Liver Of Septic Mice Research Articles

Development of novel liver-targeting glucocorticoid prodrugs

BackgroundGlucocorticoids (GCs) are widely used in the treatment of inflammatory liver diseases and sepsis, but GC’s various side effects on extrahepatic tissues limit their clinical benefits. Liver-targeting GC therapy may have multiple advantages over systemic GC therapy. The purpose of this study was to develop novel liver-targeting GC prodrugs as improved treatment for inflammatory liver diseases and sepsis. MethodsA hydrophilic linker or an ultra-hydrophilic zwitterionic linker carboxylic betaine (CB) was used to bridge cholic acid (CA) and dexamethasone (DEX) to generate transporter-dependent liver-targeting GC prodrugs CA-DEX and the highly hydrophilic CA-CB-DEX. The efficacy of liver-targeting DEX prodrugs and DEX were determined in primary human hepatocytes (PHH), macrophages, human whole blood, and/or mice with sepsis induced by cecal ligation and puncture. ResultsCA-DEX was moderately water soluble, whereas CA-CB-DEX was highly water soluble. CA-CB-DEX and CA-DEX displayed highly transporter-dependent activities in reporter assays. Data mining found marked dysregulation of many GR-target genes important for lipid catabolism, cytoprotection, and inflammation in patients with severe alcoholic hepatitis. These key GR-target genes were similarly and rapidly (within 6 h) induced or down-regulated by CA-CB-DEX and DEX in PHH. CA-CB-DEX had much weaker inhibitory effects than DEX on endotoxin-induced cytokines in mouse macrophages and human whole blood. In contrast, CA-CB-DEX exerted more potent anti-inflammatory effects than DEX in livers of septic mice. ConclusionsCA-CB-DEX demonstrated good hepatocyte-selectivity in vitro and better anti-inflammatory effects in vivo. Further test of CA-CB-DEX as a novel liver-targeting GC prodrug for inflammatory liver diseases and sepsis is warranted.

MFG-E8 has guiding significance for the prognosis and treatment of sepsis

Sepsis remains a significant clinical challenge. Ferroptosis is involved in the pathogenesis of sepsis. Ferroptosis is associated with oxidative stress, and excessive oxidative stress is suppressed by milk fat globule epidermal growth factor 8 (MFG-E8) under various conditions. However, the role of MFG-E8 in sepsis-induced ferroptosis and oxidative stress is still unclear. First, we collected blood samples from patients with sepsis and detected the expression of serum MFG-E8. Then, the relationship between serum concentrations of MFG-E8 and disease severity was detected. Finally, the effects of MFG-E8 treatment on ferroptosis and oxidative stress in the livers of septic mice were determined. The expression of serum MFG-E8 in healthy subjects was notably higher than that in septic patients. In addition, when nonsurvivors and survivors of sepsis were compared, MFG-E8 levels were considerably lower in the former. The ROC curve for MFG-E8 was also generated. The area under the curve for MFG-E8 was 0.768 (95% confidence interval [CI] 0.627–0.909, p = 0.003). The patients were separated into two groups based on the MFG-E8 cut-off value of 3.86 ng/mL. According to the Kaplan‒Meier survival analysis, patients with low MFG-E8 levels had a significantly decreased 28-day survival rate compared with patients with high MFG-E8 levels. High MFG-E8 levels were substantially related to a decreased risk of death, as demonstrated by the Cox proportional hazard model that we utilized. In addition, compared with sham mice, septic mice exhibited liver and kidney damage, and MFG-E8 may have protective effects. The survival study indicated that MFG-E8 could effectively improve the survival rate of septic mice. Treatment with MFG-E8 suppresses oxidative stress and ferroptosis in the livers of septic mice. Serum MFG-E8 levels are lower in septic patients and are negatively related to disease severity. Treatment with MFG-E8 suppresses oxidative stress and ferroptosis in the livers of septic mice, contributing to significantly improved survival in septic mice. These findings showed that MFG-E8 could be a new sepsis predictive biomarker. MFG-E8 may have therapeutic potential in the treatment of sepsis.

