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.

BACKGROUND

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.

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.

Objectives

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.

Design

Prospective, controlled, and randomized animal study.

Setting

A research institute laboratory.

Subjects

Male C57BL/6J mice (20–25g).

Interventions

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.

Conclusions

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.

Background

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.

Methods

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.

Results

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.

Conclusions

There is a possible association between the ACE I/D polymorphism genotype and the mortality risk of ALI/ARDS in Asians.

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.

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.

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.

Background

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.

Methods

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.

Results

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%.

Conclusions

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.

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.

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.

Background

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.

Methods

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.

Results

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.

Conclusion

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.

Aims

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.

Significance

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.

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.

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.

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.

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.

The threat of nuclear terrorism has led to growing worldwide concern about exposure to radiation. Acute radiation syndrome, or radiation sickness, develops after whole-body or a partial-body irradiation with a high dose of radiation. In the terrorist radiation exposure scenario, however, radiation victims likely suffer from additional injuries such as trauma, burns, wounds or sepsis. Thus, high-dose radiation injuries and appropriate therapeutic interventions must be studied. Despite advances in our understanding of the pathophysiology of radiation injury, very little information is available on the therapeutic approaches to radiation combined injury. In this review, we describe briefly the pathological consequences of ionizing radiation and provide an overview of the animal models of radiation combined injury. We highlight the combined radiation and sepsis model we recently established and suggest the use of ghrelin, a novel gastrointestinal hormone, as a potential therapy for radiation combined injury.

In sepsis, phagocytosis and killing of bacteria by phagocytes are important. Similarly, the clearance of accumulating apoptotic cells is critical in maintaining normal immunity. Upon maturation, peritoneal macrophages (PM) become a major source of proinflammatory cytokines, while losing their efficacy of phagocytosis. However, the underlying mechanism remains unknown. Here we investigated the differential effects of apoptotic thymocytes (AoTC) on TNF-α release in immature thioglycolate-elicited PM (TGPM) and mature resident PM (RPM) in vitro by culturing them with or without AoTC and/or LPS. MFG-E8 expression was assessed by Western blot and the ability to engulf AoTC was determined histologically. Cytokine secretion was measured by ELISA. MAP kinase phosphorylation was assessed by Western blot. Mature RPM express less than 50% of TGPM MFG-E8 levels and have a 30% lower capacity to clear AoTC. Their proinflammatory response (TNF-α release) to LPS is 5 times higher, and their capability to phagocytose is decreased along with further downregulation of MFG-E8 after LPS-stimulation. RPMs also lack phagocytosis-induced inhibition of TNF-α release after LPS stimulation. LPS-induced phosphorylation of ERK1/2, p38 and JNK is enhanced in RPM compared to TGPM. MFG-E8-mediated apoptotic cell phagocytosis results in an inhibition of MAPK and NFκB signaling pathways. Differential MAPK activation may play a role in the enhanced LPS responsiveness of RPM and the lack of MFG-E8 impedes post-phagocytic suppression of LPS-response through the inhibition of those signaling pathways. These results provide a potential mechanistic insight into the benefit of promoting apoptotic cell clearance via MFG-E8 under inflammatory conditions.

In the terrorist radiation exposure scenario, radiation victims are likely to suffer from additional injuries such as sepsis. Our previous studies have shown that ghrelin is protective in sepsis. However, it remains unknown whether ghrelin ameliorates sepsis-induced organ injury and mortality after radiation exposure. The purpose of this study is to determine whether human ghrelin attenuates organ injury and improves survival in a rat model of radiation combined injury (RCI) and, if so, the potential mechanism responsible for the benefit. To study this, adult male rats were exposed to 5-Gy whole body irradiation followed by cecal ligation and puncture (CLP, a model of sepsis) 48 h thereafter. Human ghrelin (30 nmol/rat) or vehicle (saline) was infused intravenously via an osmotic minipump immediately after radiation exposure. Blood and tissue samples were collected at 20 h after RCI (68 h after irradiation or 20 h after CLP) for various measurements. To determine the longterm effect of human ghrelin after RCI, the gangrenous cecum was removed at 5 h after CLP and 10-d survival was recorded. In addition, vagotomy or sham vagotomy was performed in sham and RCI animals immediately prior to ghrelin administration, and various measurements were performed at 20 h after RCI. Our results showed that serum levels of ghrelin and its gene expression in the stomach were decreased markedly at 20 h after RCI. Administration of human ghrelin attenuated tissue injury markedly, reduced proinflammatory cytokine levels, decreased tissue myeloperoxidase activity, and improved survival after RCI. Furthermore, elevated plasma levels of norepinephrine (NE) after RCI were reduced significantly by ghrelin. However, vagotomy prevented ghrelin’s beneficial effects after RCI. In conclusion, human ghrelin is beneficial in a rat model of RCI. The protective effect of human ghrelin appears to be attributed to re-balancing the dysregulated sympathetic/parasympathetic nervous systems.

