LPS-induced Apoptosis Research Articles

Recombinant Klotho protein protects pulmonary alveolar epithelial cells against sepsis-induced apoptosis by inhibiting the Bcl-2/Bax/caspase-3 pathway.

Inflammation-induced apoptosis of alveolar type II epithelial cells is a primary contributor to sepsis-induced acute respiratory distress syndrome (ARDS). Klotho is a single-pass transmembrane protein with anti-inflammatory and anti-apoptotic effects. However, the role and mechanism of Klotho in the development of ARDS remains unknown. This study aimed to investigate the effect of Klotho on sepsis-induced apoptosis in human pulmonary alveolar epithelial cells (HPAEpiCs) together with the potential mechanism. Cecal ligation and puncture (CLP) were performed to generate an in vivo sepsis model, and HPAEpiCs were treated with lipopolysaccharide (LPS) to mimic sepsis in vitro. Both models were administered recombinant Klotho protein. The morphology of the lung tissue was observed, and apoptotic cells and cell viability were detected. Interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA), while the expression of Bcl-2, Bax and cleaved caspase-3 was detected with western blotting. Klotho reversed the CLP-induced decrease in mouse survival in vivo (p < 0.001) and increased inflammatory cell infiltration and inflammatory substance exudation in the lung tissue of mice with sepsis (both p < 0.001). Klotho also suppressed apoptosis (p < 0.001) as demonstrated by IL-1β, IL-6 and TNF-α expression (all p < 0.001), and Bcl-2/Bax/caspase-3 pathway activation (p < 0.001). Klotho pretreatment significantly prevented LPS-induced apoptosis in vitro (p < 0.001), as demonstrated by IL-1β, IL-6 and TNF-α upregulation (all p < 0.001); and Bcl-2/Bax/caspase-3 pathway activation in HPAEpiCs (p < 0.001). This study demonstrated that Klotho can ameliorate acute lung injury (ALI) induced by sepsis by inhibiting inflammatory responses and exerting anti-apoptotic effects by suppressing Bcl-2/Bax/caspase-3 pathway activation.

PTPRO inhibits LPS-induced apoptosis in alveolar epithelial cells

Acute lung injury (ALI) and its severe manifestation, acute respiratory distress syndrome (ARDS), represent critical clinical syndromes with multifactorial origins, notably stemming from sepsis within intensive care units (ICUs). Despite their high mortality rates, no selective cure is available beside ventilation support. Apoptosis plays a complex and pivotal role in the pathophysiology of acute lung injury. Excessive apoptosis of alveolar epithelial and microvascular endothelial cells can lead to disruption of lung epithelial barrier integrity, impairing the body's ability to exchange blood and gas. At the same time, apoptosis of damaged or dysfunctional cells, including endothelial and epithelial cells, can help maintain tissue integrity and accelerate recovery from organ pro-inflammatory stress. The balance between pro-survival and pro-apoptotic signals in lung injury determines patient outcomes, making the modulation of apoptosis an area of intense research in the quest for more effective therapies. Here we found that protein tyrosine phosphatase receptor type O (PTPRO), a poorly understood receptor-like protein tyrosine phosphatase, is consistently upregulated in multiple tissue types of mice under septic conditions and in the lung alveolar epithelial cells. PTPRO reduction by its selective short-interfering RNA (siRNA) leads to excessive apoptosis in lung alveolar epithelial cells without affecting cell proliferation. Consistently PTPRO overexpression by a DNA construct attenuates apoptotic signaling induced by LPS. These effects of PTPTO on cellular apoptosis are dependent on an ErbB2/PI3K/Akt/NFκB signaling pathway. Here we revealed a novel regulatory pathway of cellular apoptosis by PTPRO in lung alveolar epithelial cells during sepsis.

Identification of tumor necrosis factor receptor 1 and its association with LPS-induced apoptosis in Onchidium reevesii

