Volatile Flavor Profile and Sensory Properties of Vegetable Soybean.

The volatile flavor profiles and sensory properties of different vegetable soybean varieties popularized and cultivated in China for 20, 10, and 2 years (TW292, X3, and SX6, respectively) were investigated. Nutrient composition analysis revealed that TW292 had a high soluble protein and soluble sugar content but low fat content. The total free amino acid content (15.43 mg/g) and umami free amino acid content (6.08 mg/g) of SX6 were significantly higher (p < 0.05) than those of the other varieties. An electronic tongue effectively differentiated between the umami and sweetness characteristics of the vegetable soybeans. Differences in sensory evaluation results were mainly reflected in texture and taste. A total of 41 volatile compounds were identified through HS-SPME-GC-MS, and the main flavor compounds were 1-octen-3-ol, hexanal, (Z)-2-heptenal, 2-octene, nonanal, (Z)-2-decenal, and 3,5-octadien-2-one. However, the volatile composition of different vegetable soybean varieties exhibited large variability in type and relative contents. Considerable differences in nutritional, organoleptic, and aroma characteristics were found among different varieties. The results of this study will provide a good basis for the assessment and application of the major vegetable soybean varieties grown in China.

Cardiospecific Overexpression of ATPGD1 (Carnosine Synthase) Increases Histidine Dipeptide Levels and Prevents Myocardial Ischemia Reperfusion Injury.

BACKGROUND Myocardial ischemia reperfusion (I/R) injury is associated with complex pathophysiological changes characterized by pH imbalance, the accumulation of lipid peroxidation products acrolein and 4-hydroxy trans-2-nonenal, and the depletion of ATP levels. Cardioprotective interventions, designed to address individual mediators of I/R injury, have shown limited efficacy. The recently identified enzyme ATPGD1 (Carnosine Synthase), which synthesizes histidyl dipeptides such as carnosine, has the potential to counteract multiple effectors of I/R injury by buffering intracellular pH and quenching lipid peroxidation products and may protect against I/R injury. METHODS AND RESULTS We report here that ß-alanine and carnosine feeding enhanced myocardial carnosine levels and protected the heart against I/R injury. Cardiospecific overexpression of ATPGD1 increased myocardial histidyl dipeptides levels and protected the heart from I/R injury. Isolated cardiac myocytes from ATPGD1-transgenic hearts were protected against hypoxia reoxygenation injury. The overexpression of ATPGD1 prevented the accumulation of acrolein and 4-hydroxy trans-2-nonenal-protein adducts in ischemic hearts and delayed acrolein or 4-hydroxy trans-2-nonenal-induced hypercontracture in isolated cardiac myocytes. Changes in the levels of ATP, high-energy phosphates, intracellular pH, and glycolysis during low-flow ischemia in the wild-type mice hearts were attenuated in the ATPGD1-transgenic hearts. Two natural dipeptide analogs (anserine and balenine) that can either quench aldehydes or buffer intracellular pH, but not both, failed to protect against I/R injury. CONCLUSIONS Either exogenous administration or enhanced endogenous formation of histidyl dipeptides prevents I/R injury by attenuating changes in intracellular pH and preventing the accumulation of lipid peroxidation derived aldehydes.

Carnosine Supplementation Enhances Post Ischemic Hind Limb Revascularization.

High (millimolar) concentrations of the histidine containing dipeptide - carnosine (ß-alanine-L-histidine) are present in the skeletal muscle. The dipeptide has been shown to buffer intracellular pH, chelate transition metals, and scavenge lipid peroxidation products; however, its role in protecting against tissue injury remains unclear. In this study, we tested the hypothesis that carnosine protects against post ischemia by augmenting HIF-1α angiogenic signaling by Fe2+ chelation. We found that wild type (WT) C57BL/6 mice, subjected to hind limb ischemia (HLI) and supplemented with carnosine (1g/L) in drinking water, had improved blood flow recovery and limb function, enhanced revascularization and regeneration of myocytes compared with HLI mice placed on water alone. Carnosine supplementation enhanced the bioavailability of carnosine in the ischemic limb, which was accompanied by increased expression of proton-coupled oligopeptide transporters. Consistent with our hypothesis, carnosine supplementation augmented HIF-1α and VEGF expression in the ischemic limb and the mobilization of proangiogenic Flk-1+/Sca-1+ cells into circulation. Pretreatment of murine myoblast (C2C12) cells with octyl-D-carnosine or carnosine enhanced HIF-1α protein expression, VEGF mRNA levels and VEGF release under hypoxic conditions. Similarly pretreatment of WT C57/Bl6 mice with carnosine showed enhanced blood flow in the ischemic limb following HLI surgery. In contrast, pretreatment of hypoxic C2C12 cells with methylcarcinine, a carnosine analog, lacking Fe2+ chelating capacity, had no effect on HIF-1α levels and VEGF release. Collectively, these data suggest that carnosine promotes post ischemic revascularization via augmentation of pro-angiogenic HIF-1α/VEGF signaling, possibly by Fe2+ chelation.

