Dual Lactate Clearance in the Viability Assessment of Livers Donated After Circulatory Death With Ex Situ Normothermic Machine Perfusion.

Perfusate lactate clearance (LC) is considered one of the useful indicators of liver viability assessment during normothermic machine perfusion (NMP); however, the applicable scope and potential mechanisms of LC remain poorly defined in the setting of liver donation after circulatory death. METHODS: The ex situ NMP of end-ischemic human livers was performed using the OrganOx Metra device. We further studied the extracellular signal-regulated kinases (phospho-extracellular signal-regulated kinase1/2 [pERK1/2]) pathway and several clinical parameters of these livers with successful LC (sLC, n = 5) compared with non-sLC (nLC, n = 5) in the perfusate (<2.2 mmol/L at 2 h, n = 5, rapid retrieval without normothermic regional perfusion). RESULTS: We found the pERK1/2 level was substantially higher in the nLC livers than in the sLC livers (n = 5) at 2- and 6-h NMP (P = 0.035 and P = 0.006, respectively). Immunostaining showed that upregulation of pERK1/2 was in both the hepatocytes and cholangiocytes in the nLC livers. Successful LC was associated with a marginally higher glycogen restoration than nLC at 2 h NMP (n = 5, P = 0.065). Furthermore, bile lactate levels in all sLC livers were cleared into the normal range at 6 h NMP, whereas in the nLC group, only 2 livers had lower bile lactate levels, and the other livers had rising bile lactate levels in comparison with the corresponding perfusate lactate levels. The necrosis scores were higher in the nLC than in the sLC livers (n = 5) at 0- and 6-h NMP (P = 0.047 and P = 0.053, respectively). CONCLUSIONS: The dual LC in perfusate and bile can be helpful in evaluating the hypoxic injury of hepatocytes and cholangiocytes during the NMP of donation after circulatory death in liver donors.

Cytoprotection of rat hepatocytes by desipramine in a model of simulated ischemia/reperfusion.

We investigated the cytoprotective effect of desipramine (DMI) during in vitro simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 µM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.

Critical Roles of Calpastatin in Ischemia/Reperfusion Injury in Aged Livers.

Ischemia/reperfusion (I/R) injury unavoidably occurs during hepatic resection and transplantation. Aged livers poorly tolerate I/R during surgical treatment. Although livers have a powerful endogenous inhibitor of calpains, calpastatin (CAST), I/R activates calpains, leading to impaired autophagy, mitochondrial dysfunction, and hepatocyte death. It is unknown how I/R in aged livers affects CAST. Human and mouse liver biopsies at different ages were collected during in vivo I/R. Hepatocytes were isolated from 3-month- (young) and 26-month-old (aged) mice, and challenged with short in vitro simulated I/R. Cell death, protein expression, autophagy, and mitochondrial permeability transition (MPT) between the two age groups were compared. Adenoviral vector was used to overexpress CAST. Significant cell death was observed only in reperfused aged hepatocytes. Before the commencement of ischemia, CAST expression in aged human and mouse livers and mouse hepatocytes was markedly greater than that in young counterparts. However, reperfusion substantially decreased CAST in aged human and mouse livers. In hepatocytes, reperfusion rapidly depleted aged cells of CAST, cleaved autophagy-related protein 5 (ATG5), and induced defective autophagy and MPT onset, all of which were blocked by CAST overexpression. Furthermore, mitochondrial morphology was shifted toward an elongated shape with CAST overexpression. In conclusion, CAST in aged livers is intrinsically short-lived and lost after short I/R. CAST depletion contributes to age-dependent liver injury after I/R.

Antiulcer Activity of Steamed Ginger Extract against Ethanol/HCl-Induced Gastric Mucosal Injury in Rats.

Ginger (Zingiber officianale), the most widely consumed species, is traditionally used as a folk medicine to treat some inflammatory diseases in China and Korea. However, the functional activity of steamed ginger extract on gastric ulcers has not been previously explored. The present study aimed to investigate antiulcer activity of steamed ginger extract (GGE03) against ethanol (EtOH)/HCl-induced gastric ulcers in a rat model. GGE03 (100 mg/kg) was orally administered for 14 days to rats before oral intubation of an EtOH/HCl mixture to induce gastric damage. Pretreatment with GGE03 markedly protected the formation of microscopic pathological damage in the gastric mucosa. Further, administration of GGE03 significantly increased mucosal total nitrate/nitrite production in gastric tissues, and elevated total GSH content, catalase activity and superoxide dismutase (SOD) expression as well as decreasing lipid peroxidation and myeloperoxidase (MPO) activity. Underlying protective mechanisms were examined by assessing inflammation-related genes, including nuclear factor-κB (NF-κB), prostaglandin E2 (PGE2), and pro-inflammatory cytokines levels. GGE03 administration significantly reduced the expression of NF-κB and pro-inflammatory cytokines. Our findings suggest that GGE03 possesses antiulcer activity by attenuating oxidative stress and inflammatory responses.

Resolvin D1 activates the sphingosine-1-phosphate signaling pathway in murine livers with ischemia/reperfusion injury.

