Effect Of Ezetimibe On Prevention Of Cholesterol Gallstones In C57BL/6 Mice With Lithogenic Diet Feeding

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 MethodsI Animal modelMale 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 targets1. Experiment of the effect of EZET on cholesterol gallstone formationWere 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 functionWere 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 metabolismWere 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 methods1.10% 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.ResultsⅠEstablishment of C57BL/6 mouse model of CGDThe 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 miceThe 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 mouseIn 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 mouseHistological 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.Conclusions1. 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.