| ObjectiveAtherosclerosis(AS)is a process of chronic proliferative vascular remodeling caused by the interaction of arterial lipid deposition and immune inflammation.The pathogenesis of AS is very complicated.Researchers have put forward many theories,such as lipid infiltration and endothelial injury,but they can not explain the formation mechanism of AS in an all-round way.In 1999,Ross and et al put forward that "AS is a chronic inflammatory reaction caused by the interaction of monocyte macrophages and the pathogenic lipoproteins of the arterial wall".This view is widely recognized,which provides a new direction for the study of the mechanism of AS.Macrophage is one of the most important immune cells involved in inflammation,can raise the target blood vessel injury in various inflammatory mediators of stimulation,and then through the low density lipid protein and cell debris,a large number of inflammatory cytokines the phagocytic oxidative modification effect of AS occurrence,development and extinction.Studies have shown that macrophages can be polarized into proinflammatory M1 subtypes and anti inflammatory M2 subtypes in different microenvironments.The increase of macrophage to M1 polarization and the decrease of M2 polarization are considered to be one of the pathological bases of the development of AS.However,the key molecules that regulate the polarization of macrophages have not been elucidated.Cellular repressor of E1A-stimulated genes(CREG)is a small molecule(220 amino acids)glycoprotein,which locates mainly in peri endoplasmic reticulum and lysosome.CREG is widely expressed in differentiated and mature tissues.It can promote the differentiation of cardiomyocytes,vascular smooth muscle cells and endothelial cells,and is considered to be an important regulator of cell differentiation homeostasis.In addition,previous studies in our laboratory have found that CREG can antagonize the development of AS and inhibit the inflammatory reaction of macrophages,but the mechanism is still unclear.Based on the above clues,this study suggests that CREG may be a key factor in the regulation of macrophage polarization.To test this hypothesis,we used mononuclear macrophage system to establish macrophage polarization model in vitro,clarify the role of CREG in macrophage polarization,and preliminarily explore its mechanism.The implementation of this study will provide experimental and theoretical clues for the new polarization mechanism of macrophage,and may provide new targets for the prevention and control of AS.Methods(1)The culture of mouse macrophage cell line RAW264.7 in vitro,with lipopolysaccharide(LPS,100ng/mL)+ interferon-gamma(IFN-gamma 20ng/mL)to induce the activation of classical polarization(M1),with interleukin 4(20ng/mL)induced the activation to replace(M2)the establishment of macrophage polarization.The uninduced cells were used as the control group.Flow cytometry,ELISA,Western blot and quantitative PCR were used to detect the proportion of M1 and M2 macrophages and the expression of biomarkers,and to verify whether the macrophage polarization model was successfully established.Further Western blot,quantitative PCR and immunofluorescence staining to detect M1 type and M2 type macrophage marker CREG and lysosomal markers including lysosome associated membrane protein 1(LAMP-1),lysosome associated membrane protein 2(LAMP-2)and lysosomal integral membrane protein 2(LIMP-2),positive correlation between CREG expression and macrophage polarization and lysosomal markers.(2)The low expression of CREG and overexpression of macrophage were used to establish the model of adenovirus infection and siRNA gene silencing method.Flow cytometry was used to detect M1 type and M2 type macrophage proportion,expression of Western blot method for detection of M1 type,M2 type macrophage markers expression and lysosomal markers,a clear causal relationship CREG and macrophage polarization.