TRPA1 channel contributes to myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) results in the generation of free radicals, accumulation of lipid peroxidation-derived unsaturated aldehydes, variable angina (pain), and infarction. The transient receptor potential ankyrin 1 (TRPA1) mediates pain signaling and is activated by unsaturated aldehydes, including acrolein and 4-hydroxynonenal. The contribution of TRPA1 (a Ca2+-permeable channel) to I/R-induced myocardial injury is unknown. We tested the hypothesis that cardiac TRPA1 confers myocyte sensitivity to aldehyde accumulation and promotes I/R injury. Although basal cardiovascular function in TRPA1-null mice was similar to that in wild-type (WT) mice, infarct size was significantly smaller in TRPA1-null mice than in WT mice (34.1 ± 9.3 vs. 14.3 ± 9.9% of the risk region, n = 8 and 7, respectively, P < 0.05), despite a similar I/R-induced area at risk (40.3 ±8.4% and 42.2 ± 11.3% for WT and TRPA1-null mice, respectively) after myocardial I/R (30 min of ischemia followed by 24 h of reperfusion) in situ. Positive TRPA1 immunofluorescence was present in murine and human hearts and was colocalized with connexin43 at intercalated disks in isolated murine cardiomyocytes. Cardiomyocyte TRPA1 was confirmed by quantitative RT-PCR, DNA sequencing, Western blot analysis, and electrophysiology. A role of TRPA1 in cardiomyocyte toxicity was demonstrated in isolated cardiomyocytes exposed to acrolein, an I/R-associated toxin that induces Ca2+ accumulation and hypercontraction, effects significantly blunted by HC-030031, a TRPA1 antagonist. Protection induced by HC-030031 was quantitatively equivalent to that induced by SN-6, a Na+/Ca2+ exchange inhibitor, further supporting a role of Ca2+ overload in acrolein-induced cardiomyocyte toxicity. These data indicate that cardiac TRPA1 activation likely contributes to I/R injury and, thus, that TRPA1 may be a novel therapeutic target for decreasing myocardial I/R injury. NEW & NOTEWORTHY Transient receptor potential ankyrin 1 (TRPA1) activation mediates increased blood flow, edema, and pain reception, yet its role in myocardial ischemia-reperfusion (I/R) injury is unknown. Genetic ablation of TRPA1 significantly decreased myocardial infarction after I/R in mice. Functional TRPA1 in cardiomyocytes was enriched in intercalated disks and contributed to acrolein-induced Ca2+ overload and hypercontraction. These data indicate that I/R activation of TRPA1 worsens myocardial infarction; TRPA1 may be a potential target to mitigate I/R injury.

Fibrin-mediated integrin signaling plays a critical role in hepatic regeneration after partial hepatectomy in mice.

UNLABELLED:  Background. The regenerative capacity of the liver is critical for proper responses to injury. Fibrin extracellular matrix (ECM) deposition is a common response to insult and contributes to inflammatory liver injury. However, the role of this matrix in hepatic regeneration has not been determined. OBJECTIVE: The purpose of this study was first to determine the role of fibrin ECM in hepatic regeneration followed by the role of the fibrin-binding αvß3 integrin in mediating this effect. MATERIAL AND METHODS: C57Bl/6J (WT) or PAI-1 knockout (KO) mice underwent 70% partial hepatectomy (PHx); plasma and histologic indices of regeneration were determined, as well as expression of key genes involved in hepatic regeneration. RESULTS: PHx promoted transient fibrin deposition by activating coagulation and concomitantly decreasing fibrinolysis. Inhibiting fibrin deposition, either by blocking thrombin (hirudin) in WT mice or by knocking out PAI-1, was associated with a decrease in hepatocyte proliferation after PHx. This strongly suggested a role for fibrin ECM in liver regeneration. To investigate if αvß3 integrin mediates this action, we tested the effects of the anti-αvß3 cyclic peptide RGDfV in animals after PHx. As was observed with inhibition of fibrin deposition, competitive inhibition of αvß3 integrin delayed regeneration after PHx, while not affecting fibrin deposition. These effects of RGDfV correlated with impaired angiogénesis and STAT3 signaling, as well as transient endothelial dysfunction. In conclusion, these data suggest that αvß3 integrin plays an important role in coordinating hepatocyte division during liver regeneration after PHx via crosstalk with fibrin ECM.