Koumine regulates macrophage M1/M2 polarization via TSPO, alleviating sepsis-associated liver injury in mice

BackgroundTranslocator protein (TSPO) is an 18-kDa transmembrane protein found primarily in the mitochondrial outer membrane, and it is implicated in inflammatory responses, such as cytokine release. Koumine (KM) is an indole alkaloid extracted from Gelsemium elegans Benth. It has been reported to be a high-affinity ligand of TSPO and to exert anti-inflammatory and immunomodulatory effects in our recent studies. However, the protective effect of KM on sepsis-associated liver injury (SALI) and its mechanisms are unknown. Purpose: To explore the role of TSPO in SALI and then further explore the protective effect and mechanism of KM on SALI. MethodsThe effect of KM on the survival rate of septic mice was confirmed in mouse models of caecal ligation and puncture (CLP)-induced and lipopolysaccharide (LPS)-induced sepsis. The protective effect of KM on CLP-induced SALI was comprehensively evaluated by observing the morphology of the mouse liver and measuring liver injury markers. The serum cytokine content was detected in mice by flow cytometry. Macrophage polarization in the liver was examined using western blotting. TSPO knockout mice were used to explore the role of TSPO in sepsis liver injury and verify the protective effect of KM on sepsis liver injury through TSPO. ResultsKM significantly improved the survival rate of both LPS- and CLP-induced sepsis in mice. KM has a significant liver protective effect on CLP-induced sepsis in mice. KM treatment ameliorated liver ischaemia, improved liver pathological injuries, and decreased the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and proinflammatory cytokines in serum. Western blotting results showed that KM inhibited M1 polarization of macrophages and promoted M2 polarization. In TSPO knockout mice, we found that TSPO knockout can improve the survival rate of septic mice, ameliorate liver ischaemia, improve liver pathological injuries, and decrease the levels of ALT, AST, and LDH. In addition, TSPO knockout inhibits the M1 polarization of macrophages in the liver of septic mice and promotes M2 polarization and the serum levels of proinflammatory cytokines. Interestingly, in TSPO knockout septic mice, these protective effects of KM were no longer effective. ConclusionsWe report for the first time that TSPO plays a critical role in sepsis-associated liver injury by regulating the polarization of liver macrophages and reducing the inflammatory response. KM, a TSPO ligand, is a potentially desirable candidate for the treatment of SALI that may regulate macrophage M1/M2 polarization through TSPO in the liver.

IL-1R1 blockade attenuates liver injury through inhibiting the recruitment of myeloid-derived suppressor cells in sepsis

Myeloid-derived suppressor cells (MDSCs) mobilize and migrate from bone marrow to peripheral tissues or immune organs, which is associated with poor prognosis in sepsis. Intervention of MDSCs might be a potential target for the effective treatment of sepsis. In the present study, we demonstrated that IL-1R1 blockade with either recombinant human IL-1R antagonist Anakinra or IL-1R1 deficiency had a protective effect on the liver injury in septic mice. The possible mechanism was that Anakinra treatment and IL-1R1 knockout inhibited the migration of MDSCs to the liver in sepsis, thus attenuating the immune suppression of MDSCs on effector T cells characterized with the decrease in proportion of CD4+ and CD8+ T cells. Furthermore, the switch from pro-inflammatory M1 macrophage to anti-inflammatory M2 phenotype and the ability of bacterial clearance in the liver of septic mice were enhanced obviously by Anakinra and IL-1R1 deficiency, which contributes to the attenuated liver injury. Taken together, these findings provide new ideas for revealing the relationship between IL-1R1 and MDSCs in sepsis, thereby providing a potentially effective target for ameliorating septic liver injury.

P2X7 receptor deletion attenuates oxidative stress and liver damage in sepsis.

