Attenuation of hemorrhage-associated lung injury by adjuvant treatment with C23, an oligopeptide derived from cold-inducible RNA-binding protein.
ABSTRACT: BACKGROUND:Hemorrhagic shock (HS) is an important cause of mortality. HS is associated with an elevated incidence of acute lung injury and acute respiratory distress syndrome, significantly contributing to HS morbidity and mortality. Cold-inducible RNA-binding protein (CIRP) is released into the circulation during HS and can cause lung injury. C23 is a CIRP-derived oligopeptide that binds with high affinity to the CIRP receptor and inhibits CIRP-induced phagocyte secretion of TNF-?. This study was designed to determine whether C23 is able to attenuate HS-associated lung injury. METHODS:C57BL/6 mice were subjected to controlled hemorrhage leading to a mean arterial pressure of 25 ± 3 mm Hg for 90 minutes. Mice were then volume-resuscitated for 30 minutes with normal saline solution alone (vehicle) or plus adjuvant treatment with C23 (8 mg/kg BW). At 4.5 hours after resuscitation, the blood and lungs were harvested. RESULTS:Serum levels of organ injury markers lactate dehydrogenase, aspartate aminotransferase were significantly elevated in hemorrhaged mice receiving vehicle and were reduced by 51.3% and 52.2% in mice adjuvantly treated with C23, respectively. Similarly, lung mRNA levels of IL-1?, TNF-?, and IL-6, and lung myeloperoxidase activity were elevated after HS and reduced by 66.1%, 54.4%, 69.7%, and 24.3%, respectively, in mice treated with C23. Adjuvant treatment with C23 also decreased the lung histology score by 33.9%, lung extravasation of albumin carrying Evans blue dye by 36.8%, and the protein level of intercellular adhesion molecule-1, and indicator of vascular endothelial cell activation, by 40.3%. CONCLUSION:Together, these results indicate that adjuvant treatment with the CIRP-derived oligopeptide C23 is able to improve lung inflammation and vascular endothelial activation secondary to HS, lending support to the development of CIRP-targeting adjuvant treatments to minimize lung injury after HS.
Project description:INTRODUCTION:Neonatal sepsis remains a leading cause of infant mortality. Cold-inducible RNA binding protein (CIRP) is an inflammatory mediator that induces TNF-? production in macrophages. C23 is a CIRP-derived peptide that blocks CIRP from binding its receptor. We therefore hypothesized that treatment with C23 reduces systemic inflammation and protects the lungs in neonatal sepsis. METHODS:Sepsis was induced in C56BL/6 mouse pups (5-7?days) by intraperitoneal injection of adult cecal slurry (0.525?mg/g body weight, LD100). One hour later pups received retroorbital injection of C23 (8?mg/kg) or vehicle (normal saline). Ten hours after sepsis induction, blood and tissues were collected for analysis. RESULTS:C23 treatment resulted in a 58% and 69% reduction in serum levels of proinflammatory cytokines IL-6 and IL-1?, respectively, and a 40% and 45% reduction of AST and LDH, as compared to vehicle-treated septic pups. In the lungs, C23 treatment reduced expression of cytokines IL-6 and IL-1? by 78% and 74%. In addition, the mRNA level of neutrophil chemoattractants KC and MIP-2 was reduced by 84% and 74%, respectively. These results corresponded to a reduction in histologic lung injury score. Vehicle-treated pups scored 0.49?±?0.19, while C23 treatment reduced scores to 0.29?±?0.12 (p?<?0.05; Max?=?1). Apoptosis in the lungs, measured by TUNEL assay, was also decreased by 53% with C23 treatment (p?<?0.05). CONCLUSIONS:Inhibition of CIRP with C23 treatment is protective in septic neonatal mice as demonstrated by reduced inflammatory markers systemically and in the lung. Therefore, C23 has promising therapeutic potential in treatment of neonatal sepsis. LEVEL OF EVIDENCE:Level I.
