Injury In Septic Mice Research Articles

CircROCK1 Promotes septic myocardial injury through regulating miR-96-5p/OXSR1 axis.

A recent high-throughput sequencing showed that circular RNA Rho-associated kinase 1 (circROCK1) is abnormally highly expressed in sepsis, but whether it is involved in sepsis development remains unclear. The objective of this study was to investigate the biological function of circROCK1 in sepsis-induced myocardial injury and reveal its potential downstream molecular mechanism. Real-time reverse transcriptase-polymerase chain reaction was applied to detect circROCK1 and miR-96-5p expressions in the serum of septic patients. Spearman correlation analysis examined the correlation between circROCK1 and the clinicopathological characteristics of septic patients. The Cecal puncture and ligation (CLP) method was used to establish an in vivo sepsis model. circROCK1 and miR-96-5p expressions in mice were modified by injection of lentivirus or oligonucleotide. The left ventricular systolic pressure, left ventricular end-diastolic pressure, and the maximum increase/decrease rate of left ventricular pressure were checked. ELISA was applied to detect inflammatory factors levels as well as myocardial injury markers levels. Hematoxylin and eosin staining was performed to observe pathological changes in myocardial tissues, and Western blot examined phosphorylated nuclear factor (NF)-κB and oxidative stress-responsive 1 (OXSR1) expression. Dual luciferase reporter experiment was conducted to confirm the targeting relationship between circROCK1, OXSR1, and miR-96-5p. circROCK1 and OXSR1 were highly expressed in sepsis and miR-96-5p was under-expressed. circROCK1 was positively correlated with serum creatinine, C-reactive protein, procalcitonin, and sequential organ failure assessment scores in septic patients. Silencing circROCK1 could improve the diastolic and systolic function of CLP mice, as well as myocardial damage, reduce myocardial tissue edema and necrosis, and inhibit inflammatory factor level and phosphorylated NF-κB expression. Down-regulating miR-96-5p promoted myocardial injury in CLP mice. Silencing circROCK1 and miR-96-5p inhibited and promoted OXSR1 expression, respectively. Both circROCK1 and OXSR1 had a targeting relationship with miR-96-5p. CircROCK1 promotes myocardial injury in septic mice by regulating the miR-96-5p/OXSR1 axis, and it can be used as a potential target for treating septic myocardial dysfunction.

Shenhuangdan decoction alleviates sepsis-induced lung injury through inhibition of GSDMD-mediated pyroptosis

Ethnopharmacological relevanceSepsis-induced lung injury is closely associated with it remarkable morbidity and mortality. Shenhuangdan (SHD) decoction, a traditional Chinese medicine prescription, has been clinically proven to be an effective treatment for sepsis. However, the mechanism of SHD decoction in treating sepsis remains unclear. Aim of the studyThis study aimed to evaluate the therapeutic effect of SHD decoction on sepsis-induced lung injury and its underlying mechanism. Materials and methodsIn this study, we established a mouse model of sepsis by cecum ligation and puncture (CLP) surgery. Firstly, seven-day survival analysis and histological staining of lung tissue were used to evaluate the therapeutic effect of SHD decoction on lung injury in septic mice. Multifactor microarray was used to detect cytokine expression changes in serum and bronchoalveolar lavage fluid (BALF). Subsequently, the main components in medicated serum of SHD decoction were inspected by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The material basis of SHD decoction and potential interaction mechanisms were analysed by systemic pharmacology. To confirm the reliability of network pharmacology for predicting outcomes, we used molecular docking techniques to identify interactions between the core components and targets of SHD. Finally, TUNEL staining, immunofluorescence and western blotting were used to explore the mechanism of SHD decoction through the inhibition of GSDMD-mediated pyroptosis in septic mice. ResultsSHD was found to be effective in reducing the mortality and alleviating lung pathological damage in septic mice. Multifactor microarray results showed that SHD can reduce the expression of inflammation factors (IL-18, IL-1β, IL-5, IL-6 and TNF-α) in serum and BALF of septic mice. There were 22 major blood-entry components detected by UPLC-MS/MS. Then, combined with the network pharmacological analysis, it is evident that the main components of SHD for sepsis are Renshen-ginsenoside Rh2, Danshen-tanshinone IIA and Dahuang-rhein. The main targets were IL-1β and caspase-1, which were related to GSDMD-mediated pyroptosis signalling pathway. Molecular docking exhibited that Renshen-ginsenoside Rh2, Danshen-tanshinone IIA and Dahuang-rhein can closely bind to GSDMD, IL-1β and caspase-1. In addition, TUNEL staining and immunohistochemistry demonstrated that SHD alleviated the expression of GSDMD protein. The western blotting showed that SHD significantly inhibited the protein expression levels of NLRP3, GSDMD, GSDMD-N, cleved caspase-1, caspase-1 and ASC in lung tissue. ConclusionsOur study revealed that SHD improves CLP-induced lung injury by blocking the GSDMD-mediated pyroptosis signalling pathway in septic mice. This study provides evidence to support that SHD had a potential therapeutic effect on sepsis.

