依折麦布预防饮食诱导的C57BL/6鼠胆囊胆固醇结石形成的实验研究
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摘要
胆囊结石病是由于胆囊胆汁生化学失衡引发的临床疾病,80%以上为胆固醇结石病(CGD)。目前公认的CGD治疗方法是胆囊切除术,但该种治疗方法具有创伤性,存在发生胆管损伤等严重并发症的风险,治疗费用较高。因此,更加透彻地认识CGD的发病机制,寻找有效的、无创的预防和治疗CGD的方法越来越必要。
关于CGD发病机制的研究已从Admirand和Small基于热力学的生物化学理论解释转移到多种因素综合作用的学说,如胆固醇过量分泌及所伴随的胆汁非生理性过饱和、胆囊的运动功能下降及所伴随的胆汁淤积、胆汁中胆固醇结晶形成及促成核因子和抗成核因子作用的失衡等,而且与CGD发病有关的脏器研究也扩展到肝脏、胆囊和肠道。虽然CGD这种受环境和遗传因素交互影响的多因素疾病的发病机制迄今还不十分明晰,但公认的是胆汁中胆固醇的非生理性过饱和是CGD发病的标志性病理生理改变。然而,胆汁胆固醇的过量分泌、小肠胆固醇的吸收、胆固醇的逆向转运和体内胆固醇的合成这些胆固醇代谢过程中的重要环节在CGD发病中的作用和相互影响机制还有许多研究的空白领域。
研究表明肠腔中饮食来源和胆汁来源的胆固醇的获得和小肠固醇吸收的效能与结石易感群体的成石状态密切相关,因此抑制了胆固醇小肠吸收这一过程就可能对CGD的预防和治疗起到有益作用。
依折麦布(EZET)是美国先灵葆雅公司新近研发并获得美国FDA批准上市的一种口服、强效的降脂药物。EZET选择性抑制小肠粘膜细胞刷状缘对胆固醇的摄取和吸收,将胆固醇限制在肠腔内随粪便排出体外,从而降低血循环中胆固醇的水平,进而降低小肠中的胆固醇向肝脏中的转运。已经证实Niemann-Pick C1 like1蛋白(npc111)是EZET的作用靶点。
本研究旨在建立致石饮食诱发的C57BL／6鼠胆囊胆固醇结石动物模型,并在此基础上观察EZET对致石饮食诱发的C57BL／6鼠胆囊胆固醇结石形成、胆囊运动功能和肝脏脂质代谢的影响。
实验方法
一、动物模型的建立
雄性C57BL／6鼠,8-10周龄,体质量11-16g,对照组(20只)饲以普通饲料(胆固醇含量0.02%(wt/wt));致石组(20只)饲以高胆固醇／高脂肪／胆酸致石饲料(含2%胆固醇、0.5%胆酸和15%脂肪)。喂饲8周后取材。
二、实验对象分组和给药
1、EZET对胆囊胆固醇结石形成影响实验
随机分为4组：(1)致石饮食+蒸馏水组(EZET-0):20只；(2)致石饮食+EZET(1mg/kg/d)组(EZET-1):20只；(3)致石饮食+EZET(5mg/kg/d)组(EZET-2):20只；(4)致石饮食+EZET(10mg/kg/d)组(EZET-3):20只。
2、EZET对胆囊运动功能影响实验
随机分为5组：(1)普通饮食+蒸馏水组(CON)：5只；(2)致石饮食+蒸馏水组(EZET-0):5只;(3)致石饮食+EZET(1mg/kg/d)组(EZET-1):5只;(4)致石饮食+EZET(5mg/kg/d)组(EZET-2):5只；(5)致石饮食+EZET(10mg/kg/d)组(EZET-3):5只。
3、EZET对肝脏脂质代谢影响实验
随机分为3组：(1)普通饮食+蒸馏水组(CON)：20只；(2)致石饮食+蒸馏水组(LITH):20只；(3)致石饮食+EZET(5mg/kg/d)组(EZET):20只。
三、标本采集和实验方法
1、10%水合氯醛腹腔注射麻醉后,取腹部正中切口,记录成石情况,测量胆囊长径和宽径,收集胆囊胆汁,胆总管置PE管收集肝胆汁并计量,切取胆囊组织、全部肝脏(称重)、空肠起始段1cm,下腔静脉穿刺取血。
2、按椭圆形公式计算胆囊体积,计算肝胆汁分泌速率。
3、红外光谱溴化钾压片法,对采集的结石成分进行定性分析。
4、胆囊胆汁涂片后于偏光显微镜下观察。
5、采用全自动生化分析仪检测胆汁中胆固醇、磷脂和胆汁酸的浓度并计算胆固醇饱和指数(CSI)。
6、透射电镜下观察肝脏细胞超微结构。
7、颈静脉置管后以17nmol/Kg体重给予外源性CCK-8,收集每10min肝胆汁计量统计。
8、胆囊组织HE染色和饱和苦味酸-天狼猩红染色,光镜下和偏光显微镜下观察组织学改变和胆囊壁胶原沉积。
9、采用全自动生化分析仪检测血清中胆固醇、甘油三酯、胆汁酸和谷丙转氨酶的含量。
10、肝脏组织油红O染色,光镜下观察肝脏脂质沉积情况。
11、肝脏组织HE染色和脂肪浸润评分。
12、采用Western blot方法检测肝脏组织主要肝胆转运蛋白abcb11、abcg5、abcg8和abcb4蛋白的表达；检测空肠粘膜上皮npc111蛋白的表达。
实验结果
一、C57BL／6鼠胆囊胆固醇结石动物模型的建立
肉眼观察：对照组小鼠成石率为0(0／19)。致石组成石率为100%(18／18)。结石经溴化钾压片红外光谱检测证实为胆固醇结石。
对照组小鼠胆囊体积为(22.6±8.35)μl,致石组胆囊体积为(63.2±10.6)μl,二者相比具有统计学差异(t=12.98,p<0.01)。对照组小鼠肝胆汁的分泌速率为(1.12±0.06)μl/min/g liver,致石组为(1.32±0.07)μl/min/g liver,二者相比具有统计学差异(t=6.66,P<0.01)。
偏光显微镜下观察：对照组小鼠胆囊胆汁内未见胆固醇结晶,致石组可见胆固醇—水合物结晶、晶体和结石。
对照组胆囊胆汁中胆固醇的浓度为5.79 mmol/L,致石组为18.67 mmol/L,远高于对照组；对照组胆囊胆汁中磷脂的浓度为29.69 mmol/L,致石组为28.99mmol/L,两组数值接近；对照组胆囊胆汁中胆汁酸的浓度为154.46 mmol/L,致石组为138.69 mmol/L,较对照组略低；致石组胆囊胆汁中CSI明显高于对照组(1.71vs 0.58),致石组CSI>1,为胆固醇过饱和胆汁。
透射电镜下观察：致石组与对照组相比小鼠肝细胞内出现大量小泡形脂滴,并呈现肝细胞损伤的改变。
二.EZET预防致石饮食诱导的C57BL／6鼠胆囊胆固醇结石的形成
EZET-0组成石率为100％(18/18)。EZET-1组成石率44.4%(8／18)。EZET-2组成石率为10.5%(2／19)。EZET-3组成石率为0(0／17)。
偏光显微镜下观察,EZET-0组小鼠胆囊胆汁内多见真结石。EZET-1组小鼠胆囊胆汁内多见不完整的未定型的泥沙样结石边缘有散在的胆固醇—水合物结晶突出或胆固醇晶体形成。EZET-2组小鼠胆囊胆汁内多见散在胆固醇结晶。EZET-3组小鼠胆囊胆汁内未见胆固醇结晶。
EZET-0组肝胆汁的分泌速率为(1.32±0.07)μl/min/g liver；EZET-1组肝胆汁的分泌速率为(1.43±0.05)μl/min/g liver；EZET-2组肝胆汁的分泌速率为(1.53±0.12)μl/min/g liver；EZET-3组肝胆汁的分泌速率为(1.73±0.08)μl/min/g liver,各组相比具有统计学差异(P<0.05)。
EZET-0组胆囊胆汁中胆固醇的浓度为18.67 mmol/L:EZET-1组为12.43mmol/L:EZET-2组为9.65 mmol/L;EZET-3组为4.42 mmol/L,逐渐下降。EZET-0组胆囊胆汁中磷脂的浓度为28.99 mmol/L;EZET-1组为28.23 mmol/L;EZET-2组为30.14 mmol/L;EZET-3组为33.82 mmol/L,数值接近。EZET-0组胆囊胆汁中胆汁酸的浓度为138.69 mmol/L;EZET-1组为138.26 mmol/L:EZET-2组为145.98mmol/L;EZET-3组为169.21 mmol/L,有逐渐增加趋势。