(3)The expression of CREG protein in RAW264.7 cells,a macrophage model with low expression of LAMP-1 using siRNA technology.Flow cytometry was used to detect M1 and M2.Western blot method to detect the proportion of macrophages,M1 and M2 type macrophage markers,CREG protein and lysosomal marker expression,to determine whether CREG affected by lysosomes pathways that regulate macrophage polarization.Results(1)The results of flow cytometry showed that,compared to the control group and non induced,LPS+IFN-gamma induced macrophages to M1 polarization,PE-F4/80 and FITC-CD16/32 double labeled M1 macrophages proportion(83.18 ± 3.07)%;IL-4 to M2 induced macrophage polarization,PE-F4/ 80,FITC-CD206 double labeled M2 the proportion of macrophages(42.79 ± 2.70)%.Western blot and ELISA results showed that M1 macrophages highly expressed iNOS protein and secreted proinflammatory cytokines MCP-1 and TNF-a lot,while M2 type macrophages expressed high CD206 protein and secreted anti-inflammatory factors IL-10 and TGF-beta 1.These results suggest that we have successfully established a model of macrophage polarization.In addition,quantitative PCR and Western blot results showed that compared with the control group,CREG and lysosomal markers(LAMP-1,LAMP-2 and LIMP-2)expression of mRNA and protein levels decreased in the M1 polarization group,while the increase in the M2 polarization group,there is a positive correlation between CREG and lysosome level and macrophage polarization.(2)Western blot results showed that CREG expression increased significantly after adenovirus infected macrophages,while CREG expression decreased significantly after siRNA transfected macrophages,indicating that we successfully established macrophages with over expression and low expression of CREG.Over expression of CREG in macrophage group:(1)compared with the control group GFP adenovirus infection,the flow cytometry results showed that after induced by LPS+IFN-gamma,PE-F4/80,PerCP-CD16/32 double positive M1 macrophages decreased [(56.91 ± 3.31)% vs.(73.94 ± 2.37)%,P< 0.05];after induced by IL-4,PE-F4/80 and PerCP-CD206 double positive M2 macrophages increased [(43.93 ± 2.11)% vs.(28.65 ± 1.97)%,P<0.05].(2)Western blot results showed that the expression of M1 macrophage marker iNOS protein was significantly decreased after induction by LPS+IFN-gamma,and the expression of CD206 protein of M2 macrophage marker increased significantly after IL-4 induction.These results suggest that overexpression of CREG inhibits the M1 polarization of macrophages and promotes the M2 polarization of macrophages.The low expression of CREG in macrophage group:(1)compared with Control group siRNA,the flow cytometry results showed that after induced by LPS+IFN-gamma,PE-F4/80,PerCP-CD16/32 double positive M1 macrophages increased [(73.94 ± 2.37)% vs(90.30 ± 2.31)%,P<0.05];after induced by IL-4,PE-F4/80,PerCP-CD206 double M2 positive macrophages decreased the proportion of [(25.23 ± 0.17)% vs(17.06 ± 1.81)%,P<0.05].(2)The results of Western blot showed that the expression of iNOS protein of M1 macrophage markers increased after LPS+IFN-gamma induction.These results suggest that low expression of CREG can promote M1 type polarization of macrophages.In addition,we also found that in overexpressing CREG macrophage group,lysosomal markers LAMP-1 and LAMP-2 protein increased significantly after LPS+IFN-gamma and IL-4 induction,while in lysosomal markers LAMP-1 and LAMP-2 decreased in low expression CREG macrophages.These results suggest that lysosomes may be involved in the regulation of CREG on the polarization of macrophages.(3)In order to investigate whether CREG can play a role in regulating lysosome,we used siRNA LAMP-1 to knock down LAMP-1 in overexpressing CREG macrophages and observe whether CREG’s function was blocked.(1)Western blot results showed that the CREG protein in adenovirus-CREG group increased significantly,and the expression of LAMP-1 protein in siRNA LAMP-1 group decreased significantly,indicating that LAMP-1 was knocked down successfully in macrophages overexpressing CREG.Compared with group Control siRNA,iNOS expression in macrophage marker M1 increased(P<0.05)in LAMP-1 siRNA group,while CD206 expression in M2 macrophage marker decreased(P<0.05).(2)The flow cytometry results showed that,compared with Control siRNA in the LAMP-1 group,siRNA group,PerCP-CD16/32 M1 positive macrophages significantly increased the proportion of [(15.40 ± 1.86)% vs(37.33 ± 2.91)%,P<0.05],PerCP-CD206 positive macrophages decreased significantly [(12.67 ± 1.20)% vs(5.33 ± 0.44)%,P<0.05].These results suggest that the reduction of LAMP-1 in macrophages can block the effect of CREG,and LAMP-1 may be a target for downstream CREG.ConclusionsCREG is an important molecule that regulates the M2 polarization of macrophages,and its mechanism may be mediated by the effect of lysosome.This study provides a new experimental basis for the molecular mechanism of macrophage polarization,and is expected to provide new targets for the prevention and treatment of AS. |
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