Apoptosis In Sepsis Research Articles

Identification of apoptosis-immune-related gene signature and construction of diagnostic model for sepsis based on single-cell sequencing and bulk transcriptome analysis.

Sepsis leads to multi-organ dysfunction due to disorders of the host response to infections, which makes diagnosis and prognosis challenging. Apoptosis, a classic programmed cell death, contributes to the pathogenesis of various diseases. However, there is much uncertainty about its mechanism in sepsis. Three sepsis gene expression profiles (GSE65682, GSE13904, and GSE26378) were downloaded from the Gene Expression Omnibus database. Apoptosis-related genes were obtained from the Kyoto Encyclopedia of Genes and Genomes Pathway database. We utilized LASSO regression and SVM-RFE algorithms to identify characteristic genes associated with sepsis. CIBERSORT and single cell sequencing analysis were employed to explore the potential relationship between hub genes and immune cell infiltration. The diagnostic capability of hub genes was validated across multiple external datasets. Subsequently, the animal sepsis model was established to assess the expression levels of hub genes in distinct target organs through RT-qPCR and Immunohistochemistry analysis. We identified 11 apoptosis-related genes as characteristic diagnostic markers for sepsis: CASP8, VDAC2, CHMP1A, CHMP5, FASLG, IFNAR1, JAK1, JAK3, STAT4, IRF9, and BCL2. Subsequently, a prognostic model was constructed using LASSO regression with BCL2, FASLG, IRF9 and JAK3 identified as hub genes. Apoptosis-related genes were closely associated with the immune response during the sepsis process. Furthermore, in the validation datasets, aside from IRF9, other hub genes demonstrated similar expression patterns and diagnostic abilities as observed in GSE65682 dataset. In the mouse model, the expression differences of hub genes between sepsis and control group revealed the potential impacts on sepsis-induced organ injury. The current findings indicated the participant of apoptosis in sepsis, and apoptosis-related differentially expressed genes could be used for diagnosis biomarkers. BCL2, FASLG, IRF9 and JAK3 might be key regulatory genes affecting apoptosis in sepsis. Our findings provided a novel aspect for further exploration of the pathological mechanisms in sepsis.

NUFIP1-engineered exosomes derived from hUMSCs regulate apoptosis and neurological injury induced by propofol in newborn rats

BackgroundPropofol can increase neurotoxicity in infants but the precise mechanism is still unknown. Our previous study revealed that nuclear FMR1 interacting protein 1 (NUFIP1), a specific ribophagy receptor, can alleviate T cell apoptosis in sepsis. Yet, the effect of NUFIP1-engineered exosomes elicited from human umbilical cord blood mesenchymal stem cells (hUMSCs) on nerve injury induced by propofol remains unclear. This study intended to investigate the effect of NUFIP1-engineered exosomes on propofol-induced nerve damage in neonatal rats. MethodsFirstly, NUFIP1-engineered exosomes were extracted from hUMSCs serum and their identification was conducted using transmission electron microscopy (TEM), Flow NanoAnalyzer, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB). Subsequently, the optimal exposure duration and concentration of propofol induced apoptosis were determined in SH-SY5Y cell line using WB. Following this, we co-cultured the NUFIP1-engineered exosomes in the knockdown group (NUFIP1-KD) and overexpression group (NUFIP1-OE) with SH-SY5Y cells and assessed their effects on the apoptosis of SH-SY5Y cells using terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay, Hoechst 33258 staining, WB, and flow cytometry, respectively. Finally, NUFIP1-engineered exosomes were intraperitoneally injected into neonatal rats, and their effects on the learning and memory ability of neonatal rats were observed through the righting reflex and Morris water maze (MWM) test. Hippocampi were extracted from different groups for hematoxylin-eosin (HE) staining, immunohistochemistry, immunofluorescence, and WB to observe their effects on apoptosis in neonatal rats. ResultsTEM, Flow NanoAnalyzer, qRT-PCR, and WB analyses confirmed that the exosomes extracted from hUMSCs serum exhibited the expected morphology, diameter, surface markers, and expression of target genes. This confirmed the successful construction of NUFIP1-KD and NUFIP1-OE-engineered exosomes. Optimal exposure duration and concentration of propofol were determined to be 24 hours and 100 µg/ml, respectively. Co-culture of NUFIP1 engineered exosomes and SH-SY5Y cells resulted in significant up-regulation of pro-apoptotic proteins Bax and c-Caspase-3 in the KD group, while anti-apoptotic protein Bcl-2 was significantly decreased. The OE group showed the opposite trend. TUNEL apoptosis assay, Hoechst 33258 staining, and flow cytometry yielded consistent results. Animal experiments demonstrated that intraperitoneal injection of NUFIP1-KD engineered exosomes prolonged the righting reflex recovery time of newborn rats, and MWM tests revealed a significant diminution in the time and number of newborn rats entering the platform. HE staining, immunohistochemistry, immunofluorescence, and WB results also indicated a significant enhancement in apoptosis in this group. Conversely, the experimental results of neonatal rats in the OE group revealed a certain degree of anti-apoptotic effect. ConclusionsNUFIP1-engineered exosomes from hUMSCs have the potential to regulate nerve cell apoptosis and mitigate neurological injury induced by propofol in neonatal rats. Targeting NUFIP1 may hold great significance in ameliorating propofol-induced nerve injury.

