Mouse Model Of Sepsis Research Articles

Rhoifolin protects cecal ligation and puncture induced sepsis mice model by regulating inflammatory pathway

Sepsis is a systemic infection affects several organs, which needs novel therapy for the management of it, thus protective effect of Rhoifolin was estimated against sepsis. Cecal ligation and puncture (CLP) method was used to induce sepsis and thereafter mice were treated with rhoifolin (20 and 40 mg/kg, i.p.) for one week. Food intake and survival rate was determined sepsis mice, moreover liver function test and cytokines was estimated in the serum of sepsis mice. In the lung tissue homogenate, oxidative stress parameters were determined, histopathological analysis was performed in liver and lung tissue of sepsis mice. Food intake and percentage of survival was improved in rhoifolin treated group than sham group. Level of liver function enzyme and cytokine was reduced significantly in the serum of rhoifolin treated sepsis mice. Treatment with rhoifolin ameliorates the altered oxidative stress parameters, and mRNA expression of Toll-like receptor 4 (TLR-4) in lung tissue of sepsis mice. Histopathological changes were also reverse in rhoifolin treated group than sham group of mice. In conclusion, result of report indicates Rhoifolin treatment reduces oxidative stress and inflammation in CLP induced sepsis mice, as it regulates TLR4/MyD88/NF-κB pathway.

SuPAR links a dysregulated immune response to tissue inflammation and sepsis-induced acute kidney injury.

Acute kidney injury (AKI) secondary to sepsis results in poor outcomes and conventional kidney function indicators lack diagnostic value. Soluble urokinase plasminogen activator receptor (suPAR) is an innate immune-derived molecule implicated in inflammatory organ damage. We characterized the diagnostic ability of longitudinal serum suPAR levels to discriminate severity and course of sepsis-induced AKI (SI-AKI) in 200 critically ill patients meeting Sepsis-3 criteria. The pathophysiologic relevance of varying suPAR levels in SI-AKI was explored in a polymicrobial sepsis model in WT, (s)uPAR-knockout, and transgenic suPAR-overexpressing mice. At all time points studied, suPAR provided a robust classification of SI-AKI disease severity, with improved prediction of renal replacement therapy (RRT) and mortality compared with established kidney biomarkers. Patients with suPAR levels of greater than 12.7 ng/mL were at highest risk for RRT or death, with an adjusted odds ratio of 7.48 (95% CI, 3.00-18.63). suPAR deficiency protected mice against SI-AKI. suPAR-overexpressing mice exhibited greater kidney damage and poorer survival through inflamed kidneys, accompanied by local upregulation of potent chemoattractants and pronounced kidney T cell infiltration. Hence, suPAR allows for an innate immune-derived and kidney function-independent staging of SI-AKI and offers improved longitudinal risk stratification. suPAR promotes T cell-based kidney inflammation, while suPAR deficiency improves SI-AKI.

Beneficial herb-drug interaction of rhein in Jinhongtang and Imipenem/Cilastatin mediated by organic anion transporters

