Mechanism Of Chronic Intermittent Hypoxia Induced Hepatocyte Dysregulated Autophagy In The Initiation And Development Of NAFLD

Obstructive sleep apnea(OSA)is a sleep disorder characterized by repeated arousals and intermittent hypoxia caused by the repeated collapse of the upper airway during sleep.Non-alcoholic fatty liver disease(NAFLD)is a common chronic liver disease with accumulation of hepatic triglycerides and is closely related to insulin resistance(IR),type 2 diabetes mellitus(T2DM).Chronic intermittent hypoxia(CIH)is an important pathophysiological characteristics of OSA,also is an important risk factor involved in the initiation and development of NAFLD.Previous studies mostly focused on the association between hypoxia-induced factor and NAFLD,while it remains unclear whether CIH-induced imbalance of cell homeostasis is involved in the initiation and development of NAFLD.Autophagy is a cellular adaptive response to stress condition,through different signaling pathways control cell fate,the basic autophagy was benefit for cell survival,while dysregulated autophagy promotes cell apoptosis.The role of autophagy in the pathogenesis of NAFLD is controversial,and the point of CIH-induced autophagy for cell survival are inconsistent in different tissues and organs.Therefore,this study investigated whether CIH-induced dysregulated autophagy,which was involved in the initiation and development of NAFLD.Objective:To explore the association between CIH-induced dysregulated autophagy and NAFLD,and to explore how dysregulated autophagy promotes the incidence insulin resistance of liver.Methods:1.In vivo experiment:Primary hepatocyte and AML12 cells were selected to construct a cellular model of intermittent hypoxia.The expression of autophagy related protein(LC3B/A,p62),endoplasmic reticulum stress(ERS)-related protein(ATF6,IRE1?,p-IRE1?,XBP1,EIF2?,P-EIF2?),and lipid synthesis related gene(SCD-1)were tested.2)Primary hepatocyte were stimulated with oleic acid(OA)and exposed with IH,GSH,SOD activity and the level of MDA were tested,the protein expression of MFN2 was detected.2.In vitro experiment:(1)Liver lipid metabolism:6-week-old male C57BL/6J mice were randomly divided into three groups:CD+Air group,CD+CIH group and CD+CIH+CQ group.The body weight,liver weight,serum and liver triglyceride cholesterol(TG/TC),serum glutamic-oxalacetic transaminase(ALT/AST)were detected.Liver inflammatory cells infiltrating,lipid accumulation and liver fibrosis were detected by HE staining,oil red staining and Masson staining,respectively.Transmission electron microscopy(TEM)was used to observe the changes of ER structure and lipid droplet distribution.The expression of ERS-related,autophagy-,apoptosis-,lipid synthesis-were further detected.(2)The 6-week-old male C57BL/6J mice were fed with high fat diet(HFD)and normal diet(CD)for12 months,and then randomly divided into three groups:normoxic group,CIH treatment for 12weeks,and CIH+CQ treatment for one week.Insulin tolerance test(ITT)was performed on HFD mice after CIH exposure,and the m RNA expression of oxidative stress markers(HO-1,i NOS,NRF2)in liver of mice was measured by q-PCR.Mitochondrial endoplasmic reticulum associated membrane(MAMs)was detected by the expression of MFN2 protein and transmission electron microscopy observation.Mitochondrial fission and damage were detected by the protein expression of Drp1 and Cyot C and transmission electron microscopy(TEM)observation.Results:In vivo experiment:1)The protein level of LC3B/A ratio in primary hepatocyte and AML12 cells increased significantly after IH exposure,the protein level of ERS-related(IRE1a P-IRE1A ATF6)was increased,which were correlated with the LC3B/A ratio.Meanwhile,the protein level of SCD-1 was significantly increased compared with control group,and was correlated with the change of LC3B/A ratio.2)Compared with normoxic group,the GSH,SOD activity in primary hepatocyte exposed to IH after OA stimulation/non-OA stimulation was significantly decreased,and MDA was significantly increased,suggesting that IH induced oxidative stress in liver cells.The expression of MFN2 protein was significantly increased after IH exposure in primary hepatocyte and LO₂cells.In vitro experiment:(1)Resluts in liver lipid metabolism:1)The liver weight,the ratio of liver to body weight,serum TG and TC were increased after CIH exposure,while decreased after CQ treatment.Similarly,the results of serum ALT and AST were in line with serum TG.2)Oil red O staining showed liver steatosis,TEM exhibited an increase in the number of lipid droplets in liver cells,HE staining showed inflammatory infiltration and cell damage,the expression of pro-inflammatory factor(TNF-?IL-6 IL1-?)m RNA were increased after CIH exposure.While the number of lipid droplets and the expression of pro-inflammatory factor m RNA decreased in CD+CIH+CQ group.Masson staining showed no obvious fibrosis among three groups.3)Compared with normoxia group,CIH induced the expression of autophagy related protein(Beclin-1,LC3B/A),ERS-(p-IRE1A/IRE1a,p-EIF2A/e IF2a,ATF6),apoptosis-(cleaved-Caspase-3/caspase-3),while these protein expression were decreased in CIH+CQ group.CIH induced an increase in the m RNA and protein expression of FASn,ACC,SCD-1,while these protein and m RNA expression were decreased in CIH+CQ group.(2)Liver insulin resistance:1)The blood glucose of both CD and HFD mice increased significantly after CIH exposure,and decreased after CQ intervention;ITT showed significant insulin resistance in HFD+CIH group.The levels of oxidative stress markers(HO-1,i NOS,NRF2)m RNA of liver were increased in CD and HFD mice after CIH exposure.2)After CIH exposure,the protein expression of MFN2 were increased in liver of CD and HFD mice,and MAMs enrichment,mitochondrial swelling and mitochondrial ridge disappearance.However,the protein expression of MFN2 was decreased,MAMs enrichment alleviated,and mitochondrial structure recovered after CQ treatment.3)After CIH exposure,the protein expression of Drp1 and Cyot C were increased in liver of CD and HFD mice,and mitochondrial volume decreased,fragmentation,and mitochondrial swelling.However,the protein expression of Drp1 and Cyot C were decreased,mitochondrial fragmentation was alleviated,and mitochondrial morphology recovered.Conclusion:1.CIH induced dysregulated autophagy of hepatocytes and promote hepatocyte ERS;2.CIH induced dysregulated autophagy promoted liver lipid synthesis,liver lipid accumulation,and further promoted liver steatosis;3 CIH induce dyregulated autophagy promote MAMs enrichment,excessive mitochondrial fission,and induced liver oxidative stress and inflammation,and promote liver insulin resistance.