The Association Of Obstructive Sleep Apnea Hypopnea Syndrome With Liver Injury And The Underlying Mechansims

Purpose: Obstructive sleep apnea hypopnea syndrome(OSAHS)is a common sleep breathing disorder which can cause multiple organ injury.Some studies have indicated that the liver is one of the target organs for systemic damage caused by OSAHS.OSAHS often coexists with obesity,which is an important risk factor for liver injury.Some studies suggested that the association between OSAHS and liver injury became non-significant after adjusting for confounding factors such as obesity.Overall,the sample size of most of the related studies is small and the conclusions yielded are not consistent.Animal studies have shown that chronic intermittent hypoxia(CIH)led to liver injury and increased hepatic oxidative stress,suggesting that oxidative stress might be involved in OSAHS-related liver injury.However,the exact mechanisms are still unclear.Ferroptosis is a form of regulated cell death resulting from iron accumulation and lipid peroxidation.Ferroptosis has been implicated in pathophysiologic process such as liver,heart,and kidney ischemia/reperfusion(I/R)injury.Considering that the recurrent intermittent hypoxia(IH)in OSAHS closely mimics what is seen in I/R injury,we hypothesized that the related mechanisms were dys-regulated and then contributed to the occurrence of ferroptosis in OSAHS-related liver injury.Therefore,this study was mainly focused on the association of OSAHS with liver injury and the underlying mechanisms.Firstly,we evaluated the correlation between OSAHS and liver injury by collecting and analyzing a large sample of clinical data;secondly,we explored the role of ferroptosis in CIH-induced liver injury and potential molecular mechanisms by vitro and vivo experiments.Methods: 1.1085 patients,who were referred to our sleep centers because of symptoms of snoring with or without witness episodes of sleep apnea and daytime sleepiness between November 2015 to November 2019,were included to the study.They were divided into control group,mild to moderate OSAHS group,and severe OSAHS group.Spearman correlation was used to analyze the correlation between sleep parameters and liver injury,and multivariate logistic regression was used to explore independent risk factors for liver injury.These clinical data were further stratified according to obesity,gender,and age,to explore the correlation between OSAHS and liver injury in different subgroups.2.14 male SD rats were randomly allocated to either the normoxia control(NC)or CIH group.Rats were exposed to intermittent hypoxia(IH)for 8 weeks in CIH group.Liver function,histological changes,and markers of oxidative stress(malondialdehyde)were evaluated.The protein levels of hypoxia-inducible factor-1α(HIF-1α),nuclear factor E2-related factor 2(Nrf2),Acyl-Co A synthetase long-chain family member 4(ACSL4),and glutathione peroxidase 4(GPX4)in liver were examined by western blot analysis.3.IH BRL-3A cells model was established.Cell viability was measured at different IH exposure time thereafter.The appropriate IH exposure period was chosen for further cell experiments.Ferroptosis was assessed by detecting cell viability,reactive oxygen species(ROS),total iron content,and protein expression of GPX4 and ferritin heavy chain 1(FTH1).CCK8 and western blot analysis were used to detect cell viability and protein expression,respectively.Ferroptosis was evaluated for BRL-3A cells treated with ferroptosis inhibitor Ferrostatin-1 under IH exposure.Finally,ferroptosis was evaluated for BRL-3A cells with Nrf2 knockdown under IH exposure.4.24 male C57 mice were randomly allocated to NC,CIH group,or CIH plus N-acetylcysteine(NAC).The period of CIH exposure was 12 weeks.Liver function,histological changes,liver iron content,and markers of oxidative stress(malondialdehyde,glutathione)were evaluated.The protein levels of Nrf2,FTH1,and GPX4 in liver were examined by western blot analysis.Results: Liver function parameters including alamine aminotransferase(ALT),aspartate aminotransferase(AST),ALT/AST,gamma glutamyltransferase value increased significantly with the increase of OSAHS severity(p<0.05).The three groups did not differ in terms of total bilirubin or alkaline phosphatase(p>0.05).Spearman correlation analysis showed that oxygen desaturation index(ODI),apnea hyponea index(AHI),the percentage of sleep time with Sp O2 <90%(T90%),lowest oxyhemoglobin saturation(La SO2),average oxyhemoglobin saturation(average Sp O2)were significantly correlated with ALT and AST.Multivariate logistic regression showed that higher body mass index(BMI),triglycerides,fasting blood