| Objective: The incidence of gallstone in the world about 10% to 15%,which can lead to inflammation,biliary obstruction and even the formation of tumors,seriously affecting people’s health.With the deepening of modern medical understanding of the mechanism of gallstone formation and complex physiological functions of the gallbladder,and the development and progress of endoscopic and minimally invasive surgery,the method of preserving the gallbladder while only taking the stone treatment has been gradually realized.In recent years,we carried out endoscopic ultrasonography-assisted precision positioning technology,with metal stent,the wall between the stomach/duodenal wall and the gallbladder to establish fistula,and through this fistula to remove gallstone under the endoscopic vision.However,it takes 8 to 10 days to establish a stable fistula through the scaffold,which may result in the juice,food and gastrointestinal bacteria in the upper digestive tract entering the gall bladder due to the connection of the digestive tract and the gallbladder Gallbladder mucosal injury,changes in the composition of gallbladder microorganisms,resulting in impaired gallbladder function.To verify these possible risks,we conducted animal experiments to study the changes in the composition of the microorganism in gallbladders and the effect on the recurrence of stones after gastrointestinal-gallbladder stent placements.Methods: Ten healthy Bama miniature pigs weighing 30 ± 5kg were selected.The gallbladder contraction test showed that the gallbladder functioned well.The gallbladder was punctured with EUS guided gallbladder before gallbladder anastomosis.After 2 weeks,performing gallbladder cleaning to simulate the process of removing the stones.Closing upper digestive tract wall fistula by the hemostatic clip.Another two weeks later,the animals were executed by lethal injection for bile.The collected bile for the following experiments: 1,changes in bacterial community using dilution plate counting bacteria culture,different types of bacteria identified by mass spectrometry.2,16 S rDNA was extracted from bile for high-throughput sequencing,comparing the composition of microorganisms before and after the twogroups.3,β-glucuronidase activity in bile Determination by colorimetry.4.Detection of bacterial active substances such as phospholipase A2(PLA2),Helicobacter pylori cytotoxin-related protein A(Hp-CagA),mucin 5B(MUC5B)and mucins 5AC(MUC5AC)in bile were all performed by ELISA.Result:1.All experimental animals underwent endoscopic ultrasound-guided gastrostastic anastomosis and simulated gallbladder lithotomy.There were no obvious adverse reactions,normal appetite and activity,normal bowel movements,and no significant body weight loss.2.A total of 3 bile samples were cultured before and after surgery.No viable bacteria were found in 3 cases before surgery.Bacterial culture results of the bile samples were positive after 2 weeks of stent removal.The positive rate of E.coli was 100%.About6.09-7.61 cfu/ml was counted on blood agar plates,about 6.05-7.19 log10cfu/ml was counted on MacConkey plates,followed by 5.30 log10cfu/ml of Staphylococcus epidermidis and 5.40 log10cfu/ml of Enterococcus faecalis in one case of bile samples.3.Analysis of 10 pairs of Clean Reads of bile samples before and after surgery using Uparse software for clustering,the default consistency of 97% of the sequences clustered into OTUs,the comparison of the species classification of each sample or each group,and screening of special attention Compare species.The highest abundance in the preoperative group was Phyllobacterium(31.53%),followed by Streptococcus(13.20%),Enterococcus(10.03%),Acinetobacter spp.(5.35%),Escherichia-Shigella(4.82%),Pseudomonas(2.90%),Bacteroides(1.06%),Fusobacterium(0.14%),Bilophila(0.01%),and Terrisporobacter(0.01%).The abundance of Escherichia-Shigella in group B was significantly higher than that in group A(39.27%),and the genus of the second highest abundance was: Bacteroides(16.20%),shuttle Fusobacterium(6.90%),Chrysosporium(3.74%),Terrisporobacter(2.10%),and the abundance of other species decreased.4.The group composition of the bile samples in the preoperative group was higher than that in the group 2 weeks after the stent removal.Beta diversity index analysis showed no statistically significant differences between the preoperative group and thetwo-week post-stent removal group sample composition(T-test P = 0.750,Wilcox rank sum test P = 0.860).5.The top 6 species that were significantly increased in the postoperative group after screening by Metastat analysis were: Zavarzinia,Anaeroplasma,Nitrosospira,BS11_gut_group,Ruminofilibacter and Salinimicrobium.These bacteria come from the soil and the rumen of ruminants,proving that gastro-cystic anastomosis stents can cause stomach bacteria to enter the gallbladder.6.Bacterial metabolite activity was compared between 10 pairs of bile samples before and after surgery.The preoperative bile exogenous β-glucuronidase activity(24.53± 25.86)U/mgprot and postoperative(10.56 ± 8.39)U/mgprot were not different.Statistically significant(P>0.05);Bile phospholipase A2 concentration before surgery(324.10±52.62)ng/m L,Postoperatively(270.65±44.25)ng/mL,the difference was statistically significant(P<0.05);Bile Hp-CagA concentration was(29.53 ± 32.38)ng/mL,postoperative(27.03 ± 43.64)ng/mL,the difference was not statistically significant(P>0.05);bile MUC 5B concentration was(6.87 ± 0.78)ng/m L before operation.After operation,the difference was statistically significant(5.19 ± 0.98)ng/mL(P<0.05);the preoperative bile MUC5 AC concentration was(103.98±50.51)ng/mL,postoperatively(133.83±21.99)ng/mL,difference No statistical significance(P>0.05).Conclusion: After two weeks of EUS-guided upper gastrointestinal-Gallbladder anastomosis,the content of bacteria in the gallbladder increased and the original colony structure changed.Although there was a change in the bile microenvironment,the main substances causing stone-related activity of bacteria did not increase,Therefore,the impact of surgery on the recurrence of gallstone disease smaller.EUS-guided upper gastrointestinal-gallbladder anastomosis in the treatment of gallstones,gallbladder inflammation to relieve the treatment effect is worthy of promotion. |
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