Western diet enhances hepatic inflammation in mice exposed to cecal ligation and puncture

Background  

Obese patients display an exaggerated morbidity during sepsis. Since consumption of a western-style diet (WD) is a major factor for obesity in the United States, the purpose of the present study was to examine the influence of chronic WD consumption on hepatic inflammation in mice made septic via cecal ligation and puncture (CLP). Feeding mice diets high in fat has been shown to enhance evidence of TLR signaling and this pathway also mediates the hepatic response to invading bacteria. Therefore, we hypothesized that the combined effects of sepsis and feeding WD on TRL-4 signaling would exacerbate hepatic inflammation. Male C57BL/6 mice were fed purified control diet (CD) or WD that was enriched in butter fat (34.4% of calories) for 3 weeks prior to CLP. Intravital microscopy was used to evaluate leukocyte adhesion in the hepatic microcirculation. To demonstrate the direct effect of saturated fatty acid on hepatocytes, C3A human hepatocytes were cultured in medium containing 100 μM palmitic acid (PA). Quantitative real-time PCR was used to assess mRNA expression of tumor necrosis factor-alpha (TNF-α, monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), toll-like receptor-4 (TLR-4) and interleukin-8 (IL-8).     

Cyclooxygenase-2 deficiency leads to intestinal barrier dysfunction and increased mortality during polymicrobial sepsis

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2(-/-) and COX-2(+/+) BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD(2), or vehicle and stimulated with cytokines. COX-2(-/-) mice developed exaggerated bacteremia and increased mortality compared with COX-2(+/+) mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD(2) attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.     

Short-term high fat feeding increases organ injury and mortality after polymicrobial sepsis

The purpose of this study was to examine the effect of short-term high fat feeding on the inflammatory response in polymicrobial sepsis. Male C57BL/6 mice at 6 weeks of age were randomized to a high-fat diet (HFD) (60% kcal fat) or control diet (CD) (16% kcal fat) for 3 weeks. After 3 weeks of feeding, sepsis was induced by cecal ligation and puncture (CLP) and animals were monitored for survival. In a separate experiment, after 3 weeks of feeding mice underwent CLP and were sacrificed at various time points thereafter. Tissue was collected for biochemical studies. Mice fed a HFD gained more weight and had a greater fat mass compared to CD-fed mice. Mice on a HFD had a lower probability of survival and more severe lung injury compared with CD-fed mice following sepsis. Myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, was increased in the lung and liver after CLP in HFD-fed mice compared with CD (P < 0.05). The plasma cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were increased in both groups after CLP, however, TNF-α and IL-6 levels were lower in HFD mice at 3 h after CLP compared with CD and consistent with lung, but not liver, messenger RNA (mRNA) expression. Leptin levels were higher in HFD-fed mice at 18 h after sepsis compared to baseline levels (P < 0.05). Polymicrobial sepsis increased hepatic nuclear factor-κB (NF-κB) activation in HFD-fed mice after CLP vs. CD-fed mice. Short duration high fat feeding increases mortality and organ injury following polymicrobial sepsis. These effects correspond to changes in NF-κB.     

MiR-146b protect against sepsis induced mice myocardial injury through inhibition of Notch1

Myocardial dysfunction is a major cause of death in sepsis. MicroRNA-146b (miR-146b) has been reported to be related to myocardial disease. However, the role of miR-146b in sepsis as well as myocardial injury is still unclear. Septic cardiac dysfunction in mice was induced by cecal ligation and puncture (CLP) and miR-146b was found increased significantly in the myocardium tissue of CLP mice. It was found that up-regulation of miR-146b by agomiR injection suppressed expression of IL-1β in mice as well as myocardium apoptosis in CLP mice. However, suppression of miR-146b by antagomiR injection had inverse effects. Notch1 was identified as a target gene of miR-146b by bioinformatics analysis. And it was verified that in cardiomyocytes, the decrease of miR146b led to increase of both the mRNA and protein level of Notch1 and vice versa. In septic mice serum stimulated cardiomyocytes, up-regulation of miR-146b decreased the level of Notch1 and Hes1. The knockout of Notch1 in transgenic mice showed that the deficiency of Notch1 improved myocardial injury induced by CLP operation. The apoptosis of cardiomyocytes was relieved and the expression of IL-1β was decreased. In conclusion, miR-146b targets to Notch1 and protected cardiomyocytes against inflammation and apoptosis.     

