Effect Of Soy Proten On Expression Of The Cholesterol Metabolism Ralated Gene In Hypercholesterolemic Rats

SubjectData from epidemiologic studies demonstrate that hypercholesterolemia is the main risk factor of atherosclerosis and coronary heart disease, which threaten life and health of human bebing severely. Therefore, control and decrease of blood cholesterol level is important for preventing cerebrovascular and cardiovascular disease, especially for preventing the incidence and development of coronary heart disease.Both animal trials and human clinical studies have proved that soy protein, as high - quality vegetable protein, has obvious effect on decreasing blood cholesterol level. Many hypothesis were reported to explain the mechanisms of cholesterol reduction effect by soy protein , but up to now, the accurate mechanism is not clear completely.Both 3 - hydroxy - 3 - methylglutaryl - CoA reductase ( HMG - CoA R) , the rate - limiting enzyme in cholesterol biosynthesis pathway,and cholesterol 7a -hydroxylase(CYP7A) , the rate - limiting enzyme in the conversion of cholesterol to bile acids, play crucial roles in regulating the balance of cholesterol. In this study, to discuss whether soy protein lower choleserol by altering the gene expression of HMG - CoA R and CYP7A, and to disclose the molecule mechanism of cholesterol reduction for the better utilization of soy protein .Materials and Methods1. Development of hypercholesterolemic ratsAfter feeding with ordinary laboratory diet for one week, 60 male Wistarrats, aged four - week, were divided randomly into two groups : 50 rat were fed rich cholesterol diet (5% fat, 1% cholesterol, 0.25% bile salt) for inducing hypercholesterolemia model ; 10 rat were continually fed ordinary laboratory diet as negative control group. After 14 days , blood of rats were sampled to measure serum lipids.2. TreatmentsAccording to serum total cholesterol level and body weight, rats were divided into 4 groups:20% soy protein group(A) ,30% soy protein group(B) ,20% casein group(C)and 30% casein group(D). These rats were fed purified diets containing soy and casein. After 56 days, blood and liver of rats were sampled to measure serum lipids and gene expression, respectively.3. Mensuration3.1 Serum lipid determination: blood samples one ml were collected from the orbital vein. The samples were kept in water of 37 T! for 30 min , serum was prepared by contrifugation at 5000r/min for 15 min. Total cholesterol ( TC ) , tri-glyceride ( TG) , low density lipoprotein cholesterol ( LDL - C ) , high density lipoprotein cholesterol(HLD — C) concentrations in the serum were determined enzymatically using biochemical autoanalyser.3. 2 Determination of liver mRNA expression; total mRNA was isolated with Trizol reagent. The reverse transcription - polymerase chain reaction ( RT -PCR) was performed to assess the mRNA levels of HMG -CoA R,CYP7A, u-sing TaKaRa RNA PCR kit,and glyceraldehyde -3 - phosphate dehydrogenase ( GAPDH) was used as invariant control. RT was carried out under the following conditions:30T: for 10 min,42t: for 30 min,99T: for 5 min,50t: for 5 min, one cycle; PCR was carried out under the following conditions:94T for 30 s, 60t for 30 8,72^ for 1 min^Ot for 5 min,24 cycles, 12X for 10 min. The ten |xl of PCR product was run on a 2% agarose gel stained with EB and photographed under UV transilluminator. The relative quantity of mRNA was analyzed by densitometry scanning with FluorChem Stand alone V2.0 software.4. Statistical anlysisAll data were analyzed with one - way ANOVA by SPSS statistic software.Results1. Development of hypercholesterolemic ratsAfter rich cholesterol diet had been fed for 14 days ,TC concentration of rats (4.55 ± 1.25mmol/L) was almost 2 - fold higher than rats fed ordinary laboratory diets(2.30 ±0.18mmol/L).2. Feed intake and body weight gain of rat during treatmentFeed intakes of all groups were similar. Body weight gain and feed efficiency ratio of casein protein group were higher than that of soy protein group, but the difference was not statistically significant.3. Plasma lipidsCompared with the rats in casein group, TC, HDL - C and LDL - C levels of soy protein group were significantly low(P < 0.05) after treatment was given for 56 days. Whereas,the changes of TG were not significant in these groups( P > 0. 05).4. Effect of soy protein on hepatic gene expressionCompared with casein group,CYP7A mRNA expressions of 20% and 30% soy protein groups were significantly higher(33.0% and 35.2% , respectively, P <0.05) ;HMG-CoA R mRNA expressions of 20% soy protein group was significantly lower(54. 9% of control, P <0. 01) ,that of 30% soy protein group was only lower 0.09%.Conclusion1. Soy protein increase CYP7A mRNA gene expression of hypercholesterolemic rats, up - regulating CYP7 A activity and stimulating cholesterol conversion to bile acids , which decrease plasma cholesterol concentration.2. Soy protein doesn't affect HMG - CoA gene expression directly. When intake of soy protein is high, CYP7A gene expression were remarkably increased , significantly lowering TC concentration by promoting cholesterol conve-rtion to bile acids, that stimulate HMG - CoA gene expression by the feedback...