Ethnopharmacological relevanceJinhongtang as a traditional Chinese medicine (TCM) formula, has been widely used as a clinical adjuvant in the treatment of acute abdominal diseases and sepsis. Clinical benefits of the concurrent use of Jinhongtang and antibiotics have been observed, however, the mechanism has not been fully understood. Aim of the studyThe present study aimed to explore the effect of Jinhongtang on the antibacterial activity of Imipenem/Cilastatin and to clarify the underlying mechanism of herb-drug interaction (HDI). Materials and methodsA mouse model of sepsis induced by Staphylococcus aureus (S. aureus) was used to evaluate the pharmacodynamic interaction in vivo. In vitro antibacterial activity of Imipenem/Cilastatin was studied by determining minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Pharmacokinetic interaction was investigated by pharmacokinetic studies in rats and uptake assays using OAT1/3-HEK293 cells. The main constituents ingested into blood of rats were qualitatively identified by UHPLC-Q-TOF-MS. ResultsMice treated by Imipenem/Cilastatin and Jinhongtang exhibited higher survival rate, lower bacteria load and less inflammation in blood and lung tissues, compared with those treated by Imipenem/Cilastatin alone after injection of S. aureus. However, MIC and MBC of Imipenem/Cilastatin against S. aureus in vitro were not significantly changed in the presence of Jinhongtang. On the contrary, Jinhongtang increased the plasma concentration of Imipenem and decreased its urinary excretion in rats. CLr of Imipenem was reduced by 58.5%, while its half-life (t1/2) was prolonged for approximate 1.2 times after coadministered Jinhongtang. Furthermore, the extracts of Jinhongtang, single herb in the prescription, and main absorbable constituents inhibited cellular uptake of probe substrates and Imipenem by OAT1/3-HEK293 cells to different extents. Among them, rhein exhibited the strongest inhibition capacity with IC50 values of 0.08 ± 0.01 μM (OAT1) and 2.86 ± 0.28 μM (OAT3). Moreover, coadministration of rhein also significantly enhanced the antibacterial activity of Imipenem/Cilastatin in sepsis mice. ConclusionConcomitant administration of Jinhongtang enhanced antibacterial activity of Imipenem/Cilastatin in sepsis mice induced by S. aureus through reducing renal elimination of Imipenem via inhibition of OATs. Our investigation provided the insight of Jinhongtang as an effective supplement to enhance the antibacterial activity of Imipenem/Cilastatin and can be useful for future clinical studies.

Fucoxanthin Alleviates Lipopolysaccharide-Induced Inflammation and Immunosuppression in RAW264.7 Macrophages.

Fucoxanthin (FX) has been reported to reduce mortality in mouse models of sepsis, but its exact cause remains to be determined. In the present study, we evaluated the immunomodulatory properties of FX in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Our results showed that FX could not only suppress the immune activation responses caused by LPS primary stimulation, but also antagonize LPS restimulation-induced immunosuppression in macrophages. The immunomodulatory capabilities of FX was mainly demonstrated by regulating the production of the inflammatory mediator under different LPS stimuli. Furthermore, we found that adenosine monophosphate-activated protein kinase (AMPK) activation was required for FX's anti-inflammatory and anti-immunosuppressive activities. Our results complement existing data supporting the clinical potential for FX in treating sepsis.

SPI1-mediated autophagy of peripheral blood monocyte cells as a mechanism for sepsis based on single-cell RNA sequencing.

Autophagy has been documented to participate in immune responses and inflammatory diseases, but the mechanistic actions of monocyte autophagy in sepsis remain largely unknown. This study intends to analyze the mechanism of autophagy of peripheral blood monocyte cells (PBMCs) in sepsis based on single-cell RNA sequencing (scRNA-seq). The scRNA-seq data of PBMC samples from sepsis patients were downloaded from the GEO database, followed by identification of cell marker genes, key pathways and key genes. The bioinformatics analysis showed that the PBMC samples of sepsis patients mainly contained 9 immune cell types, among which three types of monocytes showed significant changes in cell numbers in sepsis patients. Of note, the highest autophagy score was found in the intermediate monocytes. The Annexin signaling pathway was a key pathway for the communication between monocytes and other cells. More importantly, SPI1 was predicted as a key gene in the autophagy phenotype of intermediate monocytes, and SPI1 might suppress ANXA1 transcription. The high expression of SPI1 in sepsis was confirmed by RT-qPCR and Western blot analysis. Dual luciferase reporter gene assay verified that SPI1 could bind to the promoter region of ANXA1. Furthermore, it was found that SPI1 might affect monocyte autophagy in the mouse model of sepsis through regulation of ANXA1. In conclusion, we provide insight into the mechanism underlying the septic potential of SPI1, which enhances monocyte autophagy by inhibiting the transcription of ANXA1 in sepsis.

Inhibition of DDX3X alleviates persistent inflammation, immune suppression and catabolism syndrome in a septic mice model.

