The Study On The Relationship Between The Intestinal Barrier And The Pigment Gallstone Formation

PrefaceThe intestinal barrier function has drawn more and more attention in recent years for its important role in many diseases such as burn, wound, pancreatitis, severe infection, shock, et al. More and more diseases were found influenced by intestinal barrier during their development. But few studies have been performed on the relationship between the intestinal barrier and the pathogenesis of cholelithiasis. In our preliminary experiments about the pigment gallstone, we found some signs of the intestinal barrier function disorder during the pigment gallstone formation which may suggest the potential role in its pathogenesis. This study was undertaken in guinea pig model and cholelithiasis patients to investigate the possible action and mechanism of the intestinal barrier function in the pigment gallstone pathogenesis. The experiment was arranged though a clue;intestinal barrier injury — bacteria translocation - enzyme in bile change — gallstone formation. A series of indexes about the intestinal barrier and bacterial translocation were observed such as: plasma DAO level, serum endotoxin level, morphology and permeability of intestine mucosa, et al. Glutamine was used as an intestine mucosa protector. We hope to investigate the pathogenesis of pigment gallstonein our experiment though a new angle: intestinal barrier.MethodsEighty guinea pigs (weighing 200 ~ 250g) were divided randomly into normal group ( CON) , pigment gallstone group (PS) and intestinal mucosa protection group (GLN). Normal forage, pigment gallstone - forming forage and pigment gallstone - forming forage with supplemental intestinal mucosa protector (glutamine) were given to each group. The urine, bile, intestine tissue and abdominal lymph node were collected for detection after eight weeks. The gallstone - forming rate, morphology of intestinal mucosa, intestinal permeability, serum endotoxin, bacteria in abdominal lymph node and biliaryfj - glucuronidase were detected.32 patients with cholelithiasis and 27 patients with non - gastrointestinal diseases were collected from May 2005 to July 2005 in the second affiliated hospital china medical university. According to the type of the gallstone, patients were divided into three groups;cholesterol group (CS group, n = 16) , pigment stone group ( PS group, n = 16) and control group ( CON group, n = 27). There were not signs of acute bile duct infectious disease, obstructive jaundice or any other gastrointestinal diseases among these patients. The samples of plasma and serum were collected and assayed.In animal group, the gallstone can be identified easily during operation and the categories of the gallstones were confirmed by infrared spectrometry. The ile-um tissues 5cm away from the cecum was isolated and immediately fixed in 30g/ L glutaraldehyde for 15min and then dehydrated and dyed according to the standard sample preparation procedure for electron microscopy. The tight junction between adjoining cells, the microvilli on the cell surface and the intracellu-lar ultrastructure were observed with a JEM - 100CX II transmission electron microscope. Some other Mucosa tissue of ileum segment was fixed in arsinic dimethyl buffer containing 30g/L lanthanum nitrate and 30g/L glutaraldehyde. The distribution of lanthanum granules in junctions between adjacent cells, intracel-lular or intercellular spaces was examined with a JEN - 100CX H transmission e-lectron microscope, as a index of intestinal mucosa permeability. The activity of plasma DAO was detected by spectrophotometric method. The urine samples were collected after oral administration of lactulose jxl) and mannitol (M) and the proportionality of L/M were measured by high performance liquid chromato-gram. For detecting the endotoxin concentration, serum samples were collected and then measured by dynamic turbidity method on BET -32B endotoxin detector. In cholelithiasis patients, activity of plasma DAO and level of serum endotoxin were detected through the same method above. Guinea pig gallbladder bile was collected by puncture during operation. The activity of endogenous and exogenous p - glucuronidase in the bile samples were measured according to the modified fishman method. Bacteria in abdominal lymph node were detected by PCR method with taking 16SrRNA as the target gene which is common in most bactercias. Data are expressed as mean ± SD. All analyses were undertaken u-sing x and t analysis by SPSS 11.5 software. A p value of ^ 0. 05 was accepted as the level of statistical significance.ResultsThe incidence of Gallstone formation was 73. 