Chronic intermittent hypoxia exacerbates the pathogenesis of non-alcoholic fatty liver disease : a mechanistic study in rats and HepG2 cells

Abstract

The obstructive sleep apnea (OSA) syndrome is characterized by recurrent obstruction of the upper airway during sleep leading to intermittent hypoxia (IH). Non-alcoholic fatty liver disease (NAFLD) is the most common chronic fatty liver disease, which ranges from steatosis to non-alcoholic steatohepatitis (NASH) and liver cirrhosis. The prevalence of OSA is high in patients with NAFLD. Emerging evidence suggests that OSA may be involved in the progression of NAFLD. However, the mechanistic effect of IH on the pathogenesis of NAFLD remains elusive. It was hypothesized that IH could aggravate oxidative stress, inflammation and apoptosis induced by high fat diet (HFD) at an initial stage of pathogenesis of NAFLD in the rat liver. After HFD and IH treatment, remarkably elevated levels of liver injury (ALT, AST and histological study), inflammatory mediators (IL-6, TNF-α and IL-1β), oxidative stress, apoptosis and autophagy were observed in the co-treatment group than other groups. Besides, NADPH oxidase (NOX4) and hypoxia-inducible factor 1α (HIF-1α) expression levels were aggravated by IH in the co-treatment group, suggesting a significant effect of IH on aggravating the early pathogenesis of NAFLD. Up to date, the underlying mechanism of oxidative stress and reactive oxygen species (ROS) on the pathogenesis of NAFLD aggravated by IH is still unknown. We hypothesized that IH aggravates inflammation and apoptosis induced by sodium palmitate (SP) in HepG2 cells via oxidative stress. MnTBAP chloride is a cell permeable superoxide dismutase (SOD) mimetic and ROS scavenger. Results showed that the levels of ROS production, oxidative stress, lipid accumulation, inflammation, apoptosis, NOX4 and HIF-1α were aggravated in the IH and SP co-treatment group. These deteriorated effects of IH could be attenuated by the antioxidant MnTBAP, suggesting the IH exacerbates the pathogenesis of NAFLD induced by palmitate via oxidative stress. NOX is an important source of ROS. Apocynin effectively inhibits the activity of NOX. Our results demonstrated apocynin significantly mitigated the deteriorated effects of IH on the lipid accumulation, inflammation and apoptosis induced by palmitate in HepG2 cells. Moreover, the elevated expression levels of HIF-1α and HIF-2α in the co-treated group were also attenuated by apocynin, suggesting a vicious cycle mediated by the HIF/NOX cascade. Thus, NADPH oxidase could worsen the oxidative stress exacerbated by IH which aggravates the pathogenesis of NAFLD. Further, results from knockdown of NOX4 with siRNA also showed that IH aggravates oxidative stress induced by palmitate via NOX4. Toll-like receptor 4 (TLR4) could be activated by fatty acids and induce inflammation. Our results showed that TLR4 knockdown attenuated the inflammation aggravated by IH in the co-treated group, suggesting IH-induced oxidative stress aggravates inflammation induced by sodium palmitate in HepG2 cells via TLR4. Our studies also showed caveolin1 knockdown attenuated the lipid accumulation aggravated by IH in the cells treated with palmitate. In conclusion, NADPH oxidase especially NOX4 could worsen the oxidative stress induced by IH which aggravates the pathogenesis of NAFLD. Caveolin1-mediated lipid accumulation, TLR4-induced inflammatory response and a vicious cycle mediated by the HIF/NOX cascade might play crucial roles in the pathogenesis of NAFLD.