The Protective Role of Interleukin-33 in Myocardial Ischemia and Reperfusion Is Associated with Decreased HMGB1 Expression and Up-Regulation of the P38 MAPK Signaling Pathway

Interleukin-33 (IL-33) plays a protective role in myocardial ischemia and reperfusion (I/R) injury, but the underlying mechanism was not fully elucidated. The present study was designed to investigate whether IL-33 protects against myocardial I/R injury by regulating both P38 mitogen-activated-protein kinase (P38 MAPK), which is involved in one of the downstream signaling pathways of IL-33, and high mobility group box protein 1 (HMGB1), a late pro-inflammatory cytokine. Myocardial I/R injury increased the level of IL-33 and its induced receptor (sST) in myocardial tissue. Compared with the I/R group, the IL-33 group had significantly lower cardiac injury (lower serum creatine kinase (CK), lactate dehydrogenase (LDH), and cTnI levels and myocardial infarct size), a suppressed inflammatory response in myocardial tissue (lower expression of HMGB1, IL-6, TNF-α and INF-γ) and less myocardial apoptosis (much higher Bcl-2/Bax ratio and lower cleaved caspase-3 expression). Moreover, IL-33 activated the P38 MAPK signaling pathway (up-regulating P-P38 expression) in myocardial tissue, and SB230580 partially attenuated the anti-inflammatory and anti-apoptosis effects of IL-33. These findings indicated that IL-33 protects against myocardial I/R injury by inhibiting inflammatory responses and myocardial apoptosis, which may be associated with the HMGB1 and P38 MAPK signaling pathways.     

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Phosphorylation by Protein Kinase Cδ (PKCδ) Inhibits Mitochondria Elimination by Lysosomal-like Structures following Ischemia and Reoxygenation-induced Injury

After cardiac ischemia and reperfusion or reoxygenation (I/R), damaged mitochondria propagate tissue injury by promoting cell death. One possible mechanism to protect from I/R-induced injury is the elimination of damaged mitochondria by mitophagy. Here we identify new molecular events that lead to mitophagy using a cell culture model and whole hearts subjected to I/R. We found that I/R induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) association with mitochondria and promotes direct uptake of damaged mitochondria into multiorganellar lysosomal-like (LL) structures for elimination independently of the macroautophagy pathway. We also found that protein kinase C δ (PKCδ) inhibits GAPDH-driven mitophagy by phosphorylating the mitochondrially associated GAPDH at threonine 246 following I/R. Phosphorylated GAPDH promotes the accumulation of mitochondria at the periphery of LL structures, which coincides with increased mitochondrial permeability. Either inhibition of PKCδ or expression of a phosphorylation-defective GAPDH mutant during I/R promotes a reduction in mitochondrial mass and apoptosis, thus indicating rescued mitophagy. Taken together, we identified a GAPDH/PKCδ signaling switch, which is activated during oxidative stress to regulate the balance between cell survival by mitophagy and cell death due to accumulation of damaged mitochondria.     

IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.     

C3a Receptor Antagonist Ameliorates Inflammatory and Fibrotic Signals in Type 2 Diabetic Nephropathy by Suppressing the Activation of TGF-β/smad3 and IKBα Pathway

Objective

Diabetic nephropathy (DN) is a serious complication for patients with diabetes mellitus (DM). Emerging evidence suggests that complement C3a is involved in the progression of DN. The aim of this study was to investigate the effect of C3a Receptor Agonist (C3aRA) on DN and its potential mechanism of action in rats with type 2 diabetes mellitus (T2DM).

Methods

T2DM was induced in SD rats by a high fat diet (HFD) plus repeated low dose streptozocin (STZ) injections. T2DM rats were treated with vehicle or C3aRA for 8 weeks. Biochemical analysis, HE and PAS stains were performed to evaluate the renal function and pathological changes. Human renal glomerular endothelial cells (HRGECs) were cultured and treated with normal glucose (NG), high glucose (HG), HG+C3a, HG+C3a+C3aRA and HG+C3a+BAY-11-7082 (p-IKBα Inhibitor) or SIS3 (Smad3 Inhibitor), respectively. Real-time PCR, immunofluorescent staining and western blot were performed to detect the mRNA and protein levels, respectively.