Role of nitric oxide in the expression of hepatic vascular stress genes in response to sepsis

This study examined the role of nitric oxide (NO) on the expression of the hepatic vasoregulatory gene during polymicrobial sepsis. Aminoguanidine (AG, 100 mg/kg) or Nomega-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg) was injected intraperitoneally at 0, 3, 6, 10, and 20 h after a cecal ligation and puncture (CLP). The heart rate increased 24 h after the CLP, and this increase was attenuated by L-NAME and further attenuated by AG. The mean arterial pressure in the CLP animals did not change significantly 24 h after the onset of sepsis but was increased after the L-NAME injection. Sepsis increased the serum aminotransferase levels, which were attenuated by AG but augmented by L-NAME. CLP increased the mRNA level of the ET-1 and ETB receptors in the liver. This increase was prevented by AG but augmented by L-NAME. The level of iNOS and HO-1 mRNA expression were increased by CLP, which was prevented by both AG and L-NAME. The level of TNF-alpha and COX-2 mRNA expression increased after CLP, and was attenuated by AG. These results show that iNOS and eNOS are regulated differently in sepsis. While eNOS appears to have a protective role in liver microcirculation, the strong upregulation of iNOS might contribute to a microvascular dysfunction and hepatic injury.     

Caveolin-1 regulates nitric oxide-mediated matrix metalloproteinases activity and blood-brain barrier permeability in focal cerebral ischemia and reperfusion injury

The roles of caveolin-1 (cav-1) in regulating blood-brain barrier (BBB) permeability are unclear yet. We previously reported that cav-1 was down-regulated and the production of nitric oxide (NO) induced the loss of cav-1 in focal cerebral ischemia and reperfusion injury. The present study aims to address whether the loss of cav-1 impacts on BBB permeability and matrix metalloproteinases (MMPs) activity during cerebral ischemia-reperfusion injury. We found that focal cerebral ischemia-reperfusion down-regulated the expression of cav-1 in isolated cortex microvessels, hippocampus, and cortex of ischemic brain. The down-regulation of cav-1 was correlated with the increased MMP-2 and -9 activities, decreased tight junction (TJ) protein zonula occludens (ZO)-1 expression and enhanced BBB permeability. Treatment of N(G) -nitro-L-arginine methyl ester [L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor] reserved the expression of cav-1, inhibited MMPs activity, and reduced BBB permeability. To elucidate the roles of cav-1 in regulating MMPs and BBB permeability, we used two approaches including cav-1 knockdown in cultured brain microvascular endothelial cells (BMECs) in vitro and cav-1 knockout (KO) mice in vivo. Cav-1 knockdown remarkably increased MMPs activity in BMECs. Meanwhile, with focal cerebral ischemia-reperfusion, cav-1 deficiency mice displayed higher MMPs activities and BBB permeability than wild-type mice. Interestingly, the effects of L-NAME on MMPs activity and BBB permeability was partly reversed in cav-1 deficiency mice. These results, when taken together, suggest that cav-1 plays important roles in regulating MMPs activity and BBB permeability in focal cerebral ischemia and reperfusion injury. The effects of L-NAME on MMPs activity and BBB permeability are partly mediated by preservation of cav-1.     

Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression

IL-6 is known to be an important pro- and anti-inflammatory cytokine, which is up-regulated during sepsis. Our previous work has suggested a role for IL-6 in the up-regulation of C5aR in sepsis. We reported earlier that interception of C5a or C5aR results in improved outcomes in experimental sepsis. Using the cecal ligation/puncture (CLP) model in mice, we now demonstrate that treatment with anti-IL-6 Ab (anti-IL-6) results in significantly improved survival, dependent on the amount of Ab infused. CLP animals showed significantly increased binding of 125I-labeled anti-C5aR to organs when compared to either control mice at 0 h or CLP animals infused with normal rabbit 125I-labeled IgG. Binding of 125I-labeled anti-C5aR to lung, liver, kidney, and heart was significantly decreased in anti-IL-6-treated animals 6 h after CLP. RT-PCR experiments with mRNA isolated from various organs obtained 3, 6, and 12 h after CLP demonstrated increased C5aR mRNA expression during the onset of sepsis, which was greatly suppressed in CLP mice treated with anti-IL-6. These data suggest that IL-6 plays an important role in the increased expression of C5aR in lung, liver, kidney, and heart during the development of sepsis in mice and that interception of IL-6 leads to reduced expression of C5aR and improved survival.     