EZET-0组胆管胆汁中胆固醇的浓度为(4.67±0.28) mmol/L;EZET-1组为(3.17±0.41)mmol/L;EZET-2组为(2.25±0.51)mmol/L;EZET-3组为(1.76±0.38)mmol/L,依次显著下降。EZET-0组胆管胆汁中磷脂的浓度为(8.26±0.45)mmol/L; EZET-1组为(7.25±0.46) mmol/L;EZET-2组为(5.63±0.62) mmol/L;EZET-3组为(5.18±0.95) mmol/L。EZET-0组胆管胆汁中胆汁酸的浓度为(24.67±1.01)mmol/L;EZET-1组为(24.17±1.08)mmol/L;EZET-2组为(23.44±1.63)mmol/L:EZET-3组为(23.39±1.47) mmol/L。
Western blot结果显示,EZET-0组小鼠空肠粘膜上皮npclll蛋白表达水平最高,随着EZET给药量的增大,npclll蛋白表达水平逐渐下降。
三、EZET改善了致石饮食诱导的胆囊胆固醇结石病C57BL／6鼠胆囊运动功能
CON组胆囊体积为(22.02±4.35)μl。EZET-0组胆囊体积为(62.76±8.11)μl,EZET-1组胆囊体积为(48.84±7.66)μl, EZET-2组胆囊体积为(34.74±5.96)μl,EZET-3组胆囊体积为(23.84±5.76)μl,各组均显著低于EZET-0组(p<0.05)。
CCK-8干预实验结果显示：EZET可以明显提高小鼠对CCK-8的反应性,并且随着EZET剂量的增大,胆汁流量曲线下面积逐渐增大,提示胆汁分泌量明显增加。
镜下观察发现：与CON组相比,EZET-0组小鼠胆囊组织出现显著的粘膜增生和固有层结缔组织的扩张,出现显著的胶原沉积；EZET-1、EZET-2和EZET-3组小鼠胆囊组织的上述改变较EZET-0组稍轻但不明显。
四、EZET减轻了致石饮食诱导的胆囊胆固醇结石病C57BL／6鼠肝脏的脂质沉积
肝脏的组织学观察和油红O染色观察发现：EZET明显减轻了致石饮食诱导的小鼠肝脏的脂肪沉积和损伤改变。EZET显著降低了肝脏脂肪浸润评分。
EZET显著降低了致石饮食诱导的血中胆固醇水平的升高；显著降低了致石饮食诱导的血中胆汁酸水平的升高；同时显著降低了致石饮食诱导的血中谷丙转氨酶水平的升高；但对血中甘油三酯水平无影响。
Western blot结果显示：致石饮食并未显著影响胆酸转运子abcbll蛋白的表达,然而,EZET组的abcbll的表达水平较LITH组升高,提示EZET干预后abcbll蛋白的表达上调；研究还发现致石饮食显著增加了胆固醇转运蛋白abcg5和abcg8蛋白的表达,但EZET并没有显著改变abcg5和abcg8蛋白的表达；研究发现致石饮食并未显著影响磷脂转运子abcb4蛋白的表达,同时EZET也没有显著改变abcb4蛋白的表达。
1、高脂饮食诱导的C57BL／6小鼠胆囊胆固醇结石动物模型经济可靠,成模率高,可用于胆囊胆固醇结石病的动物实验研究。该模型建立的病理生理基础是高脂饮食诱导的胆汁中胆固醇的非生理性过饱和。
2、EZET对高脂饮食诱导的C57BL／6小鼠胆囊胆固醇结石形成具有预防作用,其机制是通过EZET降低胆囊胆固醇结石C57BL／6小鼠胆汁中胆固醇的非生理性过饱和实现的。EZET可能是通过降低小鼠空肠粘膜上皮刷状缘npc111蛋白的表达进而减少肠道胆固醇的吸收而实现上述作用的。
3、EZET对致石饮食诱导的胆囊胆固醇结石C57BL／6小鼠的胆囊运动功能具有保护作用。
4、EZET能够明显减轻致石饮食诱导的胆囊胆固醇结石C57BL／6鼠肝脏的脂肪沉积和损伤。EZET能够增加致石饮食诱导的胆囊胆固醇结石C57BL／6小鼠肝脏胆汁酸转运蛋白abcb11的表达,但对胆固醇转运蛋白abcg5、abcg8和磷脂转运蛋白abcb4的表达无影响。
Gallstone disease, of which more than 80% was cholesterol gallstone disease (CGD), was caused by the biochemical imbalances of gallbladder bile. Currently accepted treatment of CGD is cholecystectomy, but this treatment was traumatic, there were serious complications such as bile duct injury occurring and the higher treatment costs. Therefore, a more thorough understanding of the pathogenesis of CGD to find effective, non-invasive method of prevention and treatment of CGD was increasingly necessary.
Research on the pathogenesis of CGD from Admirand and Small theoretical explanation based on the thermodynamics of biochemical has transferred to the doctrine of the role of a variety of factors, such as excessive cholesterol bile secretion and the accompanying non-physiological saturation, the decrease of gallbladder motor function accompanied by cholestasis, bile cholesterol crystal formation and the imbalances of nucleation factors and anti-nucleation factors, but also the organ of CGD research of pathogenesis extended to the liver, gall bladder and intestine. Although the pathogenesis of CGD, a multifactorial disease, was the interaction by environmental and genetic factors, has so far not entirely clear. But we acknowledged that the non-physiological supersaturation of the bile cholesterol is a sign of the pathophysiological changes of CGD. However, there are many gaps in the field of research that excess cholesterol secretion of the bile, intestinal cholesterol absorption, reverse cholesterol transport, and synthesis and metabolism of cholesterol in vivo has an important role in the pathogenesis of CGD.
Studies have shown that the availability of dietary and biliary sources of cholesterol in the intestine is closely related to the stone susceptibility, thereby blocking the intestinal absorption of cholesterol in this process is likely to play a useful role in prevention and treatment of CGD.