Sepsis is a severe disease characterized by an uncontrolled systemic inflammation and consequent organ dysfunction generated in response to an infection. Extracellular ATP acting through the P2X7 receptor induces the maturation and release of pro-inflammatory cytokines (i.e., IL-1β) and the production of reactive nitrogen and oxygen species that lead to oxidative tissue damage. Here, we investigated the role of the P2X7 receptor in inflammation, oxidative stress, and liver injury in sepsis. Sepsis was induced by cecal ligation and puncture (CLP) in wild-type (WT) and P2X7 knockout (P2X7-/-) mice. The oxidative stress in the liver of septic mice was assessed by 2',7'-dichlorofluorescein oxidation reaction (DCF), thiobarbituric acid-reactive substances (TBARS), and nitrite levels dosage. The status of the endogenous defense system was evaluated through catalase (CAT) and superoxide dismutase (SOD) activities. The inflammation was assessed histologically and by determining the expression of inflammatory cytokines and chemokines by RT-qPCR. We observed an increase in the reactive species and lipid peroxidation in the liver of septic WT mice, but not in the liver from P2X7-/- animals. We found an imbalance SOD/CAT ratio, also only WT septic animals. The number of inflammatory cells and the gene expression of IL-1 β, IL-6, TNF-α, IL-10, CXCL1, and CXCL2 were higher in the liver of WT septic mice in comparison to P2X7-/- septic animals. In summary, our results suggest that the P2X7 receptor might be a therapeutic target to limit oxidative stress damage and liver injury during sepsis.

Serum irisin levels are decreased in patients with sepsis, and exogenous irisin suppresses ferroptosis in the liver of septic mice.

BackgroundSepsis remains a major health issue without an effective therapy. Ferroptosis, an iron‐dependent programmed cell death, has been proposed to be related to the pathogenesis of sepsis. Irisin, a myokine released during exercise, improves mitochondrial function under various conditions. Ferroptosis is closely related to mitochondrial function. However, the role of irisin in sepsis‐induced ferroptosis and mitochondrial dysfunction in the liver remained unknown. Thus, we hypothesize that irisin treatment suppresses ferroptosis and improves mitochondrial function in sepsis.MethodsTo study this, we first explored the role of serum irisin levels in patients with sepsis, and then determined the effect of irisin administration on ferroptosis and mitochondrial function in the liver of septic mice.ResultsSerum irisin levels were decreased and negatively correlated with the APACHE II scores in patients with sepsis. In mice subjected to cecal ligation and puncture (CLP), exogenous irisin administration suppressed ferroptosis, inhibited inflammatory response, decreased reactive oxygen species (ROS) production, restored abnormal mitochondrial morphology, and increased mtDNA copy number and adenosine triphosphate (ATP) content. The effect of irisin on ferroptosis was confirmed in LPS‐treated hepatocytes and CLP‐induced septic mice. Inhibition of glutathione peroxidase 4 (GPX4), a central regulator of ferroptosis, reduced irisin's protective effects in LPS‐treated hepatocytes and CLP‐induced septic mice, while blocking the irisin receptor with RGD peptide or Echistain decreased irisin‐induced GPX4 expression.ConclusionsSerum irisin levels are decreased and negatively correlated with disease severity in patients with sepsis, and irisin treatment suppresses ferroptosis and restores mitochondrial function in experimental sepsis. Irisin may offer therapeutic potential in the management of sepsis.

Erythropoietin prevents liver injury in septic mice

In the present study, the effect of erythropoietin in liver of septic mice was investigated. Sepsis was induced by caecal ligation and puncture model. After 20+2 hrs of surgery, blood and liver samples were collected. Plasma TNF-α, IL-1β, Serum AST, ALT and Liver lipid peroxidation, myeloperoxidase, thiols was estimated and histopathology was performed. Post-treatment with erythropoietin ameliorated the sepsis induced changes in hepatic and systemic inflammatory changes in mice.