miR-135b-5p抑制脓毒症引起的小鼠急性肺损伤(ALI)的作用及机制*

目的: 探究miR-135b-5p在小鼠脓毒症(sepsis)引起的急性肺损伤(ALI)模型中的表达水平及其对小鼠肺部炎症反应和细胞焦亡的影响。方法: 将C57BL/6小鼠随机分为6组,每组8只,通过盲肠结扎穿刺法(CLP)手术构建CLP诱导的脓毒症小鼠模型:腹腔注射0.1 mg/kg的巴比妥麻醉,腹部纵向切开暴露盲肠,结扎盲肠并用注射器针头进行穿孔,挤出部分肠道内容物后缝合伤口。假手术组(Sham组)开腹后不做任何处理缝合伤口,无CLP手术处理。治疗组分为CLP+NC mimic组,CLP+miR-135b-5p mimic组,CLP+NC mimic+empty vector组,CLP+消皮素D (GSDMD)组,CLP+miR-135b-5p mimic+GSDMD组。治疗组小鼠在CLP手术前一周皮下注射200 μl溶解于生理盐水的NC mimic(200 nmol/L),miR-135b-5p mimic(200 nmol/L),empty vector(100 nmol/L),GSDMD vector(100 nmol/L),每天注射1次,连续一周。术后24 h采用二氧化碳窒息法实施安乐死。采用qRT-PCR检测小鼠肺组织样本中miR-135b-5p和GSDMD mRNA的表达水平;苏木精-伊红(HE)染色检测小鼠肺组织形态和损伤状态;采用5 ml生理盐水冲洗小鼠右肺3次,每次持续约3~5 min,收集肺泡灌洗液(BALF),酶联免疫吸附实验(ELISA)检测小鼠肺泡灌洗液(BALF)中GSDMD、白介素1β(IL-1β)和白介素18(IL-18)的表达水平;蛋白免疫印迹法检测小鼠肺组织内含NLR家族PYRIN域蛋白3(NLRP3),半胱氨酸天冬氨酸蛋白水解酶1(caspase 1)以及切割后的N-端GSDMD端蛋白结构域(cleaved-GSDMD-N)的表达水平。双荧光素酶报告基因检测系统验证miR-135b-5p与GSDMD的靶向结合关系。结果: 与对照组相比,CLP组小鼠肺组织中有大量的炎症细胞浸润,肺泡损伤,细胞间质水肿及肺泡塌陷等病理特征,小鼠肺组织内细胞焦亡相关蛋白(NLRP3,caspase-1和GSDMD)的表达水平明显增加(P<0.01),但miR-135b-5p的表达水平明显下调(P<0.01);与CLP组相比,超表达miR-135b-5p能够明显抑制CLP诱导的小鼠肺组织内细胞焦亡(P<0.01),靶向抑制GSDMD的表达水平(P<0.01);超表达GSDMD能够逆转超表达miR-135b-5p对肺组织细胞焦亡的抑制作用(P<0.01),超表达miR-135b-5p能够通过靶向GSDMD抑制小鼠BALF中IL-1β及IL-18的表达水平(P<0.01)。结论: miR-135b-5p靶向下调GSDMD抑制细胞焦亡,改善脓毒症引起的ALI,为脓毒症诱导的ALI治疗提供了潜在的治疗靶点和理论依据。     

Protective effect of TRPV1 against renal fibrosis via inhibition of TGF-β/Smad signaling in DOCA-salt hypertension

