Objective To investigate liver regeneration after transplantation of microencapsulated hepatocytes in rats with acute liver failure (ALF). Methods ALF rat model was established by intraperitoneal injection of D-galactosamine (D-GalN). After 18 h, rats were randomized into control group ( Ⅰ ), free hepatoeyte transplantation group ( Ⅱ ) and the microencapsulated hepatecyte transplantation group (Ⅲ). Six rats for each group were randomly selected and sacrificed at 6, 12, 24, 36, 48, 72, 120, 168 and 240 h after ALF induced and blood samples from inferior vena cava were collected. Liver functions were tested in blood samples, and the expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. Results Ten-day survival rates of 3 groups were 26.7% (4/15), 40.0% (6/15) and 73. 3% (11/15), respectively (x2 = 9. 349,P = 0. 009). Survival rate of group Ⅲ was significantly higher than that of group Ⅰ and Ⅱ. Levels of ALT and AST in each group increased significantly at 6 h after ALF induced, and peaked between 48 ～ 72 h. Levels of ALT and AST in group Ⅱ and Ⅲ declined from 36 h, which was more significant in group Ⅲ. Tbil levels in group Ⅰ gradually increased after ALF induced and peaked at 72 h. Tbil in group Ⅱ and Ⅲ declined from 48 h, which was more markedly in group Ⅲ. In normal rats, the expression of PCNA protein was almost negative, but it was strongly expressed in ALF rats and peaked at 48 h. The number of positive cells in group Ⅲ was higher than that in group Ⅰ and Ⅱ, and the differences were of statistical signifieance. Conclusion The transplantation of microencapsulated hepatocytes can promote the regeneration of liver, and it can improve the liver function and prognosis in rats with ALF.

Objective To study the expression of vascular endothelial growth factor(VEGF)in hepatic tissue of acute liver failure(ALF)rats after transplantation of microencapsulated hepatocytes.Methods The ALF model of rats was induced by D-galactosamine(D-Gal)1400 mg/kg.Then the ALF rats were divided into three groups 18 h after injection of D-Gal:ALF model group,free hepatocyte transplantation group,and microencapsulated hepatocyte transplantation group,50 rats in each group,respectively.Each group was divided into 7 subgroups,and the rats were sacrificed at 24,36,48,72,120,168 and 240 h respectively after injection of D-Gal.The expression of proliferating cell nuclear antigen(PCNA) and VEGF in liver tissue of ALF rats was detected by immunohistochemistry method.VEGF mRNA in liver tissue was detected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR).Comparison among groups was done by one way ANOVA.Results Compared with ALF model group,the expression of PCNA protein and in free hepatocyet transplantation group in microencapsulated hepatocyte transplantation group began to increase obviously at 36 h after injection of D-Gal(F=26.26,P<0.05).Compare to that of ALF model and free hepatocyte transplantation group,the expression of VEGF protein in microencapsulated hepatocyte transplantation group increased significantly at 36 h after injection of D-Gal(F=25.44,P<0.05),which decreased slower and was still higher at 240 h after injection of D-Gal(F=220.25,P<0.05).Compared with ALF model group,the expression of VEGF mRNA in free hepatocyte transplantation group increased obviously at 24 h(48.0±1.9 vs 56.7±9.1;F=3.54,P<0.05).And that in microencapsulated hepatocyte transplantation group was higher than free hepatocyte transplantation group at 48 h(100.7±1.9 vs 94.5±1.4;F=47.82,P<0.05),which was still significantly higher than free hepatocyte transplantation group at 240 h after injection of D-Gal(F=21.70,P<0.05). Conclusion Transplantation of microencapsulated hepatocytes could upregulate the expression of VEGF in liver tissues and promote the regeneration of hepatocytes in ALF rats.

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.

Objective To investigate the effect of rapamycin(RPM) on the gene expression of Toll-like receptor (TLR)-4 by inhibiting Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in rats with acute liver failure (ALF).Methods The ALF model of rats was induced by D-galactosamine (D-GalN) 800 mg/kg and lipopolysaccharide (LPS) 8 μg.The blood and liver tissue samples were collected at 2,6,12,24 and 48 h after D-GalN/LPS injection.SD rats were randomly divided into three groups:normal control group (n=6),ALF group (n=30) and RPM treatment group (n=30).The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at each time points were tested.The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were tested by enzyme-linked immunosorbent assay (ELISA),and the mRNA expressions of TLR-4 in liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR).The data analysis was performed using t test.Results The levels of TNF-α and IL-6 were increased markedly 2 h after GalN/LPS iniection and peaked at 6 h.Treatment with RPM could significantly inhibit the levels of TNF-α and IL-6.The TLR-4 mRNA expression levels in liver tissues at 6,12,24,48 h in ALF group were 0.745±0.135,1.092±0.175,1.115±0.152 and 0.812±0.13,respectively;those in RPM group were 0.545±0.118,0.798±0.124,0.857±0.109 and 0.595±0.152,respectively.The differences between two groups at each time points were all significant (t=2.726,3.349,3.382 and 2.567,respectively,all P＜0.05).In addition,TLR-4 mRNA expression was positively correlated with ALT and AST levels(r=0.722 and 0.712,respectively,both P＜0.01).Conclusions Inhibition of JAK/STAT pathway can markedly down regulate TLR-4 gene expression in liver tissue of rats with ALF,which indicates that JAK/STAT pathway may be involved in the regulation of TLR-4 mRNA expression during ALF.

