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

introductionCardiovascular disease has become the leading cause of death among Chinese Adults.Dyslipidemia especially hypercholesterolemia is one of the primary factors in the development of cardiovascular disease.Therefore,the prevention and control of dyslipidemia in particular hypercholesterolemia is very important to reduce the occurrence and development of cardiovascular disease morbidity and improve the prognosis.It have been documented that serum cholesterol levels in rats can be decreased by perilla seed oil,but the mechanism has remained unclear.Liver plays a central role in the cholesterol homeostasis in the body.At first,70%～80%cholesterol in the body is synthesized in liver,and this pathway is regulated by 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-CoA R).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.Therefore,Wistar rats were fed diets containing perilla seed oil of different doses,To observe the effects of perilla seed oil on the serum cholesterol level,the expression of HMG-CoA R and CYP7A1 mRNA,to elucidate the molecular mechanism of perilla seed oil on cholesterol metabolism in rats.Materials and Methods1.AnimalsAfter acclimatization periods,45 male Wistar rats were randomly divided into 5 groups according to body weight:basal control group,lard group,perilla oil groupⅠ, perilla oil groupⅡand perilla 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 perilla oil groupⅠ,perilla oil groupⅡand perilla oil groupⅢwere prepared by replacing 6%,10%and 15%lard in the high fat diet with perilla seed 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)×100.⑤visceral 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 R and CYP7A1:total mRNA was extracted from 30～50mg liver tissue using RNAout reagent.The reverse transcription-polymerase chain reaction(RT-PCR)was performed to asses the mRNA levels of HMG-CoA R 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℃10min,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℃10 min.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 ANOVA by SPSS 11.5 statistic software.Results1.general conditions and body weight gainDuring the experiment,there were no abnormal situation in each group except one rat of perilla oil groupⅢdied.Body weight gain of rats in lard group was the highest and in perilla oil groupⅢwas the lowest,but no statistical significant changes were observed among the five groups.The feed intake of perilla oil groupⅢare lower than those of the other groups(p＜0.05).2.serum lpidsCompared with lard group,perilla oil groupⅠ,perilla oil groupⅡand perilla oil groupⅢhad lower serum TC levels(p＜0.05),perilla oil groupⅡand perilla oil groupⅢhad lower serum TG and HDL-C levels(p＜0.05).The HDL-C levels in rats of perilla oil groupⅠ,perilla oil groupⅡand perilla oil groupⅢalso lower than those of basal control group(p＜0.05).3.Liver indices and visceral fat/body weight ratioCompared with basal control group,all the other group had higher liver indices (p＜0.01),and the liver/body weight ratio of perilla oil groupⅢare also higher than those of lard group(p＜0.05).mesenteric fat/body weight ratio in rats of perilla oil groupⅡare higher than those of other groups(p＜0.05).4.hepatic pathological changesThe livers of the basal control group rats had a fresh dark-red color,the livers in rats of the other groups were enlarged,had a yellowish color and greasy cut surface. Under the light microscopy,there is no steatosis of hepatocyte in basal control group, but there are a little of fat vacuoles in rat liver tissue of other groups.5.Hepatic HMG-CoA R and CYP7A1 mRNAThere is no significant difference in HMG-CoA R mRNA and CYP7A1 mRNA expression among all groups.Conclusions1.Serum TC levels in rats can be decreased by different doses of perilla seed oil, but take into account other factors,the low dose perilla seed oil(6%)is the best one to maintain normal levels of serum lipids in rats.2.The hypocholesterolemic effect of perilla seed oil is not produced by affect the HMG-CoA R and CYP7A1 mRNA expression in liver.