CLP-induced Sepsis Research Articles

Alpha-Chymotrypsin Protects Against Acute Lung, Kidney, and Liver Injuries and Increases Survival in CLP-Induced Sepsis in Rats Through Inhibition of TLR4/NF-κB Pathway.

Inflammation and oxidative stress play a major role in the development of sepsis and its associated complications, leading to multiple organ failure and death. The lungs, liver, and kidneys are among the early affected organs correlated with mortality in sepsis. Alpha-chymotrypsin (α-ch) is a serine protease that exerts anti-inflammatory, anti-edematous, and anti-oxidant properties.PurposeThis study was undertaken to elucidate if the anti-inflammatory and anti-oxidant effects of α-ch observed in previous studies can alleviate lung, liver, and kidney injuries in a cecal ligation and puncture (CLP)-induced sepsis model, and thus decrease mortality.Materials and MethodsSeptic animals were given α-ch 2 h post CLP procedure. Sepsis outcomes were assessed in the lungs, liver, and kidneys. Separate animal groups were investigated for a survival study.ResultsCLP resulted in 0% survival, while α-chymotrypsin post-treatment led to 50% survival at the end of the study. Administration of α-chymotrypsin resulted in a significant attenuation of sepsis-induced elevated malonaldehyde (MDA) and total nitrite/nitrate (NOx) levels. In addition, there was a significant increase in reduced glutathione (GSH) content and superoxide dismutase (SOD) activity in the lungs, liver, and kidneys. Administration of α-ch reduced elevated tissue expression of toll-like receptor-4 (TLR4), nuclear factor kappa-B (NF-κB), myeloperoxidase (MPO), and inducible nitric oxide synthase (iNOS). Alpha-chymotrypsin resulted in a significant reduction in serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Alpha-chymotrypsin attenuated the rise in serum creatinine, cystatin C, blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels that was observed in the septic group. In addition, α-ch significantly reduced the lung wet/dry weight ratio, total protein content, and leukocytic counts in bronchoalveolar lavage fluid (BALF). Histopathological examination of the lungs, liver, and kidneys confirmed the protective effects of α-ch on those organs.Conclusionα-ch has protective potential against sepsis through lowering tissue expression of TLR4, NF-κB, MPO, and iNOS leading to decreased oxidative stress and inflammatory signals induced by sepsis. This effect appeared to alleviate the damage to the lungs, liver, and kidneys and increase survival in rats subjected to sepsis.

Circ-Katnal1 Enhances Inflammatory Pyroptosis in Sepsis-Induced Liver Injury through the miR-31-5p/GSDMD Axis.

Background Sepsis is a systemic inflammatory response that can elicit organ dysfunction as well as circulatory diseases in serious cases. When inflammatory responses are especially dysregulated, severe complications can arise, including sepsis-induced liver injury. Various microRNAs along with circular (circ) RNAs are involved in inflammatory responses; nevertheless, their functions in regulating sepsis-induced liver injury remain unknown. The cecal ligation and puncture (CLP) procedure can induce liver injury as well as polymicrobial sepsis. Methods In this study, CLP was used to induce liver injury as well as polymicrobial sepsis. Then, liver function, inflammatory cytokine expression, and hepatic histopathology were evaluated. High-throughput sequencing was employed to investigate the abnormal hepatic circRNA expression after CLP. Raw264.7 cells were utilized to simulation an in vitro sepsis inflammation model with LPS induce. The relative mRNA as well as protein levels of TNF-α, IL-1β, and IL-6 was explored by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays. We explored functional connections among circRNAs, miR-31-5p, and gasdermin D (GSDMD) using dual-luciferase reporter assays. Western blot was employed to test GSDMD, caspase-1, and NLRP3 expression in mice and cell models. Results Our results showed that CLP-induced sepsis promoted liver injury via increasing inflammatory pyroptosis. The abnormal expression of circ-Katnal1 played an important role in CLP-induced sepsis. Downregulating circ-Katnal1 suppressed LPS-induced inflammatory pyroptosis in Raw264.7 cells. Bioinformatics and luciferase reporter results confirmed that miR-31-5p and GSDMD were downstream targets of circ-Katnal1. Inhibiting miR-31-5p or upregulating GSDMD reversed the protective effects of silencing circ-Katnal1. Conclusion Taken together, circ-Katnal1 enhanced inflammatory pyroptosis in sepsis-induced liver injury through the miR-31-5p/GSDMD axis.

Luteolin alleviates vascular dysfunctions in CLP-induced polymicrobial sepsis in mice.