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.

Signal transducer and activator of transcription 6 mediates skeletal muscle cell injury in septic mice by regulating ferroptosis

To explore the effect of signal transducer and activator of transcription 6 (STAT6) on ferroptosis in skeletal muscle cells in sepsis model and its potential mechanism. Twenty-four 8-week-old male specific pathogen free Kunming mice were divided into normal control group, sham group, sepsis model group and STAT6 inhibitor pretreatment group according to random number table method with 6 mice in each group. A mouse sepsis model was reproduced by cecal ligation and perforation (CLP). In the sham group, the skin of mice was sutured after exposing the cecum tissue. In the STAT6 inhibitor pretreatment group, 10 mg/kg AS1517499 was injected intraperitoneally 1 hour before model reproduction. The sham group and the model group were intraperitoneally injected with the same volume of normal saline. Mice in the normal control group did not receive any operation or drug intervention. The mice were sacrificed 24 hours after model reproduction, and the muscle tissue of hind limb was obtained under sterile condition. Hematoxylin-eosin (HE) staining was used to observe the histopathology with optical microscope, and mitochondrial morphological changes were observed by transmission electron microscopy after double staining with uranium acetate lead citrate. The ferroptosis marker proteins expressions of chitinase-3-like protein 1 (CHI3L1), cyclooxygenase-2 (COX-2), acyl-CoA synthetase long-chain family member 4 (ACSL4), ferritin heavy chain 1 (FTH1), and glutathione peroxidase 4 (GPx4) were detected by Western blotting. Under the optical microscope, the morphology and structure of skeletal muscle tissues in the normal control and sham groups were normal. In the model group, the structure of skeletal muscle tissues was loose, the muscle fiber became smaller and atrophic, inflammatory cell infiltration and even muscle fiber loss were found. Compared with the model group, the structure of skeletal muscle tissues was tight and skeletal muscle atrophy was improved in the STAT6 inhibitor pretreatment group. The ultrastructure of skeletal muscle cell in the normal control and sham groups was normal under transmission electron microscope. The ultrastructure characteristics of skeletal muscle in the model group showed that cell membrane was broken and blister, mitochondria became smaller and membrane density increased, the mitochondrial crista decreased or disappeared, the mitochondrial outer membrane was broken, and the nucleus was normal in size but lacked chromatin condensation. Compared with the model group, the STAT6 inhibitor pretreatment group had a significant improvement in the ultrastructure of muscle cells. Compared with the normal control and sham groups, the protein expressions of CHI3L1, COX-2, ACSL4 and FTH1 in the muscle of the model group were significantly increased, while the protein expression of GPx4 was decreased significantly, indicating that the skeletal muscle cells in the mouse sepsis model showed characteristic mitochondrial injury and abnormal expression of ferroptosis markers. Compared with the model group, the protein expressions of CHI3LI, COX-2, ACSL4 and FTH1 in the STAT6 inhibitor pretreatment group were significantly decreased [CHI3L1 protein (CHI3L1/GAPDH): 0.70±0.08 vs. 0.97±0.09, COX-2 protein (COX-2/GAPDH): 0.61±0.03 vs. 0.83±0.03, ACSL4 protein (ACSL4/GAPDH): 0.75±0.04 vs. 1.02±0.16, FTH1 protein (FTH1/GAPDH): 0.49±0.06 vs. 0.76±0.13, all P < 0.05], while the protein expression of GPx4 was significantly increased (GPx4/GAPDH: 1.14±0.29 vs. 0.53±0.03, P < 0.05). Sepsis can induce ferroptosis in skeletal muscle cells of mice. STAT6 may mediate ferroptosis in mouse skeletal muscle cells by regulating the expressions of COX-2, ACSL4, FTH1 and GPx4, thereby inducing skeletal muscle cell injury in sepsis.

