM3) in HSCs was investigated through gain- and loss-of-function experiments, utilizing Chrm3ΔHSC mice and AAV-mediated Chrm3 overexpression driven by the alpha-smooth muscle actin (Acta2) promoter. Results: Buche mRNA expression in livers and enzymatic activity in serum are downregulated in patients and mouse models with liver fibrosis, and show a negative correlation with F stage and ACTA2 expression.

Pharmacological inhibition or depletion of BuChE in hepatocytes results in increased HSC activation and aggravates liver fibrosis in various mouse models, whereas its overexpression exerts the opposite effect, indicating a causative role of BuChE downregulation in liver diseases. BuChE is predominantly expressed in hepatocytes in the liver, which can be reduced by TGFβ treatment. Notably, BuChE enzymatic activity is required to prevent HSC activation and fibrogenesis in homeostatic liver.

As consequence of the lost BuChE metabolic activity, levels of released unmetabolized ACh are increased, which acts as DAMP to activate HSCs via ChRM3 signaling. This finding was confirmed using Chrm3ΔHSC mice, which exhibit diminished HSC activation and liver fibrosis. Conversely, ChRM3 overexpression in HSCs is promoting the fibrogenic process.

Conclusion: Hepatocyte injury disrupts BuChE activity, causing local ACh accumulation and subsequent HSC activation via ChRM3 signaling. This novel cholinergic circuit drives fibrosis and represents a promising new target for therapeutic intervention.