P: 5 Let7f Expression Is Upregulated in the Frontal Cortex During Acute Liver Failure and Contributes to Hepatic Encephalopathy via Downregulation of IGF1 Expression

Abstract

BACKGROUND: Hepatic encephalopathy (HE) is a neurological complication that arises due to loss of liver function and is associated with increased blood-brain barrier (BBB) permeability, neuroinflammation and subsequent onset of cognitive decline. We have previously shown that increased circulating TGFβ levels induce BBB permeability and contribute to the development of HE. Associated with the aberrant TGFβ signaling during HE was an upregulation of the microRNA Let7f, a known modulator of IGF1 expression in the brain. However, whether Let7f contributes to the development of HE is unknown. The aims of this study were to assess the expression of Let7f in a mouse model of Type A HE and to determine its involvement in the neurological complications of acute liver failure (ALF). METHODS: C57Bl/6 mice were injected with azoxymethane (AOM) to induce ALF and HE. In parallel, mice were given an intracerebroventricular infusion of a Let7f antagomir or recombinant IGF1 (rIGF1) for 3 days prior to AOM injection. Cognitive impairment was monitored by reflex response assessment at various time points. Neuromuscular deficits were assessed using a grip strength meter, and a digigait analysis system was utilized to measure ataxia. Liver damage was assessed by hematoxylin and eosin staining and serum chemistry. IGF1, Let7f 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 Let7f mimic and treated with vehicle or rIGF1 for 4 to 24 hr. The expression and secretion of IGF1 and the proinflammatory chemokine, CCL2 was assessed by qPCR and EIA. RESULTS: Mice injected with AOM had increased Let7f and decreased IGF1 expression in the frontal cortex. Treatment with a Let7f antagomir attenuated the i) suppression of cortical IGF1, ii) neuroinflammation, and iii) neurological and neuromuscular deficits of AOM-treated mice. Specific targeting of IGF1 expression by Let7f was demonstrated in vitro, where treatment of neurons with a Let7f mimic suppressed IGF1 expression and secretion. Furthermore, treatment of neurons with Let7f mimic increased the expression of CCL2, which could be attenuated with the co-treatment with rIGF1. Lastly, infusion of rIGF1 to restore the dampened IGF1 signaling attenuated the neurological and neuromuscular deficits, as well as the neuroinflammation observed in AOM-treated mice. CONCLUSIONS: Elevated cortical Let7f expression contributes to the pathogenesis of HE in AOM-treated mice via mechanisms involving the suppression of IGF1 expression. These deleterious effects of Let7f during HE can be reversed by inhibiting Let7f expression or by increasing IGF1 concentration in the brain.