In vivo monitoring of dynamic interaction between neutrophil and human umbilical cord blood-derived mesenchymal stem cell in mouse liver during sepsis

BackgroundSepsis is a global inflammatory disease that causes death. It has been reported that mesenchymal stem cell (MSC) treatment can attenuate inflammatory and septic symptoms. In this study, we investigated how interactions between neutrophils and human umbilical cord blood (hUCB)-MSCs in the liver of septic mice are involved in mitigating sepsis that is mediated by MSCs. Accordingly, we aimed to determine whether hUCB-MSC application could be an appropriate treatment for sepsis.MethodsTo induce septic condition, lipopolysaccharide (LPS) was intraperitoneally (i.p.) injected into mice 24 h after the intravenous (i.v.) injection of saline or hUCB-MSCs. To determine the effect of hUCB-MSCs on the immune response during sepsis, histologic analysis, immunoassays, and two-photon intravital imaging were performed 6 h post-LPS injection. For the survival study, mice were monitored for 6 days after LPS injection.ResultsThe injection (i.v.) of hUCB-MSCs alleviated the severity of LPS-induced sepsis by increasing IL-10 levels (p < 0.001) and decreasing mortality (p < 0.05) in septic mice. In addition, this significantly reduced the recruitment of neutrophils (p < 0.001) to the liver. In hUCB-MSC-treated condition, we also observed several distinct patterns of dynamic interactions between neutrophils and hUCB-MSCs in the inflamed mouse liver, as well as vigorous interactions between hepatic stellate cells (HSCs or ito cells) and hUCB-MSCs. Interestingly, hUCB-MSCs that originated from humans were not recognized as foreign in the mouse body and consequently did not cause graft rejection.ConclusionsThese distinct interaction patterns between innate immune cells and hUCB-MSCs demonstrated that hUCB-MSCs have beneficial effects against LPS-induced sepsis through associations with neutrophils. In addition, the immunomodulatory properties of hUCB-MSCs might enable immune evasion in the host. Taken together, our results suggest the prospects of hUCB-MSCs as a therapeutic tool to inhibit inflammation and alleviate pathological immune responses such as sepsis.

Tracking of GFP-labeled unrestricted somatic stem cells transplanted in the sepsis mouse model

Cell-based therapy provides a promising approach for the treatment of sepsis and related disorders. Fate determination of transplanted cells is the most essential issue for cell therapist. Optical imaging is the reliable, time and cost-effective system for cell tracking. The present study was aimed to apply an optical imaging system for monitoring of GFP-labeled unrestricted somatic stem cells in the sepsis animal model. In vivo imaging showed the most accumulation of intravenously injected cells into the lungs and liver of septic mice. Thereafter, the imaging data were more approved by flow cytometry and immunohistochemistry staining. Cellular localization in septic lungs and liver that observed by optical imaging technique may offer beneficial evidence for designing of sepsis clinical trials.

2'-O-Galloylhyperin Isolated From Pyrola incarnata Fisch. Attenuates LPS-Induced Inflammatory Response by Activation of SIRT1/Nrf2 and Inhibition of the NF-κB Pathways in Vitro and Vivo.

2′-O-galloylhyperin, a major compound of Pyrola incarnata Fisch., possesses a variety of biological and pharmacological activities, including anti-oxidative and anti-inflammatory activities. Nevertheless, the underlying molecular mechanisms of 2′-O-GH in microbial infection and sepsis are not clear. In this study, we investigated the anti-inflammatory effects of 2′-O-GH. We found that 2′-O-GH significantly reduced the production of TNF-α, IL-6, and nitric oxide (NO), suppressed the expression levels of iNOS, blocked the translocation of NF-κB from the cytosol to nucleus, and decreased the MAPK activation in LPS-activated RAW 264.7 cells. 2′-O-GH also enhanced the nuclear translocation of Nrf2 and up-regulated the expression of heme oxygenase-1 (HO-1) and SIRT1. In addition, the administration of 2′-O-GH attenuated the TNF-α and IL-6 production in the serum, infiltration of inflammatory cells, liver tissue damage, and the mortality rate of LPS-challenged mice. Moreover, 2′-O-GH significantly upregulated Nrf2 and SIRT1 expression and inhibited the inflammatory responses in the liver of septic mice. The collective data indicate that 2′-O-GH could potentially be a novel functional food candidate in the treatment of sepsis.

