reasons for the occurrence and development of as include lipid aggregation , endothelial injury , and inflammation . in humans , endothelial dysfunction is one of the first detectable vascular alterations in the evolution of atherosclerosis , and its presence also correlates well with future cardiovascular events . endothelial injury is one of the primary pathogenesis in the initiation and progression of atherosclerosis . nitric oxide ( no ) is a potential regulating factor of protected internal environment homeostasis and prevented apoptosis . no generated continuously can prevent apoptosis of endothelial cells and protect endothelial cells from damage , which is beneficial to prevent occurrence and development of as . during injury , therefore , arginine may reduce the development of atherosclerosis in rats fed a high - fat diet by protecting endothelial function . the present study , in which rats were fed a high - fat diet supplemented with arginine and their vasculum was detected , confirms this hypothesis . cd36 can adhere to and assist macrophages in phagocytizing oxygenized low - density lipoproteins ( oxldls ) . arginine potentially contributes to the reduction of platelet aggregation and the adhesion of mononuclear cells to the endothelium in hypercholesterolemic subjects . this study aimed to define the role of cd36 in as rats treated with arginine by detecting the expression of cd36 . simvastatin is used in many clinical studies , so we choose simvastatin as the positive control lipid - lowering drug . twenty adult male and 20 adult female sprague - dawley rats , aged 8 weeks and weighing 20020 g , were supplied by the shanghai super b&k laboratory animal co. , ltd . , all rats were housed under standard conditions at 25c with a 12/12 h light - dark cycle and free access to water . this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health . the animal use protocol was reviewed and approved by the institutional animal care and use committee ( iacuc ) of fudan university . the rats were randomly divided into 4 groups : control group ( 10 ) , fat diet group ( 10 ) , simvastatin group ( 10 ) , and arginine group ( 10 ) . control group rats were fed with fat - free food for 12 weeks ; the other 3 groups rats were fed a high - fat diet ( 94.3% basal feed + 3% lard ( purchased in the market ) + 2% cholesterol ( shanghai technical service industry and biological engineering co. , ltd . , shanghai , china ) + 0.5% sodium cholate ( shanghai technical service industry and biological engineering co. , ltd . , shanghai , china ) + 0.2% propylthiouracil ( shanghai zhaohui pharmaceutical co. , ltd . , shanghai , china ) and were intramuscularly injected with vitamin d3 ( harbin mt agricultural veterinary medicine co. , ltd . , harbin , china ) ( 310 u / kg / moon ) for 12 weeks . the arginine group rats were given arginine hydrochloride injections ( 20 ml : 5 g , shanghai sine jin zhu pharmaceutical co. , ltd . , the dosage is chosen according to its usual dosage 10 g / day in 60 kilogram human and conversion formula of human - rat 1:6.17 . the simvastatin group rats were given distilled water of the same volume with the simvastatin ( hangzhou msd pharmaceutical co. , ltd . , hangzhou , china ) by gavage ( 4 mg / kg / d ) . the dosage is chosen according to its usual dosage of 40 mg / day in 60-kilogram humans and conversion formula of human - rat 1:6.17 . the control and fat diet group rats were given distilled water of the same volume for 12 weeks by gavage . then , routine dehydration , paraffin embedding , he staining , and microscopic examination were performed . routine paraffin sectioning , dewaxing , and hydration using 3% hydrogen peroxide were performed to remove endogenous peroxidase . approximately 50 l ( 1:100 ) of rabbit anti - rat cd36 antibody ( wuhan eiaab science co. , ltd . , wuhan , china ) was added , and the mixture was incubated at 25c overnight . then , approximately 50 l of goat anti - rabbit igg antibody ( wuhan eiaab science co. , ltd . , wuhan , china ) was added , and the mixture was incubated at 37c for 60 min and colored using diaminobenzidine ( tiangen biotech co. , ltd . , equal amounts of protein were electrophoresed on a 10% sds - polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membranes . after blocking with 2% bsa in tris - buffered saline at room temperature for 1 h , the membranes were incubated with an antibody against rabbit anti - rat cd36 antibody ( wuhan eiaab science co. , ltd . , the membranes were then washed with pbs and incubated with horseradish peroxidase - conjugated goat anti - rabbit igg antibody ( wuhan eiaab science co. , ltd . , total rna from mononuclear cells was isolated using trizol - a reagent ( tiangen biotech co. , ltd . , beijing , china ) and cdna was synthesized with 4 l total rna using the quantscript rt kit ( tiangen biotech co. , ltd . , beijing , china ) according to the manufacturer s