EXTRACELLULAR CIRP INHIBITS NEUTROPHIL APOPTOSIS TO PROMOTE ITS AGING BY UPREGULATING SERPINB2 IN SEPSIS.

Background: Sepsis reduces neutrophil apoptosis. As the result, neutrophils may become aged, exacerbating inflammation and tissue injury. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern to promote inflammation and tissue injury in sepsis. SerpinB2, a serine protease inhibitor, has been shown to inhibit apoptosis. We hypothesize that eCIRP upregulates SerpinB2 to promote aged neutrophil subset by inhibiting apoptosis in sepsis. Methods: We stimulated bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice with 1 μg/mL of recombinant mouse CIRP (i.e., eCIRP) and assessed cleaved caspase-3 and SerpinB2 by western blotting. Apoptotic neutrophils were assessed by Annexin V/PI. Bone marrow-derived neutrophils were stimulated with 1 μg/mL eCIRP and treated with or without PAC-1 (caspase-3 activator) and aged neutrophils (CXCR4 hi CD62L lo ) were assessed by flow cytometry. To induce sepsis, we performed cecal ligation and puncture in WT or CIRP -/- mice. We determined the percentage of aged neutrophils and SerpinB2 + neutrophils in blood and spleen by flow cytometry. Results: We found that cleaved caspase-3 levels were increased at 4 h of PBS treatment compared with 0 h but decreased by eCIRP treatment. Extracellular cold-inducible RNA-binding protein reduced apoptotic cells after 20 h of treatment. Extracellular cold-inducible RNA-binding protein also increased the frequencies of aged neutrophils compared with PBS after 20 h, while PAC-1 treatment reduced aging in eCIRP-treated BMDNs. Extracellular cold-inducible RNA-binding protein significantly increased the expression of SerpinB2 at protein levels in BMDNs at 20 h. In WT mice, the frequencies of aged and SerpinB2 + neutrophils in blood and spleen were increased after 20 h of cecal ligation and puncture, while in CIRP -/- mice, aged and SerpinB2 + neutrophils were significantly decreased compared with WT mice. We also found that aged neutrophils expressed significantly higher levels of SerpinB2 compared with non-aged neutrophils. Conclusions: eCIRP inhibits neutrophil apoptosis to increase aged phenotype by increasing SerpinB2 expression in sepsis. Thus, targeting eCIRP could be a new therapeutic strategy to ameliorate inflammation caused by neutrophil aging in sepsis.

Ginkgolic acid promotes inflammation and macrophage apoptosis via SUMOylation and NF-κB pathways in sepsis.

