The Study On The Interaction Between The Bacteria And The Host With Biliary Tract Infection

PrefacePrimary intrahepatic brown pigment stones are the most common cause of biliary tract infection in our country.At the presence of brown pigment stones,most of patients are asymptomatic with bacteria in bile.Under the condition of acute biliary tract obstruction and high pressure of bilary tract,patients with intrahepatic brown pigment stones usually suffer from the acute cholangitis of severe type with charcot triad.Previous studies have shown the pathogens which caused bilary tract infection were combination of Gram-negative bacteria,Gram-positive bacteria and anareo bacteria.Among of them,Gram-negative bacteria such as E.coli was the most common pathogen.As far,more sthdies about bacteriology of bialiry tract infection focused on bacterial culture of bile or stones,drug sensitive tests and some enzyme secreted by them et al.The detail characteristics of bacteria such as fimbriae of E.coli and the mechanism of the interactions between bacteria and host defense system are not well known.It's important for us to understand the role of bacteria and the host defense system playing in bilary tract infection.Thus,we can know much deeply about the origin of bacteria in biliary tract,the pathway they invad into biliary tract from gut,the site of bacteria act with mucosa of biliay tract and the innate and adaptive host defense system against bacteria and their products.We can treat and prevent the severe bilary tract infection by blocking some points on the way of interaction between bacteria and the host defense system.In this study,we examined the the expression of P fimbriae and papG in E.coli isolated from infect biliary tract and compared it with that in E.coli in normal flora to learn the adhere characteristics of E.coli originated from bilary tract.We detected the the expression of NF-κB,TLR-4 and TLR-2 in mutltiple organs in acute cholangitis of severe type to investigate the response of host defense system against the pathogens. Materials and Method1.Materials1.1 34 E.coli isolated from bile of patients with biliary tract infection obtained from the microbiological lab of Shengjing hospital(China Medical University)and 8 E.coli isolated from stool of healthy volunteers were prepared.MRHA was perfomed to examin the expression of P fimbirae and papGⅠ,papGⅡ,papGⅢwere detected by RT-PCR.1.2 Wistar mouse,160-220g,male or female.Animal models of ACST were made as experiment group,n=36;Animal models of single biliary tract obstruction were made as control group,n=182.Specimen collection2.1 Blood,liver,lung and enterol tissues were prepared at 1h,6h and 24h after sugery2.2 Blood was kept in-20℃after centrifugation;Part of tissues were conserved in -80℃for RT-PCR and Western-Blot.The other part were fixed in 5%paraform for pathology and immumo histology chemistry.3.Examination objects3.1 The expression of p fimbirae:exmained the the expression of p fimbirae of 42 E.coli by MRHA.Observed by eyes at time or overnight in 4℃,if the agglutination was≥"++",it was considered as positive result.3.2 The detection of papG gene:the expression of papGⅠ,papGⅡ,papGⅢwas perfomed by PCR3.3 The expression of Cytokines in blood:TNF-α,IL-10,IL-4 level in blood in different time point were detected by ELISA3.4 Pathology examination:the pathology examination of liver,lung and enterol were performed by HE stain.3.5 The expression of NF-κB protein:distribution of NF-κB was observed in liver,lung and enterol tissue of ACST or control group by IHC.Quantative expression of NF-κB in liver was perfomed by Western-Blot.3.6 The expression of NF-κB mRNA:NF-κB mRNA in different tissues were examined by RT-PCR3.7 The expression of TLR-2,TLR-4 protein:distribution of TLR-2,TLR-4 was observed in liver,lung and enterol tissue of ACST or control group by IHC.Quantative expression of TLR-2,TLR-4 was perfomed by Westem-Blot.3.8 The expression of TLR-2,TLR-4 mRNA:TLR-2,TLR-4 mRNA in different tissues were examined by RT-PCRResult1.The expression of p fimbirae:the expression of p fimbirae in E.coli