Role And Mechanism Of Low Salt Diet In Atherosclerosis Of ApoE^-/- Mice

ObjectiveAtherosclerosis （AS） is one of the most common and severecardiovascular disease, but its pathophysiology mechanism remains unclear. Ina long time, high salt diet has been considered one of risk factors forhyperthension, and both physicians and patients accepted the view that low saltdiet is beneficial for cardiovascular health. However, a recent study found thatlow salt diet could promote the devepolement of AS. Two conflicting viewpointsconcerning low and high salt diet attracted wide attention, so the relationshipbetween salt intake and AS should be given a new recognition. The aim of ourstudy is to investigate the role of low salt diet in AS and further elucidate itspossible mechanism.MethodsIn this study, male ApoE-/-mice, aged10weeks, weighted20to25g, wereused to establish the model of atherosclerosis. Firstly,72mice were randomlydivided into3groups according to amount of salt intake, i.e. low salt diet group(LS diet,0.03%NaCl and6%fat, n=24), normal salt diet group (NS diet,0.3%NaCl and6%fat, n=24) and high salt diet group (HS diet,3%NaCl and6%fat,n=24). All the3groups of mice were fed for12weeks. The data of body weight,blood pressure and salt intake were collected. Intima-media thickness of carotidartery, ascending aorta and brachiocephalic atery were detected by ultrasoundVevo2100. Serum angiotensinII (AngII) and monocyte chemotactic protein1 (MCP-1) were determined by ELISA, and the lipid level was assessed bylipoprotein detection kit.The whole aorta was isolated and underwent oil red Oand HE staining to observe the atherosclerotic plaque formation; Image-ProPlus6.0was used to measure the area of plaques and statistical analysis wasperformed to assess the severity of AS; Immunohistochemical and Massonstaining were carried out to evaluate the aortic remodeling and the nature of theplaques. We are expected to determine the role of low salt diet inatherosclerosis by implementation of the above studies. In addition, a group oflow salt diet combined with angiotensin converting enzyme inhitbitor (ACEI)drugs-captopril (LS+ACEI diet, captopril,50mg·kg-1day-1) was supplementedon the basis of low salt diet group (LS diet) and normal salt diet group (NS diet).AS, inflammation, and blood lipid were evulated as mentioned above.Immunofluorescence staining was performed to assess endothelial injury.Western Blot was used to detect expression of angiotensin II type1receptor(AT1R), angiotensin converting enzyme2(ACE2) and the low densitylipoprotein receptor (LDLR) in aorta, determining whether these molecules wereinvolved in salt induced atherosclerosis. Finally, human umbilical veinendothelial cells (HUVECs) were treated with AngII (10-7mol/L) in vitro.Immunofluorescence staining and western blot were used to observe thelocation and expression of LDLR, exploring the mechanism of AngII toendothelial cell.ResultsDuring12weeks of feeding, LS diet group had higher mortality rate (P <0.05), and the death was mainly attributed to severe heart failure and plaquerupture. After12weeks, the survived mice in LS diet group presented withhigher serum AngII leve（l217.21±14.73pg/ml vs122.70±3.87pg/ml，P＜0.05）,higher MCP-1level（785.33±62.45pg/ml vs225.2±64.26pg/ml，P＜0.05），higher total cholesterol （TC）(12.70±0.66mmol/L vs10.13±0.54mmol/L，P＜ 0.05）and lo w d e n s i t y l i p o p rotein（LDL）3.60±1.45mmol/L vs2.33±0.89mmol/L，P＜0.05），and lower high density lipoprotein（HDL）leve（l1.79±0.84mmol/L vs2.45±0.70mmol/L，P＜0.05）. In addition, AS is worse in LS dietgroup than the NS diet and HS diet group (P <0.05) by Vevo2100andhistochemistry staining, whereas there was no significant statistical difference inthe latter2groups. Histochemistry staining showed that the atheroscleroticplaque in LS diet group had larger lipid core, and thinner fibrous cap with moreinflammatory cells and less collagen ingredients. In contrast, HS diet group hadno obvious plaque but thickened vascular wall. In the LS diet group, expressionof AT1R increased, while ACE2and LDLR decreased, and the endothelial cellswere seriously damaged in the aorta. Furhtermore, ACEI intervention reducedthe expression of AngII level and effectively relieved the severity of AS and anddyslipidemia. After treating HUVECs with AngII, LDLR translocated formmembrane to cytoplasm, and its expression was decressed at the same time.ConclusionsIn ApoE-/-mice, a low salt with normal fat diet could lead to dyslipidemia,increased inflammation and endothelial damage, thus promoting AS anddevelopment of unstable plaque, contributing to high mortality. The increasedAngII induced by low salt diet may be the underlying mechanism. IncreasedAngII accelerated LDLR translocation from membrane to cytoplasm and inhibitits expression in endothelial cells, leading to severe endothelial damage, whichis regarded as an initriatior of AS. In addition, long-term high salt diet could leadto significant vascular remodeling and less AS in salt insensitive mice comparedto low salt diet group. In conculsion, low salt diet promoted development of ASand formation of unstable plaque through AngII induced endothelial injury.Results from our study may provide theoretical basis for establishment of lowerlimit of low salt diet, and may drive the formation of correct concept of saltintake.