Resolvin D2 Enhances Postischemic Revascularization While Resolving Inflammation.

BACKGROUND: Resolvins are lipid mediators generated by leukocytes during the resolution phase of inflammation. They have been shown to regulate the transition from inflammation to tissue repair; however, it is unknown whether resolvins play a role in tissue revascularization following ischemia. METHODS: We used a murine model of hind limb ischemia (HLI), coupled with laser Doppler perfusion imaging, microcomputed tomography, and targeted mass spectrometry, to assess the role of resolvins in revascularization and inflammation resolution. RESULTS: In mice undergoing HLI, we identified resolvin D2 (RvD2) in bone marrow and skeletal muscle by mass spectrometry (n=4-7 per group). We also identified RvD2 in skeletal muscle biopsies from humans with peripheral artery disease. Monocytes were recruited to skeletal muscle during HLI and isolated monocytes produced RvD2 in a lipoxygenase-dependent manner. Exogenous RvD2 enhanced perfusion recovery in HLI and microcomputed tomography of limb vasculature revealed greater volume, with evidence of tortuous arterioles indicative of arteriogenesis (n=6-8 per group). Unlike other treatment strategies for therapeutic revascularization that exacerbate inflammation, RvD2 did not increase vascular permeability, but reduced neutrophil accumulation and the plasma levels of tumor necrosis factor-α and granulocyte macrophage colony-stimulating factor. In mice treated with RvD2, histopathologic analysis of skeletal muscle of ischemic limbs showed more regenerating myocytes with centrally located nuclei. RvD2 enhanced endothelial cell migration in a Rac-dependent manner, via its receptor, GPR18, and Gpr18-deficient mice had an endogenous defect in perfusion recovery following HLI. Importantly, RvD2 rescued defective revascularization in diabetic mice. CONCLUSIONS: RvD2 stimulates arteriogenic revascularization during HLI, suggesting that resolvins may be a novel class of mediators that both resolve inflammation and promote arteriogenesis.

Genetic Deficiency of Glutathione S-Transferase P Increases Myocardial Sensitivity to Ischemia-Reperfusion Injury.

RATIONALE: Myocardial ischemia-reperfusion (I/R) results in the generation of oxygen-derived free radicals and the accumulation of lipid peroxidation-derived unsaturated aldehydes. However, the contribution of aldehydes to myocardial I/R injury has not been assessed. OBJECTIVE: We tested the hypothesis that removal of aldehydes by glutathione S-transferase P (GSTP) diminishes I/R injury. METHODS AND RESULTS: In adult male C57BL/6 mouse hearts, Gstp1/2 was the most abundant GST transcript followed by Gsta4 and Gstm4.1, and GSTP activity was a significant fraction of the total GST activity. mGstp1/2 deletion reduced total GST activity, but no compensatory increase in GSTA and GSTM or major antioxidant enzymes was observed. Genetic deficiency of GSTP did not alter cardiac function, but in comparison with hearts from wild-type mice, the hearts isolated from GSTP-null mice were more sensitive to I/R injury. Disruption of the GSTP gene also increased infarct size after coronary occlusion in situ. Ischemia significantly increased acrolein in hearts, and GSTP deficiency induced significant deficits in the metabolism of the unsaturated aldehyde, acrolein, but not in the metabolism of 4-hydroxy-trans-2-nonenal or trans-2-hexanal; on ischemia, the GSTP-null hearts accumulated more acrolein-modified proteins than wild-type hearts. GSTP deficiency did not affect I/R-induced free radical generation, c-Jun N-terminal kinase activation, or depletion of reduced glutathione. Acrolein exposure induced a hyperpolarizing shift in INa, and acrolein-induced cell death was delayed by SN-6, a Na(+)/Ca(++) exchange inhibitor. Cardiomyocytes isolated from GSTP-null hearts were more sensitive than wild-type myocytes to acrolein-induced protein crosslinking and cell death. CONCLUSIONS: GSTP protects the heart from I/R injury by facilitating the detoxification of cytotoxic aldehydes, such as acrolein.

