PYR-41, A Ubiquitin-Activating Enzyme E1 Inhibitor, Attenuates Lung Injury in Sepsis.

During sepsis, systemic inflammation is observed and is associated with multiple organ failure. Activation of NF-κB is crucial for inducing inflammation, which is controlled by degradation of inhibitor molecules (IκB). The ubiquitination proteasome pathway is responsible for the regulation of protein turnover. In this study, we hypothesized that administration of 4[4-(5-nitro-furan-2-ylmethylene)-3, -dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41), an inhibitor of ubiquitination, could reduce inflammation and organ injury in septic mice. PYR-41 prevented the reduction of IκB protein levels and inhibited release of tumor necrosis factor (TNF)-α in mouse macrophage RAW264.7 cells at 4 h after lipopolysaccharide stimulation dose-dependently. Male C57BL/6 mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. PYR-41 (5 mg/kg) or dimethyl sulfoxide in saline (vehicle) was injected intravenously immediately after CLP. At 20 h after CLP, PYR-41 treatment significantly decreased serum levels of proinflammatory cytokines (TNF-α, interleukin [IL]-1ß, and IL-6) and organ injury markers (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). PYR-41 significantly improved microscopic structure, and reduced myeloperoxidase activity, number of apoptotic cells and caspase-3 degradation in the lungs of septic mice. The reduced protein levels of IκB in the lungs after CLP were restored by PYR-41 treatment. PYR-41 inhibited the expression of cytokines (IL-1ß and IL-6), chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2), and inflammatory mediators (cyclooxygenase-2 and inducible nitric oxide synthase) in the lungs of septic mice. Importantly, PYR-41 significantly increased 10-day survival in septic mice from 42% to 83%. Therefore, targeting ubiquitination by PYR-41 to inhibit NF-κB activation may represent a potential strategy of sepsis therapeutics.

Heat Stress Modulates Both Anabolic and Catabolic Signaling Pathways Preventing Dexamethasone-Induced Muscle Atrophy In Vitro.

It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid-induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid-induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel-like factor 15 (KLF15). Heat stress recovered the dexamethasone-induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress-induced protection against glucocorticoid-induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. J. Cell. Physiol. 232: 650-664, 2017. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

MFG-E8 inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG­E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG­E8 attenuates neutrophil migration. Recombinant human MFG­E8 (rhMFG­E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL­60, was treated with rhMFG­E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin­8 (IL­8) as the chemoattractant. Surface CXCR2 and intracellular G protein­coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen­activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG­E8 resulted in a significant inhibition of dHL­60 cell migration in a dose­dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG­E8­treated dHL­60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL­60 cells, treatment with rhMFG­E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10­30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL­60 cell migration which was significantly inhibited treatment with rhMFG­E8. Furthermore, blocking the MFG­E8 receptors, αvß3/αvß5­integrins, by anti­αv­integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG­E8­induced inhibition of dHL­60 cell migration. Finally, treatment of the dHL­60 cells with SB203580 and PD98059 neutralized the rhMFG­E8­induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG­E8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

Receptor-interacting protein kinase 3 deficiency inhibits immune cell infiltration and attenuates organ injury in sepsis.

INTRODUCTION: Sepsis is defined as a systemic hyper-inflammatory immune response, with a subsequent immune-suppressive phase, which leads to multiple organ dysfunction and late lethality. Receptor-interacting protein kinase 3 (RIPK3)-dependent necrosis is implicated in driving tumor necrosis factor alpha (TNF-α)- and sepsis-induced mortality in mice. However, it is unknown if RIPK3 deficiency has any impact on immune cell trafficking, which contributes to organ damage in sepsis. METHODS: To study this, male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice on C57BL/6 background were subjected to sham operation or cecal ligation and puncture (CLP)-induced sepsis. Blood and tissue samples were collected 20 hours post-CLP for various measurements. RESULTS: In our severe sepsis model, the mean survival time of Ripk3-/- mice was significantly extended to 68 hours compared to 41 hours for WT mice. Ripk3-/- mice had significantly decreased plasma levels of TNF-α and IL-6 and organ injury markers compared to WT mice post-CLP. In the lungs, Ripk3-/- mice preserved better integrity of microscopic structure with reduced apoptosis, and decreased levels of IL-6, macrophage inflammatory protein (MIP)-2 and keratinocyte-derived chemokine (KC), compared to WT. In the liver, the levels of MIP-1, MIP-2 and KC were also decreased in septic Ripk3-/- mice. Particularly, the total number of neutrophils in the lungs and liver of Ripk3-/- mice decreased by 59.9% and 66.7%, respectively, compared to WT mice post-CLP. In addition, the number of natural killer (NK) and CD8T cells in the liver decreased by 64.8% and 53.4%, respectively, in Ripk3-/- mice compared to WT mice post-sepsis. CONCLUSIONS: Our data suggest that RIPK3 deficiency modestly protected from CLP-induced severe sepsis and altered the immune cell trafficking in an organ-specific manner attenuating organ injury. Thus, RIPK3 acts as a detrimental factor in contributing to the organ deterioration in sepsis.

