| Objective NAG-1/GDF15（or NAG-1）has a wide range of physiological effects,such as inhibiting obesity,reducing of inflammatory cytokines,and is also associated with the development of cardiovascular diseases.However,its specific role and mechanism in the development of atherosclerosis remains unclear.In this study,ApoE-/-mice and LDLR-/-mice were used to study the effects of NAG-1 on fat accumulation and body weight changes in atherosclerotic model mice fed with high fat and high level of cholesterol.We will also determine the effects of NAG-1 on aortic plaque formation,to elucidate whether NAG-1inhibits the development of atherosclerosis in these two animal models.In addition,we will examine whether NAG-1 regulates the expression of lipid metabolism and inflammatory cytokines in liver tissue and serum.These results will help to elucidate the specific mechanisms by which NAG-1 regulate the development of atherosclerosis,whethe through suppression of inflammation and improvement of lipid metabolism and other pathways.In addition,our results will help to elucidate the association of NAG-1 withthe development of atherosclerosis for clinical relevance,and to providetheoretical basisfor the prevention and treatment of atherosclerosis by targeting NAG-1.Methods（1）LDLR-/-NAG-1mice and ApoE-/-NAG-1 mice were obtained by hybridization of NAG-1 transgenic mice with LDLR-/-or ApoE-/-,respectively.Genotyping was performed to obtain the ideal mice model.LDLR-/-mice were divided into two groups:LDLR-/-（control）and LDLR-/-NAG-1（experimental）.ApoE-/-mice were divided into ApoE-/-（control）and ApoE-/-NAG-1（experimental）two groups.（2）High-fat and high-cholesterol diets were fed to all micefor 12 consecutive weeks,animals were weighed weekly and food intake was measured weekly.（3）Blood lipids were measured by blood biochemistry in two atherosclerotic mice and the differences in blood lipid levels between the two atherosclerotic mice were compared.（4）The morphological observations and quantification of the plaque area were performed upon Oil red O staining to compare the size of the plaque area between the two groups of atherosclerotic mice and to analyze the effect of NAG-1 on plaque formation.（5）H&E staining and immunohistochemistry of aortic root were performed to compare the differences of lipid deposition in the aortic roots and the expression of related proteins in two atherosclerotic mice.（6）qRT-PCR method was used to detect the expression of NAG-1 on hepatic lipid metabolism and inflammation related factors in two atherosclerotic mice,and detect the expression of NAG-1 receptor GFRAL in mouse hindbrain.Results（1）LDLR-/-and ApoE-/-third-generation homozymous mice were successfully obtained,and LDLR-/-NAG-1 mice and ApoE-/-NAG-1 mice were obtained.The mice were healthy and the survival rate was 100%.（2）During 12 weeks of high fat and high cholesterol continuous feeding,the body weight of LDLR-/-NAG-1 mice was significantly lower than that of LDLR-/-mice.And some tissue weights,especially the weight of adipose tissue,there is a significant reduction in LDLR-/-NAG-1 mice.However,in ApoE-/-mice,except for the significant difference in the first week,there was no significant difference in weight between the two groups of mice during the 12-week high-fat and high-cholesterol feeding.In particular,with time,there is a trend that body weight of two groups of mice approached to similar.Therefore,there was no significant difference in body weights between the two groups at the end of the study.（3）Blood biochemistry was used to detect the levels of blood lipids in two atherosclerotic mice.It was found that the TG,TC,and LDL-C of LDLR-/-NAG-1 mice were significantly lower than those of LDLR-/-mice.Whereas,in ApoE-/-mice,there were no significant differences in blood lipid levels between NAG-1 expression and NAG-1 null ApoE-/-mice.（4）The comparison of plaque area between two groups of atherosclerotic mice revealed that in LDLR-/-mice,LDLR-/-NAG-1 mice aortic plaque area was significantly reduced compared with LDLR-/-mice,and there was a significant difference.However,in ApoE-/-mice,there was no significant difference in aortic plaque area between the two groups.（5）By H&E staining and immunohistochemistry,we found that lipid accumulation and lesions in the aortic root of LDLR-/-NAG-1 mice were much lower than in LDLR-/-mice.In ApoE-/-mice,there was no significant difference in the plaque area and pathological changes of the aorta between the two groups of mice.（6）By qRT-PCR method,we detected the expression of lipid metabolism and inflammation-related genes in the liver of the two atherosclerotic mice models that may be regulatedby NAG-1 expression.Our results indicate that in LDLR-/-mice,NAG-1 may accelerate reversecholesterol transport,reduce cholesterol accumulation and regulate cholesterol transport related factors such as ABCA1,ABCG1,SRB-1,LPL,SCD-1,Angptl3,ApoF,ApoA1,etc.In addition,NAG-1 may regulate the expressionof fatty acid metabolism related factors including CPT-1,ACOX-1,PPARα,CD36,FASn,etc.in LDLR-/-mice,which resulting in changing fatty acid metabolism,accelerating lipolysis and reducing triglyceride accumulation andinflammatory cytokines,such as TNF-α,IL-1β,and IL-6.Overexpression of NAG-1 may reduce endothelial damage and reduce immune responses,thereby protect the endothelial system and improve atherosclerotic lesions in experimental mice.However,there was no significant difference in the expression of NAG-1 receptor GFRAL in the brain tissue of both mice model.Conclusion Our study showed that NAG-1 can significantly reduce body weight and blood lipid levels in LDLR-/-mice and reduce atherosclerotic plaque area in LDLR-/-mice,thereby inhibiting the development ofatherosclerosis in LDLR-/-mice.However,NAG-1 had no significant effect on changes in body weight and development of atherosclerosis in ApoE-/-mice.NAG-1 may reduce cholesterol accumulation by increasing the expression of cholesterol transport-related factors,thereby reducing TC levels in LDLR-/-mice and decreasing LDL-C levels.We could speculate that NAG-1 regulate fatty acid metabolism and thus reduces TG levels in serum of LDLR-/-mice;and may reduce endothelial damage by reducing inflammatory response-related factors,thereby reducing atherosclerotic plaque area and ultimately inhibit the development of atherosclerosisin LDLR-/-mice.The different results of the two atherosclerotic mice may be due to the different effects of NAG-1 on the body weight of the two mice,suggesting that NAG-1 may play a different role in the two atherosclerotic mice.In future studies,more clinical and basic research are needed to clarify the role of NAG-1 in the development of atherosclerosis and its regulatory mechanisms,and ultimately to diagnose,prevent and treatatherosclerosis and other cardiovascular diseases that targeting NAG-1. |
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