Ezetimibe (EZET), an oral, potent lipid-lowering drugs manufactured by Schering-Plough Inc., developed recently in the United States and received U.S. FDA clearance to market. EZET selectively inhibited the cholesterol uptake and absorption of small intestinal brush border cells, and limited cholesterol to the intestinal lumen with the feces excreted, thereby reducing the level of cholesterol in the blood circulation, thus reducing cholesterol in the small intestine to the liver trans-shipment. Studies have confirmed that Niemann-Pick C1 like 1 protein (npc111) is the target of EZET.
This study was designed to establish lithogenic diet-induced C57BL/6 mouse model of cholesterol gallstone, and on this basis to observe the effect of EZET to the lithogenic diet-induced cholesterol gallstone formation, gallbladder motor function and lipid metabolism in liver.
Materials and Methods
I Animal model
Male C57BL/6 mice,8-10 weeks of age, body mass 11～16g, the control group (20 rats) were fed with normal diet (cholesterol content of 0.02% (wt/wt)); lithogenic group (20 rats) were fed with high cholesterol/high fat/cholic acid-lithogenic diet (containing 2% cholesterol,0.5% cholic acid and 15% fat). The mice were harvested after 8 weeks feeding.
II Experimental groups and drug delivery targets
1. Experiment of the effect of EZET on cholesterol gallstone formation
Were randomly divided into four groups:(1) lithogenic diet+distilled water group (EZET-0):20 individuals; (2) lithogenic diet+EZET (lmg/kg/d) group (EZET-1):20 individuals; (3) lithogenic diet+EZET (5mg/kg/d) group (EZET-2):20 individuals; (4) lithogenic diet+EZET (10mg/kg/d) group (EZET-3):20 individuals.
2. Experiment of the effect of EZET on gallbladder motor function
Were randomly divided into five groups:(1) chow diet+distilled water group (CON):5 individuals; (2) lithogenic diet+distilled water group (EZET-0):5 individuals; (3) lithogenic diet+EZET (1mg/kg/d) group (EZET-1):5 individuals; (4) lithogenic diet+EZET (5mg/kg/d) group (EZET-2):5 individuals; (5) lithogenic diet+ EZET (10mg/kg/d) group (EZET-3):5 individuals.
3. Experiment of the effect of EZET on liver lipid metabolism
Were randomly divided into 3 groups:(1) chow diet+distilled water group (CON): 20 individuals; (2) lithogenic diet+distilled water group (LITH):20 individuals; (3) lithogenic diet+EZET (5mg/kg/d) group (EZET):20 individuals.
ⅢSpecimen collection and experimental methods
1.10% chloral hydrate intraperitoneal injection for anesthesia, take the middle abdominal incision, recording the prevalence of gallstones, measurement of gallbladder length and width diameters, collection of gallbladder bile, common bile duct PE tube set to collect the liver bile, gallbladder tissues, all of liver (weighing) and initial 1cm segment of the jejunum were collected, the inferior vena cava puncture to take blood.
2. Calculate the gallbladder volume and the liver bile secretion rate.
3. Infrared spectra analysis of stone composition by potassium bromide tablet method.
4. Gallbladder bile after smears were observed under polarizing microscope.
5. Using automatic biochemical analyzer detected bile cholesterol, phospholipids and bile acid concentrations and calculated cholesterol saturation index (CSI).
6. Ultrastructure of liver cell was observed under transmission electron microscope.
7.17nmol/Kg of exogenous CCK-8 was induced via jugular vein catheter, and collected bile of each 10min for measurement.
8. HE staining and saturated picric acid-Sirius red staining of gallbladder tissue, and histological changes and collagen deposition of the gallbladder wall were observed under the microscope and polarized light microscope.
9. Using automatic biochemical analyzer detected serum cholesterol, triglycerides, bile acids and alanine aminotransferase levels.
10. Oil red O staining of the liver, and light microscope observation of liver lipid deposition.
11. HE staining of liver tissues and fatty infiltration score.
12. Using Western blot method for detection of the major hepatobiliary transporter protein abcbll, abcg5, abcg8 and abcb4 protein expression in liver tissue, and detection of the expression of npc111 protein in intestinal epithelia.
Results
ⅠEstablishment of C57BL/6 mouse model of CGD
The observation by naked eye:the prevalence of gallstones in control group mice was 0 (0/19). The prevalence of gallstones in lithogenic group mice was 100%(18/18). Stones were confirmed to be cholesterol stones.
Gallbladder volumes in control group of mice was (22.6±8.35)μl, and lithogenic group was (63.2±10.6)μl, there was statistical difference between the two groups(t= 12.98, p<0.01). Liver bile secretion rate in control group mice was (1.12±0.06)μl/min/g liver, lithogenic group was (1.32±0.07)μl/min/g liver, there was statistical difference between the two groups(t= 6.66, P<0.01).
Polarizing microscope observation:the control group mice have no cholesterol crystals in gallbladder bile, and in lithogenic group cholesterol monohydrate crystals and stones can be seen.
In control group, cholesterol concentration of gallbladder bile was 5.79 mmol/L, and lithogenic group was 18.67 mmol/L. In control group, the phospholipids concentration of gallbladder bile was 29.69 mmol/L, and lithogenic group was 28.99 mmol/L. In control group, the bile acids concentration of gallbladder bile was 154.46 mmol/L, and lithgenic group was 138.69 mmol/L, is slightly lower than the control group. The CSI of gallbladder bile in lithogenic group was significantly higher (1.71 vs 0.58), as the cholesterol-supersaturated bile.
Observation under transmission electron microscope:compared with the control group, the liver cells of lithogenic group mouse showed a large number of small bubbles form lipid droplets and showed changes in liver cell injury.
II EZET prevented lithogenic diet-induced formation of cholesterol gallstone in C57BL/6 mice
The prevalence of gallstones in EZET-0 group was 100%(18/18), EZET-1 was 44.4% (8/18), EZET-2 was 10.5% (2/19), and EZET-3 was 0 (0/17).
Polarizing microscope observation:In EZET-0 group, true stones in gallbladder bile were more common seen. In EZET-1 mice, incomplete, undifferentiated silt-like stones were more common seen in gallbladder bile. In EZET-2 mice, sporadic gallbladder bile cholesterol crystals were more common seen. In EZET-3 mice, no cholesterol crystal was seen within the gallbladder bile.
The liver bile secretion rate of EZET-0 group was (1.32±0.07)μl/min/g liver; EZET-1 group was (1.43±0.05)μl/min/g liver; EZET-2 group was (1.53±0.12)μl/min/g liver; EZET-3 groupwas (1.73±0.08)μl/min/g liver, compared each group with a statistically significant difference (p<0.05).
In EZET-0 group, cholesterol concentration of gallbladder bile was 18.67 mmol/L; EZET-1 group was 12.43 mmol/L; EZET-2 group was 9.65 mmol/L; and EZET-3 group was 4.42 mmol/L. In EZET-0 group, phospholipids concentration in gallbladder bile was 28.99 mmol/L; EZET-1 group was 28.23 mmol/L; EZET-2 group was 30.14 mmol/L; and EZET-3 group was 33.82 mmol/L. In EZET-0 group, bile acid concentration of gallbladder bile was 138.69 mmol/L; EZET-1 group was 138.26 mmol/L; EZET-2 group was 145.98 mmol/L; and EZET-3 group was 169.21 mmol/L, there is a gradual increase trend.