Luteolin, a naturally occurring flavonoid, is thought to have health-promoting properties as a part of human diet and has been reported to possess a wide range of pharmacological activities. Therefore, the present study was undertaken to evaluate the effect of luteolin pre-treatment on vascular dysfunctions in sepsis induced by caecal ligation and puncture (CLP) in the mouse model. Mice were divided into four groups: sham, luteolin plus sham, CLP, and luteolin plus CLP. Luteolin was administered (0.2mg/kg body weight) intraperitoneally one hour (h) before CLP surgery in mice. 20 ± 2h post CLP surgery, the isolated thoracic aorta of mice was assessed for its vascular reactivity to noradrenaline (NA) and acetylcholine (ACh). To explore the underlying mechanism, aortic mRNA expressions of α1D adrenoceptors, eNOS and iNOS were investigated. In mice with CLP-induced sepsis luteolin pre-treatment markedly increased the survival time and attenuated serum lactate level. The CLP group manifested the reduced vascular reactivity to NA and this deficit was restored by luteolin pre-treatment. However, luteolin pre-treatment did not improve α1D adrenoceptors down-regulation observed in septic mice aorta. In the presence of 1400W, the NA contractile response was significantly restored in CLP mice aortic tissue in comparison with the respective control of septic mice and further enhanced in the presence of luteolin. Luteolin reduced the iNOS mRNA expression and iNOS-derived nitrite production. Pre-treatment with luteolin restored the endothelial dysfunction in septic mice aorta by improving eNOS mRNA expression and enhanced eNOS-derived nitric oxide (NO) production in septic mice aorta and aortic iNOS gene expression and inducible NO production. The present study suggests that the vasoplegic state to NA in aorta was restored through the iNOS pathway and endothelial dysfunction was reversed via eNOS and NO production pathway.

PTP1B inhibitors protect against acute lung injury and regulate CXCR4 signaling in neutrophils.

Acute lung injury (ALI) can cause acute respiratory distress syndrome (ARDS), a lethal condition with limited treatment options and currently a common global cause of death due to COVID-19. ARDS secondary to transfusion-related ALI (TRALI) has been recapitulated preclinically by anti–MHC-I antibody administration to LPS-primed mice. In this model, we demonstrate that inhibitors of PTP1B, a protein tyrosine phosphatase that regulates signaling pathways of fundamental importance to homeostasis and inflammation, prevented lung injury and increased survival. Treatment with PTP1B inhibitors attenuated the aberrant neutrophil function that drives ALI and was associated with release of myeloperoxidase, suppression of neutrophil extracellular trap (NET) formation, and inhibition of neutrophil migration. Mechanistically, reduced signaling through the CXCR4 chemokine receptor, particularly to the activation of PI3Kγ/AKT/mTOR, was essential for these effects, linking PTP1B inhibition to promoting an aged-neutrophil phenotype. Considering that dysregulated activation of neutrophils has been implicated in sepsis and causes collateral tissue damage, we demonstrate that PTP1B inhibitors improved survival and ameliorated lung injury in an LPS-induced sepsis model and improved survival in the cecal ligation and puncture–induced (CLP-induced) sepsis model. Our data highlight the potential for PTP1B inhibition to prevent ALI and ARDS from multiple etiologies.

Tim-3 Blockade Decreases the Apoptosis of CD8+ T Cells and Reduces the Severity of Sepsis in Mice

IntroductionThe T cell immunoglobulin and mucin domain 3 (Tim-3) mediated immunosuppressive pathway has been shown to play an essential role in the development of sepsis. However, the influence of Tim-3 blockade during sepsis and the possible effects on T cells’ function remains largely unknown. Our study investigates the role of Tim-3 in cecal ligation and puncture (CLP)-induced sepsis in mice. MethodsSepsis was induced in C57BL/6 male mice via CLP. The expression of Tim-3 in CD8+ T cells after CLP challenge was measured. A dose of 50 μg anti-Tim-3 antibodies was injected intraperitoneally 30 min after surgery. Postoperative survival, bacterial clearance in the blood and peritoneal lavage fluid, cytokine secretion in the blood, and lung and liver histology were evaluated. In addition, the apoptosis of immune cells in the spleen and thymus was examined, respectively. ResultsTim-3 expression was elevated in the splenic CD8+ T cells of septic mice. At the early stage of CLP-induced sepsis, blocking Tim-3 with anti-Tim-3 antibodies reduced the severity of sepsis. The anti-Tim-3 antibodies alleviated the morphology of lung and liver injuries in septic mice. The anti-Tim-3 antibodies also reduced the severity of the inflammatory responses and lymphocyte apoptosis in septic mice. ConclusionsAnti-Tim-3 antibodies might be a potential therapeutic strategy for sepsis.

