Pathogenesis Of Abnormal Hepatic Lipid Metabolism Induced By Chronic Intermittent Hypoxia In Rats And The Therapeutic Effect Of N-acetylcysteine

Obstructive sleep apnea(OSA)affects multiple body systems.Its pathophysiological basis is chronic intermittent hypoxia(CIH)/reoxygenation.This leads to nocturnal hypoxemia and the generation of oxygen free radicals through ischemia-reperfusion injury,with the onset of both local and systemic inflammatory responses that cause liver,lung,and cardiovascular damage.Clinical and basic studies have confirmed that severity of hypoxia in OSA patients is closely related to abnormal hepatic lipid metabolism,fatty liver formation,and elevated blood lipid levels.Animal models have indicated that CIH can cause injury in multiple organs in rats,with different organs showing varying sensitivities to CIM.In this study,we aimed to investigate the molecular and cellular effects of CIH on hepatic lipid metabolism in rats and its underlying pathogenesis.We also investigated its mechanism of action by drug intervention experiments to provide a theoretical basis for the clinical treatment and prevention of OSA syndrome.Current studies on the protective effect of NAC focused mainly on the respiratory,cardiovascular,and central nervous system aspects.NAC(N-acetylcysteine)as an antioxidant,within the prescribed scope can be safely used in humans and animals,has obvious effect to stop the production of oxygen free radicals,remove the existing free radicals,remove the existing free radicals,remove the existing free radicals,regulate cell metabolism,prevent cell damage,regulation of gene expression and signal transduction system,etc.At present,studies on NAC mainly focus on the respiratory system,cardiovascular system and other aspects,and there are few reports on the role of NAC in the digestive system such as liver.Since OSAHS has a multi-system impact on human body and CIH has a comprehensive impact on animals,we speculate that metabolic abnormalities and damage may occur in rat liver induced by CIH.Since NAC has positive an d extensive therapeutic effects,we speculate that it may also have positive protective and therapeutic effects in liver.The main purpose of this study was to explore the damage mechanism of rat liver under CIH and the therapeutic effect of NAC.Due to the limitation of ethical and moral factors,the basic research of pathogenesis and pathophysiology is impossible to experiment with people themselves.Simply by relying on accumulation of experience and knowledge in clinical medicine,there are very great limitations.Therefore it is very important to conduct experimental study.The establishment of a model of chronic intermittent hypoxia(CIH)is essential for animal studies on OSA.Since the first report of a rat hypoxia-reoxygenation model by Fletcher in 1992,many similar animal models have been developed during the last 20 years,each with their own characteristics,but it is still explored and developed.Through many years of research and with the support of an electronic equipment engineer,we have established a complete set of chronic intermittent hypoxia models appropriate for animal and cell studies.This set of chronic intermittent hypoxia models helped us successfully complete several experimental studies.It is a reliable and effective experimental platform for research.Part I Establishment and significance of an animal model of chronic intermittent hypoxiaObjective:Rats were selected as experimental animals for establishing an animal model of chronic intermittent hypoxia to provide a reliable experimental platform for basic research on OSA.Methods Preparation of hypoxia chamber system:A color touch screen and programmable logic controller(PLC)automatic control and an automated data acquisition and monitoring system were used to create the equipment needed to establish the experimental model.The hypoxia chamber consists of the following three main components:main unit section,hypoxia chamber and gas line control.Model establishment:The 30 rats were randomly divided using the random number table into four groups with 15 rats each:normoxic control(CON)and CIH model(CIH).Group CON was been put into the normoxic control chamber.The rats were exposed for one week to the same experimental conditions of 23 ?,50%humidity,and normal feeding.The hypoxia chamber was set to a low oxygen concentration of 5%for 40 s and a normoxic period of 40 s(both with a buffering period of 5 s)in a hypoxia-normoxia-hypoxia cycle for 8 h daily from 09:00 to 17:00 for nine weeks.Sample collection and processing:Rats were given a normal feeding.After 9 weeks,the rats were dissected,and liver samples were collected for hematoxylin and eosin(HE)staining,transmission electron microscopy,and Oil Red "O" staining.The HE stained tissue sections were mounted prior to observation under a light microscope.Electron microscopy staining was subjected to observation under a TEM.Oil Red "O"stained tissue sections were mounted