磷脂酶A2与NF-κB活化在大鼠急性坏死性胰腺炎肺损伤中的作用

目的:探讨磷脂酶A2 (PLA2 )与NF -κB活化在大鼠急性坏死性胰腺炎肺损伤中的作用。方法:Wistar大鼠随机分为假手术组、模型组、PLA2 抑制剂组。每组按不同时点(3h、6h、12h) ,分别测定血清淀粉酶、脂肪酶、PLA2 、TNFα、IL - 1、IL -6水平;测定肺组织MPO、PLA2 、TNFα、IL - 1、IL - 6水平;行肺、胰腺组织病理学检查;化学发光ELISA法检测肺泡灌洗液巨噬细胞NF -κB(P65)表达情况;每组12只用于观察2 4h死亡率。结果:PLA2 抑制剂组血清淀粉酶、脂肪酶、PLA2 ,肺组织及血清TNFα、IL - 1、IL - 6水平显著低于模型组(P值均小于0 0 1) ;肺组织MPO、PLA2 水平亦降低;肺泡灌洗液巨噬细胞NF -κB活性显著下降,同时肺脏及胰腺病理损害明显减轻;2 4h死亡率明显降低。结论:PLA2 可通过激活肺组织NF -κB ,上调多种细胞因子的表达而产生肺损害作用;阻断磷脂酶A2 可有效地减轻胰腺炎肺损伤     

Carbon Monoxide Protects Rat Lung Transplants From Ischemia-Reperfusion Injury via a Mechanism Involving p38 MAPK Pathway

Carbon monoxide (CO) provides protection against oxidative stress via anti‐inflammatory and cytoprotective actions. In this study, we tested the hypothesis that a low concentration of exogenous (inhaled) CO would protect transplanted lung grafts from cold ischemia‐reperfusion injury via a mechanism involving the mitogen‐activated protein kinase (MAPK) signaling pathway. Lewis rats underwent orthotopic syngeneic or allogeneic left lung transplantation with 6 h of cold static preservation. Exposure of donors and recipients (1 h before and then continuously post‐transplant) to 250 ppm CO resulted in significant improvement in gas exchange, reduced leukocyte sequestration, preservation of parenchymal and endothelial cell ultrastructure and reduced inflammation compared to animals exposed to air. The beneficial effects of CO were associated with p38 MAPK phosphorylation and were significantly prevented by treatment with a p38 MAPK inhibitor, suggesting that CO's efficacy is at least partially mediated by activation of p38 MAPK. Furthermore, CO markedly suppressed inflammatory events in the contralateral naïve lung. This study demonstrates that perioperative exposure of donors and recipients to CO at a low concentration can impart potent anti‐inflammatory and cytoprotective effects in a clinically relevant model of lung transplantation and support further evaluation for potential clinical use.     

Redox Modulation Protects Islets From Transplant-Related Injury

OBJECTIVE

Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant.

RESEARCH DESIGN AND METHODS

The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation.

RESULTS

Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection.

