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1.  Human Ghrelin Mitigates Intestinal Injury and Mortality after Whole Body Irradiation in Rats 
PLoS ONE  2015;10(2):e0118213.
Widespread use of ionizing radiation has led to the realization of the danger associated with radiation exposure. Although studies in radiation countermeasures were initiated a half century ago, an effective therapy for a radiomitigator has not been identified. Ghrelin is a gastrointestinal hormone, and administration of ghrelin is protective in animal models of injuries including radiation combined injury. To test whether ghrelin can be protective in whole body irradiaton (WBI) alone, male Sprague Dawley (SD) rats were treated with human ghrelin (20 nmol/rat) daily for 6 days starting at either 24 h or 48 h after 10 Gray (Gy) WBI and survival outcome was examined. The 10 Gy WBI produced a LD70/30 model in SD rats (30% survival in 30 days). The survival rate in rats treated with ghrelin starting at 24 h was significantly improved to 63% and when treatment was initiated at 48 h, the survival remained at 61%. At 7 days post WBI, plasma ghrelin was significantly reduced from the control value. Ghrelin treatment starting at 24 h after WBI daily for 6 days improved histological appearance of the intestine, reduced gut permeability, serum endotoxin levels and bacterial translocation to the liver by 38%, 42% and 61%, respectively at day 7 post WBI. Serum glucose and albumin were restored to near control levels with treatment. Ghrelin treatment also attenuated WBI-induced intestinal apoptosis by 62% as evidenced by TUNEL staining. The expression of anti-apoptotic cell regulator Bcl-xl was decreased by 38% in the vehicle and restored to 75% of the control with ghrelin treatment. Increased expression of intestinal CD73 and pAkt were observed with ghrelin treatment, indicating protection of the intestinal epithelium after WBI. These results indicate that human ghrelin attenuates intestinal injury and mortality after WBI. Thus, human ghrelin can be developed as a novel mitigator for radiation injury.
PMCID: PMC4325005  PMID: 25671547
2.  Cold-inducible RNA-binding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis 
Nature medicine  2013;19(11):10.1038/nm.3368.
Excessive production of proinflammatory mediators is observed in patients undergoing hemorrhagic and septic shock. Here, we report the detection of cold-inducible RNA-binding protein (CIRP) in the blood of surgical ICU individuals. In animal models of hemorrhage and sepsis, CIRP is up-regulated in several organs and released into the circulation. Under hypoxic stresses, CIRP in macrophages is translocated from the nucleus to the cytosol and actively released. Recombinant CIRP stimulates TNF-α and HMGB1 release in macrophages as well as induces inflammatory responses and causes tissue injury in animals. Antisera to CIRP attenuate shock-induced inflammation, tissue injury, and lethality. Extracellular CIRP's activity is mediated through the TLR4/MD2 complex. Surface plasmon resonance analysis indicates that CIRP binds to the TLR4/MD2 complex as well as to individual TLR4 and MD2. The human CIRP amino-acid segment 106-125 binds to MD2 with high affinity. Collectively, CIRP is a new proinflammatory mediator of shock.
PMCID: PMC3826915  PMID: 24097189
3.  Milk Fat Globule-EGF Factor VIII Ameliorates Liver Injury after Hepatic Ischemia-Reperfusion 
The Journal of surgical research  2012;180(1):e37-e46.
Hepatic ischemia-reperfusion (I/R) injury is a serious clinical complication that may compromise liver function because of extensive hepatocyte loss. Therefore, the development of novel and effective therapies for hepatic I/R is critical for the improvement of patient outcome. It has been previously shown that administration of milk fat globule-EGF factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, exerts significant beneficial effects under acute inflammatory conditions through multiple physiological processes associated with tissue remodeling.
To determine whether administration of recombinant human (rh) MFG-E8 attenuates liver injury in an animal model of hepatic I/R. Male adult rats were subjected to 70% hepatic ischemia for 90 min, followed by reperfusion. At the beginning of reperfusion, rats were treated intravenously with normal saline (Vehicle) or rhMFG-E8 (160 μg/kg) over a period of 30 min. MFG-E8 levels and various measurements were assessed 4 h after reperfusion. In addition, survival study was conducted in MFG-E8−/− and rhMFG-E8-treated wild-type (WT) mice using a total hepatic ischemia model.
Liver and plasma MFG-E8 protein levels were significantly decreased after hepatic I/R. Administration of rhMFG-E8 significantly improved liver injury, suppressed apoptosis, attenuated inflammation and oxidative stress, and down-regulated NF-κB pathway. We also noticed that rhMFG-E8 treatment restored the down-regulated PPARγ expression after hepatic I/R. MFG-E8−/− mice showed deterioration on survival and, in contrast, rhMFG-E8-treated WT mice showed a significant improvement of survival compared with vehicle-treated WT mice.
MFG-E8-mediated multiple physiological events may represent an effective therapeutic option in tissue injury following an episode of hepatic I/R.
PMCID: PMC3401639  PMID: 22487387
MFG-E8; hepatic ischemia-reperfusion; inflammation; apoptosis; NF-κB; PPARγ
4.  Conserved Bacterial RNase YbeY Plays Key Roles in 70S Ribosome Quality Control and 16S rRNA Maturation 
Molecular cell  2012;49(3):427-438.
