Effect of heme oxygenase-1-mediated atorvastatin on macrophage polarization and cholesterol accumulation / 中国组织工程研究

ABSTRACT

BACKGROUND:Studies have shown that atorvastatin can up-regulate the expression of heme oxygenase-1 and enhance the anti-inflammation and anti-oxidative damage ability of cells.However,whether atorvastatin can regulate macrophage polarization,inhibit inflammation and reduce cholesterol accumulation by inducing heme oxygenase-1 expression remains unclear. OBJECTIVE:To investigate the effect of atorvastatin on polarization,inflammation and cholesterol content of oxidized low-density lipoprotein stimulated RAW264.7 macrophages by inducing heme oxygenase-1 expression and its related mechanism. METHODS:Firstly,RAW264.7 cells were randomly divided into six groups and incubated with different concentrations of atorvastatin for 24 hours.The expression of heme oxygenase-1 protein and cell activity were detected to explore the optimal dose of atorvastatin for subsequent studies.RAW264.7 cells were randomly divided into control group,atorvastatin group and heme oxygenase-1 inhibition group.Cells were preincubated with pure medium,atorvastatin 20 μmol/L and atorvastatin 20 μmol/L + zinc protoporphyrin IX 10 μmol/L for 24 hours,and then oxidized low-density lipoprotein 50 mg/L was added for 48 hours.The polarization of macrophages was detected by flow cytometry.The secretion of inflammatory factors such as transforming growth factor β,interleukin 10,interleukin 1β,and tumor necrosis factor α was detected by ELISA.The expression levels of heme oxygenase-1,LC3II,LC3I,P62,PPARγ and ABCA1 were detected by western blot assay.The intracellular cholesterol content was measured with the oxidose method and the accumulation degree of intracellular lipid droplets was evaluated by oil red O staining. RESULTS AND CONCLUSION:(1)Atorvastatin could induce the expression of heme oxygenase-1 protein in macrophages in a dose-dependent manner.(2)Oxidized low-density lipoprotein could induce macrophages to polarize towards M1,secrete proinflammatory factors,and increase the accumulation of intracellular cholesterol.(3)Compared with the control group,the heme oxygenase-1 protein expression of macrophages was increased after atorvastatin intervention,and the cells turned to M2-type polarization and mainly secreted anti-inflammatory factors such as transforming growth factor-β and interleukin-10.PPARγ,ABCA1,LC3II/I and other signal molecules reflecting cholesterol efflux and autophagy increased,and the contents of intracellular cholesterol and lipid droplets decreased significantly(P<0.05).(4)The heme oxygenase-1 inhibition group treated with zinc protoporphyrin IX significantly reversed the above changes in the atorvastatin group.(5)The results have shown that atorvastatin may promote the polarization of macrophages stimulated by oxidized low-density lipoprotein to M2 type and inhibit inflammation by up-regulating the expression of heme oxygenase-1 and by up-regulating PPARγ/ABCA1 signaling pathway and enhancing autophagy.Atorvastatin can increase the outflow of intracellular cholesterol and reduce the accumulation of intracellular lipids.