| PrefaceThe role of bacteria in the formation of pigment gallstones attracts the attention of many researchers. Normal bile juice is sterile. The bacteria in bile duct almost come from the endogenous infection of the gut. These kinds of bacteria can produce the same β-glucuronidase( β-G) as that of E-coli, which can catalyze combined bilirubin ( CB) to uncombined bilirubin ( UCB) which binds to calcium to form pigment stones. Up to date, it is avowedly accepted by the clinic that bacteria plays an important role in the pathogenesis of pigment gallstones.As to the source of bacteria, it is still controversial. A large number of studies and clinical observation show that intestinal bacterial translocation results from the dysfunction of intestinal barrier(IB) and increase of intestinal permeability (IP).Endotoxin (ET) is lipid polysaccharide on the cellular wall of Gram negative bacilli, especially in intestinal bacilli. When the function of IB is in disorder, ET can penetrate the intestinal mucosa to the blood circulation and lead to endotoxemia. Detect the ET level in the plasma is an important method to leam about the function of IB.Diamine oxidase ( DAO) exists in the mucosa or valium layer of mammary animals and is regarded as the marker enzyme of the intestinal mucosa. Its activity is closely related to the synthesis of nuclear acid and protein in the cells. Under physical state, the activity of DAO in the plasma is very low. In the case of IB dysfunction, the DAO released from the intestinal mucosa cells enters the blood and leads to the rise of its plasma concentration.This study aims to study the changes of plasma ET and DAO to discuss theeffect of intestinal mucosa barrier function on the formation of pigment gallstones in hamsters fed on low protein and high cellulose diet.MaterialsI. Animals20 adult female hamsters of 15 weeks old, with body weight 130 ~ 150g (supplied by the Animal Room of the Second Affiliated Hospital of China Medical University)II. Main instrument and reagents(1) sterile operation instrument ( supplied by the Animal Room of the Second Affiliated Hospital of China Medical University)(2) MODELDS-671 electrical weigh-bridge(made in Dalian)(3) KUBOTA5200 automatic centrifuge(made in Japan)(4) mini-sucker tube and blood sample collection tube (supplied by the He-matology Department of the Second Affiliated Hospital of China Medical University)(5)BET-32B bacterial endotoxin detector (made in Tianjin University)(6) BET-32B bacterial endotoxin detector specialized de-thermion tube and tip (purchased from Tianjin Fulansi Electric Trade Company)(7) EasyBET3. 2 auto-analysis software (supplied by the Bacterial Department of the Second Affiliated Hospital of China Medical University)(8) thermostat ( supplied by the Bacterial Department of the Second Affiliated Hospital of China Medical University)(9)DU-7 SPECTROPHOTOMETER BECKMAN(made in USA) (10)ZH-2BLENDER(made in Tianjin University)(11) casine (purchased from Medical and Pharmaceutical Company Shenyang ) , compound vitamins ( purchased from Sanhao Drug Store) , 1 % choline chloride (presented by Zhengda Animal Food Company) , cellulose (presented by Shenyang NO. 1 Drug Factory) , peanut oil, unrefined salt and sugar (bought at market)(12) 1% sodium heparin solution (supplied by the Hematology Departmentof the Second Affiliated Hospital of China Medical University)(13) bacterial endotoxin standardized sample ,tachypleus amebocyte lysate,NO. land 2 blood disposal and sterile injection water(purchased from Economicand Technological Development Zone Zhanjiang China)(14)0.2M PBS(supplied by 304 Hospital of PLA, Beijing) (15)CADAVERINE DIHYDROCHLORIDE, DIAMINE OXIDASE and 0-DIANISIDINE( supplied by 304 Hospital of PLA, Beijing)(16) PEROXIDASE( supplied by 304 Hospital of PLA, Beijing)MethodsI. Preparation of lithogenous dietThe diet was prepared according to Yang Wenqi' s method with slight changes.II. Animal divisionEach hamster was numbered from lto 20 respectively. 20 hamsters were fed on standardized diet previously. After the blood samples were collected, low protein and high cellulose diet was given for 6 weeks.HI. Preparation of samplesBlood samples were collected before the lithogenous diet was given and after the diet was given for 6 weeks. The concentration of blood endotoxin and diam-ine oxidase were measured by dynamic turbidity method and flicker photometer method respectively.