Study On Lipid Metabolism Regulation Of Alveolar Macrophage In Silicosis By PPAR?-CD36 Signaling Pathway

Objectives The study established a foam cell model after silica(SiO2)stimulation by culturing rat alveolar macrophage lines NR8383 cells in vitro.The aim of this research was to explore the possible role on lipid metabolic regulation and control mechanism in alveolar macrophage by peroxidase scion activated receptor(PPAR?)-CD36 pathways,in order to provide new ideas for the prevention and treatment of silicosis.Methods Rat alveolar macrophage NR8383 cells were cultured in vitro,cell proliferation toxicity detection kit(MTS)was used to determine the concentration of SiO2 and ox-LDL stimulation;cells were randomly divided into the control group(conventional culture cells),SiO2 group(50?g/ml SiO2 stimulation),ox-LDL group(50?g/ml ox-LDL stimulation)and model group(50?g/ml SiO2 and ox-LDL stimulation).The effect of ox-LDL on cell viability was detected by MTS.Lipid accumulation was observed by oil red O staining.Total cholesterol(TC)and free cholesterol(FC)was detected by TC and FC kits,and calculated the cholesterol ester(CE)expression.The mRNA and protein expressions of PPAR? and CD36 were detected by real-time fluorescence quantitative PCR(RT-PCR)and Western blotting,respectively.The fluorescence intensity of PPAR? and CD36 were observed under Laser confocal microscope.The expression of TGF-? in supernatants was tested by enzyme-linked immunosorbent assay(ELISA).The macrophage foam cell model was established and treated with SSO inhibitor to block the CD36 pathway.The cells were randomly divided into four groups: SSO inhibitor group(5?M SSO group),solvent control group(DMSO group),SSO experimental group(SSO + SiO2 + ox-LDL)and model group(DMSO + SiO2 + ox-LDL)were cultured for 36 h.The above experimental methods were used to observe the expression of CD36 and PPAR? and the foaming status.The PPAR? signal pathway was blocked by GW9662,a specific inhibitor of PPAR?,and was divided into GW9662 inhibitor group(5?M GW9662 group),solvent control group(DMSO group),GW9662 experimental group(GW9662+SiO2+ox-LDL group)and model group(DMSO+ SiO2+ox-LDL group),incubated for 36h;the same above experimental indicators were observed and tested.Results 1 MTS determine the concentration of SiO2 and ox-LDL: after SiO2 stimulated for 36 h,the cell viability was significantly decreased(p<0.05)after 50?g/ml of SiO2.The pretreatment concentration of SiO2 was 50?g/ml;When ox-LDL was added,the cell survival rate reached the maximum at 50?g/ml compared with the control,and the difference was statistically significant(p<0.05).The ox-LDL concentration was 50?g/ml.Oil red O staining showed that compared with the control group,a large number of red lipid droplets and phagocytic SiO2 particles were found in the cytoplasm of SiO2 group,ox-LDL group and model group,of which the model group was more obvious.TC and FC in model group were higher than those in control group,SiO2 group and ox-LDL group(p<0.05),and CE/TC was significantly greater than 50%(p<0.05).Compared with control group,SiO2 group and ox-LDL group,the protein and gene expression of PPAR? and CD36 in model group were significantly increased(p<0.05).5 Immunofluorescence observed in the model group intracellular PPAR? and CD36 expression of fluorescence intensity were significantly higher than the control group,SiO2 group and ox-LDL group.Compared with model group,the amount of TGF-? released by cells in control group,SiO2 group and ox-LDL group was lower,and the differences were statistically significant.7 Inhibitor SSO blocking CD36 pathway,the experimental results show: foam cell model with SSO inhibitor,intracellular CD36 mRNA and protein were significantly reduced(p<0.05);compared with the model group,intracellular PPAR? mRNA expression was significantly depressed(p<0.05),while the protein had no statistical significance(p<0.05).Under confocal laser scanning microscope,the fluorescence intensity of CD36 in SSO group was significantly lower than that in model group.The contents of TC and CE in the model group were significantly lower than those in the model group(p<0.05).There was no significant difference between the SSO inhibitor group and the solvent control group(p<0.05),and the CE/TC values were less than 50%.Compared with the model group,the expression of TGF-? in the supernatant of SSO group was significantly decreased(p<0.05).8 Inhibitor GW9662 blocked the PPAR? pathway.Compared with the model group,the expression of CD36 mRNA and protein in GW9662 group was significantly decreased with the decrease of PPAR? expression(p<0.05).Further it was confirmed that GW9662 experimental group intracellular PPAR? red fluorescence was weaker than the model group by laser confocal microscopy confirmed,CD36 fluorescence intensity decreased accordingly.Compared with model group,the content of TC in experimental group,inhibitor group and solvent control group decreased significantly(p<0.05),and CE/TC in experimental group,inhibitor group and solvent control group were less than 50%(p<0.05).At the same time,the number of positive foam cells in the experimental group decreased significantly.Compared with the model group,the expression of TGF-? in the supernatant of GW9662 group was significantly decreased(p<0.05).Conclusions 1 SiO2 could promote macrophages dyslipidemia,exacerbate cell foaming,also release cytokines TGF-?.2 PPAR? may participate in the expression of CD36 induced by free SiO2.3 Free SiO2 enhanced ox-LDL-induced cellular lipid metabolism disorder by PPAR?-CD36 pathway,producing foam cells and involved in pulmonary fibrosis.