中链脂肪酸对小鼠胆固醇代谢的调节及其机制研究
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摘要
目的
1.探索并证实中链脂肪酸(medium-chain fatty acids, MCFAs)具有改善高胆固醇饲料诱导的高胆固醇血症小鼠胆固醇代谢的作用。
2.探讨中链脂肪酸(辛酸和癸酸)上述作用的可能机制，证实其具有促进粪中性固醇和粪胆汁酸排泄作用。
3.探讨中链脂肪酸(辛酸和癸酸)促进粪中性固醇和粪胆汁酸排泄可能的作用机制。
方法
1. C57BL/6J小鼠高胆固醇血症模型的建立
4周龄雄性C57BL/6J小鼠102只，用普通饲料适应喂养1周后，随机挑选出10只小鼠继续喂养普通饲料(AIN-96G)，设为对照组(Control)，余92只小鼠喂饲富含胆固醇饲料(CR组)，饲料含蛋白19.07%、脂肪13.63%、碳水化合物48.18%、胆固醇1%和胆盐0.3%。喂养2周后，禁食不禁水12h，颌下静脉采血0.2mL，测定血清总胆固醇(totalcholesterol, TC)和总胆汁酸(total bile acids, TBA)含量。
2. MCFAs对C57BL/6J高胆固醇血症小鼠胆固醇代谢的调节作用
采用前述高胆固醇血症模型小鼠按照空腹血清TC水平和空腹体重随机分6组(每组9只)，分别采用含2%的辛酸(C8:0)、癸酸(C10:0)、油酸(C18:1)、α-亚麻酸(C18:3)、棕榈酸(C16:0)和硬脂酸(C18:0)的富含胆固醇饲料喂养12周，期间记录饲料消耗量和小鼠体重。在实验6周颌下静脉采血0.2mL检测血TC、TBA、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-c)和高密度脂蛋白胆固醇(high-densitylipoprotein cholesterol, HDL-c)水平；实验12周麻醉后处死小鼠，腹主动脉取血，检测血TC、TBA、LDL-c、HDL-c、高密度脂蛋白(high-density lipoprotein, HDL)和载脂蛋白A1(apolipoprotein A1, apo-A1)水平，解剖分离肝脏和附睾周围白色脂肪组织，取出后用生理盐水洗净吸干后称重，肝组织匀浆后检测甘油三酯(triacylglycerol, TG)、TC、磷脂（phospholipid, PL）、LDL-c和HDL-c水平。
3. MCFAs促进C57BL/6J高胆固醇血症小鼠粪便内中性固醇和胆汁酸排泄的研究
按上述方法喂养小鼠，各组中随机取5只小鼠，分别在实验开始前和实验结束前放入代谢笼，单笼喂养3天，收集并记录各自饲料消耗和粪便排出量。收集后的粪便冷冻干燥后称重、粉碎，分别用气相-质谱联用法和液相-质谱联用法检测粪便内中性固醇和胆汁酸含量，并计算其3天的总排出量。
4. MCFAs调节C57BL/6J高胆固醇血症小鼠肝脏胆固醇代谢的机制研究
按上述方法喂养小鼠，实验12周结束后，处死小鼠取肝脏组织，分别用Real-timeRT-PCR法和Western blotting法检测其中的羟甲基戊二酸单酰辅酶A还原酶(hydroxymethyl glutaryl coenzyme A reductase, HMG-CR)、胆固醇7α羟化酶(cholesterol7α-hydroxylase, CYP7A1)、肝X受体(liver X receptor, LXR)和法尼酯受体(farnesoid Xreceptor, FXR)的mRNA转录水平和蛋白水平情况。
结果
1. C57BL/6J小鼠高胆固醇血症模型的建立
高胆固醇饲料喂养2周后，CR组(92只)小鼠空腹血清TC水平基本呈正态分布，其TC水平高于对照组2个标准差(2SD)以上的小鼠共64只，峰值集中分布在2.78mmol/L到4.22mmol/L之间，即高于对照组均值(2.3mmol/L)的2SD到8SD之间，共计58只，占全部CR组的63%，高胆固醇血症小鼠模型建立成功。
2. MCFAs对C57BL/6J高胆固醇血症小鼠血脂及胆固醇代谢的调节作用
1)实验结束(12周)时：C8:0、C10:0和C18:3组小鼠的空腹体重和肝脏组织重均显著低于C16:0组(P<0.05)和C18:0组(P<0.01)；C8:0、C10:0、C18:1、C16:0和C18:3组小鼠BMI、Lee’s指数均显著低于C18:0组(P<0.05)。
2)实验结束(12周)时：C8:0组(P<0.01)、C10:0组(P<0.05)及C18:3组(P<0.01)的血清TC水平显著低于C16:0组； C8:0、C10:0和C18:3组的血清LDL-c水平显著低于C16:0组(P<0.05)；C8:0、C10:0及C18:3组的HDL-c/LDL-c比值显著高于C18:1、C16:0及C18:0组；C8:0组(P<0.05)、C10:0组(P<0.01)、C18:1组(P<0.05)及C18:3组(P<0.05)血清TG水平显著低于C16:0组；C8:0组(P<0.05)血清HDL水平显著高于C18:1组、C16:0组和C18:0组。
3)实验结束(12周)时：各组间肝脏组织中的TC、LDL-c、HDL-c、TG和LP水平没有显著差异(P>0.05)。
3. MCFAs促进C57BL/6J高胆固醇血症小鼠粪便内中性固醇和胆汁酸排泄的研究
1)小鼠粪便中的胆固醇是粪便中性固醇的主要排出形式，约占97%；胆酸(cholic acid,CA)是粪便总胆汁酸的主要排出形式，占75%以上。
2)研究结束时，C8:0组(P<0.05)小鼠粪便中的胆固醇排出量及中性固醇总排出量均显著高于C16:0组和C18:0组； C8:0组小鼠粪便中的二氢胆固醇排出量显著高于C16:0组(P<0.05)。
3)研究结束时，粪便中游离胆汁酸(unconjugated bile acids, UCBAs)排泄情况如下：①C8:0组(P<0.05)的熊脱氧胆酸(ursodeoxycholic acid, UDCA)和石胆酸(lithocholicacid, LCA)排泄量显著高于C16:0组和C18:0组，CA及总UCBAs的排泄量显著高于C16:0、C18:0和C18:1组(P<0.05)，并且结束时CA排泄量显著高于自身在实验开始的排泄量(P<0.01)；②C10:0组(P<0.05)的CA排泄量显著高于C18:0和C18:1组，而总UCBAs的排泄量显著高于C16:0和C18:0组；③C18:1组(P<0.05)的LCA排泄量显著高于C16:0和C18:0组；④C18:3组(P<0.05)的UDCA排泄量显著高于C16:0和C18:0组，而CA及总UCBA的排泄量显著高于C18:0组。
