Cecal Ligation And Puncture Surgery Research Articles

Deletion of MAPL ameliorates septic cardiomyopathy by mitigating mitochondrial dysfunction.

Mitochondrial dysfunction is a critical factor in the pathogenesis of septic cardiomyopathy (SCM). Mitochondrial anchored protein ligase (MAPL), a small ubiquitin-like modifier (SUMO) E3 ligase, plays a significant role in mitochondrial function. However, the role of MAPL in SCM remains unclear. To investigate the role of MAPL in SCM, cardiomyocyte-specific MAPL knockout mice were generated. A cecal ligation and puncture (CLP) procedure was employed to induce a sepsis-like condition. The expression of MAPL in heart tissues and H9C2 cardiomyocytes was elevated following CLP challenge or lipopolysaccharide (LPS) stimulation. MAPL deficiency ameliorated CLP-induced cardiac injury, dysfunction, and inflammation, and also improved the survival rate of mice following CLP operation. Additionally, MAPL deficiency or knockdown inhibited LPS-induced cardiomyocyte apoptosis, improved mitochondrial structural abnormalities, and increased ATP production. Furthermore, MAPL knockdown mitigated LPS-induced reductions in mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production. Mechanistically, the expression of dynamin-related protein 1 (drp1) in the mitochondria of heart tissues or H9C2 cardiomyocytes was elevated under septic conditions. Accordingly, the SUMOylation of drp1 in heart tissues or H9C2 cardiomyocytes was increased under sepsis conditions, which was reduced by MAPL knockout or knockdown. Our results reveal that MAPL promotes cardiac injury/dysfunction and inflammation in SCM. Deficiency or knockdown of MAPL alleviates SCM by reducing drp1 SUMOylation as well as drp1-mediated mitochondrial dysfunction. These findings suggest that targeting MAPL may represent a therapeutic strategy for patients with SCM.

Senescence landscape in the liver following sepsis and senolytics as potential therapeutics.

Senescence, caused by cell-cycle arrest, is a hallmark of aging. Senescence has also been described in embryogenesis, wound healing, and acute injuries. Sepsis is characterized by a dysregulated host response to infection, leading to organ dysfunction and mortality. Most of the pathophysiology of human sepsis is recapitulated in the mouse model of polymicrobial sepsis, developed by cecal ligation and puncture (CLP). In this report, we demonstrate a rapid onset of cellular senescence in the liver of mice subjected to CLP-induced sepsis, characterized by the upregulation of p21, p53, and other senescence markers, including SA-βgal. Using RNAscope, confocal microscopy, and flow cytometry, we further confirm the emergence of p21-expressing senescence phenotype in the liver 24 h after sepsis induction. Senescence was observed in several cell types in the liver, including hepatocytes, endothelial cells, and macrophages. We determined the landscape of senescence phenotype in murine sepsis by single-cell sequencing, which further ascertained that this cell fate is not confined to any particular cell type but displays a heterogeneous distribution. Furthermore, we observed a significant reduction in mortality following sepsis when mice were treated with senolytics, a combination of dasatinib and quercetin, before the CLP surgery. Our experiments unequivocally demonstrated a rapid development of cellular senescence with sepsis and, for the first time, described the senescence landscape in the sepsis liver and the possible role of senescent cells in the worsening outcome following sepsis.

Protective Effects of Crotonis Semen Extract against Sepsis through NF-κB Pathway Inhibition.

Sepsis is an inflammatory condition causing organ failure due to an uncontrolled immune response to infection and remains a significant challenge. Crotonis Semen has displayed various pharmacological effects, yet its potential in protecting against sepsis and the mechanisms involved remains largely unclear. Here, we explored the antiseptic properties of Crotons Semen extract (CSE) in both LPS-stimulated J774 macrophages and mice subjected to sepsis through Cecal ligation and Puncture (CLP) or LPS induction. We found that CSE enhanced survival rates in mouse models with acute sepsis induced by CLP operation and LPS injection. Administering CSE also reduced levels of enzymes indicating organ damage, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK), in septic mice. Furthermore, CSE lowered the serum levels of inflammatory mediators and cytokines, such as NO, TNF-α, IL-1β, and IL-6, in septic mice. In LPS-stimulated J774 macrophages, CSE reduced the expression of pro-inflammatory proteins, including iNOS and COX-2. Moreover, CSE inhibited the phosphorylation of IκBα and IKK, key components of the NF-κB signaling pathway, thereby reducing inflammatory mediators and cytokines. These results demonstrate CSE's protective effects against sepsis through NF-κB pathway disruption, indicating its potential as a therapeutic option for acute inflammatory conditions.

