TGFβ1 Suppresses Neuronal IGF1 via a Let-7F-Dependent Mechanism During Azoxymethane-Induced Hepatic Encephalopathy in Mice

Abstract

Background and Aim: Hepatic encephalopathy (HE) is a neurological complication that arises after a loss of liver function. HE is associated with increased cerebral edema, neuroinflammation and the onset of cognitive decline. We have previously shown that there is increased expression and secretion of transforming growth factor β1 (TGFβ1) from hepatocytes during the azoxymethane (AOM) model of acute liver failure and HE. Increased circulating TGFβ1 was shown to promote blood-brain barrier permeability and also led to progressive neurological complications via increased activation of its receptor TGFβR2. Insulin-like growth factor 1 (IGF1) is a neuroprotective peptide that can be suppressed by TGFβ1 signaling in other organs. IGF1 expression is regulated by a number of microRNAs, including Let-7f, which is positively regulated by TGFβ1. Therefore, we hypothesize that circulating hepatic-derived TGFβ1 suppresses neural IGF1 via a mechanism involving Let-7f during HE, which subsequently exacerbates neurological decline. Methods: In order to determine the exact contribution of TGFβ1 liver-brain axis signaling in this model, male C57Bl/6 (WT) mice, hepatocyte-specific TGFβ1 knockout mice (floxed TGFβ1 × albumin-Cre) or neuron-specific TGFβR2 knockout mice (floxed TGFβR2 × Thy1-Cre) were injected with the hepatotoxin azoxymethane (AOM). In parallel, WT mice were treated with an anti-TGFβ neutralizing antibody 1 h prior to AOM, or were infused with rIGF1 into the lateral ventricle for 3 days prior to AOM injection. Cognitive impairment was monitored and livers, serum and whole brains were collected at coma. IGF1, TGFβ1, Let-7f and proinflammatory cytokine expression were assessed by immunoblotting, immunohistochemistry and/or qPCR. Microglia were stained by IBA1 and cortex field staining and cell morphology were assessed. In vitro, mouse neurons were transfected with a specific Let-7f inhibitor prior to treatment with rTGFβ1 and the expression of Let-7f, IGF1 and CCL2 were measured. Results: Mice injected with AOM had increased Let-7f and suppressed cortical IGF1 expression. Strategies to inhibit TGFβ1 signaling in the brain (ie hepatocyte-specific TGFβ1 knockout, neuron-specific TGFβR2 knockout or pretreatment with TGFβ neutralizing antibody) attenuated the (i) induction of Let-7f expression, (ii) suppression of cortical IGF1, (iii) neuroinflammation and (iv) neurological decline when compared to WT mice treated with AOM alone. In vitro, treatment of neurons with rTGFβ1 increased the expression of Let-7f and reduced the expression of IGF1 in a Let-7f-dependent manner, while also inducing CCL2 expression. Lastly, rIGF1 infusion in the brain delayed AOM-induced neurological decline and reduced neuroinflammation. Conclusion: Elevated TGFβ1 signaling during HE leads to increased cortical Let-7f expression, decreased IGF1 expression and worse outcomes in AOM-treated mice. These deleterious effects can be reversed by inhibiting neuronal TGFβ1 signaling or by increasing IGF1 concentrations in the brain, indicating that elevated TGFβ1 and suppressed IGF1 contribute to the progression of HE. The authors have none to declare.