Excessive inflammation and increased apoptosis of macrophages contribute to organ damage and poor prognosis of sepsis. Ginkgolic acid (GA) is a natural constituent extracted from the leaves of Ginkgo biloba, that can regulate inflammation and apoptosis. The present study aims to investigate the potential effect of GA in treating sepsis and its possible mechanisms. Here, a classic septic mice model and a lipopolysaccharide (LPS)-induced RAW 264.7 inflammation model were established. Cytokines in serum and culture supernatant were detected by ELISA, and the mRNA levels of them were examined by PCR. Hematoxylin and eosin (H&E) staining was performed to determine histopathological changes in liver, lung and kidney. Bacterial burden in the blood, peritoneal lavage fluids (PLFs) and organs were observed on Luria-Bertani agar medium. Flow cytometry and western blotting was used to detect apoptosis and the expression level of apoptosis related molecules, respectively. Moreover, the levels of SUMOylation were detected by western blotting. The activity of NF-κB p65 was assessed by immunofluorescence staining and western blotting. The result showed that GA promoted inflammatory responses, reduced bacterial clearance, aggravated organ damage, and increased mortality in septic mice. GA increased apoptosis in peritoneal macrophages (PMs) and RAW 264.7 cells. Meanwhile, GA inhibited SUMOylation and increased the nuclear translocation of NF-κB p65 as well as its phosphorylation level. Collectively, GA promotes inflammation and macrophage apoptosis in sepsis, which may be mediated by inhibiting the SUMOylation process and increasing NF-κB p65 activity.

Nuclear fragile X mental retardation-interacting protein 1-mediated ribophagy protects T lymphocytes against apoptosis in sepsis.

Ribophagy is a selective autophagic process that specifically degrades dysfunctional or superfluous ribosomes to maintain cellular homeostasis. Whether ribophagy can ameliorate the immunosuppression in sepsis similar to endoplasmic reticulum autophagy (ERphagy) and mitophagy remains unclear. This study was conducted to investigate the activity and regulation of ribophagy in sepsis and to further explore the potential mechanism underlying the involvement of ribophagy in T-lymphocyte apoptosis. The activity and regulation of nuclear fragile X mental retardation-interacting protein 1 (NUFIP1)-mediated ribophagy in T lymphocytes during sepsis were first investigated by western blotting, laser confocal microscopy and transmission electron microscopy. Then, we constructed lentivirally transfected cells and gene-defective mouse models to observe the impact of NUFIP1 deletion on T-lymphocyte apoptosis and finally explored the signaling pathway associated with T-cell mediated immune response following septic challenge. Both cecal ligation and perforation-induced sepsis and lipopolysaccharide stimulation significantly induced the occurrence of ribophagy, which peaked at 24h. When NUFIP1 was knocked down, T-lymphocyte apoptosis was noticeably increased. Conversely, the overexpression of NUFIP1 exerted a significant protective impact on T-lymphocyte apoptosis. Consistently, the apoptosis and immunosuppression of T lymphocytes and 1-week mortality rate in NUFIP1 gene-deficient mice were significantly increased compared with those in wild-type mice. In addition, the protective effect of NUFIP1-mediated ribophagy on T lymphocytes was identified to be closely related to the endoplasmic reticulum stress apoptosis pathway, and PERK-ATF4-CHOP signaling was obviously involved in downregulating T-lymphocyte apoptosis in the setting of sepsis. NUFIP1-mediated ribophagy can be significantly activated to alleviate T lymphocyte apoptosis through the PERK-ATF4-CHOP pathway in the context of sepsis. Thus, targeting NUFIP1-mediated ribophagy might be of importance in reversing the immunosuppression associated with septic complications.

MTOR pathway mediates endoplasmic reticulum stress-induced CD4+ T cell apoptosis in septic mice

Endoplasmic reticulum stress (ERS) has been well documented to participate in the pathophysiological processes of apoptosis in many diseases. Inhibition of ERS ameliorates pathological organ injury. However, the upstream signaling pathways and molecular regulatory mechanisms of which are still unknown. mTOR, an evolutionarily conserved protein kinase, is a key regulator of apoptosis. Hence, in this study, a classical cecal ligation and puncture (CLP) sepsis model was constructed by using the T cell-specific knockout mTOR and TSC1 (Tuberous Sclerosis Complex, the inhibitor of mTOR signaling pathway) mice to explore the underlying signaling pathway and molecular mechanism of host immune imbalance caused by apoptosis in sepsis. We found that mTOR may modulate septic T cell apoptosis by regulating Akt–IRE1–JNK pathway. To further clarify the possible mechanism, the specific inhibitors of PI3K-Akt and IRE1–JNK were used to intervene in mice before/after CLP, respectively. By analyzing the proteins of mTOR-ERS signaling pathway and the expression of apoptosis-related proteins and genes, we found that mTOR mediated the ER stress induced CD4+ T cell apoptosis in Septic mice by negatively regulating the Akt–IRE1–JNK-Caspase 3 signaling cascades. These results indicate that mTOR–Akt–IRE1α–JNK signaling pathway mediated the Endoplasmic reticulum stress induced CD4+ T cell apoptosis in Septic mice.