The sea slug (Onchidium reevesii) inhabits intertidal environments and face environmental pressures and disease threats. The immune system of mollusks plays a crucial role in their defense against pathogens and maintaining homeostasis. The molecular mechanisms underlying its immune defense and response to pathogens remain largely unexplored. This study aimed to isolate and identify the cDNA sequence of tumor necrosis factor receptor 1 (TNFR1) in the sea slug. The obtained cDNA sequence was named OrTNFR1. Like other members of the TNF receptor superfamily, OrTNFR1 contains a signal peptide, TNF homologous domain, and death-related domain. qPCR results showed that OrTNFR1 was highly expressed in the hepatopancreas, blood, and intestines of sea slug. This indicates that OrTNFR1 plays a role in the immune-related tissues of sea slug and is consistent with the broad expression of TNFR1 in bivalves. To investigate the inducible expression pattern and downstream effects of OrTNFR1 in response to lipopolysaccharide (LPS) stimulation, we studied the functional role of the OrTNFR1 gene by stimulating with LPS in vivo and suppressing OrTNFR1 expression using siRNA technology. Our findings demonstrate that LPS stimulation rapidly up-regulates the expression of OrTNFR1 in the hepatopancreas, blood, and intestine of the sea slug. Moreover, si-TNFR1 effectively inhibits LPS-induced TNFR1 expression and its downstream genes. Additionally, we discovered that inhibiting OrTNFR1 reduces LPS-induced cell apoptosis and the expression of apoptosis-associated protein Caspase.TNFR1 is involved in the immune defense of the sea slug, and blocking TNFR1 effectively attenuates LPS-induced cell apoptosis through the TNFR1 pathway. This finding suggests that inhibiting TNFR1 in the sea slug may serve as a potential approach to reduce cell apoptosis. As a shell-less living in harsh intertidal environments, TNFR1 likely participates in its innate immunity. Therefore, studying the TNFR1 pathway contributes to understanding the innate immune signaling pathway in the sea slug, further elucidating host defense against pathogens and apoptotic functions.

BMAL1 alleviates myocardial damage in sepsis by activating SIRT1 signaling and promoting mitochondrial autophagy

BackgroundBrain and muscle arnt-like protein-1 (BMAL1) deficiency is associated with myocardial dysfunction and suppressed sirtuin 1 (SIRT1). However, whether BMAL1 promotes mitophagy via SIRT1 to alleviate myocardial injury in sepsis remains unknown. MethodsAn in vitro myocardial injury model was established using lipopolysaccharide (LPS)-treated H9C2 cells. Knockdown or overexpression of genes was performed using plasmid transfection. Gene and protein expression was assessed by qRT-PCR and Western blot, respectively. Cell proliferation was evaluated using cell counting kit-8, and cellular apoptosis and reactive oxygen species (ROS) levels were analyzed using flow cytometry. An in vivo myocardial injury model of sepsis was established by cecal ligation and puncture in rats. Myocardial function was characterized by analyzing the damage-associated proteins, inflammatory factors, ejection fraction, and fraction shortening. ResultssgBMAL1 significantly decreased BMAL1 levels and remarkably increased the sensitivity of H9C2 cells to LPS stimulation, consequently enhancing LPS-induced apoptosis, inflammation, and ROS levels. These effects were further attenuated by BMAL1 overexpression. BMAL1 knockdown inhibited the expression of SIRT1 and mitophagy-associated proteins. SIRT1 overexpression reversed the enhancement of shBMAL1 on cell proliferation and inflammation. In the rat model of sepsis, BMAL1 overexpression decreased the myocardial injury-associated proteins to recover the myocardial function and suppressed inflammatory activities by promoting mitophagy via SIRT1. ConclusionBMAL1 enhances mitophagy dependent on SIRT1, thereby alleviating myocardial injury in sepsis.

Regulatory mechanism of miR-20a-5p in neuronal damage and inflammation in lipopolysaccharide-induced BV2 cells and MPTP-HCl-induced Parkinson's disease mice.

Parkinson's disease (PD) is a prevailing neurodegenerative disorder increasingly affecting the elderly population. The involvement of microRNAs (miRNAs) in PD has been confirmed. We sought to explore the molecular mechanism of miR-20a-5p in PD. Lipopolysaccharide (LPS)-induced BV2 cell model and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP-HCl)-induced PD mouse model were established. miR-20a-5p, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and IL-10 expression in BV2 cells was examined by reverse transcription - quantitative polymerase chain reaction. Cell viability was assessed by MTT assay. The apoptotic rate and levels of Bcl-2, Bax, cleaved caspase-3, and signal transducer and activator of transmission (STAT)3 were examined by flow cytometry and Western blot. Bioinformatics software predicted the potential binding sites of miR-20a-5p and STAT3. Dual-luciferase experiment verified the binding relationship. Iba1-positive and tyrosine hydroxylase (TH)-positive cell numbers in substantia nigra pars compacta were detected by immunohistochemistry. The effect of miR-20a-5p on motor function in MPTP-induced PD mice was detected by Rota-rod test, Pole test, Traction test and Beam-crossing task. miR-20a-5p was under-expressed in LPS-induced BV2 cells. Overexpression of miR-20a-5p increased the viability of LPS-induced BV2 cells and reduced apoptosis rates. Moreover, overexpression of miR-20a-5p reduced cleaved caspase-3, Bax, iNOS, IL-6, and TNF-α and increased Bcl-2 and TGF-β1, and IL-10. miR-20a-5p targeted STAT3. STAT3 overexpression partially reversed miR-20a-5p overexpression-mediated effects on LPS-induced BV2 cell viability, apoptosis, and inflammatory responses. miR-20a-5p overexpression inhibited MPTP-induced STAT3 and α-synuclein levels, microglia activation, and inflammatory response, and reduced the loss of TH-positive cells in mice. miR-20a-5p overexpression ameliorated MPTP-induced dyskinesia in PD model mice. miR-20a-5p alleviates neuronal damage and suppresses inflammation by targeting STAT3 in PD.