Skeletal Muscle Produces Acute Phase Proteins in Response to Polymicrobial Sepsis

Skeletal muscle has been implicated as an endocrine organ, as observed by the secretion of cytokines in response to multiple stimuli such as exercise, heat stress and infection. However, whether skeletal muscles can produce acute phase proteins (APPs) in response to polymicrobial sepsis is controversial. This is relevant since APPs play an important role in the transition from innate to adaptive immunity and, thus far, the liver has been considered the primary source of these proteins. Our aim was to determine whether skeletal muscles are capable of producing APPs in response to polymicrobial sepsis.METHODSWe injected (IP) 37 female C57BL/6J mice with cecal slurry (1.6 g/ml) and harvested the soleus, gastrocnemius and livers at 6, 12, and 24 h after the injection. Respective muscles and livers from non‐septic mice were used as controls. Specimens were then homogenized and analyzed for protein content. Immunoblot assays were then conducted using polyclonal antibodies for, serum amyloid a1 (SAA1), fibrinogen, lipocalin, and c‐reactive protein (CRP). For quantification, membranes were scanned for fluorescence intensity and normalized for total protein.RESULTSWe observed elevated expression of fibrinogen, lipocalin and SAA1 in the livers of septic mice across all time points in comparison to non‐septic mice. We observed an increased signal for SAA1 and fibrinogen in skeletal muscle of septic mice. Notably, the signal intensity was higher in soleus in comparison to gastrocnemius muscle, which suggests a muscle‐specific response. We did not observe any signal for lipocalin, regardless of the muscle type. We also detected the presence of CRP in the soleus muscles of septic mice. Our results suggest that the cecal slurry model of septic shock induces an acute phase response in both liver and skeletal muscle. Though skeletal muscles produce a smaller spectrum of known APPs, these may contribute to the transition from innate to adaptive immunity, and the overall response to severe bacterial infection.Support or Funding InformationSupported by NIH RO1GM118895‐01 and the BK and Betty Stevens Endowment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of MicroRNA 195 Prevents Apoptosis and Multiple-Organ Injury in Mouse Models of Sepsis.

MicroRNAs (miRs) are a class of short RNA molecules, which negatively regulate gene expression. The levels of circulating miR-15 family members are elevated in septic patients and may be associated with septic death. This study investigated whether inhibition of miR-195, a member of the miR-15 family, provided beneficial effects in sepsis. Sepsis was induced by injection of feces into the peritoneum in mice. miR-195 was upregulated in the lung and liver of septic mice. Silencing of miR-195 increased the protein levels of BCL-2, Sirt1, and Pim-1; prevented apoptosis; reduced liver and lung injury; and improved the survival in septic mice. Silencing of miR-195 provided similar protection in lipopolysaccharide-induced endotoxemic mice. In endothelial cells, upregulation of miR-195 induced apoptosis, and inhibition of miR-195 prevented lipopolysaccharide-induced apoptosis. miR-195 repressed expression of its protein targets, BCL-2, Sirt1, and Pim-1. Furthermore, overexpression of Pim-1 prevented apoptosis induced by lipopolysaccharide and miR-195 mimic. Inhibition of Pim-1 attenuated the protective effects of miR-195 silencing in septic mice. Silencing of miR-195 reduced multiple-organ injury and improved the survival in sepsis, and the protective effects of miR-195 inhibition were associated with upregulation of Bcl-2, Sirt1, and Pim-1. Thus, inhibition of miR-195 may represent a new therapeutic approach for sepsis.

Blockade of the JNK signalling as a rational therapeutic approach to modulate the early and late steps of the inflammatory cascade in polymicrobial sepsis.