Role of Thrombospondin‑1 in sepsis‑induced myocardial injury.

Sepsis often causes myocardial injury with a high mortality. The aim of the present study was to investigate the effects of thrombospondin‑1(THBS1) on myocardial cell injury, oxi‑dative stress and apoptosis in sepsis. The expression of THBS1 mRNA in lipopolysaccharide(LPS)‑induced mouse primary cardiomyocytes was detected by reverse transcription‑quantitative PCR(RT‑qPCR). A eukaryotic small interfering(si)RNA expression vector was constructed and transfected into myocardial cells to knockdown THBS1 mRNA expression, which was confirmed by RT‑qPCR. Four in vitro experimental groups were used: i)Control, ii)LPS, iii)THBS1 siRNA(siTHBS1) and iv)siTHBS+LPS. ELISA was used to detect cardiac troponinI(cTnI), pro‑brain natriuretic peptide(proBNP), reactive oxygen species(ROS), caspase‑3, IL‑6 and TNF‑α. Invivo mouse sepsis models were also established, and H&E, TUNEL and caspase‑3 staining were used to evaluate myocardial cell injury and apoptosis. Clinical samples were collected to analyze the serum THBS1levels and to associate this with the prognosis of patients with sepsis‑induced myocardial injury. The expression level of THBS1 mRNA in myocardial cells induced by LPS was increased, and the serum THBS1level in patients with myocardial injury in sepsis was also significantly increased compared with patients without sepsis‑induced myocardial injury. In the siTHBS1‑treated mice with myocardial injury, the levels of cTnI and proBNP were significantly decreased, the levels of the inflammatory cytokinesIL‑6 and TNF‑α were significantly decreased, ROS were significantly decreased, caspase‑3 was significantly decreased, and myocardial cell apoptosis was also reduced, compared with the LPSgroup. Data from the present study suggested that THBS1 may be closely related to the biological behavior of myocardial cells and may be a therapeutic target for myocardial injury in sepsis.

Increased Death of Peripheral Blood Mononuclear Cells after TLR4 Inhibition in Sepsis Is Not via TNF/TNF Receptor-Mediated Apoptotic Pathway

Background Apoptosis is one of the causes of immune depression in sepsis. Pyroptosis also occurs in sepsis. The toll-like receptor (TLR) 4 and receptor for advanced glycation end products (RAGE) have been shown to play important roles in apoptosis and pyroptosis. However, it is still unknown whether TLR4 inhibition decreases apoptosis in sepsis. Methods Stimulated peripheral blood mononuclear cells (PBMCs) with or without lipopolysaccharides (LPS) and high-mobility group box 1 (HMGB1) were cultured with or without TLR4 inhibition using monoclonal antibodies from 20 patients with sepsis. Caspase-3, caspase-8, and caspase-9 activities were measured. The expression of B cell lymphoma 2 (Bcl2) and Bcl2-associated X (Bax) was measured. The cell death of PBMCs was detected using a flow cytofluorimeter. Results After TLR4 inhibition, Bcl2 to Bax ratio elevated both in LPS and HMGB1-stimulated PBMCs. The activities of caspase-3, caspase-8, and caspase-9 did not change in LPS or HMGB1-stimulated PBMCs. The cell death of LPS and HMGB1-stimulated CD8 lymphocytes and monocytes increased after TLR4 inhibition. The cell death of CD4 lymphocytes was unchanged. Conclusion The apoptosis did not decrease, while TLR4 was inhibited. After TLR4 inhibition, there was an unknown mechanism to keep cell death in stimulated PBMCs in patients with sepsis.

Rosmarinic acid ameliorates septic-associated mortality and lung injury in mice via GRP78/IRE1α/JNK pathway.