Mitochondrial aldehyde dehydrogenase 2 alleviates septic liver injury by inhibiting ferroptosis in mouse model

To observe the ferroptosis triggered by in different pathways during cecal ligation and puncture (CLP)-induced liver injury in septic mice, and to investigate whether mitochondrial aldehyde dehydrogenase 2 (ALDH2) can alleviate sepsis-induced liver injury by inhibiting ferroptosis. Sixty 8-week-old male C57BL/6J mice were randomly divided into sham operation group (Sham group), CLP group, ferroptosis inhibitor ferrostain-1 (Fer-1) group, ALDH2-specific agonist Alda-1 group, iron chelator deferasirox Fe3+ chelate (DXZ) group and dimethyl sulfoxide (DMSO) group, with 10 mice in each group. The septic liver injury was induced by CLP in mice model. In the Sham group, only laparotomy was performed without ligation and puncture of the cecum. 10 mL/kg 5% DMSO, 5 mg/kg Fer-1, 50 mg/kg DXZ and 10 mg/kg Alda-1 were injected intraperitoneally 1 hour before CLP in the DMSO, Fer-1, DXZ and Alda-1 groups respectively. At 24 hours after operation, eyeball blood and liver tissue were collected from anesthetized mice. The hepatic structure and inflammatory infiltration were observed by hematoxylin-eosin (HE) staining. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) in serum, the levels of hepatic malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) were detected. Western blotting was used to detect the protein expressions of ALDH2, ferroptosis-related proteins glutathione peroxidase 4 (GPX4), ferroptosis suppressor protein 1 (FSP1) and transferrin receptor 1 (TFR1) in liver tissue. Compared with Sham group, the mice in CLP group showed varying degrees of congestion, disorganized hepatocyte arrangement, inflammatory cell infiltration at 24 hours after operation. Compared with the CLP group, the mice in the Fer-1 group, DXZ group and Alda-1 group liver morphology, liver injury and inflammatory cell infiltration was improved. Compared with Sham group, the serum levels of ALT and AST, the contents of MDA and ROS, and the expression of TFR1 protein in CLP group were significantly increased, while the activity of SOD and the expressions of ALDH2, GPX4 and FSP1 protein in CLP group were significantly decreased. Compared with CLP group, serum ALT and AST levels in Fer-1, DXZ and Alda-1 groups were significantly decreased [ALT (U/L): 45.76±10.81, 37.30±2.98, 36.40±12.75 vs. 73.06±12.20, AST (U/L): 61.57±2.69, 52.41±6.92, 56.05±8.29 vs. 81.59±5.46, all P < 0.05], and the contents of MDA, ROS and TFR1 protein expression in liver tissue were significantly decreased [MDA (μmol/L): 0.60±0.10, 0.57±0.18, 0.83±0.39 vs. 1.61±0.30, ROS (fluorescence intensity): 270.34±9.64, 276.02±62.33, 262.05±18.55 vs. 455.38±36.07, TFR1/GAPDH: 0.90±0.04, 1.01±0.09, 0.55±0.08 vs. 1.18±0.06, all P < 0.05], and the SOD activity and ALDH2, GPX4 and FSP1 protein expressions in liver tissue were significantly increased [SOD (kU/g): 88.77±8.20, 88.37±4.47, 93.43±7.24 vs. 50.27±3.57, ALDH2/GAPDH: 1.10±0.15, 1.02±0.07, 1.14±0.07 vs. 0.70±0.04, GPX4/GAPDH: 1.02±0.12, 0.99±0.08, 1.05±0.19 vs. 0.71±0.10, FSP1/GAPDH: 1.06±0.24, 1.02±0.08, 0.93±0.09 vs. 0.66±0.03, all P < 0.05]. There was no significant difference in the parameters between DMSO group and CLP group. Both GPX4 and FSP1 mediated ferroptosis are involved in liver injury in septic mice. Activation of ALDH2 and inhibition of ferroptosis can alleviatehepatic injury. ALDH2 may play a protective role by regulating FSP1 and GPX4 mediated ferroptosis.

Geldanamycin ameliorates multiple organ dysfunction and microthrombosis in septic mice by inhibiting the formation of the neutrophil extracellular network by activating heat shock factor 1 HSF1.

Sepsis and septic shock are common critical illnesses in the intensive care unit with a high mortality rate. Geldanamycin (GA) has a broad spectrum of antibacterial and antiviral activity and has inhibitory effects on various viruses. However, whether GA affects sepsis due to infections remains unknown. In this study, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine in serum; neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in the urine, cytokines (tumour necrosis factor alpha, interleukin-1β and interleukin-6) in the bronchoalveolar lavage fluid and myeloperoxidase in the lung tissues were measured using enzyme-linked immunosorbent assay kits. Pathological injury was measured by hematoxylin and eosin staining and neutrophils were measured by flow cytometry analysis; related expressions were analysed by qPCR, western blot and immunofluorescence assay. The results showed that GA significantly ameliorated cecum ligation and puncture (CLP)-triggered liver, kidney and lung injury in septic mice. In addition, we found that GA dose-dependently inhibited microthrombosis and alleviated coagulopathy in septic mice. Further molecular mechanism analysis suggests that GA may act through upregulation of heat shock factor 1 and tissue-type plasminogen activator. In conclusion, our study elucidated the protective effects of GA in a mouse model established using CLP, and the results reveal that GA may be a promising agent for sepsis.

