Characterization Of Intestinal Bifidobacterium Species In Rats With Acute Rejection After Liver Transplantation

ObjectiveThe aim of this study was using DGGE and RT-PCR to characterize the composition of intestinal Bifidobacterium in rats with acute rejection after liver transplantation.Methods24Lewis male rats and42BN rats were divided to five groups:①Control group (n=6):The control group was subjected to the surgical procedures described below without Orthotopic liver transplantation (OLT);②Isograft group (n=6):OLT was carried out between BN rats;③Allograft group (n=6):OLT was carried out from Lewis to BN rats;④Antibiotic group (n=6):Allograft group with2ml gentamycin by intragastric administration (4×10∧A4U)⑤Probiotic group (n=6):Allograft group with2ml bifico by intragastric administration (2.0×10∧8CFU/ml).Transplantation was performed using the modified kamada’s two cuff technique to build acute rejection model in rats. Fresh feces were collected at1day before OLT (T1), and1week after OLT (T2),2weeks after OLT (T3). On the15th day after the OLT, all animals were sacrificed after halothane anesthesia. Blood was collected from the portal vein for measurement of liver function and plasma endotoxin. The tissue samples from the liver were fixed in10%neutral formalin for later histological study by light microscopy. Nested-PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) was applied to analyze the diversity of the genus Bifidobacterium and then the most common bands in the profiles were identified by cloning and the predominantly observed species were selected for enumeration by real-time PCR.Results 1) PCR-DGGE fingerprint indicated that, gut Bifidobacterium structures of the rats in the four groups showed no significant difference at T1(1day before the OLT) except Probiotic group. However, the diversity of Probiotic group significant reduced contrast with Allograft group at T1. Contrast with Control group and Isograft group, the diversity of Allograft group significant reduced at T2. Contrast with Allograft group, the diversity of Antibiotc group increased but Probiotic reduced at T2. At T3, the diversity of gut Bifidobacterium structures of the rats in the three groups (Control、 Isograft and Allograft group) showed no significant difference. Contrast with Allograft group, the diversity of Antibiotc group increased but Probiotic reduced.2) At Tl, the copies number of every species Bifidobacterium is similar among the Control, Isograft and Allograft group. The copies number of B. spp, B. longum, B. catenulatum and B. bifidum were induced in Antibiotic group, while Bifidobacterium spp, B. longum, B. catenulatum were increased in Probiotic group. At T2, the copies number of B. detium were increased in Isograft group and Allograft group, while Bifidobacterium spp, B. catenulatum and B. animalis were less commonly detected in Allograft group than in Control group. The copies number of Bifidobacterium spp, B. longum, B. catenulatum and B. animalis were induced in Antibiotic group than in Allograft group, but Bifidobacterium spp, B. longum and B. animalis were increased in Probiotic group. At T3, the copies number of B. detium were increased in Isograft group and Allograft group, while B. catenulatum and B. animalis were less commonly detected in Allograft group than in Control group. The copies number of Bifidobacterium spp, B. longum, B. catenulatum and B. infant were induced in Antibiotic group than in Allograft group, but Bifidobacterium spp, B. longum, B. catenulatum and B. animalis were increased in Probiotic group.Conclusion1) The structure of Bifidobacterium in rat with acute rejection after liver transplantation was changed, mainly manifested as the diversity of Bifidobacterium reduced, and Bifidobacterium spp, B.longum, B. catenulatum reduced but B. dentium increased.2) The diversity of fecal Bifidobacterium significantly reduced, meanwhile the copies of Bifidobacterium spp, B. logum, B. catenulatum, B. animalis reduced in antibiotic group. Conversely, the diversity of fecal Bifidobacterium significantly increased, meanwhile the copies of Bifidobacterium spp, B. logum, B. catenulatum, B. animalis increased in Probiotic group.