DDX3X is involved in various pathological processes such as infection, immunity and cell death. This study aimed to investigate the effect of RK-33, a specific inhibitor of DDX3X, on the progression of sepsis to persistent inflammation, immune suppression and catabolism syndrome(PICS). The septic mice model was established using caecal ligation and perforation (CLP). The mice were randomly divided into four groups: sham group, sham+RK-33 group (20mg/kg, intraperitoneal injection, once a day), CLP group and CLP+RK-33 group (20mg/kg, intraperitoneal injection, once a day). The number of inflammatory cells in the peripheral blood, spleen and bone marrow was calculated, and inflammatory cytokines were detected using an enzyme-linked immunosorbent assay. The septic mice's body weight and skeletal muscle mass were measured, and skeletal muscle tissues were examined using eosin staining. Western blotting was performed to detect the expression levels of MuRF1, atrogin1 and NLRP3 in the skeletal muscle of septic mice. Additionally, reactive oxidative species, superoxide dismutase and malondialdehyde were measured using commercial kits. RK-33 reduced inflammatory cell counts and cytokine levels in CLP mice, ameliorated the decline in CD4 and CD8 T cells and prevented the loss of body weight and skeletal muscle mass in septic mice. Additionally, RX-33 reduced oxidative stress in the skeletal muscle of septic mice. In the established sepsis mouse model, RK-33 alleviated inflammation and oxidative stress, ameliorated CLP-induced immunosuppression and skeletal muscle atrophy and improved survival. These findings suggest that RK-33 could be a novel potential therapeutic agent for preventing the progression of sepsis to PICS.

The Novel Compound SUL-138 Counteracts Endothelial Cell and Kidney Dysfunction in Sepsis by Preserving Mitochondrial Function

Sepsis is defined as a dysregulated host response leading to organ dysfunction, which may ultimately result in the patient's death. Mitochondrial dysfunction plays a key role in developing organ dysfunction in sepsis. In this study, we explored the efficacy of the novel mitochondrial protective compound, SUL-138, in sepsis models in HUVECs and mice. In LPS-challenged HUVECs, SUL-138 preserved mitochondrial membrane potential and oxygen consumption and limited mitochondrial oxidative stress, resulting in increased survival at 48 h. Further, SUL-138 dampened the LPS-induced expression of IL-1β, but not of NLRP3, and IL-18 in HUVECs. Sepsis in mice induced by cecal ligation and puncture (CLP) led to a lower mitochondrial membrane potential and increased levels of mitochondrial oxidative stress in the kidney, which SUL-138 limited. In addition, SUL-138 mitigated the CLP-induced increase in kidney dysfunction markers NGAL and urea. It dampened the rise in kidney expression of IL-6, IL-1β, and ICAM-1, but not TNF-α and E-selectin. Yet, SUL-138 limited the increase in plasma levels of IL-6 and TNF-α of CLP mice. These results demonstrate that SUL-138 supports mitochondrial function, resulting in a limitation of systemic inflammation and preservation of kidney function.

Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Bazedoxifene attenuates intestinal injury in sepsis by suppressing the NF-κB/NLRP3 signaling pathways

Acute inflammatory injury is the primary cause of sepsis, leading to various organ failures. Bazedoxifene (BAZ) has been proven to have anti-inflammatory effects. However, its effects on sepsis-induced intestinal injury are unclear. Here, we demonstrated the beneficial effects of BAZ on intestinal injury and explored the underlying mechanisms using cecal ligation and perforation (CLP)-mediated sepsis mouse model and in vitro cultured intestinal epithelial MODE-K cells. We found that BAZ elevated the survival rate of septic mice and attenuated CLP-triggered intestinal damage. BAZ inhibited intestinal inflammation and restored the impaired intestinal barriers in CLP mice. The mechanistic study in lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-stimulated MODE-K cells showed that BAZ significantly downregulated the expression of NOD-like receptor protein 3 (NLRP3), interleukin-1β (IL-1β), caspase-1, and gasdermin D (GSDMD), and markedly reduced the phosphorylation of molecules in the nuclear factor kappa B (NF-κB) pathway. Moreover, BAZ prominently rescued the decreased viability of MODE-K cells and reduced lactate dehydrogenase (LDH) release upon LPS/ATP challenge. However, BAZ did not affect the inflammasome assembly, as evidenced by the lack of changes in ASC (apoptosis speck-like protein containing a CARD) speck formation. Our results suggest that BAZ relieves inflammation and intestinal barrier function disruption by suppressing the NF-κB/NLRP3 signaling pathways. Therefore, BAZ is a potential therapeutic candidate for treating intestinal injury in sepsis.