Quality control of ribosomes is critical for cellular function since protein mistranslation leads to severe physiological consequences. We report the first evidence of a ribosome quality control system in bacteria that operates at the level of 70S to remove defective ribosomes. YbeY, a previously unidentified endoribonuclease, and the exonuclease RNase R act together by a process mediated specifically by the 30S ribosomal subunit, to degrade defective 70S ribosomes but not properly matured 70S ribosomes or individual subunits. Furthermore, there is essentially no fully matured 16S rRNA in a ΔybeY mutant at 45°C, making YbeY the first endoribonuclease to be implicated in the critically important processing of the 16S rRNA 3' terminus. These key roles in ribosome quality control and maturation indicate why YbeY is a member of the minimal bacterial gene set and suggest that it could be a potential target for antibacterial drugs.
PMCID: PMC3570609  PMID: 23273979
ribosome quality control; rRNA maturation; RNase
5.  Cold-Inducible RNA-Binding Protein Is an Important Mediator of Alcohol-Induced Brain Inflammation 
PLoS ONE  2013;8(11):e79430.
Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP−/−) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP−/− mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP−/− mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.
PMCID: PMC3815202  PMID: 24223948
6.  Milk Fat Globule-EGF Factor 8 Attenuates Neutrophil Infiltration in Acute Lung Injury via Modulation of CXCR2 
Excessive neutrophil infiltration to the lungs is a hallmark of acute lung injury (ALI). Milk fat globule-EGF factor 8 (MFG-E8) was originally identified for phagocytosis of apoptotic cells. Subsequent studies revealed its diverse cellular functions. However, whether MFG-E8 can regulate neutrophil function to alleviate inflammation is unknown. We therefore aimed to reveal MFG-E8 roles in regulating lung neutrophil infiltration during ALI. To induce ALI, C57BL/6J wild-type (WT) and Mfge8−/− mice were intra-tracheally injected with LPS (5 mg/kg). Lung tissue damage was assessed by histology and the neutrophils were counted by a hemacytometer. Apoptotic cells in lungs were determined by TUNEL, while caspase-3 and MPO activities were assessed spectrophotometrically. CXCR2 and GRK2 expressions in neutrophils were measured by flow cytometry. Following LPS challenge, Mfge8−/− mice exhibited extensive lung damage due to exaggerated infiltration of neutrophils and production of TNF-α, MIP-2 and MPO. Increased number of apoptotic cells was trapped into the lungs ofMfge8−/− mice than WT mice, which may be due to insufficient phagocytosis of apoptotic cells or increased occurrence of apoptosis through the activation of caspase-3. In vitro studies using MIP-2 mediated chemotaxis, revealed higher migration of neutrophils of Mfge8−/− mice than WT mice via increased surface exposures to CXCR2. Administration of recombinant mouse (rm)MFG-E8 reduces neutrophil migration through up-regulation of GRK2, and down-regulation of surface CXCR2 expression. Conversely, these effects could be blocked by anti-αv-integrin antibodies. These studies clearly indicate the importance of MFG-E8 in ameliorating neutrophil infiltration and suggest MFG-E8 as a novel therapeutic potential for ALI.
PMCID: PMC3382007  PMID: 22634615
MFG-E8; Neutrophil; LPS; MIP-2; CXCR2; GRK2; αvβ3-integrin
7.  Sphingosine kinase-1 mediates endotoxemia-induced hyperinflammation in aged animals 
Molecular Medicine Reports  2013;8(2):645-649.
Sepsis is a serious issue in the geriatric population due to its association with high mortality rates in the elderly. The increase in mortality in the elderly correlates with inflammation. We have previously demonstrated that the inflammatory response is exacerbated in a rodent endotoxemia model of sepsis in aged rats compared with young rats. However, the molecular mediators associated with this hyperinflammatory response in aged rats have not been completely determined. Sphingosine kinase-1 (Sphk-1), an enzyme present in neutrophils and macrophages, regulates proinflammatory responses associated with endotoxemia and sepsis. To determine whether Sphk-1 is a molecular mediator associated with the observed hyperinflammatory response in aging, Sphk-1 mRNA expression was examined in hepatic tissues of young and aged rats subjected to endotoxemia. A significant increase in Sphk-1 mRNA was observed in endotoxemic aged rats compared with young rats. This increase was correlated with a significant increase in TNF-α mRNA levels in the liver. CD14 is a receptor component for lipopolysaccharide (LPS) and therefore, CD14 mRNA expression in hepatic tissues of endotoxemic young and aged rats was examined. Of note, CD14 mRNA was significantly upregulated in endotoxemic aged rats. Sphk-1 mRNA expression was significantly elevated in LPS-treated Kupffer cells and this increase correlated with an increase in CD14 mRNA expression. Results of the present study indicated that increased Sphk-1 expression in the liver in response to endotoxemia mediates the hyperinflammatory state observed in aged animals.
PMCID: PMC3776707  PMID: 23817990
endotoxemia; aged; sphingosine kinase-1; hyperinflammation; sepsis
8.  Recombinant human MFG-E8 attenuates cerebral ischemic injury: its role in anti-inflammation and anti-apoptosis 
Neuropharmacology  2011;62(2):890-900.
