ABSTRACT:Objective To investigate changes in the neutrophils in rats with D-galactosamine (D-GalN)-induced acute liver failure (ALF)and to explore the therapeutic effect of interventions treatment of neutrophils on ALF.Methods Liver function,the expressions of inflammatory cytokines TNF-αand IL-1β,and the changes of neutrophils in the peripheral blood and the liver were observed in rats with D-GalN (intraperitoneal injection)-induced ALF.SD rats were randomly divided into three groups when treated with intervention of neutrophils:control group,ALF group (intraperitoneal injection of D-GalN),and treatment group (intravenous injection of anti-PMN serum via tail vein 24 h before modeling).Biochemical analysis was used to detect serum ALT,AST, TBIL and blood ammonia.Hematology analyzer was applied to analyze the number and percentage of peripheral blood neutrophils.The number of neutrophils in the liver was evaluated by immunohistochemistry.Liver RT-PCR was adopted to detect the mRNA expression of inflammatory cytokines TNF-αand IL-1β.Results We found that 6 h after D-GalN injection,serum ALT,AST,TBIL and blood ammonia in ALF rats were significantly increased (P <0.05).The mRNA expression levels of inflammatory cytokines TNF-αand IL-1βin the liver reached the peak at 6 h after modeling (P <0.001),and it was still notably higher at 24 h than before modeling (P <0.001 ).The number and percentage of peripheral blood neutrophils and the number of neutrophils in the liver were all markedly increased 12 h after modeling (P <0.001 ),and the increase continued at least until 24 h (P <0.001 ).24 h after intravenous injection of anti-PMN serum via tail vein,ALF rats had a distinct decrease in the number of peripheral blood neutrophils and neutrophils in the liver 24 h after modeling (P <0.001).Meanwhile,serum ALT,AST,TBIL and blood ammonia were all greatly decreased compared with those in ALF group (P <0.05);a significant reduction of hepatocyte apoptosis was observed.Also,the expressions of TNF-α and IL-1β in the liver were remarkably decreased after treatment (P <0.05).Conclusion Neutrophils accumulated in peripheral blood and liver of rats with D-GalN-induced ALF.The treatment of anti-PMN serum may have a therapeutic effect on liver function and immune microenvironment in ALF rats.

Objective To establish a porcine model of liver failure after different percent hepatectomy.Methods The porcine models of liver failure 75%,85%,95% hepatectomy were developed and the living conditions and survival time were recorded.The blood samples of pre-surgery,post-hepatectomy d1,d3,d5 and post-hepatectomy 1 week,2 weeks,and 3 weeks were collected for hepatic function analysis.Histological examination of liver tissues was performed using HE staining.Liver injury histology was interpreted and scored in the terminal samples.Results The average survival time of pigs with post-hepatectomy liver failure after 75%,85%,95% hepatectomy was 19.0±5.6 days,17.3±5.5 days,1.3±1.5 days,respectively.Their pathological scores were 5.67±0.52,8.17±0.82 and 8.50±0.71,respectively.With the increase of percent hepatic resection,the incidence of hepatic failure was increasing.ALT,AST,ALP,LDH and TBA were dramatically increased in the pigs after 85% hepatectomy.Conclusions The pig model of acute liver failure by 85% hepatectomy is successfully established,which can cause typical acute liver failure in Bama miniature pigs.

Objective To investigate the safety and therapeutic effects ot a novel multi-layer flat-plate bioartificial liver (BAL) for patients with liver failure.Methods Thirty-eight patients with liver failure from Dec.2010 to Dec.2011 were treated with a novel BAL based on multi-layer flatplate bioreactor and the co-cultured cells of the porcine hepatocytes and mesenchymal stem cells.A total of 48 treatments was performed,4 h each time.The clinical signs and symptoms,liver function,ammonia,coagulation function and complete blood count were evaluated before,during and after the treatment.DNA in the collected PBMCs was extracted for PCR with PERV specific primers and the porcine specific primer Sus scrofa cytochrome B.The RT activity was detected as well.Levels of xenoantibodies (IgG,IgM) were determined by using ELISA kit. Titers of complement were quantified by CH50 kit.Results All treatment procedures were completed successfully without any adverse reaction. All samples presented negative PERV DNA and RT activity. The levels of antibodies were similar before and after treatment.Treatment was associated with a temporary decline in levels of complement,and then the levels were recovered quickly.The clinical symptoms such as acratia,anorexia and abdominal distension were improved.The stage of hepatic encephalopathy in 16 patients was decreased. The liver function and ammonia was reduced disproportionately. Seven patients in all were bridged to liver transplantation,2 patients died and 2 patients gave up the treatment,and the others were turned better.After the outcome judgment according to the standard developed by the Artificial Liver Group,and Chinese Association of Infectious and Parasitic Diseases,there were 9 patients with clinical healing,25 patients with improvement and 4 patients with no effect,and the cure-improvement rate was 89.5％.Conclusion The novel multi-layer flat-plate BAL could be used as a safe and effective therapy for patients with liver failure.