Using an Aluminum Hydroxide-Chitosan Matrix Increased the Vaccine Potential and Immune Response of Mice against Multi-Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, immobile, aerobic nosocomial opportunistic coccobacillus that causes pneumonia, septicemia, and urinary tract infections in immunosuppressed patients. There are no commercially available alternative antimicrobials, and multi-drug resistance is an urgent concern that requires emergency measures and new therapeutic strategies. This study evaluated a multi-drug-resistant A. baumannii whole-cell vaccine, inactivated and adsorbed on an aluminum hydroxide-chitosan (mAhC) matrix, in an A. baumannii sepsis model in immunosuppressed mice by cyclophosphamide (CY). CY-treated mice were divided into immunized, non-immunized, and adjuvant-inoculated groups. Three vaccine doses were given at 0D, 14D, and 28D, followed by a lethal dose of 4.0 × 108 CFU/mL of A. baumannii. Immunized CY-treated mice underwent a significant humoral response, with the highest IgG levels and a higher survival rate (85%); this differed from the non-immunized CY-treated mice, none of whom survived (p < 0.001), and from the adjuvant group, with 45% survival (p < 0.05). Histological data revealed the evident expansion of white spleen pulp from immunized CY-treated mice, whereas, in non-immunized and adjuvanted CY-treated mice, there was more significant organ tissue damage. Our results confirmed the proof-of-concept of the immune response and vaccine protection in a sepsis model in CY-treated mice, contributing to the advancement of new alternatives for protection against A. baumannii infections.

Cannabinoid WIN55,212-2 reprograms monocytes and macrophages to inhibit LPS-induced inflammation.

Chronic or uncontrolled activation of myeloid cells including monocytes, macrophages and dendritic cells (DCs) is a hallmark of immune-mediated inflammatory disorders. There is an urgent need for the development of novel drugs with the capacity to impair innate immune cell overactivation under inflammatory conditions. Compelling evidence pointed out cannabinoids as potential therapeutic tools with anti-inflammatory and immunomodulatory capacity. WIN55,212-2, a non-selective synthetic cannabinoid agonist, displays protective effects in several inflammatory conditions by mechanisms partially depending on the generation of tolerogenic DCs able to induce functional regulatory T cells (Tregs). However, its immunomodulatory capacity on other myeloid cells such as monocytes and macrophages remains incompletely understood. Human monocyte-derived DCs (hmoDCs) were differentiated in the absence (conventional hmoDCs) or presence of WIN55,212-2 (WIN-hmoDCs). Cells were stimulated with LPS, cocultured with naive T lymphocytes and their cytokine production and ability to induce T cell responses were analysed by ELISA or flow cytometry. To evaluate the effect of WIN55,212-2 in macrophage polarization, human and murine macrophages were activated with LPS or LPS/IFNγ, in the presence or absence of the cannabinoid. Cytokine, costimulatory molecules and inflammasome markers were assayed. Metabolic and chromatin immunoprecipitation assays were also performed. Finally, the protective capacity of WIN55,212-2 was studied in vivo in BALB/c mice after intraperitoneal injection with LPS. We show for the first time that the differentiation of hmoDCs in the presence of WIN55,212-2 generates tolerogenic WIN-hmoDCs that are less responsive to LPS stimulation and able to prime Tregs. WIN55,212-2 also impairs the pro-inflammatory polarization of human macrophages by inhibiting cytokine production, inflammasome activation and rescuing macrophages from pyroptotic cell death. Mechanistically, WIN55,212-2 induced a metabolic and epigenetic shift in macrophages by decreasing LPS-induced mTORC1 signaling, commitment to glycolysis and active histone marks in pro-inflammatory cytokine promoters. We confirmed these data in ex vivo LPS-stimulated peritoneal macrophages (PMΦs), which were also supported by the in vivo anti-inflammatory capacity of WIN55,212-2 in a LPS-induced sepsis mouse model. Overall, we shed light into the molecular mechanisms by which cannabinoids exert anti-inflammatory properties in myeloid cells, which might well contribute to the future rational design of novel therapeutic strategies for inflammatory disorders.

UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis.

Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.

Screening of potential immune-related genes expressed during sepsis using gene sequencing technology

To screen potential pivotal targets in sepsis through peripheral blood. Septic patients (n = 23) and healthy volunteers (n = 10) were enrolled according to SEPSIS 3.0. Peripheral blood was collected within 24 h of enrollment, RNA-seq was performed on the peripheral blood. The sequencing data was screened for DEGs (p < 0.01; logFC ≥ 2). PPI, WGCNA and survival curve analysis were used to identify potential targets. Then, 5 PBMC samples were conducted by single-cell sequencing for cell lineage location. Finally, mouse sepsis model and clinic samples were performed to verify the targets gene using RNA-seq and RT-PCR, respectively. Compared to the control group, 1007 DEGs were found in septic group. BCL9L, BCL11B, CD247, CD96, MAFG and SAMD3 were in the core of network. These six genes correlated to the survival rate of septic patients and they were mainly expressed in T cells, except that MAFG was located in monocyte cell. The expression levels of six key genes were confirmed by animal and clinical samples. BCL9L, BCL11B, CD247, CD96 and SAMD3 were decreased in sepsis and mainly expressed in the T cell; while MAFG increased in sepsis and localizes to monocytes. These genes may be therapeutic targets for sepsis.

Liposomes loaded with quercetin for resolution of lung inflammation in a lipopolysaccharide-induced mouse model of sepsis

Quercetin (QU) has been widely used as a dietary supplement and proved useful to treat lung diseases. However, the therapeutic potential of QU may be restricted because of its low bioavailability and poor water solubility. In this study, we investigated the effects of developed QU-loaded liposomes on macrophage-mediated lung inflammation. In vivo, a mouse model of sepsis induced by lipopolysaccharide challenge was used to detect the anti-inflammatory effects of liposomal QU. Hematoxylin/eosin staining and immunostaining were utilized to reveal pathological damage and leukocyte infiltration into the lung tissues. Quantitative reverse transcription-polymerase chain reaction and immunoblotting were used to determine cytokine production in the mouse lungs. In vitro, mouse RAW 264.7 macrophages were treated with free QU and liposomal QU. Cell viability assay and immunostaining were utilized to detect cytotoxicity and distribution of QU in the cells. The in vivo results showed that liposomal encapsulation promoted the inhibitory effects of QU on lung inflammation. Liposomal QU decreased mortality in septic mice with no obvious toxicity on vital organs. Mechanistically, the anti-inflammatory effects of liposomal QU were associated with inhibition of nuclear factor-kappa B-dependent cytokine production and inflammasome activation in macrophages. Collectively, the results showed that QU liposomes mitigated lung inflammation in septic mice through inhibition of macrophage inflammatory signaling.

Rational Design of Stapled Antimicrobial Peptides to Enhance Stability and In Vivo Potency against Polymicrobial Sepsis.

In this work, we sought to develop a TP4-based stapled peptide that can be used to counter polymicrobial sepsis. First, we segregated the TP4 sequence into hydrophobic and cationic/hydrophilic zones and substituted the preferred residue, lysine, as the sole cationic amino acid. These modifications minimized the intensity of cationic or hydrophobic characteristics within small segments. Then, we incorporated single or multiple staples into the peptide chain, bracketing the cationic/hydrophilic segments to improve pharmacological suitability. Using this approach, we were able to develop an AMP with low toxicity and notable in vivo efficacy. IMPORTANCE In our in vitro studies, one dual stapled peptide out of the series of candidates (TP4-3: FIIXKKSXGLFKKKAGAXKKKXIKK) showed significant activity, low toxicity, and high stability (in 50% human serum). When tested in cecal ligation and puncture (CLP) mouse models of polymicrobial sepsis, TP4-3 improved survival (87.5% on day 7). Furthermore, TP4-3 enhanced the activity of meropenem against polymicrobial sepsis (100% survival on day 7) compared to meropenem alone (37.5% survival on day 7). Molecules such as TP4-3 may be well suited for a wide variety of clinical applications.

