Retinoic acid receptor α promotes autophagy to alleviate liver ischemia and reperfusion injury.

AIM: To study the role of autophagy and the relationship between retinoic acid receptor α (RARα) and autophagy in liver ischemia and reperfusion (IR) injury. METHODS: All-trans retinoic acid (ATRA) was administered to mice for two weeks before operation. Reverse transcription-polymerase chain reaction and Western blot were used to detect the expression levels of related factors. To demonstrate the role of RARα, LE540, a RARα inhibitor, was used to treat hepatocytes injured by H2O2 in vitro. RESULTS: ATRA pretreatment noticeably diminished levels of serum alanine aminotransferase and aspartate aminotransferase as well as the degree of histopathological changes. Apoptosis was also inhibited, whereas autophagy was promoted. In vitro, RARα was inhibited by LE540, which resulted in decreased autophagy and increased apoptosis. Similarly, the expression of Foxo3a and p-Akt was downregulated, but Foxo1 expression was upregulated. CONCLUSION: This research provides evidence that ATRA can protect the liver from IR injury by promoting autophagy, which is dependent on Foxo3/p-Akt/Foxo1 signaling.

Clinicopathological significance and prognostic value of the expression of the cancer stem cell marker CD133 in hepatocellular carcinoma: a meta-analysis.

To conduct a meta-analysis to assess the association between CD133 expression and clinicopathological significance and prognostic value in hepatocellular carcinoma patients. Studies were identified via an electronic comprehensive literature search through the Pubmed, Chinese CNKI, and Wanfang databases. This meta-analysis was performed using Stata statistical software version 12.0. The outcomes included various clinicopathological and survival parameters (P < 0.05 was consider to indicate a statistical significance). A total of 21 studies comprising 2592 patients were included in this meta-analysis. CD133 overexpression was significantly associated with a series of clinicopathological parameters, such as low tumor differentiation (pooled odds ratio (OR) = 2.26, 95% CI: 1.59-3.21, P < 0.00001), advanced tumor stage (pooled OR = 2.17, 95% CI: 1.70-2.77, P < 0.00001), vascular invasion (pooled OR = 2.06, 95% CI: 1.25-3.39, P = 0.005), and vascular thrombosis (pooled OR = 1.47, 95% CI: 1.08-1.99, P = 0.015). However, CD133 expression was not correlated with hepatitis, cirrhosis, α-fetoprotein level, tumor number, tumor size, encapsulation, or metastasis. Regarding survival outcome, CD133 overexpression was significantly correlated with poor overall survival (pooled hazard ratio (HR) = 2.01, 95% CI: 1.45-2.80, P = 0.00002) and poor disease-free survival (pooled HR = 1.82, 95% CI: 1.45-2.29, P < 0.00001). This meta-analysis indicated that CD133 overexpression is significantly associated with clinicopathological factors and poorer survival outcome.

N-acetylcysteine attenuates reactive-oxygen-species-mediated endoplasmic reticulum stress during liver ischemia-reperfusion injury.

AIM: To investigate the effects of N-acetylcysteine (NAC) on endoplasmic reticulum (ER) stress and tissue injury during liver ischemia reperfusion injury (IRI). METHODS: Mice were injected with NAC (300 mg/kg) intraperitoneally 2 h before ischemia. Real-time polymerase chain reaction and western blotting determined ER stress molecules (GRP78, ATF4 and CHOP). To analyze the role of NAC in reactive oxygen species (ROS)-mediated ER stress and apoptosis, lactate dehydrogenase (LDH) was examined in cultured hepatocytes treated by H2O2 or thapsigargin (TG). RESULTS: NAC treatment significantly reduced the level of ROS and attenuated ROS-induced liver injury after IRI, based on glutathione, malondialdehyde, serum alanine aminotransferase levels, and histopathology. ROS-mediated ER stress was significantly inhibited in NAC-treated mice. In addition, NAC treatment significantly reduced caspase-3 activity and apoptosis after reperfusion, which correlated with the protein expression of Bcl-2 and Bcl-xl. Similarly, NAC treatment significantly inhibited LDH release from hepatocytes treated by H2O2 or TG. CONCLUSION: This study provides new evidence for the protective effects of NAC treatment on hepatocytes during IRI. Through inhibition of ROS-mediated ER stress, NAC may be critical to inhibit the ER-stress-related apoptosis pathway.