Excessive inflammation and apoptosis contribute to the pathogenesis of ischemic stroke. MFG-E8 is a 66-kDa glycoprotein that has shown tissue protection in various models of organ injury. However, the potential role of MFG-E8 in cerebral ischemia has not been investigated. We found that levels of MFG-E8 protein in the brain were reduced at 24 h after cerebral ischemia. To assess the potential role of MFG-E8 in cerebral ischemia, adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO). At 1 h post-stroke onset, an intravenous administration of 1 ml saline as vehicle or 160 μg/kg BW recombinant human MFG-E8 (rhMFG-E8) as treatment was given. The optimal dose of rhMFG-E8 was obtained from previous dose-response organ protection in rat sepsis studies. Neurological scores were determined at 24 h and 48 h post-MCAO. Rats were sacrificed thereafter and brains rapidly removed and analyzed for infarct size, histopathology, and markers of inflammation and apoptosis. Compared with saline vehicle, rhMFG-E8 treatment led to significant decreases in sensorimotor and vestibulomotor deficits, and infarct size at 24 h and 48 h post-MCAO. Measures associated with improved outcome included reduced microglial inflammatory cytokine secretion, adhesion molecules and neutrophil influx, cleaved caspase-3, and upregulation of peroxisome proliferator activated receptor-γ (PPAR-γ), and Bcl-2/Bax ratio leading to decreased apoptosis. Thus, rhMFG-E8 treatment is neuroprotective against cerebral ischemia through suppression of inflammation and apoptosis.
PMCID: PMC3262883  PMID: 21964436
MFG-E8; cerebral ischemia; inflammation; apoptosis; neuroprotection
9.  Cyclic arginine-glycine-aspartate attenuates acute lung injury in mice after intestinal ischemia/reperfusion 
Critical Care  2013;17(1):R19.
Intestinal ischemia is a critical problem resulting in multiple organ failure and high mortality of 60 to 80%. Acute lung injury (ALI) is a common complication after intestinal ischemia/reperfusion (I/R) injuries and contributes to the high mortality rate. Moreover, activated neutrophil infiltration into the lungs is known to play a significant role in the progression of ALI. Integrin-mediated interaction is involved in neutrophil transmigration. Synthetic peptides containing an arginine-glycine-aspartate sequence compete with adhesive proteins and inhibit integrin-mediated interaction and signaling. Thus, we hypothesized that the administration of a cyclic arginine-glycine-aspartate peptide (cRGD) inhibited neutrophil infiltration and provided protection against ALI induced by intestinal I/R.
Ischemia in adult male C57BL/6 mice was induced by fastening the superior mesenteric artery with 4-0 suture. Forty-five minutes later, the vascular suture was released to allow reperfusion. cRGD (5 mg/kg body weight) or normal saline (vehicle) was administered by intraperitoneal injection 1 hour prior to ischemia. Blood, gut, and lung tissues were collected 4 hours after reperfusion for various measurements.
Intestinal I/R caused severe widespread injury to the gut and lungs. Treatment with cRGD improved the integrity of microscopic structures in the gut and lungs, as judged by histological examination. Intestinal I/R induced the expression of β1, β2 and β3 integrins, intercellular adhesion molecule-1, and fibronectin. cRGD significantly inhibited myeloperoxidase activity in the gut and lungs, as well as neutrophils and macrophages infiltrating the lungs. cRGD reduced the levels of TNF-α and IL-6 in serum, in addition to IL-6 and macrophage inflammatory protein-2 in the gut and lungs. Furthermore, the number of TUNEL-staining cells and levels of cleaved caspase-3 in the lungs were significantly lowered in the cRGD-treated mice in comparison with the vehicle mice.
Treatment with cRGD effectively protected ALI and gut injury, lowered neutrophil infiltration, suppressed inflammation, and inhibited lung apoptosis after intestinal I/R. Thus, there is potential for developing cRGD as a treatment for patients suffering from ALI caused by intestinal I/R.
PMCID: PMC4057497  PMID: 23360591
10.  Human Ghrelin Protects Animals from Renal Ischemia-Reperfusion Injury through the Vagus Nerve 
Surgery  2011;151(1):37-47.
Acute kidney injury (AKI) secondary to renal ischemia and reperfusion (I/R) injury is widely prevalent. Ghrelin, a stomach-derived peptide, has been shown to be anti-inflammatory. The purpose of this study was to examine whether human ghrelin has any beneficial effects after renal I/R injury and if so, whether ghrelin’s action in renal I/R injury is mediated by the vagus nerve.
Male adult rats were subjected to renal I/R by bilateral renal pedicle clamping for 60 min, treated intravenously with human ghrelin (4 nmol/rat) or normal saline (vehicle) immediately following reperfusion. After 24 h, the animals were euthanized, and samples were harvested. In eparate groups, subdiaphragmatic vagotomy prior to renal I/R was performed, treated with human ghrelin or vehicle, and at 24 h, blood and organs were harvested.