MiR-926-3p influences myocardial injury in septic mice through regulation of mTOR signaling pathway by targeting TSC1.

The purpose of this study is to investigate the influence of miR-926-3p on myocardial injury and its mechanisms. An animal model of sepsis was constructed by CLP, and animals were randomly divided into 4 groups: C group, miR-926-3p inhibitor group, CLP + NC group, and CLP + miR-926-3p inhibitor group. Compared with those in C group, echocardiographic parameters remarkably declined in CLP + NC group. Compared with CLP + NC group, miR-926-3p inhibitor group indicated elevated echocardiographic parameters in mice, pathological improvement tendency of myocardial tissues and distinct reduction in cardiomyocyte apoptosis. It could be observed by electron microscopy that the number of lysosomes in miR-926-3p inhibitor group was greatly increased relative to CLP + NC group. Immunofluorescence exhibited that the number of green fluorescent puncta was significantly higher in miR-926-3p inhibitor group as compared to that in CLP + NC group. The autophagic flow was verified by observing the relative expression of LC3II at different times. The results of Western blotting manifested that miR-926-3p inhibitor up-regulated mTOR-related protein expressions and down-regulated the protein expression of p-mTOR. LPS was adopted to induce cardiomyocyte injury in vitro, and the results confirmed that, identical to in vivo experiments, miR-926-3p inhibitor was able to up-regulate the protein expressions of mTOR-related protein and down-regulate p-mTOR protein expression in cardiomyocytes. After addition of MHY1485, The expression of mTOR-related proteins changes in each group. Inhibition of miR-926-3p enhances autophagy through regulation of the mTOR signaling pathway, thus ameliorating myocardial injury in septic mice.

Protective Effect of Mesenchymal Stem Cell Transplantation on Intestinal Injury in Septic Mice and Its Mechanism

To explore the protective effect of placenta-derived mesenchymal stem cells (P-MSCs) transplantation on intestinal injury in septic mice and its mechanism. A total of 24 mice were randomly assigned to 3 groups, a sham operation group, a sepsis group that underwent cecal ligation and puncture (CLP) procedure, and a group that received CLP and P-MSCs treatment. Hereinafter, the three groups are referred to as the Sham group, the CLP group, and the CLP+P-MSCs group. For the mice in the Sham group, the abdomen was cut open and the cecum was exposed and then placed back in the abdomen. CLP was performed in the other two groups to establish the sepsis model. Mice in the Sham and the CLP groups received 0.1 mL of 0.9% NaCl injection in the tail vein 1 hour after operation, while mice in the CLP+P-MSCs group received 2×10 5 P-MSCs infusion 1 hour after operation. Intestinal and blood specimens were collected from the mice in each group 24 hours after P-MSCs transplantation. Hematoxylin and eosin (HE) staining of the intestinal tissue was performed for pathological evaluation. The serum concentrations of D-lactic acid, diamine oxidase (DAO), endotoxin, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and transforming growth factor (TGF)-β were determined by enzyme linked immunosorbent assay (ELISA). The gene expression of the relevant inflammatory factors in the small intestinal tissue was determined by real-time fluorescence polymerase chain reaction. The expression of zonula occludens protein-1 (ZO-1) and occludin protein in the intestine was determined by Western blot, the infiltration of intestinal macrophages was determined by immunohistochemical method, and the polarization of macrophages was determined by immunofluorescence. The exogenous transplantation of P-MSCs could form colonies in the injured intestines of septic mice. Compared with those of the CLP group, the intestinal injury of the CLP+P-MSCs group was significantly alleviated, the serum concentrations of D-lactic acid, DAO, endotoxin, IL-1β, IL-6, and TNF-α were significantly decreased ( P<0.05), while the serum concentrations of IL-10 and TGF-β were significantly increased ( P<0.05), the expression levels of IL-1 β, TNF-α and IL-6 genes in the intestinal tissue were significantly decreased ( P<0.05), while the expression levels of IL-10 and TGF-β genes were significantly increased ( P<0.05), and the expression of ZO-1 and occludin proteins in the intestine was also significantly increased ( P<0.05). In addition, the distribution of macrophages in the intestinal tissue of the CLP+P-MSCs group decreased significantly and the macrophages showed a tendency for M2 polarization. Exogenous transplantation of P-MSCs can significantly reduce inflammatory injury and improve the intestinal barrier function in septic mice with intestinal injury. Reduction in the infiltration of macrophages and promotion of the polarization of macrophages from M1 to M2 may be the mechanisms underlying the reduction of inflammation.

Berberine Alleviates Acute Lung Injury in Septic Mice by Modulating Treg/Th17 Homeostasis and Downregulating NF-κB Signaling.

