The Mechanisms Underlying Nf-κB Pathway In The Lipid Metabolism Of Macrophages Exposed To Polystyrene Nanoplastics

Objective:To determine the effect of polystyrene nanoplastics(PS-NPs)on lipid level in human macrophages;to clarify the effect of PS-NPs exposure on the expression level of NF-κB pathway and lipid metabolism related genes mRNA and protein in human macrophages,and to explore the mechanism of NF-κB pathway in the effect of PS-NPs on the lipid metabolism of macrophages.Methods:THP-1 cells were cultured in RPMI 1640 medium(containing 10%fetal bovine serum)in vitro,and 200 ng/mL PMA was used to differentiate THP-1 into macrophages.The macrophages of THP-1 source were exposed to 100 nm PS-NPs of different concentrations,and were divided into control group(0 μg/mL),low dose group(100μg/mL),medium dose group(200 μg/mL)and high dose group(400 μg/mL)according to the exposure concentration of PS-NPs.The NF-κB pathway was inhibited by the NFκB inhibitor BAY11-7082,and the inhibition efficiency was determined by Q-PCR.The experiment was divided into blank control group(complete culture solution),solvent control group(0.1%DMSO),PS-NPs exposure group(200 μg/mL),NF-κB inhibitor group(BAY11-7082)and PS-NPs(200 μg/mL)+NF-κB inhibitor group(BAY11-7082).All the above groups were poisoned for 48 hours and then collect cell and culture supernatant.The survival rate of macrophages was measured by CCK-8 method;the lipid level in macrophages was detected by oil red O staining and oil red O extraction;the levels of TG,TC,HDL-C and LDL-C in macrophages were detected by colorimetry;the expression level of IκBα,NF-κB p65,SREBP2,LDLR and HMGCR gene mRNA in macrophages was detected by Q-PCR;Western blot was used to determine the expression protein level of p-IκBα,IκBα,NF-κB p-p65,NF-κB p65,SREBP2,LDLR and HMGCR in macrophages.The levels of TNF-α,IL-1β and IL-18 in macrophages were detected by ELISA;IBM SPSS 24.0 is used for statistical analysis,and the measurement normal data is expressed with "x±S".One-way ANOVA was used to compare the inter-group differences of various indicators.The LSD method was used for comparison between two groups,Pearson correlation analysis was used to analyze the correlation between inflammatory factor levels and lipid metabolism related proteins.Test level was α=0.05。Results:1.The survival rate of macrophages in the 800μg/mL dose group exposed to 100nm PS-NPs was higher than that in the control group(P<0.05).2.After 48 hours of PS-NPs exposure,the levels of TC,TG,HDL-C and LDL-C and the levels of TNF-α and IL-1β increased with the exposure dose(P<0.05).3.After 48 hours of PS-NPs exposure,the levels of IκBα,NF-κB p65,SREBP2,LDLR and HMGCR mRNA expression in the middle-dose group was significantly higher than that in the control group(P<0.05).Compared with the control group,the expression level of p-IκBα,IκBα protein in the low-dose group increased significantly(P<0.05).The expression level of NF-κB p-p65,SREBP2,LDLR and HMGCR protein in the middle dose group was significantly higher than that in the control group(P<0.05).4.After 48 hours of PS-NPs exposure,the intracellular TNF-α level in the middle dose group was significantly higher than that in the low dose group(P<0.05).The level of intracellular IL-1β in low and medium dose group was significantly higher than that in control group(P<0.05).The level of IL-18 in the medium dose group was significantly higher than that in the low dose group(P<0.05).5.NF-κB inhibitor BAY11-7082 significantly inhibited the increase of TG,TC,LDL-C and LDL-C levels of macrophages caused by PS-NPs(P<0.05);NF-κB inhibitor significantly inhibited the increase of TNF-α、IL-1β and IL-18 levels in macrophages induced by PS-NPs(P<0.05);NF-κB inhibitor significantly inhibited the increase of SREBP2,LDLR,HMGCR mRNA and protein expression in macrophages induced by PS-NPs(P<0.05).Conclusions:1.PS-NPs exposure can increase the lipid level in macrophages,and there is a certain dose-effect relationship.2.Exposure to PS-NPs can upregulate the genes expression of IκBα and NF-κB p65 in macrophages,activating the NF-κB signaling pathway in macrophages.3.Exposure to PS-NPs can upregulate the expression of lipid metabolism related genes SREBP2,LDLR,and HMGCR in macrophages,leading to lipid accumulation in macrophages.4.PS-NPs exposure can trigger macrophage inflammatory response,and there is a certain dose-effect relationship.5.Inhibition of NF-κB pathway can significantly inhibit the inflammatory response in macrophages induced by PS-NPs,and downregulate the expression of lipid metabolism related genes,inhibiting the elevation of lipid levels.