protocol . pcr resulted in specific amplificates according to the cdna using superreal premix ( sybr green ) ( tiangen biotech co. , ltd . , the reactions were run using the following program : 95c for 15 min , 40 cycles of 95c for 10 s , and 60c for 32 s. the sequences of primers specific for cd36 ( sense : 5-aggaagtggcaaagaatagcag-3 , antisense : 5-acagacagtgaaggctcaaaga-3 ) and beta - actin ( sense : 5-cccatctatgagggttacgc-3 , antisense : 5-tttaatgtcacgcacgattt-3 ) were synthesized by sangon biotech ( shanghai , china ) . the overall response to increasing concentrations of tgf - b1 in monocytic cells comparisons of cells obtained from the same donor and incubated either under control conditions or in the presence of tgf - b1 were made using the matched - pairs signed rank wilcoxon test . the data among the several groups were analyzed by one - factor analysis of variance and the bonferroni method for multiple comparisons or the kruskal - wallis rank test . in all analyses , twenty adult male and 20 adult female sprague - dawley rats , aged 8 weeks and weighing 20020 g , were supplied by the shanghai super b&k laboratory animal co. , ltd . , all rats were housed under standard conditions at 25c with a 12/12 h light - dark cycle and free access to water . this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health . the animal use protocol was reviewed and approved by the institutional animal care and use committee ( iacuc ) of fudan university . the rats were randomly divided into 4 groups : control group ( 10 ) , fat diet group ( 10 ) , simvastatin group ( 10 ) , and arginine group ( 10 ) . control group rats were fed with fat - free food for 12 weeks ; the other 3 groups rats were fed a high - fat diet ( 94.3% basal feed + 3% lard ( purchased in the market ) + 2% cholesterol ( shanghai technical service industry and biological engineering co. , ltd . , shanghai , china ) + 0.5% sodium cholate ( shanghai technical service industry and biological engineering co. , ltd . , shanghai , china ) + 0.2% propylthiouracil ( shanghai zhaohui pharmaceutical co. , ltd . , shanghai , china ) and were intramuscularly injected with vitamin d3 ( harbin mt agricultural veterinary medicine co. , ltd . , harbin , china ) ( 310 u / kg / moon ) for 12 weeks . the arginine group rats were given arginine hydrochloride injections ( 20 ml : 5 g , shanghai sine jin zhu pharmaceutical co. , ltd . , shanghai , china ) by gavage ( 1 g / kg / d ) . the dosage is chosen according to its usual dosage 10 g / day in 60 kilogram human and conversion formula of human - rat 1:6.17 . the simvastatin group rats were given distilled water of the same volume with the simvastatin ( hangzhou msd pharmaceutical co. , ltd . , hangzhou , china ) by gavage ( 4 mg / kg / d ) . the dosage is chosen according to its usual dosage of 40 mg / day in 60-kilogram humans and conversion formula of human - rat 1:6.17 . the control and fat diet group rats were given distilled water of the same volume for 12 weeks by gavage . the aortic root and arch were fixed with 10% formalin . then , routine dehydration , paraffin embedding , he staining , and microscopic examination were performed . routine paraffin sectioning , dewaxing , and hydration using 3% hydrogen peroxide were performed to remove endogenous peroxidase . approximately 50 l ( 1:100 ) of rabbit anti - rat cd36 antibody ( wuhan eiaab science co. , ltd . , wuhan , china ) was added , and the mixture was incubated at 25c overnight . then , approximately 50 l of goat anti - rabbit igg antibody ( wuhan eiaab science co. , ltd . , wuhan , china ) was added , and the mixture was incubated at 37c for 60 min and colored using diaminobenzidine ( tiangen biotech co. , ltd . , equal amounts of protein were electrophoresed on a 10% sds - polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membranes . after blocking with 2% bsa in tris - buffered saline at room temperature for 1 h , the membranes were incubated with an antibody against rabbit anti - rat cd36 antibody ( wuhan eiaab science co. , ltd . , the membranes were then washed with pbs and incubated with horseradish peroxidase - conjugated goat anti - rabbit igg antibody ( wuhan eiaab science co. , ltd . , wuhan , china ) for 1 h at room temperature . total rna from mononuclear cells was isolated using trizol - a reagent ( tiangen biotech co. , ltd . , beijing , china ) and cdna was synthesized with 4 l total rna using the quantscript rt kit ( tiangen biotech co. , ltd . , pcr resulted in specific amplificates according to the cdna using superreal premix ( sybr green ) ( tiangen biotech co. , ltd . , the reactions were run using the following program : 95c for 15 min , 40 cycles of 95c for 10 s , and 60c for 32 s. the sequences of primers specific for cd36 ( sense : 5-aggaagtggcaaagaatagcag-3 , antisense : 5-acagacagtgaaggctcaaaga-3 ) and beta - actin ( sense : 5-cccatctatgagggttacgc-3 , antisense : 5-tttaatgtcacgcacgattt-3 ) were synthesized by sangon biotech ( shanghai , china ) . the overall response to increasing concentrations of tgf - b1 in monocytic cells was tested using a regression analysis . comparisons of cells obtained from the same donor and incubated either under control conditions or in the presence of tgf - b1 were made using the matched - pairs signed rank wilcoxon test . the data among the several groups were analyzed by one - factor analysis of variance and the bonferroni method for multiple comparisons or the kruskal - wallis rank test . in all analyses , after 12 weeks of being fed a high - fat diet , aortal intimal thickening and lipid deposition in the middle media developed in rats in the fat diet and arginine groups , but not those in the control and simvastatin groups . the thickness of aortal intima , media , and i / m significantly decreased in arginine group rats compared with the fat diet group rats ( p<0.05 , table 1 , figure 1 ) . cd36 expression with immunohistochemistry ( membrane and cytoplasm of positive cells were stained brown ) was up - regulated in fat diet group rats compared to the control group and down - regulated in arginine and simvastatin group rats compared with fat diet group rats ( figure 2 ) . the cd36 mrna expression of mononuclear cells was up - regulated in fat diet group rats compared with the control group and down - regulated in arginine group rats compared with the fat diet group . however , the cd36 mrna expression was up - regulated in simvastatin group rats compared with both the control group and the fat diet group ( figures 3 , 4b ) . the cd36 protein expression of mononuclear cells was up - regulated in fat diet group rats compared with the control group . no significant difference was found in the cd36 protein expression of mononuclear cells among rats in the control , arginine , and simvastatin groups ( figure 4a ) . after 12 weeks of being fed a high - fat diet , aortal intimal thickening and lipid deposition in the middle media developed in rats in the fat diet and arginine groups , but not those in the control and simvastatin groups . the thickness of aortal intima , media , and i / m significantly decreased in arginine group rats compared with the fat diet group rats ( p<0.05 , table 1 , figure 1 ) . cd36 expression with immunohistochemistry ( membrane and cytoplasm of positive cells were stained brown ) was up - regulated in fat diet group rats compared to the control group and down - regulated in arginine and simvastatin group rats compared with fat diet group rats ( figure 2 ) . the cd36 mrna expression of mononuclear cells was up - regulated in fat diet group rats compared with the control group and down - regulated in arginine group rats compared with the fat diet group . however , the cd36 mrna expression was up - regulated in simvastatin group rats compared with both the control group and the fat diet group ( figures 3 , 4b ) . the cd36 protein expression of mononuclear cells was up - regulated in fat diet group rats compared with the control group . no significant difference was found in the cd36 protein expression of mononuclear cells among rats in the control , arginine , and simvastatin groups ( figure 4a ) . clinical manifestations include myocardial infarction , aortic stenosis , aortic aneurysm , cerebral ischemia , cerebral hemorrhage , ischemic nephropathy , ischemic enteropathy , and peripheral artery disease . the complex causes of as development include lipid accumulation , endothelial damage , and inflammation . adiponectin may protect the aorta from atherosclerosis injury by reducing lesion formation size in the aortic root and reducing tc , tg , and ldl - c in serum . the normal regulative balance of the vascular endothelium is altered , including a reduction of no synthesis and secretion , increased endothelin release , increased expression of endothelial adhesion molecules , and disorder of endothelial antithrombotic function . no is not only a vasodilator substance , but it can also prevent platelet adhesion and aggregation , prevent leukocyte adhesion and migration to the arterial wall , and inhibit smooth muscle cell proliferation , which is crucial for as development . in circulation , no is highly permeable , and a small molecule generated from arginine is catalyzed by nos . .   showed that the use of an arginine diet can prevent vascular fatty streak formation induced by a fat diet , elevation of von willebrand factor ( vwf ) , and endothelial damage . .   