Resolvins (Rvs) are endogenous lipid mediators that promote resolution of inflammation and return to homeostasis. We previously reported that RvD1 both facilitates M2 macrophage polarization of Kupffer cells (KCs) and efferocytosis and modulates thioredoxin 2-mediated mitochondrial quality control in liver ischemia/reperfusion (IR) injury. However, the specific cellular or molecular targets of RvD1 remain poorly understood. Sphingosine-1-phosphate (S1P), the natural sphingolipid ligand for a family of G protein-coupled receptors (S1P1-S1P5), regulates lymphocyte circulation and various immune responses. Here we investigated the role of RvD1 in IR-induced hepatocellular damage with a focus on S1P signaling. Male C57BL/6 mice were subjected to partial hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with RvD1 (15 µg/kg, i.p.) 1 h prior to ischemia and immediately before reperfusion. To deplete KCs, liposome clodronate was administered (100 µL/mice, i.v.) 24 h prior to ischemia. Mice were pretreated with VPC23019 (100 µg/kg, i.p.), an antagonist for S1P1/S1P3 10 min prior to initial RvD1 treatment. Exogenous RvD1 attenuated IR-induced hepatocellular damage as evidenced by serum HMGB1 release. RvD1 attenuated the decrease in hepatic S1P concentration induced by IR. KC depletion by liposome clodronate did not alter the effect of RvD1 on sphingosine kinases (SKs) and S1P receptors, suggesting independency of KCs. Moreover, in purified hepatocytes of mice exposed to IR, mRNA expression of SK1, SK2, S1P1, and S1P3 decreased significantly, and this was attenuated by RvD1. Finally, VPC23019 pretreatment abolished the hepatoprotective effects of RvD1 in serum HMGB1 release. Our findings suggest that RvD1 protects the liver against IR injury by activating S1P signaling.

Pterostilbene protects against acetaminophen-induced liver injury by restoring impaired autophagic flux.

An overdose of acetaminophen (APAP) causes liver injury through formation of N-acetyl-p-benzoquinoneimine, which overproduces reactive oxygen species (ROS). Autophagy maintains cellular homeostasis and is regulated by generation of ROS. Pterostilbene (PTE) has been shown to have antioxidant and anti-inflammatory properties. In this study, we investigated the protective mechanisms of PTE against APAP-induced liver injury, focusing on autophagy. ICR mice were intraperitoneally (i.p.) treated with 400 mg/kg of APAP. PTE (15, 30, and 60 mg/kg, i.p.) and chloroquine (CQ, 60 mg/kg, i.p.) were injected 1 h after APAP treatment. Blood and liver tissues were isolated 6 h after APAP treatment. PTE decreased serum aminotransferase activities and hepatic oxidative stress; this protective effect was abolished by CQ. APAP impaired autophagic flux, as evidenced by increased microtubule-associated protein-1 light chain 3-II and p62 protein expression; this impaired autophagic flux was restored by PTE, while CQ abolished this effect. APAP decreased beclin-1 and autophagy related protein 7 protein expressions, while PTE attenuated these decreases. PTE increased the lysosome-associated membrane protein-2 protein expression and decreased the mammalian target of rapamycin and Unc-51 like autophagy activating kinase 1 phosphorylation. Our findings suggest that PTE protects against APAP-induced hepatotoxicity by enhancing autophagic flux.

BST106 Protects against Cartilage Damage by Inhibition of Apoptosis and Enhancement of Autophagy in Osteoarthritic Rats.

Chrysanthemum zawadskii var. latilobum (CZ) has been used as a traditional medicine in Asian countries for the treatment of inflammatory diseases. Recently, CZ extract was shown to inhibit differentiation of osteoclasts and provide protection against rheumatoid arthritis. The aim of this study was to investigate the molecular mechanisms of BST106, the ethanol extract of CZ, for cartilage protection in monosodium iodoacetate (MIA)-induced osteoarthritis (OA), particularly focusing on apoptosis and autophagy. BST106 (50, 100, and 200 mg/kg) was orally administered once daily to MIA-induced OA rats. Swelling, limping, roentgenography, and histomorphological changes were assessed 28 d after MIA injection. Biochemical parameters for matrix metalloproteinase (MMP), apoptosis, and autophagy were also assessed. BST106 ameliorated the severity of swelling and limping after MIA injection. Roentgenographic and histomorphological examinations revealed that BST106 reduced MIA-induced cartilage damage. BST106 decreased MIA-induced increases in MMP-2 and MMP-13 mRNA levels. Increased levels of serum cartilage oligomeric matrix protein and glycosaminoglycan release were attenuated by BST106. Furthermore, BST106 suppressed the protein expression of proapoptotic molecules and increased the protein expression of autophagosome- and autolysosome-related molecules. These findings indicate that BST106 protects against OA-induced cartilage damage by inhibition of the apoptotic pathway and restoration of impaired autophagic flux.

Genipin protects the liver from ischemia/reperfusion injury by modulating mitochondrial quality control.