PKCε contributes to chronic ethanol-induced steatosis in mice but not inflammation and necrosis.

BACKGROUND: Protein kinase C epsilon (PKCε) has been shown to play a role in experimental steatosis by acute alcohol. The "two-hit" hypothesis implies that preventing steatosis should blunt more advanced liver damage (e.g., inflammation and necrosis). However, the role of PKCε in these pathologies is not yet known. The goal of this current work was to address this question in a model of chronic alcohol exposure using antisense oligonucleotides (ASO) against PKCε. METHODS: Accordingly, PKCε ASO- and saline-treated mice were fed high-fat control or ethanol (EtOH)-containing enteral diets for 4 weeks. RESULTS: Chronic EtOH exposure significantly elevated hepatic lipid pools as well as activated PKCε. The PKCε ASO partially blunted the increases in hepatic lipids caused by EtOH. Administration of PKCε ASO also completely prevented the increase in the expression of fatty acid synthase, and tumor necrosis factor α caused by EtOH. Despite these protective effects, the PKCε ASO was unable to prevent the increases in inflammation and necrosis caused by chronic EtOH. These latter results correlated with an inability of the PKCε ASO to blunt the up-regulation of plasminogen activator inhibitor-1 (PAI-1) and the accumulation of fibrin. Importantly, PAI-1 has been previously shown to more robustly mediate inflammation and necrosis (vs. steatosis) after chronic EtOH exposure. CONCLUSIONS: This study identifies a novel potential mechanism where EtOH, independent of steatosis, can contribute to liver damage. These results also suggest that PAI-1 and fibrin accumulation may be at the center of this PKCε-independent pathway.

Zinc deficiency exacerbates diabetic down-regulation of Akt expression and function in the testis: essential roles of PTEN, PTP1B and TRB3.

Since zinc (Zn) plays an important role in the spermatogenesis and Zn deficiency exacerbated diabetes-induced testicular apoptosis, the present study investigated the effect of Zn deficiency on diabetes-induced testicular Akt-mediated glucose metabolism changes and inflammation. Zn deficiency was induced by chronic treatment of normal and diabetic mice with the Zn chelator N,N,N',N', tetrakis (2-pyridylmethyl) ethylenediaminepentaethylene (TPEN). After diabetes onset induced by streptozotocin, both diabetic and age-matched control mice were given TPEN intraperitoneally for 4 months. Western blotting assay revealed that Akt-mediated glucose metabolism signaling was down-regulated in the diabetic testis and was further decreased in diabetic mice with Zn deficiency, reflected by reduced phosphorylation of both Akt and GSK-3ß and increased phosphorylation of glycogen synthase along with a disarrangement of fatty acid metabolism (increased expression of PPAR-α and decreased adenosine-monophosphate-activated protein kinase phosphorylation). Testicular expressions of plasminogen activator inhibitor-1 and intracellular adhesion molecule-1 as inflammatory factors were increased in the TPEN or diabetes-alone group, but not additive in the group of diabetes with Zn deficiency. A mechanistic study showed that Akt negative regulators phosphatase and tensin homology deleted on chromosome 10 (PTEN), protein tyrosine phosphatases 1B and Tribbles 3 all increased in diabetic testis and further increased in the testis of diabetic mice with Zn deficiency. These studies suggest that Zn deficiency significantly exacerbated diabetic down-regulation of Akt expression and function, most likely by up-regulation of Akt negative regulators. Therefore, prevention of Zn deficiency for diabetic patients is important in order to avoid the exacerbation of diabetic inhibition of glucose metabolism in the testis.

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis.

PAI-1 has been shown to be both profibrotic and antifibrotic in animal models of hepatic fibrosis. Although these models have similarities to human fibrotic liver disease, no rodent model completely recapitulates the clinical situation; indeed, transaminase values in most models of hepatic fibrosis are much higher than in chronic liver diseases in humans. Here, wild-type and PAI-1(-/-) mice were administered AngII (500 ng/kg/min) for 4 weeks. ECM accumulation was evaluated by Sirius red staining, hydroxyproline content, and fibrin and collagen immunostaining. Induction of pro-fibrotic genes was assessed by real-time RT-PCR. Despite the absence of any significant liver damage, AngII infusion increased the deposition of hepatic collagen and fibrin ECM, with a perisinusoidal pattern. PAI-1(-/-) mice were protected from these ECM changes, indicating a causal role of PAI-1 in this fibrosis model. Protection in the knockout strain correlated with a blunted increase in αSMA, and elevated activities of matrix metalloproteinases (MMP2, MMP9). These data suggest that PAI-1 plays a critical role in mediating fibrosis caused by AngII and lends weight-of-evidence to a pro-fibrotic role of this protein in liver. Furthermore, the current study proposes a new model of 'pure' hepatic fibrosis in mice with little inflammation or hepatocyte death.