UCP2 knockout exacerbates sepsis-induced intestinal injury by promoting NLRP3-mediated pyroptosis

Sepsis-induced intestinal injury is a common complication that increases the morbidity and mortality associated with sepsis. UCP2, a mitochondrial membrane protein, is involved in numerous cellular processes, including metabolism, inflammation, and pyroptosis. According to our previous studies, UCP2 expression increases in septic intestinal tissue. However, its function in intestinal damage is not known. This work investigated UCP2′s role in intestinal injury caused by sepsis. A sepsis mouse model was established in wild-type and UCP2-knockout (UCP2-KO) animals using cecal ligation and puncture (CLP). MCC950, an NLRP3 inflammasome inhibitor, was injected intraperitoneally 3 h before CLP surgery. Overall, significantly higher levels of UCP2 were observed in the intestines of septic mice. UCP2-KO mice subjected to CLP exhibited exacerbated intestinal damage, characterized by enhanced mucosal erosion, inflammatory cell infiltration, and increased intestinal permeability. Furthermore, UCP2 knockout significantly increased oxidative stress, inflammation, and pyroptosis in the CLP mouse intestines. Interestingly, MCC950 not only inhibited pyroptosis but also reversed inflammation, oxidative stress as well as damage to intestinal tissues as a result of UCP2 knockout. Our results highlighted the protective functions of UCP2 in sepsis-associated intestinal injury through modulation of inflammation and oxidative stress via NLRP3 inflammasome-induced pyroptosis.

M1 Macrophage-Derived Exosomal MiR-155 Enhances Autophagy in Sepsis-Triggered Acute Kidney Injury

Sepsis-induced Acute kidney injury (SA-AKI), a common complication in sepsis, significantly impacts patients’ health and quality of life. M1 macrophages have been demonstrated to release inflammatory mediators that exacerbate kidney injury. MiR-155 has been implicated in promoting inflammation and damage during sepsis while reducing miR-155 levels alleviates SA-AKI. However, the relationship between miR-155 and M1 macrophage-derived exosomes in regulating autophagy during SA-AKI remains unclear. In this study, we aim to investigate the relationship between M1 macrophage-derived exosomes and miR-155 in regulating autophagy during SA-AKI. A mouse model of SA-AKI was established by performing cecal ligation and puncture (CLP) surgery. Additionally, the HK-2 cell line was utilized to establish a sepsis cell model by inducing lipopolysaccharide (LPS). We demonstrated that the mice model of SA-AKI exhibited renal injury along with enhanced autophagy, inflammation response, and macrophage polarization after CLP surgery. M1 macrophages attenuated cell viability and enhanced autophagy in LPS-treated HK-2 cells. Additionally, M1 macrophage-derived exosomes were observed to enhance autophagy in LPS-treated HK-2 cells. Furthermore, we confirmed an increased expression of miR-155 in M1 macrophage-derived exosomes. Furthermore, exosome-mediated miR-155 enhanced autophagy in LPS-treated HK-2 cells. In conclusion, this study provides the first evidence that exosomal miR-155 derived from M1 macrophages enhances autophagy in SA-AKI. These findings suggest that targeting exosomal miR-155 could be a promising therapeutic strategy for SA-AKI.

Maresin 1 alleviates neuroinflammation and cognitive decline in a mouse model of cecal ligation and puncture.

