Induction of autophagy reduces ischemia/reperfusion injury in steatotic rat livers.

BACKGROUND: Steatotic livers are particularly vulnerable to ischemia/reperfusion injury (IRI). One of the reasons is an underlying impairment of autophagy. Autophagy is regulated by glycogen synthase kinase 3b (GSK3b) and extracellular signal-regulated kinases (ERK1/2) pathways. Both of them are target proteins of a cell-protective drug, lithium chloride. Lithium chloride treatment reduces IRI in many organs including liver. Therefore, we aimed to investigate the effect of lithium chloride treatment on autophagy induction in steatotic rat livers. We also wanted to evaluate the related cell-protective effects on the enhanced hepatic IRI. MATERIALS AND METHODS: After inducing hepatic steatosis, rats were injected with lithium chloride or normal saline for 3 d before being subjected to 70% selective warm ischemia for 60 min. After reperfusion, rats were observed for 30 min, 6, 24, and 48 h. RESULTS: Lithium chloride appeared to protect hepatocytes from IRI via its ability to induce autophagy by modulation of both GSK3b and ERK1/2 pathways. Hepatic damage was significantly decreased in the treatment group as indicated by a reduced inflammatory response, less apoptosis, less necrosis, and lower liver enzyme levels. CONCLUSIONS: Simultaneous modulation of GSK3b and ERK1/2 pathways might be an interesting strategy to reduce IRI in steatotic livers with an impairment of autophagy.

Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury.

Ischemia/reperfusion injury (IRI) occurs inevitably in liver transplantations and frequently during major resections, and can lead to liver dysfunction as well as systemic disorders. High-mobility group box 1 (HMGB1) plays a pathogenic role in hepatic IRI. In the normal liver, HMGB1 is located in the nucleus of hepatocytes; after ischemia reperfusion, it translocates to the cytoplasm and it is further released to the extracellular space. Unlike the well-explored functions of nuclear and extracellular HMGB1, the role of cytoplasmic HMGB1 in hepatic IRI remains elusive. We hypothesized that cytoplasmic HMGB1 interacts with binding proteins involved in the hepatocellular response to IRI. In this study, binding proteins of cytoplasmic HMGB1 during hepatic IRI were identified. Liver tissues from rats with warm ischemia reperfusion (WI/R) injury and from normal rats were subjected to cytoplasmic protein extraction. Co-immunoprecipitation using these protein extracts was performed to enrich HMGB1-protein complexes. To separate and identify the immunoprecipitated proteins in eluates, 2-dimensional electrophoresis and subsequent mass spectrometry detection were performed. Two of the identified proteins were verified using Western blotting: betaine-homocysteine S-methyltransferase 1 (BHMT) and cystathionine γ-lyase (CTH). Therefore, our results revealed the binding of HMGB1 to BHMT and CTH in cytoplasm during hepatic WI/R. This finding may help to better understand the cellular response to IRI in the liver and to identify novel molecular targets for reducing ischemic injury.

G-CSF pretreatment aggravates LPS-associated microcirculatory dysfunction and acute liver injury after partial hepatectomy in rats.

Liver dysfunction is a serious complication in the early phase following major liver resection or liver transplantation and might be aggravated by the translocation of bacteria and lipopolysaccharide (LPS). As a preventive strategy, granulocyte colony-stimulating factor (G-CSF) is prophylactically applied in patients who are subjected to major surgery. However, we previously demonstrated that G-CSF can induce LPS sensitization. In this study, we aimed to evaluate the effects of G-CSF pretreatment on hepatic microcirculatory disturbances and postoperative liver dysfunction after 70 % partial hepatectomy (PH) in rats. PH alone was well tolerated by all animals (100 % survival rate, slight liver damage and inflammation). LPS application after 70 % PH caused moderate inflammation, microcirculatory disturbances and hepatic damage and led to a 24-h survival rate of 30 % after the operations. In the G-CSF-LPS-PH group, all of the rats died within 4 h with severe inflammatory responses and liver damage (i.e., pronounced erythrocyte congestion and neutrophil infiltration). Portal hypertension and microcirculatory disorders (i.e., inhomogeneous perfusion, sinusoidal dilatation and reductions on functional capillary density) were more pronounced in the G-CSF-LPS-PH group. In conclusion, increased circulating LPS levels were associated with an imbalanced inflammatory response and microcirculatory dysfunction that preceded liver damage and subsequent dysfunction following surgery. G-CSF-pretreatment aggravated microcirculatory disturbances and liver damage, which might have been related to G-CSF-induced LPS sensitization.