In EZET-0 group, cholesterol concentration of bile duct bile was (4.67±0.28) mmol/L; EZET-1 group was (3.17±0.41) mmol/L; EZET-2 group was (2.25±0.51) mmol/L; and EZET-3 group was (1.76±0.38) mmol/L. In EZET-0 group, phospholipid concentration in bile duct bile was (8.26±0.45) mmol/L; EZET-1 group was (7.25±0.46) mmol/L; EZET-2 group was (5.63±0.62) mmol/L; and EZET-3 group was (5.18±0.95) mmol/L. In EZET-0 group, bile acid concentration of the bile duct bile was (24.67±1.01) mmol/L; EZET-1 group was (24.17±1.08) mmol/L; EZET-2 group was (23.44±1.63) mmol/L; and EZET-3 group was (23.39±1.47) mmol/L.
Western blot results showed that, EZET-0 mice had the highest levels of protein expression of npclll. With the increasing amount of EZET administration, npclll protein expression level decreased gradually.
ⅢEZET improve the gallbladder motor function of lithogenic diet-induced cholesterol gallstone C57BL/6 mouse
In CON group, gallbladder volume was (22.02±4.35)μl, EZET-0 group was (62.76±8.11)μl, EZET-1 group was (48.84±7.66)μl, EZET-2 group was (34.74±5.96)μl, and EZET-3 group was (23.84±5.76)μl, each group was significantly lower than EZET-0 group (p<0.05).
Results of CCK-8 interference experiment showed that EZET can significantly improve the reactivity to CCK-8 of mice, and with EZET dose increased, the areas under the curve gradually increased, indicating a marked increase in bile flow.
Microscopic observation:Compared with the CON group, gallbladder tissues of EZET-0 mice showed significant proliferation and expansion of connective tissue of mucosal lamina propria and marked collagen deposition; the above-mentioned changes of gallbladder tissues in EZET-1, EZET-2 and EZET-3 group mice was slightly lighter than EZET-0 group, but not obvious.
IV EZET reduced lipid deposition in the liver of lithogenic diet-induced cholesterol gallstone C57BL/6 mouse
Histological observation and oil red O staining of the liver observation:EZET significantly reduced the lithogenic diet-induced fatty liver changes, lipids deposition and damage in mice. EZET significantly reduced the scores of liver fatty infiltration.
EZET significantly reduces the lithogenic diet-induced elevated blood cholesterol levels; significantly reduces the lithogenic diet-induced elevated blood bile acids levels; while significantly reducing the lithogenic diet-induced elevated blood alanine aminotransferase levels; but no influence on blood triglyceride levels.
Western blot results showed that:lithogenic diet did not significantly affect abcbll bile acid transporter protein expression, however, in EZET group, expression level of abcbll was higher than the LITH group. Study also found that lithogenic diet significantly increased the cholesterol transport protein abcg5 and abcg8 protein expressions, but no significant change in EZET group; Study found that lithogenic diet did not significantly affect abcb4 phospholipid transporter protein expression, while no significant change in EZET group.
Conclusions
1. high-fat diet-induced C57BL/6 mice animal models of cholesterol gallstones was economic, reliable, and highly successful, which can be used for gallbladder cholesterol gallstone animal experiments. The establishment of the model is based on the pathophysiology of high-fat diet-induced non-physiological supersaturation of bile cholesterol.
2. EZET has a preventive effect on cholesterol gallstone formation in high-fat diet-induced C57BL/6 mice, and the mechanism is that EZET lowers non-physiological bile cholesterol supersaturation in C57BL/6 mice. EZET may effect in mice by reducing the npclll expression at brush border of intestinal mucosa thereby reducing intestinal absorption of cholesterol to achieve the above effect.
3. Administration of EZET to lithogenic diet-induced cholesterol gallstone C57BL /6 mice has a protective effect of gallbladder motor function.
4. EZET can significantly reduce the fat deposition and damage of the liver in lithogenic diet-induced cholesterol gallstone C57BL/6 mouse. EZET can increase the expression of bile acid transporter protein abcbll in the liver of lithogenic diet-induced cholesterol gallstone C57BL/6 mouse, but has no effect on the expression of cholesterol transfer protein abcg5, abcg8, and phospholipid transfer protein abcb4.
关于CGD发病机制的研究已从Admirand和Small基于热力学的生物化学理论解释转移到多种因素综合作用的学说,如胆固醇过量分泌及所伴随的胆汁非生理性过饱和、胆囊的运动功能下降及所伴随的胆汁淤积、胆汁中胆固醇结晶形成及促成核因子和抗成核因子作用的失衡等,而且与CGD发病有关的脏器研究也扩展到肝脏、胆囊和肠道。虽然CGD这种受环境和遗传因素交互影响的多因素疾病的发病机制迄今还不十分明晰,但公认的是胆汁中胆固醇的非生理性过饱和是CGD发病的标志性病理生理改变。然而,胆汁胆固醇的过量分泌、小肠胆固醇的吸收、胆固醇的逆向转运和体内胆固醇的合成这些胆固醇代谢过程中的重要环节在CGD发病中的作用和相互影响机制还有许多研究的空白领域。
研究表明肠腔中饮食来源和胆汁来源的胆固醇的获得和小肠固醇吸收的效能与结石易感群体的成石状态密切相关,因此抑制了胆固醇小肠吸收这一过程就可能对CGD的预防和治疗起到有益作用。