Project description:Cold-inducible RNA-binding protein (CIRP) is a novel sepsis inflammatory mediator and C23 is a putative CIRP competitive inhibitor. Therefore, we hypothesized that C23 can ameliorate sepsis-associated injury to the lungs and kidneys. First, we confirmed that C23 dose-dependently inhibited TNF-? release, I?B? degradation, and NF-?B nuclear translocation in macrophages stimulated with CIRP. Next, we observed that male C57BL/6 mice treated with C23 (8?mg/kg BW) at 2?h after cecal ligation and puncture (CLP) had lower serum levels of LDH, ALT, IL-6, TNF-?, and IL-1? (reduced by ?39%) at 20?h after CLP compared with mice treated with vehicle. C23-treated mice also had improved lung histology, less TUNEL-positive cells, lower serum levels of creatinine (34%) and BUN (26%), and lower kidney expression of NGAL (50%) and KIM-1 (86%). C23-treated mice also had reduced lung and kidney levels of IL-6, TNF-?, and IL-1?. E-selectin and ICAM-1 mRNA was significantly lower in C23-treated mice. The 10-day survival after CLP of vehicle-treated mice was 55%, while that of C23-treated mice was 85%. In summary, C23 decreased systemic, lung, and kidney injury and inflammation, and improved the survival rate after CLP, suggesting that it may be developed as a new treatment for sepsis.
Project description:Extracellular cold-inducible RNA-binding protein (CIRP) functions as damage-associated molecular pattern and has been demonstrated to be responsible in part for the damage occurring after renal ischemia-reperfusion (I/R). A short peptide derived from CIRP, named C23, binds to myeloid differentiation factor 2, a Toll-like receptor 4 coreceptor. We hypothesize that C23 reduces renal ischemia-reperfusion (RIR) injury by blocking CIRP. We observed that pretreatment with C23 significantly decreased the levels of recombinant mouse CIRP-induced tumor necrosis factor-? (TNF-?) in a dose-dependent fashion in cultured macrophages. C57BL/6 mice were subjected to bilateral renal pedicle clamps for 35?min to induce ischemia, followed by reperfusion for 24 h and harvest of blood and renal tissue. C23 peptide (8?mg/kg) or vehicle was injected intraperitoneally at the beginning of reperfusion. Plasma TNF-?, interleukin 1 beta (IL-1?), and IL-6 levels were decreased in C23-treated RIR mice as compared with vehicle-treated mice by 74%, 85%, and 68%, respectively. Expressions of TNF-? and keratinocyte chemoattractant in the kidneys from C23-treated mice were decreased by 55% and 60%, respectively. Expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in the kidney of C23-treated mice were significantly reduced by 46% and 55%, respectively. Renal tissue histological assessments revealed significant reduction in damage score by 44% in C23-treated mice. Finally, a survival study revealed a significant survival advantage with a 70% survival rate in C23 group vs. 37% in vehicle group. Thus, C23 has potential as a novel therapy for the patients suffering from I/R-induced renal injury.
Project description:Cold-inducible RNA-binding protein (CIRP), released into the circulation during sepsis, causes lung injury via an as yet unknown mechanism. Since endoplasmic reticulum (ER) stress is associated with acute lung injury (ALI), we hypothesized that CIRP causes ALI via induction of ER stress. To test this hypothesis, we studied the lungs of wild-type (WT) and CIRP knockout (KO) mice at 20?h after induction of sepsis by cecal ligation and puncture (CLP). WT mice had significantly more severe ALI than CIRP KO mice. Lung ER stress markers (BiP, pIRE1?, sXBP1, CHOP, cleaved caspase-12) were increased in septic WT mice, but not in septic CIRP KO mice. Effector pathways downstream from ER stress - apoptosis, NF-?B (p65), proinflammatory cytokines (IL-6, IL-1?), neutrophil chemoattractants (MIP-2, KC), neutrophil infiltration (MPO activity), lipid peroxidation (4-HNE), and nitric oxide (iNOS) - were significantly increased in WT mice, but only mildly elevated in CIRP KO mice. ER stress markers were increased in the lungs of healthy WT mice treated with recombinant murine CIRP, but not in the lungs of TLR4 KO mice. This suggests CIRP directly induces ER stress via TLR4 activation. In summary, CIRP induces lung ER stress and downstream responses to cause sepsis-associated ALI.
Project description:C23 is an abundant and multi-functional protein, which plays an important role in various biological processes, including ribosome biogenesis and maturation, cell cycle checkpoints and transcriptional regulation [1, 2]. However, the role of C23 in controlling tumorigenesis has not been well defined. Here we report that C23 is highly expressed in cancer cells and the elevated expression of C23 facilitates cancer cell proliferation in vitro and tumor xenograft growth in vivo. Notably, C23 binds to p53 through its GAR domain and suppresses the transcriptional activity of p53 under DNA damage and hypoxia. Moreover, the GAR domain is critical for C23-mediated tumor cell proliferation both in vitro and in vivo. Our findings reveal a novel role of C23 in tumorigenesis and suggest that C23 may represent a potential therapeutic target for treating malignancy.