Exacerbation of diabetes-induced testicular apoptosis by zinc deficiency is most likely associated with oxidative stress, p38 MAPK activation, and p53 activation in mice.

Since diabetes induces testicular oxidative damage and cell death, and zinc (Zn) plays an important role in the spermatogenesis, the objective of the present study was to define the effects of Zn deficiency on diabetes-induced testicular apoptosis and associated mechanisms. Zn deficiency was induced by chronic treatment of normal and diabetic mice with N,N,N',N'-tetrakis (2-pyridylemethyl) ethylenediamine (TPEN) chelation. After diabetes onset, mice were given intraperitoneally TPEN at 5mg/kg daily for four months, which, like diabetes, induced a significant decrease in testicular Zn level. TUNEL staining revealed that testicular apoptosis was significantly increased along with an increased Bax/Bcl-2 ratio, in diabetic mice and TPEN-treated normal mice. Zn deficiency significantly exacerbated diabetes-induced testicular apoptosis, along with significantly increased oxidative and nitrosative damage and down-regulation of antioxidant Nrf2 expression. Increased oxidative stress was associated with an increase in activation of p38 MAPK and p53 protein in diabetic testis, which was worsened in the testes of diabetic mice with Zn deficiency. Diabetes also induced a significant increase in endoplasmic reticulum stress and associated cell death, which was not affected by Zn deficiency. These results suggest that like diabetes, chronic depletion of Zn with TPEN induces testicular oxidative stress and damage, along with the activation of p38 MAPK and p53 signaling and mitochondria-related apoptotic cell death. Therefore, prevention of Zn deficiency for diabetic patients is important in order to avoid the exacerbation of diabetic effects on testicular cells death.

PAI-1 plays a protective role in CCl4-induced hepatic fibrosis in mice: role of hepatocyte division.

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein that has been shown to play a role in experimental fibrosis caused by bile duct ligation (BDL) in mice. However, its role in more severe models of hepatic fibrosis (e.g., carbon tetrachloride; CCl(4)) has not been determined and is important for extrapolation to human disease. Wild-type or PAI-1 knockout mice were administered CCl(4) (1 ml/kg body wt ip) 2x/wk for 4 wk. Plasma (e.g., transaminase activity) and histological (e.g., Sirius red staining) indexes of liver damage and fibrosis were evaluated. Proliferation and apoptosis were assessed by PCNA and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively, as well as by indexes of cell cycle (e.g., p53, cyclin D1). In contrast to previous studies with BDL, hepatic fibrosis was enhanced in PAI-1(-/-) mice after chronic CCl(4) administration. Indeed, all indexes of liver damage were elevated in PAI-1(-/-) mice compared with wild-type mice. This enhanced liver damage correlated with impaired hepatocyte proliferation. A similar effect on proliferation was observed after one bolus dose of CCl(4), without concomitant increases in liver damage. Under these conditions, a decrease in phospho-p38, coupled with elevated p53 protein, was observed; these results suggest impaired proliferation and a potential G(1)/S cell cycle arrest in PAI-1(-/-) mice. These data suggest that PAI-1 may play multiple roles in chronic liver diseases, both protective and damaging, the latter mediated by its influence on inflammation and fibrosis and the former via helping maintain hepatocyte division after an injury.

Fibrin accumulation plays a critical role in the sensitization to lipopolysaccharide-induced liver injury caused by ethanol in mice.