Results

T2DM rats showed worse renal morphology and impaired renal function compared with control rats, including elevated levels of serum creatinine (CREA), blood urea nitrogen (BUN) and urine albumin excretion (UACR), as well as increased levels of C3a, C3aR, IL-6, p-IKBα, collagen I, TGF-β and p-Smad3 in the kidney of T2DM rats and C3a-treated HRGECs. In contrast, C3aRA treatment improved renal function and morphology, reduced CREA, UACR and the intensity of PAS and collagen I staining in the kidney of T2DM rats, and decreased C3a, p-IKBα, IL-6, TGF-β, p-Smad3 and collagen I expressions in HRGECs and T2DM rats.

Conclusion

C3a mediated pro-inflammatory and pro-fibrotic responses and aggravated renal injury in T2DM rats. C3aRA ameliorated T2DN by inhibiting IKBα phosphorylation and cytokine release, and also TGF-β/Smad3 signaling and ECM deposition. Therefore, complement C3a receptor is a potential therapeutic target for DN.     

Knockout of Toll-Like Receptors 2 and 4 Prevents Renal Ischemia-Reperfusion-Induced Cardiac Hypertrophy in Mice

We investigated whether the pathways linked to Toll-like receptors 2 and 4 (TLRs) are involved in renal ischemia-reperfusion (I/R)-induced cardiac hypertrophy. Wild type (WT) C57BL/6J, TLR2^-/- and TLR4^-/- mice were subjected to left kidney ischemia for 60 min followed by reperfusion for 5, 8, 12 and 15 days. Proton density magnetic resonance showed alterations in the injured kidney from WT mice, together with signs of parenchymal edema and higher levels of vimentin mRNA, accompanied by: (i) small, but significant, increase in serum urea after 24 h, (ii) 100% increase in serum creatinine at 24 h. A serum peak of inflammatory cytokines occurred after 5 days of reperfusion. Heart weight/body weight and heart weight/tibia length ratios increased after 12 and 15 days of reperfusion, respectively. Cardiac hypertrophy markers, B-type natriuretic peptide (BNP) and α-actin, left ventricle mass, cardiac wall thickness and myocyte width increased after 15 days of reperfusion, together with longer QTc and action potential duration. Cardiac TLRs, MyD88, HSP60 and HSP70 mRNA levels also increased. After 15 days of reperfusion, absence of TLRs prevented cardiac hypertrophy, as reflected by similar values of left ventricular cardiac mass and heart weight/body weight ratio compared to the transgenic Sham. Renal tissular injury also ameliorated in both knockout mice, as revealed by the comparison of their vimentin mRNA levels with those found in the WT on the same day after I/R. The I/R TLR2^-/- group had TNF-α, IFN-γ and IL-1β levels similar to the non-I/R group, whereas the TLR4^-/- group conserved the p-NF-κB/NF- κB ratio contrasting with that found in TLR2^-/-. We conclude: (i) TLRs are involved in renal I/R-induced cardiac hypertrophy; (ii) absence of TLRs prevents I/R-induced cardiac hypertrophy, despite renal lesions seeming to evolve towards those of chronic disease; (iii) TLR2 and TLR4 selectively regulate the systemic inflammatory profile and NF- κB activation.     

IL-33 Attenuates Anoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis by Inhibition of PKCβ/JNK Pathway

Background

Interleukin-33 (IL-33) is a new member of the IL-1 cytokine family. The objectives of present study are to assess whether IL-33 can protect cardiomyocytes from anoxia/reoxygenation (A/R)-induced injury and the mechanism involved in the protection.

