Interaction Between Hepatic Graft Injury And Gut Microbiota

Currently, liver transplantation (LT) has been accepted as an established therapy for various end-stage liver diseases including liver cirrhosis, liver cancer and liver failure for more than three decades, and also aradical therapy for primary liver cancer.Nowadays, the survival rate of patients following LT approximately is 90% for 1 year and 75% for 5 years.According to the statistics from United Network for Organ Sharing (UNOS), there have been 128 894 patients following liver transplantationonly in United States by August 8,2014. In China, there have been a total of 26600 patients with liver transplantations, and China has become a second country with the most patients with liver transplantation, secondly to United States. More than 300 thousands patients are waiting for liver transplantation in China, and China has become a country that has been a most potential demander worldwide.In "recent years, intestinal microbiota has been considered as the most important micro-ecosystem and equivalent to a major metabolic "organ" that has a symbiotic relationship with the body. The human intestine accommodates nearly 400 different species of bacteria and comprises 1013 to 1014 microorganisms whose collective genome contains at least 100 times as large as human beings genome. The vast majority of these microbes (10 to 100 trillion) inhabit human gastrointestinal tract, play a crucial role on human physiology and nutrition, and are essential for human survival and life. Although intestinal microbiota plays crucial roles on various beneficial functions including digestion of complex plant polysaccharides, maturity of initial immune system and resistance to the invasion of enteric pathogens, it has been associated with a spectrum of human diseases, such as inflammatory bowel diseases, liver cirrhosis and hepatocellular carcinoma.The imbalance of intestinal microbiota can promote the onset and development of variety of diseases, including liver diseases. Due to the intimate anatomical and functional relationship between the gut and the liver, intestinal organisms and their metabolites interact with the host and play an important role in liver inflammation, chronic liver fibrosis, liver cirrhosis and cancer progression through liver-gut circulation and microbiota-liver axis.Hepatic injury or diseases such as hepatic I/R injury, alcoholic steatohepatitis and liver cirrhosis always follow alterations in intestinal permeability and microbial composition. Meanwhile, under disease condition, intestinal microbial imbalance can aggravate liver injury and chronic inflammatory disease, ultimately promoting hepatocellular carcinoma.During LT, the recipients inevitably suffer from the dual ischemia-reperfusion (I/R) injuries from liver and intestine. These injuries are closely associated with many postoperative complications, and they likely lead to endogenous infections, increasing postoperative mortality rate. Meanwhile, hepatic graft I/R injury and rejection injury are the two main causes of graft dysfunction. Both "liver-gut" axis and "microbiota-liver axis" closely link liver function with gut function, thus hepatic graft injury is closely associated with gut microbial alterations. Therefore, studying the interaction between hepatic graft I/R injury and gut microbial alterations, as well as the relationship between acute rejection and gut microbial alteration, has a significant influence on improving graft function, alleviating rejection response, decreasing infection rate, reducing chronic graft dysfunction, improving long-term survival and life quality for patients following liver transplantation.In this study, we focus on the interactions between hepatic graft injuries and gut microbial alterations, divided two parts to detailedly illustrate that the improvement of hepatic graft ischemia-reperfusion injury promotes gut microbial restoration following liver transplantation; and that Interaction between development of acute rejection in hepatic graft and alterations of gut microbiota following liver transplantation.Part 1 Improvement of hepatic graft ischemia-reperfusion injury promotes gut microbial restoration following liver transplantationObjective:Ischemia-reperfusion (I/R) injury is associated with intestinal microbial dysbiosis. The "gut-liver axis" closely links gut function and liver function in health and disease. Ischemic preconditioning (IPC) has been proven to reduce I/R injury in the surgery. This study aims to explore the effect of IPC on intestinal microbiota and to analyze characteristics of microbial structure shift following liver transplantation (LT).Methods:The LT animal models of liver and gut IPC were established. Hepatic graft function was assessed by histology and serum ALT/AST. Intestinal barrier function was evaluated by mucosal ultrastructure, serum endotoxin, bacterial translocation, fecal sIgA content and serum TNF-α. Intestinal bacterial populations were determined by quantitative PCR. Microbial composition was characterized by DGGE and specific bacterial species were determined by sequence analysis.Principal Findings:Liver IPC improved hepatic graft function expressed as ameliorated graft structure and reduced ALT/AST levels. After administration of liver IPC, intestinal mucosal ultrastructure was improved, serum endotoxin and bacterial translocation mildly decreased, fecal sIgA content increased, and serum TNF-α decreased. Moreover, liver IPC promoted microbial restorations mainly through restoring Bifidobacterium spp., Clostridium clusters Ⅺ and Clostridium cluster ⅩⅣab on bacterial genus level. DGGE profiles indicated that liver IPC increased microbial diversity and species richness, and cluster analysis demonstrated that microbial structures were similar and clustered together between the NC group and Liver-IPC group. Furthermore, the phylogenetic tree of band sequences showed key bacteria corresponding to 10 key band classes of microbial structure shift induced by liver IPC, most of which were assigned to Bacteroidetes phylum.Conclusion:Liver IPC can not only improve hepatic graft function and intestinal barrier function, but also promote restorations of intestinal microbiota following LT, which may further benefit hepatic graft by positive feedback of the "gut-liver axis".Part 2 Interaction between development of acute rejection in hepatic graft and alterations of gut microbiota following liver transplantationObjective:Acute rejection (AR) remains a life-threatening complication after orthotopic liver transplantation (OLT) and there are few available diagnostic biomarkers clinically for AR. This study aims to identify intestinal microbial profile and explore potential application of microbial profile as a biomarker for AR after OLT.Methods:The OLT models in rats were established. Hepatic graft histology, ultra-structure, function and intestinal barrier function were tested; Ileocecal contents were collected for intestinal microbial analysis.Results:Hepatic graft suffered from the I/R injury on day 1, initial AR on day 3 and severe AR on day 7 after OLT. RT-qPCR results showed that genus Faecalibacterium prausnitzii and Lactobacillus were decreased, while Clostridium bolteae was increased during AR. Notably, cluster analysis of DGGE profiles showed the 7AR and 3AR groups clustered together with 73.4% similarity, suggesting that intestinal microbiota was more sensitive than hepatic function in responding to AR. Microbial diversity and species richness were decreased duringAR. Phylogenetic tree analysis showed that most of decreased key bacteria belonged to phylum Firmicutes, while increased key bacteria belonged to phylum Bacteroidetes. Moreover, intestinal microvilli loss and tight junction damage were noted, and intestinal barrier dysfunction during AR presented a decrease of fecal sIgA and increase of blood bacteremia, endotoxin and TNF-a.Conclusion:We dynamically detailed intestinal microbial characterization and found a high sensitivity of microbial change, suggesting that intestinal microbial variation may predict AR in early phase after OLT and become an assistant therapeutic target to improve rejection after OLT.