A common complication of sepsis is acute lung injury (ALI), which is associated with an acute onset, rapid disease changes, and high mortality. Regulatory T (Treg) and T helper 17 (Th17) cells comprise CD4+ T cell subsets, which strongly influence inflammation during ALI. In this study, we investigated the effect of berberine (BBR), an antioxidant, anti-inflammatory, and immunomodulatory drug, on the inflammatory response and immune state in mice with sepsis. A mouse model of cecal ligation and puncture (CLP) was established. The mice were intragastrically administered 50 mg/kg BBR. We used histological techniques to evaluate inflammatory tissue injury and flow cytometry for analyzing Treg/Th17 levels. We also assessed NF-κB signaling pathways by Western blotting assays and immunofluorescence staining. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the content of cytokines. Treatment with BBR considerably mitigated lung injury while improving survival, post-cecal ligation, and puncture (CLP). Treatment with BBR ameliorated pulmonary edema and hypoxemia in septic mice and inhibited the NF-κB signaling pathway. BBR also increased Treg cells and decreased Th17 proportions in the spleen and lung tissue of CLP-treated mice. Blocking Treg cells weakened the protective effect of BBR on sepsis-associated lung injury. Overall, these results suggested that BBR is a potential therapeutic agent for sepsis.

Lycorine protects against septic myocardial injury by activating AMPK-related pathways.

Cardiac dysfunction is a common complication in patients with sepsis triggering high morbidity and mortality. Lycorine (LYC), the main effective monomer component extracted from Lycoris bulbs, possesses antiviral, anti-inflammatory, analgesic, liver protection properties. In this study, the effect of LYC pre- and post-treatment as well as the underlying mechanism were evaluated in the cecal ligation and puncture (CLP) model of Balb/c mice. The survival rate, anal temperature, sepsis score, blood biochemical/routine indicators, cardiac function, sepsis-related pathophysiological processes, and AMPK signaling in septic mice were observed by echocardiography, histological staining, western blot, qPCR, and etc. LYC pretreatment attenuated myocardial injury in septic mice by improving survival rate, sepsis score, blood biochemical/routine indicators, cardiac function and structure, inhibiting inflammation and oxidative stress, improving mitochondrial function, modulating endoplasmic reticulum stress, and activating AMPK pathway. In particular, AMPK deficiency and AMPK inhibitor (Compound C) partially reversed the protective effects of LYC in septic mice. In addition, LYC posttreatment also has slight protective phenotypes on septic myocardial injury, but the effect is not as ideal as pretreatment. Taken together, these findings suggest that LYC may be a potential drug for the treatment of sepsis.

RBM3 is associated with acute lung injury in septic mice and patients via the NF-κB/NLRP3 pathway.

Sepsis refers to host response disorders caused by infection, leading to life-threatening organ dysfunction. RNA-binding motif protein 3 (RBM3) is an important cold-shock protein that is upregulated in response to mild hypothermia or hypoxia. In this study, we aimed to investigate whether RBM3 is involved in sepsis-associated acute lung injury (ALI). Intraperitoneal injection of LPS (10mg/kg) was performed in wild type (WT) and RBM3 knockout (KO, RBM3-/-) mice to establish an in vivo sepsis model. An NLRP3 inflammasome inhibitor, MCC950 (50mg/kg), was injected intraperitoneally 30min before LPS treatment. Serum, lung tissues, and BALF were collected 24h later for further analysis. In addition, we also collected serum from sepsis patients and healthy volunteers to detect their RBM3 expression. The results showed that the expression of RBM3 in the lung tissues of LPS-induced sepsis mice and the serum of patients with sepsis was significantly increased and positively correlated with disease severity. In addition, RBM3 knockout (KO) mice had a low survival rate, and RBM3 KO mice had more severe lung damage, inflammation, lung cell apoptosis, and oxidative stress than WT mice. LPS treatment significantly increased the levels of nucleotide binding and oligomerization domain-like receptor family 3 (NLRP3) inflammasomes and mononuclear cell nuclear factor-κB (NF-κB) in the lung tissues of RBM3 KO mice. However, these levels were only slightly elevated in WT mice. Interestingly, MCC950 improved LPS-induced acute lung injury in WT and RBM3 KO mice but inhibited the expression of NLRP3, caspase-1, and IL-1β. In conclusion, RBM3 was overexpressed in sepsis patients and LPS-induced mice. RBM3 gene deficiency aggravated sepsis-associated ALI through the NF-κB/NLRP3 pathway.

Neutrophil Extracellular Traps Induce Alveolar Macrophage Pyroptosis by Regulating NLRP3 Deubiquitination, Aggravating the Development of Septic Lung Injury.