依折麦布(EZET)是美国先灵葆雅公司新近研发并获得美国FDA批准上市的一种口服、强效的降脂药物。EZET选择性抑制小肠粘膜细胞刷状缘对胆固醇的摄取和吸收,将胆固醇限制在肠腔内随粪便排出体外,从而降低血循环中胆固醇的水平,进而降低小肠中的胆固醇向肝脏中的转运。已经证实Niemann-Pick C1 like1蛋白(npc111)是EZET的作用靶点。
本研究旨在建立致石饮食诱发的C57BL／6鼠胆囊胆固醇结石动物模型,并在此基础上观察EZET对致石饮食诱发的C57BL／6鼠胆囊胆固醇结石形成、胆囊运动功能和肝脏脂质代谢的影响。
实验方法
一、动物模型的建立
雄性C57BL／6鼠,8-10周龄,体质量11-16g,对照组(20只)饲以普通饲料(胆固醇含量0.02%(wt/wt));致石组(20只)饲以高胆固醇／高脂肪／胆酸致石饲料(含2%胆固醇、0.5%胆酸和15%脂肪)。喂饲8周后取材。
二、实验对象分组和给药
1、EZET对胆囊胆固醇结石形成影响实验
随机分为4组：(1)致石饮食+蒸馏水组(EZET-0):20只；(2)致石饮食+EZET(1mg/kg/d)组(EZET-1):20只；(3)致石饮食+EZET(5mg/kg/d)组(EZET-2):20只；(4)致石饮食+EZET(10mg/kg/d)组(EZET-3):20只。
2、EZET对胆囊运动功能影响实验
随机分为5组：(1)普通饮食+蒸馏水组(CON)：5只；(2)致石饮食+蒸馏水组(EZET-0):5只;(3)致石饮食+EZET(1mg/kg/d)组(EZET-1):5只;(4)致石饮食+EZET(5mg/kg/d)组(EZET-2):5只；(5)致石饮食+EZET(10mg/kg/d)组(EZET-3):5只。
3、EZET对肝脏脂质代谢影响实验
随机分为3组：(1)普通饮食+蒸馏水组(CON)：20只；(2)致石饮食+蒸馏水组(LITH):20只；(3)致石饮食+EZET(5mg/kg/d)组(EZET):20只。
三、标本采集和实验方法
1、10%水合氯醛腹腔注射麻醉后,取腹部正中切口,记录成石情况,测量胆囊长径和宽径,收集胆囊胆汁,胆总管置PE管收集肝胆汁并计量,切取胆囊组织、全部肝脏(称重)、空肠起始段1cm,下腔静脉穿刺取血。
2、按椭圆形公式计算胆囊体积,计算肝胆汁分泌速率。
3、红外光谱溴化钾压片法,对采集的结石成分进行定性分析。
4、胆囊胆汁涂片后于偏光显微镜下观察。
5、采用全自动生化分析仪检测胆汁中胆固醇、磷脂和胆汁酸的浓度并计算胆固醇饱和指数(CSI)。
6、透射电镜下观察肝脏细胞超微结构。
7、颈静脉置管后以17nmol/Kg体重给予外源性CCK-8,收集每10min肝胆汁计量统计。
8、胆囊组织HE染色和饱和苦味酸-天狼猩红染色,光镜下和偏光显微镜下观察组织学改变和胆囊壁胶原沉积。
9、采用全自动生化分析仪检测血清中胆固醇、甘油三酯、胆汁酸和谷丙转氨酶的含量。
10、肝脏组织油红O染色,光镜下观察肝脏脂质沉积情况。
11、肝脏组织HE染色和脂肪浸润评分。
12、采用Western blot方法检测肝脏组织主要肝胆转运蛋白abcb11、abcg5、abcg8和abcb4蛋白的表达；检测空肠粘膜上皮npc111蛋白的表达。
实验结果
一、C57BL／6鼠胆囊胆固醇结石动物模型的建立
肉眼观察：对照组小鼠成石率为0(0／19)。致石组成石率为100%(18／18)。结石经溴化钾压片红外光谱检测证实为胆固醇结石。
对照组小鼠胆囊体积为(22.6±8.35)μl,致石组胆囊体积为(63.2±10.6)μl,二者相比具有统计学差异(t=12.98,p<0.01)。对照组小鼠肝胆汁的分泌速率为(1.12±0.06)μl/min/g liver,致石组为(1.32±0.07)μl/min/g liver,二者相比具有统计学差异(t=6.66,P<0.01)。
偏光显微镜下观察：对照组小鼠胆囊胆汁内未见胆固醇结晶,致石组可见胆固醇—水合物结晶、晶体和结石。
对照组胆囊胆汁中胆固醇的浓度为5.79 mmol/L,致石组为18.67 mmol/L,远高于对照组；对照组胆囊胆汁中磷脂的浓度为29.69 mmol/L,致石组为28.99mmol/L,两组数值接近；对照组胆囊胆汁中胆汁酸的浓度为154.46 mmol/L,致石组为138.69 mmol/L,较对照组略低；致石组胆囊胆汁中CSI明显高于对照组(1.71vs 0.58),致石组CSI>1,为胆固醇过饱和胆汁。
透射电镜下观察：致石组与对照组相比小鼠肝细胞内出现大量小泡形脂滴,并呈现肝细胞损伤的改变。
二.EZET预防致石饮食诱导的C57BL／6鼠胆囊胆固醇结石的形成
EZET-0组成石率为100％(18/18)。EZET-1组成石率44.4%(8／18)。EZET-2组成石率为10.5%(2／19)。EZET-3组成石率为0(0／17)。
偏光显微镜下观察,EZET-0组小鼠胆囊胆汁内多见真结石。EZET-1组小鼠胆囊胆汁内多见不完整的未定型的泥沙样结石边缘有散在的胆固醇—水合物结晶突出或胆固醇晶体形成。EZET-2组小鼠胆囊胆汁内多见散在胆固醇结晶。EZET-3组小鼠胆囊胆汁内未见胆固醇结晶。
EZET-0组肝胆汁的分泌速率为(1.32±0.07)μl/min/g liver；EZET-1组肝胆汁的分泌速率为(1.43±0.05)μl/min/g liver；EZET-2组肝胆汁的分泌速率为(1.53±0.12)μl/min/g liver；EZET-3组肝胆汁的分泌速率为(1.73±0.08)μl/min/g liver,各组相比具有统计学差异(P<0.05)。
EZET-0组胆囊胆汁中胆固醇的浓度为18.67 mmol/L:EZET-1组为12.43mmol/L:EZET-2组为9.65 mmol/L;EZET-3组为4.42 mmol/L,逐渐下降。EZET-0组胆囊胆汁中磷脂的浓度为28.99 mmol/L;EZET-1组为28.23 mmol/L;EZET-2组为30.14 mmol/L;EZET-3组为33.82 mmol/L,数值接近。EZET-0组胆囊胆汁中胆汁酸的浓度为138.69 mmol/L;EZET-1组为138.26 mmol/L:EZET-2组为145.98mmol/L;EZET-3组为169.21 mmol/L,有逐渐增加趋势。
EZET-0组胆管胆汁中胆固醇的浓度为(4.67±0.28) mmol/L;EZET-1组为(3.17±0.41)mmol/L;EZET-2组为(2.25±0.51)mmol/L;EZET-3组为(1.76±0.38)mmol/L,依次显著下降。EZET-0组胆管胆汁中磷脂的浓度为(8.26±0.45)mmol/L; EZET-1组为(7.25±0.46) mmol/L;EZET-2组为(5.63±0.62) mmol/L;EZET-3组为(5.18±0.95) mmol/L。EZET-0组胆管胆汁中胆汁酸的浓度为(24.67±1.01)mmol/L;EZET-1组为(24.17±1.08)mmol/L;EZET-2组为(23.44±1.63)mmol/L:EZET-3组为(23.39±1.47) mmol/L。
Western blot结果显示,EZET-0组小鼠空肠粘膜上皮npclll蛋白表达水平最高,随着EZET给药量的增大,npclll蛋白表达水平逐渐下降。
三、EZET改善了致石饮食诱导的胆囊胆固醇结石病C57BL／6鼠胆囊运动功能
CON组胆囊体积为(22.02±4.35)μl。EZET-0组胆囊体积为(62.76±8.11)μl,EZET-1组胆囊体积为(48.84±7.66)μl, EZET-2组胆囊体积为(34.74±5.96)μl,EZET-3组胆囊体积为(23.84±5.76)μl,各组均显著低于EZET-0组(p<0.05)。
CCK-8干预实验结果显示：EZET可以明显提高小鼠对CCK-8的反应性,并且随着EZET剂量的增大,胆汁流量曲线下面积逐渐增大,提示胆汁分泌量明显增加。
镜下观察发现：与CON组相比,EZET-0组小鼠胆囊组织出现显著的粘膜增生和固有层结缔组织的扩张,出现显著的胶原沉积；EZET-1、EZET-2和EZET-3组小鼠胆囊组织的上述改变较EZET-0组稍轻但不明显。
四、EZET减轻了致石饮食诱导的胆囊胆固醇结石病C57BL／6鼠肝脏的脂质沉积
肝脏的组织学观察和油红O染色观察发现：EZET明显减轻了致石饮食诱导的小鼠肝脏的脂肪沉积和损伤改变。EZET显著降低了肝脏脂肪浸润评分。
EZET显著降低了致石饮食诱导的血中胆固醇水平的升高；显著降低了致石饮食诱导的血中胆汁酸水平的升高；同时显著降低了致石饮食诱导的血中谷丙转氨酶水平的升高；但对血中甘油三酯水平无影响。
Western blot结果显示：致石饮食并未显著影响胆酸转运子abcbll蛋白的表达,然而,EZET组的abcbll的表达水平较LITH组升高,提示EZET干预后abcbll蛋白的表达上调；研究还发现致石饮食显著增加了胆固醇转运蛋白abcg5和abcg8蛋白的表达,但EZET并没有显著改变abcg5和abcg8蛋白的表达；研究发现致石饮食并未显著影响磷脂转运子abcb4蛋白的表达,同时EZET也没有显著改变abcb4蛋白的表达。
1、高脂饮食诱导的C57BL／6小鼠胆囊胆固醇结石动物模型经济可靠,成模率高,可用于胆囊胆固醇结石病的动物实验研究。该模型建立的病理生理基础是高脂饮食诱导的胆汁中胆固醇的非生理性过饱和。
2、EZET对高脂饮食诱导的C57BL／6小鼠胆囊胆固醇结石形成具有预防作用,其机制是通过EZET降低胆囊胆固醇结石C57BL／6小鼠胆汁中胆固醇的非生理性过饱和实现的。EZET可能是通过降低小鼠空肠粘膜上皮刷状缘npc111蛋白的表达进而减少肠道胆固醇的吸收而实现上述作用的。
3、EZET对致石饮食诱导的胆囊胆固醇结石C57BL／6小鼠的胆囊运动功能具有保护作用。
4、EZET能够明显减轻致石饮食诱导的胆囊胆固醇结石C57BL／6鼠肝脏的脂肪沉积和损伤。EZET能够增加致石饮食诱导的胆囊胆固醇结石C57BL／6小鼠肝脏胆汁酸转运蛋白abcb11的表达,但对胆固醇转运蛋白abcg5、abcg8和磷脂转运蛋白abcb4的表达无影响。
Gallstone disease, of which more than 80% was cholesterol gallstone disease (CGD), was caused by the biochemical imbalances of gallbladder bile. Currently accepted treatment of CGD is cholecystectomy, but this treatment was traumatic, there were serious complications such as bile duct injury occurring and the higher treatment costs. Therefore, a more thorough understanding of the pathogenesis of CGD to find effective, non-invasive method of prevention and treatment of CGD was increasingly necessary.
