| Objective1. 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.Methods1. Hypercholesterolemia model in C57BL/6J MouseOne 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 withhypercholesterolemiaThe 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 hypercholesterolemiaThe 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 miceThe 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.Results1. Hypercholesterolemia model in C57BL/6J MouseTwo 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 withhypercholesterolemia1) 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 hypercholesterolemia1) 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 mice1) 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).Conclusions1) 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. |
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