RG100204, A Novel Aquaporin-9 Inhibitor, Reduces Septic Cardiomyopathy and Multiple Organ Failure in Murine Sepsis

Sepsis is caused by systemic infection and is a major health concern as it is the primary cause of death from infection. It is the leading cause of mortality worldwide and there are no specific effective treatments for sepsis. Gene deletion of the neutral solute channel Aquaporin 9 (AQP9) normalizes oxidative stress and improves survival in a bacterial endotoxin induced mouse model of sepsis. In this study we described the initial characterization and effects of a novel small molecule AQP9 inhibitor, RG100204, in a cecal ligation and puncture (CLP) induced model of polymicrobial infection. In vitro, RG100204 blocked mouse AQP9 H2O2 permeability in an ectopic CHO cell expression system and abolished the LPS induced increase in superoxide anion and nitric oxide in FaO hepatoma cells. Pre-treatment of CLP-mice with RG100204 (25 mg/kg p.o. before CLP and then again at 8 h after CLP) attenuated the hypothermia, cardiac dysfunction (systolic and diastolic), renal dysfunction and hepatocellular injury caused by CLP-induced sepsis. Post-treatment of CLP-mice with RG100204 also attenuated the cardiac dysfunction (systolic and diastolic), the renal dysfunction caused by CLP-induced sepsis, but did not significantly reduce the liver injury or hypothermia. The most striking finding was that oral administration of RG100204 as late as 3 h after the onset of polymicrobial sepsis attenuated the cardiac and renal dysfunction caused by severe sepsis. Immunoblot quantification demonstrated that RG100204 reduced activation of the NLRP3 inflammasome pathway. Moreover, myeloperoxidase activity in RG100204 treated lung tissue was reduced. Together these results indicate that AQP9 may be a novel drug target in polymicrobial sepsis.

PTX3 Protects Intestinal Mucosal Barrier Damage in Sepsis Through Toll-Like Receptor Signaling Pathway.

This study aims to confirm the protective effect of Pentraxin 3 (PTX3) on intestinal mucosal barrier damage in sepsis in animal and cell models and explore its mechanism. Analysis of the GSE147775 gene set revealed that the level of PTX3 was upregulated in the lipopolysaccharide (LPS)-induced rat sepsis model. The mice sepsis model was established by cecal ligation perforation (CLP), and the cell inflammation model was induced by LPS. Cell apoptosis and the expression of apoptosis-related protein were detected by flow cytometry and Western blotting. The PTX3 level was significantly upregulated in the mice sepsis model. Intestinal mucosal barrier damage was aggravated and inflammatory factor expression was upregulated after PTX3 downregulation in sepsis mice. After upregulation of PTX3, intestinal mucosal barrier damage was alleviated and inflammatory factor expression was decreased in sepsis mice. Further data mining suggested that the anti-inflammatory effect of PTX3 might be realized through inhibition of the toll-like receptor (TLR) signaling pathway. Moreover, compared with the LPS group, downregulation of PTX3 increased cell apoptosis and the levels of BCL2-associated X (Bax), myeloperoxidase (MPO), tumor necrosis factor-alfa (TNF-α), interleukin 1 beta (IL-1β), and interferon-gamma (IFN-γ), and decreased the levels of B-cell lymphoma-2 (Bcl-2), zona occludens (ZO)-1, and occludin. On the contrary, overexpression of PTX3 reduced cell apoptosis and the levels of Bax, MPO, TNF-α, IL-1β, and IFN-γ. Moreover, downregulation of PTX3 reversed the inhibitive effects on cell apoptosis and inflammation and promotive effects on the levels of Zo-1 and occludin induced by CLI-095 (a TLR signaling pathway inhibitor). In the CLP-induced mice sepsis model and LPS-induced cell inflammation model, PTX3 inhibits inflammatory response and reduces intestinal mucosal barrier damage through the TLR signaling pathway.

Trichinella spiralis cystatin alleviates polymicrobial sepsis through activating regulatory macrophages