Renal I/R injury caused significant increases in the serum levels of tissue injury markers as compared to sham operation. Human ghrelin treatment significantly attenuated serum creatinine and BUN, by 55% and 53%, and liver enzymes (AST and ALT) by 20% and 24%, respectively as compared to vehicle-treated groups. Tissue water contents, plasma and kidney IL-6 and kidney MPO activity were reduced. Bcl-2/Bax ratio was increased, and histology of the kidneys was improved. More importantly, prior vagotomy abolished ghrelin’s protective effect in tissue injury markers and tissue water contents in renal I/R injured animals.
Human ghrelin treatment in renal I/R injured rats attenuated systemic and kidney specific inflammatory responses. The protection of human ghrelin in renal I/R injury was mediated by the vagus nerve. These data suggest ghrelin can be developed as a novel treatment for patients with AKI induced by renal I/R injury.
PMCID: PMC3237861  PMID: 21943641
acute kidney injury; renal ischemia-reperfusion; ghrelin; inflammation
11.  Recombinant Human MFG-E8 Attenuates Intestinal Injury and Mortality in Severe Whole Body Irradiation in Rats 
PLoS ONE  2012;7(10):e46540.
The gastrointestinal (GI) syndrome component of acute radiation syndrome (ARS) results from depletion of immature parenchymal stem cells after high dose irradiation and contributes significantly to early mortality. It is associated with severe, irreparable damage in the GI tract and extremely low survival. There is a need for the development of viable mitigators of whole body irradiation (WBI) due to the possibility of unexpected high level radiation exposure from nuclear accidents or attacks. We therefore examined the effect of recombinant human milk fat globule-EGF factor 8 (rhMFG-E8) in mitigating damage after WBI. Male Sprague-Dawley rats were exposed to 10 Gy WBI using Cesium-137 as the radiation source. The animals in the treatment group received rhMFG-E8 (166 µg/kg BW) subcutaneously once a day with the first dose given 6 h after WBI. Blood and tissue samples from the ileum were collected after 3 days of treatment. A separate cohort of animals was treated for 7 days and the 21 day mortality rate was determined. Treatment with rhMFG-E8 significantly improved the survival from 31% to 75% over 21 days. Furthermore, rhMFG-E8 treatment resulted in a 36% reduction in the radiation injury intestinal mucosal damage score, corresponding to visible histological changes. MFG-E8 gene expression was significantly decreased in WBI-induced animals as compared to sham controls. Treatment with rhMFG-E8 increased p53 and p21 expression by 207% and 84% compared to untreated controls. This was accompanied by an 80% increase in the expression of anti-apoptotic cell regulator Bcl-2. p53 and p21 levels correlate with improved survival after radiation injury. These cell regulators arrest the cell after DNA damage and enable DNA repair as well as optimize cell survival. Taken together, these results indicate that rhMFG-E8 ameliorates the GI syndrome and improves survival after WBI by minimizing intestinal cell damage and optimizing recovery.
PMCID: PMC3466304  PMID: 23056336
12.  Protective effect of milk fat globule-EGF factor VIII after renal ischemia-reperfusion injury in mice 
Critical care medicine  2011;39(9):2039-2047.
Renal ischemia-reperfusion (I/R) injury causes acute renal failure and the hallmarks of renal I/R injury are inflammation, apoptosis, necrosis, and capillary dysfunction. Milk fat globule-EGF factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, is produced by immune cells and reported to participate in multiple physiological processes associated with tissue remodeling. We have recently shown that MFG-E8 treatment attenuates organ injury, inflammatory responses, and survival after sepsis through the enhancement of phagocytosis of apoptotic cells. The purpose of this study was to determine whether administration of MFG-E8 attenuates renal I/R injury.
Prospective, controlled, and randomized animal study.
A research institute laboratory.
Male C57BL/6J mice (20–25g).
Renal I/R injury with bilateral renal pedicle clamping for 45 minutes, followed by reperfusion. A recombinant murine MFG-E8 (rmMFG-E8; 0.4 µg/20g) was given intraperitoneally at the beginning of reperfusion.
Measurements and Main Results
MFG-E8 levels, organ injury variables, inflammatory responses, histology, apoptosis, and capillary functions were assessed at 1.5 and 20 hours after reperfusion. A 60-hour survival study was conducted in MFG-E8−/− and rmMFG-E8-treated wild-type (WT) mice. After renal I/R injury, MFG-E8 mRNA and protein expressions were significantly decreased in the kidneys and spleen. Treatment with rmMFG-E8 recovered renal dysfunction, significantly suppressed inflammatory responses, apoptosis, necrosis, and improved capillary functions in the kidneys. In the survival study, MFG-E8−/− mice showed a significant deterioration and, in contrast, rmMFG-E8-treated WT mice showed a significant improvement of survival compared with vehicle-treated WT mice.
MFG-E8 can be developed as novel treatment for renal I/R injury. This protective effect appears to be mediated through the enhancement of apoptotic cell clearance and improvement of capillary functions in the kidneys.
PMCID: PMC3158289  PMID: 21666453
MFG-E8; renal ischemia-reperfusion injury; inflammation; apoptosis; necrosis; capillary function
13.  Association between insertion/deletion polymorphism in angiotensin-converting enzyme gene and acute lung injury/acute respiratory distress syndrome: a meta-analysis 
BMC Medical Genetics  2012;13:76.
A previous meta-analysis reported a positive association between an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme gene (ACE) and the risk of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Here, we updated this meta-analysis and additionally assessed the association of this polymorphism with ALI/ARDS mortality.