Dermatomyositis associated with gallbladder carcinoma: A case report.

Patients with gallbladder carcinoma can present with a variety of paraneoplastic syndromes, including Cushing's syndrome, hypercalcemia, acanthosis nigricans, bullous pemphigoid, dermatomyositis and the sign of Leser-Trélat. Surgical resection of the primary tumor results in resolution of these paraneoplastic syndromes. We present a 67-year old female with facial and cervical erythema who was initially diagnosed with dermatomyositis. However, an abdominal computed tomography (CT) and positron emission tomography-CT scan was suspicious for gallbladder carcinoma with lymph node metastasis. After surgical resection, her dermatomyositis was resolved. This case demonstrates that dermatomyositis may be a manifestation of preexisting gallbladder carcinoma.

Inhibition of GSK-3beta ameliorates hepatic ischemia-reperfusion injury through GSK-3beta/beta-catenin signaling pathway in mice.

BACKGROUND: Glycogen synthase kinase (GSK)-3beta/beta-catenin signaling regulates ischemia-reperfusion (I/R)-induced apoptosis and proliferation, and inhibition of GSK-3beta has beneficial effects on I/R injury in the heart and the central nervous system. However, the role of this signaling in hepatic I/R injury remains unclear. The present study aimed to investigate the effects and mechanism of GSK-3beta/beta-catenin signaling in hepatic I/R injury. METHODS: Male C57BL/6 mice (weighing 22-25 g) were pretreated with either SB216763, an inhibitor of GSK-3beta, or vehicle. These mice were subjected to partial hepatic I/R. Blood was collected for test of alanine aminotransferase (ALT), and liver specimen for assays of phosphorylation at the Ser9 residue of GSK-3beta, GSK-3beta activity, axin 2 and the anti-apoptotic factors Bcl-2 and survivin, as well as the proliferative factors cyclin D1 and proliferating cell nuclear antigen, and apoptotic index (TUNEL). Real-time PCR, Western blotting and immunohistochemical staining were used. RESULTS: SB216763 increased phospho-GSK-3beta levels and suppressed GSK-3beta activity (1880+/-229 vs 3280+/-272 cpm, P<0.01). ALT peaked at 6 hours after reperfusion. Compared with control, SB216763 decreased ALT after 6 hours of reperfusion (4451+/-424 vs 7868+/-845 IU/L, P<0.01), and alleviated hepatocyte necrosis and vacuolization. GSK-3beta inhibition led to the accumulation of beta-catenin in the cytosol (0.40+/-0.05 vs 1.31+/-0.11, P<0.05) and nucleus (0.62+/-0.14 vs 1.73+/-0.12, P<0.05), beta-catenin further upregulated the expression of axin 2. Upregulation of GSK-3beta/beta-catenin signaling increased Bcl-2, survivin and cyclin D1. Serological and histological analyses showed that SB216763 alleviated hepatic I/R-induced injury by reducing apoptosis (1.4+/-0.2% vs 3.6+/-0.4%, P<0.05) and enhanced liver proliferation (56+/-8% vs 19+/-4%, P<0.05). CONCLUSION: Inhibition of GSK-3beta ameliorates hepatic I/R injury through the GSK-3beta/beta-catenin signaling pathway.

Methylprednisolone inhibits activated CD4+ T cell survival promoted by toll-like receptor ligands.