BackgroundSepsis is a life-threateningorgandysfunction caused by the cytokine storm induced by the severe bacterial infection. Excessive inflammatory responses are responsible for the lethal organ damage during the early stage of sepsis. Helminth infection and helminth-derived proteins have been identified to have the ability to immunomodulate the host immune system by reducing inflammation against inflammatory diseases. Trichinella spiralis cystatin (Ts-Cys) is a cysteine protease inhibitor with strong immunomodulatory functions on host immune system. Our previous studies have shown that excretory-secretory proteins of T. spiralis reduced sepsis-induced inflammation and Ts-Cys was able to inhibit macrophages to produce inflammatory cytokines. Whether Ts-Cys has a therapeutic effect on polymicrobial sepsis and related immunological mechanism are not yet known. MethodsSepsis was induced in BALB/c mice using cecal ligation and puncture (CLP), followed by intraperitoneal injection of 15 µg recombinant Ts-Cys (rTs-Cys). The therapeutic effect of rTs-Cys on sepsis was evaluated by observing the 72-hour survival rates of CLP-induced septic mice and the acute injury of lung and kidney through measuring the wet/dry weight ratio of lung, the levels of blood urea nitrogen (BUN) and creatinine (Cr) in sera and the tissue section pathology. The potential underlying mechanism was investigated using mouse bone marrow-derived macrophages (BMDMs) by observing the effect of rTs-Cys on LPS-stimulated macrophage polarization. The expression of genes associated with macrophage polarization in BMDMs and tissues of septic mice was measured by Western Blotting and qPCR. ResultsIn this study, we demonstrated the treatment with rTs-Cys alleviated CLP-induced sepsis in mice with significantly reduced pathological injury in vital organs of lung and kidney and reduced mortality of septic mice. The further study identified that treatment with rTs-Cys promoted macrophage polarization from classically activated macrophage (M1) to alternatively activated macrophage (M2) phenotype via inhibiting TLR2/MyD88 signal pathway and increasing expression of mannose receptor (MR), inhibited pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and increased regulatory anti-inflammatory cytokines (IL-10 and TGF-β) in sera and tissues (lung and kidney) of mice with polymicrobial sepsis. ConclusionsOur results demonstrated that rTs-Cys had a therapeutic effect on sepsis through activating regulatory macrophages possibly via suppressing TLR2/MyD88 signal pathway. We also identified that rTs-Cys-induced M2 macrophage differentiation was associated with increased expression of MR on the surface of macrophages. Our results underscored the importance of MR in regulating macrophages during the treatment with rTs-Cys, providing another immunological mechanism in which helminths and their derived proteins modulate the host immune system. The findings in this study suggest that rTs-Cys is a potential therapeutic agent for the prevention and treatment of sepsis and other inflammatory diseases.

The anti-inflammatory and antioxidant effects of Montelukast on lung sepsis in adult mice.

One of the most complex clinical challenges facing medical practice is sepsis-induced lung dysfunction resulting from polymicrobial sepsis. Although many therapeutic approaches have been used in such clinical challenges, there is still further need for a new effective therapeutic approach. The objective of this study was to investigate if Montelukast could protect the lungs during polymicrobial sepsis by regulating inflammatory markers and the oxidative stress pathways. Twenty-four mature male Swiss-albino mice aged 8–12 weeks, with a weight of 20–30 g, were randomized into 4 equal groups (n=6), sham (laparotomy without cecal ligation and puncture (CLP)), CLP (laparotomy with CLP), vehicle 1 (equivalent volume of DMSO 1 hour prior to CLP), Montelukast (10 mg/kg IP 1 hour prior to CLP). Lung tissue pro-inflammatory mediators IL-6, IL-1β, IL-17, LTB-4 12(S) HETE, and oxidative stress were assessed using ELISA. The levels of F2 isoprostane were considerably greater in the sepsis group (p<0.05) as compared to the sham group, while Montelukast was significantly lower (p<0.05) in these inflammatory mediators and oxidative stress as compared to the sepsis group. Histologically, the lung tissue damage was significant (p<0.05) in all mice in the sepsis group, while Montelukast significantly reduced lung tissue injury (p<0.05). The current findings indicated that Montelukast could attenuate lung dysfunction during CLP-induced polymicrobial sepsis in male mice through their modulating effects on pro-inflammatory and oxidative stress downstream signalling pathways and subsequently decrease lung tissue cytokine concentrations (IL-1β, IL-6, IL-17, LTB-4, and 12(S)HETE).

Sepsis-Induced Gut Dysbiosis Mediates the Susceptibility to Sepsis-Associated Encephalopathy in Mice.