(1) Blood sample collection before the lithogenous diet was given Mini-sucker tubes were soaked in the 1% sodium heparin solution overnight and gotten rid of the thermion. Blood samples were collected by puncture of the orbital vein of the hamsters with the mini-sucker tubes. 1.5ml blood could be collected into the specialized de-thermion tube, then protected at room temperature for 2 hours and centrifugalized at 3500rpm. The upper serum 0.5ml was collected and stored at -20 for ET analysis. After the blood samples were collected, all the animals were given normal diet continuously. 2 days later, if the hamsters were still alive, another 1.5ml blood sample was collected and prepared by the samemethods for DAO analysis.(2) If the hamsters were still alive after the two times of blood collection, 2 days were given for refreshment before the lithogenous diet was given. The body weight of each hamster was recorded every week. The lithogenous time lasted for 6 weeks.(3)6 weeks later, if the hamsters were still alive, blood samples for ET and DAO were collected again with the same method. Cholecystectomy was performed to search for gallstones. If so, the stones were washed with water, dried naturally and stored carefully.IV. Determination of serum ET concentration(1) Preparation of standardized curve1. Turned on the computer, dotted on EasyBET3.2 software, selected the i-tem of preparation of standardized curve and went on according to the software instruction.2.200 EU bacterial endotoxin standardized sample was added to 2ml sterile injection water and blended for 15min.3.0.9ml water and 0. lml standardized sample were added to the tube and blended for 30s. 0. lml of the solution was taken out into the 2nd tube and blended for 30s, and so on. The ET contents of the 4 tubes were; 10EU,lEU,0.1 EU,0.01EU.4. Got another 4 tubes, added 0. lml tachypleus amebocyte lysate and 0.2 ml diluted standardized sample and blended for 5s. Put it into the BET-32B bacterial endotoxin detector for dynamic analysis of 3600s.5. Afer the analysis, the preparation of standardized curve was completed.(2) Samples adding1. Open the computer, select the original standardized curve and determine the setting index2.0.5ml NO. 1 blood disposal and 0.5ml sample were mixed and centrifu-galized at 3200rpm for 10min.3.0.5ml of the 2 times diluted solution was mixed with 0.5ml NO. 2 blood disposal. After 6 minutes' water bathing at 65 ~70℃ ,the solution was centrifu-galized at 4200 rpm to get the four times diluted solution.4.0.5ml nonObacterial injection water was mixed with the four times diluted solution to get the eight times diluted solution.5.0.1ml tachypleus amebocyte lysate was added to 0.2ml of the eight times diluted solution and then blended for 5min. Put it into the corresponding location of the machine and observed for 3600s.6. When the time was due, stopped the detection manually and chose the selected standardized curve and got the content of ET.V. Determination of serum DAO concentration(1) Preparation of 0. 2M PBS(2) Preparation of standardized samples80mg DAO standardized sample was added into lml water. 0. 5ml of the mixture was taken out into another tube and added 0.5 ml water into it again and so on until 5 standardized tubes were prepared, which contained DAO 80mg/ ml,40 mg/ml,20 mg/ml,10 mg/ml,5 mg/ml( lmg =0.08u).(3) Sample adding, 3. 8ml for each sample l.Got 0.2M PBS 3.0ml PH 7. 2.2.0. 1ml peroxidase was added into the tube and mixed evenly.3. 0. lml 0-dianisidine were added into the tube above and mixed evenly.4.0. 5ml serum sample or DAO standardized sample was added.5.0. lml cadaverine dihydrochloride was added.(4) After the adding of the samples, the tube was immersed into 37℃ water bathing for 30min.(5) 30 minutes later, the sample was taken out and placed into DU-7 SPECTROPHOTOMETER and obtained the OD value at 426nm. Then chose the selected standardized curve and got the content of DAO.VI. Statistical analysisAll the datas were expressed as Mean ± SD. SPSS11. 5 software was used for analysis. P value less than 0. 05 was considered to be of significant difference and P value less than 0.01 was considered to be of great significant difference.ReslutsI. The growth and stone formation of hamstersAfter the first time of blood collection, 4 hamsters died. Another 4 died during the lithogenesis with a mortality rate of 25%. The body weight of the hamsters decreased during the experiment rather than increase with loss of hair in some malnourished ones. Pigment gallstones were found in 11 hamsters at the end of the experiment with a lithogenous rate of 91. 