Cecal ligation and puncture (CLP) is an experimental polymicrobial sepsis induced systemic inflammation that leads to acute organ failure. Aim of our study was to evaluate the effects of SP600125, a specific c-Jun NH2-terminal kinase (JNK) inhibitor, to modulate the early and late steps of the inflammatory cascade in a murine model of CLP-induced sepsis. CB57BL/6J mice were subjected to CLP or sham operation. Animals were randomized to receive either SP600125 (15 mg/kg) or its vehicle intraperitoneally 1 hour after surgery and repeat treatment every 24 hours. To evaluate survival, a group of animals was monitored every 24 hours for 120 hours. Two other animals were sacrificed 4 or 18 hours after surgical procedures; lung and liver samples were collected for biomolecular and histopathologic analysis. The expression of p-JNK, p-ERK, TNF-α, HMGB-1, NF-κB, Ras, Rho, Caspase 3, Bcl-2, and Bax was evaluated in lung and liver samples; SP600125 improved survival, reduced CLP induced activation of JNK, NF-κB, TNF-α, and HMGB-1, inhibited proapoptotic pathway, preserved Bcl-2 expression, and reduced histologic damage in both lung and liver of septic mice. SP600125 protects against CLP induced sepsis by blocking JNK signalling; therefore, it can be considered a therapeutic approach in human sepsis.

Identification of mitochondrial deficits and melatonin targets in liver of septic mice by high-resolution respirometry

AimsPrevious data showed that melatonin maintains liver mitochondrial homeostasis during sepsis, but neither the mechanisms underlying mitochondrial dysfunction nor the target of melatonin are known. Main methodsHere, we analyzed mitochondrial respiration in isolated mouse liver mitochondria with different substrate combinations (glutamate/malate, glutamate/malate/sucinate or succinate/rotenone) to identify mitochondrial defects and melatonin targets during sepsis. Other bioenergetic parameters including a+a3, b, and c+c1 content, mitochondrial mass, and mitochondrial supercomplexes formation were analyzed. Mitochondrial function was assessed during experimental sepsis induced by cecal ligation and puncture (CLP) in livers of 3mo. C57BL/6 mice at early and late phases of sepsis, i.e., at 8 and 24h after sepsis induction. Key findingsSeptic mice showed mitochondrial injury with a decrease in state 3, respiratory control rate, mitochondrial mass, and cytochrome b and c+c1 content, which was prevented by melatonin treatment. Mitochondrial dysfunction in sepsis was mainly linked to complex I damage, because complex II was far less impaired. These mitochondria preserved the respiratory supramolecular organization, maintaining their electron transport system capacity. SignificanceThis work strengthens the use of substrate combinations to identify specific respiratory defects and selective melatonin actions in septic mitochondria. Targeting mitochondrial complex I should be a main therapeutical approach in the treatment of sepsis, whereas the use of melatonin should be considered in the therapy of clinical sepsis.

Melatonin modifies cellular stress in the liver of septic mice by reducing reactive oxygen species and increasing the unfolded protein response

Background & aimsMelatonin's hepatoprotective actions have numerously been demonstrated in the past but the underlying molecular mechanisms are widely unknown. For a better understanding of melatonin's effects on hepatic stress response this study aimed to elucidate alterations in oxidative stress, unfolded protein response and acute phase response in septic mice. MethodsMale C3H/HeN mice underwent sham operation or cecal ligation and incision and remained anesthetized for 5h. Production of reactive oxygen species was determined by electron spin resonance spectroscopy. Protein and mRNA expression levels were determined by western blot analysis and quantitative real-time PCR, respectively. ResultsProduction of reactive oxygen species was strongly increased in the aorta and liver after 5h of polymicrobial sepsis which was entirely inhibited by treatment with melatonin. SOD-1 levels did not differ between the groups. Sepsis also induced the upregulation of VCAM-1 and ICAM-1 independent of melatonin treatment but probably regulated via ERK1/2 signaling. Melatonin triggered the transcriptional upregulation of PERK in septic animals which seems to be independent on ERK1/2 signaling and NR4A1 activation. Melatonin therapy also engendered an increased expression of CHOP, but apoptosis was not initiated. Furthermore, sepsis reduced the expression of the transcription factor CREBH which was entirely suppressed by melatonin. ConclusionsThis study gives new insight into the mechanisms by which melatonin might confer its hepatoprotective actions during polymicrobial sepsis. The results clearly show the melatonin-mediated amelioration of oxidative stress as well as alterations in the cellular stress mechanisms via the unfolded protein response and the acute phase response.