Resveratrol Prevents Vibrio vulnificus-Induced Sepsis by Attenuating Necroptosis

ObjectiveThis study investigated how the natural phytophenol and potent SIRT1 activator resveratrol (RSV) regulate necroptosis during Vibrio vulnificus (V. vulnificus)-induced sepsis and the potential mechanism. MethodsThe effect of RSV on V. vulnificus cytolysin (VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays. Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V. vulnificus-induced sepsis mouse model. ResultsRSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells. RSV also inhibited the inflammatory response, had a protective effect on histopathological changes, and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages, lung, spleen, and liver tissues of V. vulnificus-induced septic mice in vivo. Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V. vulnificus-induced septic mice. RSV also improved the survival of V. vulnificus-induced septic mice. ConclusionOur findings collectively demonstrate that RSV prevented V. vulnificus-induced sepsis by attenuating necroptosis, highlighting its potency in the clinical management of V. vulnificus-induced sepsis.

Anti-inflammatory effect of Danhong injection through inhibition of GSDMD-mediated pyroptosis

BackgroundPyroptosis is an inflammatory form of cell death that has been implicated in various infectious and non-infectious diseases. Gasdermin family proteins are the key executors of pyroptotic cell death, thus they are considered as novel therapeutic targets for inflammatory diseases. However, only limited gasdermin specific inhibitors have been identified to date. Traditional Chinese medicines have been applied in clinic for centuries and exhibit potential in anti-inflammation and anti-pyroptosis. We attempted to find candidate Chinese botanical drugs which specifically target gasdermin D (GSDMD) and inhibit pyroptosis. MethodsIn this study, we performed high-throughput screening using a botanical drug library to identify pyroptosis specific inhibitors. The assay was based on a cell pyroptosis model induced by lipopolysaccharides (LPS) and nigericin. Cell pyroptosis levels were then evaluated by cell cytotoxicity assay, propidium iodide (PI) staining and immunoblotting.We then overexpressed GSDMD-N in cell lines to investigate the direct inhibitory effect of the drug to GSDMD-N oligomerization. Mass spectrometry studies were applied to identify the active components of the botanical drug. Finally, a mouse model of sepsis and a mouse model of diabetic myocardial infarction were constructed to verify the protective effect of the drug in disease models of inflammation. ResultsHigh-throughput screening identified Danhong injection (DHI) as a pyroptosis inhibitor. DHI remarkably inhibited pyroptotic cell death in a murine macrophage cell line and bone marrow-derived macrophages. Molecular assays demonstrated the direct blockade of GSDMD-N oligomerization and pore formation by DHI. Mass spectrometry studies identified the major active components of DHI, and further activity assays revealed salvianolic acid E (SAE) as the most potent molecule among these components, and SAE has a strong binding affinity to mouse GSDMD Cys192. We further demonstrated the protective effects of DHI in mouse sepsis and mouse myocardial infarction with type 2 diabetes. ConclusionThese findings provide new insights for drug development from Chinese herbal medicine like DHI against diabetic myocardial injury and sepsis through blocking GSDMD-mediated macrophage pyroptosis.

Embelin attenuates lipopolysaccharide-induced acute kidney injury through the inhibition of M1 macrophage activation and NF-κB signaling in mice

Septic acute kidney injury (AKI) is commonly associated with renal dysfunction and high mortality in patients. Owing to the rapid and violent occurrence of septic AKI with inflammation, there are no effective therapies to clinically treat it. Embelin, a natural product, has a potential regulatory role in immunocytes. However, the role and mechanism of embelin in septic AKI remains unknown. This study aimed to elucidate the role of embelin in macrophage regulation in lipopolysaccharide (LPS)-induced septic AKI. Embelin was intraperitoneally administered to mice after LPS injection. And bone marrow-derived macrophages (BMDMs) were subsequently isolated from the mice to explore the immunomodulatory role of embelin in macrophages. We found that embelin attenuated renal dysfunction and pathological renal damage in the LPS-induced sepsis mouse model. Molecular docking predicted that embelin could bind to phosphorylated NF-κB p65 at the ser536 site. Embelin inhibited the translocation of NF-κB p65 via phosphorylation at ser536 in LPS-induced AKI. It also reduced the secretion of IL-1β and IL-6 and increased the secretion of IL-10 and Arg-1 of BMDMs and mice after LPS stimulation, indicating that embelin suppressed macrophage M1 activation in LPS-induced AKI. Therefore, embelin attenuated LPS-induced septic AKI by suppressing NF-κB p65 at ser536 in activated macrophages. This study preclinically suggests a therapeutic role of embelin in septic AKI.