4)研究结束时，粪便中牛磺酸结合型胆汁酸(taurine conjugated bile acids, TCBAs)排泄情况如下：①C8:0组(P<0.05)的牛磺脱氧胆酸(taurodeoxycholic acid, TDCA)排泄量显著高于C16:0、C18:0和C18:1组，牛磺鹅脱氧胆酸(taurochenodeoxycholic acid,TCDCA)及总TCBAs的排泄量显著高于C18:0组；②C10:0组(P<0.05)的TDCA排泄量显著高于C18:0组，TCDCA及总TCBAs的排泄量显著高于C16:0和C18:0组；③C18:1组(P<0.05)的TDCA排泄量显著低于C16:0组，TCDCA排泄量显著高于C16:0和C18:0组，总TCBAs的排泄量显著高于C18:0组；④C18:3组(P<0.05)的TCDCA和TDCA排泄量都显著高于C18:0组。
5)研究结束时，粪便中甘氨酸结合型胆汁酸(glycine conjugated bile acids, GCBAs)排泄情况如下：C18:1组(P<0.05)小鼠粪便中甘氨鹅脱氧胆酸(glycochenodeoxycholic acid,GCDCA)及总GCBAs排泄量显著高于其他各组。
6)实验结束时：①按照胆汁酸合成经典途径中的CA及其与氨基酸结合系列产物统计，C8:0和C10:0组的排泄量显著高于C16:0、C18:0和C18:1组(P<0.01)；其中C8:0组还显著高于C18:3组(P<0.05)。②按照胆汁酸合成经典途径中的脱氧胆酸(deoxycholic acid, DCA)及其与氨基酸结合系列产物统计，C8:0组显著高于C16:0、C18:0和C18:1组(P<0.01)，并显著高于C18:3组(P<0.05)；C10:0组显著高于C18:0和C18:1组(P<0.01)。③按照只由经典合成途径生成的胆汁酸总排泄量统计，C8:0和C10:0组均显著高于C16:0、C18:0和C18:1组(P<0.01)，其中C8:0组还显著高于C18:3组(P<0.05)。
7)实验结束时：①按照胆汁酸合成替代途径中的鹅脱氧胆酸(chenodeoxycholic acid,CDCA)及其与氨基酸结合系列产物统计，C10:0组的排泄量显著高于C16:0、C18:0和C18:3组(P<0.01)，C8:0组显著高于C18:0组(P<0.05)。②按照胆汁酸合成替代途径中的LCA及其与氨基酸结合系列产物统计，C8:0组显著高于C16:0和C18:0组(P<0.01)；C10:0组显著高于C16:0和C18:0组(P<0.05)。③按照只可由替代合成途径生成的胆汁酸总排泄量进行统计，C8:0和C10:0组均显著高于C16:0和C18:0组(P<0.01)，其中C10:0组还显著高于C18:3组(P<0.05)。
8)实验结束时：总初级胆汁酸排泄量比较发现C8:0和C10:0组均显著高于C16:0、C18:0和C18:1组(P<0.01)，其中C8:0组还显著高于C18:3组(P<0.05)；总次级胆汁酸排泄量比较发现C8:0组显著高于C16:0、C18:0和C18:1组(P<0.01)，以及显著高于C18:3组(P<0.05)，而C10:0组显著高于C16:0组(P<0.05)和C18:0组(P<0.01)。
9) C8:0和C10:0组粪便中总胆汁酸排泄量均显著高于C16:0、C18:0和C18:1组(P<0.05)，并且C18:3组也显著高于C18:0组(P<0.05)。
4. MCFAs调节C57BL/6J高胆固醇血症小鼠肝脏胆固醇代谢的机制研究
1)实验结束时，各组小鼠肝脏组织中HMG-CR的mRNA转录和蛋白表达水平均无显著差异(P>0.05)。
2)实验结束时，C8:0和C10:0组小鼠肝组织中的CYP7A1的mRNA转录和蛋白表达水平均显著高于C16:0和C18:0组(P<0.05)；C18:1和C18:3组小鼠肝组织中的CYP7A1的mRNA转录水平显著高于C18:0组(P<0.05)。
3)实验结束时，C8:0组小鼠肝组织中LXR的mRNA转录水平显著高于C18:0组(P<0.05)，蛋白表达水平显著高于C18:1和C18:3组(P<0.01)；C10:0组小鼠肝组织中的LXR的mRNA转录(P<0.05)和蛋白表达水平(P<0.01)显著高于C16:0和C18:0组。C18:3组肝组织中LXR的蛋白表达水平显著高于C16:0和C18:0组(P<0.01)。
4)实验结束时，各组小鼠肝脏组织中FXR的mRNA转录水平均无显著差异(P>0.05)；C8:0和C10:0组的FXR的蛋白表达水平显著低于C16:0和C18:0组(P<0.01)。
结论
1.给予雄性C57BL/6J小鼠富含胆固醇饲料(脂肪占13.63%，蛋白质占19.07%，碳水化合物占48.18%，胆固醇占1%，胆盐占0.3%)喂养2周后，可诱导60%的小鼠空腹血清胆固醇水平显著升高，此方法复制的高胆固醇血症小鼠模型可作为后续研究的动物模型。
2.长期给予MCFAs饲料喂养12周后，与LCFAs相比，MCFAs可显著降低高胆固醇血症小鼠体重、Lee’s指数、BMI和肝脏组织重，改善血清TG、TC、LDL-c和HDL-c水平，但并不影响肝脏脂蛋白水平。
3. MCFAs改善血清TG、TC、LDL-c和HDL-c水平主要途径之一是促进了粪便中胆固醇和胆汁酸的排泄，其中辛酸主要通过促进粪胆固醇和粪胆酸排泄从而降低血清TC和LDL-c水平；癸酸主要通过促进粪便中粪胆酸排泄从而降低血清TC和LDL-c水平。
4. MCFAs通过上调肝脏组织中LXR的转录和蛋白表达水平，同时下调FXR的蛋白表达水平，从而上调CYP7A1酶的转录和蛋白表达，进而促进了粪便的中性固醇和胆汁酸的排泄；同时并不影响肝脏组织中的HMG-CR酶的转录和蛋白表达。MCFAs对肝脏中控制胆固醇合成和代谢的两个关键限速酶的上述作用共同促进了机体内的胆固醇经由粪便以中性固醇和胆汁酸的形式大量排出，进而降低血清TC和LDL-c水平。
Objective
1. To investigate and confirm that medium-chain fatty acids (MCFAs) can effectivelyimprove cholesterol metabolism in mice with hypercholesterolemia induced by acholesterol-rich diet.
2. To test our working hypothesis that MCFAs (caprylic acid and capric acid) regulatecholesterol metabolism by enhancing the excretion of faecal neutral steroid and bileacids.