Objective To investigate the effect of endotoxin tolerance(ETT) on the rats with acute liver failure (ALF) and Janus kinase/signal transducer and activator of transcription (JAK/ STAT) signal transduction pathway. Methods S-D male rats were divided randomly into three groups: control group, ALF model group anti ETT group, lipopolysacharide (LPS) 0.1 mg/kg(ETT groups) or saline(ALF groups)was administered by five consecutive intraperitoneal injections at 24 h intervals. On the sixth day all animals were treated with intraperitoneal injection of D-galactosamine (D-GaIN) 800 mg/kg and LPS 8 μg/rat. The blood was gathered from portal vein and livers were take out before and 2, 6,12, 24 and 48 h after the injection of D-GalN/LPS. Liver function and liver histopathology of each group were observed. The gene expressions of STAT3 and SOCS3 in the livers were measured by semi-quantitative reverse transcriptionpolymerase chain reaction(RT-PCR). The tumor necrosing factor(TNF)-α and interleukin(IL)-6 level were determined by enzyme-linked immunosorbent assay(ELISA). The data analysis was performed by using t test. Results The histological damage in the liver tissue was significantly milder in ETT group compared to ALF group, but still severer than that of control group (TNF-α: 6 h: t=2. 670,P<0.05,12 h: t=3. 604,24 h: t=6. 426, 48 h: t=3. 274,all P<0.01;IL-6:6 h: t=2. 333,P<0. 05,12 h: t=4. 266, 24 h: t=8. 063,48 h: t=4. 177, all P<0. 01). The gene expressions of STAT3 and SOCS3 in the liver were increased significantly in ALF group, however, in ETT group the expression of STAT3 was inhibited while the expression of SOCS3 was increased and much higher than those in ALF groups. Conclusions LPS pretreatment can induce ETT in rats, which will reduce the expression of TNF-α and IL-6. In ETT groups, the gene expression of STAT3 is lower while the gene expression of SOCS3 is higher compared to those in ALF groups. It suggests that JAK/STAT pathway may involve in mechanisms of ETT.

Objective To investigate the changes of fractalkine (FKN) in rat model of acute liver failure (ALF) and the role of FKN in liver inflammatory injury.Methods SD rats were divided into tWO groups:6 in normal group and 36 in model group.D-galactosamine(D-Gal) was used to induce ALF in model group.The sera and hepatic tissue samples were collected at 12,24,48,72,120 andl68 h.After D-Gal injection.FKN mRNA and nuclear factor(NF)-kB mRNA in hepatic tissue samples were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 12 h were(208.3±43.5)U/L and (375.2±117.3)lJ/L,respectively,which were both significantly higher than those in normal group[(31.8±2.9)U/L and (90.8±3.1)U/L](t=-9.912 and-5.935,respectively,both P<0.01);the levels of ALT and AST peaked at 72 h after D-Gal injection.The levels of FKN mRNA(O.086±0.009)in model group at 12 h were significantly higher than those (O.044±0.009) in normal group(t=-7.999.P<0.01),and peaked at 72 h (O.333±0.033),then decreased obviously at 120 h. The levels of NF-KB mRNA in the liver of normal rats were very little;and the levels in model group were increased gradually over time,then peaked at 72 h (O.583±0.i01,t=-12.607,P<0.01).FKN mRNA and NF0kB mRNA were positively correlated (r=0.760,P<0.01).Conclusion The FKN expression may play all important role in liver inflammatory injury in rat model of acute liver failure, which could provide a new approach for ALF therapy.