Alginate Oligosaccharide Alleviates Lipopolysaccharide-Induced Apoptosis and Inflammatory Response of Rumen Epithelial Cells through NF-κB Signaling Pathway.

AOS alleviates inflammatory responses; however, whether it exerts an effect on the rumen or regulates rumen inflammatory reaction remains unknown. In this study, firstly, the ovine ruminal epithelial cells (ORECs) were treated with 0, 200, 400, 600, and 800 µg/mL AOS, hoping to explore whether AOS hurt cell health. The results showed that compared with the AOS-0 group, the AOS-400 group could significantly increase (p < 0.05) cell viability, reduce (p < 0.05) reactive oxygen species (ROS) and interleukin (IL)-6 content, and have no adverse effect on cells. Secondly, we used LPS to construct an in vitro inflammatory model of rumen epithelial cells and then explored the protective role of AOS on rumen epithelial cells. The study was divided into three groups: the control group (CON), LPS, and LPS + AOS. The results demonstrated that the LPS + AOS group significantly increased the cell viability and reduced the ROS level in comparison with the LPS group (p < 0.05). Pretreatment with AOS also repressed (p < 0.05) the secretion of IL-1β, IL-6, IL-8, and immunoglobulin (Ig)A from ORECs in the culture medium following LPS. In terms of tight junction (TJ) proteins, AOS treatment also significantly increased (p < 0.05) the zonula occludens 1 (ZO-1) and Occludin expression. The apoptosis rate, Caspase3, Caspase9, BAD, and BCL-2/BAX were decreased (p < 0.05) after AOS treatment, and the expression of BCL-2 was increased (p < 0.05). In addition, the expressions of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were inhibited (p < 0.05) with the addition of AOS. At the protein level, pretreatment of AOS decreased (p < 0.05) the expression of MyD88 and the phosphorylation level of inhibitor κB α (IκBα) after the LPS challenge. Taken together, our results indicated that AOS could alleviate the LPS-induced apoptosis and inflammatory response of rumen epithelial cells through the NF-κB signaling pathway, which may be a promising strategy for treating apoptosis and inflammation in sheep breeding.

Spock2 Functions as a Key Time-Series Gene of Endothelial Cells in Sepsis-Induced Cardiomyopathy.

The study aimed to investigate the pathogenesis of sepsis-induced cardiomyopathy, a leading cause of mortality in septic patients. Transcriptome data from cecal ligation and puncture-induced septic mice were analyzed at different time points (24, 48, and 72 hours) using GSE171546 data. Through weighted gene co-expression network analysis, time series, and differential expression analyses, key time-series differentially expressed genes were identified. In addition, single-cell sequencing data (GSE207363) were used for both differential and pseudotime analyses to pinpoint differentially expressed genes specific to endothelial cells. The study highlighted Spock2, S100a9, S100a8, and Xdh as differential genes specific to endothelial cells in a time-dependent manner. Immunofluorescence validation confirmed the increased expression of SPOCK2 in the endothelial cells of cecal ligation and puncture-induced septic mice. Furthermore, in vitrostudies showed that deletion of Spock2 significantly increased LPS-induced apoptosis and necrosis in human umbilical vein endothelial cells. In conclusion, SPOCK2 expression was increased in septic cardiac endothelial cells and LPS-induced human umbilical vein endothelial cells and may play a protective role.

STING-deficiency in lung resident mesenchymal stromal cells contributes to the alleviation of LPS-induced lung injury

Acute respiratory distress syndrome (ARDS) is characterized by acute diffuse inflammatory lung injury with a high mortality rate. Mesenchymal stromal cells (MSC) are pluripotent adult cells that can be extracted from a variety of tissues, including the lung. Lung-resident MSC (LR-MSC) located around vascular vessels and act as important regulators of lung homeostasis, regulating the balance between lung injury and repair processes. LR-MSC support the integrity of lung tissue by modulating immune responses and releasing trophic factors. Studies have reported that the STING pathway is involved in the progression of lung injury inflammation, but the specific mechanism is unclear. In this study, we found that STING deficiency could ameliorate lipopolysaccharides (LPS)-induced acute lung injury, STING knockout (STING KO) LR-MSC had an enhanced treatment effect on acute lung injury. STING depletion protected LR-MSC from LPS-induced apoptosis. RNA-sequencing and Western blot results showed that STING KO LR-MSC expressed higher levels of MSC immunoregulatory molecules, such as Igfbp4, Icam1, Hgf and Cox2, than WT LR-MSC. This study highlights that LR-MSC have a therapeutic role in acute lung injury, and we demonstrate that STING deficiency can enhance the immunomodulatory function of LR-MSC in controlling lung inflammation. Thus, STING can be used as an intervention target to enhance the therapeutic effect of MSC.