3. To clarify a possible mechanism for MCFAs (caprylic acid and capric acid) to increasethe excretion of faecal neutral steroid and bile acids.
Methods
1. Hypercholesterolemia model in C57BL/6J Mouse
One hundred and two C57BL/6J male mice, aged about4weeks, were used and were feda normal diet (AIN-96G) to adapt circumstance for one week.10mice were randomly chosento be a normal control and feed the normal diet. The other mice were fed a cholesterol-richdiet (CR). The CR diet contains19.07%protein,13.63%fat,48.18%carbohydrate,1%cholesterol and0.3%bile acids. After2weeks feeding, all mice were deprived of diet, butnot water, for overnight fasting (at least12h).0.2mL blood samples were collected frommandibular venous plexus for determination of levels of total cholesterol (TC) and total bileacids (TBA).
2. Effects of MCFAs on cholesterol metabolism in C57BL/6J mice withhypercholesterolemia
The hypercholesterolemia model in C57BL/6J mice was established according to themethod of the first part of the experiment, and the hypercholesterolemia mice were randomlyassigned to six groups (9mice in each group) according to their fasting blood TC levels andwere fed six different experimental diets containing2%caprylic acid (C8:0),2%capric acid(C10:0),2%palmitic acid (C16:0),2%stearic acid (C18:0),2%oleic acid (C18:1), or2%α-linolenic acid (C18:3), respectively for consecutive12weeks. During the study duration,diet intakes and body weights were measured every week. After6weeks, fasting bloodsamples were collected from the mandibular venous plexus, and serum TC, low-densitylipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) andHDL-c/LDL-c ratio were measured or calculated. At the end of the study, all mice weredeprived of diets, but not water, for overnight fasting (at least12h). In the followingmorning after the fasting, the mice were anesthetized, and blood samples were collected fromthe aorta ventralis for measurement of fasting serum TC, HDL-c, LDL-c, high-densitylipoprotein (HDL) and apolipoprotein-A1(apo-A1). Livers and epididymal fat pads wereexcised, rinsed with icy saline and weighed. Livers were homogenized for measurement of TC, triacylglycerol (TG), phospholipid (PL), LDL-c and HDL-c.
3. Effect of MCFAs on the excretion of faecal neutral steroids and bile acids inC57BL/6J mice with hypercholesterolemia
The mice model and diet used were the same to the methods of the second part of theexperiment, and5mice were chosen randomly from each group for their recording dietintakes and sampling faeces output. Daily diet intakes and faeces of each mouse wererecorded and collected by using the metabolic cage for consecutive3days at the start and endof the study. Faecal neutral steroids and bile acids were measured by gaschromatography-mass spectrometry (GCMS) and liquid chromatography-mass spectrometry(LCMS) respectively, and the total amounts of the excretions in3days were calculated.
4. Investigation of the mechanism of lowering cholesterol by MCFAs in the liver ofC57BL/6J hypercholesterolemia mice
The mice were fed and killed according to the methods of the second part of theexperiment, livers were used for measurement of mRNA transcription and protein expressionlevel of hydroxymethyl glutaryl coenzyme A reductase (HMG-CR), cholesterol7α-hydroxylase (CYP7A1), liver X receptor (LXR) and farnesoid X receptor (FXR) by theReal-time RT-PCR and Western blotting analysis, respectively.
Results
1. Hypercholesterolemia model in C57BL/6J Mouse
Two weeeks after feeding the CR diet, Gaussian distribution of levels of serum TC of themice in CR groups (92mice) were observed,64of them were higher than the average levelplus2standard deviation (SD) of the control group, and most of them (58mice) were rangedfrom2.78to4.22mmol/L (i.e.2SD to8SD), which are up to63%among the CR groups.We assumed that hypercholesterolemia model in C57BL/6J mice fed CR diet wassuccessfully established.
2. Effects of MCFAs on cholesterol metabolism in C57BL/6J mice withhypercholesterolemia
1) At the end of study, the fasting body weight and liver weight of the mice in the C8:0,C10:0or C18:3groups were significantly lower than those in the C16:0(P<0.05) or C18:0(P<0.01) groups. The BMI, Lee’s indices of the mice in the C8:0, C10:0, C18:1, C16:0orC18:3groups were significantly lower than those in the C18:0group (P<0.05).
2) At the end of study, the level of serum TC in the C8:0(P<0.01), C10:0(P<0.05) orC18:3(P<0.01) groups were significantly lower than that in the C16:0group. The level ofserum LDL-c in the C8:0, C10:0or C18:3groups were significantly lower than those in theC16:0(P<0.05) group. The ratio of serum HDL-c/LDL-c in the C8:0, C10:0or C18:3groupswere significantly higher than those in the C18:1, C16:0or C18:0groups (P<0.05). The levelof serum TG in the C8:0(P<0.05), C10:0(P<0.01), C18:1(P<0.05) or C18:3(P<0.05)groups were significantly lower than those in the C16:0diet group. The level of serumHDL-c in the C8:0(P<0.05) group was significantly higher than that in the C18:1, C16:0orC18:0diet groups.
3) At the end of study, the concentrations of TC, LDL-c, HDL-c, TG and LP of liverhomogenates were no significantly differences among all the groups (P>0.05).
3. Effect of MCFAs on the excretion of faecal neutral steroids and bile acids inC57BL/6J mice with hypercholesterolemia
1) Cholesterol (up to97%) and cholic acid (more than75%) were the main components ofneutral steroids and bile acids, respectively in mice faeces.
2) At the end of study, the faecal excretion of cholesterol and total neutral steroids in theC8:0group were significantly higher than those in the C16:0or C18:0groups (P<0.05). Thefaecal excertion of cholestanol in the C8:0group was significantly higher than that in theC16:0group (P<0.05).
3) At the end of study, the changes in faecal excretion of unconjugated bile acids (UCBAs)were as follows:①The faecal excretion of ursodeoxycholic acid (UDCA) and lithocholicacid (LCA) in the C8:0group were significantly higher than those in the C16:0or C18:0groups (P<0.05), the faecal excretion of cholic acid (CA) and total UCBAs in the C8:0groupwere significantly higher than those in the C16:0, C18:0or C18:1groups (P<0.01), and thefaecal excretion of CA in the C8:0group at the end of the study was significantly higher thanthat at the start of the study (P<0.01);②The faecal excretion of CA in the C10:0group wassignificantly higher than that in the C18:1or C18:0groups (P<0.05), and the faecal excretionof total UCBAs in the C10:0group was significantly higher than that in the C16:0or C18:0groups (P<0.05);③The faecal excretion of LCA in the C18:1group was significantlyhigher than that in the C16:0or C18:0groups (P<0.05).④The faecal excretion of UDCAin the C18:3group was significantly higher than that in the C16:0or C18:0groups (P<0.05),and the faecal excretion of CA and total UCBAs in the C18:3group were significantly higherthan those in the C18:0group (P<0.05).
