Infection Of Pancreatobiliary Reflux On Gallbladder

Pancreatobiliary maljunction (PBM) is a congenital anomaly. Since the action of the sphincter of Oddi (SO) does not functionally affect the junction, continuous reciprocal reflux between pancreatic juice and bile occurs. As the hydropressure within the pancreatic duct is usually greater than that in the bile duct, pancreatobiliary reflux (PBR) often occures. The activated of pancreatic enzymes, secondary bile acids and mutagenic substances result in multiple pathological changes such as hyperplasia, metaplasia, dysplasia and gene mutations may occure, and even malignancies of gallbladder or bile duct. It reveals that PBR may also exist in nomal pancreatobiliary junction, which is termed as functional pancreatobiliary maljunction, including high confluence of pancreatobiliary ducts (HCPBD) and occult PBR. Significant PBR almost exists in all HCPBD, with high incidence of gallbladder carcinoma. Moreover, occult PBR is also related to gallbladder carcinoma, reported by Japanese experts. However, it is still unclear about mechanism of PBR in functional pancreatobiliary maljunction, its relation with SO or the effect of endoscopic sphincterotomy on it.So far the treatment of PBR has been mainly on surgery since no effective medication is confirmed. Cholecystectomy is adopted in PBM, followed by hepaticojejunostomy with Roux-en-Y in dilated type to prevent malignancies. Prophylactic cholecystectomy for HCPBD is recommended in Japan, while there is still no therapeutic consensus on occult PBR. Traditional Chinese medicine has not involved in this field. The aim of this study is to explore the impact of PBR in normal pancreatobiliary junction on gallbladder, SO function and endoscopic sphincterotomy (EST) on PBR, to explain its etiology and pathogenesis with traditional Chinese medicine theory and verify it in PBR models.Methods:1. Clinical research(1) Patients with gallbladder diseases hospitalized for cholecystectomy underwent abdominal ultrosonography to evaluate the thickness of gallbladder wall and inner layer and gallbladder wall bloodflow. Bile was sampled to detect BA and cultivate bacteria. All patients were assigned into PBR group and control group according to the BA level. Resected gallbladder specimens were examined histopathologically and immunohistochemically. Data of the two groups were compared. (2) Consecutive patients who received SO manometry (SOM) during therapeutic ERCP for biliary tract diseases were assigned into three groups:normal SO basal pressure group, elevated SO pressure group and EST group. Data of SO basal pressure, BA and bacteria culture findings were analyzed and compared between the three groups.2. Animal experiments(1)Seven-week-old female Syrian golden hamsters were subjected to cholecystoduodenostomy and dissection of common duct at the distal end to create PBR model. Four weeks after operation, the surviving ones were randomly assigned into operation group and operation with N-Nitrosobis(2-oxopropyl)amine (BOP) group. Those in the latter group were subcutaneously injected BOP weekly for9successive weeks. Meanwhile, hamsters without operation were assigned into blank control group and simple BOP group. The simple BOP group was injected BOP weekly four weeks after the experiment for9weeks. All animals were killed13weeks later. The body weight, spleen index and pathological changes of gallbladder between the groups were compared.(2) Hamster PBR models were established. Four weeks later, the surviving ones were randomly assigned into four groups:the operation group without further intervention, low dose of Coicis Semen oil group (1.25g/kg Coicis Semen oil taken orally per day), middle dose group (2.5g/kg per day) and high dose group (5.0g/kg per day) until sacrifice. All animals were killed28weeks after experiment. The body weight, spleen index and pathological changes of gallbladder between the groups were compared.Results:1.Clinical research(1) Comparison of gallbladder pathological changes between the PBR group (n=24) and the control group (n=30):The thickness of gallbladder wall and inner layer, the bloodflow of gallbladder wall and the positive rate of bacteria culture were not different between the two groups (p>0.05). There was no significance both in frequency of inflammation, hyperplasia, metaplasia and in expression of p53between them (p>0.05) either, while higher presence of dysplasia and higher expression of COX2and Ki-67were seen in PBR group (p<0.05). (2) All patients were enrolled and assigned into three groups:normal SO basal pressure group (n=21), elevated pressure group (n=47) and EST group (n=15). There was no difference in positive rate of bacteria culture, proportion of PBR patients and their BA value between the normal pressure group and the elevated pressure group. However in the latter group, negative correlation was seen between SO pressure and BA (r=-0.43). The BA value in patients with common bile duct wider than15mm was significantly higher than that in patients with common bile duct less than15mm. Furthermore, significant difference in both the proportion of elevated BA patients (P<0.05) and the positive rate of bacteria culture was seen between the intact papilla patients in the former two groups and those in EST group (P<0.05).2. Animal experiments(1) A total of21hamsters were operated.4weeks later,12surviving ones were randomly assigned into operation group and operation with BOP group with6in each group. Other10without operation were assigned into blank control group and simple BOP group,5in each group. When they were killed13weeks later, both body weight and spleen index were significantly higher in the blank control group than in the other three groups (P<0.05).There was no difference in liver function between the groups (P>0.05).Pathological findings:The gallbladder epithelium in both the blank control group and simple BOP group was normal, composed of monolayer cubic or columnar cells. No occurrence of hyperplasia, metaplasia, dysplasia or tumors was seen in these two groups, while in the operation group operation with BOP group, the number of hamsters with hyperplasia was significantly more than the former two groups (P<0.05). The total number with dysplasia or cancer in operation with BOP group was significantly more than that in the blank control group or simple BOP group.(2) The impact of different dose of Coicis Semen oil on PBR models:40hamsters were operated.4weeks later,39surviving ones were randomly assigned into operation group (n=9), low dose group (n=10), middle dose group(n=10) and high dose group(n=10). No difference in body weight was seen between the groups (P>0.05) on sacrifice. The spleen index in Coicis Semen oil groups was significantly higher than that in operation group. So with the moderate or high dose group versus the low dose one(P<0.05). The difference in liver function between the groups was not significant (P>0.05)Pathological findings:The incidence of hyperplasia and dysplasia in both middle and high dose groups were far more less than operation group (P<0.05). The incidence of hyperplasia in middle or high dose was less than that in low dose group. However with further increase in dose, no increasing therapeutic effect was seen. Although the frequency of dysplasia in low dose group was not different from that of the operation group, its degree was seemingly less serious.Conclusions:1. In patients with occult PBR, dysplasia and active proliferation in gallbladder epithelium more commonly occurred which might relate to the progress to malignancy.2. There was no direct relation between the elevated basal SO pressure and PBR, but marked increase in diameter of common bile duct can lead to a rise in BA. Moreover, although EST can bring about PBR and bile bacterial infection, it may facilitate the outflow of the refluent pancreatic juice by relieving cholestasis.3. A PBR model was established successfully and the epithelial lesions such as hyperplasia, metaplasia and dysplasia in gallbladder were induced, laying foundations for further investigation of pathogenesis and chemoprevention of PBR.4. We managed to explain the etiology and pathogenesis of PBR with the theory of traditional Chinese medicine, which was that phlegm-dampness condensation due to stagnation of gallbaladder Qi, as well as deficiency of vial QI. The results of our study proved that Coicis Semen oil had inhibitory effect on gallbladder malignant tendency related to PBR in animal models by decreasing hyperplasia and dysplasia, and also enhanced their immunity. With the feature of safety with little side effects, its long-term application was feasible. Moreover, middle dose and high dose had similar effect for the hamster models.