glucose,and OSAHS were independent risk factors for liver injury.After adjusting for confounding factors,the risk of liver injury in both mild to moderate OSAHS group and severe OSAHS group was higher than that of control group,by 0.705 times(OR=1.705,95%CI=1.105-2.631,p=0.016)and 1.394 times(OR=2.394,95%CI=1.490-3.848,p<0.001),respectively.2.Subgroup analysis based on gender suggested that higher waist circumference,triglycerides,Epworth sleepiness scale(ESS)scores,and OSAHS were independent risk factors for liver injury in the male group(p<0.05).In the female group,higher BMI and fasting blood glucose were independent risk factors for liver injury(p<0.05),while OSAHS was not an independent risk factor for liver injury(p>0.05).Subgroup analysis based on BMI suggested that higher fasting blood glucose and OSAHS were independent risk factors for liver injury in the obese group(p<0.05).In the non-obese group,higher BMI,triglycerides,and OSAHS were independent risk factors for liver injury(p<0.05).Subgroup analysis based on age suggested that antihypertensive and OSAHS were independent risk factors for liver injury in the elderly group(p<0.05).In the non-elderly group,higher BMI,fasting blood glucose,ESS scores,and OSAHS were independent risk factors for liver injury(p<0.05).3.Compared with the NC group,the serum ALT and AST of the CIH group were significantly increased.Normal hepatic architecture was observed in the liver tissues of NC group.There was significant liver injury evidenced by discernible swelled cells,necrosis,and infiltrated inflammatory cells(lymphocyte)in CIH group.The serum and liver malondialdehyde level of rats in the CIH group were significantly higher than NC group.CIH exposure significantly reduced the GPX4 protein expression and increased ACSL4 protein expression in rat liver.In addition,the protein expression of Nrf2 in the liver of the CIH group was significantly lower than that of the NC group,while the liver HIF-1α protein expression level in the CIH group was significantly higher than that of NC group.4.The IH exposure time was significantly correlated with reduction of cell viability.48 h IH treatment significantly induced a decrease in cell viability,an increase in lipid ROS level and total iron content,and a decrease in GPX4 and FTH1 protein expression levels in BRL-3A cells.A significant increase in Nrf2 protein expression also was observed after 48 h IH exposure.Treatment with the ferroptosis inhibitor Ferrostatin-1significantly reversed IH-induced ferroptosis of BRL-3A cells,which evidenced by the increase of cell viability,decrease of lipid ROS level and total iron content,and increase of GPX4 and FTH1 protein expression levels.Silencing Nrf2 significantly augmented IH-induced ferroptosis of BRL-3A cells,which evidenced by a decrease of cell viability,an increase in lipid ROS levels and total iron content,and a decrease in GPX4 and FTH1 protein expression levels.5.NAC treatment significantly alleviated the elevation of serum ALT and AST,and liver pathological injury caused by CIH in mice.The serum and liver malondialdehyde levels and total liver iron content of CIH group were significantly higher than NC group.When compared with CIH group,these were signicantly decreased in CIH plus NAC group.The signicant reduction of serum and liver glutathione levels were observed in CIH group,and NAC treatment resulted in a significant increase of serum and liver glutathione levels.NAC treatment significantly reversed the reduction of protein expression of Nrf2,GPX4,and FTH1 caused by CIH in mice.Conclusions: 1.OSAHS was an independent risk factor for liver injury.After adjusting for confounding factors,the risk of liver injury in both mild to moderate OSAHS group and severe OSAHS group was higher than that of control group,by 0.705 times and1.394 times,respectively.2.OSAHS was an independent risk factor for liver injury in male population,however,this was not observed in female population.OSAHS was an independent risk factor for liver injury,which was not affected by age or BMI.3.The CIH animal model showed that CIH caused rat liver injury,increase of liver and systemic oxidative stress,occurrence of liver ferroptosis,and decrease of liver Nrf2 protein expression.4.The IH cells model indicated that IH caused BRL-3A cells ferroptosis and ferroptosis inhibitor could reverse the IH-induced ferroptosis.IH led to increased expression of Nrf2 in BRL-3A cells.Silencing Nrf2 augmented the IH-induced ferroptosis in BRL-3A cells.5.NAC could reverse CIH-induced hepatic ferroptosis and liver injury in mice,which might be mediated by enhancing glutathione content and expression of Nrf2.