Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells

AIM:To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver allograft.METHODS:Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs, respectively. GMCSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A),GM-CSF-propagated DCs (group B), GM-CSF+IL-4-propagated DCs (group C), and GM-CSF+NF-κB decoy ODNs(group D) or scrambled ODNs -propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation.DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24h, 48h, 72h and 84h,respectively. Liver graft-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with ^51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84h in group C rats and 48h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ production.At the time of the maximal regeneration of liver allograft,the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-y production were detected in group D rats. The apoptotic index of hepatocytes, the activityof liver- resident NK cells, the hepatic TFN-γ mRNA expression level and the serum IFN-γ level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.CONCLUSION:The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified Des may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liverresident NK cells and the reduced IFN-γ production.     

Hyperbaric oxygenation promotes regeneration of biliary cells and improves cholestasis in rats

AIM:To investigate the effects of hyperbaric oxygenation(HBO) on regeneration of the biliary ductal system and postoperative cholestasis in hepatectomized rats.METHODS:HBO was performed in Wistar rats daily starting 12 h after a 70% partial hepatectomy.Regenerated liver weight,serum parameters and the proliferating cell nuclear antigen labeling index of hepatocytes and biliary ductal cells were measured.Hepatocyte growth factor(HGF),c-Met and transforming growth factor(TGF) β-1 mRNA expression levels were a...     

Expression of tumor necrosis factor-alpha converting enzyme in liver regeneration after partial hepatectomy

AIM：To study the expression of tumor necrosis factor-alpha converting enzyme （TACE） and evaluate its significance in liver regeneration after partial hepatectomy in vivo.
METHODS： Male SD rats underwent 70% partial hepatectomy. The remaining liver and spleen tissue samples were collected at indicated time points after hepatectomy. TACE expression was investigated by Western blotting, immunohistochemistry, and serial section immunostaining.
RESULTS： Expression of TACE in liver and spleen tissues after partial hepatectomy was a time-dependent alteration, reaching a maximal level between 24 and 48 h and remaining elevated for more than 168 h. TACE protein was localized to mononuclear cells （MNC）, which infiltrated the liver from the spleen after hepatectomy. The kinetics of TACE expression was in accordance with the number of TACE-staining MNCs and synchronized with those of transforming growth factor-α （TGFα）. In addition, TACE-staining MNC partially overlapped with CD3^＋ T lymphocytes.
CONCLUSION： TACE may be involved in liver regeneration by pathway mediated with TGFα-EGFR in the cellcycle progressive phase in vivo. TACE production and effect by paracrine may be a pathway of involvement in liver regeneration for the activated CD3^＋ T lymphocytes.     

Augmented regeneration of partial liver allograft induced by nuclear factor-kB decoy oligodeoxynucleotides-modified dendritic cells

AIM:To investigate the effect of NF-κB decoyoligodeoxynuleotides(ODNs)-modified dendritic cells(DCs)on regeneration of partial liver allograft.METHODS:Bone marrow(BM)-derived DCs from SD ratswere propagated in the presence of GM-CSF or GM-CSF IL-4 to obtain immature DCs or mature DCs,respectively.GM-CSF-propagated DCs were treated with double-strand NF-~Bdecoy ODNs containing two NF-κB binding sites or scrambledODNs.Allogeneic(SD rat to LEW rat)50% partial livertransplantation was performed.Normal saline(group A),GM-CSF-propagated DCs(group B),GM-CSF IL-4-propagated DCs(group C),and GM-CSF NF-κB decoy ODNs(group D)or scrambled ODNs-propagated DCs(group E)were injected intravenously into recipient LEW rats 7 daysprior to liver transplantation and immediately aftertransplantation.DNA synthesis(BrdU labeling)and apoptosisof hepatocytes were detected with immunostaining andTUNEL staining postoperative 24 h,48 h,72 h and 84 h,respectively.Liver graft-resident NK cell activity,hepaticIFN-γ mRNA expression and recipient serum IFN-γ level atthe time of the maximal liver allograft regeneration weremeasured with ~(51)Cr release assay,semiquantitative RT-PCRand ELISA,respectively.RESULTS:Regeneration of liver allograft was markedlypromoted by NF-κB decoy ODNs-modified immature DCsbut was significantly suppressed by mature DCs,the DNAsynthesis of hepatocytes peaked at postoperative 72 h ingroup A,group B and group E rats,whereas the DNAsynthesis of hepatocytes peaked at postoperative 84 h ingroup C rats and 48 h in group D rats,respectively.Themaximal BrdU labeling index of hepatocytes in group D ratswas significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedlysuppressed but mature DCs markedly promoted apoptosisof hepatocytes,liver-resident NK cell activity,hepatic IFN~mRNA expression and recipient serum IFN-γ production.Atthe time of the maximal regeneration of liver allograft,theminimal apoptosis of hepatocytes,the minimal activity ofliver-resident NK cells,the minimal hepatic IFN-γ mRNAexpression and serum IFN-γ production were detected ingroup D rats.The apoptotic index of hepatooltes,the activity of liver-resident NK cells,the hepatic IFN-γ mRNA expressionlevel and the serum IFN-γ level in group D rats were significantlylower than that in the other groups rats at the time of themaximal regeneration of liver allograft.CONCLUSION:The data suggest that the augmentedregeneration of partial liver allograft induced by NF-κB decoyODNs-modified DCs may be attributable to the reducedapoptotic hepatocytes,the suppressed activity of liver-resident NK cells and the reduced IFN-γ production.     