We searched electronic databases through October 2011 for the terms “angiotensin-converting enzyme gene”, “acute lung injury”, and “acute respiratory distress syndrome,” and reviewed all studies that reported the relationship of the I/D polymorphism in ACE with ALI/ARDS in humans. Seven studies met the inclusion criteria, comprising 532 ALI/ARDS patients, 3032 healthy controls, and 1432 patients without ALI/ARDS. We used three genetic models: the allele, dominant, and recessive models.
The ACE I/D polymorphism was not associated with susceptibility to ALI/ARDS for any genetic model. However, the ACE I/D polymorphism was associated with the mortality risk of ALI/ARDS in Asian subjects ( Pallele < 0.0001, Pdominant = 0.001, Precessive = 0.002). This finding remained significant after correction for multiple comparisons.
There is a possible association between the ACE I/D polymorphism genotype and the mortality risk of ALI/ARDS in Asians.
PMCID: PMC3459791  PMID: 22938636
Angiotensin-converting enzyme (ACE) gene; Acute lung injury (ALI); Acute respiratory distress syndrome (ARDS); Meta-analysis
14.  Enhancing apoptotic cell clearance mitigates bacterial translocation and promotes tissue repair after gut ischemia-reperfusion injury 
A key aspect of intestinal ischemia/reperfusion (I/R) injury is the increased occurrence of apoptotic cell death in the gut. Insufficient clearance of apoptotic cells leads to increased inflammation and impaired tissue repair. Our recent studies have shown that administration of milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a crucial molecule for apoptotic cell clearance, reduces apoptosis and inflammation under various disease conditions. The purpose of this study was to determine whether MFG-E8 reduces bacterial translocation and promotes tissue repair in a mouse model of gut I/R. Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 90 min in male adult mice. After removing the clip, recombinant murine MFG-E8 (rmMFG-E8) (0.4 μg/20 g BW) or normal saline (Vehicle) was intraperitoneally injected. At 4 h after reperfusion, apoptosis in the gut was measured by TUNEL staining. The mesenteric lymph node (MLN) complex was homogenized and plated on chocolate agar plates for bacterial culture. Neutrophil infiltration was assessed by examining myeloperoxidase (MPO) activity in the gut. Vascular endothelial growth factor (VEGF) levels in the gut, an indicator of tissue repair, were measured by western blotting. Out results showed that TUNEL-positive staining in the gut increased significantly in gut I/R vehicle-treated mice. Treatment with rmMFG-E8 markedly suppressed the number of apoptotic cells. Bacterial translocation to the MLN was minimal in sham mice, but was extensive in gut I/R vehicle-treated mice. rmMFG-E8 treatment significantly reduced bacterial translocation to the MLN. Similarly, gut I/R induced a significant increase in intestinal MPO activities in vehicle-treated mice. rmMFG-E8 treatment markedly reduced the increase in intestinal MPO activities after gut I/R. Intestinal levels of VEGF decreased significantly at 4 h after gut I/R. rmMFG-E8 treatment significantly increased intestinal VEGF levels. Thus, enhancing apoptotic cell clearance by rmMFG-E8 mitigates bacterial translocation, inhibits neutrophil infiltration and promotes tissue repair after gut I/R. Enhancing apoptotic cell clearance can be a novel concept in the treatment of gut I/R injury.
PMCID: PMC3573750  PMID: 22751701
milk fat globule-epidermal growth factor-factor 8; gut ischemia/reperfusion; apoptosis; bacterial translocation; vascular endothelial growth factor
15.  Upregulation of Kupffer cell α2A–adrenoceptors and downregulation of MKP-1 mediate hepatic injury in chronic alcohol exposure 
Alcohol-induced liver disease is associated with unacceptable morbidity and mortality. When activated, Kupffer cells (KCs), the resident macrophages in the liver, release proinflammatory cytokine TNF-α, a key mediator of hepatic damage. Although chronic alcohol causes increase in norepinephrine (NE) release leading to hepatic dysfunction, the mechanism of NE-induced hepatic injury in chronic alcohol exposure has not been elucidated. This study was conducted to determine whether chronic alcohol exposure increases NE and upregulates KC α2A-adrenoceptors (α2A-AR) to cause TNF-α release. We also examined the role of mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1) in this process. Male adult rats were fed the Lieber-DeCarli liquid diet containing alcohol as 36% of total calories. The animals were sacrificed after 6 weeks and blood and liver samples were harvested for further analysis. KCs from healthy male rats were cultured with alcohol for 7 days, and cells then harvested for RNA and protein analyses. Chronic alcohol exposure resulted in hepatic damage. Alcohol caused a 276% increase in circulating NE and 86% increase in TNF-α in the liver. There was a 75% and 62% decrease in MKP-1 mRNA and protein levels in the liver, respectively. In-vitro experiments revealed 121% and 98% increase in TNF-α and α2A-AR mRNA levels with alcohol exposure, respectively, and a 32% decrease in MKP-1 mRNA compared to controls. In summary, chronic alcohol exposure elevates NE and upregulates KC α2A-AR to release TNF-α. Alcohol induced downregulation of MKP-1 leads to further release of TNF-α and hepatic injury.
