| With the improvement of living standard, requirement for the quality of livestock products is much higher, especially for pork products. As a result, how to reduce fat deposition and improve meat quality has become a new problem which troubled cultivation industry. Previous study reported that supplementation of functional additive, such as phyto-extracts, could effectively alter fatty acid composition, depress abdominal fat sediment and improve meat flavor. Studies in human and experimental animals indicated that the isoflavones which widely exist in soybean could reduce blood fat level and depress fat deposition, but had no obviously side effect. These results have demonstrated the potential of isoflavones in regulating body fat composition and improve carcass quality. Daidzein, a main isoflavone in soybean, can be degraded into equol which is more bioactive than daidzein by gastrointestinal microflora. Daidzein and equol both had the similar structure to endogenous estrogen and they could play estrogenic role in vivo. This paper was conducted to investigate the effects of single supplementation of daidzein, equol and co-supplementation of daidzein and equol producing bacteria zx-7in soybean-free diets on growth performance, blood fat level and liver lipid metabolism in SD rat, and to clarify whether equol producing bacteria zx-7was able to increase the effects of daidzein on body fat level and lipid metabolism, furthermore to evaluate the regulation mechanism and estrogen receptor dependency of daidzein and equol on cholesterol metabolism through cell culture in vitro. This thesis was described in the following three sections:Section1:To investigate the effects of single supplementation of daidzein, equol and co-supplementation of daidzein and equol producing bacteria zx-7in soybean-free diets on growth performance, blood fat level, liver fat content and lipid metabolism correlated enzymes activity,30female SD rats with an average body weight of211±9g were selected and randomly allotted to5groups. The5groups were:control group, soybean free diet with introgastric administration of1mL of sterile purified water per day; estradiol group, soybean free diet with introgastric administration of0.2mg of estrodiol per day; daidzein group, soybean free diet with introgastric administration of10mg of daidzein per day; equol group, soybean free diet with introgastric administration of10mg of equol per day; daidzein and equol producing bacteria group, soybean free diet with introgastric administration of10mg of daidzein per day and1ml of equol producing bacteria culture fluid on the day of1st,2nd,3rd and21st. All rats were slaughtered on the42nd day to collect blood, muscle and organ samples to determine body fat content, blood fat level, liver fat content and activities of lipid metabolism correlated enzymes. During the whole trial period, the initial and final body weight of each rat and the feed intake of each cage were weighted to calculate the average daily gain (ADG), average daily feed intake (ADFI) and feed:gain ratio (F:G). The results showed that the body weight, liver weight and body fat content in treatment groups were variously reduced comparing to control group, among them only body weight in equol group reached significant level (P<0.05). There were no remarkable difference on ADFI and F:G in all groups (P>0.05). Daidzein supplementation significantly reduced total cholesterol (TC) and the ratio of low density lipoprotein cholesterol to high density lipoprotein cholesterol (LDL-c/HDL-c) in blood (P<0.05). Equol supplementation obviously lowered TC, LDL-c/HDL-c and nonesterified fatty acids (NEFA)(P<0.05), while increased blood triglyeride (TG) content significantly (P<0.05). Co-supplementation of daidzein and equol producing bacteria zx-7significantly decreased blood content of TC and LDL (P<0.05). All treatments obviously decreased the content of liver TG (P<0.05), while equol significantly decreased contents of liver TC, TG and NEFA (P<0.05). All treatments remarkably increased Total Lipase (TL=LPL+HL) activity and depressed Malate Dehydrogenase (MDH) activity (P<0.05). In conclusion, supplementation of daidzein and equol had the trend of depressing body weight growth, reducing liver fat content and improving the transportation and utilization of lipid in rat. As the end metabolic product of daidezein, equol showed higher regulative activity in this experiment. Introgastric administration of equol-producing bacteria zx-7can effectively improve the regulative activity of daidzein in lipid metabolism, especially in cholesterol metabolism.Section2:In order to clarify the regulative mechanism of daidzein and equol on cholesterol metabolism, healthy adult’s hepatocyte HL-7702incubated in different concentration of daidzein and equol was used to evaluate expression of cholesterol metabolism correlated genes. Results firstly showed a dosage effect of daidzein and equol on proliferation of healthy adult’s hepatocyte HL-7702. MTT results showed that0.1and1μmol/L of daidzein and equol could remarkably increase cell growth activity (P<0.01).10and50μmol/L of daidzein and10μmol/L of equol had no significant effect on proliferation of HL-7702, while50μmol/L of equol significantly depressed the proliferation of HL-7702. Results with realtime PCR showed that50μmol/L of daidzein and equol could significantly increase the expression of LDL-R and HMGCR gene (P<0.01), and equol showed greater effect than daidzein. For the expression of SREBP-2gene, daidzein and equol showed slight enhancement (P>0.05). These results indicated that the effects of daidzein and equol on hepatocyte cholesterol metabolism may through regulating multiple gene expression such as LDL-R, SREBP-2and HMGCR. Different gene had different doesage susceptibility. Low concentration of daidzein and equol could depress HMGCR gene expression which was responsible for TC synthesis, and increase SERBP-2gene expression which was best known to up-regulate genes involved in cholesterol biosynthesis and uptake. Moreover, higer concentration of the daidzein and equol could up-regulate LDL-R mRNA expression and then promote the clearance of cholesterol in the blood. Nevertheless, these effects may depend on appropriate drug concentration and treatment time.Section3:To investigate the estrogen receptor dependence of effects of daidzein and equol on LDL-R, SERBP-2and HMGCR gene expression, healthy adult’s hepatocyte HL-7702were treated with daidzein, equol and estrogen receptor antagonist (ICI182,780). Based on the results of section2, treatments were as follows:LDL-R gene:Control,50μmol/L Daidzein,50μmol/L Equol,50μmol/L Daidzein+50μmol/L ICI182,780,50μmol/L Equol+50μmol/L ICI182,780; SREBP-2gene:Control,0.1μmol/L Daidzein,0.1μmol/L Equol,0.1μmol/L Daidzein+0.1μmol/L ICI182,780,0.1μmol/L Equol+0.1μmol/L ICI182,780; HMGCR gene:Control,0.1μmol/L Daidzein,0.1μmol/L Equol,0.1μmol/L Daidzein+0.1μmol/L ICI182,780,0.1μmol/L Equol+0.1μmol/L ICI182,780. After24h incubation, cells were collected to evaluate LDL-R, SREBP-2and HMGCR gene expression using Real-time PCR. The results showed that the enhancement of50μmol/L of daidzein and equol on intracellular LDL-R expression could completely eliminate by estrogen receptor antagonist (50μmol/L ICI182,780), which indicated that the up-regulation effect of daidzein and equol addition on LDL-R expression might depend on the activation of estrogen receptor. Low concentration of daidzein and equol both could slightly increase intracellular SREBP-2expression (P>0.05), and estrogen receptor antagonist (ICI182,780) could partially eliminate the effect. This result suggested that the regulation effect of daidzein and equol on SREBP-2expression might not only depend on estrogen receptor. Low concentration of daidzein and equol depressed intracellular HMGCR expression obviously (P<0.01). Supplementation of ICI182,780could partially remove this depression (P>0.05). This result indicated that the inhibition effect of daidzein and equol on HMGCR expression might mediated by estrogen receptor. |
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