4) At the end of study, the changes in faecal excretion of taurine conjugated bile acids(TCBAs) were as follows:①The faecal excretion of taurodeoxycholic acid (TDCA) in theC8:0group was significantly higher than that in the C16:0, C18:0or C18:1groups (P<0.05),the faecal excretion of taurochenodeoxycholic acid (TCDCA) and total TCBAs in the C8:0group were significantly higher than those in the C16:0or C18:0groups (P<0.05);②Thefaecal excretion of TDCA in the C10:0group was significantly higher than that in the C18:0group (P<0.05), and the faecal excretion of TCDCA and total TCBAs in the C10:0diet groupwere significantly higher than those in the C16:0or C18:0groups (P<0.05);③The faecalexcretion of TDCA in the C18:1group was significantly lower than that in the C16:0group(P<0.05), the faecal excretion of TCDCA in the C18:1group was significantly higher thanthat in the C16:0or C18:0groups (P<0.05), and the faecal excretion of total TCBAs in theC18:1group was significantly higher than that in the C18:0group (P<0.05);④The faecalexcretions of TCDCA and TDCA in the C18:3group were significantly higher than those inthe C18:0group (P<0.05).
5) At the end of study, the changes in faecal excretion of glycine conjugated bile acids (GCBAs) were as follows: The faecal excretion of glycochenodeoxycholic acid (GCDCA)and total GCBAs in the C18:1group were significantly higher than those in the any othergroups (P<0.05).
6) At the end of study, the changes of faecal excretion of bile acids which synthesis fromclassical pathway were as follows:①The faecal excretion of all the bile acids related to CA,which in the C8:0or C10:0groups were significantly higher than that in the C16:0, C18:0orC18:1groups (P<0.01), and that in the C8:0group was also significantly higher than that inthe C18:3group (P<0.05);②The faecal excretion of all the bile acids related todeoxycholic acid (DCA), which in the C8:0group was significantly higher than that in theC16:0, C18:0or C18:1groups (P<0.01), as well as C18:3group (P<0.05). And it in theC10:0group was also significantly higher than that in the C18:0or C18:1groups (P<0.05);③The faecal excretion of all the bile acids that only synthesis from classical pathway,which in the C8:0or C10:0groups were significantly higher than that in the C16:0, C18:0orC18:1groups (P<0.01), and that in the C8:0group was also significantly higher than that inthe C18:3group (P<0.05).
7) At the end of study, the changes in faecal excretion of bile acids which synthesis fromalternative pathway were as follows;①The faecal excretion of all the bile acids related tochenodeoxycholic acid (CDCA), which in the C10:0group was significantly higher than thatin the C16:0, C18:0or C18:3groups (P<0.01), while that in the C8:0group was significantlyhigher than that in the C18:0group (P<0.05);②The faecal excretion of all the bile acidsrelated to LCA, which in the C8:0groups was significantly higher than that in the C16:0orC18:0groups (P<0.01), while that in the C10:0group was significantly higher than that inthe C16:0or C18:0groups (P<0.05);③The faecal excretion of all the bile acids that onlysynthesized from alternative pathway in the C8:0or C10:0groups were significantly higherthan those in the C16:0or C18:0groups (P<0.01), furthermore, that in the C10:0group wasalso significantly higher than that in the C18:3group (P<0.05).
8) At the end of study, the faecal excretion of total primary bile acids in the C8:0or C10:0groups were significantly higher than those in the C16:0, C18:0or C18:1groups (P<0.01),furthermore, that in the C8:0diet group was significantly higher than that in the C18:3group(P<0.05). While the faecal excretion of total secondly bile acids in the C8:0groups wassignificantly higher than that in the C16:0, C18:0or C18:1group (P<0.01), and that in theC10:0group was significantly higher than that in the C16:0(P<0.05) or C18:0(P<0.01)groups.
9) At the end of study, the faecal excretion of total bile acids in the C8:0or C10:0groupswere significantly higher than those in the C16:0, C18:0or C18:1group (P<0.05), and that inthe C18:3group was significantly higher than that in the C18:0group (P<0.05).
4. Investigation of a possible mechanism of lowering cholesterol by MCFAs in the liverof C57BL/6J hypercholesterolemia mice
1) At the end of study, neither the mRNA transcriptions nor the protein expressions of HMG-CR in mice livers were different among all groups (P>0.05).
2) At the end of study, the mRNA transcriptions and protein expressions of CYP7A1inmice livers in the C8:0or C10:0group were significantly higher than those in the C16:0and C18:0group (P<0.05), and the mRNA transcriptions of CYP7A1in the C18:1orC18:3group were significantly higher than those in the C18:0group (P<0.05).
3) At the end of study, the mRNA transcriptions of LXR in mice livers in the C8:0groupwas significantly higher than that in the C18:0group (P<0.05). The mRNAtranscriptions (P<0.05) and protein expressions (P<0.01) of LXR in mice livers in theC10:0group were significantly higher than those in the C16:0or C18:0group. Theprotein expressions of LXR in mice livers in the C18:3group was significantly higherthan that in the C16:0or C18:0group (P<0.05).
4) At the end of study, the mRNA transcriptions of FXR in mice livers was no differenceamong all groups (P>0.05). While the protein expressions of FXR in mice livers in theC8:0or C10:0group were significantly lower than those in the C16:0and C18:0group(P<0.01).
Conclusions
1) Among more than60%of the C57BL/6J mice fed cholesterol-rich(CR) diet (whichcontains13.63%fat,19.07%protein,48.18%carbohydrate,1%cholesterol and0.3%bile acids) for2weeks, their serum cholesterol levels were significantly increased. Thismethod can be used for hypercholesteremia mice model.
2) Compared with LCFAs, which were fed in CR diet in C57BL/6J male mice, MCFAs caneffectively reduce body weight, Lee’s index, BMI and liver weight, and improve theserum TC, TG, LDL-c and HDL-c level, but not lipid profiles in liver of the mice.
3) Improvement of the serum lipid profiles by MCFAs might be mainly due to theirenhancement of excretion of faecal neutral steroids and bile acids. C8:0enhanced mainlythe excretion of feacel cholesterol and CA, and C10:0enhanced mainly the excretion offeacel CA.
4) MCFAs can increase the mRNA transcription and protein expression of CYP7A1, whichwas considered to be due to both the enhancement of the mRNA transcription and proteinexpression of LXR and the inhibition of the protein expression of FXR in liver. MCFAsdid not regulate the mRNA transcription and protein expression of HMG-CR. Thesynergetic effects of the two rate-limiting enzymes of cholesterol synthesis in liverenhanced the cholesterol excretion through the feacel neutral steroids and bile acids andfinally reduced the serum TC and LDL-c level.
