Role of probiotics in hepatic ischemia‐reperfusion injury

See article in J. Gastroenterol. Hepatol. 2006; 21: 647–656. Hepatic ischemia-reperfusion (IR) injury is associated with temporary clamping of the hepatoduodenal ligament during liver surgery, hypoperfusion shock and graft failure after liver transplantation. Although the precise mechanisms that lead to IR have not been elucidated, substantial evidence exists regarding the participation of diverse factors. These include: (i) loss of calcium homeostasis;1, 2 (ii) reactive oxygen and nitrogen species generation;1, 3 (iii) changes in microcirculation;4 (iv) Kupffer cell activation;5-7 and (v) complement activation.1, 8,9 It has been demonstrated that hepatic IR injury occurs from biphasic responses involving the initial phase and subsequent phase.10 The event occurring in the initial phase is believed to be characterized by the production of reactive oxygen species (ROS), whereas the subsequent phase is typified by the accumulation of inflammatory cells. In the subsequent phase, the accumulation of neutrophils is observed. The activated neutrophils play a pivotal role in the later phase of hepatic IR injury.11 Currently, there is no treatment available for IR injury. Experimental approaches that have been performed in animal models of liver transplantation include antibodies to leukocyte/endothelial cell adhesion sites, inhibitors of Kupffer cell activation, and various antioxidant measures.12 Antioxidants play a fundamental role in the prevention of degenerative diseases. Between the tested antioxidants we can mention alpha-tocopherol,13 alopurinol,14 superoxide dismutase (SOD) and catalase enzymes.15 They act as scavengers of ROS and metal chelators that protect human cells and reduce oxidative damage. There are several promising approaches to prevent the initial phase of hepatic IR injury induced by occlusion of the portal vein for 30 min followed by a 1 h reperfusion mice model. However, they have also been used in the late phase injury mediated by infiltrating neutrophils as a preventive agent against injury.15, 16 The hypothesis that restoring the intestine population with Bifidobacterium and Lactobacillus could ameliorate intestinal epithelial cell mucosal structure to enhance intestinal barrier function and thus decrease intestinal permeability was proposed. In this issue of the Journal, Xing et al.17 speculate that with probiotic pretreatment, plasma endotoxin levels and bacterial translocations could decrease. A probiotic is defined classically as a viable microbial dietary supplement that beneficially affects the host through its effects in the intestinal tract. This definition, however, was initially intended for use with animal feed products. For human nutrition, the following definition has been proposed: ‘a live microbial food ingredient that is beneficial to health’.18 The origins of Xing et al.'s hypothesis deserve further considerations. First, in a previous work, Xing et al.19 demonstrated that hepatic IR injury caused a significant increase in serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were accompanied by elevation of malondialdehyde (MDA) concentration and reduced SOD activity in the liver. These results suggested that liver injury was mediated by ROS. These results are in accordance with those reported by Matsui et al.,20 where ALT levels were significantly increased after reperfusion and immediately before the increase of hepatic myeloperoxidase activity. Second, another interesting point in the Xing et al. study19 was the finding that portal venous congestion could slow down the intestinal flow, causing anoxia in the intestinal wall and several mucosa lesions, and subsequent coordinated motor dysfunction that delays intestinal transit. A third reason for considering that probiotics can be used as a pretreatment in IR liver injury comes from Xing et al.'s findings.19 They found high counts of aerobic and low counts of anaerobic bacterial populations, including Bifidobacteria and Lactobacillium, in the same IR model. Regarding this finding it is important to describe the following concepts. Besides their major functions in digestion and absorption of nutrients, the intestines also act as a barrier to prevent microorganisms and toxins contained within the lumen from spreading to distant tissues and organs. Failure of intestinal barrier function often occurs in many clinical conditions, including hemorrhagic shock, severe burn injury and major surgery for critical illness (e.g. hepatectomy, liver transplantation), resulting in increased intestinal permeability and subsequent translocation of bacteria or/and endotoxin from gut. It is clear that increased gut permeability and bacteria with or without endotoxin translocation plays a key role in the development of severe complications such as systemic inflammatory response syndrome, sepsis, multiple organ dysfunction syndrome and multiple organ failure. Therefore it is important to identify any intestinal injuries following a variety of insults, including liver