Effect And Potential Mechanisms Of TREM2 On Cholesterol Efflux And Inflammatory Phenotype Of Foam Macrophages Under High-Glucose Condition

Background and Objective:Atherosclerosis cardiovascular disease(ASCVD)is the primary disease that harms human health at present.The pathological basis of atherosclerosis is cholesterol accumulation in arterial wall and chronic inflammatory state.Diabetes mellitus is an independent risk factor for the occurrence and development of atherosclerosis,which is characterized by the disorder of blood glucose and blood lipid metabolism.ASCVD is the main cause of death in diabetic patients.Atherosclerosis of diabetes occurs early and diffusely,and cholesterol accumulation and inflammatory phenotype in the lesions are particularly prominent,which is the difficulty of treatment at present.Our previous research has confirmed that high glucose can promote the aggregation and phenotypic transformation of inflammatory monocyte subsets and macrophages in plaques,and promote the occurrence and development of diabetic atherosclerosis,but the mechanism needs to be further clarified.Recently,it has been found that the trigger receptor-2(TREM2)expressed on myeloid cells is the main signal hub for regulating the disorder of lipid metabolism and inflammatory response,which induces different cell phenotypes and functions at the cellular level in different environments and participates in inducing phagocytosis,lipid metabolism and inflammatory response.Therefore,this study established a foam macrophage model induced by oxidized low density lipoprotein(OX-LDL),and explored the effects of TREM2 on cholesterol efflux and inflammatory phenotype of foam cells under high glucose conditions and its related regulatory mechanisms.Research Methods:1.Cultured mouse peritoneal macrophages RAW264.7,and after adhering to the wall,the cells were incubated with D-glucose media containing 5.5m M(normal concentration),15 m M(medium concentration)and 35 m M(high concentration)for 24 hours,and then the foam macrophage model was established by using the above three different sugar concentration media containing 50ug/ml Ox-LDL for 24 hours;2.Oil Red O staining was used to observe the morphological changes of cells,and the contents of cholesterol in macrophages under normal glucose concentration(control group)and foam macrophages under normal glucose concentration,medium glucose concentration and high glucose concentration were detected;3.NBD cholesterol combined with fluorescent enzyme-labeled instrument was used to detect the cholesterol outflow of foam macrophages in different sugar concentration groups;4.The mRNA of foam macrophages was extracted,and the mRNA expression levels of TREM2 and cholesterol efflux related genes: ATP-binding cassette transporter A1(ABCA1)and ATP-binding cassette transporter G1(ABCG1)in different sugar concentration groups were measured by real-time fluorescence quantitative technique(RT-q PCR).5.The protein of foam macrophage in different sugar concentration groups was extracted,and the expressions of TREM2,ATP-binding cassette transporter A1(ABCA1)and ATP-binding cassette transporter G1(ABCG1)in each group were determined by Western blotting technology.6.To further observe the changes of inflammatory phenotype of foam macrophages when exposed to a high glucose concentration of 35mmol/L.RT-q PCR technique was used to detect the inflammatory phenotype of macrophages under high glucose conditions and revealed a shift in mRNA levels of MI-type markers such as TNF-α,IL-1β,and Arg-1,as well as M2-type markers like TGF-β.7.Protein of foam macrophage in different sugar concentration groups was extracted,and the changes of PI3K/AKT signaling pathway related proteins PI3 K,p-PI3 K,Akt and p-Akt protein levels in each group were determined by Western blotting technology;8.Transfection of TREM2-siRNA knocks down the expression level of TREM2 mRNA and protein in foam macrophages,and the cell mRNA and protein are extracted.The expression levels of TREM2,ABCA1 and ABCG1 mRNA and protein in each group were measured by RT-QCPR and Western blotting techniques,and the regulatory effect of TREM2 on cholesterol outflow from foam macrophages under high glucose conditions was observed.9.Knock down that expression level of TREM2 mRNA in foam macrophage by transfecting TREM2-siRNA,extract cell mRNA,and detecting M1 markers: TNF-α and IL-1β,M2 markers: Arg-1,TGF-β transcription level changes by RT-q PCR technology,to observe the regulatory effect of TREM2 on inflammatory phenotype of foam macrophages under high glucose conditions.10.Transfect TREM2-siRNA,in comparison to the NC group with high glucose,observe the expression changes of PI3 K,p-PI3 K,AKT and p-Akt proteins related to the PI3K/AKT pathway in foam macrophages,and explore the possible regulatory effect of TREM2 on PI3K/AKT pathway proteins under high glucose.Results:1.After the foam macrophage was modeled,it was found that the cholesterol uptake of foam macrophage increased under the condition of high glucose by oil red O staining and quantitative analysis of lipid accumulation.The absorption rate of cholesterol in the control group was lower than that of the foam macrophage group with normal sugar concentration(P≤0.05).However,when Ox-LDL(50ug/ml)was induced with a rise in sugar concentration,the cholesterol absorption rate of middle and high sugar concentration groups was significantly higher than that of the foam macrophage group with normal sugar concentration(P < 0.01).Moreover,the absorption rate of cholesterol