1.探索并证实中链脂肪酸(medium-chain fatty acids, MCFAs)具有改善高胆固醇饲料诱导的高胆固醇血症小鼠胆固醇代谢的作用。
2.探讨中链脂肪酸(辛酸和癸酸)上述作用的可能机制，证实其具有促进粪中性固醇和粪胆汁酸排泄作用。
3.探讨中链脂肪酸(辛酸和癸酸)促进粪中性固醇和粪胆汁酸排泄可能的作用机制。
方法
1. C57BL/6J小鼠高胆固醇血症模型的建立
4周龄雄性C57BL/6J小鼠102只，用普通饲料适应喂养1周后，随机挑选出10只小鼠继续喂养普通饲料(AIN-96G)，设为对照组(Control)，余92只小鼠喂饲富含胆固醇饲料(CR组)，饲料含蛋白19.07%、脂肪13.63%、碳水化合物48.18%、胆固醇1%和胆盐0.3%。喂养2周后，禁食不禁水12h，颌下静脉采血0.2mL，测定血清总胆固醇(totalcholesterol, TC)和总胆汁酸(total bile acids, TBA)含量。
2. MCFAs对C57BL/6J高胆固醇血症小鼠胆固醇代谢的调节作用
采用前述高胆固醇血症模型小鼠按照空腹血清TC水平和空腹体重随机分6组(每组9只)，分别采用含2%的辛酸(C8:0)、癸酸(C10:0)、油酸(C18:1)、α-亚麻酸(C18:3)、棕榈酸(C16:0)和硬脂酸(C18:0)的富含胆固醇饲料喂养12周，期间记录饲料消耗量和小鼠体重。在实验6周颌下静脉采血0.2mL检测血TC、TBA、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-c)和高密度脂蛋白胆固醇(high-densitylipoprotein cholesterol, HDL-c)水平；实验12周麻醉后处死小鼠，腹主动脉取血，检测血TC、TBA、LDL-c、HDL-c、高密度脂蛋白(high-density lipoprotein, HDL)和载脂蛋白A1(apolipoprotein A1, apo-A1)水平，解剖分离肝脏和附睾周围白色脂肪组织，取出后用生理盐水洗净吸干后称重，肝组织匀浆后检测甘油三酯(triacylglycerol, TG)、TC、磷脂（phospholipid, PL）、LDL-c和HDL-c水平。
3. MCFAs促进C57BL/6J高胆固醇血症小鼠粪便内中性固醇和胆汁酸排泄的研究
按上述方法喂养小鼠，各组中随机取5只小鼠，分别在实验开始前和实验结束前放入代谢笼，单笼喂养3天，收集并记录各自饲料消耗和粪便排出量。收集后的粪便冷冻干燥后称重、粉碎，分别用气相-质谱联用法和液相-质谱联用法检测粪便内中性固醇和胆汁酸含量，并计算其3天的总排出量。
4. MCFAs调节C57BL/6J高胆固醇血症小鼠肝脏胆固醇代谢的机制研究
按上述方法喂养小鼠，实验12周结束后，处死小鼠取肝脏组织，分别用Real-timeRT-PCR法和Western blotting法检测其中的羟甲基戊二酸单酰辅酶A还原酶(hydroxymethyl glutaryl coenzyme A reductase, HMG-CR)、胆固醇7α羟化酶(cholesterol7α-hydroxylase, CYP7A1)、肝X受体(liver X receptor, LXR)和法尼酯受体(farnesoid Xreceptor, FXR)的mRNA转录水平和蛋白水平情况。
结果
1. C57BL/6J小鼠高胆固醇血症模型的建立
高胆固醇饲料喂养2周后，CR组(92只)小鼠空腹血清TC水平基本呈正态分布，其TC水平高于对照组2个标准差(2SD)以上的小鼠共64只，峰值集中分布在2.78mmol/L到4.22mmol/L之间，即高于对照组均值(2.3mmol/L)的2SD到8SD之间，共计58只，占全部CR组的63%，高胆固醇血症小鼠模型建立成功。
2. MCFAs对C57BL/6J高胆固醇血症小鼠血脂及胆固醇代谢的调节作用
1)实验结束(12周)时：C8:0、C10:0和C18:3组小鼠的空腹体重和肝脏组织重均显著低于C16:0组(P<0.05)和C18:0组(P<0.01)；C8:0、C10:0、C18:1、C16:0和C18:3组小鼠BMI、Lee’s指数均显著低于C18:0组(P<0.05)。
2)实验结束(12周)时：C8:0组(P<0.01)、C10:0组(P<0.05)及C18:3组(P<0.01)的血清TC水平显著低于C16:0组； C8:0、C10:0和C18:3组的血清LDL-c水平显著低于C16:0组(P<0.05)；C8:0、C10:0及C18:3组的HDL-c/LDL-c比值显著高于C18:1、C16:0及C18:0组；C8:0组(P<0.05)、C10:0组(P<0.01)、C18:1组(P<0.05)及C18:3组(P<0.05)血清TG水平显著低于C16:0组；C8:0组(P<0.05)血清HDL水平显著高于C18:1组、C16:0组和C18:0组。
3)实验结束(12周)时：各组间肝脏组织中的TC、LDL-c、HDL-c、TG和LP水平没有显著差异(P>0.05)。
3. MCFAs促进C57BL/6J高胆固醇血症小鼠粪便内中性固醇和胆汁酸排泄的研究
1)小鼠粪便中的胆固醇是粪便中性固醇的主要排出形式，约占97%；胆酸(cholic acid,CA)是粪便总胆汁酸的主要排出形式，占75%以上。
2)研究结束时，C8:0组(P<0.05)小鼠粪便中的胆固醇排出量及中性固醇总排出量均显著高于C16:0组和C18:0组； C8:0组小鼠粪便中的二氢胆固醇排出量显著高于C16:0组(P<0.05)。
3)研究结束时，粪便中游离胆汁酸(unconjugated bile acids, UCBAs)排泄情况如下：①C8:0组(P<0.05)的熊脱氧胆酸(ursodeoxycholic acid, UDCA)和石胆酸(lithocholicacid, LCA)排泄量显著高于C16:0组和C18:0组，CA及总UCBAs的排泄量显著高于C16:0、C18:0和C18:1组(P<0.05)，并且结束时CA排泄量显著高于自身在实验开始的排泄量(P<0.01)；②C10:0组(P<0.05)的CA排泄量显著高于C18:0和C18:1组，而总UCBAs的排泄量显著高于C16:0和C18:0组；③C18:1组(P<0.05)的LCA排泄量显著高于C16:0和C18:0组；④C18:3组(P<0.05)的UDCA排泄量显著高于C16:0和C18:0组，而CA及总UCBA的排泄量显著高于C18:0组。