prior to light microscopy and quantification using the Image-Pro Plus software.Results 1.Morphological changes:HE staining in the CIH group showed lipid accumulation and lipid vacuole formation leading to hepatocyte steatosis.Microscopy of the ultrastructure showed large clusters of fat droplets and the appearance of large numbers of autophagosomes and lysosomes,focal accumulation of glycogen in the rough endoplasmic reticulum,and sparse microvilli in the bile canaliculi(see labels in figure).These were rarely seen in the CON group.Hepatocyte morphology in the CIH group underwent obvious changes in this model.2.Oil Red O staining results:Red granules are lipid-like substances.The difference in IOD/Area between the CIH group and the CON group was statistically significat(p<0.05).Hepatocytes in the CIH group were obviously stained red,whereas hepatocytes in the CON group stained mildly red,showing the difference between the two groups of samples produced by the model.3.CIH animal model establishment results:Observation and study of the hepatocyte morphology of rats in the CIH group and the CON group showed that there was obvious cell injury,supporting the pathological signs of liver tissue undergoing functional impairment under CIH and also confirming that the CIH model we have established is effective and reliable..Conclusion1.A rat model of CIH in OSA was successfully established.2.The model rats exhibited the pathophysiological characteristics of OSA.3.The established model is as a reliable experimental platform for further OSA research.Part II Pathogenesis of abnormal hepatic lipid metabolism induced by chronic intermittent hypoxia in rats and the therapeutic effect of N-acetylcysteineObjective The pathogenesis of chronic intermittent hypoxia(CIH)-induced abnormal hepatic lipid metabolism in rats remains unclear.Here,we investigated the therapeutic effect of N-acetylcysteine(NAC)on abnormal hepatic lipid metabolism.Material and Methods Model establishment:The 60 rats were randomly divided using the random number table into four groups with 15 rats each:normoxic control(CON),CIH model(CIH),CIH + 0.9%NaCl model control(CIH+NS),and CIH +N-acetylcysteine treatment(CIH+NAC).The rats were exposed for one week to the same experimental conditions of 23 ?,50%humidity,and normal feeding.The hypoxia chamber was set to a low oxygen concentration of 5%for 40 s and a normoxic period of 40 s(both with a buffering period of 5 s)in a hypoxia-normoxia-hypoxia cycle for 8 h daily from 09:00 to 17:00 for nine weeks.Rats in groups CIH+NAC and CIH+NS were injected intraperitoneally with NAC + NS(20 mg/Kg/d,1%)and an equal volume of NS daily 15 min prior to hypoxic treatment for a total of nine weeks,respectively.Their initial and final body weights(g)were recorded.Detection of histological samples:The rats were given a normal feeding.After 9 weeks of experiment,the rats were weighed and their pathological changes with regard to the hepatic lipid metabolism were observed via HE and Oil Red "O" staining while their hepatocyte ultrastructures were observed under electron microscopy.The oxidative stress level of hepatocytes was assessed using Dihydroethidium(DBE)probe.Besides,the expression level of nuclear factor-kappa B(NF-?B)and inflammatory:cytokines(IL-1?,IL-6 and TNFa)in each group were determined via Western blotting,Real-time PCR and immunohistochemistry(IHC)testing while the serum lipoprotein lipase(LPL)was examined using enzyme-linked immunosorbent assay(ELISA)and blood lipid(TG and TC)levels were examined using Peroxidase Antiperoxidase Method(PAP).Results Compared with the normoxic control group(CON group),rats in the hypoxic model group(CIH group)showed a significant body weight gain(P<0.05)with abnormal hepatic lipid metabolism,lipid vacuolization,accumulation of lipid droplets,as well as formation of abundant autophagosomes and lysosomes.Besides,the oxidative stress,inflammation level and blood lipid levels increased significantly(P<0.05)whereas the LPL level reduced(P<0.05)significantly.Compared with the model control group(CIH+NS group),rats in treatment group(CIH+NAC group)had a normal body weight gain(P<0.05)with an improved lipid metabolism,reduced lipid droplets,alleviated ultrastructural injuries,decreased oxidative stress and inflammation level(P<0.05),as well as elevated LPL level and reduced blood lipid level(P<0.05).Conclusions1.Rat liver tissue generates ROS via oxidative stress under CIH.2.CIH activates NF-?B signaling and regulates cytokine expression.3.CIH upregulates serum TG and TC levels,and downregulates LPL levels.4.CIH causes hepatocyte lipid accumulation and injury,and abnormal body weight gain.5.The harmful effects of CIH on rat liver are possibly associated with the reactive oxygen species(ROS)/NF-?B signaling pathway.6.NAC is capable of attenuating lipid metabolism alterations and abnormal body weight gain in the CIH rat model,via a possible mechanism related to inhibition of ROS/NF-?B signaling.