UNLABELLED: The early stages of alcohol-induced liver injury involve chronic inflammation. Whereas mechanisms by which this effect is mediated are not completely understood, it is hypothesized that enhanced sensitivity to circulating lipopolysaccharide (LPS) contributes to this process. It has recently been shown that ethanol induces activation of plasminogen activator inhibitor-1 (PAI-1). PAI-1 causes fibrin accumulation in liver by inhibiting degradation of fibrin (fibrinolysis). LPS also enhances fibrin accumulation by activating the coagulation cascade. It was therefore hypothesized that ethanol will synergistically increase fibrin accumulation caused by LPS, enhancing liver damage. Accordingly, the effect of ethanol pretreatment on LPS-induced liver injury and fibrin deposition was determined in mice. Ethanol enhanced liver damage caused by LPS, as determined by plasma parameters and histological indices of inflammation and damage. This effect was concomitant with a significant increase in PAI-1 expression. Extracellular fibrin accumulation caused by LPS was also robustly increased by ethanol preexposure. Coadministration of the thrombin inhibitor hirudin or the MEK (mitogen-activated protein kinase) inhibitor U0126 significantly attenuated the enhanced liver damage caused by ethanol preexposure; this protection correlated with a significant blunting of the induction of PAI-1 caused by ethanol/LPS. Furthermore, thrombin/MEK inhibition prevented the synergistic effect of ethanol on the extracellular accumulation of fibrin caused by LPS. Similar protective effects on fibrin accumulation were observed in tumor necrosis factor receptor 1 (TNFR-1)(-/-) mice or in wild-type injected with PAI-1-inactivating antibody. CONCLUSION: These results suggest that enhanced LPS-induced liver injury caused by ethanol is mediated, at least in part, by fibrin accumulation in livers, mediated by an inhibition of fibrinolysis by PAI-1. These results also support the hypothesis that fibrin accumulation may play a critical role in the development of early alcohol-induced liver injury.

PKCepsilon plays a causal role in acute ethanol-induced steatosis.

Steatosis is a critical stage in the pathology of alcoholic liver disease (ALD), and preventing steatosis could protect against later stages of ALD. PKCepsilon has been shown to contribute to hepatic steatosis in experimental non-alcoholic fatty liver disease (NAFLD); however, the role of PKCepsilon in ethanol-induced steatosis has not been determined. The purpose of this study was to therefore test the hypothesis that PKCepsilon contributes to ethanol-induced steatosis. Accordingly, the effect of acute ethanol on indices of hepatic steatosis and insulin signaling were determined in PKCepsilon knockout mice and in wild-type mice that received an anti-sense oligonucleotide (ASO) to knockdown PKCepsilon expression. Acute ethanol (6g/kg i.g.) caused a robust increase in hepatic non-esterified free fatty acids (NEFA), which peaked 1h after ethanol exposure. This increase in NEFA was followed by elevated diacylglycerols (DAG), as well as by the concomitant activation of PKCepsilon. Acute ethanol also changed the expression of insulin-responsive genes (i.e. increased G6Pase, downregulated GK), in a pattern indicative of impaired insulin signaling. Acute ethanol exposure subsequently caused a robust increase in hepatic triglycerides. The accumulation of triglycerides caused by ethanol was blunted in ASO-treated or in PKCepsilon(-/-) mice. Taken together, these data suggest that the increase in NEFA caused by hepatic ethanol metabolism leads to an increase in DAG production via the triacylglycerol pathway. DAG then subsequently activates PKCepsilon, which then exacerbates hepatic lipid accumulation by inducing insulin resistance. These data also suggest that PKCepsilon plays a causal role in at least the early phases of ethanol-induced liver injury.

New role of resistin in lipopolysaccharide-induced liver damage in mice.

Studies in rodents suggest that the adipocytokine resistin causes insulin resistance via impairing normal insulin signaling. However, in humans, resistin may play a more important role in inflammation than in insulin resistance. Whether resistin contributes to inflammation in rodents is unclear. Therefore, the purpose of the present study was to determine the effect of resistin exposure on the basal and stimulated [lipopolysaccharide (LPS)] inflammatory response in mouse liver in vivo. Resistin alone had no major effects on hepatic expression of insulin-responsive genes, either in the presence or absence of LPS. Although it had no effect alone, resistin significantly enhanced hepatic inflammation and necrosis caused by LPS. Resistin increased expression of proinflammatory genes, e.g., plasminogen activator inhibitor (PAI)-1, and activity of mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase 1/2, caused by LPS, but had little effect on anti-inflammatory gene expression. Resistin also enhanced fibrin deposition (an index of hemostasis) caused by LPS. The increase in PAI-1 expression, fibrin deposition, and liver damage caused by LPS + resistin was almost completely prevented either by inhibiting the coagulation cascade, hirudin, or by blocking MAP kinase signaling, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene], indicating that these pathways play a causal role in observed enhanced liver damage caused by resistin. Taken together, the augmentation of LPS-induced liver damage caused by resistin seems to involve, at least in part, up-regulation of hepatic inflammation via mechanisms most likely involving the coagulation cascade and fibrin accumulation. These data also suggest that resistin may have proinflammatory roles in mouse liver independent of its effects on insulin signaling, analogous to previous work in humans.

