ABSTRACT
Oxidative stress plays a major part in the pathogenesis of drug-induced liver injury. Yet, overcoming it with other xenobiotics impose additional risks. In this study, we consider the use of natural-occurring and purified Vitamin E analogs as hepatoprotective agents. Vitamin E is well-known for its intrinsic antioxidant property even though the differential effect of specific analogs of tocopherol (TP) and tocotrienol (T3) is still not ascertained. This study investigates the protective effect of T3 analogs (α-, δ-, γ-) in comparison with α-TP followed by assessing the underlying mechanisms of the cytoprotective T3 analog(s) in two xenobiotics-induced liver injury models using (1) acetaminophen (APAP)- and (2) hydrogen peroxide (H2O2). Both α-TP and α-T3 exerted cytoprotective effects while only lower concentration of γ-T3 was effective in inhibiting both toxicants induced injury. α-TP/α-T3 protected hepatocytes from APAP and H2O2-induced liver injury through arresting free radicals and inhibiting oxidative stress (inhibition of reactive oxygen species, lipid peroxidation and mitochondrial permeability transition). There was also demonstrable inhibition of the apoptotic pathway (inhibition of caspse-3 activity and overexpression of Bcl-XL), accompanied with an induction of liver regeneration (PCNA and NF-kB). The cellular uptake of α-T3 was higher than α-TP at the same treatment dosage after 24h. Overall, α-T3 seems to be a more potent hepatoprotective analog among the tocotrienols and α-TP at the same in vitro treatment dosage. In summary, these results suggest that α-TP/α-T3 elicit hepatoprotective effects against toxicants-induced damage mainly through activation of antioxidant responses at an early stage to prevent the exacerbation of injury.
Subject(s)
Antioxidants/pharmacology , Hepatocytes/drug effects , Mitochondria/drug effects , Protective Agents/pharmacology , Tocopherols/pharmacology , Vitamin E/pharmacology , Acetaminophen/antagonists & inhibitors , Acetaminophen/toxicity , Animals , Caspase 3/genetics , Caspase 3/metabolism , Cell Line, Transformed , Cell Survival/drug effects , Gene Expression Regulation , Hepatocytes/cytology , Hepatocytes/metabolism , Hydrogen Peroxide/antagonists & inhibitors , Hydrogen Peroxide/toxicity , Interleukin-6/genetics , Interleukin-6/metabolism , Lipid Peroxidation/drug effects , Mice , Mitochondria/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress/drug effects , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
INTRODUCTION: Mesenchymal stem cell-conditioned medium (MSC-CM) has been shown to have protective effects against various cellular-injury models. This mechanism of protection, however, has yet to be elucidated. Recently, exosomes were identified as the active component in MSC-CM. The aim of this study is to investigate the effect of MSC-derived exosomes in an established carbon tetrachloride (CCl4)-induced liver injury mouse model. This potential effect is then validated by using in vitro xenobiotic-induced liver-injury assays: (1) acetaminophen (APAP)- and (2) hydrogen peroxide (H2O2)-induced liver injury. METHODS: The exosomes were introduced concurrent with CCl4 into a mouse model through different routes of administration. Biochemical analysis was performed based on the blood and liver tissues. Subsequently the exosomes were treated in APAP and H2O2-toxicants with in vitro models. Cell viability was measured, and biomarkers indicative of regenerative and oxidative biochemical responses were determined to probe the mechanism of any hepatoprotective activity observed. RESULTS: In contrast to mice treated with phosphate-buffered saline, CCl4 injury in mice was attenuated by concurrent-treatment exosomes, and characterized by an increase in hepatocyte proliferation, as demonstrated with proliferating cell nuclear antigen (PCNA) elevation. Significantly higher cell viability was demonstrated in the exosomes-treated group compared with the non-exosome-treated group in both injury models. The higher survival rate was associated with upregulation of the priming-phase genes during liver regeneration, which subsequently led to higher expression of proliferation proteins (PCNA and cyclin D1) in the exosomes-treated group. Exosomes also inhibited the APAP- and H2O2-induced hepatocytes apoptosis through upregulation of Bcl-xL protein expression. However, exosomes do not mitigate hepatocyte injury via modulation of oxidative stress. CONCLUSIONS: In summary, these results suggest that MSC-derived exosomes can elicit hepatoprotective effects against toxicants-induced injury, mainly through activation of proliferative and regenerative responses.
Subject(s)
Chemical and Drug Induced Liver Injury/pathology , Exosomes , Hepatocytes/cytology , Liver Regeneration/physiology , Mesenchymal Stem Cells/metabolism , Acetaminophen/toxicity , Animals , Blotting, Western , Carbon Tetrachloride/toxicity , Cell Proliferation , Chemical and Drug Induced Liver Injury/etiology , Disease Models, Animal , Humans , Hydrogen Peroxide/toxicity , Male , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Heat shock protein 27 (Hsp27) is a molecular chaperone protein which regulates cell apoptosis by interacting directly with the caspase activation components in the apoptotic pathways. With the assistance of the Tat protein transduction domain we directly delivered the Hsp27 into the myocardial cell line, H9c2 and demonstrate that this protein can reverse hypoxia-induced apoptosis of cells. In order to characterize the contribution of Hsp27 in blocking the two major apoptotic pathways operational within cells, we exposed H9c2 cells to staurosporine and cobalt chloride, agents that induce mitochondria-dependent (intrinsic) and -independent (extrinsic) pathways of apoptosis in cells respectively. The Tat-Hsp27 fusion protein showed a greater propensity to inhibit the effect induced by the cobalt chloride treatment. These data suggest that the Hsp27 predominantly exerts its protective effect by interfering with the components of the extrinsic pathway of apoptosis.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Caspases/metabolism , Gene Products, tat/metabolism , HSP27 Heat-Shock Proteins/immunology , Myocardium/metabolism , Recombinant Fusion Proteins/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/immunology , Caspases/genetics , Caspases/immunology , Cell Line , Cobalt/pharmacology , Cytoprotection , Gene Products, tat/genetics , Gene Products, tat/immunology , HSP27 Heat-Shock Proteins/genetics , HSP27 Heat-Shock Proteins/metabolism , Humans , Hypoxia/metabolism , Mitochondria, Heart/drug effects , Mitochondria, Heart/metabolism , Myocardium/immunology , Myocardium/pathology , Recombinant Fusion Proteins/immunology , Staurosporine/pharmacology , Transduction, GeneticABSTRACT
Myocardial infarction causes a high rate of morbidity and mortality worldwide, and heat shock proteins as molecular chaperones have been attractive targets for protecting cardiomyoblasts under environmental stimuli. In this study, in order to enhance the penetration of heat shock protein 27 (HSP27) across cell membranes, we fused HSP27 with transcriptional activator (TAT) derived from human immunodeficiency virus (HIV) as a protein transduction domain (PTD). We loaded the fusion protein (TAT-HSP27) into microsphere/hydrogel combination delivery systems to control the release behavior for prolonged time periods. We found that the release behavior of TAT-HSP27 was able to be controlled by varying the ratio of PLGA microspheres and alginate hydrogels. Indeed, the released fusion protein maintained its bioactivity and could recover the proliferation of cardiomyoblasts cultured under hypoxic conditions. This approach to controlling the release behavior of TAT-HSP27 using microsphere/hydrogel combination delivery systems may be useful for treating myocardial infarction in a minimally invasive manner.