4)研究结束时，粪便中牛磺酸结合型胆汁酸(taurine conjugated bile acids, TCBAs)排泄情况如下：①C8:0组(P<0.05)的牛磺脱氧胆酸(taurodeoxycholic acid, TDCA)排泄量显著高于C16:0、C18:0和C18:1组，牛磺鹅脱氧胆酸(taurochenodeoxycholic acid,TCDCA)及总TCBAs的排泄量显著高于C18:0组；②C10:0组(P<0.05)的TDCA排泄量显著高于C18:0组，TCDCA及总TCBAs的排泄量显著高于C16:0和C18:0组；③C18:1组(P<0.05)的TDCA排泄量显著低于C16:0组，TCDCA排泄量显著高于C16:0和C18:0组，总TCBAs的排泄量显著高于C18:0组；④C18:3组(P<0.05)的TCDCA和TDCA排泄量都显著高于C18:0组。
5)研究结束时，粪便中甘氨酸结合型胆汁酸(glycine conjugated bile acids, GCBAs)排泄情况如下：C18:1组(P<0.05)小鼠粪便中甘氨鹅脱氧胆酸(glycochenodeoxycholic acid,GCDCA)及总GCBAs排泄量显著高于其他各组。
6)实验结束时：①按照胆汁酸合成经典途径中的CA及其与氨基酸结合系列产物统计，C8:0和C10:0组的排泄量显著高于C16:0、C18:0和C18:1组(P<0.01)；其中C8:0组还显著高于C18:3组(P<0.05)。②按照胆汁酸合成经典途径中的脱氧胆酸(deoxycholic acid, DCA)及其与氨基酸结合系列产物统计，C8:0组显著高于C16:0、C18:0和C18:1组(P<0.01)，并显著高于C18:3组(P<0.05)；C10:0组显著高于C18:0和C18:1组(P<0.01)。③按照只由经典合成途径生成的胆汁酸总排泄量统计，C8:0和C10:0组均显著高于C16:0、C18:0和C18:1组(P<0.01)，其中C8:0组还显著高于C18:3组(P<0.05)。
7)实验结束时：①按照胆汁酸合成替代途径中的鹅脱氧胆酸(chenodeoxycholic acid,CDCA)及其与氨基酸结合系列产物统计，C10:0组的排泄量显著高于C16:0、C18:0和C18:3组(P<0.01)，C8:0组显著高于C18:0组(P<0.05)。②按照胆汁酸合成替代途径中的LCA及其与氨基酸结合系列产物统计，C8:0组显著高于C16:0和C18:0组(P<0.01)；C10:0组显著高于C16:0和C18:0组(P<0.05)。③按照只可由替代合成途径生成的胆汁酸总排泄量进行统计，C8:0和C10:0组均显著高于C16:0和C18:0组(P<0.01)，其中C10:0组还显著高于C18:3组(P<0.05)。
8)实验结束时：总初级胆汁酸排泄量比较发现C8:0和C10:0组均显著高于C16:0、C18:0和C18:1组(P<0.01)，其中C8:0组还显著高于C18:3组(P<0.05)；总次级胆汁酸排泄量比较发现C8:0组显著高于C16:0、C18:0和C18:1组(P<0.01)，以及显著高于C18:3组(P<0.05)，而C10:0组显著高于C16:0组(P<0.05)和C18:0组(P<0.01)。
9) C8:0和C10:0组粪便中总胆汁酸排泄量均显著高于C16:0、C18:0和C18:1组(P<0.05)，并且C18:3组也显著高于C18:0组(P<0.05)。
4. MCFAs调节C57BL/6J高胆固醇血症小鼠肝脏胆固醇代谢的机制研究
1)实验结束时，各组小鼠肝脏组织中HMG-CR的mRNA转录和蛋白表达水平均无显著差异(P>0.05)。
2)实验结束时，C8:0和C10:0组小鼠肝组织中的CYP7A1的mRNA转录和蛋白表达水平均显著高于C16:0和C18:0组(P<0.05)；C18:1和C18:3组小鼠肝组织中的CYP7A1的mRNA转录水平显著高于C18:0组(P<0.05)。
3)实验结束时，C8:0组小鼠肝组织中LXR的mRNA转录水平显著高于C18:0组(P<0.05)，蛋白表达水平显著高于C18:1和C18:3组(P<0.01)；C10:0组小鼠肝组织中的LXR的mRNA转录(P<0.05)和蛋白表达水平(P<0.01)显著高于C16:0和C18:0组。C18:3组肝组织中LXR的蛋白表达水平显著高于C16:0和C18:0组(P<0.01)。
4)实验结束时，各组小鼠肝脏组织中FXR的mRNA转录水平均无显著差异(P>0.05)；C8:0和C10:0组的FXR的蛋白表达水平显著低于C16:0和C18:0组(P<0.01)。
结论
1.给予雄性C57BL/6J小鼠富含胆固醇饲料(脂肪占13.63%，蛋白质占19.07%，碳水化合物占48.18%，胆固醇占1%，胆盐占0.3%)喂养2周后，可诱导60%的小鼠空腹血清胆固醇水平显著升高，此方法复制的高胆固醇血症小鼠模型可作为后续研究的动物模型。
2.长期给予MCFAs饲料喂养12周后，与LCFAs相比，MCFAs可显著降低高胆固醇血症小鼠体重、Lee’s指数、BMI和肝脏组织重，改善血清TG、TC、LDL-c和HDL-c水平，但并不影响肝脏脂蛋白水平。
3. MCFAs改善血清TG、TC、LDL-c和HDL-c水平主要途径之一是促进了粪便中胆固醇和胆汁酸的排泄，其中辛酸主要通过促进粪胆固醇和粪胆酸排泄从而降低血清TC和LDL-c水平；癸酸主要通过促进粪便中粪胆酸排泄从而降低血清TC和LDL-c水平。
4. MCFAs通过上调肝脏组织中LXR的转录和蛋白表达水平，同时下调FXR的蛋白表达水平，从而上调CYP7A1酶的转录和蛋白表达，进而促进了粪便的中性固醇和胆汁酸的排泄；同时并不影响肝脏组织中的HMG-CR酶的转录和蛋白表达。MCFAs对肝脏中控制胆固醇合成和代谢的两个关键限速酶的上述作用共同促进了机体内的胆固醇经由粪便以中性固醇和胆汁酸的形式大量排出，进而降低血清TC和LDL-c水平。
Objective
1. To investigate and confirm that medium-chain fatty acids (MCFAs) can effectivelyimprove cholesterol metabolism in mice with hypercholesterolemia induced by acholesterol-rich diet.
2. To test our working hypothesis that MCFAs (caprylic acid and capric acid) regulatecholesterol metabolism by enhancing the excretion of faecal neutral steroid and bileacids.