To investigate the effects of the transient receptor potential vanilloid type 1 (TRPV1) channel on renal extracellular matrix (ECM) protein expression including collagen deposition and the transforming growth factor β (TGF-β)/Smad signaling pathway during salt-dependent hypertension, wild-type (WT) and TRPV1-null (TRPV1?/?) mutant mice were uninephrectomized and given deoxycorticosterone acetate (DOCA)-salt for 4 wks. TRPV1 gene ablation exaggerated DOCA-salt-induced impairment of renal function as evidenced by increased albumin excretion (μg/24 h) compared with WT mice (83.7 ± 7.1 versus 28.3 ± 4.8, P < 0.05), but had no apparent effect on mean arterial pressure (mmHg) as determined by radiotelemetry (141 ± 4 versus 138 ± 3, P > 0.05). Morphological analysis showed that DOCA-salt-induced glomerulosclerosis, tubular injury and macrophage infiltration (cells/mm2) were increased in TRPV1?/? compared with WT mice (0.74 ± 0.08 versus 0.34 ± 0.04; 3.14 ± 0.26 versus 2.00 ± 0.31; 68 ± 5 versus 40 ± 4, P < 0.05). Immunostaining studies showed that DOCA-salt treatment decreased nephrin but increased collagen type I and IV as well as phosphorylated Smad2/3 staining in kidneys of TRPV1?/? compared with WT mice. Hydroxyproline assay and Western blot showed that DOCA-salt treatment increased collagen content (μg/mg dry tissue) and fibronectin protein expression (%β-actin arbitrary units) in the kidney of TRPV1?/? compared with WT mice (26.7 ± 2.7 versus 17.4 ± 1.8; 0.93 ± 0.07 versus 0.65 ± 0.08, P < 0.05). Acceleration of renal ECM protein deposition in DOCA-salt-treated TRPV1?/? mice was accompanied by increased TGF-β1, as well as phosphorylation of Smad2/3 protein expression (%β-actin arbitrary units) compared with DOCA-salt-treated WT mice (0.61 ± 0.07 versus 0.32 ± 0.05; 0.57 ± 0.07 versus 0.25 ± 0.05; 0.71 ± 0.08 versus 0.40 ± 0.06, P < 0.05). These results show that exaggerated renal functional and structural injuries are accompanied by increased production of ECM protein and activation of the TGF-β/Smad2/3 signaling pathway. These data suggest that activation of TRPV1 attenuates the progression of renal fibrosis possibly via suppression of the TGF-β and its downstream regulatory signaling pathway.     

Factor XI Deficiency Alters the Cytokine Response and Activation of Contact Proteases during Polymicrobial Sepsis in Mice

Sepsis, a systemic inflammatory response to infection, is often accompanied by abnormalities of blood coagulation. Prior work with a mouse model of sepsis induced by cecal ligation and puncture (CLP) suggested that the protease factor XIa contributed to disseminated intravascular coagulation (DIC) and to the cytokine response during sepsis. We investigated the importance of factor XI to cytokine and coagulation responses during the first 24 hours after CLP. Compared to wild type littermates, factor XI-deficient (FXI^-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1β and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in wild type mice 24 hours post-CLP, but not in FXI^-/- mice, supporting the impression of a reduced inflammatory response in the absence of factor XI. Surprisingly, there was little evidence of DIC in mice of either genotype. Plasma levels of the contact factors factor XII and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation. However, factor XII and PK levels were not reduced in FXI^-/- animals, indicating factor XI deficiency blunted contact activation. Intravenous infusion of polyphosphate into WT mice also induced changes in factor XII, but had much less effect in FXI deficient mice. In vitro analysis revealed that factor XIa activates factor XII, and that this reaction is enhanced by polyanions such polyphosphate and nucleic acids. These data suggest that factor XI deficiency confers a survival advantage in the CLP sepsis model by altering the cytokine response to infection and blunting activation of the contact (kallikrein-kinin) system. The findings support the hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.     

Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)-/- mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS-/- and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS-/- mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS-/- mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS-/- mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS-/- mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.     