transplantation. Conversely, D(–)-lactate is a product of bacterial fermentation from the human gastrointestinal tract. Many tissues in mammalians do not produce it and in some their metabolism is very slow. Sun et al.21 investigated the changes of plasma D(–)-lactate and lipopolysaccharide (LPS) levels and their correlation in gut IR. They suggested that the plasma D(–)-lactate level could be a useful marker to evaluate intestinal injury and endotoxemia. D(–)-lactate is produced by some bacteria including Klebsiella, Escherichia coli, Lactobacillus species, and Bacteroides species.21 In this issue, Xing et al.17 performed an elegant study in which they evaluated the effects of the administration of two different strains of Lactobacillus and Bifidobacterium on alterations of microflora, plasma endotoxin levels and liver injury induced by hepatic IR in rats. These authors designed a study that included six animal groups: one control group treated with saline solution only (sham group), one IR-induced group without treatment, one IR-induced group in presence of B. Catenalatum ZYB0401 (BT), one IR-induced group with L. Fermentum ZYL0401 (LACT), one IR-induced group with both bacteria BT/LACT, and one IR-induced group treated with gentamicin. To test whether or not administration of probiotics or gentamicin could prevent IR-induced liver injury, ALT and AST levels were measured. Xing et al. found that AST and ALT levels were increased significantly in the IR-induced group versus the control group (sham group). However, when bacteria and gentamicin were administered, both profoundly reduced serum ALT levels in the BIF and BIF/LACT groups, while AST levels increased significantly in the LACT group. To evaluate the effect of bacteria and gentamicin on endotoxemia, plasma endotoxin levels were tested. Xing et al. proposed that pretreatment in the BIF, LACT and BIF/LACT groups could reduce plasma endotoxin levels significantly, but at the same time gentamicin pretreatment had no effect on endotoxin levels. MDA levels were used as a marker of free radical mediated lipid peroxidation injury. MDA levels in the BIF and BIF/LACT groups were significantly lower than those in the IR-induced group, while the values did not reach significant difference in the LACT group. ROS are usually generated during aerobic cellular metabolism and they are neutralized by diverse antioxidant mechanisms. According to other authors,15 hepatic disorders could be diminished by treatment with SOD. During the IR insult, the mucosa is injured and intestinal permeability is increased. However, in this study supplementation with probiotics had different effects: (i) probiotics induced a curative effect on the IR model, augmenting barrier function of the gut, modifying various bacterial populations as Gram-negative aerobic bacteria (such as E. coli) and partially restoring the intestinal anaerobic bacteria population; and (ii) probiotics induced elevated mucus and secretory immunoglobulin A (IgA) production, which are also considered to be involved in the reinforcement of the intestinal barrier.22 In addition to these findings, promising targets are gastrointestinal functions that are associated with a balanced colonic microflora, that control nutrient bioavailability (ions in particular), that modify gastrointestinal immune activity, or that are mediated by the endocrine activity of the gastrointestinal system. A less well documented hypothesis is the claim of beneficial stimulation of the immune system. The gut, with its massive blood supply, is the immune system's primary contact with the outside world, and gut bacteria seem to play a role in teaching the immune system to differentiate between dangerous invaders and non-hostile changes. Although some studies suggest that probiotics can affect features of the immune system, other reports have shown that these changes are beneficial to health. However, in previous studies it has been observed that infants supplemented with a strain of Lactobacillus casei have enhanced concentrations of circulating IgA, which correlates with shortened duration of rotavirus-induced diarrhea.23 Indeed, it is known that phagocytic activity is involved with natural immunity and that phagocytes are implicated in antibody immune responses acting as antigen-presenting cells. The results of this study are indeed interesting but a note of caution should be considered. In the next few years, many others studies will help to determine the actual beneficial and safety properties of probiotics. One potential problem is that many probiotic strains have genes that allow them to resist antibiotics, which they might pass on to pathogenic bacteria. Studies to test for any direct toxic effects that probiotics may have are needed. In this case a complete genome databank would make it much easer to select species for specific probiotic effects. Foremost is the issue of safety, so further studies to restore liver damage will be necessary to find the safest and best effect with a specific bacterial strain before probiotics can be used in medical practice.