Research on the pathogenesis of CGD from Admirand and Small theoretical explanation based on the thermodynamics of biochemical has transferred to the doctrine of the role of a variety of factors, such as excessive cholesterol bile secretion and the accompanying non-physiological saturation, the decrease of gallbladder motor function accompanied by cholestasis, bile cholesterol crystal formation and the imbalances of nucleation factors and anti-nucleation factors, but also the organ of CGD research of pathogenesis extended to the liver, gall bladder and intestine. Although the pathogenesis of CGD, a multifactorial disease, was the interaction by environmental and genetic factors, has so far not entirely clear. But we acknowledged that the non-physiological supersaturation of the bile cholesterol is a sign of the pathophysiological changes of CGD. However, there are many gaps in the field of research that excess cholesterol secretion of the bile, intestinal cholesterol absorption, reverse cholesterol transport, and synthesis and metabolism of cholesterol in vivo has an important role in the pathogenesis of CGD.
Studies have shown that the availability of dietary and biliary sources of cholesterol in the intestine is closely related to the stone susceptibility, thereby blocking the intestinal absorption of cholesterol in this process is likely to play a useful role in prevention and treatment of CGD.
Ezetimibe (EZET), an oral, potent lipid-lowering drugs manufactured by Schering-Plough Inc., developed recently in the United States and received U.S. FDA clearance to market. EZET selectively inhibited the cholesterol uptake and absorption of small intestinal brush border cells, and limited cholesterol to the intestinal lumen with the feces excreted, thereby reducing the level of cholesterol in the blood circulation, thus reducing cholesterol in the small intestine to the liver trans-shipment. Studies have confirmed that Niemann-Pick C1 like 1 protein (npc111) is the target of EZET.
This study was designed to establish lithogenic diet-induced C57BL/6 mouse model of cholesterol gallstone, and on this basis to observe the effect of EZET to the lithogenic diet-induced cholesterol gallstone formation, gallbladder motor function and lipid metabolism in liver.
Materials and Methods
I Animal model
Male C57BL/6 mice,8-10 weeks of age, body mass 11～16g, the control group (20 rats) were fed with normal diet (cholesterol content of 0.02% (wt/wt)); lithogenic group (20 rats) were fed with high cholesterol/high fat/cholic acid-lithogenic diet (containing 2% cholesterol,0.5% cholic acid and 15% fat). The mice were harvested after 8 weeks feeding.
II Experimental groups and drug delivery targets
1. Experiment of the effect of EZET on cholesterol gallstone formation
Were randomly divided into four groups:(1) lithogenic diet+distilled water group (EZET-0):20 individuals; (2) lithogenic diet+EZET (lmg/kg/d) group (EZET-1):20 individuals; (3) lithogenic diet+EZET (5mg/kg/d) group (EZET-2):20 individuals; (4) lithogenic diet+EZET (10mg/kg/d) group (EZET-3):20 individuals.
2. Experiment of the effect of EZET on gallbladder motor function
Were randomly divided into five groups:(1) chow diet+distilled water group (CON):5 individuals; (2) lithogenic diet+distilled water group (EZET-0):5 individuals; (3) lithogenic diet+EZET (1mg/kg/d) group (EZET-1):5 individuals; (4) lithogenic diet+EZET (5mg/kg/d) group (EZET-2):5 individuals; (5) lithogenic diet+ EZET (10mg/kg/d) group (EZET-3):5 individuals.
3. Experiment of the effect of EZET on liver lipid metabolism
Were randomly divided into 3 groups:(1) chow diet+distilled water group (CON): 20 individuals; (2) lithogenic diet+distilled water group (LITH):20 individuals; (3) lithogenic diet+EZET (5mg/kg/d) group (EZET):20 individuals.
ⅢSpecimen collection and experimental methods
1.10% chloral hydrate intraperitoneal injection for anesthesia, take the middle abdominal incision, recording the prevalence of gallstones, measurement of gallbladder length and width diameters, collection of gallbladder bile, common bile duct PE tube set to collect the liver bile, gallbladder tissues, all of liver (weighing) and initial 1cm segment of the jejunum were collected, the inferior vena cava puncture to take blood.
2. Calculate the gallbladder volume and the liver bile secretion rate.
3. Infrared spectra analysis of stone composition by potassium bromide tablet method.
4. Gallbladder bile after smears were observed under polarizing microscope.
5. Using automatic biochemical analyzer detected bile cholesterol, phospholipids and bile acid concentrations and calculated cholesterol saturation index (CSI).
6. Ultrastructure of liver cell was observed under transmission electron microscope.
7.17nmol/Kg of exogenous CCK-8 was induced via jugular vein catheter, and collected bile of each 10min for measurement.
8. HE staining and saturated picric acid-Sirius red staining of gallbladder tissue, and histological changes and collagen deposition of the gallbladder wall were observed under the microscope and polarized light microscope.
9. Using automatic biochemical analyzer detected serum cholesterol, triglycerides, bile acids and alanine aminotransferase levels.
10. Oil red O staining of the liver, and light microscope observation of liver lipid deposition.
11. HE staining of liver tissues and fatty infiltration score.
12. Using Western blot method for detection of the major hepatobiliary transporter protein abcbll, abcg5, abcg8 and abcb4 protein expression in liver tissue, and detection of the expression of npc111 protein in intestinal epithelia.
