Cold inducible RNA-binding protein (CIRP) is a nuclear protein which has been recently identified as a novel inflammatory mediator in hemorrhagic shock and sepsis. We hypothesized that CIRP acts as a potent inflammatory mediator in hepatic ischemia-reperfusion (I/R), and thus blocking CIRP protects against I/R-induced liver injury. Male C57BL/6 mice were subjected to 70% hepatic ischemia by microvascular clamping of the hilum of the left and median liver lobes for 60 min, followed by reperfusion. Anti-CIRP antibody (1 mg/kg body weight) or vehicle (normal saline) in 0.2 mL was injected via the internal jugular vein at the beginning of the reperfusion. Blood and liver tissues were collected 24 h after I/R for various measurements and a 10-day survival study was performed. CIRP released into the circulation was significantly increased 24 h after hepatic I/R. Anti-CIRP antibody treatment markedly reduced hepatocellular damage markers and significantly improved the liver microarchitecture. Anti-CIRP also reduced the systemic and local inflammation demonstrated by attenuation in both serum and hepatic levels of interleukin 6. The expression of neutrophil-attracting chemokine as well as liver neutrophil infiltration was reduced by anti-CIRP treatment. Anti-CIRP also dramatically decreased the amount of apoptosis and nitrosative stress, evidenced by decrease in TUNEL staining and inducible nitric oxide synthase and cyclooxygenase-2 levels, respectively. Finally, the 10-day survival rate was increased from 37.5% in the vehicle group to 75% in the anti-CIRP treatment group. Thus, targeting CIRP offers potential therapeutic implications in the treatment of hepatic I/R injury.
IntroductionSepsis refers to severe systemic inflammation leading to acute lung injury (ALI) and death. Introducing novel therapies can reduce the mortality in ALI. Osteopontin (OPN), a secretory glycoprotein produced by immune reactive cells, plays a deleterious role in various inflammatory diseases. However, its role in ALI caused by sepsis remains unexplored. We hypothesize that treatment with an OPN-neutralizing antibody (anti-OPN Ab) protects mice against ALI during sepsis.MethodsSepsis was induced in 8-week-old male C57BL/6 mice by cecal ligation and puncture (CLP). Anti-OPN Ab or non-immunized IgG as control, at a dose of 50 μg/mouse, was intravenously injected at the time of CLP. After 20 hours, the expression of OPN and proinflammatory cytokines in tissues and plasma was examined by real-time PCR, Western blot, and ELISA. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) and the lung myeloperoxidase (MPO) levels were determined by colorimetric assays. Lung damage and neutrophil infiltrations were determined by histological H&E and Gr-1 staining, respectively. The effect of recombinant mouse OPN (rmOPN) on human neutrophil-like cell (HL-60) migration was performed by Boyden chamber assays and the involvement of intracellular signaling molecules in HL-60 cells was revealed by Western blot.ResultsAfter 20 hours of sepsis, mRNA and protein levels of OPN were significantly induced in lungs, spleen, and plasma. Treatment with an anti-OPN Ab in septic mice significantly reduced the plasma levels of ALT, AST, and LDH, and the proinflammatory cytokines IL-6, IL-1β and the chemokine MIP-2, compared with the vehicle group. Similarly, the lung mRNA and protein expressions of proinflammatory cytokines and chemokine were greatly reduced in anti-OPN Ab-treated animals. The lung histological architecture, MPO and neutrophil infiltration were significantly improved in anti-OPN Ab-treated mice compared with the vehicle animals. Treatment of rmOPN in HL-60 cells significantly increased their migration, in vitro. The neutrophils treated with rmOPN remarkably increased the levels of phospho focal adhesion kinase (pFAK), phospho extracellular signal-regulated kinase (pERK) and phospho p38.ConclusionsOur findings clearly demonstrate the beneficial outcomes of anti-OPN Ab treatment in protecting against ALI, implicating a novel therapeutic strategy in sepsis.
SRT1720 treatment enhanced energy metabolism by stimulating mitochondrial biogenesis as well as decreasing nitrosative stress and inflammation, thereby attenuating I/R-induced renal injury.
Objective Hepatic ischemia-reperfusion (I/R) is a major clinical problem with limited treatment options. The pathophysiology of hepatic I/R is characterized by mitochondrial dysfunction and cellular energy deficits. Sirtuin 1 (Sirt1) is an energy-sensing enzyme known to modulate mitochondrial biogenesis. We hypothesized that pharmacologic activation of Sirt1 is protective after hepatic I/R injury. Design Animal study. Setting University-based experimental laboratory. Subjects Wild-type C57BL/6 mice. Interventions C57BL/6 mice were subjected to 60-min partial hepatic I/R and post-treated with Sirt1 activator, SRT1720 (20 mg/kg), or vehicle. Blood and liver were collected at 24 h after I/R for analyses of hepatic injury, ATP levels, mitochondrial mass, autophagy, inflammation, and oxidative stress. H4IIE hepatoma cells and rat primary hepatocytes were incubated with oxyrase to induce hypoxia followed by reoxygenation (H/R) in the presence or absence of SRT1720 for assessment of mitochondrial mass, mitochondrial membrane potential, and autophagy. Measurements and Main Results SRT1720 restored the reduction in mitochondrial mass, enhanced autophagy, and preserved ATP levels in the liver after I/R, which was associated with a decrease in I/R-induced hepatic injury, apoptosis, and necrosis. I/R-induced inflammation was also significantly reduced by SRT1720 as measured by systemic and hepatic cytokine and chemokine levels as well as a decrease in neutrophil infiltration to the liver. Further, oxidative stress was markedly attenuated in the SRT1720-treated mice, compared to the vehicle. SRT1720 treatment increased ATP levels and survival of cultured hepatocytes after H/R. SRT1720 not only increased the mitochondrial mass but also increased mitochondrial membrane potential per cell in cultured hepatocytes after H/R. Moreover, SRT1720 prevented the H/R-induced mitochondrial depolarization and resulted in an enhancement of autophagy in cultured hepatocytes after H/R. Conclusions Pharmacologic stimulation of Sirt1 attenuates liver injury after hepatic I/R by restoring mitochondrial mass and membrane potential, which is associated with the enhancement of autophagy.
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