DT390-triTMTP1, a novel fusion protein of diphtheria toxin with tandem repeat TMTP1 peptide, preferentially targets metastatic tumors.

Peptide-based therapies have emerged as one of the most promising therapeutics strategy in cancer-targeted therapy. Using our laboratory newly identified peptide TMTP1 and diphtheria toxin, we developed a new fusion protein that showed remarkable ability to target highly metastatic tumors. Fusion protein toxins were generated by fusing the first 390 amino acids of diphtheria toxin [truncated diphtheria toxin (DT390)] to different repeats of peptide TMTP1 (DT390-TMTP1, DT390-biTMTP1, and DT390-triTMTP1). Efficacies of the recombinant fusion proteins on tumor growth and metastasis were evaluated in vitro and in vivo. DT390-triTMTP1 showed the most powerful toxicity against cancer, which led to tumor growth retardation or regression and prolonged survival of human prostate cancer PC-3M-1E8 subcutaneously bearing or gastric cancer MKN-45 orthotopic nude mice. Increased TUNEL and caspase-3 staining and reduced ki67 staining in tumor cells suggested that the anticancer effects of DT390-triTMTP1 were through selectively inducing apoptosis and inhibiting proliferation of cancer cells. In a murine model of human orthotopic gastric carcinoma, DT390-biTMTP1 significantly inhibited metastases to liver and spleen, while DT390-triTMTP1 not only totally suppressed metastasis but also reduced primary tumors by 66.6%. In the biodistribution test, DT390-triTMTP1 was observed to home to tumor tissue rapidly and lasted over 48 h, with only a transient appearance in liver and kidney immediately after injection. Thus, our present study provided a novel recombinant fusion protein DT390-triTMTP1 with preferential targeting and high cytotoxicity, which may be a promising strategy for the targeted therapy of cancer metastasis.

Sodium thiosulfate refuels the hepatic antioxidant pool reducing ischemia-reperfusion-induced liver injury.

Ischemia-reperfusion injury is a critical liver condition during hepatic transplantation, trauma, or shock. An ischemic deprivation of antioxidants and energy characterizes liver injury in such cases. In the face of increased reactive oxygen production, hepatocytes are vulnerable to the reperfusion driving ROS generation and multiple cell-death mechanisms. In this study, we investigate the importance of hydrogen sulfide as part of the liver's antioxidant pool and the therapeutic potency of the hydrogen sulfide donors sodium sulfide (Na2S, fast releasing) and sodium thiosulfate (STS, Na2S2O3, slow releasing). The mitoprotection and toxicity of STS and Na2S were investigated on isolated mitochondria and a liver perfusion oxidative stress model by adding text-butyl hydroperoxide and hydrogen sulfide donors. The respiratory capacity of mitochondria, hepatocellular released LDH, glutathione, and lipid-peroxide levels were quantified. In addition, wild-type and cystathionine-γ-lyase knockout mice were subjected to warm selective ischemia-reperfusion injury by clamping the main inflow for 1 h followed by reperfusion of 1 or 24 h. A subset of animals was treated with STS shortly before reperfusion. Glutathione, plasma ALT, and lipid-peroxide levels were investigated alongside mitochondrial changes in structure (electron microscopy) and function (intravital microscopy). Liver tissue necrosis quantified 24 h after reperfusion indicates the net effects of the treatment on the organ. STS refuels and protects the endogenous antioxidant pool during liver ischemia-reperfusion injury. In addition, STS-mediated ROS scavenging significantly reduced lipid peroxidation and mitochondrial damage, resulting in better molecular and histopathological preservation of the liver tissue architecture. STS prevents tissue damage in liver ischemia-reperfusion injury by increasing the liver's antioxidant pool, thereby protecting mitochondrial integrity.

[Expression and clinical significance of Sema4C in esophageal cancer, gastric cancer and rectal cancer].