CONCLUSIONS

Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.Hypoxia is the leading cause of β-cell death during islet isolation and transplantation (1), with the highest percentage of islet graft loss and dysfunction occurring just days after transplantation (2,3). Because islets are a cellular transplant, devoid of intrinsic vasculature (1,4), they are exceptionally susceptible to ischemia-reperfusion injury. Islets are also increasingly vulnerable because they have inherently decreased antioxidant capacity (5–10), making them prone to oxidative/nitrosative/free radical damage. The antigen-independent complexities of islet transplantation increase the incidence of primary graft nonfunction and β-cell death, thus requiring protection for islets at early stages of the transplant procedure (11).In addition to antigen-independent innate-mediated inflammatory injury, islet allografts are also plagued by the antigen-dependent T-cell mediated alloimmune response, which necessitates immunosuppressive drugs for allograft survival. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response, although most are harmful to islets and do not protect well from reactive oxygen species and inflammation during islet isolation and ischemia-reperfusion injury (12–14). In their review, Balamurugan et al. (13) concluded that successful islet transplantation in type 1 diabetes necessitates islet-sparing immunosuppressive agents that combat recurrent autoimmunity with low islet toxicity. Predominantly, the field of islet transplantation is devoid of cytoprotective agents that promote islet survival and function by inhibiting nonspecific innate-mediated inflammation during islet isolation and early inflammatory events in islet transplantation (11,13,15–19).The first phase of immunity involves innate immune activation and subsequent proinflammatory signals required for optimal adaptive immune function (20–22), yet the majority of immunosuppressive drugs only target adaptive immune function (17,23), the second phase of immunity. A nontoxic, cell-permeable catalytic antioxidant (CA) redox modulator, FBC-007 [manganese(II) tetrakis (N-ethylpyridium-2-yl)porphyrin], is able to depress free radical and cytokine production by antigen-presenting cells (24) and T cells in transgenic and allospecific mouse models (20,25). Additionally, redox modulation inhibits cytotoxic lymphocyte target cell lysis by reducing the production of intracellular cytolytic molecules (perforin and granzyme B) in a mixed leukocyte reaction without toxicity (25), preserves and promotes human islet function in vitro (15,16), prevents the transfer of diabetes into young NOD.scid mice (26), and inhibits innate-immune nuclear factor (NF)-κB activation (24). Thus, islet-sparing agents, which decrease the production of free radicals and, therefore, inflammatory cytokines, may have a positive impact on islet function post-transplant.Because islet transplantation can benefit from agents that inhibit early inflammatory cascades to preserve islet function (18), we hypothesize that redox modulation holds potential as a therapy in islet transplantation to decrease the incidence of β-cell primary nonfunction. To further test the effects of redox modulation using CA we treated human islets with streptozotocin (STZ) in vitro and treated mice in vivo with STZ, both in the presence or absence of CA, to mimic antigen-independent free radical damage and inflammation of post-transplant ischemia-reperfusion injury. To examine the effects of islet-directed CA treatment on innate-mediated (antigen-independent) primary islet nonfunction in vivo, we performed syngeneic (175 islets/recipient), suboptimal syngeneic (100 islets/recipient), allogeneic (300 islets/recipient), and xenogeneic (400–500 islets/recipient) islet transplants to assess islet function. Additionally, we performed (300 islets/recipient) islet transplants in diabetic recipients to assess islet function in the presence or absence of systemic redox modulation in an allogeneic transplant setting inclusive of both innate (antigen-independent) and adaptive (antigen-dependent) immune responses. Our results demonstrate that islet-directed and systemically delivered redox modulation, administered in the absence of an additional immunosuppressive regimen, preserve islet function post-transplant.     