Inflammation in the central nervous system plays a crucial role in the occurrence and development of sepsis-associated encephalopathy. This study aims to explore the effects of maresin 1 (MaR1), an anti-inflammatory and pro-resolving lipid mediator, on sepsis-induced neuroinflammation and cognitive impairment. Mice were randomly assigned to 4 groups: A sham group (sham operation+vehicle), a cecal ligation and puncture (CLP) group (CLP operation+vehicle), a MaR1-LD group (CLP operation+1 ng MaR1), and a MaR1-HD group (CLP operation+10 ng MaR1). MaR1 or vehicle was intraperitoneally administered starting 1 h before CLP operation, then every other day for 7 days. Survival rates were monitored, and serum inflammatory cytokines [tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6] were measured 24 h after operation using enzyme-linked immunosorbent assay (ELISA). Cognitive function was assessed 7 days after operation using the Morris water maze (MWM) test and novel object recognition (NOR) task. The mRNA expression of TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS), IL-4, IL-10, and arginase 1 (Arg1) in cortical and hippocampal tissues was determined by real-time reverse transcription PCR (RT-PCR). Western blotting was used to determine the protein expression of iNOS, Arg1, signal transducer and activator of transcription 6 (STAT6), peroxisome proliferator-activated receptor gamma (PPARγ), and phosphorylated STAT6 (p-STAT6) in hippocampal tissue. Microglia activation was visualized via immunofluorescence. Mice were also treated with the PPARγ antagonist GW9662 to confirm the involvement of this pathway in MaR1's effects. CLP increased serum levels of TNF-α, IL-1β, and IL-6, and reduced body weight and survival rates (all P<0.05). Both 1 ng and 10 ng doses of MaR1 significantly reduced serum TNF-α, IL-1β, and IL-6 levels, improved body weight, and increased survival rates (all P<0.05). No significant difference in efficacy was observed between the 2 doses (all P>0.05). MWM test and NOR task indicated that CLP impaired spatial learning, which MaR1 mitigated. However, GW9662 partially reversed MaR1's protective effects. Real-time RT-PCR results demonstrated that, compared to the sham group, mRNA expression of TNF-α, IL-1β, and iNOS significantly increased in hippocampal tissues following CLP (all P<0.05), while IL-4, IL-10, and Arg1 showed a slight decrease, though the differences were not statistically significant (all P>0.05). Compared to the CLP group, both 1 ng and 10 ng MaR1 decreased TNF-α, IL-1β, and iNOS mRNA expression in hippocampal tissues and increased IL-4, IL-10, and Arg1 mRNA expression (all P<0.05). Immunofluorescence results indicated a significant increase in Iba1-positive microglia in the hippocampus after CLP compared to the sham group (P<0.05). Administration of 1 ng and 10 ng MaR1 reduced the percentage area of Iba1-positive cells in the hippocampus compared to the CLP group (both P<0.05). Western blotting results showed that, compared to the CLP group, both 1 ng and 10 ng MaR1 down-regulated the iNOS expression, while up-regulated the expression of Arg1, PPARγ, and p-STAT6 (all P<0.05). However, the inclusion of GW9662 counteracted the MaR1-induced upregulation of Arg1 and PPARγ compared to the MaR1-LD group (all P<0.05). MaR1 inhibits the classical activation of hippocampal microglia, promotes alternative activation, reduces sepsis-induced neuroinflammation, and improves cognitive decline.

Lactobacillus rhamnosus GG improves cognitive impairments in mice with sepsis.

Survivors of sepsis may encounter cognitive impairment following their recovery from critical condition. At present, there is no standardized treatment for addressing sepsis-associated encephalopathy. Lactobacillus rhamnosus GG (LGG) is a prevalent bacterium found in the gut microbiota and is an active component of probiotic supplements. LGG has demonstrated to be associated with cognitive improvement. This study explored whether LGG administration prior to and following induced sepsis could ameliorate cognitive deficits, and explored potential mechanisms. Female C57BL/6 mice were randomly divided into three groups: sham surgery, cecal ligation and puncture (CLP), and CLP+LGG. Cognitive behavior was assessed longitudinally at 7-9d, 14-16d, and 21-23d after surgery using an open field test and novel object recognition test. The impact of LGG treatment on pathological changes, the expression level of brain-derived neurotrophic factor (BDNF), and the phosphorylation level of the TrkB receptor (p-TrkB) in the hippocampus of mice at two weeks post-CLP (16d) were evaluated using histological, immunofluorescence, immunohistochemistry, and western blot analyses. The CLP surgery induced and sustained cognitive impairment in mice with sepsis for a minimum of three weeks following the surgery. Compared to mice subjected to CLP alone, the administration of LGG improved the survival of mice with sepsis and notably enhanced their cognitive functioning. Moreover, LGG supplementation significantly alleviated the decrease in hippocampal BDNF expression and p-TrkB phosphorylation levels caused by sepsis, preserving neuronal survival and mitigating the pathological changes within the hippocampus of mice with sepsis. LGG supplementation mitigates sepsis-related cognitive impairment in mice and preserves BDNF expression and p-TrkB levels in the hippocampus.

Alpha1-antitrypsin improves survival in murine abdominal sepsis model by decreasing inflammation and sequestration of free heme.

Excessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis. Polymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT-plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)-or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC). All AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro. Data from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.

Blockade of sympathetic ganglia improves vascular dysfunction in septic shock.

