Gli1 activation and protection against hepatic encephalopathy is suppressed by circulating transforming growth factor β1 in mice

Abstract

Background & Aims Hepatic encephalopathy (HE) is a neurologic disorder that develops during liver failure. Few studies exist investigating systemic-central signaling during HE outside of inflammatory signaling. The transcription factor Gli1, which can be modulated by hedgehog signaling or transforming growth factor β1 (TGFβ1) signaling, has been shown to be protective in various neuropathies. We measured Gli1 expression in brain tissues from mice and evaluated how circulating TGFβ1 and canonical hedgehog signaling regulate its activation. Methods Mice were injected with azoxymethane (AOM) to induce liver failure and HE in the presence of Gli1 Vivo-morpholinos (to mediate knockdown), the hedgehog inhibitor cyclopamine, smoothened Vivo-morpholinos, a smoothened agonist, or TGFβ-neutralizing antibodies. Molecular analyses were used to assess Gli1, hedgehog signaling, and TGFβ1 signaling in the liver and brain of AOM mice and HE patients. Results Gli1 expression was increased in brains of AOM mice and in HE patients. Intra-cortical infusion of Gli1 Vivo-morpholinos exacerbated the neurologic deficits of AOM mice. Measures to modulate hedgehog signaling had no effect on HE neurological decline. Levels of TGFβ1 increased in the liver and serum of mice following AOM administration. TGFβ neutralizing antibodies slowed neurologic decline following AOM administration without significantly affecting liver damage. TGFβ1 inhibited Gli1 expression via a SMAD3-dependent mechanism. Conversely, inhibiting TGFβ1 increased Gli1 expression. Conclusions Cortical activation of Gli1 protects mice from induction of HE. TGFβ1 suppresses Gli1 in neurons via SMAD3 and promotes neurologic decline. Strategies to activate Gli1 or inhibit TGFβ1 signaling might be developed to treat patients with HE.

Bile Acid Signaling Is Involved in the Neurological Decline in a Murine Model of Acute Liver Failure

“…These stages have been previously described in detail. 17 Expression of the bile acid transporter ASBT, the bile acid nuclear receptor FXR, and its cofactor SHP was significantly increased after AOM injection in the frontal cortex before the onset of neurological symptoms compared with control tissue ( Figure 5A and Supplemental Figure S1A), an effect that was observed through to major neurological decline. Interestingly, the expression of these key bile acid signaling components was dramatically decreased at the time of coma ( Figure 5A and Supplemental Figure S1A).…”

“…17,18 Cerebral edema was assessed in all mice using the wet/dry weight method described by Baskaya et al 20 Water content was expressed as a percentage of brain weight, calculated as follows: ([wet weight À dry weight]/wet weight) Â 100%. Increased brain water content of 1% to 2% in mice is indicative of cerebral edema and is characterized by increased intracranial pressure.…”

“…Primary cortical neurons were isolated as previously described, 17 allowed to differentiate, and treated with 10 mmol/L DCA in the presence or absence of 10 mmol/L FXR antagonist guggulsterone for 24 hours. FXR nuclear translocation was assessed by immunofluorescence microscopy, as described previously, 30 and neurons were counterstained with the nuclear stain DAPI.…”

Neuroinflammatory Signals during Acute and Chronic Liver Diseases

“…Speciically, TGFβ has both anti-inlammatory and proinlammatory efects on various immune cells in the body, including microglial activation. Increased levels of TGFβ have been demonstrated in the liver and serum in the AOM model of acute liver failure [38]. The authors demonstrated that peripheral TGFβ has implications on microglial activation [39].…”

“…The authors demonstrated that peripheral TGFβ has implications on microglial activation [39]. Speciically, systemic treatment of mice with a neutralizing anti-TGFβ antibody, that did not signiicantly alter the underlying liver damage, but inhibited the actions of circulating TGFβ delayed the neurological decline observed in AOM-induced acute liver failure [38], and atenuated the morphological changes in Iba-1 positive microglia [39]. However, whether liver-derived TGFβ is acting directly on microglia to regulate the neuroinlammatory response in these models of HE, or whether the changes in microglial activation are an indirect efect of the protective efect of anti-TGFβ neutralizing antibodies remains to be established.…”