Contribution of the sympathetic hormone epinephrine to the sensitizing effect of ethanol on LPS-induced liver damage in mice.

It is well known that ethanol preexposure sensitizes the liver to LPS hepatotoxicity. The mechanisms by which ethanol enhances LPS-induced liver injury are not completely elucidated but are known to involve an enhanced inflammatory response. Ethanol exposure also increases the metabolic rate of the liver, and this effect of ethanol on liver is mediated, at least in part, by the sympathetic hormone, epinephrine. However, whether or not the sympathetic nervous system also contributes to the sensitizing effect of ethanol preexposure on LPS-induced liver damage has not been determined. The purpose of this study was therefore to test the hypotheses that 1) epinephrine preexposure enhances LPS-induced liver damage (comparable to that of ethanol preexposure) and that 2) the sympathetic nervous system contributes to the sensitizing effect of ethanol. Accordingly, male C57BL/6J mice were administered epinephrine for 5 days (2 mg/kg per day) via osmotic pumps or bolus ethanol for 3 days (6 g/kg per day) by gavage. Twenty-four hours later, mice were injected with LPS (10 mg/kg ip). Both epinephrine and ethanol preexposure exacerbated LPS-induced liver damage and inflammation. Concomitant administration of propranolol with ethanol significantly attenuated the sensitizing effect of ethanol on LPS-induced liver damage. These data support the hypothesis that the sympathetic nervous system contributes, at least in part, to the mechanism of the sensitizing effect of ethanol. These results also suggest that sympathetic tone may contribute to the initiation and progression of alcoholic liver disease.

Subhepatotoxic exposure to arsenic enhances lipopolysaccharide-induced liver injury in mice.

Exposure to arsenic via drinking water is a serious health concern in the US. Whereas studies have identified arsenic alone as an independent risk factor for liver disease, concentrations of arsenic required to damage this organ are generally higher than found in the US water supply. The purpose of the current study was to test the hypothesis that arsenic (at subhepatotoxic doses) may also sensitize the liver to a second hepatotoxin. To test this hypothesis, the effect of chronic exposure to arsenic on liver damage caused by acute lipopolysaccharide (LPS) was determined in mice. Male C57Bl/6J mice (4-6 weeks) were exposed to arsenic (49 ppm as sodium arsenite in drinking water). After 7 months of exposure, animals were injected with LPS (10 mg/kg i.p.) and sacrificed 24 h later. Arsenic alone caused no overt hepatotoxicity, as determined by plasma enzymes and histology. In contrast, arsenic exposure dramatically enhanced liver damage caused by LPS, increasing the number and size of necroinflammatory foci. This effect of arsenic was coupled with increases in indices of oxidative stress (4-HNE adducts, depletion of GSH and methionine pools). The number of apoptotic (TUNEL) hepatocytes was similar in the LPS and arsenic/LPS groups. In contrast, arsenic pre-exposure blunted the increase in proliferating (PCNA) hepatocytes caused by LPS; this change in the balance between cell death and proliferation was coupled with a robust loss of liver weight in the arsenic/LPS compared to the LPS alone group. The impairment of proliferation after LPS caused by arsenic was also coupled with alterations in the expression of key mediators of cell cycle progression (p27, p21, CDK6 and Cyclin D1). Taken together, these results suggest that arsenic, at doses that are not overtly hepatotoxic per se, significantly enhances LPS-induced liver injury. These results further suggest that arsenic levels in the drinking water may be a risk modifier for the development of chronic liver diseases.

Early growth response-1 contributes to galactosamine/lipopolysaccharide-induced acute liver injury in mice.