Sepsis/septic shock activates the sympathetic nervous system (SNS) to deal with the infection stress. However, an imbalanced or maladaptive response due to excessive or uncontrolled activation characterizes autonomic dysfunction. Our hypothesis was that reducing this excessive activation of the autonomic nervous system would impact positively in sepsis. Using ganglionic blockers as a pharmacological approach, the main aim of the present report was to assess the role of ganglionic transmission in the vascular dysfunction associated with sepsis.Sepsis was induced in rats by cecal ligation and puncture (CLP). One hour after CLP surgery, rats were treated subcutaneously with hexamethonium (15 mg/kg; ganglionic blocker), pentolinium (5 mg/kg; a blocker with a higher selectivity for sympathetic ganglia compared to hexamethonium), or vehicle (PBS). Basal blood pressure and the response to adrenergic agonists were evaluated at 6 and 24 h after CLP surgery. Reactivity to vasoconstrictors, nitric oxide (NO) synthase 2 (NOS-2) expression, IL-1 and TNF plasma levels, and density of α1 adrenergic receptors were evaluated in the aorta 24 h after CLP.Septic shock resulted in hypotension and hyporesponsiveness to norepinephrine and phenylephrine, increased plasma cytokine levels and NOS-2 expression in the aorta, and decreased α1 receptor density in the same vessel. Pentolinium but not hexamethonium recovered responsiveness and α1 adrenergic receptor density in the aorta. Both blockers normalized the in vivo response to vasoconstrictors, and reduced plasma IL-1 and NOx levels and NOS-2 expression in the aorta.Blockade of ganglionic sympathetic transmission reduced the vascular dysfunction in experimental sepsis. This beneficial effect seems to be, at least in part, due to the preservation of α1 adrenergic receptor density and to reduced NOS-2 expression and may lead to adjuvant ways to treat human sepsis.

Menaquinone-4 Alleviates Sepsis-Associated Acute Lung Injury via Activating SIRT3-p53/SLC7A11 Pathway.

Sepsis-associated acute lung injury (SI-ALI) is triggered by various direct or indirect noncardiogenic factors affecting the alveolar epithelium and capillary endothelial cells. Menaquinone-4 (MK-4), a major component of vitamin K, plays a crucial role as an antioxidant by effectively neutralizing reactive oxygen species (ROS) and safeguarding critical biomolecules from oxidative harm within cells. However, the specific mechanisms and clinical implications of MK-4 in SI-ALI are unclear and require further study. Cecal ligation and puncture (CLP) surgery is a commonly used method to induce sepsis in C57BL/6N wild-type mice, and the mice were administered MK-4 at a dosage of 200 mg/kg/day and 3-TYP at 5 mg/kg/day via intraperitoneal injection for 3 days, or erastin (5 mg/kg) 0.5hours before CLP surgery. The mice were sacrificed 24hours after CLP surgery, and blood and lung tissue samples were collected. Pathological changes in the lung tissue and oxidative stress levels were detected. The expression levels of Sirt3, acetylated lysine, p53, SLC7A11 ALOX12 and ferroptosis-related proteins were determined. ligation and puncture (CLP). In this study, we observed that the lung inflammation was associated with reduced Sirt3 expression and increased acetylated lysine levels. The progression of SI-ALI was mitigated by MK-4 through its role in upregulating Sirt3 expression. MK-4 achieved antioxidant effects by downregulating ROS and inflammatory factor levels. Mechanistically, MK-4 inhibited the p53/SLC7A11 signalling pathway in ferroptosis by inhibiting the acetylation of p53, independent of p53 levels. In addition, MK-4 inhibited ferroptosis independent of GPX4. These findings indicate that MK-4 is a promising novel therapeutic agent for treating SI-ALI and possibly sepsis. These experiments revealed that MK-4 acts as a ferroptosis suppressor, increasing the expression of Sirt3, inhibiting the p53/SLC7A11 signalling pathway, and reducing oxidative stress and inflammatory responses, thereby exerting a protective effect against ALI in sepsis.

Aurantio-obtusin exerts an anti-inflammatory effect on acute kidney injury by inhibiting NF-κB pathway.

Acute kidney injury (AKI) is one of the major complications of sepsis. Aurantio-obtusin (AO) is an anthraquinone compound with antioxidant and anti-inflammatory activities. This study was developed to concentrate on the role and mechanism of AO in sepsis-induced AKI. Lipopolysaccharide (LPS)-stimulated human renal proximal tubular epithelial cells (HK-2) and BALB/c mice receiving cecal ligation and puncture (CLP) surgery were used to establish in vitro cell model and in vivo mouse model. HK-2 cell viability was measured using MTT assays. Histological alterations of mouse renal tissues were analyzed via hematoxylin and eosin staining. Renal function of mice was assessed by measuring the levels of serum creatinine (SCr) and blood urea nitrogen (BUN). The concentrations of pro-inflammatory cytokines in HK-2 cells and serum samples of mice were detected using corresponding ELISA kits. Protein levels of factors associated with nuclear factor kappa-B (NF-κB) pathway were measured in HK-2 cells and renal tissues by Western blotting. AO exerted no cytotoxic effect on HK-2 cells and AO dose-dependently rescued LPS-induced decrease in HK-2 cell viability. The concentrations of pro-inflammatory cytokines were increased in response to LPS or CLP treatment, and the alterations were reversed by AO treatment. For in vivo experiments, AO markedly ameliorated renal injury and reduced high levels of SCr and BUN in mice underwent CLP operation. In addition, AO administration inhibited the activation of NF-κB signaling pathway in vitro and in vivo. In conclusion, AO alleviates septic AKI by suppressing inflammatory responses through inhibiting the NF-κB pathway.