ABSTRACTSepsis-associated encephalopathy (SAE) is common in septic patients and is associated with adverse outcomes. The gut microbiota has been recognized as a key mediator of neurological disease development. However, the exact role of the gut microbiota in regulating SAE remains elusive. Here, we investigated the role of the gut microbiota in SAE and its underlying mechanisms. Cecal ligation and puncture (CLP) was conducted to induce sepsis in mice. Neurological scores were recorded to distinguish SAE-resistant (SER) (score of >6 at 36 h postoperatively) from SAE-susceptible (SES) (score of ≤6 at 36 h postoperatively) mice. 16S rRNA gene sequencing and metabolomics analyses were used to characterize the gut microbiota in the two groups. Fecal microbiota transplantation was performed to validate the role of the gut microbiota in SAE progression. The gut microbiota was more severely disrupted in SES mice than in SER mice after sepsis modeling. Interestingly, mice receiving postoperative feces from SES mice exhibited more severe cortical inflammation than mice receiving feces from SER mice. Indole-3-propionic acid (IPA), a neuroprotective molecule, was more enriched in feces from SER mice than in feces from SES mice. IPA alleviated CLP-induced anxiety and spatial memory impairment in septic mice. Moreover, IPA markedly inhibited NLRP3 inflammasome activation and interleukin-1β (IL-1β) secretion in lipopolysaccharide-stimulated microglia. These responses were attenuated after antagonizing the aryl hydrocarbon receptor. Our study indicates that the variability in sepsis-induced gut dysbiosis mediates the differential susceptibility to SAE in CLP-induced experimental sepsis mice, and microbially derived IPA is possibly involved in SAE development as a neuroprotective compound.IMPORTANCE The bidirectional interactions between the gut microbiota and sepsis-associated encephalopathy (SAE) are not well characterized. We found that the gut microbiota was more severely disturbed in SAE-susceptible (SES) mice than in SAE-resistant (SER) mice after sepsis modeling. Mice gavaged with postoperative feces from SES mice exhibited more severe neuroinflammation than mice gavaged with feces from SER mice. The gut microbiota from SER mice enriched a neuroprotective metabolite, IPA, which appeared to protect mice from SAE. The potential underlying mechanism of the protective effect of IPA may be mediated via the inhibition of NLRP3 inflammasome activation and IL-1β secretion in microglia. These anti-inflammatory effects of IPA may be regulated by aryl hydrocarbon receptors. These results enhance our understanding of the role of the intestinal microbiota in sepsis. In particular, gut microbiota-derived IPA may serve as a potential therapeutic agent to prevent neuroinflammation in SAE.

CCL25 Inhibition Alleviates Sepsis-Induced Acute Lung Injury and Inflammation.

PurposeAcute lung injury (ALI) is a common clinical syndrome with high mortality. The chemokine ligand 25 (CCL25) is involved in inflammation, leukocyte trafficking and immunoregulation. However, the role and mechanism of CCL25 in ALI are not fully understood yet. The aim of this study was to explore the relationship between acute lung injury and CCL25.Patients and MethodsIn this study, we first examined chemokine expression in sepsis patients and found that serum CCL25 expression levels were relatively high in sepsis patients compared to healthy individuals. Based on this, we designed in vitro and in vivo experiments to verify the validity of the theory. In vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs). In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis.ResultsIn vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs) and found significantly higher expression of CCL25 by enzyme-linked immunosorbent assay. Inhibition of CCL25 resulted in a significant decrease in the expression of inflammatory cytokines in HPMECs. In addition, we found that CCL25 promoted increased endothelial permeability by reducing the expression of tight junction proteins and was associated with activation of the P38 MAPK pathway by measuring the transepithelial electrical resistance and fluorescence intensity of fluorescein isothiocyanate. Results from luciferase assays and chromatin immunoprecipitation assays showed that inhibition of NF-κB activity in HPMECs decreased CCL25 expression, but addition of recombinant CCL25 increased cell permeability and inflammatory cytokine expression. In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis. We found that inhibition of CCL25 significantly reduced inflammatory cytokine expression in a CLP-induced sepsis model, thereby alleviating lung tissue damage in mice.ConclusionOur study suggests that CCL25 contributed to the development of ALI by modulating the functions of microvascular endothelial cells.

Panax notoginseng Alleviates Sepsis-Induced Acute Kidney Injury by Reducing Inflammation in Rats.

Background Sepsis is defined as a host inflammatory response to infection that can result in end-organ dysfunction. One of the most common consequences of sepsis is acute kidney injury (AKI). Panax notoginseng powder (PNP) has been previously reported to protect against overactive inflammation process. However, the potential effect of PNP on septic AKI is poorly described. The current study was conducted to investigate the protective effects of PNP in septic AKI rats. Methods A model of septic AKI was established on male SD rats by using the cecal ligation and puncture procedure. PNP was administrated by gavage after the cecal ligation and puncture (CLP) procedure, and the mice were sacrificed at 6, 12, and 72 h after induction of sepsis. The serum and kidney samples were collected and assayed for biochemical tests, histopathological staining, inflammation, and apoptosis-related gene/protein expression. In addition, 15 rats in each group were used to calculate the 7-day survival rate. Results CLP-induced kidney injury was observed by the histopathological score, which markedly was attenuated by PNP treatment. Consistently, PNP intervention significantly alleviated the elevated levels of serum creatinine and blood urea nitrogen in CLP-induced sepsis rats. The CLP procedure also triggered proinflammatory cytokine production and increased the expression of various inflammation-related proteins in the kidneys. However, PNP inhibited the renal expression of IL-18, IL-1β, TNF-α, and IL-6 to substantially improve inflammatory response. Mechanistically, CLP induced the increase of the NF-κB p65 level in the injured kidneys, while PNP notably inhibited the corresponding protein expression. Conclusion PNP attenuated kidney inflammation to protect against CLP-induced septic AKI in rats via inhibiting the NF-κB signaling pathway.