Leishmanial lipid suppresses the bacterial endotoxin-induced inflammatory response with attenuation of tissue injury in sepsis

The use of live, attenuated, or genetically modified microbes or their cellular component(s) or metabolites has begun to emerge as a potential new approach in medicinal research to deliver biologically active entities. Thus, advancing our knowledge of such microbe-mediated therapy may suggest new avenues for therapeutic intervention in many diseases. We had earlier reported that the total lipid of attenuated Leishmania donovani suppressed the inflammatory responses in rheumatoid arthritis patients. Our present study reveals that the pLLD, isolated from pathogenic L. donovani, decreases the inflammatory level of bacterial endotoxin in stimulated mouse macrophages, as also in the in vivo murine system. It exerts the activity by reducing the level of different mediators, such as cytokine-chemokine(s). It also suppresses the expression of the ubiquitous transcription factor NF-κBp65 in stimulated macrophage cells, improves the endotoxin-associated liver damage, reduces the vascular permeability factors, such as VEGF, and suppresses the expression of cell adhesion molecules, including ICAM-1, VCAM-1, PECAM-1, P-selectin, and E-selectin, in liver of septic mice. These findings indicate that pLLD may prove to be a potential anti-inflammatory agent and protect from endotoxin-induced sepsis in hepatic impairment.

Complete Activation of Autophagic Process Attenuates Liver Injury and Improves Survival in Septic Mice

The accumulation of autophagosomes in the terminal step of the autophagic process has recently emerged as a potentially maladaptive process in the septic heart and lung. However, the role of autophagy in the septic liver has not been ascertained. This study was investigated by first examining the entire sequence of the autophagic process in the liver of septic mice. Second, a novel pharmacotherapeutic approach was utilized to treat sepsis with autophagy enhancer/inhibitor. Sepsis was induced by cecal ligation and puncture (CLP). C57BL/6 mice received autophagy enhancer carbamazepine (CBZ), autophagy inhibitor 3-methyladenine (inhibition of autophagosomal formation), or chloroquine (impairment of autophagosomal clearance). We found that the whole autophagic process was activated at 4 h after CLP; however, it did not proceed to completion during the 4- to 24-h time period, as indicated by accumulated autophagosomes and decreased autophagic flux. Carbamazepine, which induced complete activation of the autophagic process, improved CLP survival. This protective effect was also associated with decreased cell death, inflammatory responses, and hepatic injury. However, disruption of autophagosomal clearance with chloroquine abolished the above protective effects in CBZ-treated CLP mice. 3-Methyladenine, which resulted in inhibition of the autophagosomal formation, did not show any above beneficial effects in CLP mice. Impaired autophagosome-lysome fusion resulting in incomplete activation of autophagy may contribute to sepsis-induced liver injury. Treatment with CBZ may serve a protective role in the septic liver, possibly through the effect of complete activation of autophagic process.

Dexamethasone Down-Regulates the Expression of microRNA-155 in the Livers of Septic Mice

To investigate the expression of microRNA-155 (miRNA-155) in the livers of mice with lipopolysaccharide (LPS)-induced sepsis and to determine the role of dexamethasone (DXM) in the regulation of miRNA-155 expression, we pretreated mice with or without DXM prior to LPS exposure. Our study demonstrated that the expression of miRNA-155 and inflammatory factors increased in the liver tissues of mice with LPS-induced sepsis and that DXM down-regulated their expression in a dose-dependent manner. Moreover, DXM alone inhibited the expression of miRNA-155 to below the baseline level, but did not impact the expression of inflammatory factors, suggesting that the down-regulation of miRNA-155 by DXM may partially, but not completely, depend on the suppression of pro-inflammatory cytokines by DXM. Our data indicate that the overexpression of miRNA-155 in the livers of mice with LPS-induced sepsis may play an important role in the pathological processes of sepsis and that the down-regulation of miRNA-155 by DXM may be a novel mechanism regulating inflammation and immunity.