Early growth response (Egr)-1 is a transcription factor that regulates genes involved in inflammation, innate and adaptive immunity, coagulation, and wound healing; however, little is known about the role of Egr-1 in acute liver injury. We tested the hypothesis that Egr-1 is involved in acute liver injury induced by galactosamine/lipopolysaccharide (GalN/LPS). GalN/LPS exposure biphasically increased hepatic egr-1 mRNA accumulation at 1 h and again at 4-5.5 h after treatment in wild-type mice. Within 4-5.5 h after GalN/LPS exposure, wild-type mice exhibited histological evidence of hepatocyte injury, cell death, and extensive areas of hemorrhage, as well as increased plasma alanine aminotransferase activities. In contrast, these parameters were largely attenuated in egr-1(-/-) mice. The initial expression of tumor necrosis factor-alpha, macrophage inflammatory protein-2, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1 mRNA or protein was equivalent between genotypes at 1 h after GalN/LPS administration. However, at subsequent time points, hepatic expression of these genes was decreased in egr-1(-/-) compared with wild-type mice. In addition, neutrophil extravasation from hepatic sinusoids into the liver parenchyma was decreased in egr-1(-/-) compared with wild-type mice 4 h after GalN/LPS. Whereas caspase-3 activation and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei were detected in wild-type mice at 4 and 5.5 h after GalN/LPS administration, respectively, these markers of apoptosis were delayed in egr-1(-/-) mice. Delayed development of apoptosis was associated with an extension of survival by 1 h in egr-1(-/-) compared with wild-type mice. These data demonstrate that Egr-1 plays an important role in acceleration of hepatic inflammation, apoptosis, and subsequent mortality in GalN/LPS-induced acute liver injury.

[Study on microsurgical procedures for pterygium operation and its therapeutic effects].

OBJECTIVE: To study the microsurgical procedures for the treatment of large primary pterygium and their therapeutic effects. To observe the recurrence rate, the changes of visual acuity after microsurgery and the pathological relationships between pterygium and cornea/sclera under the surgical microscope. METHODS: Forty-six eyes of forty-one patients with pterygium which invading the cornea over the pupil border were included. Pterygium was dissected by various methods under surgical microscope. The pathological relationship between the pterygium and cornea/sclera was observed. The lengths of the pterygium head and its three parts were measured. Degenerative Tenon's capsule was removed totally and the wound was covered by rotated conjunctival flaps. These patients were followed-up for 12.0 - 50.2 months (median: 22.4 months). Changes in visual acuity and recurrence rate after operation were observed. RESULTS: The average length of the total pterygium heads was (6.3 +/- 0.4) mm. The head was divided into three parts: the apical, loose and adhesive parts. The apical part was located at the top of the pterygium head with a length of (1.7 +/- 0.4) mm. The tissue of apical part was compact, hard, translucent and adhered to the cornea tissue. The adhesive part was a band in front of the anterior border of the limbus and paralleled to the limbus. The width of adhesive part was (0.9 +/- 0.1) mm and was tightly adhered to the cornea. The loose part lied between the apical and the adhesive part. The length of which was (3.6 +/- 0.4) mm and could be separated from the cornea easily. The neck and the body parts of pterygium could be separated easily from the limbus and sclera. Non-corrected visual acuity averaged 0.3 (ranged from finger count to 0.7) before the operation and averaged 0.7 (ranged from finger count to 1.5) 1 month postoperatively (Wilcoxon signed rank test u = 5.435, P < 0.01). Pterygium relapsed in 5 eyes with a recurrence rate of 11% (5/46). CONCLUSIONS: There is a regular pathological relationship between the pterygium and the cornea/sclera under surgical microscope, which is fundamental for the microsurgery of the pterygium. Extensively degenerated Tenon's capsule should be removed totally and the defect should be covered by rotated conjunctival flaps. The recurrence rate is low and the visual acuity increases significantly after the operation.

Metformin prevents alcohol-induced liver injury in the mouse: Critical role of plasminogen activator inhibitor-1.

BACKGROUND & AIMS: The biguanide drug metformin has recently been found to improve steatosis and liver damage in animal models and in humans with nonalcoholic steatohepatitis. METHODS: The aim of the present study was to determine whether metformin also prevents steatosis and liver damage in mouse models of acute and chronic alcohol exposure. RESULTS: Acute ethanol exposure caused a >20-fold increase in hepatic lipids, peaking 12 hours after administration. Metformin treatment significantly blunted the ethanol effect by >60%. Although metformin is a known inducer of AMP kinase (AMPK) activity, the hepatoprotective property of metformin did not correlate with activation of AMPK or of AMPK-dependent pathways. Instead, the protective effects of metformin correlated with complete prevention of the upregulation of plasminogen activator inhibitor (PAI)-1 caused by ethanol. Indeed, a similar protective effect against acute alcohol-induced lipid accumulation was observed in PAI-1-/- mice. Hepatic fat accumulation caused by chronic enteral ethanol feeding was also prevented by metformin or by knocking out PAI-1. Under these conditions, necroinflammatory changes caused by ethanol were also significantly attenuated. CONCLUSIONS: Taken together, these findings suggest a novel mechanism of action for metformin and identify a new role of PAI-1 in hepatic injury caused by ethanol.