Acute lung injury (ALI) is the major complication of sepsis, and no effective treatment is available now. Recently, rosmarinic acid (RA), a water-soluble polyphenolic phytochemical, exerts a potential role on ALI with anti-inflammation, and antioxidant properties. However, there is still no evidence on its protective effect on cell apoptosis in sepsis. Here, we investigated the protective effect of RA in septic-associated mortality and lung injury based on apoptosis. Male C57BL/6 mice were administered with lipopolysaccharide (LPS) (15 mg/kg, ip) to establish ALI mice model. Preteatment of RA (20 or 40 mg/kg, ip) was performed once daily for five consecutive days. The mortality was monitored for seven days after injection of LPS. RA (40 mg/kg) significantly decreased mortality and alleviated septic-associated lung injury. Meanwhile, RA significantly reversed LPS induced decrease in serum T-aoc level and superoxide dismutase (SOD) activity, and increase in malondialdehyde (MDA) activity. Furthermore, RA pretreatment significantly inhibited lung cell apoptosis, as well as decreased p53 level in sepsis mice. Finally, the LPS induced activation of GRP78/IRE1α/JNK pathway was suppressed by RA pretreatment. These findings indicated that RA could be beneficial to septic-associated lung injury through anti-apoptosis effect.

MicroRNA-181b Inhibits Inflammatory Response and Reduces Myocardial Injury in Sepsis by Downregulating HMGB1.

MicroRNAs (miRNAs) are short endogenous noncoding RNAs regulating protein translation. However, the specific mechanism by which miR-181b influences sepsis via high-mobility group box-1 protein (HMGB1) still remains unknown. Thus, the aim of this study is to investigate the mechanism of miR-181b in regulating inflammatory response in sepsis-induced myocardial injury through targeting high-mobility group box-1 protein (HMGB1). Through cecal ligation and puncture (CLP), the rat model of sepsis was established. Then, the effect of altered expression of miR-181b and HMGB1 on cardiomyocytes was investigated. The positive expression rate of HMGB1, concentration of inflammatory factors, and serum myocardial enzyme of myocardial tissues were determined. Besides, the binding site between miR-181b and HMGB1 was determined by bioinformatics information and dual-luciferase reporter gene assay. The expression of related genes in cells of each group was determined by RT-qPCR and western blot analysis, and the apoptosis rate of transfected cells in each group was determined by TUNEL assay. HMGB1 expression and inflammatory factors were significantly increased in myocardial tissue of rats with sepsis. Cell morphology and the infiltration of inflammatory cells were significantly improved by overexpression of miR-181b. miR-181b directly targeted HMGB1, and downregulation of HMGB1 reduced inflammatory factors and myocardial injury and inhibited cardiomyocyte apoptosis in sepsis. This present study suggests that miR-181b decreased inflammatory factors and reduced myocardial injury in sepsis through downregulation of HMGB1. Thus, a better understanding of this process may aid in the development of novel therapeutic agents in sepsis.

VSIG4(+) peritoneal macrophages induce apoptosis of double-positive thymocyte via the secretion of TNF-\u03b1 in a CLP-induced sepsis model resulting in thymic atrophy

Thymic atrophy in sepsis is a critical disadvantage because it induces immunosuppression and increases the mortality rate as the disease progresses. However, the exact mechanism of thymic atrophy has not been fully elucidated. In this study, we discovered a novel role for VSIG4-positive peritoneal macrophages (V4(+) cells) as the principal cells that induce thymic atrophy and thymocyte apoptosis. In CLP-induced mice, V4(+) cells were activated after ingestion of invading microbes, and the majority of these cells migrated into the thymus. Furthermore, these cells underwent a phenotypic shift from V4(+) to V4(−) and from MHC II(low) to MHC II(+). In coculture with thymocytes, V4(+) cells mainly induced apoptosis in DP thymocytes via the secretion of TNF-α. However, there was little effect on CD4 or CD8 SP and DN thymocytes. V4(−) cells showed low levels of activity compared to V4(+) cells. Thymic atrophy in CLP-induced V4(KO) mice was much less severe than that in CLP-induced wild-type mice. In addition, V4(KO) peritoneal macrophages also showed similar activity to V4(−) cells. Taken together, the current study demonstrates that V4(+) cells play important roles in inducing immunosuppression via thymic atrophy in the context of severe infection. These data also suggest that controlling the function of V4(+) cells may play a crucial role in the development of new therapies to prevent thymocyte apoptosis in sepsis.

