Bone Marrow Mesenchymal Stem Cells Transplantationalleviate Rats Acute Liver Failure By Increase The Expression Of HO-1

Research background:Acute liver failure is a type of clinical syndrome which caused by a variety of factors, along with the liver function abnormal, hepatic encephalopathy, blood coagulation dysfunction, jaundice and multiple organ failure. At present, the mortality rate of medical treatment is still stand at 50-80%; Liver transplantation is the most effective method to deal with acute liver failure, but liver donor shortage, rejection, expensive cost are restrict wide use of liver transplantation. Recent studies shown that mesenchymal stem cell transplantation has achieved remarkable effect in acute liver failure by promoting liver cell regeneration which provides a new direction for the treatment of acute liver failure. Neutrophils are an important part of the innate immune system which could accumulated in necrosis tissues through secreting inflammatory cytokines and oxidative burst, leading to extensive tissue necrosis. Heme oxygenase is the rate limiting enzyme of heme. HO can degraded heme into carbon monoxide, free iron and biliverdin. HO-1 exists widely in body with anti-oxidation, anti-apoptosis, immune adjustment function. Some studies have showed that MSC could increased expression of HO-1 in vivo. But the relationship of HO-1, neutrophil and MSC has not been invested. D-galactosamine and lipopolysaccharide were used to induce rats acute liver failure model. The aim of study is to investigate whether MSC inhibit the accumulation and activity of neutrophil by increase the expression of HO-1.Research Methods:Part one:72 SD rats were randomly divided into control group, acute liver failure (ALF) group and MSC treatment group.0.8g·kg-1 D-gal and 20·gkg-1 LPS were injected intraperitoneally to inducing acute liver failure model. 1×106/ml MSC from SD rat bone marrow were transplanted into blood vessel. The level of ALT, AST and T-bil were detected at time point 24h,48h,72h,120h and 148h after induction. (1) automatic biochemical analyzer was used to detect rat NH3, ALT and AST level (n=14); (2) rats survival rates were detected (n=10); (3)microscopy was used to observe rat liver sections; (4)flow cytometry was used to identify mesenchymal stem cells (CD29, CD34, CD44, CD45, CD90).Part 2:120 SD rats were divided randomly into 5 groups:control group, acute liver failure (ALF) group and MSC treatment (ALF+MSC) group, ALF+MSC+ zinc protoporphyrin (ZnPP, HO-1 antagonist) group and hemin (HO-1 inducer) therapy (ALF+Hemin) group (n=24). Physiological saline was injected in control group; 0.8g·kg-1 D-gal and 20 g·kg-1 LPS were injected intraperitoneally to inducing acute liver failure model; 1×106/ml MSC from SD rat bone marrow were transplanted into blood vessel in ALF+MSC group.50 μmol·kg-1 Znpp was injected into rats in ALF+MSC+ZnPP group.40 μmol·kg-1 hemin was given at 6h after ALF induction in ALF+Hemin group. Automatic biochemical analyzer was used to detect rat NH3, ALT and AST level at 24h,48h,72h,120h and 148h after induction. Immunohistochemical stain was used to detecte neutrophil (MPO), liver regeneration (Ki67), apoptosis (Tunel) and HO-1. The level of HO-1 and liver related pathways (p38, Erk, JNK and NF-κB) were detected with western blot. The oxidative burst of peripheral blood neutrophil was detected by flow cytometry (DHR123). The variation of MDA, MPO activity and HO-1 activity were detected. The level of IL1β、TNFα、IFNy were detected by ELISA.Results:Part one:ALF model have been successfully established with D-gal and LPS. The level of AST、ALT and T-bil increased in acute liver failure; The rats serum NH3, AST, T-bil and ALT level decreased significantly (P<0.05) in MSC transplantation group. Liver histology injury decreased after MSC treatment. The mortality in ALF group was 80% which was damped into 40% in MSC transplantation group.Part two:Western blot and immunohistochemical staining showed that HO-1 levels in the ALF+MSC group and ALF+MSC+ ZnPP group and ALF+Hemin group increased significantly (P< 0.05); HO-1 activity raised significantly in the ALF+MSC group and ALF+Hemin group, and ZnPP inhibited HO-1 activity (P< 0.05).In ALF group, the level of blood NH3, AST, ALT, IL1β, TNFα, IFNy, the number of MPO positive cells, Tunel positive cells incresed compared with Comtrol group (P<0.05); The level of MDA, MPO and peripheral blood neutrophil oxidative burst activity appeared some trend. While those results were reversed by MSC transplantation or Hemin treatment; In ALF+MSC group, the number of Ki67 positive cells increased (P<0.05); At the same time the flow cytometry showed that peripheral blood neutrophil oxidative burst increased in ALF group (P< 0.05), the level of MDA and MPO activity were elevated (P<0.05); Liver pathological damage, blood NH3, AST and ALT level was decreased (P< 0.05); MSC or Hemin treatment decreased the expression of IL1β、TNFα、IFNy, while Znpp partialy abolished BMSC protective effects. Western results show that NF kappa B pathway is involved in the HO-1 mediated protective effect.Conclusions:(1) MSC transplantation could ameliorate ALF induced inflamtory; (2) neutrophil and PMN associated oxidative burst play a important role in acute liver failure; (3) MSC Increase Expression of Heme Oxygenase-1 Leading to Anti-inflammatory Activity in Treatment of Acute Liver Failure.