Therapeutic effect of adipose tissue-mesenchymal stem cells transplantation in rats with acute liver failure

Abstract

Background and aims. Acute liver failure (ALF) is a life-threatening condition in which a sudden and massive liver injury can determines encephalopathy, coagulopathy and eventually multiorgan failure leading to death. The main cause of ALF in industrialized countries is acetaminophen (APAP) intoxication. When pharmacological treatment fails, orthotopic liver transplantation (OLT) is the standard of choice; however, the shortage of donor organs for OLT makes the need of finding alternative therapeutic options. In this study, we aimed at identifying if the transplantion of ASCs may exert a therapeutic effect in APAP-ALF and if ASCs transplantation may affect oxidative stress and JNK activation, which are key events involved in the pathogenesis of APAP-ALF. Materials and methods. ASCs were isolated from abdominal subcutaneous tissue of a healthy donor undergoing ernioplasty and were transfected with a green fluorescent protein (GFP) before transplantation.Twelve Sprague-Dawley rats, weighting 200 gr, were administrated 300 mg/Kg of APAP i.p. Four rats served as healthy controls. After 2 hour from APAP dose, six rats were transplanted 200.000 ASCs, suspended in saline, via the caudal vein, whereas six animals received only the vehicle. After 24 hours from APAP administration, rats were sacrificed and blood, liver and brain samples were collected. Plasma AST, ALT, ammonia and prothrombin time were measured; brain water content was also determined. Histological damage was evaluated by using hematoxylin-eosine staining. Indirect immunofluorescence for the identification of GFP+ cells was performed on frozen liver samples. Monoclonal antibodies for human CK-8 and vimentin were used for immunohistochemistry. Liver isoprostanes and 8-hydroxyguanosine (8-OHG) was determined by ELISA. Hepatic nitrite/nitrate was measured colorimetrically. JNK expression was evaluated by western blot. Results. Rats with APAP intoxication presented a marked increase of AST (P<0.01), ALT (P<0.01), ammonia (P<0.05) and prothrombin time (P<0.01), and increased brain water content (P<0.05) as compared to healthy animals. Transplantation of ASCs decreased AST (P<0.01), ALT (P<0.01), ammonia (P<0.05) and prothrombin time (P<0.01) to the levels observed in control rats. Consistently, brain water content of transplanted rats was similar to healthy animals and these animals did not present clinical encephalopathy as well. Liver sections of rats with APAP-ALF showed diffuse vacuolar degeneration indicating mitochondrial damage and scattered necroinflammatory foci. In rats transplanted with ASCs, liver injury was almost absent. GFP-staining demonstrated that ASCs engrafted into the liver, where they localized in the parenchyma and around sinusoids. Most cells presented a shaped fibroblast-like morphology, but few cells had a round epithelial-like aspect. Consistently, most ASCs were vimentin-positive, whereas only few cells were CK-8 positive. Rats with APAP-ALF presented a marked increase of liver isoprostanes (P<0.01), 8-OHG (P<0.01) and nitrite/nitrates (P<0.01)as compared with control animals. Tranplantation of ASCs decreased liver isoprostanes (P<0.01), 8-OHG (P<0.01) and nitrite/nitrates (P<0.01) to the levels of control rats. Finally, the expression of phospho-JNK was markedly increased in rats with APAP-ALF (P<0.01) as compared to control rats, whereas ASCs transplantation restored pospho-JNK expression to levels of healthy animals. Conclusion. In this study we demonstrated for the first time that ASCs transplantation is effective in treating APAP-induced ALF and encephalopathy in rats. ASCs engraft in the injuried liver, where exerts potent antioxidant effects and inhibits JNK activation. Further studies are warranted in order to elucidate additional molecular pathways involved in the therapeutic effect of ASCs transplantation in ALF. In our opinion, our findings provide a strong rationale for the potential use of ASCs in the clinical management of patients with ALF.