Ginsenoside Rg1 Alleviates Sepsis-Induced Acute Lung Injury by Reducing FBXO3 Stability in an m6A-Dependent Manner to Activate PGC-1α/Nrf2 Signaling Pathway.

Sepsis-induced acute lung injury (ALI) is one of the serious life-threatening complications of sepsis and is pathologically associated with mitochondrial dysfunction. Ginsenoside Rg1 has good therapeutic effects on ALI. Herein, the pharmacological effects of Rg1 in sepsis-induced ALI were investigated. Sepsis-induced ALI models were established by CLP operation and LPS treatment. HE staining was adopted to analyze lung pathological changes. The expression and secretion of cytokines were measured by RT-qPCR and ELISA. Cell viability and apoptosis were assessed by MTT assay, flow cytometry and TUNEL staining. ROS level and mitochondrial membrane potential (MMP) were analyzed using DHE probe and JC-1 staining, respectively. FBXO3 m6A level was assessed using MeRIP assay. The interactions between FBXO3, YTHDF1, and PGC-1α were analyzed by Co-IP or RIP. Rg1 administration ameliorated LPS-induced epithelial cell inflammation, apoptosis, and mitochondrial dysfunction in a dose-dependent manner. Mechanically, Rg1 reduced PGC-1α ubiquitination modification level by inhibiting FBXO3 expression m6A-YTHDF1 dependently. As expected, Rg1's mitigative effect on LPS-induced inflammation, apoptosis and mitochondrial dysfunction in lung epithelial cells was abolished by FBXO3 overexpression. Moreover, FBXO3 upregulation eliminated the restoring effect of Rg1 on CLP-induced lung injury in rats. Rg1 activated PGC-1α/Nrf2 signaling pathway by reducing FBXO3 stability in an m6A-YTHDF1-dependent manner to improve mitochondrial function in lung epithelial cells during sepsis-induced ALI progression.

GNAI3 mediated by Lin28A regulates lipopolysaccharide-induced inflammation and osteogenic differentiation in periodontal stem cells by mediating the NF-κB/NLRP3 inflammasome pathway

ObjectivesThe aim of this study was to investigate the regulatory role of G protein subunit alpha i3 (GNAI3) in periodontitis. DesignFollowing the induction of human periodontal ligament stem cells (hPDLSCs) with lipopolysaccharide (LPS), the mRNA and protein expressions of GNAI3 and Lin28A were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. The transfection efficiency of Oe-GNAI3 and sh-Lin28A was examined by virtue of RT-qPCR and western blot. With the application of ELISA and flow cytometry, the releases of inflammatory cytokines and cell apoptosis were appraised. Alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining were conducted to evaluate osteogenic differentiation. Next, the binding ability of Lin28A with GNAI3 mRNA was estimated by radioimmunoprecipitation (RIP) assay while the stability of GNAI3 mRNA was assessed utilizing RT-qPCR. Western blot was employed for the measurement of inflammation-, apoptosis- and nuclear factor-kappaB (NF-κB)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway-related proteins and osteogenic markers. ResultsThe expression of GNAI3 was down-regulated in LPS-induced hPDLSCs. After the transfection with Oe-GNAI3, the inflammation and apoptosis in LPS-induced hPDLSCs were inhibited while osteogenic differentiation was promoted. Moreover, Lin28A could stabilize GNAI3 mRNA and Lin28A knockdown significantly reduced GNAI3 expression. Further experiments verified that the inhibitory effects of GNAI3 overexpression on LPS-induced cellular inflammation and cell apoptosis as well as the promotive effects on osteogenic differentiation in hPDLSCs were all partially counteracted by Lin28A depletion, which may possibly be mediated via the regulation of the NF-κB/NLRP3 inflammasome pathway. ConclusionGNAI3 that mediated by Lin28A regulates the inflammation and osteogenic differentiation in LPS-induced hPDLSCs by mediating the NF-κB/NLRP3 inflammasome pathway.

Bovine lactoferrin inhibits inflammatory response and apoptosis in lipopolysaccharide-induced acute lung injury by targeting the PPAR-γ pathway.