Fatty acid synthase inhibitor C75 ameliorates experimental colitis.

Abnormalities of lipid metabolism through overexpression of fatty acid synthase (FASN), which catalyzes the formation of long-chain fatty acids, are associated with the development of inflammatory bowel disease (IBD). C75 is a synthetic α-methylene-γ-butyrolactone compound that inhibits FASN activity. We hypothesized that C75 treatment could effectively reduce the severity of experimental colitis. Male C57BL/6 mice were fed 4% dextran sodium sulfate (DSS) for 7 d. C75 (5 mg/kg body weight) or dimethyl sulfoxide (DMSO) (vehicle) was administered intraperitoneally from d 2 to 6. Clinical parameters were monitored daily. Mice were euthanized on d 8 for histological evaluation and measurements of colon length, chemokine, cytokine and inflammatory mediator expression. C75 significantly reduced body weight loss from 23% to 15% on d 8, compared with the vehicle group. The fecal bleeding, diarrhea and colon histological damage scores in the C75-treated group were significantly lower than scores in the vehicle animals. Colon shortening was significantly improved after C75 treatment. C75 protected colon tissues from DSS-induced apoptosis by inhibiting caspase-3 activity. Macrophage inflammatory protein 2, keratinocyte-derived chemokine, myeloperoxidase activity and proinflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1ß and IL-6) in the colon were significantly downregulated in the C75-treated group, compared with the vehicle group. Treatment with C75 in colitis mice inhibited the elevation of FASN, cyclooxygenase-2 and inducible nitric oxide synthase expression as well as IκB degradation in colon tissues. C75 administration alleviates the severity of colon damage and inhibits the activation of inflammatory pathways in DSS-induced colitis. Thus, inhibition of FASN may represent an attractive therapeutic potential for treating IBD.

FK866, a visfatin inhibitor, protects against acute lung injury after intestinal ischemia-reperfusion in mice via NF-κB pathway.

OBJECTIVE: To determine whether administration of FK866, a competitive inhibitor of visfatin, attenuates acute lung injury induced by intestinal ischemia-reperfusion (I/R). BACKGROUND: Acute lung injury, a frequent complication of intestinal I/R, is an inflammatory disorder of the lung, which is characterized by an overproduction of proinflammatory cytokines and increased permeability of the alveolar-capillary barrier, resulting in multiple organ dysfunction. Therefore, the development of novel and effective therapies for intestinal I/R is critical for the improvement of patient outcome. Visfatin, a 54-kDa secretory protein, is known as a proinflammatory cytokine and plays a deleterious role in inflammatory diseases. METHODS: Male C57BL/6J mice were subjected to intestinal I/R induced by occlusion of the superior mesenteric artery for 90 minutes, followed by reperfusion. During reperfusion period, mice were treated with vehicle or FK866 (10 mg/kg of body weight) by an intraperitoneal injection. The levels of visfatin, proinflammatory mediators, and other markers were assessed 4 hours after reperfusion. In addition, survival study was conducted in intestinal I/R mice with or without FK866 treatment. RESULTS: Plasma and lung visfatin protein levels were significantly increased after intestinal I/R. FK866 treatment significantly attenuated intestinal and lung injury by inhibiting proinflammatory cytokine production, cellular apoptosis, and NF-κB activation, hence improving survival rate. In vitro studies showed that macrophages treated with lipopolysaccharides upregulated visfatin expression, whereas FK866 inhibited proinflammatory cytokine production via modulation of the NF-κB pathway. CONCLUSIONS: Collectively, these findings implicate FK866 as a novel therapeutic compound for intestinal I/R-induced attenuates acute lung injury via modulation of innate immune functions.

[A recurrence after surgery for colon cancer with metastases of the liver and periaortic lymph nodes, with CR achieved by using bevacizumab+mFOLFOX6].

We have experienced and report here a case of postoperative recurrence of colon cancer with metastases in the liver and the periaortic lymph nodes, where we attained CR through combination therapy with bevacizumab+mFOLFOX6. The patient was a male aged 65. He had already had cecal cancer, metastasis in the para-aortic lymph nodes, and multiple hepatic metastases. The surgery involved right colon resection+partial hepatectomy as well as dissection of the para-aortic and the superior mesenteric lymph nodes. The postoperative stage was SSN3H1P0M1 (#216)-pStage IV. S-1+CPT-11 was chosen as an adjuvant therapy. The para-aortic lymph nodes at the level of renal hilus were found enlarged after 17 courses of the therapy. Hence bevacizumab+mFOLFOX6 was chosen for the first-line treatment. After 14 courses of the therapy, CR was proved on the RECIST standard. On continued chemotherapy, CR has been maintained at present, 2 years and 11 months after the surgery or 10 months after confirmation of CR.