Functional TauT Protects Against Acute Kidney Injury

Nephrotoxicity is common with the use of the chemotherapeutic agent cisplatin, but the cellular mechanisms that modulate the extent of injury are unknown. Cisplatin downregulates expression of the taurine transporter gene (TauT) in LLC-PK1 proximal tubular renal cells, and forced overexpression of TauT protects against cisplatin-induced apoptosis in vitro. Because the S3 segments of proximal tubules are the sites of both cisplatin-induced injury and adaptive regulation of the taurine transporter, we hypothesized that TauT functions as an anti-apoptotic gene and protects renal cells from cisplatin-induced nephrotoxicity in vivo. Here, we studied the regulation of TauT in cisplatin nephrotoxicity in a human embryonic kidney cell line and in LLC-PK1 cells, as well as in TauT transgenic mice. Cisplatin-induced activation of p53 repressed TauT and overexpression of TauT prevented the progression of cisplatin-induced apoptosis and renal dysfunction in TauT transgenic mice. Although cisplatin activated p53 and PUMA (a p53-responsive proapoptotic Bcl-2 family protein) in the kidneys of both wildtype and TauT transgenic mice, only wildtype animals demonstrated acute kidney injury. These data suggest that functional TauT plays a critical role in protecting against cisplatin-induced nephrotoxicity, possibly by attenuating a p53-dependent pathway.Acute kidney injury due to ischemic or toxic renal damage is a common disorder with mortality of approximately 50%.^1,2 As a highly effective chemotherapeutic agent, cisplatin has been used to treat a wide variety of solid tumors.³ However, 25% to 35% of patients experience a significant decline in renal function after the administration of a single dose of cisplatin.⁴ Several mechanisms, including oxidation, inflammation, genotoxic damage, and cell cycle arrest, have been implicated in cisplatin nephrotoxicity.^5–10Elevated levels of the tumor suppressor gene p53 have been found in the kidneys of animal models of acute kidney injury induced by cisplatin administration.¹¹ Jiang et al.¹² have demonstrated that p53 activation is an early signal in cisplatin-induced apoptosis in renal tubular cells. The Varmus group¹³ has found that transgenic mice overexpressing p53 undergo progressive renal failure through a novel mechanism by which p53 appears to alter cellular differentiation, rather than by growth arrest or the direct induction of apoptosis. These findings suggest that altered expression of certain p53 target gene(s) involved in renal development may be responsible for p53-induced progressive renal injury in p53 transgenic mice.Our studies have shown that TauT is negatively regulated by p53 in renal cells.¹⁴ Interestingly, the progressive renal injury seen in p53 transgenic mice is similar to that previously observed in the offspring of taurine-deficient cats, which showed ongoing kidney damage and abnormal renal and retinal development,¹⁵ suggesting that the taurine transporter gene is an important target of p53 during kidney development and renal injury. It is worth noting that cisplatin accumulates in cells from all nephron segments, but is preferentially taken up by the highly susceptible proximal tubule cells within the S3 segment, which is the site for renal adaptive regulation of TauT.^16,17 A recent study showed that taurine was able to attenuate cisplatin-induced nephrotoxicity and protect renal tubular cells from tubular atrophy and apoptosis.¹⁸ Therefore, downregulation of TauT by p53 may play an important role in cisplatin-induced nephrotoxicity.     

Pancreatitis-associated protein protects the lung from leukocyte-induced injury.

BACKGROUND: Severe pancreatitis is often complicated by shock and acute lung failure. Little is known about the pathophysiologic impact of the 16.6-kD lectine, named pancreatitis-associated protein (PAP), which is expressed during pancreatitis and which reduces mortality in a rat model with severe pancreatitis. Therefore, the aim of this study was to investigate the effects of PAP on the pulmonary vasculature after leukocyte activation with N-formyl-Met-Leu-Phe (fMLP). METHODS: The experiments were performed in buffer-perfused isolated rabbit lungs. Mean pulmonary artery pressure, weight gain, and thromboxane A2 synthesis of the lungs were monitored. PAP was obtained by affinity chromatography of pancreas juice from pancreatitic rats. The authors tested whether treatment with PAP (260 microg/l, n = 9; or 500 microg/l, n = 6) before fMLP injection (10(-6) M) influences mean pulmonary artery pressure and edema formation. Lungs that were treated only with fMLP (n = 6) served as controls. Additional experiments in which PAP was applied were performed to study whether PAP (260 microg/l, n = 3; 500 microg/l, n = 3; 1,000 microg/l, n = 3) itself effects lung vasculature. RESULTS: Application of fMLP resulted in an increase of mean pulmonary artery pressure (+/- SD) from 8 +/- 2 mmHg up to 26 +/-13 mmHg (P < 0.01) at a flow of 150 ml/min. Pretreatment with PAP reduced the peak pressure developed after fMLP to 15 +/- 7 mmHg (PAP 260 microg/l; P < 0.05) and to 9 +/- 4 mmHg (PAP 500 microg/l), respectively. In addition, the fMLP-induced lung weight gain of 9 +/- 7 g in the controls was prevented by pretreatment with PAP after 150 min in either concentration. In parallel to the attenuated pressure increase, thromboxane A2 release was significantly suppressed in the 260-microg/l (200 +/- 220 pmol x ml(-1) x min(-1); P < 0.01) and 500-microg/l (285 +/- 70 pmol x m(-1) x min(-1); P < 0.05) PAP groups compared with controls (1,138 +/- 800 pmol x ml(-1) x mi(-1)). Treatment with PAP alone in either concentration did not induce any changes in mean pulmonary artery pressure, weight gain, or thromboxane A2 release. CONCLUSION: Clinically relevant concentrations of PAP prevented fMLP-induced vasoconstriction and edema formation in the lung. These findings point toward a protective effect of PAP on polymorphonuclear neutrophil leukocyte-mediated lung injury.     