Objective To explore the therapeutic effects of soluble cytokines secreted by mesenchymal stem cells (MSCs) on acute liver failure (ALF).Methods MSCs isolated from Sprague-Dawley rats were determined by FACS analysis.Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray.SD rats were divided into 3 groups:(1) ALF + dulbecco's modified eagle medium (DMEM) group:1 ml DMEM was injected into SD rats after D-Gal administration;(2) ALF + MSCs group:1 ml MSCs (1 × 106) was injected into SD rats after D-Gal administration;(3) ALF + MSCs-CM group:1 ml MSCs-CM was injected into SD rats after D-Gal administration.Biochemical indicators,survival rate,histology and inflammatory factors were studied.Exogenous recombinant rat IL-10,antirat IL-10 antibody and AG490 (STAT3 signaling pathway inhibitor) were administrated to explore the therapeutic mechanism of MSCs-CM.Results The respective serum biochemical indexes of ALF + DMEM group,ALF + MSCs group,and ALF + MSCs-CM group were:ALT (1 709.8 ± 372.1,865.5 ± 52.8,964.7 ± 414.6 U/L),AST (4234.0 ± 807.3,2440.8 ± 511.9,2739.8 ± 587.3 U/L),andTBil (79.3 ± 10.9,43.8 ± 7.0,61.2 ± 6.7 μg/L).The survival rates of the three groups were 10.0％,80.0％,and 70.0％,respectively.The levels of inflammatory factors in each group were IFN-γ (69.8 ± 4.7,46.4 ± 4.3,54.6 ± 2.4pg/ml),IL-1β (58.5 ± 7.6,40.5 ± 6.9,44.1 ± 6.0pg/ml),IL-6 (71.9 ± 16.1,38.4 ± 7.7,45.3 ± 9.0),and IL-10 (38.3 ± 6.0,75.4 ± 11.1,59.6 ± 11.9 pg/ml).Protein microarray results suggested that MSCs-CM expresses a variety of inflammatory-related cytokines,with IL-10 levels being most pronounced.IL-10 (ALT 1 126.9 ± 419.3 U/L,AST2370.8 ± 561.2 U/L) alone significantly reduced transaminase levels compared with ALF group (ALT 1 709.8 ± 372.1 U/L,AST 4234.0 ± 807.3 U/L),while anti-IL-10 antibody (ALT 1 568.5 ± 325.4 U/L,AST4043.7 ± 819.0 U/L) neutralized the therapeutic effect of MSCs-CM (ALT 964.7 ± 414.6 U/L,AST 2 739.8 ± 587.3 U/L).IL-10 could significantly increase the level of pSTAT3 in ALF rats (0.93 ± 0.03 vs 0.68 ± 0.01),while STAT3 inhibitor AG490 (0.84 ± 0.04) could decrease the expression of pSTAT3 and reverse the therapeutic effect of IL-10.Conclusion The factors released by MSCs,especially IL-10,have the potential therapeutic effect on ALF,and STAT3 signaling pathway may mediate the anti-inflammation effects of IL-10.