Lactoferrin (LF) is an iron-binding multifunctional cationic glycoprotein. Previous studies have demonstrated that LF may be a potential drug for treating acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In this study, we explored the anti-inflammatory effect and mechanism of bovine lactoferrin (bLF) in ALI using the RNA sequencing (RNA-seq) technology and transcriptome analysis. Based on the differentially expressed genes (DEGs) obtained from RNA-seq of the Lung from mouse model, the bioinformatics workflow was implemented using the BGISEQ-500 platform. The protein-protein interaction (PPI) network was obtained using STRING, and the hub gene was screened using Cytoscape. To verify the results of transcriptome analysis, the effects of bLF on Lipopolysaccharide (LPS)-induced BEAS-2B cells and its anti-reactive oxygen species (ROS), anti-inflammatory, and antiapoptotic effects were studied via Cell Counting Kit-8 (CCK-8) test, active oxygen detection test, ELISA, and western blot assay. Transcriptome analysis revealed that two hub gene modules of DEGs were screened via PPI analysis using the STRING and MCODE plug-ins of Cytoscape. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these core modules are enriched in the PPAR (peroxisome proliferator-activated receptor) and AMPK (AMP-activated protein kinase) signaling pathways. Through cell experiments, our study shows that bLF can inhibit ROS, inflammatory reaction, and LPS-induced BEAS-2B cell apoptosis, which are significantly antagonized by the PPAR-γ inhibitor GW9662. This study has suggested that the PPAR-γ pathway is the critical target of bLF in anti-inflammatory reactions and apoptosis of ALI, which provides a direction for further research.

Epigallocatechin-3-Gallate attenuates lipopolysacharide-induced pneumonia via modification of inflammation, oxidative stress, apoptosis, and autophagy

BackgroundPneumonia, the acute inflammation of lung tissue, is multi-factorial in etiology. Hence, continuous studies are conducted to determine the mechanisms involved in the progression of the disease and subsequently suggest effective treatment. The present study attempted to evaluate the effects of Epigallocatechin-3-Gallate (EGCG), an herbal antioxidant, on inflammation, oxidative stress, apoptosis, and autophagy in a rat pneumonia model.MethodsForty male Wistar rats, 5 months old and 250–290 g were divided into four groups including control, EGCG, experimental pneumonia (i/p LPS injection, 1 mg/kg), and experimental pneumonia treated with EGCG (i/p, 15 mg/kg, 1 h before and 3 h after LPS instillation). Total cell number in the bronchoalveolar lavage fluid, inflammation (TNF-a, Il-6, IL-1β, and NO), oxidative stress (Nrf2, HO-1, SOD, CAT, GSH, GPX, MDA, and TAC), apoptosis (BCL-2, BAX, CASP-3 and CASP-9), and autophagy (mTOR, LC3, BECN1) were evaluated.ResultsThe findings demonstrated that EGCG suppresses the LPS-induced activation of inflammatory pathways by a significant reduction of inflammatory markers (p-value < 0.001). In addition, the upregulation of BCL-2 and downregulation of BAX and caspases revealed that EGCG suppressed LPS-induced apoptosis. Furthermore, ECGC suppressed oxidative injury while promoting autophagy in rats with pneumonia (p-value < 0.05).ConclusionThe current study revealed that EGCG could suppress inflammation, oxidative stress, apoptosis, and promote autophagy in experimental pneumonia models of rats suggesting promising therapeutical properties of this compound to be used in pneumonia management.

Small nucleolar RNA host gene 1 silence inhibits the lipopolysaccharide-induced microglial apoptosis and inflammation.

Microglia activation is an early mediator of neuroinflammation and a major contributor to spinal damage and motor dysfunction. This study was designed to investigate the role of small nucleolar RNA host gene 1 (SNHG1) on the apoptosis and inflammatory response of microglial cell BV-2 and its underlying molecular mechanism. The C5 lamina contusion-induced mouse model of spinal cord injury (SCI) was constructed. Mouse microglia BV2 was stimulated by lipopolysaccharide (LPS) to establish the in vitro model of SCI. The quantitative reverse transcription polymerase chain reaction method was used to quantify RNA expression levels. Enzyme-linked immunosorbent assays were used to quantify concentrations of inflammatory cytokines. Protein levels were assessed by western blotting, and apoptosis was assessed by flow cytometry. Dual luciferase reporter gene assay and RNA pull-down assay were conducted to investigate the binding relationships between molecules. Upregulation of SNHG1 and downregulation of miR-195-5p were observed in the spinal cords of SCI mouse model. LPS treatment led to elevation of SNHG1 expression in BV2 cells, as well as accelerated apoptosis and inflammation. Evident mitigation of LPS-induced BV2 cell damage was observed after SNHG1 knockdown. MiR-195-5p was identified as a target of SNHG1. Inhibition of miR-195-5p restored the impact of SNHG1 knockdown on cell damage of LPS-treated BV2 cells. Furthermore, miR-195-5p can target activating transcription factor-6 (ATF6). In summary, SNHG1 knockdown ameliorates LPS-induced microglial apoptosis and inflammatory response via the miR-195-5p/ATF6 axis, providing a novel direction for SCI treatment.

Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury

Approximately 60% of septic patients developed acute kidney injury (AKI). The mortality rate of septic AKI (SA-AKI) is two to three times higher than that of septic without AKI (SA-non-AKI). The actual functions and mechanisms of CircRNAs in the pathophysiology of SA-AKI remain incompletely understood. Herein, we observed that the mmu_Circ_26986 could be induced by lipopolysaccharide (LPS) and cecum ligation and puncture (CLP) in BUMPT cell line and C57BL/6 mouse kidney, respectively. Functionally, mmu_Circ_26986 suppressed BUMPT cell apoptosis induced by LPS. Mechanistically, mmu_Circ_26986 sponged miRNA-29b-1-5p to upregulate the expression of PAK7. Overexpression of mmu_Circ_26986 ameliorated the progression of CLP-stimulated AKI through miRNA-29b-1-5p/PAK7 axis. In addition, we found that hsa_Circ_0072463, homologous to mmu_Circ_26986, suppressed LPS-induced HK-2 cells apoptosis via regulation of miRNA-29b-1-5p/PAK7 axis. Furthermore, sepsis patients with AKI had a higher level of hsa_Circ_0072463 compared to those without AKI. The sensitivity, specificity and AUC of hsa_Circ_0072463 were 78.8%, 87.9% and 0.866, respectively. Spearman's test indicated a noticeable positive correlation between plasma hsa_Circ_0072463 and serum creatinine in sepsis patients (r = 0.725). In summary, this study reveals that the mmu_Circ_26986/hsa_Circ_0072463 miRNA-29b-1-5p/PAK7 axis mediates septic AKI, and hsa_Circ_0072463 is a potential diagnostic marker for septic AKI.

Peficitinib alleviated acute lung injury by blocking glycolysis through JAK3/STAT3 pathway

Peficitinib is a selective Janus kinase (JAK3) inhibitor recently developed and approved for the treatment of rheumatoid arthritis in Japan. Glycolysis in macrophages could induce NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, thus resulting in pyroptosis and acute lung injury (ALI). The aim of our study was to investigate whether Peficitinib could alleviate lipopolysaccharide (LPS)-induced ALI by inhibiting NLRP3 inflammasome activation. Wild type C57BL/6J mice were intraperitoneally injected with Peficitinib (5 or 10 mg·kg−1·day−1) for 7 consecutive days before LPS injection. The results showed that Peficitinib pretreatment significantly relieved LPS-induced pulmonary edema, inflammation, and apoptosis. NLRP3 inflammasome and glycolysis in murine lung tissues challenged with LPS were also blocked by Peficitinib. Furthermore, we found that the activation of JAK3/signal transducer and activator of transcription 3 (STAT3) was also suppressed by Peficitinib in mice with ALI. However, in Jak3 knockout mice, Peficitinib did not show obvious protective effects after LPS injection. In vitro experiments further showed that Jak3 overexpression completely abolished Peficitinib-elicited inhibitory effects on pyroptosis and glycolysis in LPS-induced RAW264.7 macrophages. Finally, we unveiled that LPS-induced activation of JAK3/STAT3 was mediated by toll-like receptor 4 (TLR4) in RAW264.7 macrophages. Collectively, our study proved that Peficitinib could protect against ALI by blocking JAK3-mediated glycolysis and pyroptosis in macrophages, which may serve as a promising candidate against ALI in the future.

S100A9 exacerbates sepsis-induced acute lung injury via the IL17-NFκB-caspase-3 signaling pathway