BACKGROUND: Methylprednisolone (MP) can affect the survival of CD4+ T lymphocytes and plays an important role in adaptive immune responses; however, its mechanism of action is not clear. Recent studies have shown that toll-like receptors (TLRs) on CD4+ T cells can directly modulate adaptive immune responses by affecting the survival and proliferation of activated CD4+ T cells. This study aimed to investigate the relationship between MP, TLRs and activated CD4+ T cells. METHODS: We separated and purified CD4+ T cells from mice, activated them in vitro, and co-cultured them with TLR ligands, MP or inhibitors of nuclear factor-kappa B (NF-kappaB) and activator protein 1 (AP-1). We then assessed CD4+ T cell survival and proliferation and the expression of NF-kappaB and AP-1. RESULTS: Activated CD4+ T cells showed increased TLR-3 and TLR-9 mRNA expression, but polyinosinic-polycytidylic acid (poly I:C) and MP had no effect on the expression of these mRNAs. Still, poly I:C and CpG oligodeoxynucleotides (CpG DNA) increased the survival of activated CD4+ T cells, whereas MP reduced the survival of activated CD4+ T cells and could inhibit the survival effects of poly I:C and CpG DNA. The NF-kappaB essential modifier-binding domain (NBD) inhibited the survival of activated CD4+ T cells induced by poly I:C and CpG DNA, but the AP-1 inhibitor crucumin did not have the same effect. The increased expression of NF-kappaB induced by poly I:C and CpG DNA in activated CD4+ T cells could be inhibited by MP, but the same was not true for the increased expression of AP-1 induced by poly I:C and CpG DNA. Finally, the proliferation of activated CD4+ T cells was not affected by poly I:C or MP. CONCLUSION: The survival of activated CD4+ T cells is promoted by TLR ligands, but this effect is inhibited by MP.

Hepatic regenerative response in small-sized liver isografts in the rat.

BACKGROUND: To investigate hepatic regenerative response and associated mechanisms in different-size liver grafts in the rat. METHODS: Rat models of different-size-graft liver transplantation (whole, 50%-size, or 30%-size) were established, with a sham operation group serving as a control. Portal pressure, graft injury, interleukin 6 (IL-6), signal transducer and activator of transcription (Stat3), mitogen-activated protein kinase (MAPK), cyclin D1, and proliferating cell nuclear antigen (PCNA) were all assessed. RESULTS: The portal pressure was significantly higher and hepatic injury more severe in the smaller sized groups than in the whole graft group, especially in the 30%-size grafts. Hepatic IL-6 and tumor necrosis factor-alpha (TNF-alpha) levels in the two smaller sized groups were significantly higher than in the whole graft group, while IL-6 levels appeared to be negatively associated with graft sizes. Downstream markers of IL-6, Stat3 and MAPK phosphorylation, cyclin D1, and PCNA expression were also markedly increased in the small-sized grafts compared with the whole grafts, and appeared to positively correlate with early measurements of portal pressure and subsequent hepatic injury. CONCLUSION: Vigorous hepatic regeneration in small-for-size liver grafts may be associated with highly activated IL-6/Stat3 and MAPK signaling, which may in turn correlate with graft size, portal pressure, and hepatic injury.

Liver transplantation for polycystic liver with massive hepatomegaly: a case report.

A previous study has shown that liver or combined liver-kidney transplantation can be a valuable surgical technique for the treatment of polycystic liver disease. Herein, we present the case of a 35-year-old woman with polycystic liver disease, who underwent orthotopic liver transplantation (OLT) on November 11, 2008. The whole-size graft was taken from a deceased donor (a 51-year-old man who died of a heart attack). Resection in a patient with massive hepatomegaly is very difficult. Thus, after intercepting the portal hepatic vein, left hepatectomy was performed, then the vena cava was intercepted, the second and third porta hepatic isolated, and finally, right hepatectomy was performed. OLT was performed successfully. The recipient did well after transplantation. This case suggested that OLT is an effective therapeutic option for polycystic liver disease and left hepatectomy can be performed first during OLT if the liver is over enlarged.

Outcomes of living-related liver transplantation for Wilson's disease: a single-center experience in China.

