Characterization Of Intestinal Microbial Communities And Effect Of Intestinal Microbiota Alteration On Hepatic Damage In Rats With Acute Rejection After Liver Transplantation

Orthotopic liver transplantation (OLT) is a life-saving procedure for patients with end-stage liver disease such as acute or chronic hepatic failure and cirrhosis, hepatic malignancies and some rare metabolic liver-based disorders. Nevertheless, morbidity and mortality due to rejection and infectious complications remain as major problems following transplantation. Deaths are often related to sepsis, and most infectious episodes are associated with rejection. Bacterial translocation (BT)has been considered an important contributor to the development of infection and sepsis in surgical and critically ill patients. A few data are available on the effects of acute rejection (AR) on intestinal microbiota and bacterial translocation in rats after OLT. Whether and how AR after OLT change the intestinal microbial communities? Whether this change is related with BT? We speculate that alteration of intestinal microbiota plays an important role in the pathological process of liver injury in rats with AR. Whether the structure alteration of the intestinal microbiota (by antibiotic and probiotic) influences the liver function in rats with AR after the OLT and what is the possible mechanism?There was few data available on these questions. So we carried out the following two study here. Firstly, we assessed the effects of acute rejection on intestinal microbiota and the relationship with bacterial translocation in rats with OLT. Then, we analyzed whether the structure alteration of the intestinal microbioa (by antibiotic and probiotic) influence the liver function, intestinal inflammation and immune function of the body in rats with AR after the OLT.We characterized the dynamics of intestinal microbial communities in rats before and after OLT (including the Sham group, Isograft group and Allograft group) using PCR-denaturing gradient gel electrophoresis (DGGE), real-time quantitative PCR (RT-qPCR) and454prosequencing. Bacterial translocation to host organs and plasma endotoxin were also determined. Dynamic analysis of DGGE fingerprints showed that the microbiota structure of animals in the3groups was similar before the operation. But significant alterations in the composition of fecal microbiota in Allograft group were observed at1and2weeks after the OLT. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) analysis of454prosequencing revealed a similar structure before the operation, and significant segregation at2weeks after the OLT in Allograft group when compared with the other two groups at the phylum level. The proportion of phylum Firmicutes was significantly reduced in Allograft group at1week after the OLT. The proportion of phylum Firmicutes was significantly reduced, while Proteobacteria and Bacteroidetes were enriched in Allograft group at2weeks after the OLT. Lachnospiraceae and Ruminococcaceae were decreased, while potential pathogens such as Enterobacteriaceae and Bacteroidaceae were prevalent in Allograft group at the family level at2weeks after the OLT. Results from RT-qPCR confirmed that Enterobacteriaceae and Bacteroidaceae significantly increased at2weeks after the OLT. Alteration in gut microbiota was associated with the elevation of plasma endotoxin and a higher rate of BT to liver in rats with acute rejection.Dynamic analysis of DGGE fingerprints showed significant alterations in the composition of fecal microbiota in probiotic group and antibiotic group after using probiotic and antibiotic for one week after the OLT. The diversity of fecal microbiota significantly reduced in antibiotic group. Molecular analysis of the microbiota showed that antibiotic treatment has an impact on the colonization of the rat gut that is site dependent. The effect of antibiotics on the ileum and colon mucosa was more profound at2weeks after the OLT. The liver injury in probiotic group was significantly reduced compared with the other two groups. To further explain the mechanism, we examine the changes of body and gut immune function after disturbance of gut flora by antibiotic and probiotic under conditions of acute rejection. Peripheral blood mononuclear cell (PBMC) and intestinal lamina propria lymphocytes were isolated2weeks after the OLT. Peripheral CD4/CD8ratio, peripheral and intestinal Treg cells were analyzed by flow cytometry. Serum concentrations of the corresponding cytokines were measured by Bio-Plex. Results showed that intestinal CD4/CD8ratio and serum cytokines IL-2was decreased while Treg cell population was increased markedly in probiotic group. However, there was no difference in the peripheral Treg cell and CD4/CD8ratio between the three groups.Combination of above two parts of studies about the characterization of intestinal microbial communities and effect of probiotic on hepatic damage in rats with acute rejection after liver transplantation, we draw the following conclusions:(1) Rats with acute rejection exhibited elevation of plasma endotoxin and higher rate of BT. and these were associated with structure changes in the gut microbiota which dominant by overgrowth of potential pathogens such as Enterobacteriaceae and Bacteroidaceae, while Lachnospiraceae and Ruminococcaceae were reduced in Allograft group at the family level at2weeks after the OLT. It suggested that Lachnospiraceae and Ruminococcaceae would be potential probiotic for control of infection after liver transplantation.(2) The diversity of fecal microbiota significantly reduced in antibiotic group. Antibiotic treatment has an impact on the colonization of the rat gut that is site dependent. The effect of antibiotics on the ileum and colon mucosa was more profound at2weeks after the OLT.(3)The intestinal CD4/CD8ratio and serum cytokines IL-2was decreased while Treg cell population was increased markedly in probiotic group, thereby reducing liver injury in rats with acute rejection after liver transplantation.