3. To clarify a possible mechanism for MCFAs (caprylic acid and capric acid) to increasethe excretion of faecal neutral steroid and bile acids.
Methods
1. Hypercholesterolemia model in C57BL/6J Mouse
One hundred and two C57BL/6J male mice, aged about4weeks, were used and were feda normal diet (AIN-96G) to adapt circumstance for one week.10mice were randomly chosento be a normal control and feed the normal diet. The other mice were fed a cholesterol-richdiet (CR). The CR diet contains19.07%protein,13.63%fat,48.18%carbohydrate,1%cholesterol and0.3%bile acids. After2weeks feeding, all mice were deprived of diet, butnot water, for overnight fasting (at least12h).0.2mL blood samples were collected frommandibular venous plexus for determination of levels of total cholesterol (TC) and total bileacids (TBA).
2. Effects of MCFAs on cholesterol metabolism in C57BL/6J mice withhypercholesterolemia
The hypercholesterolemia model in C57BL/6J mice was established according to themethod of the first part of the experiment, and the hypercholesterolemia mice were randomlyassigned to six groups (9mice in each group) according to their fasting blood TC levels andwere fed six different experimental diets containing2%caprylic acid (C8:0),2%capric acid(C10:0),2%palmitic acid (C16:0),2%stearic acid (C18:0),2%oleic acid (C18:1), or2%α-linolenic acid (C18:3), respectively for consecutive12weeks. During the study duration,diet intakes and body weights were measured every week. After6weeks, fasting bloodsamples were collected from the mandibular venous plexus, and serum TC, low-densitylipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) andHDL-c/LDL-c ratio were measured or calculated. At the end of the study, all mice weredeprived of diets, but not water, for overnight fasting (at least12h). In the followingmorning after the fasting, the mice were anesthetized, and blood samples were collected fromthe aorta ventralis for measurement of fasting serum TC, HDL-c, LDL-c, high-densitylipoprotein (HDL) and apolipoprotein-A1(apo-A1). Livers and epididymal fat pads wereexcised, rinsed with icy saline and weighed. Livers were homogenized for measurement of TC, triacylglycerol (TG), phospholipid (PL), LDL-c and HDL-c.
3. Effect of MCFAs on the excretion of faecal neutral steroids and bile acids inC57BL/6J mice with hypercholesterolemia
The mice model and diet used were the same to the methods of the second part of theexperiment, and5mice were chosen randomly from each group for their recording dietintakes and sampling faeces output. Daily diet intakes and faeces of each mouse wererecorded and collected by using the metabolic cage for consecutive3days at the start and endof the study. Faecal neutral steroids and bile acids were measured by gaschromatography-mass spectrometry (GCMS) and liquid chromatography-mass spectrometry(LCMS) respectively, and the total amounts of the excretions in3days were calculated.
4. Investigation of the mechanism of lowering cholesterol by MCFAs in the liver ofC57BL/6J hypercholesterolemia mice
The mice were fed and killed according to the methods of the second part of theexperiment, livers were used for measurement of mRNA transcription and protein expressionlevel of hydroxymethyl glutaryl coenzyme A reductase (HMG-CR), cholesterol7α-hydroxylase (CYP7A1), liver X receptor (LXR) and farnesoid X receptor (FXR) by theReal-time RT-PCR and Western blotting analysis, respectively.
Results
1. Hypercholesterolemia model in C57BL/6J Mouse
Two weeeks after feeding the CR diet, Gaussian distribution of levels of serum TC of themice in CR groups (92mice) were observed,64of them were higher than the average levelplus2standard deviation (SD) of the control group, and most of them (58mice) were rangedfrom2.78to4.22mmol/L (i.e.2SD to8SD), which are up to63%among the CR groups.We assumed that hypercholesterolemia model in C57BL/6J mice fed CR diet wassuccessfully established.
2. Effects of MCFAs on cholesterol metabolism in C57BL/6J mice withhypercholesterolemia
1) At the end of study, the fasting body weight and liver weight of the mice in the C8:0,C10:0or C18:3groups were significantly lower than those in the C16:0(P<0.05) or C18:0(P<0.01) groups. The BMI, Lee’s indices of the mice in the C8:0, C10:0, C18:1, C16:0orC18:3groups were significantly lower than those in the C18:0group (P<0.05).
2) At the end of study, the level of serum TC in the C8:0(P<0.01), C10:0(P<0.05) orC18:3(P<0.01) groups were significantly lower than that in the C16:0group. The level ofserum LDL-c in the C8:0, C10:0or C18:3groups were significantly lower than those in theC16:0(P<0.05) group. The ratio of serum HDL-c/LDL-c in the C8:0, C10:0or C18:3groupswere significantly higher than those in the C18:1, C16:0or C18:0groups (P<0.05). The levelof serum TG in the C8:0(P<0.05), C10:0(P<0.01), C18:1(P<0.05) or C18:3(P<0.05)groups were significantly lower than those in the C16:0diet group. The level of serumHDL-c in the C8:0(P<0.05) group was significantly higher than that in the C18:1, C16:0orC18:0diet groups.
3) At the end of study, the concentrations of TC, LDL-c, HDL-c, TG and LP of liverhomogenates were no significantly differences among all the groups (P>0.05).
3. Effect of MCFAs on the excretion of faecal neutral steroids and bile acids inC57BL/6J mice with hypercholesterolemia
1) Cholesterol (up to97%) and cholic acid (more than75%) were the main components ofneutral steroids and bile acids, respectively in mice faeces.
2) At the end of study, the faecal excretion of cholesterol and total neutral steroids in theC8:0group were significantly higher than those in the C16:0or C18:0groups (P<0.05). Thefaecal excertion of cholestanol in the C8:0group was significantly higher than that in theC16:0group (P<0.05).
3) At the end of study, the changes in faecal excretion of unconjugated bile acids (UCBAs)were as follows:①The faecal excretion of ursodeoxycholic acid (UDCA) and lithocholicacid (LCA) in the C8:0group were significantly higher than those in the C16:0or C18:0groups (P<0.05), the faecal excretion of cholic acid (CA) and total UCBAs in the C8:0groupwere significantly higher than those in the C16:0, C18:0or C18:1groups (P<0.01), and thefaecal excretion of CA in the C8:0group at the end of the study was significantly higher thanthat at the start of the study (P<0.01);②The faecal excretion of CA in the C10:0group wassignificantly higher than that in the C18:1or C18:0groups (P<0.05), and the faecal excretionof total UCBAs in the C10:0group was significantly higher than that in the C16:0or C18:0groups (P<0.05);③The faecal excretion of LCA in the C18:1group was significantlyhigher than that in the C16:0or C18:0groups (P<0.05).④The faecal excretion of UDCAin the C18:3group was significantly higher than that in the C16:0or C18:0groups (P<0.05),and the faecal excretion of CA and total UCBAs in the C18:3group were significantly higherthan those in the C18:0group (P<0.05).