Objective To investigate the therapeutic effect of bone marrow mesenchymal stromal cell (BMSC) transplantation on D-galactosamine-induced acute liver failure in Sprague-Dawley (SD) rats,as well as the mechanism of neutrophils in this process.Methods A total of 39 male SD rats were divided into control group (8 rats,intraperitoneal injection of isotonic saline),model group (10 rats,intraperitoneal injection of D-galactosamine),solvent group (9 rats,tail vein injection of isotonic saline at 2 hours after intraperitoneal injection of D-galactosamine),and treatment group (12 rats,tail vein injection of MSCs at 2 hours after intraperitoneal injection of D-galactosamine).The rats were sacrificed at 24 hours after the model of D-galactosamine-induced acute liver failure was established,and the blood and liver tissue were harvested.The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and total bilirubin (TBil) were measured,and blood analysis was performed to measure the number and percentage of neutrophils in peripheral blood.Immunofluorescence assay was used to measure the expression of the neutrophil marker Ly6g in the liver,the myeloperoxidase (MPO) kit was used to measure the activity of MPO in liver,and RT-PCR was used to measure the mRNA expression of inflammatory cytokines and chemokines in the liver,i.e.,tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β),interferon-γ (IFN-γ),interleukin-10 (IL-10),CXC chemokine ligand 1 (CXCL1),and CXC chemokine ligand 2 (CXCL2).Another 64 male SD rats were randomly divided into groups,and the survival rates of rats in each group were observed for 7 days.The independent samples t-test was used for comparison between any two groups (Levene homogeneity test of variance,and the corrected t-test was used for a P value of ＜ 0.05),and the log-rank test was used for comparison of survival rates between any two groups.Results At 24 hours after acute liver failure was induced by D-galactosamine in the SD rats,there were significant increases in the liver function parameters (ALT:2884.1±541.0 U/L vs 45.4±11.0 U/L,P ＜ 0.001;AST:3634.9±755.9 U/L vs 143.9±23.7 U/L,P ＜ 0.001;TBil:44.4±8.4 μmmol/L vs 0.9±0.2 μmmol/L,P ＜ 0.001) and the number and percentage of peripheral blood neutrophils [number:(4.7±1.1)×109 vs (1.4±0.4)× 109,P ＜0.001;percentage:44.9％±8.0％ vs 18.3％±4.4％,P ＜ 0.001].A large number of neutrophils aggregated in the liver tissue,and there were significant increases in the MPO activity (4.72±1.09 U/g vs 1.13±0.24 U/g,P ＜ 0.001),inflammatory cytokines,and chemokines.Compared with the model group,the treatment group showed significant improvements in liver function (ALT:1 823.9a389.2 U/L vs 2 884.1±541.0 U/L,P ＜ 0.001;AST:2173.0±567.3U/L vs 3634.9±755.9 U/L,P ＜ 0.001;TBil:30.9±6.5 μmmol/L vs 44.4±8.4 μmmol/L,P ＜ 0.001) and survival rate (50％ vs 12.5％,P=0.023).Meanwhile,the treatment group also showed significant reductions in the number and percentage of peripheral blood neutrophils [number:(3.5±1.0)× 109 vs (4.7±1.1)×109,P =0.012;percentage:35.9％±8.9％ vs 44.9％±8.0％,P =0.021],number of neutrophils in the liver,and MPO activity (3.52±1.03 U/g vs 4.72±1.09 U/g,P =0.040),as well as significantly inhibited expression of inflammatory cytokines and chemokines (TNF-α:2.458±0.762 vs 3.778±1.046,P =0.005;IL-1β:2.498±0.547 vs 4.065 ± 0.953,P =0.002;IFN-γ:3.977±1.039 vs 5.418±1.255,P =0.025;IL-10:6.056±1.542 vs 3.368±0.952,P=0.001;CXCL1:7.988±1.911 vs 10.366±1.239,P =0.010;CXCL2:3.441±1.005 vs 4.847±1.113,P=0.019).Conclusion BMSC transplantation has a therapeutic effect on D-galactosamine-induced acute liver failure in rats,and this process is accompanied by reduced aggregation and activity of neutrophils in peripheral blood and liver.Inflammatory cytokines and chemokines may be involved in the mechanism of regulation of these two aspects.

Objective To study the synergetic effect and possible mechanism of transplanting mesenchymal stem cells (MSCs) in combination with interleukin-1 receptor antagonist (IL-1Ra) on acute liver failure (ALF).Methods MSCs transplantation combined with IL-1Ra was used for a swine model of ALF induced by 85％ total hepatectomy.The living conditions,blood samples and survival time were recorded or collected for analysis of hepatic function.Liver injury histology was analyzed.Hepatic cell regeneration and apoptosis were studied by immunohistochemistry staining of Ki67 and TUNELassays respectively.The expression levels of AKT and NF-κB were analyzed by Western blotting.Results The difference on the survival time between the model group and combined therapy group was statistically significant (P ＜ 0.05).Combined therapy displayed improvement not only in the serum biochemical conditions but also in the serum inflammatory cytokines.Furthermore,the observed hepatic histopathological score was significantly less compared to model group.In addition,the combined therapy group significantly inhibited the liver cell apoptosis and increased hepatic cell regeneration.Finally,a significant increase in AKT expression and decrease of NF-κB expression (P ＜ 0.05) were observed,which was consistent with their important roles in liver regeneration.Conclusion The combined therapy displayed a synergistic effect on liver regeneration,by promoting restoration and reconstruction of ALF,through regulation of inflammation and apoptosis signaling network.