PMCID: PMC3133744  PMID: 21575605
chronic alcohol; α2A-adrenoceptor; MKP-1; liver injury
16.  The parasympathetic nervous system in the quest for stroke therapeutics 
Stroke is a devastating neurovascular disease with limited therapeutic options. The pathogenesis of stroke involves complex interrelated molecular mechanisms including excitotoxicity, oxidative and nitrosative stress, cortical spreading depolarizations, inflammation, necrosis, and apoptosis. Successful development of stroke therapeutics depends on understanding these molecular mechanisms and how to counteract them to limit tissue damage during stroke. Activation of the parasympathetic nervous system (PNS) has been shown to antagonize a multiplicity of pathologic mechanisms. Elements of parasympathetic activation such as vagus nerve stimulation have already been used successfully in treating brain disorders such as epilepsy and depression. This review discusses the anatomical basis and molecular mechanisms involved in activation of the PNS, and assesses the strength of available evidence for the further development of this modality into a stroke therapy.
PMCID: PMC3099641  PMID: 21364605
parasympathetic; sphenopalatine; stroke; therapeutics; vagus
17.  Resuscitation of uncontrolled traumatic hemorrhage induced by severe liver injury: the use of human AM/AMBP-1 
The Journal of trauma  2010;69(6):1415-1422.
The liver is a major organ that is susceptible to injury following blunt and/or penetrating trauma to the abdomen. No specific non-operative treatment exists for traumatic hepatic injury (THI). Adrenomedullin (AM), a vasoactive peptide, combined with its binding protein (AMBP-1) is beneficial in various disease conditions. In this study, we propose to determine whether human AM combined with human AMBP-1 provides benefit in a model of THI in the rat.
Male adult rats were subjected to trauma-hemorrhage by resection of approximately 50% of total liver tissues and allowed bleeding for 15 min. Immediately thereafter, human AM (48 μg/kg BW) plus human AMBP-1 (160 μg/kg BW) was given intravenously over 30 min in 1 ml normal saline. After 4 h, the rats were euthanized, blood was collected, and tissue injury indicators were assessed. A 10-day survival study was also conducted.
At 4 h after THI, plasma AMBP-1 levels were markedly decreased. Plasma levels of liver injury indicators (i.e., AST, ALT and LDH) were significantly increased after THI. Likewise, lactate, creatinine and TNF-α levels were significantly increased following THI. Administration of human AM/AMBP-1 after THI produced significant decreases of 64%, 23% and 19% of plasma AST, ALT and LDH levels, respectively. Similarly, plasma levels of lactate, creatinine and TNF-α were also decreased by 42%, 28% and 46% following human AM/AMBP-1 treatment, respectively. In a 10-day survival study, while vehicle treatment produced 41% survival, human AM/AMBP-1 treatment improved the survival rate to 81%.
Administration of human AM/AMBP-1 significantly attenuated tissue injury and inflammation, and improved survival following THI. Thus, human AM/AMBP-1 can be developed as a novel treatment for victims with uncontrolled traumatic hemorrhage.
PMCID: PMC3139227  PMID: 21057332
traumatic hepatic injury; hemorrhage; adrenomedullin; adrenomedullin binding protein; survival
18.  Pre-Treatment of Recombinant Mouse MFG-E8 Downregulates LPS-Induced TNF-α Production in Macrophages via STAT3-Mediated SOCS3 Activation 
PLoS ONE  2011;6(11):e27685.
Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8−/− mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8−/− mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8−/− mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.
PMCID: PMC3217009  PMID: 22114683
19.  Role of Escherichia coli YbeY, a highly conserved protein, in rRNA processing 
Molecular microbiology  2010;78(2):506-518.
The UPF0054 protein family is highly conserved with homologs present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homolog, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′- and 3′-ends of 16S rRNA as well as maturation of the 5′-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.
PMCID: PMC2959132  PMID: 20807199
rRNA maturation; ribosome
20.  Attenuation of renal ischemia and reperfusion injury by human adrenomedullin and its binding protein 
The Journal of surgical research  2010;163(1):110-117.
Acute renal failure secondary to ischemia and reperfusion (I/R) injury poses a significant burden on both surgeons and patients. It carries a high morbidity and mortality rate and no specific treatment currently exists. Major causes of renal I/R injury include trauma, sepsis, hypoperfusion, and various surgical procedures. We have demonstrated that adrenomedullin (AM), a novel vasoactive peptide, combined with AM binding protein-1 (AMBP-1), which augments the activity of AM, is beneficial in various disease conditions. However, it remains unknown whether human AM/AMBP-1 provides any beneficial effects in renal I/R injury. The objective of our study therefore was to determine whether administration of human AM/AMBP-1 can prevent and/or minimize damage in a rat model of renal I/R injury.
Male adult rats were subjected to renal I/R injury by bilateral renal pedicle clamping with microvascular clips for 60 min followed by reperfusion. Human AM (12 µg/kg BW) and human AMBP-1 (40 µg/kg BW) or vehicle (52 µg/kg BW human albumin) were given intravenously over 30 min immediately following the clip removal (i.e., reperfusion). Rats were allowed to recover for 24 h post treatment, and blood and renal tissue samples were collected. Plasma levels of AM were measured using a radioimmunoassay specific for rat AM. Plasma AMBP-1 was measured by Western analysis. Renal water content and serum levels of systemic markers of tissue injury were measured. Serum and renal TNF-α levels were also assessed.