67%.II. The changes of blood ET and DAO before and after the stone formation (Table 1)Table 1 The changes of blood ET and DAO( n = 12,unit:U/ml)As to the pathogenesis of pigment stones, the β-glucuronidase(β-G) theory stated by Japanese scholar Maki et al in the 1960s is widely cited.The existence of bacteria in bile juice and stones is the most direct and objective proof of the effect of bacteria in the formation of stones. Many kinds of anaerobic and aerobic bacteria can be found in the culture of bile juice of the cholelithiasis patients. The positive ratio of bacterial culture of bile juice of pigment gallstone patients is significantly higher than that of the cholesterol gallstone patients.The bacteria that cause the infection of the biliary system almost can produce β-G, that is exogenous β-G. This kind of β-G possess more ability to catalyze CB to UCB and further promotes the formation of pigment stones.The bile juice of healthy people is sterile. The infection of biliary systemalmost arises from the endogenous infection of the gut. Theoretically, the bacteria in the gut can enter the bile duct in two ways: one is from the intestinal barrier ( IB) and the other is from the reflux from the Odd s sphincter, which is indeterminate.Endotoxin( ET) is lipid polysaccharide on the cellular wall of Gram negative bacilli, especially in intestinal bacilli. Under normal conditions, ET can not enter the blood circulation because of the integrity of the IB. When the function of IB is in disorder, ET can penetrate the intestinal mucosa into the blood circulation and lead to endotoxemia. Detect the ET level in the plasma is an important method to learn about the function of IB.Diamine oxidase ( DAO) exists in the mucosa or villus layer of mammary animals and is regarded as the marker enzyme of the intestinal mucosa. Its activity is closely related to the synthesis of nuclear acid and protein in the cells. Under physical state, the activity of DAO in the plasma is very low. In the case of IB dysfunction, the DAO released from the intestinal mucosa cells enters the blood and leads to the rise of its plasma concentration.Up to date, there are three methods to establish the animal models of pigment gallstones; ( 1) Low protein diet; ( 2 ) Operation intervention, which includes ligation of the common bile duct or construction of constriction of the bile duct; (3 ) Establishment of hemolytic animal models. In our country, low protein diet is widely used for study of formation of pigment stones in piglet and rabbit. But it is less in hamster. This study shows low protein and high cellulose diet can construct a model of pigment gallstones in female hamsters with lithogenous rate more than 90% , suggesting it is an ideal animal for the study of pigment stones.Many researchers have successfully established the pigment gallstones in a lot of animal models by low protein diet. But the underlying mechanism is still unidentified. Study of the literature in detail shows the following possible explanations. (1) Matsushiro et al. believed that low protein diet could decrease the content of glucurolactone, which leaded to the increase of UCB and formation of pigment stones. (2) Low protein diet undermined the resistance of tissue, leaded to the disorder of regeneration of bile duct epithelium and increased the chancesof infection and constriction of the bile duct. (3} Some believed that low protein diet changed the Zeta potential of glycocine-combined bile juice and the nature of mucus protein. (4) In the study of piglet models, people found that the concentration of mono-combined bilirubin (MCB) increased significantly. The solubility of MCB is 1/7 of that of di-combined bilirubin. Under certain PH value, its solubility is lower than UCB. This indicated that MCB may play a role in the formation of pigment gallstones.This study established the gallbladder pigment stones of hamsters by low protein and high cellulose diet and monitored the changes of blood ET and DAO. The results showed that increase of both indexes. Therefore, we think low protein diet can cause the dysfunction of IB, increase the IP, lead to the transloca-tion of bacteria and ET to the bile duct which produce exogenous G and facilitate the formation of pigment gallstones.Conclusion1. The content of serum endotoxin was significantly increased after low protein and high cellulose diet was given to hamsters to form pigment gallstones.2. The content of plasma diamine oxidase was significantly increased after