in foam cells in the high sugar concentration group also increased significantly(P<0.01)in comparison to the medium sugar concentration group.2.NBD cholesterol and fluorescent enzyme-linked immunosorbent assay were employed to detect the cholesterol efflux rate of foam macrophages in each sugar concentration group.In comparison to the normal sugar concentration group,foam cells in the middle and high concentration groups exhibited a significant decrease in cholesterol efflux capacity(P< 0.05),and the high concentration group’s efflux capacity was also significantly reduced compared to the middle sugar concentration group(P<0.05).However,there was no statistical difference in the outflow rate of cholesterol from foam cells in the normal sugar concentration group when compared to the control group(P >0.05).3.Taking the foam macrophage group with normal sugar concentration as the control group,it was found that the expression of TREM2 gene increased under high sugar concentration.Compared with the normal sugar concentration group,the expression level of trem2 mRNA in foam cells in the middle and high sugar concentration groups increased significantly(P<0.05),while compared with the middle sugar concentration group,the expression level of trem2 RNA in foam cells in the high sugar concentration group also increased significantly(P < 0.05).With the increase of sugar concentration,compared with the normal concentration group,the expression levels of ABCA1 and ABCG1 mRNA in foam cells in the middle and high concentration groups were significantly decreased(P < 0.01).At the same time,compared with the medium sugar concentration group,the expression levels of ABCA1 and ABCG1 mRNA in foam cells in the high sugar concentration group were also significantly decreased(P< 0.01).4.The expression of TREM2 protein increased under high glucose conditions.Compared with the normal sugar concentration group,the expression of TREM2 protein in foam cells in the middle and high sugar concentration groups increased significantly(P < 0.05),while the expression of TREM2 protein in foam cells in the high sugar concentration group also increased significantly compared with the middle sugar concentration group(P < 0.05).The protein expression levels of ABCA1 and ABCG1 decreased with the increase of sugar concentration.Compared with the normal sugar concentration group,the expression levels of ABCA1 and ABCG1 proteins in foam cells in the middle and high sugar concentration groups were significantly decreased(P < 0.05).At the same time,compared with the medium sugar concentration group,the expression levels of ABCA1 and ABCG1 proteins in foam cells in the high sugar concentration group decreased significantly(P < 0.05).5.Compared with the control group,the expression levels of M1-type markers IL-1β and TNF-αmRNA in foam macrophages in the high glucose group increased(P < 0.05);However,the mRNA expression levels of M2 markers Arg-1 and TGF-β decreased significantly(P < 0.01).6.The expression of PI3K/AKT pathway proteins in foam macrophages was inhibited by a high glucose concentration.Treatment with(5.5,15 and 35)m M D-glucose solution revealed a decrease in the expression of p-Pi3 k and p-Akt protein with an increase in sugar concentration.In contrast to the normal concentration group,the middle concentration group and high concentration group both experienced a significant decrease in expression of p-Pi3 k and p-Akt proteins after being exposed to different sugar concentrations(all P < 0.05)and the middle concentration group’s expression levels were also significantly decreased(P<0.05),implying that high glucose concentration inhibited the phosphorylation of PI3K/AKT pathway proteins in foam macrophages in a dependent manner.7.After transfection with TREM2-siRNA,compared with the high glucose negtive control group,the expression level of trem2-mRNA decreased significantly(P < 0.0001),and the mRNA expression levels of ABCA1 and ABCG1 decreased significantly(P <0.05,P < 0.001).8.After transfection with TREM2 siRNA,the expression level of TREM2 protein was significantly reduced compared to the high glucose NC group(P<0.0001);The protein expression levels of ABCA1 and ABCG1 also significantly decreased(P<0.001,P<0.01);9.After transfection with TREM2-siRNA,compared with the negative control group,the expression of TREM2 mRNA decreased significantly(P < 0.01),and the expression levels of M1 markers TNF-α and IL-1βmRNA still showed an upward trend(P < 0.05).However,the levels of M2 markers Arg-1 and TGF-βmRNA decreased significantly(P <0.01).It is suggested that TREM2 can reduce the inflammatory level of foam macrophages under high glucose by participating in the regulation of inflammatory phenotype of foam macrophages.10.After transfection with TREM2 siRNA,compared to the high glucose NC group,the expression levels of p-PI3 k and p-Akt protein were further decreased(P<0.05,P<0.001).It is suggested that TREM2 may promote the cholesterol efflux of foam macrophages by participating in the regulation of PI3K/AKT pathway.Conclusion:1.High glucose inhibits the outflow of cholesterol from foam macrophages,increase the accumulation of cholesterol in foam macrophages and promote its inflammatory phenotype,which may be related to the inhibition of ABCA1,ABCG1 and PI3K/AKT phosphorylation by high glucose.2.In the process of high glucose aggravating cholesterol aggregation and inflammatory phenotype transformation of foam macrophages,TREM2 is compensatory increase,but it is not enough to reverse this pathological process.