At 24 h after renal I/R injury, plasma levels of AM were significantly increased while plasma AMBP-1 was markedly decreased. Renal water content and systemic markers of tissue injury (e.g., creatinine, BUN, AST and ALT) were significantly increased following renal I/R injury. Serum and renal TNF-α levels were also increased post injury. Administration of human AM/AMBP-1 decreased renal water content, and plasma levels of creatinine, BUN, AST and ALT. Serum and renal TNF-α levels were also significantly decreased after AM/AMBP-1 treatment.
Treatment with human AM/AMBP-1 in renal I/R injury significantly attenuated organ injury and the inflammatory response. Thus, human AM combined with human AMBP-1 may be developed as a novel treatment for patients with acute renal I/R injury.
PMCID: PMC2922411  PMID: 20538296
Renal ischemia and reperfusion injury; adrenomedullin; adrenomedullin bindin protein; inflammation
21.  Aging-related hyperinflammation in endotoxemia is mediated by the α2A-adrenoceptor and CD14/TLR4 pathways 
Life sciences  2010;86(19-20):740-746.
Sepsis is a major cause of morbidity and mortality in the elderly population. In prior studies, we have shown that in vivo, the inflammatory response in aged animals is exaggerated as compared to young animals and that this response likely accounts for the increased morbidity and mortality. Part of this uncontrolled inflammatory response in sepsis is due to the innate immune response. However, recent studies have shown that the pathogenesis of sepsis is much more complex. The adrenergic autonomic nervous system is now thought to play a key role in modulating the inflammatory response in sepsis. In this study, we hypothesize that not only is the innate immune response enhanced in response to lipopolysaccharide (LPS) in aged animals, but that the adrenergic nervous system also plays a role in the release of excess inflammatory cytokines.
Main Methods
Male Fisher 344 rats (young: 3 months; aged: 24 months) were used. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS, 15 mg/kg BW). Splenic tissues were harvested and mRNA and protein were extracted. The protein expression of CD14 and TLR4, key mediators of LPS in the innate response, as well as alpha-2A adrenergic receptor (α2A-AR) and phosphodiesterase 4D (PDE4D), as the means by which the autonomic nervous system exerts its effects were analyzed.
Key Findings
Splenic tissue concentrations of α2A-AR, PDE4D, CD14, TLR4 were significantly increased in septic aged rats as compared to aged sham rats and septic young rats. The increased expression of α2A-AR in septic aged rats was further confirmed by immunohistochemical staining of splenic tissues.
These data support the hypothesis that not only is the innate immune response increased in aged animals during sepsis, but that there is also an upregulated response of the adrenergic autonomic nervous system that contributes to excess proinflammatory cytokine release.
PMCID: PMC2859120  PMID: 20302880
aging; inflammation; endotoxemia; sepsis; α2A-adrenoceptor; CD14; TLR4
22.  Milk Fat Globule-EGF Factor VIII in Sepsis and Ischemia-Reperfusion Injury 
Molecular Medicine  2010;17(1-2):126-133.
Sepsis and ischemia-reperfusion (I/R) injury are among the leading causes of death in critically ill patients at the surgical intensive care unit setting. Both conditions are marked by the excessive inflammatory response which leads to a lethal disease complex such as acute lung injury, systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Despite the advances in the understanding of the pathophysiology of those conditions, very little progress has been made toward therapeutic interventions. One of the key aspects of these conditions is the accumulation of apoptotic cells that have the potential to release toxic and proinflammatory contents due to secondary necrosis without appropriate clearance by phagocytes. Along with the prevention of apoptosis, that is reported to be beneficial in sepsis and I/R injury, thwarting the development of secondary necrosis through the active removal of apoptotic cells via phagocytosis may offer a novel therapy. Milk fat globule-EGF factor VIII (MFG-E8), which is mainly produced by macrophages and dendritic cells, is an opsonin for apoptotic cells and acts as a bridging protein between apoptotic cells and phagocytes. Recently, we have shown that MFG-E8 expression is decreased in experimental sepsis and I/R injury models. Exogenous administration of MFG-E8 attenuated the inflammatory response as well as tissue injury and mortality through the promotion of phagocytosis of apoptotic cells. In this review, we describe novel information available about the involvement of MFG-E8 in the pathophysiology of sepsis and I/R injury, and the therapeutic potential of exogenous MFG-E8 treatment for those conditions.