IM156, a new AMPK activator, protects against polymicrobial sepsis.

IM156, a novel biguanide with higher potency of AMP‐activated protein kinase activation than metformin, has inhibitory activity against angiogenesis and cancer. In this study, we investigated effects of IM156 against polymicrobial sepsis. Administration of IM156 significantly increased survival rate against caecal ligation and puncture (CLP)‐induced sepsis. Mechanistically, IM156 markedly reduced viable bacterial burden in the peritoneal fluid and peripheral blood and attenuated organ damage in a CLP‐induced sepsis model. IM156 also inhibited the apoptosis of splenocytes and the production of inflammatory cytokines including IL‐1β, IL‐6 and IL‐10 in CLP mice. Moreover, IM156 strongly inhibited the generation of reactive oxygen species and subsequent formation of neutrophil extracellular traps in response to lipopolysaccharide in neutrophils. Taken together, these results show that IM156 can inhibit inflammatory response and protect against polymicrobial sepsis, suggesting that IM156 might be a new treatment for sepsis.

Sex‐related differences in how hyperactive platelets exacerbate the cytokine storm in the peritoneal cavity during intra‐abdominal sepsis

Intra‐abdominal sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high morbidity and mortality. Inflammatory cytokine levels in the peritoneal cavity (PC) are much higher than in blood, and the enhanced release of these mediators is a key participant to the deleterious effects of sepsis. Platelets are well‐known for their role in modulating proinflammatory cytokine secretion. We have previously shown that in vivo blockade of P2Y12 signaling, an important regulator for platelet secretion, resulted in lower inflammatory cytokines in the blood. We hypothesize that proinflammatory cytokine levels in the peritoneal cavity will be elevated in a murine model of sepsis. To this end, male and female mice underwent a cecal ligation and puncture (CLP) procedure to induce sepsis and were treated with ticagrelor to block P2Y12 signaling. Peritoneal cavity cells and fluid (PCF) were collected 24 hrs post‐surgery to measure changes in proinflammatory cytokine levels and immune cell interactions. Our data show that during CLP‐induced sepsis, PCF exhibited very low platelet counts and platelet‐immune cell aggregates compared to sham‐treated mice irrespective of sex (*p £ 0.05, n=4). Surprisingly, despite low platelet numbers, soluble p‐selectin and PF‐4 levels were elevated, indicative of platelet secretion. Moreover, P2Y12 antagonist treatment significantly decreased platelet markers and immune cell aggregates in PCF from male but not female mice (*p £ 0.05, n=5). In conclusion, intercellular communication mediated through systemic platelet activation influences the peritoneal cavity during intra‐abdominal sepsis, even in the absence of platelets locally in a sex‐related manner.Moreover, P2Y12 antagonist treatment significantly decreased platelet markers and immune cell aggregates in PCF from male but not female mice. In conclusion, intercellular communication mediated through systemic platelet activation influences the peritoneal cavity during intra‐abdominal sepsis, even in the absence of platelets locally in a sex‐related manner.

Antagonism of Na,K‐ATPase signaling by pNaKtide can attenuate systemic inflammatory response in murine sepsis model induced by cecal ligation and puncture

IntroductionOxidative stress and associated inflammatory processes are crucial in the development and progression of sepsis, resulting in an immune response that leads to multiple organ failure or dysfunction. Hence, the strategies to limit this systemic inflammatory response might result in the development of an effective sepsis therapy. Previous studies from our lab have demonstrated the role of Na/K‐ATPase signaling in exacerbating systemic oxidative stress and inflammation and the potential role of Na/K‐ATPase signaling antagonist, pNaKtide, in ameliorating several pathophysiological abnormalities.HypothesisThe aim of this study is to demonstrate the role of Na,K‐ATPase signaling in exacerbating oxidant stress and inflammatory response noted in a murine model of sepsis. Furthermore, we aim to demonstrate the effect of systemic administration of Na,K‐ATPase signaling antagonist, pNaKtide, in experimental sepsis using cecal ligation and puncture (CLP) model, as a drug intervention against septic shock.MethodMurine sepsis was induced by CLP in male C57BL6 mice with or without pNaKtide (25 mg/kg body wt) which was administered, intraperitoneally, 24 hours before CLP procedure. Sham surgery was performed without the ligation and puncture, which served as controls for the study. All mice were assessed for Murine Sepsis Score (MSS) at baseline (before Sham or CLP), at 4hours, 8hours and 24 hours after Sham or CLP. Mice were euthanized after 24 hours of Sham or CLP surgery and tissues were collected for morphological and biochemical assessment. Blood was used to measure plasma levels of inflammatory cytokines.ResultsSystemic administration of pNaKtide demonstrated improved MSS at 24 hours following CLP surgery, as compared to CLP mice without pNaKtide. Histological assessment of lung tissues by H&amp;E staining showed significantly less congestion, edema, infiltration of inflammatory cells and hemorrhage in CLP mice administered with pNaKtide, as compared to CLP alone. pNaKtide also improved systemic inflammatory cytokines assessed by plasma levels of TNFα and MCP1, as well as improved mRNA expression of inflammatory and macrophage infiltration markers, in lung tissues of CLP mice.ConclusionOur study demonstrates that antagonism of Na,K‐ATPase signaling by pNaKtide may attenuate CLP‐induced sepsis by inhibition of inflammatory milieu noted in this pathophysiological condition. Hence, Na,K‐ATPase signaling may serve as a viable clinical target for therapeutic intervention of sepsis and associated inflammatory mechanisms.