离体兔肺缺血再灌注损伤肺表面活性蛋白变化及保护

目的 探讨最佳肺泡内氧浓度及温度对肺缺血再灌注损伤的保护作用。方法 离体兔肺在不同肺泡内氧浓度及不同温度下用非氧合血行肺动脉灌注３０分钟测定支气管包灌洗液中血浆白蛋白（ＡＬＢ），肺表面活性蛋白（ＳＰ，ＳＰ－Ａ，ＳＰ－Ｂ，ＳＰ－Ｃ）占总蛋白含量的百分比，ＰＳ的活性并进行细胞学观察。结果 肺泡内氧浓度与ＳＰ含量及活性成正比，与ＡＬＢ含量成反比（Ｐ〈０．０１）；肺泡内温度与ＡＬＢ含量成正比，与ＳＰ含量及     

Clotrimazole Protects the Liver Against Normothermic Ischemia-Reperfusion Injury in Rats

Objective

To investigate the possible antiapoptotic prosurvival role of the pregnane X receptor (PXR) in hepatic ischemia-reperfusion injury in rats using clotrimazole (CTZ), a strong PXR transactivator.

Materials and Methods

Male Sprague-Dawley rats were divided into 3 groups of 6 each: sham-treated, control, and CTZ-treated animals. Control and CTZ-treated animals were subjected to 30 minutes of normothermic ischemia of the whole liver followed by 6 hours of reperfusion. The animals were then killed, and the liver was excised and blood samples collected.

Results

Clotrimazole induced a significant increase in expression of the CYP3A gene, indicating PXR transactivation, whereas expression of the antiapoptotic Bcl-xL gene was not increased. Serum concentrations of aspartate aminotransaminase and alanine aminotransaminase were lower in CTZ-treated animals than in control animals (difference not significant). Levels of poly(adenosine diphosphate–ribose) polymerase, a caspase-3 substrate, remained significantly higher in the CTZ-treated group compared with controls (P < .05). Clotrimazole increased the expression of phospho-p 44/42 extracellular signal-regulated kinase 1,2 (P < .05). The gene expression of the heat shock proteins 27, 70 and 90 was significantly lower in CTZ-treated animals than in controls (P < .05).

Conclusion

Clotrimazole-mediated PXR transactivation protects the liver against ischemia-reperfusion apoptosis in rats. Phospho-p 44/42 extracellular signal-regulated kinase 1,2 is activated, whereas gene expression of heat shock proteins 27, 70, and 90 is downregulated by induction of PXR.     

Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP^−/−, and TxNIP^+/− mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP^−/− mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP^−/− mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms’ tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP^−/− mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O₂⁻ generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.     

Class‐Specific Histone/Protein Deacetylase Inhibition Protects Against Renal Ischemia Reperfusion Injury and Fibrosis Formation