BACKGROUND: Although orthotopic liver transplantation provides a therapeutic option for patients with Wilson's disease (WD) presenting fulminant liver failure or drug resistance, it is still unclear whether the living-related liver transplantation (LRLT) can result in long-term therapeutic effect on WD. METHODS: Here, we report a retrospective analysis of LRLT for 36 cases of WD patients. The indications for LRLT were fulminant hepatic failure in two patients and chronic advanced liver disease in 32 patients including 13 patients with Wilsonian neurologic manifestations. Two patients presented with severe Wilsonian neurologic manifestations even though their liver functions were stable. RESULTS: Results revealed that the survival of posttransplant patients or grafts at 1, 3, and 5 years was 91.7%, 83.3%, 75%, or 86.1%, 77.8%, 75%, respectively. Pretransplant intensive care unit-bound and model for end-stage liver disease score were indicated as independent factors predictive of patient survival. Patients with neurologic abnormalities showed significant improvement after liver transplant. CONCLUSION: Our results indicate LRLT is an excellent therapeutic modality for WD patients with end-stage liver disease. Better pretransplant conditions appeared to be advantageous in gaining better survival outcomes of patients undergoing LRLT.

[Surgical technique and concept in precise hepatectomy: experience of 338 cases of hepatectomy in single center].

OBJECTIVE: To evaluate the perioperative clinical outcome and predictive factors for perioperative complication morbidity and mortality. METHODS: From August 2003 to August 2008, the data of 338 cases of hepatectomy performed in the liver transplant center of the First Affiliated Hospital of Nanjing Medical University was collected in a prospective manner. The patients' perioperative clinical risk factors and results were analyzed. RESULTS: In the 338 hepatectomy cases, 255 patients (75.4%) underwent precise anatomical hepatectomy. The overall perioperative complication morbidity was 18.1%, while the perioperative mortality was 0.6%. In a total of 211 (62.4%) cases, the operation was carried out without blood transfusion. Univariate analysis revealed that cirrhotic liver, thrombocytopenia, blood loss in operation > 1000 ml, blood transfusion in operation and several other factors were closely related with the incidence rate of complication. Multivariate logistic regression analysis indicated that thrombocytopenia and perioperative blood transfusion were important independently predictive factors for the occurrence of perioperative complications in hepatectomy. CONCLUSIONS: Precise hepatectomy enables patients to obtain better clinical outcome with low complication morbidity and perioperative mortality. Reducing hemorrhage is an important factor that lead to good clinical results.

Adenoviral cardiotrophin-1 transfer improves survival and early graft function after ischemia and reperfusion in rat small-for-size liver transplantation model.

This study was to investigate the effect of donor liver adenoviral cardiotrophin-1 (CT-1) gene transfer on early graft survival and function in rat small-for-size liver transplantation. We constructed a recombinant murine CT-1 adenoviral vector. Donor rats were transduced in vivo with adenoviruses expressing CT-1 (AdCT-1) or control vector (AdEGFP). Livers were harvested 4 days later, reduced to 40% of weight, and transplanted. A syngeneic rat orthotopic liver transplantation model was performed using 40% small-for-size grafts. Graft survival, liver function, hepatic architecture change, the degree of necrosis and apoptosis, and cell survival signaling pathways were assessed. AdCT-1 pretreatment markedly improved liver function and the survival of small-for-size grafts. In the CT-1 treatment group, hepatic architecture was well protected, apoptotic and necrotic cells were reduced; anti-apoptotic protein bcl-2 was up-regulated and pro-apoptotic cleaved caspase-3 was down-regulated, cell survival signaling pathways were activated by phosphorylation of protein kinase B (Akt), extracellular-regulated kinase (ERK) and Signal transducer and activator of transcription-3 (Stat-3) after transplantation. In conclusion, donor liver adenoviral CT-1 transfer ameliorated ischemia/reperfusion injury by decreasing hepatic necrosis and apoptosis in small-for-size liver transplantation, mediated in part by activation of the Akt, ERK, and Stat-3 survival signaling pathways. These results may provide a potential clinical strategy to improve the outcome of small-for-size liver grafts.

Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) regulatory T cells ameliorates rejection of DA-to-Lewis rat liver transplantation.