Lethal effect and apoptotic DNA fragmentation in response of D-GalN-treatd mice to bacterial LPS can be suppressed by pre-exposure to minute amount of bacterial LPS： Dual role of TNF receptor 1

AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.     

Shedding of Fas ectodomain that affects apoptosis of hepatocytes occurring in regenerative liver

Background: On the basis of comprehending several membrane proteins undergoing ectodomain shedding, including tumor necrosis factor (TNF) α receptors, we want to know if Fas (CD95/APO-1), which belongs to the TNF receptor (TNFR) superfamily and transduces signals resulting in apoptosis upon binding of Fas ligand (L) or agonistic anti-Fas antibody, is shed on its ectodomain during liver regeneration Methods: After purification of the total membrane protein of hepatocytes, we analyzed Fas ectodomain shedding, using Western blotting, and determined the effect of Fas shedding on the apoptosis of hepatocytes by a statistical method relevant to apoptosis. Results: Fas protein ectodomain shedding occurred at 2, 12, and 36 h after partial hepatectomy, and its level decreased at 2 months after partial hepatectomy. The same results were gained from in-vitro cultured hepatocytes induced by serum from rats after partial hepatectomy. The sensivitity of hepatocytes to agonistic anti-Fas antibody increased significantly after they were treated with serum from rats after partial hepatectomy. However, the expression of Fas mRNA and Fas protein did not affect the liver regeneration. Conclusions: Fas ectodomain shedding might be an important mechanism in controlling hepatocyte apoptosis during liver regeneration. Received: February 25, 2002 / Accepted: May 31, 2002     

Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is an angiogenic factor with a growth-promoting effect that is thought to be restricted to vascular endothelial cells. Its essential role during liver regeneration has yet to be determined. The aim of this study was to document the effect of exogenous VEGF administration on liver regeneration in rats undergoing submaximal hepatic resections. METHODS: Adult male Sprague-Dawley rats (n = 4/group) undergoing 30% partial hepatectomy were administered 200 ng VEGF165 intravenously and were sacrificed at 24, 36, and 48 h postoperatively. Liver regeneration was monitored by measuring the restituted liver mass, proliferating cell nuclear antigen (PCNA) immunostaining, and hepatic PCNA protein by Western blot. RESULTS: Changes in restituted liver mass 48 h postsurgery were more prominent, but did not differ statistically between VEGF-treated and control rats (47% vs. 29%; p<0.06). Nevertheless, PCNA immunostaining showed increased labeling index of hepatocytes, apparent at 36 and 48 h after partial hepatectomy (38% vs. 18% [p<0.041 and 42% vs. 11% [p<0.021], respectively). Hepatic PCNA proteins measured by Western blot showed a 3-fold increase in VEGF-treated rats 48 h postsurgery compared with controls (p<0.01). CONCLUSION: Exogenous VEGF administration early after partial hepatectomy stimulates liver regeneration in rats. Whether or not VEGF165 is a direct mitogen for hepatocytes remains to be determined.     

Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells

AIM: To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs)on regeneration of partial liver allograft.METHODS: Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL4 to obtain immature DCs or mature DCs, respectively. GMCSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A),GM-CSF -propagated DCs (group B), GM-CSF+IL-4 -propagated DCs (group C), and GM-CSF+NF-κB decoy ODNs (group D) or scrambled ODNs -propagated DCs (group E)were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation. DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24 h, 48 h, 72 h and 84 h,respectively. Liver graft-resident NK cell activity, hepatic IFN-y mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with 51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72 h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84 h in group C rats and 48 h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γmRNA expression and recipient serum IFN-γ production. At the time of the maximal regeneration of liver allograft, the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-γ production were detected in group D rats. The apoptotic index of hepatocytes, the activity of liver- resident NK cells, the hepatic IFN-γ mRNA expression level and the serum IFN-γlevel in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.CONCLUSION: The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified DCs may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liverresident NK cells and the reduced IFN-γ production.     