Uncontrolled inflammation is a typical feature of sepsis-related lung injury. The key event in the progression of lung injury is Caspase-1-dependent alveolar macrophage (AM) pyroptosis. Similarly, neutrophils are stimulated to release neutrophil extracellular traps (NETs) to participate in the innate immune response. This study aims to illustrate the specific mechanisms by which NETs activate AM at the post-translational level and maintain lung inflammation. We established a septic lung injury model by caecal ligation and puncture. We found elevated NETs and interleukin-1b (IL-1β) levels in the lung tissues of septic mice. Western blot and immunofluorescence analyses was utilized to determine whether NETs promote AM pyroptosis and whether degrading NETs or targeting the NLRP3 inflammasome had protective effects on AM pyroptosis and lung injury. Flow cytometric and co-immunoprecipitation analysesverified intracellular reactive oxygen species (ROS) levels and the binding of NLRP3 and ubiquitin (UB) molecules, respectively. Increased NETs production and IL-1β release in septic mice were correlated with the degree of lung injury. NETs upregulated the level of NLRP3, followed by NLRP3 inflammasome assembly and caspase-1 activation, leading to AM pyroptosis executed by the activated fragment of full-length gasdermin D (FH-GSDMD). However, the opposite effect was observed in the context of NETs degradation. Furthermore, NETs markedly elicited an increase in ROS, which facilitated the activation of NLRP3 deubiquitination and the subsequent pyroptosis pathway in AM. Removal of ROS could promote the binding of NLRP3 and ubiquitin, inhibit NLRP3 binding to apoptosis-associated spotted proteins (ASC) and further alleviate the inflammatory changes in the lungs. In summary, these findings indicate that NETs prime ROS generation, which promotes NLRP3 inflammasome activation at the post-translational level to mediate AM pyroptosis and sustain lung injury in septic mice.

High Concentration Hydrogen Mitigates Sepsis-Induced Acute Lung Injury in Mice by Alleviating Mitochondrial Fission and Dysfunction.

Background: Multiple organ failure (MOF) is the main cause of early death in septic shock. Lungs are among the organs that are affected in MOF, resulting in acute lung injury. A large number of inflammatory factors and stress injury in sepsis can lead to alterations in mitochondrial dynamics. Numerous studies have confirmed that hydrogen can alleviate sepsis in the animal model. The purpose of this experiment was to explore the therapeutic effect of high concentration (67%) hydrogen on acute lung injury in septic mice and its mechanism. Methods: The moderate and severe septic models were prepared by cecal ligation and puncture. Hydrogen with different concentrations was inhaled for one hour at 1 h and 6 h after the corresponding surgery. The arterial blood gas of mice during hydrogen inhalation was monitored in real time, and the 7-day survival rate of mice with sepsis was recorded. The pathological changes of lung tissues and functions of livers and kidneys were measured. The changes of oxidation products, antioxidant enzymes and pro-inflammatory cytokines in lungs and serums were detected. Mitochondrial function was measured. Results: The inhalation of 2% or 67% hydrogen improves the 7-day survival rate and reduces acute lung injury as well as liver and kidney injury in sepsis. The therapeutic effect of 67% hydrogen inhalation on sepsis was related to increasing antioxidant enzyme activity, reducing oxidation products and pro-inflammatory cytokines in lungs and serums. Compared with the Sham group, mitochondrial dysfunction was alleviated in hydrogen groups. Conclusions: Hydrogen inhalation by high or low concentration can both significantly improve sepsis; however, a high concentration demonstrates a better protective effect. High concentration hydrogen inhalation can significantly improve the mitochondrial dynamic balance and reduce the lung injury in septic mice.

Low-Dose Colchicine Attenuates Sepsis-Induced Liver Injury: A Novel Method for Alleviating Systemic Inflammation.

Sepsis is a significant public health challenge. The immune system underlies the pathogenesis of the disease. The liver is both an active player and a target organ in sepsis. Targeting the gut immune system using low-dose colchicine is an attractive method for alleviating systemic inflammation in sepsis without inducing immunosuppression. The present study aimed to determine the use of low-dose colchicine in LPS-induced sepsis in mice. C67B mice were injected intraperitoneal with LPS to induce sepsis. The treatment group received 0.02mg/kg colchicine daily by gavage. Short and extended models were performed, lasting 3 and 5days, respectively. We followed the mice for biochemical markers of end-organ injury, blood counts, cytokine levels, and liver pathology and conducted proteomic studies on liver samples. Targeting the gut immune system using low-dose colchicine improved mice's well-being measured by the murine sepsis score. Treatment alleviated the liver injury in septic mice, manifested by a significant decrease in their liver enzyme levels, including ALT, AST, and LDH. Treatment exerted a trend to reduce creatinine levels. Low-dose colchicine improved liver pathology, reduced inflammation, and reduced the pro-inflammatory cytokine TNFα and IL1-β levels. A liver proteomic analysis revealed low-dose colchicine down-regulated sepsis-related proteins, alpha-1 antitrypsin, and serine dehydratase. Targeting the gut immune system using low-dose colchicine attenuated liver injury in LPS-induced sepsis, reducing the pro-inflammatory cytokine levels. Low-dose colchicine provides a safe method for immunomodulation for multiple inflammatory disorders.