Hepatic ischemia and reperfusion (IR) injury is closely linked to oxidative mitochondrial damage. Since mitochondrial quality control (QC) plays a pivotal role in the recovery of impaired mitochondrial function, mitochondrial QC has emerged as a potential therapeutic target. Genipin, an iridoid compound from Gardenia jasminoides, has been showed antioxidant and anti-inflammatory properties. In this study, we investigated the hepatoprotective mechanism of genipin against IR-induced hepatic injury, particularly focusing on mitochondrial QC. Male C57BL/6 mice underwent liver ischemia for 60min, followed by reperfusion for 6h. Genipin (100mg/kg, i.p.) or vehicle (10% Tween 80 in saline) was administrated to mice 1h before ischemia. Liver and blood samples were collected 6h after reperfusion. Hepatic IR increased hepatocellular oxidative damage and induced mitochondrial dysfunction. These phenomena were ameliorated by genipin. Hepatic IR also increased the level of mitochondrial fission, such as dynamin-related protein 1 and the level of PINK1 protein expression. In contrast, hepatic IR decreased the levels of mitochondrial biogenesis related proteins (e.g., peroxisome proliferator-activated receptor gamma coactivator 1α, nuclear respiratory factor 1, and mitochondrial transcription factor A), mitophagy related proteins (e.g., Parkin), and fusion related protein (e.g., mitofusin 2). Furthermore, hepatic IR decreased the levels of sirtuin1 protein and phosphorylation of AMP-activated protein kinase. Genipin alleviated these IR-induced changes. These data indicate that genipin protects against IR-induced hepatic injury via regulating mitochondrial QC. (225/250).

Enhanced nitric oxide-mediated autophagy contributes to the hepatoprotective effects of ischemic preconditioning during ischemia and reperfusion.

Ischemic preconditioning (IPC) protects against liver ischemia/reperfusion (I/R) injury. Autophagy is an essential cytoprotective system that is rapidly activated by multiple stressors. Nitric oxide (NO) acts as an inducer of IPC. We examined the impact of autophagy in liver IPC and its regulation by NO. Male C57BL/6 mice were subjected to 60 min of hepatic ischemia followed by 6 h of reperfusion. IPC was achieved for 10 min of ischemia followed by 10 min of reperfusion prior to sustained ischemia. N(ω)-Nitro-l-arginine methyl ester (L-NAME, 15 mg/kg, i.v., all NOS inhibitor) and aminoguanidine (AG, 10 mg/kg, i.v., iNOS inhibitor) were injected 10 min before IPC. SB203580 (10 mg/kg, i.p., p38 inhibitor) was injected 30 min before IPC. I/R increased serum alanine aminotransferase activity. IPC attenuated this increase, which was abolished by L-NAME, but not AG. Microtubule-associated protein-1 light chain 3-II levels increased and p62 protein levels decreased after I/R; these changes were augmented by IPC and abolished by L-NAME. I/R increased liver protein expression of autophagy-related protein (Atg)12-Atg5 complex and lysosome-associated membrane protein-2. IPC augmented the expression of these proteins, which were abolished by L-NAME, but not AG. IPC also augmented the level of phosphorylated p38 MAPK induced by I/R and this phosphorylation was abolished by L-NAME. Our findings suggest that IPC-mediated NO protects against I/R-induced liver injury by enhancing autophagic flux.

Opuntia ficus-indica seed attenuates hepatic steatosis and promotes M2 macrophage polarization in high-fat diet-fed mice.

Opuntia ficus-indica (L.) is a popular edible plant that possesses considerable nutritional value and exhibits diverse biological actions including anti-inflammatory and antidiabetic activities. In this study, we hypothesized that DWJ504, an extract of O ficus-indica seed, would ameliorate hepatic steatosis and inflammation by regulating hepatic de novo lipogenesis and macrophage polarization against experimental nonalcoholic steatohepatitis. Mice were fed a normal diet or a high-fat diet (HFD) for 10 weeks. DWJ504 (250, 500, and 1000 mg/kg) or vehicle (0.5% carboxymethyl cellulose) were orally administered for the last 4 weeks of the 10-week HFD feeding period. DWJ504 treatment remarkably attenuated HFD-induced increases in hepatic lipid content and hepatocellular damage. DWJ504 attenuated increases in sterol regulatory element-binding protein 1 and carbohydrate-responsive element-binding protein expression and a decrease in carnitine palmitoyltransferase 1A. Although DWJ504 augmented peroxisome proliferator-activated receptor α protein expression, it attenuated peroxisome proliferator-activated receptor γ expression. Moreover, DWJ504 promoted hepatic M2 macrophage polarization as indicated by attenuation of the M1 marker genes and enhancement of M2 marker genes. Finally, DWJ504 attenuated expression of toll-like receptor 4, nuclear factor κB, tumor necrosis factor α, interleukin 6, TIR-domain-containing adapter-inducing interferon ß, and interferon ß levels. Our results demonstrate that DWJ504 prevented intrahepatic lipid accumulation, induced M2 macrophage polarization, and suppressed the toll-like receptor 4-mediated inflammatory signaling pathway. Thus, DWJ504 has therapeutic potential in the prevention of nonalcoholic fatty liver disease.