Results
ⅠEstablishment of C57BL/6 mouse model of CGD
The observation by naked eye:the prevalence of gallstones in control group mice was 0 (0/19). The prevalence of gallstones in lithogenic group mice was 100%(18/18). Stones were confirmed to be cholesterol stones.
Gallbladder volumes in control group of mice was (22.6±8.35)μl, and lithogenic group was (63.2±10.6)μl, there was statistical difference between the two groups(t= 12.98, p<0.01). Liver bile secretion rate in control group mice was (1.12±0.06)μl/min/g liver, lithogenic group was (1.32±0.07)μl/min/g liver, there was statistical difference between the two groups(t= 6.66, P<0.01).
Polarizing microscope observation:the control group mice have no cholesterol crystals in gallbladder bile, and in lithogenic group cholesterol monohydrate crystals and stones can be seen.
In control group, cholesterol concentration of gallbladder bile was 5.79 mmol/L, and lithogenic group was 18.67 mmol/L. In control group, the phospholipids concentration of gallbladder bile was 29.69 mmol/L, and lithogenic group was 28.99 mmol/L. In control group, the bile acids concentration of gallbladder bile was 154.46 mmol/L, and lithgenic group was 138.69 mmol/L, is slightly lower than the control group. The CSI of gallbladder bile in lithogenic group was significantly higher (1.71 vs 0.58), as the cholesterol-supersaturated bile.
Observation under transmission electron microscope:compared with the control group, the liver cells of lithogenic group mouse showed a large number of small bubbles form lipid droplets and showed changes in liver cell injury.
II EZET prevented lithogenic diet-induced formation of cholesterol gallstone in C57BL/6 mice
The prevalence of gallstones in EZET-0 group was 100%(18/18), EZET-1 was 44.4% (8/18), EZET-2 was 10.5% (2/19), and EZET-3 was 0 (0/17).
Polarizing microscope observation:In EZET-0 group, true stones in gallbladder bile were more common seen. In EZET-1 mice, incomplete, undifferentiated silt-like stones were more common seen in gallbladder bile. In EZET-2 mice, sporadic gallbladder bile cholesterol crystals were more common seen. In EZET-3 mice, no cholesterol crystal was seen within the gallbladder bile.
The liver bile secretion rate of EZET-0 group was (1.32±0.07)μl/min/g liver; EZET-1 group was (1.43±0.05)μl/min/g liver; EZET-2 group was (1.53±0.12)μl/min/g liver; EZET-3 groupwas (1.73±0.08)μl/min/g liver, compared each group with a statistically significant difference (p<0.05).
In EZET-0 group, cholesterol concentration of gallbladder bile was 18.67 mmol/L; EZET-1 group was 12.43 mmol/L; EZET-2 group was 9.65 mmol/L; and EZET-3 group was 4.42 mmol/L. In EZET-0 group, phospholipids concentration in gallbladder bile was 28.99 mmol/L; EZET-1 group was 28.23 mmol/L; EZET-2 group was 30.14 mmol/L; and EZET-3 group was 33.82 mmol/L. In EZET-0 group, bile acid concentration of gallbladder bile was 138.69 mmol/L; EZET-1 group was 138.26 mmol/L; EZET-2 group was 145.98 mmol/L; and EZET-3 group was 169.21 mmol/L, there is a gradual increase trend.
In EZET-0 group, cholesterol concentration of bile duct bile was (4.67±0.28) mmol/L; EZET-1 group was (3.17±0.41) mmol/L; EZET-2 group was (2.25±0.51) mmol/L; and EZET-3 group was (1.76±0.38) mmol/L. In EZET-0 group, phospholipid concentration in bile duct bile was (8.26±0.45) mmol/L; EZET-1 group was (7.25±0.46) mmol/L; EZET-2 group was (5.63±0.62) mmol/L; and EZET-3 group was (5.18±0.95) mmol/L. In EZET-0 group, bile acid concentration of the bile duct bile was (24.67±1.01) mmol/L; EZET-1 group was (24.17±1.08) mmol/L; EZET-2 group was (23.44±1.63) mmol/L; and EZET-3 group was (23.39±1.47) mmol/L.
Western blot results showed that, EZET-0 mice had the highest levels of protein expression of npclll. With the increasing amount of EZET administration, npclll protein expression level decreased gradually.
ⅢEZET improve the gallbladder motor function of lithogenic diet-induced cholesterol gallstone C57BL/6 mouse
In CON group, gallbladder volume was (22.02±4.35)μl, EZET-0 group was (62.76±8.11)μl, EZET-1 group was (48.84±7.66)μl, EZET-2 group was (34.74±5.96)μl, and EZET-3 group was (23.84±5.76)μl, each group was significantly lower than EZET-0 group (p<0.05).
Results of CCK-8 interference experiment showed that EZET can significantly improve the reactivity to CCK-8 of mice, and with EZET dose increased, the areas under the curve gradually increased, indicating a marked increase in bile flow.
Microscopic observation:Compared with the CON group, gallbladder tissues of EZET-0 mice showed significant proliferation and expansion of connective tissue of mucosal lamina propria and marked collagen deposition; the above-mentioned changes of gallbladder tissues in EZET-1, EZET-2 and EZET-3 group mice was slightly lighter than EZET-0 group, but not obvious.
IV EZET reduced lipid deposition in the liver of lithogenic diet-induced cholesterol gallstone C57BL/6 mouse
Histological observation and oil red O staining of the liver observation:EZET significantly reduced the lithogenic diet-induced fatty liver changes, lipids deposition and damage in mice. EZET significantly reduced the scores of liver fatty infiltration.
EZET significantly reduces the lithogenic diet-induced elevated blood cholesterol levels; significantly reduces the lithogenic diet-induced elevated blood bile acids levels; while significantly reducing the lithogenic diet-induced elevated blood alanine aminotransferase levels; but no influence on blood triglyceride levels.
Western blot results showed that:lithogenic diet did not significantly affect abcbll bile acid transporter protein expression, however, in EZET group, expression level of abcbll was higher than the LITH group. Study also found that lithogenic diet significantly increased the cholesterol transport protein abcg5 and abcg8 protein expressions, but no significant change in EZET group; Study found that lithogenic diet did not significantly affect abcb4 phospholipid transporter protein expression, while no significant change in EZET group.
Conclusions
1. high-fat diet-induced C57BL/6 mice animal models of cholesterol gallstones was economic, reliable, and highly successful, which can be used for gallbladder cholesterol gallstone animal experiments. The establishment of the model is based on the pathophysiology of high-fat diet-induced non-physiological supersaturation of bile cholesterol.
2. EZET has a preventive effect on cholesterol gallstone formation in high-fat diet-induced C57BL/6 mice, and the mechanism is that EZET lowers non-physiological bile cholesterol supersaturation in C57BL/6 mice. EZET may effect in mice by reducing the npclll expression at brush border of intestinal mucosa thereby reducing intestinal absorption of cholesterol to achieve the above effect.
3. Administration of EZET to lithogenic diet-induced cholesterol gallstone C57BL /6 mice has a protective effect of gallbladder motor function.
4. EZET can significantly reduce the fat deposition and damage of the liver in lithogenic diet-induced cholesterol gallstone C57BL/6 mouse. EZET can increase the expression of bile acid transporter protein abcbll in the liver of lithogenic diet-induced cholesterol gallstone C57BL/6 mouse, but has no effect on the expression of cholesterol transfer protein abcg5, abcg8, and phospholipid transfer protein abcb4.
