Role And Mechanisms Of Endoplasmic Reticulum Stress Pathway In Liver Injury Induced By OSAHS Combined With Obesity

Objective:Obstructive sleep apnea hypopnea syndrome(OSAHS)is a serious sleep disorder.OSAHS has been confirmed to be closely related to the pathogenesis and disease progression of liver diseases,and obesity is the main risk factor of OSAHS.In this study,a mouse model of CIH combined with high fat diet(CIH+HFD)was constructed to simulate obese patients with OSAHS,to explore the effects of OSAHS combined with obesity on mouse liver damage and its possible mechanism.To explore the role of ERS in liver injury caused by OSAHS and obesity by constructing liver cell model of chronic intermittent hypoxia combined with high lipid in mice.To provide a new strategy for the treatment of liver injury caused by obesity and OSAHS.Methods:1.C57/BL6 mice were used to construct CIH and HFD mice models,and the mice were randomly divided into control group(CON group),chronic intermittent hypoxia group(CIH group),high fat diet group(HFD group)and chronic intermittent hypoxia combined with high fat diet group(HH group)according to n=10/ group.2.The weight of mice was monitored.After 14 weeks,the liver weight of mice was weighed.3.The morphological and structural changes of liver tissues in different treatment groups were observed by hematoxide-eosin(HE)staining,Caspase3 positive area was observed by immunohistochemical staining,and the expression of apoptotic protein Cleaved Caspase3 was detected by Western Blot(WB).4.The expressions of inflammatory cytokines(IL-6,TNF-α,TGF-β)in liver tissues of mice were detected by real-time quantitative PCR(RT-q PCR)and WB.5.The m RNA and protein expressions of endoplasmic reticulum stress markers(IRE1α,XBP1 s,JNK,CHOP)and apoptotic factors(Bax,Bcl-2)in liver tissues of mice in different treatment groups were detected by RT-q PCR and WB.6.The model of NCTC mouse liver cells with intermittent hypoxia and hyperlipemia was established,and NCTC cells were randomly divided into CON group,CIH group,hyperlipemia group(PA group),chronic intermittent hypoxia combined with hyperlipemia group(CIH+PA group).7.The expressions of inflammatory cytokines(IL-6,TNF-α,TGF-β)and ERS apoptotic pathway were detected by RT-q PCR and WB.8.Intervening with the ERS inhibitor 4-Phenylbutyric acid(4-PBA),NCTC cells were randomly divided into CON group,CIH+PA group,chronic intermittent hypoxia combined with hyperlipemia and 4-phenylbutyrate sodium salt group(CIH+PA+4-PBA group),the expression of inflammatory factors and ERS apoptosis pathway related genes were detected.Results:1.The mice models of CIH and HFD were successfully constructed in this experiment.The body weight of mice in CIH group was lower than that in CON group,while the body weight of mice in HFD and HH groups was higher than that in CON group,and the body weight of mice in HFD group was the most significant(P<0.05).The liver weight of the other three groups was increased compared with that of the CON group(P<0.05).2.HE staining results showed that the liver structure of CON group was normal.There were vacuoles in the cytoplasm of hepatocytes in CIH,HFD and HH groups,the cell swelling was obvious,and the liver tissue structure was obviously disorganized.Some cell disintegration was also observed in HH group.Immunohistochemical staining results showed that the positive area of Caspase3 in HH group was significantly increased compared with CON group(P<0.05).Results of WB detection showed that the expression level of Cleaved Caspase3 protein in HH group was significantly increased compared with that in CON group(P<0.05).3.RT-q PCR and WB detection results showed that compared with CON group,the expressions of inflammatory cytokines(IL-6,TNF-α and TGF-β)in liver tissues of the other three groups were increased,and the increases were most significant in HH group(P<0.05).4.The expression of genes related to ERS-apoptosis pathway in different treatment groups was further detected,and the results of RT-q PCR and WB detection showed: The expression levels of endoplasmic reticulum related genes(IRE1α,XBP1 s,JNK,CHOP)and apoptosisrelated genes(Bax,Bcl-2)in liver tissues of CIH,HFD and HH groups were increased compared with those of CON group,and the increases in HH group were the most significant(P<0.05).Compared with CON group,the relative expressions of p-JNK/JNK and Bax/Bcl-2 proteins in the liver tissues of the other three groups were also significantly up-regulated(P<0.05).5.In vitro experiment,intermittent hypoxia combined with high lipid NCTC cell model was successfully constructed.RT-q PCR and WB detection results showed that compared with CON group,m RNA and protein expression levels of inflammatory cytokines were increased in the other three groups,and CIH+PA group was the most significantly increased(P<0.05).6.The m RNA and protein expression of ERS-related genes IRE1α,XBP1 s,JNK and CHOP in NXTC cells of CIH+PA treatment group was significantly increased compared with CON group(P<0.05).The m RNA and protein expression of Cleaved Caspase3 was also increased compared with CON group.The Bax/Bcl-2 ratio in CIH+PA group was the highest(P<0.05).After adding 4-PBA,the protein expressions of ERS related genes IRE1α,XBP1 s,JNK and CHOP in 4-PBA treatment group were significantly lower than those in CIH+PA group.Protein expressions of Cleaved Caspase3 was also significantly down-regulated compared with CIH+PA group.The ratio of Bax/Bcl-2 was significantly lower than that of CIH+PA group(P<0.05).Conclusion:In this study,chronic intermittent hypoxia combined with high fat was successfully constructed in animal and cell models.It was found that chronic intermittent hypoxia combined with high fat up-regulated apoptosis through the ERS pathway,stimulated inflammatory response,and led to severe liver cell damage.