demonstrated that arginine supplementation has an appropriate endothelial protective effect in c57bl/6j mice by increasing endothelial progenitor cells ( epcs ) and vascular endothelial growth factor ( vegf ) . many experiments [ 1316 ] have indicated that arginine supplementation can repair endothelial function , improve cardiac function , reduce the incidence of arrhythmias , and relieve ischemia - reperfusion injury by no in many diseases associated with endothelial dysfunction . there is no significant difference in the efficacy of intravenous or oral arginine . in this study , the thickness of aortal intima , media , and i / m significantly decreased in arginine group rats compared with fat diet group rats . the results show that a high - fat diet with simultaneous oral arginine supplementation can reduce the development of atherosclerosis , which may be related to the arginine - no pathway that reduces vascular endothelial inflammation and protects endothelial function , thereby reducing the development of atherosclerosis . cd36 antigen is widely present in many different types of cells , such as macrophages , monocytes , platelets , and endothelial cells . oxidized low - density lipoprotein ( oxldl ) can be adhered to and phagocytized by cd36 into macrophages , which eventually become foam cells . foam cells , which constitute the core infrastructure of as , play an important role in the formation of plaque . a variety of as - induced inflammatory cytokines , such as macrophage colony - stimulating factor ( mcsf ) , -interferon ( ifn- ) , and interleukin-10 ( l-10 ) , may increase the expression of cd36 . animal experiments showed that pitavastatin can decrease the transcription of macrophages and th1 cell surface cd36 mrna and the expression of surface protein . in hypercholesterolemic patients given oral atorvastatin for 6 days , the cd36 expression on the platelet surface arginine plays a potential beneficial role in reducing platelet aggregation and monocyte and endothelial cell adhesion for hypercholesterolemic patients . in addition to the known no pathway , the effects of arginine on cd36 expression should be studied as a possible mechanism . cd36 protein expression was up - regulated in fat diet group rats compared with control group rats and down - regulated in arginine group rats compared with fat diet group rats . the expression of cd36 was detected using both western blot and quantitative real - time pcr in this experiment . according to the real - time pcr results , the cd36 mrna expression of rat blood mononuclear cells was up - regulated in fat diet group rats compared with control group rats and down - regulated in arginine group rats compared with fat diet group rats . similar expression levels of cd36 mrna were detected in rats in both the arginine and control groups . the experimental pathology confirmed that arginine can reduce atherosclerosis in fat diet rats and reduce cd36 expression in rat blood mononuclear cells and aorta . therefore , it can be concluded that arginine can reduce the occurrence and development of atherosclerosis by reducing cd36 expression in fat diet rat blood mononuclear cells and aorta . in simvastatin group rats , the cd36 mrna expression was up - regulated , but the cd36 protein expression was down - regulated compared with the fat diet group . therefore , simvastatin may have an anti - atherosclerosis effect by intervening in some mechanism in the cd36mrna downstream channel to influence expression of cd36 protein , not by down - regulating cd36mrna expression . arginine may reduce platelet aggregation and monocyte and endothelial cell adhesion and provides anti - oxidative and anti - inflammatory properties to reduce the generation of inflammatory cytokines , which reduces cd36 expression in blood mononuclear cells and aorta . this finding needs further experimental research to identify the mechanisms and pathways through which arginine affects the expression of cd36 . in addition to no , the mechanism of this phenomenon may be associated with the down - regulation of cd36 expression in the endothelia of the aorta and monocytes in blood circulation .
backgroundthe aim of this study was to investigate the effects of arginine in the development of atherosclerosis in rats fed a high - fat diet supplemented with arginine and to evaluate the role of cd36 in this process.material/methodsa total of 40 sprague - dawley rats were randomly assigned to 4 groups : control group , fat diet group , simvastatin group , and arginine group . 
 they were fed for 12 weeks and were then sacrificed . 
 immunohistochemical cd36 expression and pathology was investigated in the aorta ; cd36 expression in mononuclear cells was detected by western blot and rt-pcr.resultsthe thickness of the aortal intima , media , and i / m significantly decreased in the arginine group rats compared with those in the fat diet group ( p<0.05 ) . 
 cd36 expression was up - regulated in rats in the fat diet group compared with the control group and was down - regulated in rats in the arginine group compared with rats in the fat diet group.conclusionsthe addition of arginine has a significant effect on reducing rat atherosclerosis development , which may be attributed to both the down - regulation of cd36 expression in rat aortic endothelial and blood mononuclear cells and the no pathway .