Mechanism of Shenfu injection in suppressing inflammation and preventing sepsis-induced apoptosis in murine cardiomyocytes based on network pharmacology and experimental validation

Ethnopharmacological relevanceShenfu injection(SFI), as a famous classical Chinese patent medicine injection for the treatment of sepsis, has achieved good curative effects in clinical practice. However, its specific ingredients and molecular mechanisms is still unclear. Aim of the studyTo analyze the effective ingredients and molecular mechanisms of SFI in the treatment of sepsis via network pharmacology technology and experimental validation. Materials and methodsA total of 198 mice were used in this experiment. Septic mice model was performed by cecal ligation and puncture (CLP). First, Survival rates were calculted to screen the dosage and the treatment time window of SFI. Cardiac function was evaluated by echocardiography. The potential targets and pathways of SFI in the treatment of sepsis were predicted by network pharmacology. Myocardial tissue samples were harvest from different groups after CLP surgery. Hematoxylin-eosin (H&E) and TUNEL staining were used to examine the injury of heart. Western-blot analysis was performed to determine the protein expression of apoptosis. Meanwhile, the structural changes and mitochondrial membrane potential in the mitochondria of cardiomyocytes were also observed by transmission electron microscopy. ResultsThe Kaplan-Meier survival analysis showed that SFI significantly improved the 7-day survival rate as compared with that of CLP mice (P < 0.05). Echocardiography analysis found that LVEF and FS were significantly reduced in CLP mice compared with Sham mice, while SFI significantly increased LVEF (P < 001). Network pharmacology analysis indicated that the potential targets with higher degrees include IL2, BCL2, BAX, CASP7, BID, CASP8. Pathways with higher degrees include apoptosis, TNF signaling pathway, mitochondrial pathway apoptosis, PI3K-AKT signaling pathway. SFI treatment markedly attenuated the quantity of apoptotic cells as compared with the CLP group (P < 0.01). Western blot analysis indicated that CLP surgery decreased the expression of Bcl-2 (anti-apoptotic) but improved the protein expression of Bid, t-Bid, Cyc (pro-apoptotic) as compared with the Sham group (P < 0.01). While, SFI treatment markedly prevent the expression of Bid, t-Bid, Cyc and Caspase-9. The myocardial mitochondrial membrane potential of CLP group decreased after CLP surgery, while the mitochondrial membrane potential of SFI group increased significantly. Compared with the CLP group, in SFI group, the Z-line of the sarcomere was clear and distinguishable, and swollen mitochondria were significantly improved. ConclusionsThe present study demonstrated that SFI improved survival rate and cardiac function of septic mice mainly by suppressing inflammation and apoptosis.

GENIPOSIDE IMPROVES CLP-INDUCED SEPSIS MODEL PROGNOSIS BY UPREGULATING PPARγ TO MODULATE MONOCYTE PHENOTYPE AND CYTOKINE NETWORK.

Background : We explored the efficacy and main biological mechanism of geniposide intervention in sepsis. Methods : A sepsis model was established in male BALB/c mice through cecal ligation and puncture (CLP). Different doses of geniposide (20 or 40 mg/kg) were administered intravenously at 0 and/or 24 h after CLP surgery. The survival rate of different groups was observed. In addition, the expression levels of CD16 and major histocompatibility complex class II in monocytes were assessed using flow cytometry. The concentrations of TNF-α, IL-1β, IL-6, and IL-10 in the serum were measured by ELISA. We also observed the biological effects of geniposide on CD16 and MHC-II expression levels in RAW264.7 cells, as well as the secretion of TNF-α, IL-1β, IL-6, and IL-10 in the LPS-induced RAW264.7 cell model. The PPARγ levels were determined using western blot analysis. Results : Intravenous administration of 40 mg/kg of geniposide at 0 h after CLP significantly improved the survival outcomes in the septic mouse model, with no significant benefits from low dosing (20 mg/kg) or delayed administration (24 h). The effective dose of geniposide significantly decreased the serum cytokine TNF-α, IL-1β, IL-6, and IL-10 concentrations in septic mice ( P < 0.05). Notably, in vitro assays showed that geniposide specifically increased the IL-10 level. Geniposide significantly reduced the CD16 expression ( P < 0.05) and increased MHC-II expression in monocytes ( P < 0.05). In addition, geniposide elevated the PPARγ level in monocytes ( P < 0.05). Conclusions : High-dose early-stage geniposide administration significantly improved the survival rate in a CLP mouse sepsis model by modulating the monocyte phenotype and regulating the cytokine network (IL-6/IL-10 levels). The pharmacological mechanism of geniposide action might be exerted primarily through PPARγ upregulation.

