Study On The Effects And Mechanism Of Different Doses Of Olive Oil On The Serum Cholesterol Level In Rats

introductionWith the socio-economic development and people's lifestyle changes, cardiovascular and cerebrovascular diseases endangering people's health has become the most serious diseases.Caused by cardiovascular and cerebrovascular diseases is not entirely clear.Dyslipidemia especially hypercholesterolemia is one of the primary factors in the development of cardiovascular disease.Therefore,the maintenance of normal lipid levels is particularly important and urgent.Liver plays a central role in the cholesterol homeostasis in the body.At first, cholesterol in the body is synthesized in liver,and this pathway is regulated by 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-CoA reductase).Secondly, cholesterol is ultimately excreted via bile,bile acids are the products of cholesterol catabolism and this pathway is regulated by cholesterol 7α-hydroxylase(CYP7A1),so these two enzymes play important role in the cholesterol homeostasis in the body.Due to the effects of olive oil on blood lipids,there is much controversy,This study of different doses of olive oil to replace the corresponding proportion of high fat diet of lard,observation on blood cholesterol levels,liver HMG-CoA reductase mRNA expression and the impact of CYP7A1mRNA.To study whether or not olive oil at the transcriptional level through regulation of gene expression both affect the level of blood cholesterol.In order to clarify the olive oil on blood cholesterol levels in rats the effects of mechanisms,reasonable guidance to the residents for meals,nutrition-related chronic disease prevention to provide a scientific basis.Materials and Methods1.AnimalsChoice of 45 body weight 100g-135g in the health of adaptability of feeding male Wistar rats after 1 week,were randomly divided into 5 groups(n=9):basal control group,lard group,olive oil groupⅠ,olive oil groupⅡand olive oil groupⅢ, received formula mash for 8 weeks,basal control group was fed with standard diet,lard group was fed the high fat diet which prepared by mixing the following ingredients: standard diet,83.75%;lard,15%;cholesterol,1%;bile salt,0.25%,the diet of olive oil groupⅠ,olive oil groupⅡand olive oil groupⅢwere prepared by replacing 6%, 10%and 15%lard in the high fat diet with olive oil.2.SamplingAt the end of the experiment,a blood sample was collected via abdominal aorta from the anaesthetized rat,serum was prepared by centrifugation at 3000rpm for 15 min;epididymal,perirenal and mesenteric fat depots and liver were dissected and weighed.3.Analysis(1) Serum lipid determination:Total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol (LDL-C) concentrations in the serum were determined using ECOM-F6124 semi-automatic biochemical analyzer according to manufacturer's directions of the enzyme assay kits.(2) Calculate liver indices and visceral fat/body weight ratio:①liver index(%)= wet liver weight/body weight×100.②epididymal fat/body weight(%)=epididymal fat(g)/body weight(g)×100.③perirenal fat/body weight(%)=perirenal fat(g)/body weight×100.④mesenteric fat/body weight(%)=mesenteric fat(g)/body weight (g)×l00.⑤visceral fat/ body weight(%)=[epididymal fat(g)+perirenal fat(g)+ mesenteric fat(g)]/body weight(g)×100.(3) To observe hepatic pathological changes:color of rat livers was observed,liver tissue from rats were fixed in 10%neutral formalin processed by standard procedure for paraffin embedding and serial sections were cut,HE stained.Pathological changes of rat livers were observed under light microscope.(4) Determination of liver mRNA expression of HMG-CoA reductase and CYP7A1:total mRNA was extracted from 30mg-50mg liver tissue using RNAout reagent.The reverse transcription-polymerase chain reaction(RT-PCR) was performed to asses the mRNA levels of HMG-CoA reductase and CYP7A1,using TaKaRa RNA PCR Kit,and glyceraldehydes 3-phosphate dehydrogenase(GAPDH) was used as control.RT was carried out under the following conditions:30℃l0min,42℃30min, 99℃5min,5℃5min.PCR was carried out under the following conditions:94℃2min; 94℃30sec,54℃30sec,72℃30sec,31 cycle;72℃l0min.7μl of PCR product was run on a 2%agarose gel stained with genefinder and photographed under UV transilluminator.The relative quantity of mRNA was analyzed by densitometry scanning with Fluorchem V2.0 Stand Alone software.4.Statistical analysisDate are presented as means and standard deviations,all data were analyzed with one-way ANOVAby SPSS 13.0 statistic software.Results1.general conditions and body weight gainDuring the experiment,with the exception of olive oil had a rat in groupⅡwas killed,the remaining rats in each group the growth and development of normal,no abnormal performance.Incremental weight of rats in each group,food intake and feed efficiency between price no statistically significant difference(P＞0.05).2.serum lpidsComparison of the blood of rats in each group TC,LDL-C levels are the highest olive oil groupⅢ.TC level in the lard group,olive oil groupⅠ,olive oil groupⅡ, olive oil groupⅢwere significantly higher than those based on group(P＜0.05);olive oil groupⅡ,olive oil groupⅢlevel of LDL-C significantly higher than the basis of group(P＜0.05),olive oil groupⅢlevel of LDL-C higher than the lard group(P＜0.05). Comparison of each group the level of HDL-C,olive oil groupⅡ,Ⅲgroup was lower than that based on olive oil group(P＜0.05),olive oil lard group than in groupⅢ(P＜0.05).In the TG level to the highest olive oil groupⅡ,and infrastructure group with statistically significant differences(P＜0.05).3.Liver indices and visceral fat/body weight ratioAnd infrastructure group,other groups have increased in rat liver index(P＜0.05); olive oil groupⅢperirenal rat fat/body weight,fat clonorchis week/weight,mesenteric fat/body weight,visceral fat/body weight is lower than other groups,but during which there was no significant difference(P＞0.05);the basis of group and olive oil groupⅠ, olive oil groupⅡhas a statistically significant difference(P＜0.05).4.hepatic pathological changesThe liver of animals in each group were generally found that the basis of the color red in rats liver,edge sharpness,texture,softness,and other groups of rats the liver was a khaki-colored,blunt the edge of change,texture medium,cut surface greasy feeling. Rat liver slices by the group can be seen:the basis of group and normal rat liver cells, production of the liver in rats liver cells have a small number of changes have taken place in fat,but have not yet reached the degree of fatty liver.Different doses of olive oil were shown in rats with mild fatty liver change.5.Hepatic HMG-CoA reductase and CYP7A1 mRNAThere is no significant difference in HMG-CoA reductase mRNA and CYP7A1 mRNA expression among all groups(P＞0.05).Conclusions1.Different doses of olive oil have not been shown to improve the high-fat model of the role of serum lipids,and blood TC,LDL-C levels also increased significantly than that of lard.2.The hypocholesterolemic effect of olive oil is not produced by affect the HMG-CoA reductase and CYP7A1 mRNA expression in liver,there may be other mechanisms.