Objective To isolate and culture splenic CD11clow CD45RBhigh dendritic cells (DC) derived from endotoxin tolerance (ET) mice and investigate its biological characterization.Methods Mice weighed 20 to 25 gram were completely randomized into two groups including ET group and control group with 6 each.ET mice were modeled by intraperitoneal injection of low-dose lipopolysaccharide (LPS) for several days (pretreated with LPS 0.1 μg/mouse for 5 d).Mice in control group were given the same volume of normal saline (NS).CD11clowCD45RBhighDC were isolated from spleen by magnetic activated cell sorting (MACS).The immunological phenotypes were detected by flow cytometry.The suppressive capacity of CD11clow CD45RBhigh DC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in allogenic mixed T cells reaction.The expressions of interleukin (IL)-10 and IL-12 produced by CD11clow CD45RBhigh DC were measured by enzyme-linked immunosorbent assay (ELISA).Statistical significance was analyzed through one-way analysis of variance (ANOVA).The homogeneity of variances was detected by Levene test.If variances were homogeneous,the least significant difference (LSD) test was used.If not,Dunnett T3 test was applied.Results The consistence of CD1 1 clow CD45RBhigh DC in control group was 30 ％,reaching the amount of (5.30±0.12) × 105/mouse ;In ET group,the percentage of CD11clow CD45RBhighDC achieved 80 ％ and the production was (1.20 ± 0.13) × 106/mouse the difference was statistically significant (t=3.23,P＜0.01).The cellar morphology in two groups showed no obvious difference.Compared to expression levels of all cell phenotypes (histocompatibility complex-Ⅱ,CD40 and CD80) in normal mice,the cell surface expression levels of CD11clowCD45RBhigh DC in ET mice were much lower.The difference in two groups was statistically significant.Splenic CD11clowCD45RBhighDC derived from ET mice with cell concentration of 1∶ 10,1∶50and 1∶100 had more obvious prohibitory effects on allogenic T cells (t1∶0 =1.36,P1∶10 ＜0.01,t1∶50 =2.49,P1∶50 ＜0.01,1∶100 =1.88,Pm00 ＜0.01).Secretion of IL-10 produced by CD11clowCD45RBhighDC of ET mice was significantly increased (t1∶0=13.63,P1∶10 ＜0.01,t1∶50 =13.45,P1∶50 ＜0.01,t1∶00 =9.31,P1∶00 ＜0.01),but the expression of IL-12 was lower (t1∶0 =2.62,P1∶0 =0.02,1∶∶50 =2.74,P1∶0=0.02,t1∶100 =2.99,P1∶100 =0.01).Conclusion Splenic CD11clow CD45RBhigh DC from ET mice have weaker ability of antigen presenting and allogeneic lymphocytes proliferation stimulating than those from normal mice.

Objective To evaluate the nourishment and immune privilege effects of Sertoli cells on co-encapsulated hepatocytes. Methods The hepatocytes and Sertoli cells were encapsulated or co-encapsulated in various ratio of 100∶1、50∶1、20∶1、10∶1, and co-cultured for 21 days in vitro. The secretion of albumin and urea was analyzed, and the morphology of encapsulated cells was observed by microscopy, then to determine the best mixed ratio of hepatocytes to Sertoli cells. Splenocyte proliferation response was assessed to evaluate Sertoli cell’s immune privilege function to hepatocytes by CCK-8.Results Sertoli cells could elevate hepatocyte’s secretion of albumin and urea when they were co-encapsulated with each in appropriate ratio (P