Mesenchymal stem cells-derived extracellular vesicles carrying microRNA-17 inhibits macrophage apoptosis in lipopolysaccharide-induced sepsis

ObjectiveSepsis, as a disease affecting the microcirculation and tissue perfusion, results in tissue hypoxia and multiple organ dysfunctions. Bone mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) have been demonstrated to transfer trivial molecules (proteins/peptides, mRNA, microRNA and lipids) to alleviate sepsis. We sought to define the function of microRNA (miR)-17 carried in BMSC-EVs in sepsis. MethodsThe purity of the extracted BMSCs was identified and confirmed by detection of the surface markers by flow cytometry, followed by osteoblastic, adipogenic, and chondrocyte differentiation experiments. Subsequently, EVs were collected from the medium of BMSCs. The uptake of PKH-67-labeled BMSC-EVs or EVs carrying cy3-miR-17 by RAW264.7 cells was observed under laser confocal microscopy. Furthermore, a series of gain- and loss-of-function approaches were conducted to test the effects of LPS, miR-17 and BRD4 on the inflammatory factors (IL-1β, IL-6 and TNF-α), number of M1 macrophages and M2 macrophages, inflammatory-related signal pathway factors (EZH2, c-MYC and TRAIL), macrophage proliferation, and apoptosis in sepsis. The survival rates were measured in vivo. ResultsBMSC-EVs was internalized by the RAW264.7 cells. BDR4 was verified as a target of miR-17, while the expression pattern of miR-17 was upregulated in BMSC-EVs. MiR-17 carried by BMSC-EVs inhibited LPS-induced inflammation and apoptosis of RAW264.7 cells, but improved the viability of RAW264.7 cells. Next, in vitro experiments supported that miR-17 inhibited LPS-induced inflammation in RAW264.7 cells through BRD4/EZH2/TRAIL axis. BRD4 overexpression reversed the effects of miR-17. Moreover, the therapeutic function of BMSC-EVs carried miR-17 was verified by in vivo experiments. ConclusionsMiR-17 derived from BMSCs-EVs regulates BRD4-mediated EZH2/TRAIL axis to essentially inhibit LPS-induced macrophages inflammation.

MiR‑1184 regulates inflammatory responses and cell apoptosis by targeting TRADD in an LPS‑induced cell model of sepsis

MicroRNAs (miRs) have been reported to be potential clinical biomarkers for sepsis. miR-1184 is a multifunctional microRNA that exerts roles in the development of various diseases. However, the role of miR-1184 in children with sepsis remain unknown. In the present study, THP-1 cells were stimulated with 1 µg/ml lipopolysaccharide (LPS) for 24 h to establish an in vitro sepsis model. Reverse transcription-quantitative PCR was used to evaluate the expression of miR-1184 in clinical specimens, and of IL-6, TNF-α, IL-1β, miR-1184 and TNF receptor type 1-associated DEATH domain protein (TRADD) in cells with and without LPS treatment. Cell apoptosis was assessed using flow cytometry. Binding between miR-1184 and TRADD was predicted using bioinformatics software, and a luciferase reporter assay was performed to verify the interaction between miR-1184 and TRADD in LPS-induced THP-1 cells. In addition, western blot analysis was performed to detect TRADD and proteins associated with the NF-κB pathway. The results showed that miR-1184 was downregulated in the blood of children with sepsis and LPS-induced THP-1 cells. Overexpression of miR-1184 alleviated the LPS-induced production of inflammatory cytokines and cell apoptosis. Moreover, TRADD was verified to be a direct target of miR-1184. Upregulation of TRADD reversed the effects of miR-1184 on the LPS-induced inflammatory response and apoptosis of THP-1 cells. Furthermore, the NF-κB pathway was shown to be associated with the regulatory role of miR-1184 in sepsis. The present study provides evidence that miR-1184 exerts inhibitory effects on inflammatory responses and apoptosis in sepsis by targeting TRADD, which suggests that miR-1184 may be a novel potential target for the therapy of children with sepsis.

The role of apoptosis in sepsis

Sepsis is a life-threatening entity caused by a multiorgan dysfunction that generates a dysregulated host response to infection and, after several processes, results in immunosuppression. Today, there are several sepsis management strategies to reduce its multisystemic effect and improve survival, but none of them have shown clear efficacy. Therefore, latest studies focus on clarifying and seeking therapeutic alternatives based on the analysis of the molecular pathophysiology of sepsis, in order to enter a late period of continuous immunosuppression also known as immunoparalysis.Apoptosis is a molecular and physiological mechanism, whose homeostasis is altered by the presence of sepsis. It causes the elimination of key cells of innate and adaptive immunity, which leads to an increased risk of—and often fatal— secondary infection. Several postmortem studies have confirmed that sepsis-induced immune cell apoptosis is a leading factor in the genesis of sepsis-related immunosuppression. It is believed that therapeutic strategies aimed at regulating apoptosis could improve survival. This review article describes the pathophysiological role of the apoptotic phenomenon in sepsis and its effect on the evolution of this entity

CD11c regulates hematopoietic stem and progenitor cells under stress.