Glucan phosphate attenuates cardiac dysfunction and inhibits cardiac MIF expression and apoptosis in septic mice

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. We have previously reported that glucan phosphate (GP) significantly increased survival in a murine model of cecal ligation and puncture (CLP)-induced sepsis. In the present study, we examined the effect of GP on cardiac dysfunction in CLP-induced septic mice. GP was administered to ICR/HSD mice 1 h before induction of CLP. Sham surgically operated mice served as control. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham control. In contrast, GP administration prevented CLP-induced cardiac dysfunction. Macrophage migration inhibitory factor (MIF) has been implicated as a major factor in cardiomyocyte apoptosis and cardiac dysfunction during septic shock. CLP increased myocardial MIF expression by 88.3% (P < 0.05) and cardiomyocyte apoptosis by 7.8-fold (P < 0.05) compared with sham control. GP administration, however, prevented CLP-increased MIF expression and decreased cardiomyocyte apoptosis by 51.2% (P < 0.05) compared with untreated CLP mice. GP also prevented sepsis-caused decreases in phospho-Akt, phospho-GSK-3beta, and Bcl-2 levels in the myocardium of septic mice. These data suggest that GP treatment attenuates cardiovascular dysfunction in fulminating sepsis. GP administration also activates the phosphoinositide 3-kinase/Akt pathway, decreases myocardial MIF expression, and reduces cardiomyocyte apoptosis.     

Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of alpha-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (-/-) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant (P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 -/- mice exhibited significantly (P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.     

The Decrease on Na+, K+-ATPase Activity in the Cortex, but not in Hippocampus, is Reverted by Antioxidants in an Animal Model of Sepsis

In the present study, we investigated whether sepsis induced by cecal ligation and puncture (CLP) modifies Na⁺, K⁺-ATPase activity, mRNA expression, and cerebral edema in hippocampus and cerebral cortex of rats and if antioxidant (ATX) treatment prevented the alterations induced by sepsis. Rats were subjected to CLP and were divided into three groups: sham; CLP??rats were subjected to CLP without any further treatment; and ATX?CCLP plus administration of N-acetylcysteine plus deferoxamine. Several times (6, 12, and 24) after CLP or sham operation, the rats were killed and hippocampus and cerebral cortex were isolated. Na⁺, K⁺-ATPase activity was inhibited in the hippocampus 24?h after sepsis, and ATX treatment was not able to prevent this inhibition. The Na⁺, K⁺-ATPase activity also was inhibited in cerebral cortex 6, 12, and 24?h after sepsis. No differences on Na⁺, K⁺-ATPase catalytic subunit mRNA levels were found in the hippocampus and cerebral cortex after sepsis. ATX treatment prevents Na⁺, K⁺-ATPase inhibition only in the cerebral cortex. Na⁺, K⁺-ATPase inhibition was not associated to increase brain water content. In conclusion, the present study demonstrated that sepsis induced by CLP inhibits Na⁺, K⁺-ATPase activity in a mechanism dependent on oxidative stress, but this is not associated to increase brain water content.     

The adaptation of the blood-brain barrier to vascular endothelial growth factor and placental growth factor during pregnancy

Vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) are increased in the maternal circulation during pregnancy. These factors may increase blood-brain barrier (BBB) permeability, yet brain edema does not normally occur during pregnancy. We therefore hypothesized that in pregnancy, the BBB adapts to high levels of these permeability factors. We investigated the influence of pregnancy-related circulating factors on VEGF-induced BBB permeability by perfusing cerebral veins with plasma from nonpregnant (NP) or late-pregnant (LP) rats (n=6/group) and measuring permeability in response to VEGF. The effect of VEGF, PLGF, and VEGF-receptor (VEGFR) activation on BBB permeability was also determined. Results showed that VEGF significantly increased permeability (×10(7) μm(3)/min) from 9.7 ± 3.5 to 21.0 ± 1.5 (P<0.05) in NP veins exposed to NP plasma, that was prevented when LP veins were exposed to LP plasma; (9.7±3.8; P>0.05). Both LP plasma and soluble FMS-like tyrosine-kinase 1 (sFlt1) in NP plasma abolished VEGF-induced BBB permeability in NP veins (9.5±2.9 and 12±2.6; P>0.05). PLGF significantly increased BBB permeability in NP plasma (18±1.4; P<0.05), and required only VEGFR1 activation, whereas VEGF-induced BBB permeability required both VEGFR1 and VEGFR2. Our findings suggest that VEGF and PLGF enhance BBB permeability through different VEGFR pathways and that circulating sFlt1 prevents VEGF- and PLGF-induced BBB permeability during pregnancy.     

Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.     