low protein and high cellulose diet was given to hamsters to form pigment gallstones.3. These changes indicated the increase of intestinal mucosa permeability may play a role in pigment gallstone formation. Intestinal mucosa protective a-gents maybe effective in the prevention of relapse of gallstones.PrefaceDuring the recent decades, great progress has been made in the research on the pathogenesis, prevention and treatment of cholelithiasis. The effect of " intestine transit (IT) " has become a new field of interest in the study of the mechanism of the formation of cholesterol gallstones.It has been identified that the time of bile transit in the intestine or the whole gut is prolonged in gallstone patients. Therefore, there is a possible relationship between the dysfunction of IT and the formation of gallstones. The,dysfunction of IT can cause the stasis of bile salts in the gut, which leads to the formation of more deoxycholic acid ( DCA). Studies showed that gallstone patients group had longer IT time,higher serum DCA concentration,higher PH value in the colon cavity, more number of anaerobic bacteria in the cecum and higher activity of 7α-dehydroxylase, compared with healthy control group. It is believed that DCA can facilitate the formation of cholesterol gallstones in many ways.Moreover, IT can affect the motility of gallbladder. There is a close relationship between intestinal migrating motor complex ( MMC) and gallbladder motility. In cholesterol gallstone patients, the decrease of MMC frequency and prolongation of the cycle are common phenomenon. All these lead to the dysfunction of gallbladder empty and increase of bile concentration and then facilitate the formation of cholesterol crystals and stones.~99mTc-MDP served as a unabsorbed radioactive marker in the intestine. When it is poured into the digestive tract by stomach irrigation, its distribution in the intestine can reflect the level of IT. This study aims to explore and discuss the relationship between the dysfunction of IT and the formation of gallstones in hamsters fed on high cholesterol-contained diet by this method.MaterialsI . Animals64 male hamsters of 4 weeks old with body weigh 40 ~ 50g ( supplied by the Animal Room of the Second Affiliated Hospital of China Medical University) II. Main instrument and reagents(1) sterile operation instrument ( supplied by the Animal Room of the Second Affiliated Hospital of China Medical University){2) animal stomach irrigator ( supplied by the Animal Room of the Second Affiliated Hospital of China Medical University)(3)~99mTc vacuum tube (supplied by the Nuclear Department of the Second Affiliated Hospital of China Medical University)(4)RM905 RADIOACTIVITY METER (supplied by the Nuclear Department of the Second Affiliated Hospital of China Medical University)(5)~99mTc-MDP( supplied by the Nuclear Department of the Second Affiliated Hospital of China Medical University)(6) cholesterol of chemistry purity ( purchased from Medical and Pharmaceutical Company, Shenyang)MethodsI. Preparation of lithogenous dietCholesterol of chemistry purity was added to standardized hamster diet to obtain 1% cholesterol-contained lithogenous diet.II. Animal division and preparation of animal modelAll 64 hamsters were randomly assigned to two groups, experimental group and controlled group. Each group was then divided into four subgroups, with eight hamsters in each subgroup. High cholesterol-contained diet and standardized diet were fed to experimental group and controlled group respectively with the same grow conditions.III. Observation indexThe experiment was performed at the end of the 3rd, 4th, 5th and 6th week.(1) All the hamsters were fasted at the night before the experiment with freedom of water intake.(2)0. 5ml saline solution containing 0.75mCi ~99mTc-MDP was poured into the stomach through stomach irrigator.(3)30 minutes later, the hamsters were killed by cervical vertebrate dislocation. The gut was divided into the stomach (termed segment 1) , eight equal segments of small intestine ( segment 2 to 9) , and the cecum and colon (segment 10). Each segment, along with its luminal contents, was the placed into a ~99mTc vacuum tube for later use.(4) Cholecystectomy was performed to search for gallstones.(5) RM905 RADIOACTIVITY METER was used to count the radioactivity of each segment. Data were expressed as the percent radioactivity in a given segment ( % distribution). The amount of radioactivity measured in each individual segment was used to calculate the geometric center of infused marker according to the following equation; Geometric center = ∑ ( counts per segment x segment number/total counts).IV. Statistical analysisAll the datas were expressed as Mean ± SD. SPSS11. 