Objective To investigate the effects of bone marrow-derived stem cells (BMMSC)on the expressions of inflammatory cytokines and Toll-like receptor (TLR)4 pathway in primary hepatic stellate cells (HSC)with direct and indirect contact coculture system.Methods Purified HSC were separately treated with LPS in the concentrations of 0,50,100 and 150 g/L for 48 h.Proliferation ratio was tested with cell counting kit-8 to determine the optimal concentration.HSC in LPS were divided into three groups,including HSC alone group,cocultured with BMMSC at 1∶1 group and cocultured with transwell group at the optimal concentration.The supernatants were collected to detect the concentrations of interleukin-6 (IL-6)and tumor necrosis factor (TNF)-α.Cells were further divided into seven groups, including BMMSC without LPS group,HSC without LPS group,BMMSC with LPS group,HSC with LPS group,BMMSC in transwell system group,HSC in transwell system group,all cells in transwell system group and direct contact system group.The mRNA expressions ofα-smooth muscle actin (α-SMA) and collagen I were detected by quantitative real-time PCR,and protein expressions of TLR4,myeloid differentiation factor 88 (MyD88)and nuclear factor-κB (NF-κB)were analyzed by Western blot.Data were analyzed with one-way ANOVA analysis.Results The proliferation rate of HSC in 50,100 and 150μg/L LPS were (129.77±11 .26)%,(162.90±13.15 )% and (55 .12 ±11 .6)%,respectively compared with HSC without LPS group.The differences were statistically significant (t =5 .91 ,10.70 and 8.65 , respectively,all P <0.05).The concentrations of IL-6 and TNF-αin HSC alone group,directly/indirectly cocultured group were (252.02 ±30.94)ng/L and (148.00 ± 10.27 )ng/L,(88.52 ±6.61 )g/L and (72.63±5 .54)ng/L,(103.74±7.14)ng/L and (81 .79 ±6.92 )ng/L,respectively.The differences between HSC alone group and directly/indirectly cocultured group were significant (t=12.66 and 15 .82, 11 .81 and 12.34,respectively,all P < 0.05 ).The directly and indirectly cocultured groups were significantly different (t=3.83 and 2.53,respectively,both P <0.05 ).The mRNA expressions of α-SMA and collagen I in HSC with LPS were remarkably increased compared with HSC without LPS (t =14.16 and 11 .84,respectively,both P <0.05 )and reversed by cocultured with BMMSC (t =11 .98 and 4.47,respectively,P <0.05).All cells in transwell group expressed moreα-SMA and collagen I than the direct contact group (t=3.70 and 3.19,respectively,P <0.05 ).The TLR pathway associated protein expressions,TLR4,MyD88,and NF-κB in HSC in transwell group were significantly down-regulated compared with HSC with LPS group.And all cells in transwell system had higher level of protein expressions compared with direct contact system (P < 0.05 ).Conclusions BMMSC are effective in inhibiting HSC activation and inflammatory cytokines excretion,which may be modulated through TLR4 pathway and cell to cell contact.

Objective To investigate the therapeutic effect and possible mechanism of adenosine A2A receptor agonist (CGS21680) combined with bone marrow mesenchymal stem cells (BMMSC) transplantation in acute liver failure (ALF).Methods Fifty male C57BL/6 mice, 6-8 weeks old, were fed with standard diet for 1 week and randomly divided into 5 groups according to random number table: healthy control group (n=6), model group (n=11), BMMSC group (n=11), CGS21680/BMMSC group (n=11) and CGS21680 group (n=11).Except healthy control group, the other mice were injected with D-GalN and lipopolysaccharide (LPS) to establish ALF model.Ten hours later, CGS21680/BMMSC group and CGS21680 group were injected intraperitoneally with adenosine A2A receptor agonist CGS21680 (2.1 mg/kg).In addition, the BMMSC group and CGS21680/BMMSC group were injected BMMSC (1×10.6) through tail vein.After 24 hours, pathological changes of liver tissue was observed by hematoxylin and eosin staining.The change of proportion of mouse splenic Treg among CD4+T lymphocytes was detected by flow cytometry.Toll-like receptor (TLR)4 and nuclear factor (NF)-κB expression levels in liver tissue were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blot.One-way analysis of variance (one-way ANOVA) and SNK-q test was conducted for data analysis.Results Serum IL-6 levels were (23.67±2.97) pg/mL in healthy control group, (151.47±6.03) pg/mL in model control group, (72.10±3.74) pg/mL in BMMSC group, (53.35±2.50) pg/mL in CGS21680/BMMSC group and (84.85±3.25) pg/mL in CGS21680 group.The differences between healthy control group and the other 4 groups were all statistically significant (t=46.02, 25.51, 19.58 and 34.03, respectively, all P<0.01).Serum TNF-ɑ levels were (24.62±3.19) pg/mL in healthy control group, (102.25±2.10) pg/mL in model control group, (54.71±2.23) pg/mL in BMMSC group, (42.20±4.72) pg/mL in CGS21680/BMMSC group and (81.76±3.50) pg/mL in CGS21680 group.The differences between healthy control group and the other 4 groups were all statistically significant (t=46.49, 19.97, 7.72 and 29.57, respectively, all P<0.01).The differences of spleen Treg proportion in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=51.67, 12.22, 5.91 and 18.21, respectively, all P<0.01).The differences of TLR4 mRNA levels of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=26.31, 21.33, 13.24 and 27.14, respectively, all P<0.05).The differences of NF-κB mRNA level of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=16.56, 16.34, 7.83 and 13.11, respectively, all P<0.05).The differences of TLR4 protein level in liver tissue of healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=35.60, 10.38, 6.05 and 18.02, respectively, all P<0.05).The differences of liver NF-κB protein level in the healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=10.80, 7.30, 4.61 and 13.24, respectively, all P<0.05).Conclusions Adenosine A2A receptor agonist combined with BMMSC can significantly up-regulate the proportion of Treg cells in acute liver failure mice and inhibit the TLR4/NF-κB pathway activation, with both coordinated regulation, and further inhibit the liver inflammation.