BackgroundSepsis-induced acute lung injury (ALI) is associated with considerable morbidity and mortality in critically ill patients. S100A9, a key endothelial injury factor, is markedly upregulated in sepsis-induced ALI; however, its specific mechanism of action has not been fully elucidated. MethodsThe Gene Expression Omnibus database transcriptome data for sepsis-induced ALI were used to screen for key differentially expressed genes (DEGs). Using bioinformatics analysis methods such as Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction network analyses, the pathogenesis of sepsis-induced ALI was revealed. Intratracheal infusion of lipopolysaccharide (LPS, 10 mg/kg) induced ALI in wild-type (WT) and S100A9 knockout mice. Multiomics analyses (transcriptomics and proteomics) were performed to investigate the potential mechanisms by which S100A9 exacerbates acute lung damage. Hematoxylin-eosin, Giemsa, and TUNEL staining were used to evaluate lung injury and cell apoptosis. LPS (10 μg/mL)-induced murine lung epithelial MLE-12 cells were utilized to mimic ALI and were modulated by S100A9 lentiviral transfection. The impact of S100A9 on cell apoptosis and inflammatory responses were identified using flow cytometry and PCR. The expression of interleukin (IL)-17–nuclear factor kappa B (NFκB)–caspase-3 signaling components was identified using western blotting. ResultsSix common DEGs (S100A9, S100A8, IFITM6, SAA3, CD177, and MMP9) were identified in the six datasets related to ALI in sepsis. Compared to WT sepsis mice, S100A9 knockout significantly alleviated LPS-induced ALI in mice, with reduced lung structural damage and inflammatory exudation, decreased exfoliated cell and protein content in the lung lavage fluid, and reduced apoptosis and necrosis of pulmonary epithelial cells. Transcriptomic analysis revealed that knocking out S100A9 significantly affected 123 DEGs, which were enriched in immune responses, defense responses against bacteria or lipopolysaccharides, cytokine-cytokine receptor interactions, and the IL-17 signaling pathway. Proteomic analysis revealed that S100A9 knockout alleviated muscle contraction dysfunction and structural remodeling in sepsis-induced ALI. Multiomics analysis revealed that S100A9 may be closely related to interferon-induced proteins with tetratricopeptide repeats and oligoadenylate synthase-like proteins. LPS decreased MLE12 cell activity, accompanied by high expression of S100A9. The expression of IL-17RA, pNFκB, and cleaved-caspase-3 were increased by S100A9 overexpression and reduced by S100A9 knockdown in LPS-stimulated MLE12 cells. S100A9 knockdown decreases transcription of apoptosis-related markers Bax, Bcl and caspase-3, alleviating LPS-induced apoptosis. ConclusionsS100A9 as a key biomarker of sepsis-induced acute lung injury, and exacerbates lung damage and epithelial cell apoptosis induced by LPS via the IL-17–NFκB–caspase-3 signaling pathway.

CIRCVMA21-RELATED PATHWAY ALLEVIATES LIPOPOLYSACCHARIDE-INDUCED HK-2 CELL INJURY.

Background : It is reported that circVMA21 has an inhibition effect on sepsis-induced acute kidney injury (AKI). Therefore, the underlying molecular mechanisms of circVMA21 in AKI are worthy of further investigation. Material and Methods : Lipopolysaccharide (LPS) was used to induce HK2 cell injury. CircVMA21, miR-337-3p and ZEB2 expression was tested by qRT-PCR. Cell growth was detected by CCK8 assay, EdU assay, and flow cytometry. Protein levels were examined by western blot. The levels of inflammatory factors and oxidative stress markers were measured to evaluate cell inflammatory response and oxidative stress. RNA relationship as verified by dual-luciferase reporter assay, RIP assay, and RNA pull-down assay. Results : CircVMA21 had decreased expression in AKI patients. Overexpressed circVMA21 alleviated LPS-induced HK2 cell inflammation, apoptosis, and oxidative stress. Moreover, circVMA21 sponged miR-337-3p, and miR-337-3p targeted ZEB2. The inhibitory effect of circVMA21 on LPS-induced HK2 cell injury was reversed by miR-337-3p overexpression, and ZEB2 overexpression abolished the promotion effect of miR-337-3p on LPS-induced HK2 cell injury. Conclusions : CircVMA21 could inhibit LPS-induced HK2 cell injury via miR-337-3p/ZEB2 axis.

ENDOGENOUS β 3 -ADRENERGIC RECEPTOR ACTIVATION ALLEVIATES SEPSIS-INDUCED CARDIOMYOCYTE APOPTOSIS VIA PI3K/AKT SIGNALING PATHWAY.