Time-serial expression of toll-like receptor 4 signaling during polymicrobial sepsis in rats.

Sepsis caused by aggressive infection is a severe clinical problem with an increasing incidence worldwide. Toll-like receptors and their common adapter myeloid differentiation factor 88 (MyD88) can activate immune responses by recognizing a foreign microbe’s product. This study aimed to identify the different time expression of TLR four signaling pathway in an experimental rodent model of polymicrobial sepsis. A randomized animal study was investigated in rats with septic peritonitis induced by cecal ligation and puncture (CLP). The expressions of MyD88-dependent pathway biomarkers, including MyD88, nuclear factor-κB (NF-κB), and serum tumor necrosis factor-α (TNF-α), were analyzed and compared to the sham controls at the different time points after CLP surgery. CLP-induced sepsis increased liver MyD88 mRNA expression and protein expression compared to the control groups at 2 h after surgery. The MyD88 mRNA and protein expressions in rats with CLP-induced sepsis marked increased at 4 and 6 h, and their NF-κB activities and serum TNF-α levels also increased at 4 h after CLP surgery (both p < .05). The different serial expression of MyD88-ependent pathway during sepsis may be used as biomarkers during sepsis. These results may provide further helpful information for using pro-inflammatory biomarkers of innate immunity such as MyD88 and TNF-α in clinical sepsis or related abdominal surgical emergency in the future.

Glycyrrhizin alleviates sepsis-induced acute respiratory distress syndrome via suppressing of HMGB1/TLR9 pathways and neutrophils extracellular traps formation

BackgroundNeutrophil extracellular traps (NETs) are involved in the development of sepsis-induced acute respiratory distress syndrome (ARDS). Glycyrrhizin (GL), the main active ingredient of the traditional Chinese medicine Glycyrrhiza glabra, has anti-inflammatory, anti-viral, and immunomodulatory effects. ObjectiveThe study aims to explore the efficacy and potential mechanism of GL on sepsis-induced ARDS in mice. Materials and methodsMice were randomly divided into 3 groups: Control, CLP, and GL + CLP. Mice sepsis ARDS model was induced by cecal ligation and puncture (CLP) followed by intraperitoneal GL treatment. Then, the 7-day survival rate of mice was recorded. The lung function of mice was determined by whole-body plethysmography. Lung pathology and scores were observed by hematoxylin-eosin staining. The wet/dry ratio (W/D) of the lung was measured by weighing method. The protein concentration in bronchoalveolar lavage fluid (BALF) was measured by the BCA method. NETs formation in lung tissue was detected by immunofluorescence. Furthermore, HMGB1、TLR9、MyD88 and IL6 expression in lung tissue were detected by western blot and by quantitative real-time PCR, respectively. ResultsThe results showed that GL improved the survival rate, attenuated lung tissue injury and reduced the expression of inflammatory factors in mice with CLP-induced sepsis. Meanwhile, we confirmed that GL could inhibit TLR9 / MyD88 activation from reducing NETs formation by decreasing HMGB1 expression. The formation of NETs is regulated by HMGB1 / TLR9 / MyD88. In addition, GL improved lung function in mice with sepsis-induced ARDS. Lung function suggested that GL increased alveolar ventilation, alleviated ventilator fatigue and reduced airway resistance in mice with ARDS induced by sepsis. ConclusionsGL ameliorated sepsis-induced ARDS and reduced the NETs formation in lung tissues, which may be associated with the inhibition of the HMGB1 / TLR9 pathway.

Positive Role of Delta Neutrophil Index (DNI) as a Prodiagnostic Marker in Cecal Ligation and Puncture (CLP)-Induced Sepsis Murine Model.