Methods

Cardiomyocytes derived from either wild type or JNK1^−/− mice were challenged with an A/R with or without IL-33. Myocyte apoptosis was assessed by measuring caspase 3 activity, fragmented DNA and TUNEL staining. In addition, cardiomyocyte oxidative stress was assessed by measuring DHR123 oxidation; PKCβII and JNK phosphorylation were assessed with Western blot.

Results

Challenge of cardiomyocytes with an A/R resulted in cardiomyocyte oxidative stress, PKCβII and JNK phosphorylation, and myocyte apoptosis. Treatment of the cardiomyocytes with IL-33 attenuated the A/R-induced myocyte oxidative stress, prevented PKCβII and JNK phosphorylation and attenuated the A/R-induced myocyte apoptosis. The protective effect of the IL-33 did not show in cardiac myocytes with siRNA specific to PKCβII or myocytes deficient in JNK1. Inhibition of PKCβII prevented the A/R-induced JNK phosphorylation, but inhibition of JNK1 showed no effect on A/R-induced PKCβII phosphorylation.

Conclusions

Our results indicate that IL-33 prevents the A/R-induced myocyte apoptosis through inhibition of PKCβ/JNK pathway.     

Selective inhibition of protein kinase C β2 attenuates the adaptor P66Shc-mediated intestinal ischemia–reperfusion injury

Apoptosis is a major mode of cell death occurring during ischemia–reperfusion (I/R) induced injury. The p66^Shc adaptor protein, which is mediated by PKCβ, has an essential role in apoptosis under oxidative stress. This study aimed to investigate the role of PKCβ₂/p66^Shc pathway in intestinal I/R injury. In vivo, ischemia was induced by superior mesenteric artery occlusion in mice. Ruboxistaurin (PKCβ inhibitor) or normal saline was administered before ischemia. Then blood and gut tissues were collected after reperfusion for various measurements. In vitro, Caco-2 cells were challenged with hypoxia–reoxygenation (H/R) to simulate intestinal I/R. Translocation and activation of PKCβ₂ were markedly induced in the I/R intestine. Ruboxistaurin significantly attenuated gut damage and decreased the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Pharmacological blockade of PKCβ₂ suppressed p66^Shc overexpression and phosphorylation in the I/R intestine. Gene knockdown of PKCβ₂ via small interfering RNA (siRNA) inhibited H/R-induced p66^Shc overexpression and phosphorylation in Caco-2 cells. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66^Shc phosphorylation and this was inhibited by ruboxistaurin and PKCβ₂ siRNA. Ruboxistaurin attenuated gut oxidative stress after I/R by suppressing the decreased expression of manganese superoxide dismutase (MnSOD), the exhaustion of the glutathione (GSH) system, and the overproduction of malondialdehyde (MDA). As a consequence, ruboxistaurin inhibited intestinal mucosa apoptosis after I/R. Therefore, PKCβ₂ inhibition protects mice from gut I/R injury by suppressing the adaptor p66^Shc-mediated oxidative stress and subsequent apoptosis. This may represent a novel therapeutic approach for the prevention of intestinal I/R injury.     

Penehyclidine Hydrochloride Preconditioning Provides Cardioprotection in a Rat Model of Myocardial Ischemia/Reperfusion Injury