β 3 -adrenergic receptor (β 3 -AR) has been proposed as a new therapy for several myocardial diseases. However, the effect of β 3 -AR activation on sepsis-induced myocardial apoptosis is unclear. Here, we investigated the effect of β 3 -AR activation on the cardiomyocyte apoptosis and cardiac dysfunction in cecal ligation and puncture (CLP)-operated rats and lipopolysaccharide (LPS)-treated cardiomyocytes. We found that β 3 -AR existed both in adult rat ventricular myocytes (ARVMs) and H9c2 cells. The expression of β 3 -AR was upregulated in LPS-treated ARVMs and the heart of CLP rats. Pretreatment with β 3 -AR agonist, BRL37344, inhibited LPS-induced cardiomyocyte apoptosis and caspase-3, -8, and -9 activation in ARVMs. BRL37344 also reduced apoptosis and increased the protein levels of PI3K, p-Akt Ser473 and p-eNOS Ser1177 in LPS-treated H9c2 cells. Inhibition of PI3K using LY294002 abolished the inhibitory effect of BRL37344 on LPS-induced caspase-3, -8, and -9 activation in H9c2 cells. Furthermore, administration of β 3 -AR antagonist, SR59230A (5 mg/kg), significantly decreased the maximum rate of left ventricular pressure rise (+dP/dt) in CLP-induced septic rats. SR59230A not only increased myocardial apoptosis, reduced p-Akt Ser473 and Bcl-2 contents, but also increased mitochondrial Bax, cytoplasm cytochrome c, cleaved caspase-9, and cleaved caspase-3 levels of the myocardium in septic rats. These results suggest that endogenous β 3 -AR activation alleviates sepsis-induced cardiomyocyte apoptosis via PI3K/Akt signaling pathway and maintains intrinsic myocardial systolic function in sepsis.

Polysaccharide of Atractylodes macrocephala Koidz alleviates LPS-induced proliferation, differentiation inhibition and excessive apoptosis in chicken embryonic myogenic cells.

Lipopolysaccharide (LPS) can induce systemic inflammation and affect the growth and development of poultry. As a kind of traditional Chinese medicine, polysaccharide of Atractylodes macrocephala Koidz (PAMK) can effectively improve the growth performance of animals and improve the immunity of animal bodies. The purpose of this study was to investigate the effects of PAMK on LPS-induced inflammatory response, proliferation, differentiation and apoptosis of chicken embryonic myogenic cells. We used chicken embryonic myogenic cells as a model by detecting EdU/MYHC immunofluorescence, the expression of inflammation, proliferation, differentiation-related genes and proteins and the number of apoptotic cells in the condition of adding LPS, PAMK, belnacasan (an inhibitor of Caspase1) or their combinations. The results showed that LPS stimulation increased the expression of inflammatory factors, inhibited proliferation and differentiation, and excessive apoptosis in chicken embryonic myogenic cells, and PAMK alleviated these adverse effects induced by LPS. After the addition of belnacasan (inhibitor of Caspase1), apoptosis in myogenic cells was inhibited, and therefore, the number of apoptotic cells and the expression of pro-apoptotic genes Caspase1 and Caspase3 were increased. In addition, belnacasan inhibited the increased expression of inflammatory factors, inhibited proliferation, differentiation and excessive apoptosis in chicken embryonic myogenic cells induced by LPS. This study provides a theoretical basis for further exploring the mechanism of action of PAMK and exogenous LPS on chicken embryonic myogenic cells and lays the foundation for the development and application of green feed additives in animal husbandry industry.

Metformin switches cell death modes to soothe the apical periodontitis via ZBP1.

Apical periodontitis (AP) is a disease caused by pathogenic microorganisms and featured with the degradation of periapical hard tissue. Our recent research showed the crucial role of Z-DNA binding protein 1 (ZBP1)-mediated necroptosis and apoptosis in the pathogenesis of AP. However, the specific regulatory mechanisms of ZBP1 in AP are not fully elucidated. It was found that metformin has a regulatory role in cell necroptosis and apoptosis. But whether and how metformin regulates necroptosis and apoptosis through the ZBP1 in the context of AP remains unknown. This study provided evidence that lipopolysaccharide (LPS) promotes the synthesis of left-handed Z-nucleic acids (Z-NA), which in turn activates ZBP1. Knockout of Zbp1 by CRISPR/Cas9 technology significantly reduced LPS-induced necroptosis and apoptosis invitro. By using Zbp1-knockout mice, periapical bone destruction was alleviated. Moreover, type I interferon induced the expression of interferon-stimulated genes (ISGs), which serve as a major source of Z-NA. In addition, the RNA-editing enzyme Adenosine Deaminase RNA specific 1 (ADAR1) prevented the accumulation of endogenous Z-NA. Meanwhile, metformin suppressed the ZBP1-mediated necroptosis by inhibiting the expression of ZBP1 and the accumulation of ISGs. Metformin also promoted mitochondrial apoptosis, which is critical for the elimination of intracellular bacterial infection. The enhanced apoptosis further promoted the healing of infected apical bone tissues. In summary, these results demonstrated that the recognition of Z-NA by ZBP1 plays an important role in AP pathogenesis. Metformin suppressed ZBP1-mediated necroptosis and promoted apoptosis, thereby contributing to the soothing of inflammation and bone healing in AP.