4) At the end of study, the changes in faecal excretion of taurine conjugated bile acids(TCBAs) were as follows:①The faecal excretion of taurodeoxycholic acid (TDCA) in theC8:0group was significantly higher than that in the C16:0, C18:0or C18:1groups (P<0.05),the faecal excretion of taurochenodeoxycholic acid (TCDCA) and total TCBAs in the C8:0group were significantly higher than those in the C16:0or C18:0groups (P<0.05);②Thefaecal excretion of TDCA in the C10:0group was significantly higher than that in the C18:0group (P<0.05), and the faecal excretion of TCDCA and total TCBAs in the C10:0diet groupwere significantly higher than those in the C16:0or C18:0groups (P<0.05);③The faecalexcretion of TDCA in the C18:1group was significantly lower than that in the C16:0group(P<0.05), the faecal excretion of TCDCA in the C18:1group was significantly higher thanthat in the C16:0or C18:0groups (P<0.05), and the faecal excretion of total TCBAs in theC18:1group was significantly higher than that in the C18:0group (P<0.05);④The faecalexcretions of TCDCA and TDCA in the C18:3group were significantly higher than those inthe C18:0group (P<0.05).
5) At the end of study, the changes in faecal excretion of glycine conjugated bile acids (GCBAs) were as follows: The faecal excretion of glycochenodeoxycholic acid (GCDCA)and total GCBAs in the C18:1group were significantly higher than those in the any othergroups (P<0.05).
6) At the end of study, the changes of faecal excretion of bile acids which synthesis fromclassical pathway were as follows:①The faecal excretion of all the bile acids related to CA,which in the C8:0or C10:0groups were significantly higher than that in the C16:0, C18:0orC18:1groups (P<0.01), and that in the C8:0group was also significantly higher than that inthe C18:3group (P<0.05);②The faecal excretion of all the bile acids related todeoxycholic acid (DCA), which in the C8:0group was significantly higher than that in theC16:0, C18:0or C18:1groups (P<0.01), as well as C18:3group (P<0.05). And it in theC10:0group was also significantly higher than that in the C18:0or C18:1groups (P<0.05);③The faecal excretion of all the bile acids that only synthesis from classical pathway,which in the C8:0or C10:0groups were significantly higher than that in the C16:0, C18:0orC18:1groups (P<0.01), and that in the C8:0group was also significantly higher than that inthe C18:3group (P<0.05).
7) At the end of study, the changes in faecal excretion of bile acids which synthesis fromalternative pathway were as follows;①The faecal excretion of all the bile acids related tochenodeoxycholic acid (CDCA), which in the C10:0group was significantly higher than thatin the C16:0, C18:0or C18:3groups (P<0.01), while that in the C8:0group was significantlyhigher than that in the C18:0group (P<0.05);②The faecal excretion of all the bile acidsrelated to LCA, which in the C8:0groups was significantly higher than that in the C16:0orC18:0groups (P<0.01), while that in the C10:0group was significantly higher than that inthe C16:0or C18:0groups (P<0.05);③The faecal excretion of all the bile acids that onlysynthesized from alternative pathway in the C8:0or C10:0groups were significantly higherthan those in the C16:0or C18:0groups (P<0.01), furthermore, that in the C10:0group wasalso significantly higher than that in the C18:3group (P<0.05).
8) At the end of study, the faecal excretion of total primary bile acids in the C8:0or C10:0groups were significantly higher than those in the C16:0, C18:0or C18:1groups (P<0.01),furthermore, that in the C8:0diet group was significantly higher than that in the C18:3group(P<0.05). While the faecal excretion of total secondly bile acids in the C8:0groups wassignificantly higher than that in the C16:0, C18:0or C18:1group (P<0.01), and that in theC10:0group was significantly higher than that in the C16:0(P<0.05) or C18:0(P<0.01)groups.
9) At the end of study, the faecal excretion of total bile acids in the C8:0or C10:0groupswere significantly higher than those in the C16:0, C18:0or C18:1group (P<0.05), and that inthe C18:3group was significantly higher than that in the C18:0group (P<0.05).
4. Investigation of a possible mechanism of lowering cholesterol by MCFAs in the liverof C57BL/6J hypercholesterolemia mice
1) At the end of study, neither the mRNA transcriptions nor the protein expressions of HMG-CR in mice livers were different among all groups (P>0.05).
2) At the end of study, the mRNA transcriptions and protein expressions of CYP7A1inmice livers in the C8:0or C10:0group were significantly higher than those in the C16:0and C18:0group (P<0.05), and the mRNA transcriptions of CYP7A1in the C18:1orC18:3group were significantly higher than those in the C18:0group (P<0.05).
3) At the end of study, the mRNA transcriptions of LXR in mice livers in the C8:0groupwas significantly higher than that in the C18:0group (P<0.05). The mRNAtranscriptions (P<0.05) and protein expressions (P<0.01) of LXR in mice livers in theC10:0group were significantly higher than those in the C16:0or C18:0group. Theprotein expressions of LXR in mice livers in the C18:3group was significantly higherthan that in the C16:0or C18:0group (P<0.05).
4) At the end of study, the mRNA transcriptions of FXR in mice livers was no differenceamong all groups (P>0.05). While the protein expressions of FXR in mice livers in theC8:0or C10:0group were significantly lower than those in the C16:0and C18:0group(P<0.01).
Conclusions
1) Among more than60%of the C57BL/6J mice fed cholesterol-rich(CR) diet (whichcontains13.63%fat,19.07%protein,48.18%carbohydrate,1%cholesterol and0.3%bile acids) for2weeks, their serum cholesterol levels were significantly increased. Thismethod can be used for hypercholesteremia mice model.
2) Compared with LCFAs, which were fed in CR diet in C57BL/6J male mice, MCFAs caneffectively reduce body weight, Lee’s index, BMI and liver weight, and improve theserum TC, TG, LDL-c and HDL-c level, but not lipid profiles in liver of the mice.
3) Improvement of the serum lipid profiles by MCFAs might be mainly due to theirenhancement of excretion of faecal neutral steroids and bile acids. C8:0enhanced mainlythe excretion of feacel cholesterol and CA, and C10:0enhanced mainly the excretion offeacel CA.
4) MCFAs can increase the mRNA transcription and protein expression of CYP7A1, whichwas considered to be due to both the enhancement of the mRNA transcription and proteinexpression of LXR and the inhibition of the protein expression of FXR in liver. MCFAsdid not regulate the mRNA transcription and protein expression of HMG-CR. Thesynergetic effects of the two rate-limiting enzymes of cholesterol synthesis in liverenhanced the cholesterol excretion through the feacel neutral steroids and bile acids andfinally reduced the serum TC and LDL-c level.
引文
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