- Top of page
- Supporting Information
LIVER DISEASES ARE highly prevalent in the population worldwide. Currently, despite different alternatives that have been tested, the standard treatment for end-stage chronic liver disease that is available and effective is whole liver transplantation. However, liver transplantation has serious limitations such as donor scarcity, immunological incompatibilities, high cost, and significant morbidity and mortality associated with the procedure.[1-3] Additionally, considerable long-term side-effects have been reported.[4-7] Given the inherent limitations of this treatment, alternative therapies are urgently needed.
In recent years, cell-based therapy, especially therapy using bone marrow cells (BMC), has emerged as an alternative to improve damaged liver function. An increasing number of studies have been published showing evidence of therapeutic effects of BMC in liver diseases,[8-15] including clinical trials worldwide.[16-20]
The interest in this particular cell niche comes from previous reports showing the presence of donor-derived cells in the liver of bone marrow transplant recipients.[21, 22] This observation, which has been proven in animal models,[23, 24] showed potential cross-talk between BMC and the liver under certain conditions. Among the different cell types found in bone marrow, mesenchymal stem cells (MSC) have shown promising results in tissue regeneration.[8, 12, 14, 25] These cells can be easily isolated from the patient, cultured, expanded and used as an autologous cell-based therapy.
Although promising results have been shown, important questions remain. For example, no consensus exists about the mechanisms of liver repair by BMC infusion. This topic constitutes one of the most debated issues in regenerative medicine.
Recently, oxidative stress has been shown to be an important factor in liver diseases such as liver fibrosis, cirrhosis, viral hepatitis, hepatocellular carcinoma and others.[26-30] Oxidative stress is partly generated by reactive oxygen species (ROS), which are produced by different pathways such as NAD(P)H oxidases, xenobiotic metabolism, mitochondrial leakage and cytochrome P450 activity, which lead to hepatocyte damage through lipid peroxidation and alkylation of proteins, nucleic acids and lipids.[31-33] Although the liver itself has an efficient antioxidant defense system, sometimes this system is not sufficient to repair the damage and/or an imbalance exists between oxidative stress elimination and production. MSC were recently reported to have an antioxidant ability that may contribute to oxidative stress resolution. Importantly, NF-E2-related factor 2 (Nrf2) has emerged as a crucial transcription factor that is capable of inducing a large array of enzymes involved in oxidative stress resolution.[35, 36] Maintenance of the cellular redox balance by Nrf2 has multiple activation pathways and has been shown to be essential in combating many inflammatory diseases.[37-43] Some molecules such as all-trans retinoic acid (ATRA) and tert-butylhydroquinone (t-BHQ) have shown the ability to significantly reduce (ATRA) or induce (t-BHQ) Nrf2 functions, which modify the expression of antioxidant response element (ARE)-driven genes.[44, 45]
Thioacetamide (TAA) is one the most popular chemical toxins used worldwide to generate experimental liver injury.[46, 47] Its toxicity results from its biotransformation by a mixed-function oxidase system (e.g. cytochrome P450 enzymes and FAD monooxygenases), which leads to the formation of reactive metabolites including ROS.[48-53] ROS production resulting from TAA administration is related to the consequences of oxidative damage including lipid peroxidation.[54, 55]
Given the above concerns and the recent evidence for the effectiveness of cell-based therapy in liver diseases involving oxidative stress, we hypothesized that MSC could ameliorate the deleterious effects of TAA-induced oxidative stress injury in liver. In this study, we tested the ability of canine MSC (cMSC) to overcome TAA-induced oxidative stress in vitro and verified whether these cells could protect against oxidative stress damage in isolated hepatocytes. In addition, we evaluated whether cMSC could reduce the effects of TAA-induced chronic injury in vivo. An important note is that few studies have used cells derived from medium-sized animals. Results from such studies will be important for supporting new clinical trials.
- Top of page
- Supporting Information
MURINE EXPERIMENTAL MODELS are commonly used to test new therapies for hepatic diseases, including cell-based therapy using bone marrow-derived cells, which have shown promising results.[8-15] Among the different cell populations found in bone marrow, MSC have shown beneficial effects against liver disease.[8, 12, 14, 25, 60] Furthermore, MSC have advantages such as multiple tissue sources, fast proliferation, possible use in autologous transplantation and in vitro manipulation. Also, MSC were recently shown to promote an antioxidant response in injured liver.
Despite good results in basic studies and clinical trials,[61-63] the mechanism of action of these cells is still being discussed. Recently, many studies have linked oxidative stress and development of liver diseases such as viral hepatitis, cirrhosis, hepatocellular carcinoma and others.[26-29] Here, we examined whether the antioxidant potential demonstrated by MSC has effects in reducing TAA-induced liver injury.