PMCID: PMC3022991  PMID: 20882259
23.  The Inhibitory Effect of Ghrelin on Sepsis Induced Inflammation is Mediated by the MAPK phosphatase-1 
Hepatocellular dysfunction occurs early in sepsis and this appears to be caused by Kupffer cell derived TNF-α production from the liver as a result of the increased release of the sympathetic neurotransmitter, norepinephrine, from the gut. Ghrelin, a novel stomach-derived peptide, is downregulated in sepsis and administration of ghrelin into rodents decrease pro-inflammatory cytokines, attenuates hepatic and other organ injuries and improves survival. Ghrelin’s beneficial effect in sepsis is mediated by the inhibition of the sympathetic nervous system (SNS), as evidenced by the reduced gut-derived norepineprine (NE) release in sepsis after ghrelin treatment. Recent data suggest that MKP-1, the MAPK phosphatase-1, is involved in the innate immune responses. To determine that ghrelin’s beneficial effect in sepsis is mediated by MKP-1, rats were subjected to sepsis by cecal ligation and puncture (CLP) alone, or treated with ghrelin, beginning at 5h post-CLP and liver tissues were harvested and examined for MKP-1 mRNA and protein expression. CLP alone produced a significant decrease in MKP-1 gene expression in liver tissues at 20h after CLP (P<0.05). MKP-1 mRNA was decreased by 30–40% at 2h and 5h after CLP, but not statistically significant. MKP-1 protein expression was significantly decreased as early as 2h after CLP and remained low at 5–20h after CLP. While septic rats treated with vehicle produced significant decreases from sham rats, ghrelin treatment improved both mRNA and protein from vehicle group (0.58 ± 0.069 vs. 0.91 ± 0.16, P <0.05; 0.14 ± .027 vs. 0.22 ± 0.017, P=0.013), respectively. Since ghrelin’s inhibitory effect is mediated by the SNS, we hypothesized that NE treatment in Kupffer cells may downregulate MKP-1. Kupffer cells were treated with NE and examined for MKP-1. Treatment with NE for 60 min showed an average 46.9% decrease in MKP-1 mRNA expression compared to untreated cells (P<0.001). Likewise, NE treatment in RAW 264.7 cells produced significantly lower MKP-1 mRNA than that of control cells. To further confirm the effect of NE on MKP-1, normal rats were infused with NE for 2h through the portal vein and MKP-1 mRNA from the liver was examined. Infusion with NE produced a significant 73.7% decrease in MKP-1 mRNA. Therefore, ghrelin’s inhibitory effect on gut-derived NE release in sepsis leading to the downregulation of pro-inflammatory cytokines is mediated by MKP-1.
PMCID: PMC2797446  PMID: 19956915
Ghrelin; sepsis; norepinephrine; MKP-1; inflammation
24.  Peroxisome proliferator activated receptor-γ and traumatic brain injury 
Traumatic brain injury (TBI) represents a major health care problem and a significant socioeconomic challenge worldwide. No specific therapy for TBI is available. The peroxisome proliferator activated receptor-γ (PPAR-γ) belongs to the nuclear receptor superfamily. Although PPAR-γ was originally characterized in adipose tissue as a regulator of lipid and glucose metabolism, recent studies showed that PPAR-γ is present in most cell types and plays a central role in the regulation of adipogenesis, glucose homeostasis, cellular differentiation, apoptosis and inflammation. Here, we reviewed the current literature on the molecular mechanisms of PPAR-γ-related neuroprotection after TBI. Growing evidence has indicated that the beneficial effects of PPAR-γ activation in TBI appear to be mediated through downregulation of inflammatory responses, reduction of oxidative stress, inhibition of apoptosis, and promotion of neurogenesis. A thorough understanding of the PPAR-γ pathway will be critical to the development of therapeutic interventions for the treatment of patients with TBI.
PMCID: PMC2971540  PMID: 21072262
Peroxisome proliferator activated receptor-γ (PPAR-γ); traumatic brain injury (TBI); nuclear receptor super-family; neuroprotection; neurogenesis; oxidative stress
25.  Modulation of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in sepsis 
Previous studies demonstrated that hepatic matrix metalloproteinase-9 (MMP-9) activity increased following cecal ligation and puncture (CLP) in rats, indicating liver injury in sepsis. The activity of MMP-9 in degrading extracellular matrix is controlled by activation of proenzymes and inhibition of tissue inhibitor of MMPs (TIMP-1). To further assess the proteolytic cascade imbalance in sepsis, hepatic MMP-9 and TIMP-1 expressions were examined in CLP rats. In this study, sepsis was induced in rats by CLP, and at 10 and 20 h after sepsis induction, liver samples were collected and MMP-2, MMP-9, and TIMP-1 gene and protein expressions were evaluated by real time PCR and Western blot analysis, respectively. Gene expression of MMP-9 was increased by 6.4-fold and 3.0-fold at 10 h and 20 h after CLP as compared to sham group, respectively. Likewise, MMP-9 protein expression was also significantly increased at both time points. In contrast, MMP-2 gene expression was not altered at 10 h and 20 h after CLP as compared to sham controls. Interestingly, TIMP-1 gene expression was elevated to 89-fold and 46-fold from sham levels at 10 h and 20 h after CLP, respectively. Similarly, TIMP-1 protein levels were also significantly increased at both time points. In addition, MMP-9/TIMP-1 protein ratio was lower at both 10 h and 20 h after CLP compared to sham rats. Results demonstrated an imbalance between MMP and TIMP, with a more evident role for MMP-9 than MMP-2, and high value of TIMP-1 was particularly evident in CLP rats. Our results indicate that MMP-9 and TIMP-1 expressions are increased and they may serve as useful markers to predict the outcome of sepsis.
PMCID: PMC2929943  PMID: 20827315
MMP; TIMP-1; sepsis; MMP-9/TIMP-1 ratio; MMP-2; matrix metalloproteinase; inhibitors; liver; sepsis

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