DLK1 overexpression improves sepsis-induced cardiac dysfunction and fibrosis in mice through the TGF-β1/Smad3 signaling pathway and MMPs.

Sepsis is a serious inflammatory disease caused by bacterial infection. Cardiovascular dysfunction and remodeling are serious complications of sepsis, which can significantly affect sepsis patients' mortality. Delta-like homologue 1 (DLK1) has been reported could inhibit cardiac myofibroblast differentiation. However, the function of DLK1 in sepsis is unknown. In the present study, the DLK1 expression was first identified based on the online dataset GSE79962 analysis and cecal ligation and puncture (CLP)-induced sepsis mouse model. DLK1 expression was significantly reduced in septic heart tissues. In septic mouse heart, CLP operation decreased the fractional shortening (EF) (%) and ejection fraction (FS) (%) and caused significant edema, disordered myofilament arrangement, and degradation and necrosis in myocardial cells; CLP operation also increased collagen deposition and elevated the protein levels of fibrotic markers (α-SMA and F-actin). DLK1 overexpression in septic mice could effectively increase EF (%) and FS (%), attenuate CLP-caused ECM degradation and deposition and partially inhibit the CLP-induced TGF-β1/Smad signaling activation. In conclusion, DLK1 expression was poorly expressed in the CLP-induced septic mouse heart. DLK1 overexpression partially alleviated sepsis-induced cardiac dysfunction and fibrosis, with the involvement of the TGF-β1/Smad3 signaling pathway and MMPs.

Stattic ameliorates the cecal ligation and puncture-induced cardiac injury in septic mice via IL-6-gp130-STAT3 signaling pathway

AimSepsis-induced cardiac dysfunction is the leading cause of higher morbidity and mortality with poor prognosis in septic patients. Our recent previous investigation provides evidence of the hallmarks of signal transducer and activator of transcription3 (STAT3) activation in sepsis and targeting of STAT3 with Stattic, a small-molecule inhibitor of STAT3, has beneficial effects in various septic tissues. We investigated the possible cardioprotective effects of Stattic on cardiac inflammation and dysfunction in mice with cecal ligation and puncture (CLP)-induced sepsis. Main methodsA polymicrobial sepsis model was induced by CLP in mice and Stattic (25 mg/kg) was intraperitoneally given at one and twelve hours after CLP operation. The cecum was exposed in sham-control mice without CLP. After 18 h of surgery, electrocardiogram (ECG) for anaesthized mice was registered followed by collecting of samples of blood and tissues for bimolecular and histopathological assessments. Myeloperoxidase, a marker of neutrophil infiltration, was assessed immunohistochemically. Key findingsCLP profoundly impaired cardiac functions as evidenced by ECG changes in septic mice as well as elevation of cardiac enzymes, and inflammatory markers with myocardial histopathological and immunohistochemical alterations. While, Stattic markedly reversed the CLP-induced cardiac abnormalities and restored the cardiac function by its anti-inflammatory activities. SignificanceStattic treatment had potential beneficial effects against sepsis-induced cardiac inflammation, dysfunction and damage. Its cardioprotective effects were possibly attributed to its anti-inflammatory activities by targeting STAT3 and downregulation of IL-6 and gp130. Our investigations suggest that Stattic could be a promising target for management of cardiac sepsis and inflammation-related cardiac damage.

Intermedin (adrenomedullin 2) plays a protective role in sepsis by regulating T- and B-cell proliferation and activity.