Project description:Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern (DAMP) molecule which stimulates proinflammatory cytokine release in hemorrhage and sepsis. Under these medical conditions, disruption of endothelial homeostasis and barrier integrity, typically induced by proinflammatory cytokines, is an important factor contributing to morbidity and mortality. However, the role of CIRP in causing endothelial dysfunction has not been investigated. In this study, we show that intravenous injection of recombinant murine CIRP (rmCIRP) in C57BL/6 mice causes lung injury, evidenced by vascular leakage, edema, increased leukocyte infiltration and cytokine production in the lung tissue. The CIRP-induced lung damage is accompanied with endothelial cell (EC) activation marked by upregulation of cell-surface adhesion molecules E-selectin and ICAM-1. Using in vitro primary mouse lung vascular ECs (MLVECs), we demonstrate that rmCIRP treatment directly increases the ICAM-1 protein expression and activates NAD(P)H oxidase in MLVECs. Importantly, CIRP stimulates the assembly and activation of Nlrp3 inflammasome in MLVECs accompanied with caspase-1 activation, IL-1β release and induction of proinflammatory cell death pyroptosis. Finally, our study demonstrates CIRP-induced EC pyroptosis in the lungs of C57BL/6 mice for the first time. Taken together, the released CIRP in shock can directly activate ECs and induce EC pyroptosis to cause lung injury.
Project description: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-? mRNA and protein expressions along with IL-1? 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-? and IL-1? 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.
Project description:Neuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA-binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke.Stroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-? (TNF-?) levels were assessed by RT-PCR and Western blot analysis.CIRP is elevated along with an upregulation of TNF-? expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-?, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-? levels. Addition of recombinant murine (rm) CIRP directly induces TNF-? release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-?B signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP.Extracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia.Development of an anti-CIRP therapy may benefit patients with brain ischemia.
Project description:To verify the imapct of DSF and C23 on P. aeruginosa during infection We used microarray to compare the effects of adding either DSF alone or DSF with C23 on P. aeruginosa gene expression during mouse lung infection relative to the gene expression of P. aeruginosa in the mouse lung with no compound. Overall design: For the in vivo RNA samples, infected animals as described above were euthanized 24 h post-infection. RNA was isolated from airway homogenate pellets using Trizol according to the manufacturer's instructions. Samples were treated with DNAse, then rRNA was removed from 1 to 5 μg of total RNA using the RiboZero kit for Gram-negative bacteria (Epicentre). rRNA-depleted samples were concentrated by EtOH precipitation. cDNA synthesis and hybridization to Affymetrix GeneChip P. aeruginosa genome arrays.
Project description:Aims: Epidemiologic evidence indicates that diabetes may increase risk of breast cancer (BC) and mortality in patients with cancer. The pathophysiological relationships between diabetes and cancer are not fully understood, and personalized treatments for diabetes-associated BC are urgently needed. Results: We observed that high glucose (HG), via activation of nuclear phosphatase PP2C?, suppresses p53 function, and consequently promotes BC cell proliferation, migration, and invasion. PP2C? expression is higher in tumor tissues from BC patients with hyperglycemia than those with normoglycemia. The mechanisms underlying HG stimulation of PP2C? involve classical/novel protein kinase-C (PKC) activation and GSK3? phosphorylation. Reactive oxygen species (ROS)/NF-?B pathway also mediates HG induction of PP2C?. Furthermore, we identified a 1,5-diheteroarylpenta-1,4-dien-3-one (Compound 23, or C23) as a novel potent PP2C? inhibitor with a striking cytotoxicity on MCF-7 cells through cell-based screening assay for growth inhibition and activity of a group of curcumin mimics. Beside directly inhibiting PP2C? activity, C23 blocks HG induction of PP2C? expression via heat shock protein 27 (HSP27) induction and subsequent ablation of ROS/NF-?B activation. C23 can thus significantly block HG-triggered inhibition of p53 activity, leading to the inhibition of cancer cell proliferation, migration, and invasion. In addition, hyperglycemia promotes BC development in diabetic nude mice, and C23 inhibits the xenografted BC tumor growth. Conclusions and Innovation: Our findings elucidate mechanisms that may have contributed to diabetes-associated BC progression, and provide the first evidence to support the possible alternative therapeutic approach to BC patients with diabetes. Antioxid. Redox Signal. 30, 1983-1998.