The Role And Mechanism Of ANGPTL4 And Functional SNP T266M In Dyslipidemia Of OSAHS

Objective:Obstructive sleep apnea hypopnea syndrome（OSAHS）is a chronic sleep apnea respiratory disorder,and lipid metabolism disorder is one of the important biological indicators to determine the severity of OSAHS.Angiopoietin like proteins 4（ANGPTL4）is an important cytokine secreted by liver cells involved in lipid regulation.Previous studies have found that ANGPTL4 and its functional single nucleotide polymorphism rs1044250（SNP,T266M）are highly correlated with dyslipidemia in OSAHS patients,but the mechanism is unclear.Therefore,this study explores the role and mechanism of ANGPTL4 on lipid metabolism disorders in intermittent hypoxia models,providing new ideas for the treatment of OSAHS.Methods:1.To determine the effects of hypoxia on lipid metabolism and the expression levels of ANGPTL4 and related lipid metabolism enzymes.1.1 Use the human liver cell line Hep G2 to establish the model of cell hypoxia chamber culture,which the normal oxygen condition culture and 1%O₂ condition culture were given,respectively.The general growth of Hep G2 cells was observed under a common optical microscope;1.2 Using oil red O staining and Nile red staining to detect the accumulation of lipid in Hep G2 cells;1.3 The m RNA and protein expressions of ANGPTL4 and related lipid metabolism enzymes,acetyl-Co A carboxylase alpha（ACACA）,3-hydroxy-3-methylglutaryl coenzyme A reductase（HMGCR）,and lipoprotein lipase（LPL）,were analyzed by RT-q PCR and western blot.2.Explore how overexpression of ANGPTL4 affects the intracellular lipids of Hep G2 cells under hypoxic conditions.2.1 Construction of ANGPTL4 overexpression cell line（PGMLV-CMV-H-ANGPRL4-6×His-EF1-Zs Green1-T2A-Puro）,ANGPTL4 overexpression cell line（PGMLV-CMV-T266M-6×His-EF1-Zs Green1-T2A-Puro）and control cell line（PGMLV-CMV-EF1-Zs Green1-T2A-Puro）lentivirus;2.2 The expression of ANGPTL4 and related lipid metabolism enzymes ACACA,HMGCR and LPL in cells after hypoxia treatment was detected by RT-q PCR and western blot;2.3 Collect the overexpressing ANGPTL4 Hep G2 cells after 24 hours of hypoxia treatment,and detect the lipid accumulation in Hep G2 cells by Nile red staining;2.4 The lipid accumulation in ANGPTL4-Hep G2 cells after 24 hours of hypoxia was detected by oil red O staining.3.Identify the effect of knocking down ANGPTL4 on the intracellular lipids of Hep G2cells under hypoxic conditions.3.1 Construction of Knock-Down ANGPTL4 cell line（PGMLV-Zs Green1-Puro）and control cell line（PGMLV-SC5-Scable）was established by lentivirus;3.2 The expression of ANGPTL4 and related lipid metabolism enzymes ACACA,HMGCR and LPL in cells after hypoxia treatment was detected by RT-q PCR and western blot;3.3 Collect cells treated with hypoxia for 24 hours,then detect the accumulation of lipid in Hep G2 cells by Nile red staining;3.4 The lipid content in Hep G2 cells after 24 h hypoxia was detected by oil red O staining.4.Study about the mechanism of ANGPTL4 on lipid metabolism disorder.4.1 Cellular immunofluorescence was uesed for co-localization of ANGPTL4 and ACACA;4.2verify the interaction between ANGPTL4 and ACACA through Immuno-coprecipitation.Results:Hypoxia caused disorder of intracellular lipid metabolism,resulted in accumulation of lipid content.Compared with normoxia group,ANGPTL4 was highly expressed after hypoxic treatment,and the expression of ACACA and HMGCR protein related to lipid metabolism was increased,while the expression of LPL protein was decreased;Overexpression of ANGPTL4 in normoxic condition caused cell lipid accumulation,and the expression of ACACA and HMGCR proteins related to lipid metabolism increased,while the level of LPL protein decreased;In hypoxic state,the accumulation of lipid was increased,and the intracellular lipid content was improved after transfection with T266M.In normoxic environment,after knocking down the expression of ANGPTL4,the intracellular lipid content decreased.In hypoxic environment,the intracellular lipid content increased inversely,and the disorder of lipid metabolism intensified.Conclusion:1.Hypoxia can cause an increase in ANGPTL4 expression and induce lipid metabolism disorders.2.Overexpression of ANGPTL4 increases lipid accumulation in Hep G2 cells,and T266M can improve lipid accumulation caused by hypoxia.3.Under hypoxic conditions,ANGPTL4 upregulates ACACA to increase lipid synthesis and inhibit LPL activity to reduce lipid decomposition,leading to lipid accumulation and abnormal lipid metabolism in Hep G2 cells.This provides a theoretical basis for the mechanism research and prevention of OSAHS disease.