To investigate the impacts and related mechanisms of penehyclidine hydrochloride (PHC) on ischemia/reperfusion (I/R)-induced myocardial injury. A rat model of myocardial I/R injury was established by the ligation of left anterior descending coronary artery for 30 min followed by 3 h perfusion. Before I/R, the rats were pretreated with or without PHC. Cardiac function was measured by echocardiography. The activities/levels of myocardial enzymes, oxidants and antioxidant enzymes were detected. Evans blue/TTC double staining was performed to assess infarct size. Cardiomyocyte apoptosis was evaluated by TUNEL assay. The release of inflammatory cytokines and inflammatory mediators was detected by ELISA. Western blot was performed to analyze the expression of COX-2, IκB, p-IκB and NF-κB. Meanwhile, the rats were given a single injection of H-PHC before I/R. The effects of PHC on myocardial infarct and cardiac function were investigated after 7 days post-reperfusion. We found that PHC remarkably improved cardiac function, alleviated myocardial injury by decreasing myocardial enzyme levels and attenuated oxidative stress in a dose-dependent manner. Additionally, PHC preconditioning significantly reduced infarct size and the apoptotic rate of cardiomyocytes. Administration of PHC significantly decreased serum TNF-α, IL-1β, IL-6 and PGE₂ levels and myocardium COX-2 level. Meanwhile, the expression levels of p-IκB and NF-κB were downregulated, while IκB expression was upregulated. H-PHC also exerted long-term cardioprotection in a rat model of I/R injury by decreasing infarct size and improving cardiac function. These results suggest that PHC can efficiently protect the rats against I/R-induced myocardial injury.     

Inhaled Carbon Monoxide Protects against the Development of Shock and Mitochondrial Injury following Hemorrhage and Resuscitation

Aims

Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock. The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation.

Results

Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P<0.01). Additionally, CO limited the development of shock as determined by arterial blood pH (7.25±0.06 vs. 7.05±0.05; P<0.05), lactate levels (7.2±5.1 vs 13.3±6.0; P<0.05), and base deficit (13±3.0 vs 24±3.1; P<0.05). A dose response of CO (25–500 ppm) demonstrated protection against HS/R lung and liver injury as determined by MPO activity and serum ALT, respectively. CO limited HS/R-induced increases in serum tumor necrosis factor-α and interleukin-6 levels as determined by ELISA (P<0.05 for doses of 100–500ppm). Furthermore, inhaled CO limited HS/R induced oxidative stress as determined by hepatic oxidized glutathione:reduced glutathione levels and lipid peroxidation. In porcine HS/R, CO did not influence hemodynamics. However, CO limited HS/R-induced skeletal muscle and platelet mitochondrial injury as determined by respiratory control ratio (muscle) and ATP-linked respiration and mitochondrial reserve capacity (platelets).

Conclusion

These preclinical studies suggest that inhaled CO can be a protective therapy in HS/R; however, further clinical studies are warranted.     

The effect of taurine on renal ischemia/reperfusion injury

Summary. Ischemia-reperfusion (I/R) injury is one of the most common causes of renal dysfunction. Taurine is an endogenous antioxidant and a membrane-stabilizing, intracellular, free beta-amino acid. It has been demonstrated to have protective effects against I/R injuries to tissues other than kidney. The aim of this study was to determine whether taurine has a beneficial role in renal I/R injury. Forty Wistar-Albino rats were allocated into four groups as follows: sham, taurine, I/R, and I/R + taurine. Taurine 7.5 mg/kg was given intra-peritoneally to rats in the groups taurine and I/R + taurine. Renal I/R was achieved by occluding the renal arteries bilaterally for 40 min, followed by 6 h of reperfusion. Immediately thereafter, blood was drawn and tissue samples were harvested to measure 1) serum levels of BUN and creatinine; 2) serum and/or tissue levels of malondialdehyde (MDA), glutathione (GSH), glucose 6-phosphate dehydrogenase (G-6PD), 6-phosphogluconate dehydrogenase (6-PGD) and glutathione reductase (GSH-red); 3) renal morphology; and 4) immunohistochemical staining for P-selectin. Taurine administration reduced I/R-induced increases in serum BUN and creatinine, and serum and tissue MDA levels (p < 0.05). Additionally, taurine lessened the reductions in serum and tissue glutathione levels secondary to I/R (p < 0.05). Taurine also attenuated histopathologic evidence of renal injury, and reduced I/R-induced P-selectin immunoreactivity (p < 0.05). Overall, then, taurine administration appears to reduce the injurious effects of I/R on kidney.     