Sepsis is an emergent infectious disease and a leading cause of death despite immediate intervention. While Delta neutrophil index (DNI) and myeloperoxidase (MPO) are known as a prodiagnostic marker of sepsis, the preclinical evidence of the best marker of sepsis is unclear. For this, using a well-designed cecal ligation and puncture (CLP)-induced sepsis mouse model, we comparatively measured the level and cost-effectiveness of sepsis biomarkers such as DNI, myeloperoxidase (MPO), procalcitonin (PCT), and tumor necrosis factor-alpha (TNF-α). First, we found that the optimal time point for early detection is at 6 h, 24 h post-CLP. Strikingly, the peak level and fold change of DNI was revealed at 24 h, further showing the best fold change as compared with other biomarker levels. Given the fold change at 6, 24 h, PCT was next to DNI. Third, a cost-effectiveness survey showed that DNI was the best, with PCT next. Further, DNI level was moderate positively associated with PCT (ρ = 0.697, p = 0.012) and TNF-α (ρ = 0.599, p = 0.040). Collectively, these data indicate that DNI in CLP-induced sepsis mice is as effective as the existent inflammatory biomarkers such as MPO, PCT and TNF-α to predict the prognosis of sepsis. This might have clinically important implications that DNI is cost effective, thus quickly and rationally applying to diverse types of imminent sepsis regardless of species. This might be the first report on the validity of DNI in preclinical CLP-induced murine sepsis.

Protective role of crocin against sepsis-induced injury in the liver, kidney and lungs via inhibition of p38 MAPK/NF-κB and Bax/Bcl-2 signalling pathways

Context Crocin has been reported to have multiple bioactivities. However, the effect of crocin administration on caecal ligation and puncture (CLP)-induced sepsis remains unknown. Objective We investigated the effects of crocin on CLP-induced sepsis in mice and the underlying mechanism of action. Materials and methods Five experimental groups (n = 10) of BALB/c mice were used: control, CLP (normal saline) and CLP + crocin (50, 100 and 250 mg/kg, 30 min prior to CLP). Mice were sacrificed 24 h after CLP. Liver, kidney and lung histopathology, indicator levels, apoptotic status, pro-inflammatory cytokines and relative protein levels were evaluated. Results Compared to the CLP group, crocin treatment significantly increased the survival rate (70%, 80%, 90% vs. 30%). Crocin groups exhibited protection against liver, kidney and lung damage with mild-to-moderate morphological changes and lower indicator levels: liver (2.80 ± 0.45, 2.60 ± 0.55, 1.60 ± 0.55 vs. 5.60 ± 0.55), kidney (3.00 ± 0.71, 2.60 ± 0.55, 1.40 ± 0.55 vs. 6.20 ± 0.84) and lungs (8.00 ± 1.59, 6.80 ± 1.64, 2.80 ± 0.84 vs. 14.80 ± 1.79). The proinflammatory cytokines (IL-1β, TNF-α, IL-6 and IL-10 levels in the crocin groups) were distinctly lower and the apoptotic index showed a significant decrease. Crocin administration significantly suppressed p38 MAPK phosphorylation and inhibited NF-κB/IκBα and Bcl-2/Bax activation. Discussion and conclusions Pre-treatment with crocin confers protective effects against CLP-induced liver, kidney and lung injury, implying it to be a potential therapeutic agent.

SIRT3-AMPK signaling pathway as a protective target in endothelial dysfunction of early sepsis

Extensive vascular endothelial dysfunction usually occurs in sepsis, resulting in high mortality. The purpose of this study was therefore to investigate the role of AMP-dependent protein kinase (AMPK) in the aortic endothelial dysfunction of early sepsis in mice, and the relationship between AMPK and Sirtuin3 (SIRT3). Cecal ligation and puncture (CLP) surgery was performed to establish a mouse sepsis model, and human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) to mimic a sepsis model in vitro. We suppressed and increased the activities of AMPK with Dorsomorphin (CC) and Acadesine (AICAR), respectively. 3-TYP (SIRT3 inhibitor) and Honokiol (SIRT3 agonist) were used to alter SIRT3 activity. Then, the inflammatory and endothelial function parameters of the vascular tissue and survival rate were determined. In vivo, the expression of Ser1177 phosphorylation of endothelial nitric oxide synthase (p-eNOS), endothelium-dependent relaxation function, and survival decreased (P < 0.05), while NF-κB and NLRP3 pathways were activated in CLP-induced early sepsis (P < 0.05). Moreover, activation of AMPK significantly reversed the reduction of p-eNOS expression (P < 0.05), prevented endothelial dysfunction (P < 0.05), deactivated NF-κB and NLRP3 pathways (P < 0.05), and improved survival (P < 0.05) in septic mice. However, AMPK inhibition led to opposite effects (P < 0.05). In addition, changing the activity of AMPK had little effect on SIRT3 expression (P > 0.05), while the expression of p-AMPK varied with the inhibition or activation of SIRT3 (P < 0.05), which was further demonstrated using in vitro experiments. Together, the results showed that the SIRT3-AMPK signaling pathway played an important role in inhibiting vascular inflammation and endothelial dysfunction during early sepsis.