β2 integrins are well-known leukocyte adhesion molecules consisting of 4 members: CD11a-d. Their known biological functions range widely from leukocyte recruitment, phagocytosis, to immunological synapse formation, but the studies have been primarily focused on CD11a and CD11b. CD11c is 1 of the 4 members and is extremely homologous to CD11b. It has been well known as a dendritic cell marker, but the characterization of its function has been limited. We found that CD11c was expressed on the short-term hematopoietic stem cells and multipotent progenitor cells. The lack of CD11c did not affect the number of hematopoietic stem and progenitor cells (HSPCs) in healthy CD11c knockout mice. Different from other β2 integrin members, however, CD11c deficiency was associated with increased apoptosis and significant loss of HSPCs in sepsis and bone marrow transplantation. Although integrins are generally known for their overlapping and redundant roles, we showed that CD11c had a distinct role of regulating the expansion of HSPCs under stress. This study shows that CD11c, a well-known dendritic cell marker, is expressed on HSPCs and serves as their functional regulator. CD11c deficiency leads to the loss of HSPCs via apoptosis in sepsis and bone marrow transplantation.

LncRNA LUADT1 sponges miR-195 to prevent cardiac endothelial cell apoptosis in sepsis

BackgroundThe oncogenic role of the newly identified lncRNA LUADT1 has been revealed in lung adenocarcinoma. It was reported that LUADT1 plays a critical role in multiple human diseases. This study was carried out to investigate the role of LUADT1 in sepsis.MethodsSixty patients with sepsis and sixty healthy volunteers were recruited for this study. Plasma samples were collected from all participants. Human primary coronary artery endothelial cells were also used in this study. The expression of Pim-1, miR-195 and LUADT1 were detected by RT-qPCR. The interaction between miR-195 and LUADT1 was determined by overexpression experiments and luciferase activity assay. Cell apoptosis was detected by flow cytometry. The expression of apoptosis-related protein was detected by Western blotting.ResultsBioinformatics analysis revealed the potential interaction between LUADT1 and miR-195, which was confirmed by dual luciferase reporter assay. LUADT1 was downregulated in patients with sepsis. Moreover, LPS treatment downregulated the expression of LUADT1 in primary cardiac endothelial cells. Overexpression of LUADT1 and miR-195 did not affect the expression of each other in primary cardiac endothelial cells. Interestingly, overexpression of LUADT1 was found to upregulate the expression of Pim-1, a target of miR-195. In addition, it was found that overexpression of LUADT1 and Pim-1 reduced the enhancement effects of miR-195 on LPS-induced cardiac endothelial cell apoptosis.ConclusionIn summary, LUADT1 may protect cardiac endothelial cells against apoptosis in sepsis by regulating the miR-195/Pim-1 axis.

CaMKII-dependent ryanodine receptor phosphorylation mediates sepsis-induced cardiomyocyte apoptosis.

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca2+ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca2+ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca2+ release leading to mitochondrial Ca2+ overload, mitochondrial Ca2+ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca2+ release from SR which leads to mitochondrial Ca2+ overload and apoptosis.

Effects of Lentinan on Endothelial Cell Activity, Inflammatory Response, Endoplasmic Reticulum Stress, and Apoptosis in Sepsis