Apolipoprotein A-I attenuates renal ischemia/reperfusion injury in rats

Apolipoprotein A-I (ApoA-I), the major protein component of serum high-density lipoprotein (HDL), exhibits its anti-inflammatory activity in inflammatory responses. As renal inflammation plays an important role in ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the beneficial effect of ApoA-I on renal I/R injury in rats and the underlined mechanism. Using rats subjected to renal I/R by occlusion of bilateral renal pedicles, we found that administration of ApoA-I significantly reduced serum creatinine levels, serum TNF-α and IL-1β levels as well as tissue myeloperoxidase (MPO) activity, compared with I/R controls. Moreover, ApoA-I treatment suppresses the expression of intercellular adhesion molecules-1 (ICAM-1) and P-selectin on endothelium, thus diminishing neutrophil adherence and the subsequent tissue injury. These results showed that ApoA-I reduced I/R-induced inflammatory responses, decreased renal microscopic damage and improved renal function. It seems likely that ApoA-I protects kidney from I/R injury by inhibiting inflammatory cytokines release and neutrophil infiltration and activation.     

The protective effect of oxytocin on renal ischemia/reperfusion injury in rats

AIM: Oxytocin was previously shown to have anti-inflammatory effects in different inflammation models. The major objective of the present study was to evaluate the protective role of oxytocin (OT) in protecting the kidney against ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Male Wistar albino rats (250-300 g) were unilaterally nephrectomized, and subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. OT (1 mg/kg, ip) or vehicle was administered 15 min prior to ischemia and was repeated immediately before the reperfusion period. At the end of the reperfusion period, rats were decapitated and kidney samples were taken for histological examination or determination of malondialdehyde (MDA), an end product of lipid peroxidation; glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration. Creatinine and urea concentrations in blood were measured for the evaluation of renal function, while TNF-alpha and lactate dehydrogenase (LDH) levels were determined to evaluate generalized tissue damage. Formation of reactive oxygen species in renal tissue samples was monitored by chemiluminescence technique using luminol and lucigenin probes. RESULTS: The results revealed that I/R injury increased (p<0.01-0.001) serum urea, creatinine, TNF-alpha and LDH levels, as well as MDA, MPO and reactive oxygen radical levels in the renal tissue, while decreasing renal GSH content. However, alterations in these biochemical and histopathological indices due to I/R injury were attenuated by OT treatment (p<0.05-0.001). CONCLUSIONS: Since OT administration improved renal function and microscopic damage, along with the alleviation of oxidant tissue responses, it appears that oxytocin protects renal tissue against I/R-induced oxidative damage.     

The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

Hepatic ischemia/reperfusion (I/R) injury is an inflammation-mediated process arising from ischemia/reperfusion-elicited stress in multiple cell types, causing liver damage during surgical procedures and often resulting in liver failure. Endoplasmic reticulum (ER) stress triggers the activation of the unfolded protein response (UPR) and is implicated in tissue injuries, including hepatic I/R injury. However, the cellular mechanism that links the UPR signaling to local inflammatory responses during hepatic I/R injury remains largely obscure. Here, we report that IRE1α, a critical ER-resident transmembrane signal transducer of the UPR, plays an important role in promoting Kupffer-cell-mediated liver inflammation and hepatic I/R injury. Utilizing a mouse model in which IRE1α is specifically ablated in myeloid cells, we found that abrogation of IRE1α markedly attenuated necrosis and cell death in the liver, accompanied by reduced neutrophil infiltration and liver inflammation following hepatic I/R injury. Mechanistic investigations in mice as well as in primary Kupffer cells revealed that loss of IRE1α in Kupffer cells not only blunted the activation of the NLRP3 inflammasome and IL-1β production, but also suppressed the expression of the inducible nitric oxide synthase (iNos) and proinflammatory cytokines. Moreover, pharmacological inhibition of IRE1α′s RNase activity was able to attenuate inflammasome activation and iNos expression in Kupffer cells, leading to alleviation of hepatic I/R injury. Collectively, these results demonstrate that Kupffer cell IRE1α mediates local inflammatory damage during hepatic I/R injury. Our findings suggest that IRE1α RNase activity may serve as a promising target for therapeutic treatment of ischemia/reperfusion-associated liver inflammation and dysfunction.     

