| The mechanism of bile pigment stone formation has always been an hot topic in the field of disease of biliary tract over the world.It was reported that changes of the components of bile and bile duct infection played a central role in the bile pigment stone formation.Previous studies were always based on the Maki theory that bile infection was the center mechanism,and analyzed of the composition of bile,gallstone. It was often neglected that infection can lead to the changes of primary liver cells secretion function and original bile components.Recently,scholars carried out extensive research about pigment stones pathogenesis related to immunization,trace elements,oxygen free radicals,amino acids,free fatty acid and content of metal elements.However no breakthrough was found.But research shows that there are three necessary conditions and a central link with the formation of pigment stones.The necessary condition for pigment stones is free bile pigment and calcium increased in bile while the melting factor weakened.The unbalance between the sediment and dissolved bilirubin calcium is the central link of pigment stone.The main components of bile are cholesterol,bile acids and phospholipids,they are all excreted from the liver cell canalicular membrane into the bile.Bilirubin is transported by the liver cells into bile through the same method.Little is known about changes in the composition of bile owing to the primary state change of liver cells secretion in the process of pigment stone formation.We have observed the changes of major components in bile in the pigment stone formation process through the establishment of guinea pig model of pigment gallstone.We also have studied the changes in the expression of liver cell membrane canalicular transporter occurred in the pigment stones,aimed at further exploration about pigment stone formation mechanism from the state of primary liver secretion.In addition bile metabolomics was studied with liquid chromatographymass spectrometry.Through the comparative study of bile in guinea pigs with and failed stone formation,biliary metabolites had been clearly separated in different groups.A further analysis of metabolites contributed to the separation was performed, the initial identification of metabolites with larger difference was produced in pigment stones formation,to speculate the possible molecular formula and structure,and to explore the role of them in pigment gallstone formation.Materials and methodsAnimal modelHealthy 45 guinea pigs(weighing 200～250g) were divided randomly into two groups:normal group(CON),pigment gallstone group(PS).15 guinea pigs in CON group,and 30 in pigment gallstone group.6 to 10 guineas were in each cages,daily food was given for 4 to 6 times,and unrestricted to drinking water.Living environment was with room temperature at 20～25℃,relative humidity of 60 to 70%.After feeding adaptation for a week,normal forage and pigment gallstone-forming forage were given to each group.The period of stone permeation is eight weeks.Specimen collectionThe liver tissue,bile and gallstones were collected for detection and stored at -80℃after eight weeks.Targets detection1.The gallstone-forming rate was assessed and gallstone was determined by infra-red spectrum(IR spectrum).When the gallbladder in guinea pigs was anatomized, it can be clearly distinguished with gallstone formation by the naked eye.The qualitative composition of the calculus was determined by IR spectrum potassium bromide-disk technique,and the rate of stone formation was counted.2.Detecting the amount of total bilirubin,direct bilirubin,undirect bilirubin and total bile acid in different group with biochemical methods.3.Observing the distribution of liver cell membrane canalicular transporter with immunohistochemical method,concerning Bile Salt Export Protein(BSEP,Abcb11), divalent bile salt and bile pigment transporter protein(Multidrug resistance-associated protein,Mrp2,Abcc2),phospholipid transfer protein(Multidrug resistance protein, Mdr,Abcb4).4.Expression of liver cell membrane canalicular transporter(Abcb11,Abcc2, Abcb4) were detected with Western-Blot.Membrane protein was extracted from liver tissue specimens,and tested with SDS gel electrophoresis and ECL chemiluminescence method.5.Expression of liver cell membrane canalicular transporter mRNA(Abcb11, Abcc2,Abcg5,Abcg8) were detected with RT-PCR.RNA was extracted from liver tissue specimens.Target gene was amplified.6.Bile metabolomics was detected by LC-MS method.After the application of high-performance liquid chromatographic to separate the bile,and metabolites