Induction of HO-1 through p38 MAPK/Nrf2 signaling pathway by ethanol extract of Inula helenium L. reduces inflammation in LPS-activated RAW 264.7 cells and CLP-induced septic mice

High mobility group box 1 (HMGB1) plays a crucial mediator in the pathogenesis of many inflammatory diseases. We recently proposed that heme oxygenase-1 (HO-1) negatively regulates HMGB1 in inflammatory conditions. We investigated whether ethanol extract of Inula helenium L. (EIH) activates p38 MAPK/Nrf2/HO-1 pathways in RAW264.7 cells and reduces inflammation in CLP-induced septic mice. EIH induced expression of HO-1 protein in a time- and concentration-dependent manner. EIH significantly diminished HO-1 expression in siNrf2 RNA-transfected cells. As expected, the inhibited expression of iNOS/NO, COX-2/PGE2, HMGB1 release by EIH in LPS-activated RAW264.7 cells was significantly reversed by siHO-1RNA transfection. Furthermore, EIH not only inhibited NF-κB luciferase activity, phosphorylation of IκBα in LPS-activated cells but also significantly suppressed expression of adhesion molecules (ICAM-1 and VCAM-1) in TNF-α activated human umbilical vein endothelial cells. The induction of HO-1 by EIH was inhibited by SB203580 but not by SP600125, PD98059, nor LY294002. Most importantly, administration of EIH significantly reduced not only increase in blood HMGB1, ALT, AST, BUN, creatinine levels but also decrease macrophage infiltrate in the liver of septic mice, which were reversed by ZnPPIX, a HO-1 inhibitor. We concluded that EIH has anti-inflammatory effect via the induction of p38 MAPK-dependent HO-1 signaling pathway.

Expression and effects of microRNA-155 in the livers of septic mice

To evaluate the effects of microRNA-155 (miR-155) on liver injury in mice with sepsis. One hundred and twenty BALB/c mice were randomly divided into two groups of equal number according to random number table. Sepsis was induced by intraperitoneal injection of lipopolysaccharide (LPS,20 mg/kg). The mice were sacrificed at the time-points of 0, 2, 6, 12, 24, 48 hours. Blood and liver tissue were collected, and the levels of tumor necrosis factor- α (TNF- α ), interleukin (IL-6, IL-10) in serum and liver homogenate and alanine transaminase (ALT) in serum were determined by enzyme linked immunosorbent assay(ELISA). The injury of liver tissue was evaluated by histopathology. The expression of miR-155 in liver tissue was assessed by fluorescent quantitation reverse transcription-polymerase chain reaction (RT-PCR). The levels of TNF- α , IL-6 and IL-10 in serum and liver homogenate of septic mice increased with passage of time, and then the levels of TNF-α and IL-6 lowered after reaching the peak value, but remained higher than that of control group.TNF-α (ng/L) reached the peak value at 2 hours post-LPS-injection (serum: 1538.46 ± 102.12 vs. 64.52 ± 18.44,liver homogenate: 255.26 ± 41.23 vs. 60.21 + 13.55, both P<0.05). The level of IL-6 (μg/L) reached the peak value at 6 hours post-LPS-injection (serum: 875.33 ± 102.37 vs. 153.72 ± 20.67, liver homogenate: 9.22 + 0.82 vs. 3.35 ±0.36, both P<0.05), and that of IL-10 (ng/L) reached the peak value at 48 hours post-LPS-injection (serum: 520.13 ± 88.52 vs. 23.43 3.01, liver homogenate: 260.12 + 50.38 vs. 16.37 ± 3.71, both P<0.05). There were significant differences in above indexes between septic and control group (all P<0.05). The serum level of ALT (U/L) rose with passage of time, reaching the peak value at 48 hours post-LPS-injection (603.26 + 70.21 vs. 45.84 + 5.64, P<0.05). The values showed significant differences between septic and control group (P<0.05). A large number of leucocytic infiltration was found in liver. Hepatic tissue showed architectural distortion. Hepatocyte vacuolation and nodular necrosis were obvious at 12 hours post-LPS-injection. Relative expression of miR-155 was found to be increased at 2 hours post-LPS-injection, reaching its peak value at 12 hours post-LPS-injection [(72.96 ± 9.34)-fold of control group, P<0.05]. The increase in miR-155 expression might play an important role in the mechanism of liver injury during sepsis.