Insulin-like growth factor-II/mannose-6-phosphate receptors are increased in hepatocytes from regenerating rat liver

Insulin-like growth factor-II (IGF-II) receptor levels were determined in hepatocytes from sham-operated and two thirds hepatectomized rats. [125I]IGF-II binding to confluent cultures increased 2-fold in cells isolated 24 and 48 h after hepatectomy compared to that in cells from sham-operated rats. Receptor levels increased from 1.74 +/- 0.14 x 10(4)/cell (sham-operated) to 3.60 +/- 0.31 x 10(4)/cell (hepatectomized; P = 0.002), with no change in affinity of IGF-II binding (Ka = 6.4 x 10(9) M-1). As previously reported, receptor levels increased in cells plated at low density, but this effect was decreased in cells from hepatectomized rats (80% increase) compared to that in cells from control rats (300% increase). Serum IGF-I levels decreased 50% 24 h after partial hepatectomy (P less than 0.001), but returned to normal levels by 48 h. However, IGF-I synthesis was not decreased in hepatocytes isolated 24 h after partial hepatectomy, suggesting that decreased serum levels are due to decreased liver mass. Circulating IGF-II levels were not altered by partial hepatectomy, and IGF-II production was not detected in hepatocytes from sham-operated or hepatectomized rats. Transforming growth factor-beta is thought to terminate hepatocyte proliferation upon complete liver regeneration. In hepatocytes from sham-operated or hepatectomized rats transforming growth factor-beta totally blocked DNA synthesis, but had no effect on elevated IGF-II receptor levels after partial hepatectomy. Concomitant with increased IGF-II receptor levels, hepatocytes from hepatectomized rats were more sensitive to IGF-II stimulation of DNA synthesis. [3H]Thymidine incorporation in the presence of epidermal growth factor (50 ng/ml) was stimulated 66% by IGF-II (300 ng/ml) in control cells compared with 220% in cells from hepatectomized animals. These results suggest that IGF-II and the IGF-II receptor may play a role in liver regeneration.     

Ischemic preconditioning improves liver regeneration by sustaining energy metabolism after partial hepatectomy under ischemia in rats.

BACKGROUND: The protective effect of ischemic preconditioning (IPC) has been reported on improvement of survival, reduction of liver necrosis and enhancement of the regenerative capacity of hepatocytes after partial hepatectomy. This study was undertaken to confirm that IPC has a significant impact on regeneration of hepatocytes after partial hepatectomy in ischemically damaged liver. In addition, we sought to examine the role of adenine nucleotides in this process. METHODS: Wistar rats were subjected to 60 min of total hepatic ischemia, followed by 70% hepatectomy. The animals were subdivided into an IPC (10/15 min) group and a non-IPC (control) group. Liver function tests and arginase activity were analyzed. Hepatic adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate were measured using gradient high-performance liquid chromatography. The liver regeneration was identified using relative liver weight and proliferating cell nuclear antigen (PCNA) labeling index. RESULTS: IPC treatment improved serum liver enzymes and tissue arginase activity (P<0.05) when compared with the control group. The preconditioned livers were associated with upregulation of ATP expression and also increased tissue energy charge. Regenerated liver weight in the IPC group was significantly higher than in the control group (P<0.05). The PCNA labeling index in the remnant livers in the IPC group was also significantly increased at 24 and 48 h after partial hepatectomy (P<0.05). CONCLUSION: These results suggest that IPC-augmented liver regeneration after hepatectomy, probably due to the stabilization of energy metabolism in rats.     

Effect of serotonin receptor 2 blockage on liver regeneration after partial hepatectomy in the rat liver.

The effect of serotonin receptor 2 blockade (5-HT(2)) on liver regeneration after 30-34% and 60-70% partial hepatectomy in the rat liver was investigated. Materials and methods: Male Wistar rats were subjected to 60-70% (group I) and 30-34% (group II) partial hepatectomy. Serotonin receptor 2 blockade was exerted by intraperitoneal administration of ketanserin at different doses and time points after partial hepatectomy. The rats of all groups were killed at different time points until 96 h after partial hepatectomy. The rate of liver regeneration was evaluated by the mitotic index in hematoxylin and eosin sections, the immunochemical detection of Ki67 and proliferating cell nuclear antigens, the rate of [(3)H]-thymidine incorporation into hepatic DNA and liver thymidine kinase enzymatic activity. Results: Liver regeneration peaked at 24 and 32 h after partial hepatectomy in 60-70% hepatectomized rats. In 30-34% hepatectomized rats liver regeneration peaked at 60 h, whereas low rates of regenerative activity were observed between 24 and 72 h after partial hepatectomy. Ketanserin administration arrested liver regeneration only when administered at 16 h after 60-70% partial hepatectomy. Ketanserin also abrogated the observed peak of regenerative activity at 60 h in 30-34% hepatectomized rats when administered at 52 h after partial hepatectomy. All indices of liver regeneration were affected by ketanserin administration. Conclusions: Serotonin receptor 2 blockade can arrest liver regeneration only when administered close to G1/S transition point, and that while serotonin may be a cofactor for DNA synthesis, it does not play a role in initiation of liver regeneration.