Deletion of the Innate Immune NLRP3 Receptor Abolishes Cardiac Ischemic Preconditioning and Is Associated with Decreased Il-6/STAT3 Signaling

Objective

Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart.

Principal Findings

Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC^−/− and NLRP3^−/− mice. Deletion of NLRP3 inflammasome components ASC^−/− or NLRP3^−/− did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC^−/− hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3^−/− hearts against I/R injury. NLRP3^−/− hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1β levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1β signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3^−/− hearts when compared to WT hearts.

Conclusions

The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.     

Interleukin-19 Mediates Tissue Damage in Murine Ischemic Acute Kidney Injury

Inflammation and renal tubular injury are major features of acute kidney injury (AKI). Many cytokines and chemokines are released from injured tubular cells and acts as proinflammatory mediators. However, the role of IL-19 in the pathogenesis of AKI is not defined yet. In bilateral renal ischemia/reperfusion injury (IRI)-induced and HgCl₂-induced AKI animal models, real-time quantitative (RTQ)-PCR showed that the kidneys, livers, and lungs of AKI mice expressed significantly higher IL-19 and its receptors than did sham control mice. Immunohistochemical staining showed that IL-19 and its receptors were strongly stained in the kidney, liver, and lung tissue of AKI mice. In vitro, IL-19 upregulated MCP-1, TGF-β1, and IL-19, and induced mitochondria-dependent apoptosis in murine renal tubular epithelial M-1 cells. IL-19 upregulated TNF-α and IL-10 in cultured HepG2 cells, and it increased IL-1β and TNF-α expression in cultured A549 cells. In vivo, after renal IRI or a nephrotoxic dose of HgCl₂ treatment, IL-20R1-deficient mice (the deficiency blocks IL-19 signaling) showed lower levels of blood urea nitrogen (BUN) in serum and less tubular damage than did wild-type mice. Therefore, we conclude that IL-19 mediates kidney, liver, and lung tissue damage in murine AKI and that blocking IL-19 signaling may provide a potent therapeutic strategy for treating AKI.     

Flurbiprofen,a Cyclooxygenase Inhibitor,Protects Mice from Hepatic Ischemia/Reperfusion Injury by Inhibiting GSK-3β Signaling and Mitochondrial Permeability Transition

Flurbiprofen acts as a nonselective inhibitor for cyclooxygenases (COX-1 and COX-2), but its impact on hepatic ischemia/reperfusion (I/R) injury remains unclear. Mice were randomized into sham, I/R and flurbiprofen (Flurb) groups. The hepatic artery and portal vein to the left and median liver lobes were occluded for 90 min and unclamped for reperfusion to establish a model of segmental (70%) warm hepatic ischemia. Pretreatment of animals with flurbiprofen prior to I/R insult significantly decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), and prevented hepatocytes from I/R-induced apoptosis/necrosis. Moreover, flurbiprofen dramatically inhibited mitochondrial permeability transition (MPT) pore opening, and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that flurbiprofen markedly inhibited glycogen synthase kinase (GSK)-3β activity and increased phosphorylation of GSK-3β at Ser9, which, consequently, could modulate the adenine nucleotide translocase (ANT)–cyclophilin D (CyP-D) complex and the susceptibility to MPT induction. Therefore, administration of flurbiprofen prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through inhibition of MPT and inactivation of GSK-3β, and provides experimental evidence for clinical use of flurbiprofen to protect liver function in surgical settings in addition to its conventional use for pain relief.     