The aim of this study is to explore the protective effects of lentinan on endoplasmic reticulum stress, inflammation, and apoptosis in sepsis endothelial cells. Firstly, lentinan was extracted, purified, and analyzed. When the concentration of lentinan was in the range of 0.04–4 μM, there was no obvious effect on the morphology of HUVECs. When the concentration reached 10 M, the cells were obviously contracted and necrotic. CCK-8 cell activity experiment showed that when the concentration of lentinan reached 4 μM, the cell activity decreased significantly (P<0.001), and it was in a dose-dependent manner. Then, the cells were divided into the control group (0 μM lentinan), sepsis group, sepsis + lentinan 1.2 μM group, and sepsis + lentinan 2 μM group. Enzyme-linked immunosorbent assay showed that lentinan could significantly reduce the expression of TNF-α, IL-1β, and IL-6 in sepsis endothelial cells (P<0.001). In addition, flow cytometry and TUNEL staining showed that compared with the control group, the apoptosis of cells in the sepsis group increased significantly (P<0.001), and lentinan could inhibit apoptosis (P<0.001). In terms of mechanism research, the mRNA and protein expression of endoplasmic reticulum stress-related protein in endothelial cells were detected by real-time fluorescent quantitative PCR (qPCR) and Western blotting, respectively. It was found that the expression of SIRT1, the upstream factors of endoplasmic reticulum stress in sepsis cells, was obviously inhibited (P<0.001), and the expression of CHOP, GRP78, IRE1α, and ATF6 was significantly increased (P<0.001), However, the pretreatment of lentinan could significantly reverse the above changes (P<0.001). Besides, lentinan could also reduce the expression of phosphorylated p65 protein (the activation marker of NF-κb) and iNOS. Conclusion. When sepsis occurs, lentinan can protect endothelial cells from ERS inflammation and apoptosis induced by sepsis. Thus, lentinan is expected to be a new target for the treatment of sepsis-induced endothelial damage.

Cell-free hemoglobin increases inflammation, lung apoptosis, and microvascular permeability in murine polymicrobial sepsis.

Increased endothelial permeability is central to the pathogenesis of sepsis and leads to organ dysfunction and death but the endogenous mechanisms that drive increased endothelial permeability are not completely understood. We previously reported that cell-free hemoglobin (CFH), elevated in 80% of patients with sepsis, increases lung microvascular permeability in an ex vivo human lung model and cultured endothelial cells. In this study, we augmented a murine model of polymicrobial sepsis with elevated circulating CFH to test the hypothesis that CFH increases microvascular endothelial permeability by inducing endothelial apoptosis. Mice were treated with an intraperitoneal injection of cecal slurry with or without a single intravenous injection of CFH. Severity of illness, mortality, systemic and lung inflammation, endothelial injury and dysfunction and lung apoptosis were measured at selected time points. We found that CFH added to CS increased sepsis mortality, plasma inflammatory cytokines as well as lung apoptosis, edema and inflammation without affecting large vessel reactivity or vascular injury marker concentrations. These results suggest that CFH is an endogenous mediator of increased endothelial permeability and apoptosis in sepsis and may be a promising therapeutic target.

MiR‑195 promotes LPS‑mediated intestinal epithelial cell apoptosis via targeting SIRT1/eIF2a.

A microarray analysis of an animal model with experimental sepsis induced by caecal ligation and puncture revealed that the level of microRNA-195 (miR-195) was upregulated. However, to the best of our knowledge, the role of miR-195 in sepsis remains unknown. The present study investigated the effect of miR-195 on apoptosis in sepsis and investigated the underlying mechanism. The level of miR-195 was measured in human intestinal epithelial cells following exposure to lipopolysaccharide (LPS). Cell viability and apoptosis were detected using Cell Counting kit-8 and flow cytometry assays. The expression levels of apoptosis-associated proteins were determined using western blot analysis. In addition, a dual-luciferase reporter assay was employed to verify the association between miR-195 and sirtuin 1 (SIRT1). Furthermore, the SIRT1 inhibitor EX527 was applied to further confirm the regulatory network of miR-195/SIRT1 in LPS-induced apoptosis. It was demonstrated that LPS significantly inhibited cell viability and promoted cell apoptosis in NCM460 cells in a dose-dependent manner. In addition, miR-195 was significantly upregulated following LPS treatment. The present results revealed that silencing miR-195 prevented apoptosis and alleviated cell injury in LPS-induced NCM460 cells. Further investigation demonstrated that miR-195 bound directly to and negatively regulated SIRT1. Inhibition of SIRT1 reversed the protective effects of miR-195-silencing on the apoptosis and viability of NCM460 cells. Furthermore, silencing miR-195 prevented endoplasmic reticulum (ER) stress-induced apoptosis via a downregulation of SIRT1 and its downstream effectors, including activating transcription factor 4, C/EBP homologous protein, glucose-regulated protein 78 and growth arrest and DNA-damage protein 34, as well as the phosphorylation of eukaryotic translation initiation factor 2A. In conclusion, the present study revealed a novel mechanism by which miR-195 regulates SIRT1-mediated downstream effectors in ER stress-induced apoptosis in sepsis.