Background: NADPH oxidase-2 (Nox2)-derived oxidative stress is believed to play a pivotal role in inducing non-alcoholic fatty liver disease (NAFLD) but its behavior in children with NAFLD has never been investigated.
Aim: To assess Nox2 activation in children affected by biopsy proven NAFLD. Furthermore, the association, of Nox2 activation with gut permeability and histological characteristics, will be analyzed in this population.
Methods: In a cross-sectional study we want to evaluate NOX2 activation (as assessed by sNOX2-dp serum levels), oxidative stress (as assessed by serum isoprostanes), gut permeability (assessed by plasma zonulin) and LPS levels in patients with NAFLD (n=65) and controls (n=65). Then, we will analyze the same parameters in NAFLD children with or without non-alcoholic steatohepatitis (NASH) and the activation of Nox2 with histological characteristics (steatosis, inflammation, ballooning, fibrosis, NAFLD Activity Score (NAS)).
Expected results: preliminary data from a pilot study showed that compared to controls, NAFLD patients had higher Nox2 activity, isoprostanes, zonulin and LPS levels. These data must be confirmed by this proposed research study. Furthermore, multivariate linear regression analysis will be performed to assess the variables independently associated with sNox2-dp levels. We expect to have, within NAFLD group, significant higher levels of sNox2-dp, isoprostanes, LPS patients in patients with NASH compared to those without NASH.
This study could provides evidences in children with NAFLD about the role of Nox2 activation and the degree of liver damage. Furthermore, the study will analyse the relationship between Nox2 and LPS serum,, that could play a potential role for gut-derived LPS in eliciting systemic Nox2 activation.
This study could provide evidence that children with NAFLD have Nox2 over-activation, which is significantly associated with the degree of liver damage. The close relationship between Nox2 and LPS serum levels could suggest a potential role for gut-derived LPS in eliciting systemic Nox2 activation.
Oxidative stress is believed to play a pivotal role in NAFLD pathogenesis and in the progression from simple steatosis to NASH. Increased oxidative stress in the liver give rise to lipid peroxidation, inflammation and activation of stellate cells leading to fibrosis in NASH. According to the "two hits" theory, proposed by Day and James, after a "first hit" (liver steatosis related to insulin resistance, dyslipidemia and obesity) a "second hit", as increased oxidative stress, is required to develop NASH; however, it is still unclear why simple steatosis may progress to NASH only in some patients.
Nox2 is considered a main cellular source of ROS in humans and its activation has been associated with liver damages. Nox2 has been supposed to be involved in NAFLD pathogenesis. Thus, under conditions of induced liver diseases by bile duct ligation or by methionine-choline-deficient diet, knockout mice deficient p47phox, a cytosolic subunit of Nox2, disclosed attenuated fibrosis and not steatosis.
In accordance with previous studies performed in adults, we found, in the pilot study, increased Nox2 activity in children with NAFLD. In particular, we observed higher sNox2-dp levels in subjects with increased liver damage as those with NASH compared to those without NASH, coincidentally with overproduction of isoprostanes, a marker of oxidative stress. Furthermore, we found in the pilot study a significant correlation between Nox2 activation and histological grading of liver damage as steatosis, inflammation, ballooning, fibrosis and NAS.
Based on this, we want to investigate the mechanism accounting for Nox2-overactivation and focusing on LPS as previous study showed that LPS activates Nox2 via interaction with its receptor TLR4. Thus, increased levels of LPS have been detected in NAFLD patients and experimental studies showed a significant association between LPS and liver damage suggesting LPS as potential trigger of NAFLD2. Experimental data are in favor of this hypothesis because LPS behave as an inflammation trigger via up-regulating TNF and eventually eliciting NAFLD. In accordance with this, TLR4 antagonists reduced TLR4-induced hepatic inflammation in obese mice via reduction of NADPH activation. Furthermore, Kim et al. showed that TLR-4 elicited Nox2 activation in hepatic macrophages and that Nox2-deficient mice were protected against high-fat diet-induced hepatic steatosis and insulin resistance.
We also want to investigate the mechanism accounting for enhanced LPS and focused on changes of gut permeability, which is increased in settings associated with metabolic disorders such as diabetes mellitus and obesity. To explore this issue we will measure serum zonulin, which modulates gut permeability by disassembling the intercellular tight junctions. Experimental and clinical studies demonstrated that zonulin up-regulation plays a role in increasing gut permeability and that serum level of zonulin correlates with enhanced intestinal permeability.
This study could provide further insight about the role of oxidative stress on liver damage in NAFLD; in particular, we want to test if Nox2 is precociously activated and related to histological liver damage in children with NAFLD. Future studies must evaluate the predictive role of this marker on NAFLD development and progression.