Sepsis is the major cause of death in intensive care units. We previously found that intermedin (IMD), a calcitonin family peptide, can protect against sepsis by dynamically repairing vascular endothelial junctions and can ameliorate the inflammatory response by inhibiting the infiltration of macrophages in peripheral tissues. The effects of IMD on inflammatory and immune responses indicate that IMD may play a role in immunity. However, whether IMD affects immune cell development, differentiation and response to infection remains unclear. IMD-knockout (Adm2-/-) mice were generated in our previous work. Wild-type and IMD-KO mice were subjected to sham or cecal ligation and puncture (CLP) surgery, and bone marrow cells were obtained for RNA sequencing (RNA-Seq) analysis. The RNA-Seq results were verified by real-time RT-PCR. The effect of IMD KO or IMD rescue on the septic mice was explored using mild and severe infection models induced by CLP surgery at different levels of severity, and the survival outcomes were analyzed using Kaplan-Meier curves and the log-rank test. The mechanism underlying the effects of IMD in T/B cell proliferation and differentiation were investigated by PCR, Western blot (WB), and cell proliferation assays and flow cytometry analysis. RNA-Seq showed that IMD-KO mice exhibited a primary immunosuppression phenotype characterized by a marked decrease in the expression of T- and B-cell function-related genes. This immunosuppression made the IMD-KO mice vulnerable to pathogenic invasion, and even mild infection killed nearly half of the IMD-KO mice. Supplementation with the IMD peptide restored the expression of T/B-cell-related genes and significantly reduced the mortality rate of the IMD-KO mice. IMD is likely to directly promote T- and B-cell proliferation through ERK1/2 phosphorylation, stimulate T-cell differentiation via Ilr7/Rag1/2-controled T cell receptor (TCR) recombination, and activate B cells via Pax5, a transcription factor that activates at least 170 genes needed for B-cell functions. Together with previous findings, our results indicate that IMD may play a protective role in sepsis via three mechanisms: protecting the vascular endothelium, reducing the inflammatory response, and activating T/B-cell proliferation and differentiation. Our study may provide the first identification of IMD as a calcitonin peptide that plays an important role in the adaptive immune response by activating T/B cells and provides translational opportunities for the design of immunotherapies for sepsis and other diseases associated with primary immunodeficiency.

Poly(lactic acid)-based immunomodulatory nanoparticles promote survival in a polymicrobial mouse model of sepsis

Abstract Sepsis is a life-threatening multifactorial organ dysfunction caused by a dysregulated host response to infection. Despite being recognized as a global health priority, current therapeutic approaches for sepsis have yet to demonstrate significant improvements in patient outcomes. Our group previously demonstrated that cargo-less immunomodulatory nanoparticles (iNPs) could suppress innate immune cell proinflammatory responses and increase survival in an LPS-induced endotoxemia mouse model. In the present work, we examine the impact of poly(lactic acid)-based iNP delivery on survival and comprehensively characterize the host immune response using a clinically relevant and lethal mouse model of polymicrobial sepsis, cecal ligation and puncture (CLP). iNPs, when administered intraperitoneally in combination with broad spectrum antibiotics following CLP surgery, resulted in a significant improvement in disease symptoms and survival compared to CLP + antibiotics only. We also observed that iNP treatment reduced the levels of proinflammatory cytokines in the peritoneal fluid. Analysis of immune cell populations suggested that iNP treatment modulated macrophage, myeloid-derived suppressor cell, and inflammatory monocyte populations both locally and systemically. Our study demonstrates that local administration of iNPs promotes survival through a multimodal immunomodulatory mechanism to improve sepsis outcomes. This research was supported by startup funds from the University of Maryland School of Pharmacy, National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM142752, and the Shock Society Faculty Research Award awarded to Ryan Pearson. Nhu Truong is supported by a PhRMA Foundation Pre-Doctoral Fellowship from Washington, DC. This publication was supported by funds through the National Cancer Institute - Cancer Center Support Grant (CCSG) - P30CA134274, and Maryland Department of Health's Cigarette Restitution Fund Program

Early markers of Sepsis Cardiomyopathy in Murine Models by Echocardiography.

Strain echocardiography (SE) is a procedure for analyzing myocardial dysfunction that is known to be less dependent on preload and afterload of heart function. Unlike dimension-based parameters, like ejection fraction (EF) and fractional shortening (FS), SE measures cardiac function by tracking cardiac tissue deformation and anomalies throughout the cardiac cycle. Although SE is proven to locate myocardial ailments in various heart diseases, few studies exist regarding using SE relevant to sepsis pathophysiology. The study aimed to calculate the myocardial strain and strain rates, like longitudianl strain (LS), global radial strain (GRS), and global longitudinal strain (GLS), and to show these to be reduced earlier in cecal ligation and puncture (CLP) and lipopolysaccharide (LPS)-induced sepsis in coordination with an elevation of pro-inflammatory cytokines Methodology: Wild-type mice C57BL/6J (WT) were taken in this study and classified as CLP, LPS, and control groups. CLP surgery and LPS injection were given to induce sepsis. Endotoxemic septic shock was induced by intraperitoneal (IP) injection of LPS Escherichia coli. Echocardiography short axis views (SAX), longitudinal strain (LS), global circumferential strain (GCS), and global radial strain (GRS) were measured from the anterior and posterior positions of the septal and lateral walls of the heart. Real-time polymerase chain reaction (RT-PCR) was performed to evaluate post-CLP and LPS to analyze cardiac pro-inflammatory cytokines expressions. Inter- and intra-observer variables were tested by Bland-Altman analyses (BA). All data analysis was performed by GraphPad Prism 6 software. p<0.05 was taken as statistically significant. After 48 hours following CLP and LPS-induced sepsis, a significant declination in both longitudinal strain and strain rate (LS and LSR) was identified in the CLP and LPS groups compared to the control group. Strain depression in sepsis was linked with the up-regulation of pro-inflammatory cytokines in RT-PCR analysis. In the present study, we found myocardial strain and strain rate parameters, like LS, GRS, and GLS, to be reduced after CLP and LPS-induced sepsis in coordination with the elevation of pro-inflammatory cytokines.