originated from bilairy tract is 47.06%(16/34),significantly higher than that in E.coli originated from gut flora 0%(0/8).2.The detection of papG gene:the expression ofpapGⅠ,papGⅡ,papGⅢin E.coli originated from bilairy tract were 67.65%,44.12%,82.35%,respectively.Among of them,papGⅢis significantly higher than that in E.coli originated from gut flora 37.5%(p<0.05).3.The expression of Cytokines in blood:TNF-α,IL-4 level increased 1h after sugery in ACST mouse and reached peak at 6h,significantly higher than that in control group.They decreased at 24h.However,IL-10 reached peak at 1h,then decareased with the time.4.Pathology examination:the common bile duct of ACST rats expanded obviously, reached to 3-5mm indiameter.The liver was congested and swollen,and had many petechiaes,furthermore microabscesses could also be seen on liver surface of severe cases,and the number and size were not equal.Under light microscope,it was found that sinus hepaticus and interlobular duct expanded obviously,and there were inflammatory cell infiltration in sinus hepaticus,around of hepatocyte and portal areas. The alignment of hepatocyte was scattered and chaotic,and the hepatocyte showed significant fatty degeneration.There were also punctiform or fragmentis necrosis with inflammatory cell infiltration.The lung tissue paramental swollen and inflammatory cell infiltration.,even the abscess were observed at ACST rats.No significant change was observed in enerol.5.The expression of NF-κB protein:the positive cells were observed in hapatocyte,lung epithelium and enterol mucosa cell in ACST rat by IHC.Low expression,even if psositive,was observed in control group.NF-κB protein upregulation was observed in experimental group.It's significantly higher than that in control group at 6h(11805.67±2410.69vs9888.67±244.85 p=0.026).6.The expression of NF-κB mRNA:NF-κB mRNA in liver in ACST increased at 1h and reached peak at 6h,which is significantly higher than that in control group. (1.95±1.67 vs 0.75±0.22,p=0.001).It decreased at 24h.It's similar in lung tissue and enterol tissue.In lung 6h(1.69±0.36 vs 0.58±0.19,p=0.033),in enterol tissue 1h (0.38±0.16 vs 0.25±0.04,p=0.001)and 6h(0.86±0.34 vs 0.57±0.05,p=0.003), 24h(0.63±0.24 vs 0.25±0.05,p=0.011).7.The expression of TLR-2,TLR-4 protein:the positive cells were observed in hapatocyte,lung epithelium and enterol mucosa cell in ACST rat by IHC.Low expression,even if positive,was observed in control group.Both of them located in cytoplasm.TLR-2 in liver increased with the time and reached peak at 6h (11278.50±1066.73 vs 8434±470.57,p=0.03),slightly decreased at 24h (10964.67±883.33 vs 8288.83±212.53,p=0.008).TLR4 increased at 1h(8613.33±1999.22 vs 6406.50±224.98,p=0.05)and reached peak at 6h(11209.33±1271.09 vs 7287.33±240.78,p=0.12).8.The expression of TLR-2,TLR-4 mRNA:TLR-4mRNA in liver and enterol increased at 1h and reached at 6h,decreased at 24h.In liver 6h(14.80±2.58 vs 9.30±1.08 p=0.002),in enterol 1h(8.25±2.14 vs 5.01±0.68 p=0.05),24h (7.89±3.14 vs 6.96±1.07 p=0.001).TLR-4mRNA in lung increased at1 h,slightly decreased at 6h,(6.98±1.87 vs 5.49±0.91 p=0.043),slightly increased at 24h (8.02±1.32 vs 4.67±0.53 p=0.037).TLR-2mRNA in liver,lung and enterol is similar.Inceased at 1h(liver 1.51±0.83 vs 1.18±0.25 p=0.002),(lung 10.13±5.20 vs 5.54±1.12 p=0.001),reached at 6h(lung 11.59±5.11 vs 6.89±1.07 p=0.001),(enterol 4.78±1.83 vs 2.05±1.40,p=0.002),decrease at 24h(enterol 4.52±1.30 vs 2.59±1.36, p=0.004).Conclusion 1.The expression of p fimbirae in E.coli originated from bilairy tract is significantly higher than that in E.coli originated from gut flora.2.The expression of papGⅢin E.coli originated from bilairy tract is significantly higher than that in E.coli originated from gut flora3.P fimbirae and papGⅢplayed an important role in the bilairy trac infecrtion caused by E.coli4.NF-κB activation and upregulation in liver\lung\enterol in ACST rats correlated with TNF-a,IL-4 level in serum was important in MODS caused by ACST5.TLR-2,TLR-4 in liver\lung\enterol in ACST rats played a crutial role in the development of SIRS and MODS...