5 software was used for analysis. P value less than 0. 05 was considered to be of significant difference.ResultsI . The growth and stone formation of hamstersThe growth of the hamsters was fine during the experiment without death, diarrhea or loss of hair. Cholesterol gallstones were found in two hamsters at the end of the 4th week of the experiment.II. The value of geometric center of experimental and control group at the four time intervals (table 2)DiscussionThe incidence of cholelithiasis increases with the increase of age. The path-ogenesis of the disease has been deduced as the following: disorder of cholesterol metabolism, dysfunction of gallbladder contraction and absorption, bacterial infection and lithogenous genes et al. In the 1990s, more research focused on the intestine-gallbladder motor rule. Therefore, more attention has been paid to the relationship between intestine and the formation of cholesterol gallstones.It has been shown that, the intestinal transit (IT) time was prolonged in gallstone patients. Theoretically, the dysfunction of IT can cause the stasis of bile salts in the intestine, leading to more formation of deoxycholic acid ( DCA) . Xu Q et al. found in the hamster model that high cholesterol contained diet could induce the increase of DCA in the bile juice super-saturation of cholesterol and gallstone formation. IT prolongation was also observed. Moreover, long IT can prolong the enterohepatic cycle of bile acid and reduce the reabsorption rate of bile salts leading to the decrease of bile salts in bile juice and promoting the formation of cholesterol crystals and stones.Increase of DCA can promote the formation of gallstones in many ways. (1) DCA itself can slow down IT, thereby allowing more time for intestinal cholesterol absorption and exerting a positive feedback on its own formation. (2) DCA can enhance biliary cholesterol secretion by an effect on the hepatocytic canalic-ular membrane. (3 ) In vitro studies have revealed that DC A enhances biliary cholesterol crystallization by destabilising cholesterol-rich vesicles. (4 ) DC A can affect gallbladder motility through nerve reflex or other regulatory factors.Moreover, IT can affect the motility of gallbladder and facilitate stone formation. There is a close relationship between inter-digestive period gallbladder contraction and intestinal migrating motor complex ( MMC) and serum motilin (MTL). In cholesterol gallstone patients, the decrease of MMC frequency and prolongation of the cycle are common phenomenon. In healthy people, the gallbladder can empty the cholesterol crystals and mini-stones in time and eventually avoid the formation of gross stones. While in gallstone patients, the gallbladder can not empty the lithogenous bile juice but serve as a contributor to the formation of stones. All these lead to the dysfunction of gallbladder empty and increase of bile concentration and then facilitate the formation of cholesterol crystals and stones.In fact, the cycle of inter-digestive period MMC determines the cycle of bile salts reabsorption. IT and gallbladder empty cooperate each other and serve as the rate-limiting step of bile salts enterohepatic cycle. IT and gallbladder motility affect the size, proportion, lipid constitution and tendency of stone formation. Therefore, it is of great significance to discuss the relationship between IT and gallbladder motility in gallstone patients based on the physical relationship between them.In summary, decrease of IT leads to DCA pool increase,more cholesterol secretion and decrease of cholesterol vesicle stability; MMC cycle frequency decrease leads to dysfunction of gallbladder empty and increase of bile concentration. All these lead to the formation of cholesterol crystals and stones.~99mTc-MDP served as a unabsorbed radioactive marker in the intestine. When poured into the digestive tract by stomach irrigation, its distribution in the intestine can reflect the level of IT. This study showed high cholesterol diet significantly reduced the IT at the end of the 4th, 5th and 6th week ( P <0. 05). This showed that high cholesterol diet significantly reduced the intestinal transit function and facilitated the formation of cholesterol gallstones.
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