BACKGROUND: Adoptive transfusion of splenocytes from long-term survivors of a tolerance model of rat orthotopic liver transplantation can induce acceptance of liver allografts in a rejection model preconditioned with donor gamma-irradiation before liver transplantation. Recent studies suggest that the regulatory T cells (Treg cells) in splenocytes from long-term survivors play an important role in the induction of liver graft tolerance, but this observation was made from a rejection model preconditioned with donor gamma-irradiation; little is known about the role of Treg cells in liver graft rejection using a naive rejection model. In this study, we examined the therapeutic potential of CD4(+)CD25(+) Treg cells in a naive rejection model of rat liver transplantation. METHODS: Freshly isolated or ex vivo alloantigen-stimulated CD4(+)CD25(+) Treg cells (1 x 10(6) cells) from naive Lewis RT(1) (LEW) rats were adoptively transferred into another LEW rat on days 1 and 7 after liver transplantation from a Dark Agouti RT1(a) (DA) rat. Recipients were treated with or without oral tacrolimus (FK506) (0.1 mg/kg/day) from days 1 to 7 after transplantation. For ex vivo alloantigen-stimulation, CD4(+)CD25(+) Treg cells from LEW rats were cocultured with mitomycin C-treated DA (donor alloantigen specific) or Brown Norway (BN)(RT1(n), third party) splenocytes for 72 hours. Ex vivo alloantigen-specific CD4(+)CD25(-) T-cell proliferation responses were assessed with fresh and stimulated CD4(+)CD25(+) Treg cells. RESULTS: Freshly isolated, donor alloantigen-stimulated and third-party alloantigen- stimulated CD4(+)CD25(+) Treg cells suppressed antigen-specific CD4(+)CD25(-) T-cell proliferation ex vivo, and adoptive transfusion of these 3 kinds of CD4(+)CD25(+) Treg cells prolonged survival of the liver allografts. The group transfused with the donor alloantigen-stimulated CD4(+)CD25(+) Treg cells had the greatest mean survival among the 3 groups (fresh Treg cells, 21 +/- 2 days, n = 6; third-party alloantigen-stimulated Treg cells, 20 +/- 2 days, n = 6; donor alloantigen-stimulated Treg cells, 30 +/- 2 days, n = 6). When combined with short-term tacrolimus administration, adoptive transfusion of donor antigen-stimulated Treg cells induced the greatest survival time in recipients (greater than 60 days; n = 6). CONCLUSION: Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) Treg cells combined with short-term tacrolimus treatment may represent a new strategy for preventing rejection after liver transplantation.

Mesenchymal stem cells over-expressing hepatocyte growth factor improve small-for-size liver grafts regeneration.

Ischemia-reperfusion (I/R) associated with small-for-size liver transplantation (SFSLT) impairs liver graft regeneration. Mesenchymal stem cells (MSCs) have the capability, under specific conditions, of differentiating into hepatocytes. Hepatocyte growth factor (HGF) has potent anti-apoptotic and mitogenic effects on hepatocytes during liver injury, and has been utilized in many experimental and clinical applications. In this study, we implanted HGF-expressing MSCs into liver grafts via the portal vein, using a 30% small-for-size rat liver transplantation model. HGF, c-met expression, hepatic injury and liver regeneration were assessed after liver transplantation. Our study demonstrated that MSCs over-expressing HGF prevented liver failure and reduced mortality in rats after SFSLT. These animals also exhibited improved liver function and liver weight recovery during the early post-transplantation period. Using green fluorescent protein (GFP) gene as a marker, we demonstrated that the engrafted cells and their progeny incorporated into remnant livers and produced albumin. These findings suggest that MSCs genetically modified to over-express HGF and implanted in the liver graft, may offer a novel approach to promoting liver regeneration after small-for-size transplantations.

Protective effect of adenosine A2A receptor activation in small-for-size liver transplantation.