OBJECTIVE: To explore the expression and clinical significance of signal protein Sema4C in esophageal cancer, gastric cancer and rectal cancer. METHODS: Fifty esophageal cancer, 75 gastric cancer, 50 rectal cancer and 20 corresponding normal mucous membrane specimens, collected during the period of January 2008 to December 2010, were detected with streptavidin-peroxidase immunohistochemistry to detect the expression levels of Sema4C. And the relationships of the Sema4C expression with clinicopathological data was analyzed. RESULTS: The expression levels of Sema4C in three kinds of cancers were significantly higher than the corresponding normal mucous membranes (80.0% (n = 40) vs 20.0% (n = 4), 77.3% (n = 58) vs 25.0% (n = 5), 80.0% (n = 40) vs 15.0% (n = 3), all P = 0.000). Furthermore, the percentage of Sema4C positive cells was significantly higher in carcinoma nests of tumors with lymphatic metastasis than those without (90.3% (n = 28) vs 63.2% (n = 12), 85.0% (n = 51) vs 46.7% (n = 7), 92.0% (n = 23) vs 68.0% (n = 17), P = 0.049, 0.005, 0.034). However, no significant correlations were found between the Sema4C expression with gender, age, location of tumors, types of cancer cells, cell differentiation, tumor size, depth of invasion or tumor stage (all P > 0.05). CONCLUSION: There is a high expression of Sema4C in esophageal cancer, gastric cancer and rectal cancer. And it is strongly correlated with lymphatic metastasis. Thus Sema4C may play critical roles in the invasion and lymphatic metastasis of esophageal cancer, gastric cancer and rectal cancer.

Ischemia-Reperfusion Injury in Aged Livers-The Energy Metabolism, Inflammatory Response, and Autophagy.

Because of the lack of adequate organs, the number of patients with end-stage liver diseases, acute liver failure or hepatic malignancies waiting for liver transplantation is constantly increasing. Accepting aged liver grafts is one of the strategies expanding the donor pool to ease the discrepancy between the growing demand and the limited supply of donor organs. However, recipients of organs from old donors may show an increased posttransplantation morbidity and mortality due to enhanced ischemia-reperfusion injury. Energy metabolism, inflammatory response, and autophagy are 3 critical processes which are involved in the aging progress as well as in hepatic ischemia-reperfusion injury. Compared with young liver grafts, impairment of energy metabolism in aged liver grafts leads to lower adenosine triphosphate production and an enhanced generation of free radicals, both aggravating the inflammatory response. The aggravated inflammatory response determines the extent of hepatic ischemia-reperfusion injury and augments the liver damage. Autophagy protects cells by removal of damaged organelles, including dysfunctional mitochondria, a process impaired in aging and involved in ischemia-reperfusion-related apoptotic cell death. Furthermore, autophagic degradation of cellular compounds relieves intracellular adenosine triphosphate level for the energy depressed cells. Strategies targeting the mechanisms involved in energy metabolism, inflammatory response, and autophagy might be especially useful to prevent the increased risk for ischemia-reperfusion injury in aged livers after major hepatic surgery.

Establishment of a rat model: Associating liver partition with portal vein ligation for staged hepatectomy.

BACKGROUND: We adapted the anatomically oriented parenchyma-preserving resection technique for associating liver partition with portal vein ligation (PVL) for staged hepatectomy (ALPPS) in rats and examined the role of revascularization in intrahepatic size regulation. METHODS: We performed the procedures based on anatomic study. The ALPPS procedure consisted of a 70% PVL (occluding the left median, left lateral, and right lobes), parenchymal transection (median lobe) and partial (10%) hepatectomy (PHx; caudate lobe). The transection effect was evaluated by measuring the extent of hepatic atrophy or regeneration of individual liver lobes in the ALPPS and control groups (70% PVL and 10% PHx without transection). The survival rates after stage II resection and collateral formation within the portal vein system was examined. RESULTS: Anatomic study revealed a close spatial relationship between the demarcation line and the middle median hepatic vein. This enabled placing the transection plane without injuring the hepatic vein. Transection was achieved via stepwise clamping, followed by 2-3 parenchyma-preserving piercing sutures on both sides of the clamp. Ligated liver lobes atrophy was significantly enhanced after ALPPS compared with the control group. In contrast, both a significantly greater relative weight of the regenerated lobe and proliferation index on the first postoperative day were observed. All animals tolerated stage II-resection without complications. Portoportal collaterals were only observed in the control group. CONCLUSION: We developed an anatomically precise technique for parenchymal transection. The lack of a dense vascular network between the portalized and deportalized lobes may play an important role in accelerating regeneration and atrophy augmentation.