MicroRNA Profiles in Intestinal Epithelial Cells in a Mouse Model of Sepsis.

Sepsis is a systemic inflammatory disorder that leads to the dysfunction of multiple organs. In the intestine, the deregulation of the epithelial barrier contributes to the development of sepsis by triggering continuous exposure to harmful factors. However, sepsis-induced epigenetic changes in gene-regulation networks within intestinal epithelial cells (IECs) remain unexplored. In this study, we analyzed the expression profile of microRNAs (miRNAs) in IECs isolated from a mouse model of sepsis generated via cecal slurry injection. Among 239 miRNAs, 14 miRNAs were upregulated, and 9 miRNAs were downregulated in the IECs by sepsis. Upregulated miRNAs in IECs from septic mice, particularly miR-149-5p, miR-466q, miR-495, and miR-511-3p, were seen to exhibit complex and global effects on gene regulation networks. Interestingly, miR-511-3p has emerged as a diagnostic marker in this sepsis model due to its increase in blood in addition to IECs. As expected, mRNAs in the IECs were remarkably altered by sepsis; specifically, 2248 mRNAs were decreased, while 612 mRNAs were increased. This quantitative bias may be possibly derived, at least partly, from the direct effects of the sepsis-increased miRNAs on the comprehensive expression of mRNAs. Thus, current in silico data indicate that there are dynamic regulatory responses of miRNAs to sepsis in IECs. In addition, the miRNAs that were increased with sepsis had enriched downstream pathways including Wnt signaling, which is associated with wound healing, and FGF/FGFR signaling, which has been linked to chronic inflammation and fibrosis. These modifications in miRNA networks in IECs may lead to both pro- and anti-inflammatory effects in sepsis. The four miRNAs discovered above were shown to putatively target LOX, PTCH1, COL22A1, FOXO1, or HMGA2, via in silico analysis, which were associated with Wnt or inflammatory pathways and selected for further study. The expressions of these target genes were downregulated in sepsis IECs, possibly through posttranscriptional modifications of these miRNAs. Taken together, our study suggests that IECs display a distinctive miRNA profile which is capable of comprehensively and functionally reshaping the IEC-specific mRNA landscape in a sepsis model.

Single-cell transcriptome analysis reveals the regulatory effects of artesunate on splenic immune cells in polymicrobial sepsis

Sepsis is characterized by a severe and life-threatening host immune response to polymicrobial infection accompanied by organ dysfunction. Studies on the therapeutic effect and mechanism of immunomodulatory drugs on the sepsis-induced hyperinflammatory or immunosuppression states of various immune cells remain limited. This study aimed to investigate the protective effects and underlying mechanism of artesunate (ART) on the splenic microenvironment of cecal ligation and puncture-induced sepsis model mice using single-cell RNA sequencing (scRNA-seq) and experimental validations. The scRNA-seq analysis revealed that ART inhibited the activation of pro-inflammatory macrophages recruited during sepsis. ART could restore neutrophils’ chemotaxis and immune function in the septic spleen. It inhibited the activation of T regulatory cells but promoted the cytotoxic function of natural killer cells during sepsis. ART also promoted the differentiation and activity of splenic B cells in mice with sepsis. These results indicated that ART could alleviate the inflammatory and/or immunosuppressive states of various immune cells involved in sepsis to balance the immune homeostasis within the host. Overall, this study provided a comprehensive investigation of the regulatory effect of ART on the splenic microenvironment in sepsis, thus contributing to the application of ART as adjunctive therapy for the clinical treatment of sepsis.