Thioacetamide is a drug that is widely used in animal models. Because biotransformation of TAA produces oxidative damage associated with liver injury and this drug is usually used for systemic infusion, we examined if MSC, which are also usually injected systemically, could provide resistance to the toxic effects produced by TAA. Surprisingly, rather than resistance alone, cMSC showed a high level of tolerance to TAA (Fig. 2). Additionally, when cMSC were pretreated with ATRA or t-BHQ, they showed opposite responses regarding cytotoxicity, viability and ROS accumulation (Fig. 2). Considering that ATRA inhibits and t-BHQ induces Nrf2 effects both in vitro and in vivo,[44, 45] these results indicate that cMSC have high antioxidant activity in vitro and suggest that the Nrf2 pathway may be involved in this process. Consistent with this hypothesis, Mohammadzadeh et al. recently showed that induced overexpression of Nrf2 by MSC was able to promote reduction of cell death in hypoxia, serum deprivation and oxidative stress conditions. In this study, MSC with transient overexpression of Nrf2 presented better cell viability and reduced apoptosis levels. Moreover, Gorbunov et al. showed that MSC treated with lipopolysaccharide, which induces inflammatory responses including release of ROS, induce a number of adaptive responses including induction and nuclear translocation of redox response elements such as nuclear factor-κB and Nrf2. They suggested that the prosurvival pathways that are activated in MSC in vitro could be a part of an adaptive response employed by stromal cells under injury conditions.
A direct and specific effect of ROS in viability was ruled out using H2O2 and NAC in cultures. As expected, these molecules increased (H2O2) and decreased (NAC) intracellular ROS, but no direct relationship between viability and ROS levels was seen at the time points tested (data not shown). Additionally, to assess whether cMSC could potentially prevent oxidative stress in liver cells, we utilized a co-culture model with murine hepatocytes and cMSC. In this experiment, we found a lower ROS level in co-cultured murine hepatocytes treated with TAA (Fig. 3), suggesting a hepatoprotective effect of cMSC via antioxidant activity. Using a mouse primer for Nrf2 with no cross-reactivity against canine samples in silico, we verified the higher amount of mRNA in co-cultured hepatocytes (Fig. S1). However, unexpectedly, monocultured hepatocytes showed higher ROS levels when TAA was absent from the culture medium, suggesting that hepatocytes have a mechanism similar to cMSC in the presence of TAA. The underlying mechanisms are now under investigation.
Our above in vitro results motivated us to test cell therapy using cMSC in TAA-induced liver injury in NOD/SCID mice. In chronic TAA-induced injury, the animals that received cMSC infusions by tail vein showed better results for the biochemical parameters. The serum injury markers (ALT, AST and LDH) were reduced with successive cell infusions, suggesting protection of hepatocytes from necrosis and apoptosis (Fig. 4). Because ALT and AST are enzymes that reveal hepatocyte damage, these results strongly support our in vitro findings showing that cMSC have hepatoprotective effects against TAA-induced injury. We cannot rule out the possibility that infused cMSC may act systemically to aid the liver in its recovery. Consistent with our results and considering the possibility that Nrf2 may be involved in this process, Xu et al. demonstrated a delayed ALT decrease in sera from Nrf2-knockout mice after treatment with hepatotoxin. Because Nrf2 is crucial for induction of expression of a wide range of antioxidant genes, antioxidant activity may be essential for promoting liver regeneration.
As already discussed, oxidative stress plays an important role in liver injury, and some authors have recently demonstrated that cell-based therapy can be an effective treatment. Recently, Cho et al. have shown that MSC have an antioxidant potential to ameliorate acute liver injury induced by carbon tetrachloride. In a murine model of carbon tetrachloride-induced acute liver injury, they found increased Nrf2 activity and lower ROS, ALT and AST levels in animals treated with syngeneic MSC.
Okuyama et al. reported that transgenic mice with high expression of thioredoxin, a small redox-active protein with antioxidant effects, showed not only ameliorated liver injury but also decreased liver fibrosis.[67, 68] Consistent with this result, we showed that the possible antioxidant activity of cMSC reduced necrotic and inflammatory areas (Fig. 4d,e) and fibrosis levels by measuring of different parameters (Fig. 5). We also found higher concentration of matrix metalloproteinase 9 in liver tissues harvested from cell-treated group what can in part explain the results found in fibrosis analyses (Fig. S2).
In this present study, we confirmed that animals in the cell-treated group had better redox homeostasis by showing higher total serum antioxidant activity and lower lipid peroxidation in liver tissues (Fig. 6). The cMSC infusions seemed to sustain normal overall total antioxidant activity in these animals, which may explain the decreased lipid peroxidation (Fig. 6b), serum injury markers (Fig. 4a–c) and histological findings in vivo (Figs 4, 5). At this juncture, we can clearly see that cMSC can act efficiently in combating oxidative stress in liver.
As far as we know, this study is the first to use a complete approach (in vitro + in vivo) to evaluate the role of antioxidant activity in ameliorating liver injury using cells from a medium-sized animal. These results reveal potent antioxidant activity and hepatoprotective effects of cMSC in vitro and in vivo and support more studies examining the antioxidant activity of stem cells to combat liver diseases.
In conclusion, we showed that cMSC can protect hepatocytes by reducing ROS damage induced by TAA both in vivo and in vitro. These results suggest a potential for MSC treatment in several hepatic diseases.