was softly ionizated,the mass spectrometry analysis was performed.Statistical analysisSPSS11.5 statistical package was adopted for analysis.The statistical data of our research were all in normal distribution.The results were expressed by mean±SD. Pigment gallstone group and control group were compared and two independent samples T-test was conducted.Comparison of the rate was performed by using chi-square exact test,P＜0.05 was refered as significant difference.Result1.Establishment of guinea pig model of pigment gallstoneModel-building situation on the bile pigment stones in the guinea pig,In guinea, the gallstone can be identified easily during operation and the categories of the gallstones were confirmed by infrared spectrometry.Stone-formation rate in PS group(68.42%)was higher compared with that in control group(14.29%) after feeded for 8 weeks.Stone was identified as a pigment stones.2.Changes in the expression of liver cell membrane canalicular transporter(1) Immunohistochemistry results showed that brown paricles were distributed in the liver cells ductular membrane,suggested the expression of ductular hepatocyte lateral membrane protein.Compared with the CON group,the expressions of Abcb11 (t=5.589,P＜0.01) and Abcc2(t=5.468,P＜0.01) were decreased in PS group and shading less;the coloration of Abcb4 increased slightly,but no statistical significance. In PS group,the coloration of Abcb11(t=8.620,P＜0.01) and Abcc2(t=8.620,P＜0.01) were decreased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation.(2) RT-PCR results showed that the mRNA expressions of Abcb11(t=6.885, P＜0.01) and Abcc2(t=6.794,P＜0.01) were decreased in PS group compared with the CON group;In PS group,the mRNA expressions of Abcb11(t=2.824,P＜0.05) were also decreased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation.The mRNA expression decreased concerning with Abcc2(t=2.503,P＜0.05) and with statistical significance.The expressions of Abcg5 and Abcg8 were no statistical in mRNA level.(3)Western Blot results showed that the expressions of Abcb11(t=8.412,P＜0.01, P＜0.01) and Abcc2(t=7.894,P＜0.01) were decreased in PS group compared with the CON group;In PS group,the expressions of Abcb11(t=6.042,P＜0.01) and Abcc2 (t=9.764,P＜0.01) were also decreased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation.The expression of Abcb4 was no difference in all groups.3.Changes of major components in bileThe results of bile with biochemistry test showed that compared with the control group(CON),the guinea pigs in pigment stone group(PS) was proved with the elevation about total bilirubin(t=3.722,P＜0.01) and free bilirubin(t=3.727,P＜0.01) significant.The ratio between free bilirubin and direct bilirubin(t=3.528,P＜0.01) was also increased significantly.The total bile acid was significantly decreased in PS group(t=4.972,P＜0.01).In PS group,the total bile bilirubin(t=3.307,P＜0.01) and free bilirubin(t=3.447,P＜0.01) were also increased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation. The direct bilirubin was mildly elevated.The ratio between free bilirubin and direct bilirubin(t=3.545,P＜0.01) was also increased significantly.The total bile acid was significantly decreased in stone formation guinea pigs(t=8.040,P＜0.01).4.LC-MS detection of bile metabolomics researchCompared with failed stone formation guinea pigs,metabolites with M/Z for the quality of 483.2 amu,515.8 amu,484.2 amu,312.7 amu was significantly increased, M/Z mass number for 277.9 amu,295.0 amu,297.1 amu,506.2 amu,449.0 amu was decreased significantly.Conclusions1.The expression of liver cell membrane canalicular transporter was changed during the process of diet-induced guinea pig gallbladder bile pigment stone formation; There was a certain relevance betweenpigment gallstone formation and the abnormal expression of liver cell membrane canalicular transporter.2.During the process of diet-induced pigment gallstone formation in guinea pigs, the changes of bile composition occurred.The levels of free bilirubin,taurocholate, stearic acid were significantly increased,and total bile acid,glycocholic acid and linolenic acid are decreased.This played a role in pigment gallstone formation.3.Secretory function of liver cells was involved in the impact on pigment gallstone formation.The abnormal expression of liver cell membrane canalicular transporter lead to the changes in bile composition may be one of the mechanisms of stone formation as the secretion function of primary liver cells involved in pigment gallstone formation.4.It was feasible for bile metabolomics study with LC-MS.
|
PDF Full Text Request