Protective effects of salusin-α and salusin-β on renal ischemia/reperfusion damage and their levels in ischemic acute renal failure

Salusin-α and salusin-β are expressed in many tissues including the central nervous system, vessels and kidneys; they have been shown to decrease endoplasmic reticulum stress during heart ischemia/reperfusion (I/R) and to decrease apoptosis. We investigated the relation of salusin-α and salusin-β levels to acute ischemic renal failure. We also investigated whether these peptides are protective against renal I/R damage. Fifty-three rats were divided into six groups: control, I/R, I/R + salusin-α1, I/R + salusin-α10, I/R + salusin-β1 and I/R + salusin-β10. After removing the right kidney, the left kidney was subjected to ischemia for 1 h and reperfusion for 23 h. The treatment groups were injected subcutaneously at the beginning of ischemia with 1 or 10 μg/kg salusin-α, and 1 or 10 μg/kg salusin-β. Histopathology was assessed at the end of the experiment. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) activity and malondialdehyde (MDA) levels were measured in the kidney tissue. Serum levels of blood urea nitrogen (BUN), creatinine (Cre), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) also were measured. Levels of salusin-α and salusin-β were measured in the serum and kidney tissues of the control and I/R groups. SOD, CAT and GSH-PX activities were decreased and the levels of MDA, TNF-α, IL-6, IL-1β, BUN and Cre were increased in the I/R group compared to controls. Severe glomerular and tubular damage was apparent in the I/R group compared to controls. The level of salusin-β was decreased in the serum and kidney tissue of the I/R group compared to controls, whereas the level of salusin-α was decreased in the serum and increased in the kidney tissue. Salusin-α and salusin-β administration increased SOD and GSH-PX enzyme activation and decreased the levels of MDA, TNF-α, IL-6 and IL-1β compared to the I/R group. BUN and Cre levels were decreased in the I/R + salusin-α1 group and the level of Cre was decreased in I/R + salusin-β10 group compared to the I/R group. We demonstrated a protective effect of salusin-α and salusin-β against renal I/R damage. Changes in the levels of salusin-α and salusin-β in the I/R group suggest that these peptides may be associated with acute renal failure.     

Toll-Like Receptor 2 Mediates Ischemia-Reperfusion Injury of the Small Intestine in Adult Mice

Toll-like receptor 2 (TLR2) recognizes conserved molecular patterns associated with both gram-negative and gram-positive bacteria, and detects some endogenous ligands. Previous studies demonstrated that in ischemia-reperfusion (I/R) injury of the small intestine, the TLR2-dependent signaling exerted preventive effects on the damage in young mice, but did not have a significant effect in neonatal mice. We investigated the role of TLR2 in adult ischemia-reperfusion injury in the small intestine. Wild-type and TLR2 knockout mice at 16 weeks of age were subjected to intestinal I/R injury. Some wild-type mice received anti-Ly-6G antibodies to deplete circulating neutrophils. In wild-type mice, I/R induced severe small intestinal injury characterized by infiltration by inflammatory cells, disruption of the mucosal epithelium, and mucosal bleeding. Compared to wild-type mice, TLR2 knockout mice exhibited less severe mucosal injury induced by I/R, with a 35%, 33%, and 43% reduction in histological grading score and luminal concentration of hemoglobin, and the numbers of apoptotic epithelial cells, respectively. The I/R increased the activity of myeloperoxidase (MPO), a marker of neutrophil infiltration, and the levels of mRNA expression of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the small intestine of the wild-type mice by 3.3-, 3.2-, and 13.0-fold, respectively. TLR2 deficiency significantly inhibited the I/R-induced increase in MPO activity and the expression of mRNAs for TNF-α and ICAM-1, but did not affect the expression of COX-2 mRNA. I/R also enhanced TLR2 mRNA expression by 2.9-fold. TLR2 proteins were found to be expressed in the epithelial cells, inflammatory cells, and endothelial cells. Neutrophil depletion prevented intestinal I/R injury in wild-type mice. These findings suggest that TLR2 may mediate I/R injury of the small intestine in adult mice via induction of inflammatory mediators such as TNF-α and ICAM-1.