Semaglutide Pretreatment Induces Cardiac Autophagy to Reduce Myocardial Injury in Septic Mice.

Sepsis-induced myocardial dysfunction (SIMD) confers substantial morbidity and mortality. Semaglutide treatment has demonstrated efficacy in ameliorating sepsis-related organ damage via attenuation of inflammation, oxidative stress, and apoptotic cell death. In this study, we constructed a mouse SIMD model using cecal ligation and puncture (CLP) to explore whether semaglutide preconditioning can modulate autophagy levels and attenuate myocardial injury. C57BL/6 mice were randomly divided into six groups: sham, CLP (including CLP-6 h, CLP-12 h and CLP-24 h subgroups), semaglutide, and semaglutide+Compound-C, with five mice in each group. The latter two groups were given daily intraperitoneal injections of semaglutide for 14 days. The semaglutide+Compound-C group was given the autophagy inhibitor Compound-C intraperitoneally 1-hour before CLP surgery. After the last injection of semaglutide, SIMD mouse models were constructed by CLP surgery, while the sham group underwent a sham operation. All mice were sacrificed after surgery, and blood and myocardial specimens were collected. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory factors and myocardial injury markers in the serum, while quantitative real-time polymerase chain reaction (qRT-PCR) and western blot was used to detect the expression of autophagic markers [microtubule-associated protein 1A/1B-light chain 3B (LC3B), Beclin-1, p62] and AMP-activated protein kinase (AMPK) in myocardial tissue. Hematoxylin and eosin (H&E) staining was used to observe pathological changes in myocardial tissue. The myocardial fibers in the sham group were normal, while those in the CLP group showed disordered arrangement, interstitial edema, and a large number of infiltrating inflammatory cells. A few vacuolar changes were observed locally in the semaglutide group, and more vacuolar changes were observed in the semaglutide+Compound-C group. Autophagy was inhibited in the CLP group mice. Compared with the CLP group, the semaglutide group showed a decreased levels of inflammatory factors (tumor necrosis factor-α, interleukin-1β) and myocardial injury markers (creatine kinase isoenzyme, cardiac troponin T) in the serum, a reduced expression of autophgic substrate p62, and an increased expression of LC3II (the lipidated form of LC3I)/LC3I (microtubule-associated protein 1A/1B-light chain 3), Beclin-1, and p-AMPK (phosphorylated AMP-activated protein kinase)/AMPK in the injured myocardial tissues of mice (p < 0.05). And the protective effects of semaglutide against SIMD were partially reversed by the treatment of AMPK inhibitor Compound-C (p < 0.05). Taken together, these data indicate that semaglutide provides protection against CLP-triggered myocardial inflammation and injury, potentially by reactivating myocardial autophagy pathways via activation of AMPK signaling. Further mechanistic studies are needed to definitively elucidate the functional significance of AMPK signaling in mediating the beneficial cardiac effects of semaglutide during sepsis.

Circular RNA protein tyrosine kinase 2 aggravates pyroptosis and inflammation in septic lung tissue by promoting microRNA-766/eukaryotic initiation factor 5A axis-mediated ATP efflux

Sepsis is characterized by an acute inflammatory response to infection, often with multiple organ failures, especially severe lung injury. This study was implemented to probe circular RNA (circRNA) protein tyrosine kinase 2 (circPTK2)-associated regulatory mechanisms in septic acute lung injury (ALI). A cecal ligation and puncture-based mouse model and an lipopolysaccharides (LPS)-based alveolar type II cell (RLE-6TN) model were generated to mimic sepsis. In the two models, inflammation- and pyroptosis-related genes were measured. The degree of lung injury in mice was analyzed by hematoxylin and eosin (H&E) staining and the apoptosis was by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. In addition, pyroptosis and toxicity were detected in cells. Finally, the binding relationship between circPTK2, miR-766, and eukaryotic initiation factor 5A (eIF5A) was detected. Data indicated that circPTK2 and eIF5A were up-regulated and miR-766 was down-regulated in LPS-treated RLE-6TN cells and lung tissue of septic mice. Lung injury in septic mice was ameliorated after inhibition of circPTK2. It was confirmed in the cell model that knockdown of circPTK2 effectively ameliorated LPS-induced ATP efflux, pyroptosis, and inflammation. Mechanistically, circPTK2 mediated eIF5A expression by competitively adsorbing miR-766. Taken together, circPTK2/miR-766/eIF5A axis ameliorates septic ALI, developing a novel therapeutic target for the disease.

