Study On The Effects Of Lipid Metabolism And SIRT1, PPARα, CYP7A1 Gene On High-fat Mice With Resveratrol In Short Time

IntroductionIn recent years, our country's urban and rural residents in diet, nutritional status improved significantly. The prevalence of malnutrition and nutritional deficiencies continue to fall, however, excess nutrients caused by non-communicable chronic disease has become increasingly prominent. According to the 2002 Chinese residents in nutrition and health survey, China's population hypertension, diabetes, overweight and obesity prevalence showed a rising trend. Dyslipidemia is atherosclerosis, coronary heart disease, stroke, high blood pressure and other important risk factor for cardiovascular disease. Therefore, prevention and control of dyslipidemia of great significance.The French daily intake of fat, while cardiovascular disease morbidity and mortality significantly below other European countries. This is relevant to their daily lot of drinking red wine, red wine rich in resveratrol may have protective effects. RSV can improve the silent information regulatorl, SIRT1 activity and improve substrate affinity acetylation, is a function of SIRT1 activator. The SIRT1 enable pemxisome proliferative activated receptor, PPARs deacetylation. PPARa involved in cholesterol metabolism in the process of a key enzyme cholesterol-7α-hydroxylase, CYP7A1 regulation, Thereby affecting cholesterol homeostasis, CYP7A1 transcription is dependent on PPARa.. SIRT1 could be affected by regulating cholesterol metabolism in PPARa, increase the expression of CYP7A1 through the acceleration of cholesterol catabolism. Therefore, this study was to start from the establishment of mouse model of hyperlipidemia and to RSV as an intervention factor, observation of changes in serum cholesterol levels as well as the liver of mice SIRT1, PPARa, and CYP7A1 at the mRNA level. To explore whether RSV can increase the activity of SIRT1 and whether through SIRT1, PPARa, CYP7A1 affect lipid metabolism in mice blood cholesterol.Materials and Methods1. AnimalsAfter acclimatization periods,50male Kunming mouse weighting18-22g randomly divided into 2 groups according to body weight:Normal control group (NC group) and high fat group (HF group).The mice in NC group (n=10) were fed with standard diet. The mice in HF group(n=40) were fed with high fat diet.After 4-week high fat diet, Venous blood from the inner canthus of about 0.5ml, serum Total cholesterol, TC level, By serum TC levels will be high-fat group were randomly divided into 4 groups(n=10):HF group, RSV I group, RSVⅡgroup, RSVⅢgroup.2. TreatmentsBased group fed basic diet, HF group, RSV I group, RSV II group, RSVⅢgroup were fed with high fat diet, which prepared by mixing the following ingredients: standard diet,92%; lard,6.75%; cholesterol,1%; bile salt,0.25%and RSV I group, RSV II group, RSVⅢgroup were fed with resveratrol 5mg/(kg-bw-d),22.5 mg/(kg-bw-d),45mg/(kg-bw·d) for 6 week. At the end of the experiment, a blood sample was collected via pick eyeball from the anaesthetized mice, serum was prepared by centrifugation at 3000rpm for 15 min; liver were dissected and weighed, and we also measured sirtl mRNA and PPARa mRNA expression in the liver of the mice.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)LPO level:SOD, MDA, GSH-Px and T-AOC in the liver were determined using ELIASA according to manufacturer's directions of the enzyme assay kits.(3)To observe hepatic pathological changes:color and texture of mice livers was observed, production of a portion of liver tissue frozen section, oil red "O" staining Production of paraffin section, for HE staining to observe pathological changes in liver tissue in mice.(4) Calculate liver indices/body weight ratio:liver index (%)= wet liver weight/body weight×100.(5) Determination of liver mRNA expression:total mRNA was extracted from liver using RNA out reagent. The reverse transcription-polymerase chain reaction (RT-PCR) was performed to asses the mRNA levels of SIRT1, PPARa and CYP7A using TaKaRa RNA PCR Kit, and P-actin was used as control. The same reverse transcription reaction conditions is 30℃1Omin,42℃30min,99℃5min,5℃5min, while SIRT1 gene and PPARa gene PCR reaction conditions is 94℃2min predegeneration,33 cycle of amplification including denaturation 94℃30s, annealing 56℃30s, extension 72℃35s and then 1 cycle of extension 72℃1Omin. CYP7A1 gene PCR reaction conditions is 94℃2min; 94℃30sec,54℃30sec,72℃30sec,31 cycle; 72℃10 min. Took 5μl target gene products, plus pre-stained liquor reacted 3min, then ran on 2% agarose gel electrophoresis and photographed under gel imaging camera analyzer.4. Statistical analysisDate are presented as means and standard deviations, all data were analyzed with one-way ANOVA by SPSS 13.0 statistic software. 1.General conditions and body weight gainDuring the experiment, mice in each group in general in good condition, no abnormal growth and development of the performance. Body weight of mice in each group increments to the highest RSV I group, but other groups are not statistically significant (P> 0.05).2. Liver indicesMouse liver index in each group is not statistically significant (P> 0.05).3. Hepatic generally change and pathological changesObserved in livers of mice in each group found that in general samples, basic groups and RSV I, II groups of mice liver color red, soft texture, edges sharp, The high-fat group and the RSV III mice blunt the edge of change, texture hardens, cut surface greasy feeling. By the group of mice liver biopsy can be seen that the basis of morphologically normal liver cells in mice, almost no fatty degeneration; High-fat groups of mice are part of the liver in the liver cells of the fat changes, but have not yet reached the degree of fatty liver. The remaining three groups of mice interfere with the liver have occurred in a small number of liver cells, fatty change, does not meet the degree of fatty liver.4. Serum lpidsComparison of mice in each group TC, LDL-C levels, high-fat group and the basis of group differences were statistically significant (P＜0.05) In the TG levels to a minimum RSV II group, but other groups are not statistically significant(P>0.05). HDL-C levels in each group showed no significant difference (P>0.05).5.LPO level(1)Levels of serum anti-oxidationAll the parameters were not statistically significant(P>0.05). (2)Organization of anti-oxidant levelsMDA levels of mice in each group compared to the highest high-fat group, their differences with the other groups were statistically significant (P＜0.05); SOD, T-AOC, GSH-PX levels, the group does not have statistical significance (P> 0.05).6. Hepatic SIRT1, PPARa and CYP7A1 mRNASIRT1,PPARa and CYP7A1 mRNA expression of all groups were familiar.Conclusions1.Different doses of resveratrol had lower serum TC levels of fat model trends, and can significantly reduce LDL-C levels.2.Doses of resveratrol in different lipid model have increased levels of serum SOD trends, and can significantly reduce the MDA level of liver fat mice.3.The intervention dose to 22.5mg/(kg-bw-d) mice of resveratrol on fat lipid levels and antioxidant levels of intervention works best.4.This study did not show effect of resveratrol on mice liver fat SIRT1, PPARa, CYP7A1 mRNA expression, the result may be a short time with the intervention.