9% in PS group suggesting that the gallstone - inducing animal model was successfully established. While the incidence of gallstone formation in GLN group in which glutamine was used to protect the intestinal barrier was significantly decreased (44. 4% ) compare with that in PS group ( p < 0. 05).Compare with that of CON group, the ultrastructure of intestinal mucosa cell in PS group showed some signs of disorder;the microvilli of the cells arrayed in a disorder way, part of the tight junction broke and showed heavier e-lectron density, mitochondrion in some cells exhibit vacuolar degeneration with cristae fading, endoplasmic reticulum distention with ribosome run - off, and occasionally nucleus aberrance. In GLN group, heavier electron density along intercellular junction could also be seen but with an integrity constitution and outline , microvilli arrayed in a raritas but regular way, and slight signs of degeneration in some cells were occasionally found.The lanthanum granule is a kind of heavy metal macromolecule with a diameter of 1 ~ 4nm. Under normal condition, it can not cross though the mechanism barrier of the intestinal mucosa which is consist of the junctions between the adjacent epithelium, and it will not accumulate there. But when the permeability of the intestinal mucosa increases, the lanthanum granule will be able to cross the intercellular junction even the membrane of epithelium and enter the intercellular space or the cell. Our lanthanum nitrate tracer study showed that in PS group the high electron density lanthanum granule deposited among the mi-crovilli and intercellular space, arrayed along the outline of the cells, entered the cytoplasm of some cells. It suggested that the permeability of the intestinal mucosa and cell membrane increased and the barrier function of the intestinal mucosa was injured. In GLN group, less amount of lanthanum granule deposited among some cell microvilli, seldom lanthanum granule could be found in intercellular space, and no granule was found in cytoplasm. It suggested that the intestinal permeability was relatively good.Compare with the CON group, activity of serum DAO in PS group (0. 612 ±0. 152 U/ml) increased indicating the injury of intestine mucosa ( p <0. 05 ). While in GLN group(0. 555 ±0.172 U/ml) the numerical value of DAO activity were lower than that in PS group but it failed to achieve statistical significance.In PS group(0.062 ±0.031) , the proportionality of L/M increased suggesting a higher permeability of intestine mucosa(p < 0.05). In GLN group (0.035 ±0. 023) , the proportionality of L/M was lower than that in PS group ( p <0. 05) which suggest that glutamine exerted some protection function on intestine mucosa.In cholelithiasis patients, the activity of DAO in CS (1. 027 ±0. 877 U/ ml) group and PS group (0.950 ± 0.724 U/ml) is above the normal level (0. 602 ±0.145 U/ml) (p <0.05) , but there was no difference between them. While the serum endotoxin concentration in PS group (1.296 ± 1.015 EU/ml) was significantly higher than that in CS group(0.336 ±0. 202 EU/ml) and CON group (0.231 ±0.114 EU/ml) (p<0.01).In guinea model, Serum endotoxin level was significantly higher in PS group (1. 367 ± 0. 525EU/ml) than that in CON group (0. 077 ± 0. 043 EU/ml) (p <0.01). In GLN group (0. 156 ±0. 097EU/ml) , it is also higher than that in CON group (p <0.05) but lower than that in PS group (p <0.01).The activity of both the endogenous and exogenous ￡ - glucuronidase in PS group were higher than that in CON group ( p < 0.01 ) , and especially obvious in the exogenous enzyme. In GLN group, activity of both the two enzyme are also higher than that in CON group (p < 0. 05). In our experiments the activity ofp - glucuronidase in GLN group seemed lower than that in PS group, but no statistical significance were found within our sample amounts.The positive rate of bacteria detection by PCR in guinea pig abdominal lymph node were 30% , 80% and45% in CON group, PS group and GLN group respectively. Compare with that in PS group, the positive rate was lower in GLN group (p<0.05).ConclusionsIntestinal barrier function disorder can be found in the process of pigment gallstone formation in diet - induced pigment gallstone guinea pig model and in cholelithiasis patients. The intestinal barrier is correlated with the pathogenesis of pigment gallstone. Glutamine can improve the intestinal barrier function and decrease the rate of pigment gallstone formation. The intestinal barrier function disorder may take part in the process of promoting pigment gallstone formation through bacteria translocation , endotoxemia and affecting the activity of biliary 3 - glucuronidase.