Downregulation of the gene expression of Cyp2c29 and Cyp3a11 by cecal ligation and puncture-induced sepsis is associated with interleukin-6.

Sepsis is a pathological condition that affects the metabolism of administered drugs, leading to changes in the duration and intensity of their intended efficacies. Proinflammatory cytokines downregulate the expression of cytochrome P450s (P450s). The effects of P450 expression under inflammatory conditions have been studied using prophlogistic substances such as lipopolysaccharide; however, few studies have focused on clinical models of sepsis. Here, we show that cecal ligation and puncture (CLP), an approach for the study of human polymicrobial sepsis, leads to the expression of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNFα) at 24h after the CLP operation. Following CLP, IL-6-/- mice exhibited markedly lower survival than WT mice. In addition, CLP led to the significant downregulation of Cyp2c29 and Cyp3a11 gene expression in IL-1α-/-/β-/- (IL-1-/-) and TNFα-/- mice as well as in WT mice. In contrast, CLP elicited no significant effect on Cyp3a11 expression in IL-6-/- mice. Although CLP reduced the Cyp2c29 expression level in IL-6-/- mice, the expression of Cyp2c29 was lower in CLP-operated WT mice than in CLP-operated IL-6-/- mice. The reduction in the respective P450 protein levels and activities due to CLP-induced sepsis, reflected in the mRNA expression levels, was abolished by IL-6 depletion. Thus, CLP-induced sepsis downregulates P450 gene expression, particularly Cyp2c expression, and this effect is associated with IL-6 without affecting resistance to CLP-induced sepsis. These findings demonstrate the usefulness of CLP for studying the regulation of P450s and highlight IL-6 as a potential indicator of drug-metabolizing capacity under septic conditions.

HMGB1 mediates synaptic loss and cognitive impairment in an animal model of sepsis-associated encephalopathy

BackgroundMicroglial activation-mediated neuroinflammation is one of the essential pathogenic mechanisms of sepsis-associated encephalopathy (SAE). Mounting evidence suggests that high mobility group box-1 protein (HMGB1) plays a pivotal role in neuroinflammation and SAE, yet the mechanism by which HMGB1 induces cognitive impairment in SAE remains unclear. Therefore, this study aimed to investigate the mechanism of HMGB1 underlying cognitive impairment in SAE.MethodsAn SAE model was established by cecal ligation and puncture (CLP); animals in the sham group underwent cecum exposure alone without ligation and perforation. Mice in the inflachromene (ICM) group were continuously injected with ICM intraperitoneally at a daily dose of 10 mg/kg for 9 days starting 1 h before the CLP operation. The open field, novel object recognition, and Y maze tests were performed on days 14–18 after surgery to assess locomotor activity and cognitive function. HMGB1 secretion, the state of microglia, and neuronal activity were measured by immunofluorescence. Golgi staining was performed to detect changes in neuronal morphology and dendritic spine density. In vitro electrophysiology was performed to detect changes in long-term potentiation (LTP) in the CA1 of the hippocampus. In vivo electrophysiology was performed to detect the changes in neural oscillation of the hippocampus.ResultsCLP-induced cognitive impairment was accompanied by increased HMGB1 secretion and microglial activation. The phagocytic capacity of microglia was enhanced, resulting in aberrant pruning of excitatory synapses in the hippocampus. The loss of excitatory synapses reduced neuronal activity, impaired LTP, and decreased theta oscillation in the hippocampus. Inhibiting HMGB1 secretion by ICM treatment reversed these changes.ConclusionsHMGB1 induces microglial activation, aberrant synaptic pruning, and neuron dysfunction in an animal model of SAE, leading to cognitive impairment. These results suggest that HMGB1 might be a target for SAE treatment.