引文
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3 Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res. 1978; 19:945-955.
4 Fernandez ML. Guinea pigs as models for cholesterol and lipoprotein metabolism. J Nutr.2001; 131:10-20.
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6 Combettessouverain M, Souid IM, Parquetma, et al. The Syrian golden hamster strain LPN:a useful animal model for human cholelithiasis. J Nutr Biochem.2002; 13:226-236.
7 Souid IM, Combettessouverain M, Milliat F, et al. Hamsters predisposed to sucrose-induced cholesterol gallstones(LPN strain) are more resistant to excess dietary cholesterol than hamsters that are not sensitive to cholelithiasis induction. J Nutr.2001; 131:1803-1811.
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1 Frick W, Bauer-Schafer A, Bauer J, et al. Synthesis of a biotin-tagged photoaffinity probe of 2-azetidinone cholesterol absorption inhibitors. Bioorg Med Chem.2003; 11:1639-1642.
2 Rudel LL. Preclinical and clinical pharmacology of a new class of lipid management agents. Am J Manag Care.2002; 8:S33-S35.
3 van Heek M, Compton DS, Davis HR. The cholesterol absorption inhibitor, ezetimibe, decreases diet-induced hypercholesterolemia inmonkeys. Eur J Pharmacol.2001; 415:79-84.
4 Davis HR Jr, Compton DS, Hoos L, et al. Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in ApoE knockout mice. Arterioscler Thromb Vasc Biol.2001; 21:2032-2038.
5 Altmann SW, Davis HR Jr, Yao X, et al. The identification of intestinal scavenger receptor class B, type I(SR-B I) by expression cloning and its role in cholesterol absorption. Biochim Biophys Acta.2002; 1580:77-93.
6 van Heek M, Farley C, Compton DS, et al. Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function. Br J Pharmacol.2001; 134:409-417.
7 Davis HR Jr, Zhu LJ, Hoos LM, et al. Niemann-Pick C1 Like 1(NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole2body cholesterol homeostasis. J Biol Chem.2004; 279:33586-33592.
8 Garcia-Calvo M, Lisnock J, Bull HG, et al. The target of ezetimibe is Niemann-Pick C1 Like 1(NPC1L1). Proc Natl Acad Sci USA.2005; 102:8132-8137.
9 Duan LP, Wang HH, Ohashi A, et al. Role of intestinal sterol transporters Abcg5, Abcg8, and Npclll in cholesterol absorption in mice:gender and age effects. Am J Physiol Gastrointest Liver Physiol.2006; 290:G269-G276.
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11 Yu L, Bharadwaj S, Brown JM, et al. Cholesterol regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake. J Biol Chem.2006; 281:6616-6624.
12 Buhman KK, Accad M, Novak S, et al. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice. Nat Med.2000; 6:1341-1347.
13 Garver WS, Xie C, Repa JJ, et al. Niemann-Pick C1 expression is not regulated by the amount of cholesterol flowing through cells in the mouse. J Lipid Res.2005; 46:1745-1754.
14 Davies JP, Levy B, Ioannou YA. Evidence for a Niemann-pick C(NPC) gene family: identification and characterization of NPC1L1. Genomics.2000; 65:137-145.
15 Horton JD, Goldstein JL, Brown MS. SREBPs:activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest.2002; 109:1125-1131.
16 Repa JJ, Dietschy JM, Turley SD. Inhibition of cholesterol absorption by SCH 58053 in the mouse is not mediated via changes in the expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte. J Lipid Res.2002; 43:1864-1874.
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2 Portincasa P, Moschetta A, Colecchia A, et al. Measurement of gallbladder motor function by ultrasonography:towards standardization. Dig Liver Dis.2003; 35:S56-S61.
3 Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res. 1978; 19:945-955.
4 Fernandez ML. Guinea pigs as models for cholesterol and lipoprotein metabolism. J Nutr.2001; 131:10-20.
5 Boehler N, Riottot M, Ferezou J, et al. Antilithiasic effect of beta-cyclodextrin in LPN hamster: comparison with cholestyramine. J Lipid Res.1999; 40:726-734.
6 Combettessouverain M, Souid IM, Parquetma, et al. The Syrian golden hamster strain LPN:a useful animal model for human cholelithiasis. J Nutr Biochem.2002; 13:226-236.
7 Souid IM, Combettessouverain M, Milliat F, et al. Hamsters predisposed to sucrose-induced cholesterol gallstones(LPN strain) are more resistant to excess dietary cholesterol than hamsters that are not sensitive to cholelithiasis induction. J Nutr.2001; 131:1803-1811.
8 Mathison R, Shaffer E, Pfannkuche HJ, et al. Effects of tegaserod on bile composition and hepatic secretion in Richardson ground squirrels on an enriched cholesterol diet. Lipids Health Dis.2006; 5:15.
9 Macpherson BR, Pemsingh RS. Ground squirrel model for cholelithiasis:role of epithelial glycoproteins. Microsc Res Tech.1997; 39:39-55.
10 Schwesinger WH, Kurtin WE, Page CP, et al. Soluble dietary fiber protects against cholesterol gallstone formation. Am J Sur.1999; 177:307-310.
11 Zhao JC, Xiao LJ, Zhu H, et al. Changes of lipid metabolism in plasma, liver and bile during cholesterol gallstone formation in rabbit model. World J Gastroenterol.1998; 4:337-339.
12 Pekow CA, Weller RE, Schulte SJ, et al. Dietary induction of cholesterol gallstones in the owl monkey:preliminary findings in a new animal model. Lab Anim Sci.1995; 45:657-662.
13 Shaffer EA. Epidemiology and risk factors for gallstone disease:has the paradigm changed in the 21st century?Curr Gastroenterol Rep.2005; 7:132-140.
14 Hofmann AF. Pathogenesis of cholesterol gallstones. J Clin Gastroenterol.1998; 10:S1-S11.
15 Portincasa P, Moschetta A, Palasciano G. Cholesterol gallstone disease. Lancet.2006; 368: 230-239.
16 MC Carey, B Paigen. Epidemiology of the American Indians'burden and its likely genetic origins. Hepatology.2002; 36:781-791.
17 Johnston DE, Kaplan MM. Pathogenesis and treatment of gallstones. N Engl J Med.1993; 328: 412-421.
18 Meng CQ. Ezetimibe, Schering-Plough. Curr Opin Invest Drugs.2002; 3:427-432.
19 Nutescu EA, Shapiro NL. Ezetimibe:a selective cholesterol absorption inhibitor. Pharmacotherapy.2003; 23:1463-1474.
20 Frick W, Bauer-Schafer A, Bauer J, et al. Synthesis of a biotin-tagged photoaffinity probe of 2-azetidinone cholesterol absorption inhibitors. Bioorg Med Chem.2003; 11:1639-1642.
21 Rudel LL. Preclinical and clinical pharmacology of a new class of lipid management agents. Am J Manag Care.2002; 8:S33-S35.
22 van Heek M, Compton DS, Davis HR. The cholesterol absorption inhibitor, ezetimibe, decreases diet-induced hypercholesterolemia inmonkeys. Eur J Pharmacol.2001; 415:79-84.
23 Davis HR Jr, Compton DS, Hoos L, et al. Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in ApoE knockout mice. Arterioscler Thromb Vasc Biol.2001; 21:2032-2038.
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