The aim of the present study was to investigate the potential role of adenosine A(2A) receptor (A(2A)R) activation in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed by using 40% (range: 36-46%) liver grafts. Recipients were given either saline (control group) or CGS 21680 (2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride, a selective A(2A)R agonist), or CGS 21680+ ZM 241385 (a selective A(2A)R antagonist) immediately after reperfusion for 3 h. Compared with control group, CGS 21680 used at both low dose (0.05 microg/kg/min) and high dose (0.5 microg/kg/min) increased the survival rate from 16.7% (2/12) to 83.3% (10/12) and 66.7% (8/12), respectively. These effects correlated with improved liver function and preserved hepatic architecture. CGS 21680 effectively decreased neutrophil infiltration, suppressed pro-inflammatory (TNF-alpha, IL-1beta and IL-6) expression, promoted expression of antiapoptotic molecules, and inhibited apoptosis. The effects of CGS 21680 were prevented when ZM 241385 was co-administrated. In conclusion, the present study showed that A(2A)R activation alleviated portal hypertension, suppressed inflammatory response, reduced apoptosis, and potentiated the survival of small-for-size liver grafts. Our findings provide the rationale for a novel therapeutic approach using A(2A)R activation to maximize the availability of small-for-size liver grafts.

[Isolation and function analysis of rat CD4+ CD25+ regulatory T cells].

AIM: To investigate the isolation method and to analyze the function of rat CD4(+) CD25(+) regulatory T cells. METHODS: Lymphocytes were isolated from the rat spleens and then CD4(+) CD25(+) T cells were sorted by magnetic bead cell sorting (MACS) system. The purity and Foxp3 expression of CD4(+) CD25(+) T cells were analyzed by flow cytometry(FCM) and RT-PCR, respectively. The suppressive effect of CD4(+) CD25(+) T cells on the proliferation of CD4(+) CD25(-) T cells was analyzed by mixed lymphocyte reaction. IL-2, IFN-gamma and IL-10 levels in culture supernatant were detected by ELISA. RESULTS: The purity of CD4(+) CD25(+) T cells sorted by MACS was 86%-93%. The CD4(+) CD25(+) T cells could specifically express the Foxp3 gene as compared with CD4(+) CD25(-) T cells. In vitro CD4(+) CD25(+) T cells could suppress the proliferation of CD4(+) CD25(-) T cells and IFN-gamma, IL-2 production, but they themselves could secrete IL-10. CONCLUSION: We established an effective procedure for enrichment of CD4(+) CD25(+) regulatory T cells by MACS with satisfactory cell purity, viability and function. CD4(+) CD25(+) T cells can suppress CD4(+) CD25(-) T cells and specifically express Foxp3 gene.

[CD4(+) CD25(+) Tr cells and transcription factor Foxp3 in the naturally tolerance of rat liver transplantation].

OBJECTIVE: To investigate the role of intrahepatic CD4(+)CD25(+) T regulatory cells and Foxp3 gene in the natural tolerance in rat liver transplantation. METHODS: The orthotopic liver transplantation models of inbred rats (LEW and DA rats) were established with double-sleeve technique and the models were divided into two groups: tolerance group (TOL group, LEW-to-DA) and rejection group (REJ group, DA-to-LEW). The intrahepatic lymphocytes from each group were isolated by using density gradient centrifugation. CD4(+)CD25(+) T cells were isolated by magic cell sorting system (MACS) and identified by flow cytometry (FCM). CD4(+)CD25(+) Tr cells suppression on the proliferation of CD4(+)CD25(-) T effector cells were analyzed by cell proliferation assay in vitro. Western blot was used to detect Scurfin protein expression of CD4(+)CD25(+) Tr cells. RESULTS: CD4(+)CD25(+) Tr cells developed significantly greater in the TOL group than in the REJ group. In vitro, the spleen cells from LEW rats can irritate the proliferation of CD4(+)CD25(+) T cells more obviously than the syngeneic spleen cells. CD4(+)CD25(+) T cells could suppress the proliferation of CD4(+)CD25(-) T cells, but the inhibition was reversed by exogenous IL-2 (200 U/ml). CONCLUSIONS: The immune suppression function of CD4(+)CD25(+) Tr cell, mediated by Foxp3 gene, is one of the mechanisms in liver transplantation tolerance.