Severe sepsis is a common, expensive, and fatal condition with as many deaths annually as those from acute myocardial infarctions. The average cost per case seems to exceed $22,000. The increased morbidity and mortality attributed to sepsis could be due to the lack of our understanding of mediators and factors responsible for early cellular alterations and thus could not be intervened which result in progressive deterioration of cell and organ function and even death. It has been well documented that hepatocellular dysfunction occurs early in sepsis and it contributes to multiple organ failure and ultimately death; however the exact mechanism is poorly understood. We and others have shown that cytochrome P-450 (CYP) enzyme system, a superfamily of heme proteins responsible for the metabolism of a variety of endogenous and exogenous substances, plays a crucial role in the prevention of hepatocellular dysfunction in sepsis. In this review, we describe the alterations of CYP enzymes in the experimental model of sepsis and provide the limited information available in septic and severely injured patients. We also review the potential mechanism for the alterations of CYP enzymes in sepsis. Finally, we highlight the importance of future studies needed to understand the regulation of CYP isoforms to develop therapy for hepatocellular dysfunction in sepsis.

Introduction

Protein disulfide isomerase (PDI) is an important factor for the protein modification step in the post-translational event. PDI plays an essential role in cell survival under various stress conditions. It has been reported that PDI can serve as a negative regulator of nuclear factor-kappa-B (NF-κB) and that it can inhibit lipopolysaccharide (LPS)-induced proinflammatory cytokine production in macrophages. Thus, PDI may be an intracellular anti-inflammatory molecule. Although we have previously shown that Kupffer cell-derived proinflammatory cytokines cause liver injury in sepsis, the effect of sepsis on PDI expression as well as the effect of PDI inhibition on cytokine production have not been investigated. We therefore hypothesized that sepsis downregulates PDI expression and that the inhibition of PDI promotes proinflammatory cytokine production.

Method

Adult male rats were subjected to sepsis by cecal ligation and puncture (CLP) or endotoxemia (continuous infusion of 1 μg/kg body weight LPS by an osmotic pump) for 20 hours. Hepatic tissues were collected and PDI gene expression was determined. In additional experiments, cells from a macrophage-like cell line, RAW 264.7, were treated with 100 ng/mL LPS for 4 hours and protein expressions were measured. RAW 264.7 cells were also treated with bacitracin, a specific PDI inhibitor, for 24 hours, and tumor necrosis factor-alpha (TNF-α) gene and protein expression as well as its release in the cell supernatant were determined. To further confirm the beneficial effect of PDI in sepsis, RAW 264.7 cells were transfected with PDI short interfering RNA (siRNA) and PDI gene expression and TNF-α release were measured by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.

Results

PDI gene expression was significantly decreased by 28% and 69% at 20 hours after CLP or LPS infusion, respectively. LPS also decreased PDI protein expression by 33% in RAW 264.7 cells. Incubation of RAW 264.7 cells with bacitracin significantly increased TNF-α gene expression and TNF-α release as well as its cellular levels in a dose-dependent manner. Transfection of RAW 264.7 cells with PDI siRNA produced an average 36.8% inhibition of the PDI gene expression. This downregulation was correlated with a 3.19-fold increase in TNF-α release into the cell supernatant.

Conclusion

Taken together, these results suggest that downregulation of PDI by sepsis significantly increases proinflammatory cytokine production. Thus, prevention of PDI downregulation in sepsis may be a novel approach to attenuate hyperinflammation and to reduce tissue injury under such conditions.

Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late hypodynamic phase. Adrenomedullin (AM), a vasodilatory peptide, inhibits this transition from the early phase to the late phase. Adrenomedullin binding protein-1 (AMBP-1) enhances AM-mediated activities. The decrease of AMBP-1 levels in late sepsis reduces the vascular response to AM and produces the hypodynamic phase. Studies have indicated that the administration of LPS downregulates AMBP-1 production in the liver. Since hepatocytes are the primary source of AMBP-1 biosynthesis in the liver, we employed a co-culture strategy using hepatocyte and Kupffer cells to determine whether LPS directly or by increasing pro-inflammatory cytokines from Kupffer cells downregulates AMBP-1 production. Hepatocytes and Kupffer cells isolated from rats were co-cultured and treated with LPS for 24 hrs. LPS significantly attenuated AMBP-1 protein expression in a dose dependent manner. Since AMBP-1 is basically a secretory protein, cell supernatants from co-culture cells treated with LPS were examined for AMBP-1 protein levels. LPS treatment caused a dose related decrease in AMBP-1 protein secretion. Similarly, LPS treatment produced a significant decrease in AMBP-1 protein expression in hepatocytes and Kupffer cells cultured using transwell inserts. LPS had no direct effect on AMBP-1 levels in cultured hepatocytes or Kupffer cells alone. To confirm that the observed effects in co-culture were due to the cytokines released from Kupffer cells, hepatocytes were treated with IL-1β or TNF-α for 24 hrs and AMBP-1 expression was examined. The results indicated that both cytokines significantly inhibited AMBP-1 protein levels. Thus, pro-inflammatory cytokines released from Kupffer cells are responsible for downregulation of AMBP-1.

Ethanol has been known to cause injury to the liver and other tissues; however the molecular factors responsible for alcohol induced liver injury has not been fully understood. Recent studies indicate that reactive oxygen species (ROS) may play an important role in alcohol induced liver injury. Peroxisome proliferator activated receptor-γ (PPAR-γ)-coactivator 1α (PGC-1α) has been shown to be involved in defenses against ROS by inducing many ROS-detoxifying enzymes. However, the role of PGC-1α in alcohol induced liver injury has not been elucidated. Therefore, in this study, we examined the effect of alcohol on gene and protein expression of PGC-1α in H4-IIE cells (in vitro) and hepatic tissues (in vivo) by real-time PCR and Western blot, respectively. Our results show that exposure to 500 mM ethanol in H4-IIE cells for 24 h significantly decreased both gene and protein expression of PGC-1α. PGC-1α gene expression was significantly decreased in cells exposed to 100 ng/ml LPS or 1% hypoxia for 24 h. In addition, PGC-1α gene and protein expressions were slightly lower in hepatic tissues of rats exposed to ethanol for 15 h, at the level equivalent to the 500 mM used in culture cells, in comparison to sham rats. In contrast, serum LDH and AST levels in ethanol exposed rats were 1.9 fold and 2.8 fold higher than that of sham rats, respectively, which suggest significant organ injury in these rats following ethanol exposure. Likewise, catalase, an enzyme that hydrolyzes peroxide to water, is significantly increased in ethanol exposed H4-IIE cells which further confirms ROS generation due to ethanol exposure. Thus, our results show that oxidative stress conditions such as acute alcohol consumption, LPS or hypoxia suppresses PGC-1α expression in the liver, thereby presumably downregulates pertinent ROS-scavenging enzymes and enhances liver injury.

Hyperglycemia is common in critically ill patients and pronounced hyperglycemia may lead to complications which include severe infections, polyneuropathy, multiple organ failure and death in such patients. Sustained hyperglycemia is generally observed in patients with Type 2 diabetes. To explore sepsis-induced inflammation in Type 2 diabetes, polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in the Goto-Kakizaki (GK) rat, a spontaneous animal model of Type 2 diabetes. The Wistar-Kyoto (WKY) rats, non-diabetic inbred rats, were used as controls for the experiment. Blood glucose levels were measured at basal, 2 hr and 20 hr after CLP. At 20 hr after CLP, blood and tissue samples were collected. Plasma levels of lactate, IL-6, IL-10 and endotoxins were measured. Total RNA from liver tissues were extracted and subjected to reverse transcription-polymerase chain reaction using rat specific IL-6 primers. GK rats exhibited significantly elevated basal glucose levels compared to WKY rats. Glucose levels in septic GK rats were significantly elevated compared to WKY rats at all time points studied. While both WKY and GK rats showed significant increases in IL-6 at 20 hr after CLP, the GK rats exhibited an average 2.68-fold increase than that of WKY rats. At 20 hr after CLP, hepatic IL-6 gene expression in GK rats was 1.77-fold greater than that of WKY rats. Although, both WKY and GK rats showed significant increases in plasma lactate levels at 20 hr after CLP, the GK rats exhibited an average increase of 1.69-fold, from the already elevated basal levels, than that of WKY rats. Since the lactate levels in GK sham groups were slightly higher than that of WKY sham, the relative changes in the fold induction by CLP between strains were similar. Both WKY and GK rats showed significantly elevated endotoxin levels at 20 hr after CLP, but no statistical differences were observed between the two groups. These studies suggest that sepsis-induced inflammation is exacerbated in an animal model of Type 2 diabetes.