Renal ischemia‐reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure. Histone/protein deacetylases (HDACs) regulate gene accessibility and higher order protein structures and may alter cellular responses to a variety of stresses. We investigated whether use of pan‐ and class‐specific HDAC inhibitors (HDACi) could improve IRI tolerance in the kidney. Using a model of unilateral renal IRI, we investigated early renal function after IRI, and calculated fibrosis after IRI using an automated scoring system. We found that pan‐HDAC inhibition using trichostatin (TSA) yielded significant renal functional benefit at 24–96 hours (p < 0.001). Treated mice developed significantly less fibrosis at 30 days (p < 0.0004). Class I HDAC inhibition with MS‐275 yielded similar effects. Protection from fibrosis formation was also noted in a cold ischemia transplant model (p < 0.008) with a trend toward improved cold ischemic survival in TSA‐treated mice. These effects were not accompanied by induction of typical ischemic tolerance pathways or by priming of heat shock protein expression. In fact, heat shock protein 70 deletion or overexpression did not alter renal ischemia tolerance. Micro‐RNA 21, known to be enhanced in vitro in renal tubular cells that survive stress, was enhanced by treatment with HDACi, pointing to possible mechanism.     

前列腺素E1对大鼠肝脏缺血再灌注损伤的保护作用

目的 探讨前列腺素E1（PGE1）对肝脏 因再灌注损伤的保护作用。方法 制作常温下大鼠部分肝叶缺血再灌注模型，于缺血前经门静脉给予PGE1，45min后恢复血流灌注，并于1h后取门静脉血测定血清谷草转氨酶（GOT）、谷丙转氨酶（GPT）、乳酸脱氢酶（LDH）、肿瘤坏死因子－α（TNF－α）及内皮素1（ET－1），同时取缺血肝叶行病理组织学检查。结果 缺血再灌注组GOT、GPT、LDH及TNF－α和ET－1均明显高于正常对照组，PGE1组则明显低于缺血再灌注组。PGE1组的肝脏病理组织学改变明显轻于缺血再灌注组，并接近正常对照组。结论 PGE1对肝缺血再灌注具有保护作用。     

Remifentanil Preconditioning Protects against Ischemic Injury in the Intact Rat Heart

Background: Opioid receptors mediate cardiac ischemic preconditioning. Remifentanil is a new, potent ultra-short-acting phenylpiperidine opioid used in high doses for anesthesia. The authors hypothesize that pretreatment with this drug confers cardioprotection.

Methods: Male Sprague-Dawley rats were anesthetized and the chest was opened. All animals were subjected to 30 min of occlusion of the left coronary artery and 2 h of reperfusion. Before the 30-min occlusion, rats received either preconditioning by ischemia (ischemic preconditioning, 5-min occlusion, 5-min reperfusion x 3) or pretreatment with remifentanil, performed with the same regime (3 x 5-min infusions) using 0.2, 0.6, 2, 6, or 20 [mu]g[middle dot]kg-1[middle dot]min-1 intravenously. The experiment was repeated with naltrindole (a selective [DELTA]-opioid receptor antagonist, 5 mg/kg), nor-binaltorphimine (a selective [kappa]-OR antagonist, 5 mg/kg), or CTOP (a selective [mu]-opioid receptor antagonist, 1 mg/kg) administered before remifentanil-induced preconditioning or ischemic preconditioning, respectively. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining.

Results: There was a dose-related reduction in infarct size/area at risk after treatment with remifentanil that was similar to that seen with ischemic preconditioning. This effect was prevented or significantly attenuated by coadministration of a [mu], [kappa], or [DELTA]-opioid antagonist. The infarct-sparing effect of ischemic preconditioning was abolished by blockade of [kappa]-opioid receptors or [DELTA]-opioid receptors but not by [mu]-opioid receptors.     

Fatty Acid Synthase Blockade Protects Steatotic Livers from Warm Ischemia Reperfusion Injury and Transplantation

Cerulenin has been shown to reduce body weight and hepatic steatosis in murine models of obesity by inhibiting fatty acid synthase (FAS). We have shown that attenuating intrahepatocyte lipid content diminished the sensitivity of ob/ob mice to ischemia/reperfusion injury and improved survival after liver transplantation. The mechanism of action is by inhibition of fatty acid metabolism by downregulating PPARalpha, as well as mitochondrial uncoupling protein 2 (UCP2), with a concomitant increase in ATP. A short treatment course of cerulenin prior to I/R injury is ideal for protection of steatotic livers. Cerulenin opens the potential for expanding the use of steatotic livers in transplantation.