Rapamycin Protects Sepsis-Induced Cognitive Impairment in Mouse Hippocampus by Enhancing Autophagy

The purpose of this study is to test the hypothesis that the mammalian target of rapamycin (mTOR) signaling pathway might mediate neuroprotection in a mouse model of septic encephalopathy and also to identify the role of autophagy. Mice were subjected to cecal ligation and puncture (CLP) or a sham operation, and all 50 mice were randomly assigned to five groups: sham, CLP+ saline, CLP+ rapamycin (1, 5, 10 mg/kg) groups. Two weeks after the operation, Morris water maze was conducted for behavioral test; Nissl staining was used for observing glia infiltration; immunohistochemical staining and biochemical measures in hippocampi were performed to detect mTOR targets and autophagy indicators. Immunochemistry revealed significant loss of neurons and increased glia infiltration in hippocampus after CLP operation. Inhibition of mTOR by rapamycin rescued cognitive deficits caused by sepsis (p < 0.05). Rapamycin did not affect total mTOR targets, while phosphorylated mTOR targets (p-mTOR-Ser2448, p-p70S6k-Thr389, p-AKT-S473) decreased (p < 0.05) and autophagy indicators (LC3-II, Atg5, Atg7) were increased, and P62 was decreased in rapamycin-treated CLP mice compared with the untreated (p < 0.05) in hippocampus. Rapamycin improves learning after sepsis through enhancing autophagy and may be a potentially effective therapeutic agent for the treatment of sepsis-induced cognitive impairment.     

HMGB1 在a2A- 肾上腺素受体介导脓毒血症大鼠急性肺损伤的作用

目的:探讨HMGB1在a2A-肾上腺素受体介导脓毒血症大鼠急性肺损伤(ALI)的作用。方法:64大鼠建立盲肠结扎穿孔法(CLP)脓毒症模型,随机均分为以下两组:CLP组及CLP+马来酸钠组。各组分别于模型建立后2(T1)、6(T2)、12(T3)、24 h(T4)时检测大鼠血清TNF-、高迁移率族蛋白1(HMGB1)及IL-10含量。CLP24 h后检测肺组织干湿重比(W/D)和髓过氧化物酶活性(MPO)及HMGB1表达;并采用HE法进行肺组织学评分。结果:CLP+马来酸钠组T2时的TNF-水平明显低于CLP组(P<0.05);而HMGB1在T2、T3及T4均明显低于CLP组(P<0.05);IL-10在各个时间点比较结果差异无统计学意义(P>0.05)。CLP+马来酸钠组肺组织W/D、MPO活性、肺组织损伤评分均明显低于CLP组(P<0.05)。CLP+马来酸钠组肺组织HMGB1表达明显低于CLP组(P<0.05)。结论:HMGB1参与了ALI的病理过程,a2A-肾上腺素受体阻断可以通过抑制HMGB1从而改善ALI时的肺功能。     

Dietary glutamine supplementation modulates Th1/Th2 cytokine and interleukin-6 expressions in septic mice

Glutamine (Gln) has been demonstrated to have benefit in the modulation of systemic immunity in sepsis. However, the effects of Gln on local immunity and intra-lymphocyte cytokine expression have not been investigated in mice with gut-derived sepsis. This study evaluated the influence of a Gln-enriched diet on interleukin (IL)-6 expression in organs and Th1/Th2 type cytokine production within lymphocytes in septic mice. Male ICR mice were assigned to control and Gln groups. The control group was fed a semi-purified diet, while in the Gln group, Gln replaced part of the casein. After feeding the respective diets for 3 weeks, sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0, 6, 12 and 24h after CLP and their organs were harvested for further analysis. Results showed that IL-6 levels in the liver were decreased, whereas levels were increased in the lungs, kidneys and intestines with the progression of sepsis in both groups. Also, intra-lymphocyte interferon (IFN)-gamma expression decreased and IL-4 expression increased during sepsis. Compared to the control group, the Gln group had higher levels of IL-6 in the liver and lower levels in other organs at various time points. Lymphocyte IFN-gamma expression in the Gln group was higher, and IL-4 levels were lower than those of the control group after CLP. These results suggest that Gln supplementation decreased IL-6 production in non-hepatic organs, while reducing intra-lymphocyte IL-4 and enhancing IFN-gamma expressions. This change may reverse the Th2 type response to a more-balanced Th1/Th2 response during sepsis.