Effects of Krüppel-like factor 4 on inflammatory response and organ injury in septic mice

Effects of Krüppel-like factor 4 on inflammatory response and organ injury in septic mice

Design and synthesis of hederagenin derivatives modulating STING/NF-κB signaling for the relief of acute liver injury in septic mice

Systemic inflammatory responses often result in sepsis and inhibition of inflammation is one strategy for sepsis treatment. In this study, we designed and synthesized 32 novel hederagenin (HD) derivatives with modifications at the A-ring, C-28, and C-23 positions and screened their anti-inflammatory activities in vitro, finding multiple compounds with potential anti-inflammatory activity. Of these, compound 1 was the most effective and was used for subsequent investigations into its mechanism of action and in vivo activity. In vivo assessments of anti-inflammatory activity showed that compound 1 reduced inflammation in a mouse model of sepsis with acute liver injury caused by lipopolysaccharide (LPS). Compound 1 also inhibited STING, p-IRF3, p-TBK1, p-p65, and p-IκB proteins in cGAS-STING-associated signaling. These findings indicated that compound 1 reduced inflammation through inhibition of STING expression and hence reducing activation of STING and nuclear factor-κB (NF-κB) signaling. Our work demonstrated that compound 1 is a promising lead compound for designing and developing anti-sepsis drugs.

Phosphorylation-mediated PI3K-Art signalling pathway as a therapeutic mechanism in the hydrogen-induced alleviation of brain injury in septic mice.

Our previous studies illustrated that 2% H2 inhalation can protect against sepsis-associated encephalopathy (SAE) which is characterized by high mortality and has no effective treatment. To investigate the underlying role of protein phosphorylation in SAE and H2 treatment, a mouse model of sepsis was constructed by caecal ligation and puncture (CLP), then treated with H2 (CLP + H2 ). Brain tissues of the mice were collected to be analysed with tandem mass tag-based quantitative proteomics coupled with IMAC enrichment of phosphopeptides and LC-MS/MS analysis. In proteomics and phosphoproteomics analysis, 268 differentially phosphorylated proteins (DPPs) showed a change in the phosphorylated form in the CLP + H2 group (p < 0.05). Gene ontology analysis revealed that these DPPs were enriched in multiple cellular components, biological processes, and molecular functions. KEGG pathway analysis revealed that they were enriched in glutamatergic synapses, tight junctions, the PI3K-Akt signalling pathway, the HIF-1 signalling pathway, the cGMP-PKG signalling pathway, the Rap1 signalling pathway, and the vascular smooth muscle contraction. The phosphorylated forms of six DPPs, including ribosomal protein S6 (Rps6), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (Ywhag/14-3-3), phosphatase and tensin homologue deleted on chromosome ten (Pten), membrane-associated guanylate kinase 1 (Magi1), mTOR, and protein kinase N2 (Pkn2), were upregulated and participated in the PI3K-Akt signalling pathway. The WB results showed that the phosphorylation levels of Rps6, Ywhag, Pten, Magi1, mTOR, and Pkn2 were increased. The DPPs and phosphorylation-mediated molecular network alterations in H2 -treated CLP mice may elucidate the biological roles of protein phosphorylation in the therapeutic mechanism of H2 treatment against SAE.

NAT10 regulates neutrophil pyroptosis in sepsis via acetylating ULK1 RNA and activating STING pathway

Emerging evidence suggests that pyroptosis is involved in sepsis. However, the role of neutrophil pyroptosis in sepsis and the mechanisms remains elusive. We find that N-acetyltransferase 10 (NAT10), an acetyltransferase responsible for the N4-acetylation of Cytidine (ac4C) in mRNA, is significantly downregulated in neutrophils from septic mice. Neutrophil-specific over-expression of NAT10 improves the survival and ameliorates lung injury in septic mice by inhibiting neutrophil pyroptosis. Notably, UNC-52-like kinase 1 (ULK1) is identified as the target of NAT10 in neutrophils. The decreased expression of NAT10 resultes in the decay of ULK1 transcripts and therefore the reduced expression of ULK1. As a regulator of STING phosphorylation, the loss of ULK1 enhances the activation of STING-IRF3 signaling and subsequently the elevated pyroptosis-inducing NLRP3 inflammasome in neutrophils. While over-expression of NAT10 restrains pyroptosis in neutrophils as well as septic lethality in mice by reversing the ULK1-STING-NLRP3 axis. The decreased expression of NAT10 are also observed in sepsis patients and its correlation with clinical severity is found. Collectively, our findings disclose that NAT10 is a negative regulator of neutrophil pyroptosis and its downregulation contributes to the progress of sepsis by exacerbating pyroptosis via the ULK1-STING-NLRP3 axis, therefore revealing a potential therapeutic target for sepsis.