Objective To investigate the gene expression and regulatory mechanisms of mouse embryonic fibroblasts (MEFs) under inflammatory conditions, aiming to elucidate the role of MEFs in inflammatory responses and provide a foundation for discovering anti-inflammatory drugs that act by modulating MEF function. Methods MEFs cultured in vitro were divided into the following groups: lipopolysaccharides (LPS)-treated group, inflammatory conditioned medium (CM)-treated group, and control group, which were treated with LPS, CM, and equal volume solvent, respectively. Transcriptome sequencing was used to analyze the effects of two stimuli on gene expression profile of MEFs. Real time fluorescence quantitative PCR (RT-qPCR) was employed to verify the transcription levels of highly expressed genes of MEFs induced by CM. ELISA was performed to determine the concentrations of cytokines in cell supernatants. Finally, the regulatory effects of CM on the activation of signaling pathways in MEFs were analyzed by immunoblotting. Results Transcriptome analysis showed that both LPS and CM induced the transcription of a large number of genes in MEFs. Compared with LPS, CM potentiated the mRNA transcription of some acute phase proteins, inflammatory cytokines, chemokines, matrix metalloproteinases (MMP), prostaglandin synthetases, and colony-stimulating factors. The transcriptome analysis was verified by RT-qPCR. The results of ELISA showed that CM treatment significantly increased the secretion of interleukin 6 (IL-6), C-C motif chemokine ligand (CCL2), and C-X-C motif chemokine ligand (CXCL1) by MEFs compared with LPS. Mechanism study showed that both LPS and CM induced the phosphorylation of nuclear factor-κB p65 (NF-κB p65), p38 mitogen-activated protein kinase (p38 MAPK), extracellular regulated protein kinases 1/2 (ERK1/2), and TANK-binding kinase (TBK) in MEFs, and CM strongly stimulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MEFs. Conclusion Both LPS and CM can induce transcription and protein secretion of various inflammation-related genes in MEFs. CM can partly enhance LPS-induced activation of MEFs, and the mechanism may be related to the enhancement effect of CM on the activation STAT3 signaling pathway.
Animals ; Fibroblasts/immunology* ; Mice ; Lipopolysaccharides/pharmacology* ; Inflammation/metabolism* ; Signal Transduction/drug effects* ; Gene Expression Regulation/drug effects* ; Cytokines/genetics* ; Culture Media, Conditioned/pharmacology* ; Cells, Cultured

OBJECTIVES: Acute lung injury (ALI) is an acute respiratory failure syndrome characterized by impaired gas exchange. Due to the lack of effective targeted drugs, it is associated with high mortality and poor prognosis. Tripterygium wilfordii (TW) has demonstrated anti-inflammatory activity in the treatment of various diseases. This study aims to investigate the effects and underlying mechanisms of TW on myeloid-derived suppressor cells (MDSCs) in ALI, providing experimental evidence for TW as a potential adjuvant therapy for ALI. METHODS: Eighteen specific pathogen-free (SPF) C57BL/6 mice were randomly divided into normal control (NC; intranasal saline), lipopolysaccharide (LPS; 5 mg/kg intranasally to induce ALI), and LPS+TW (50 mg/kg TW by gavage on the first day of modeling, followed by 5 mg/kg LPS intranasally to induce ALI) groups (n=6 each). Lung injury and edema were assessed by histopathological scoring and wet-to-dry weight ratio. Cytokine levels [interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor-α (TNF-α)] in lung tissue lavage fluid were measured by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to assess the proportions of MDSCs, polymorphonuclear MDSCs (PMN-MDSCs), and monocytic MDSCs (M-MDSCs) in bone marrow, spleen, peripheral blood, and lung tissue, as well as reactive oxygen species (ROS) levels in lung tissues. Messenger RNA (mRNA) expression levels of inducible nitric oxide synthase (iNOS) and arginase-1 (ARG-1) in lung tissues were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). PMN-MDSCs sorted from the lungs of LPS-treated mice were co-cultured with splenic CD3⁺ T cells and divided into NC, triptolide (TPL)-L, and TPL-H groups, with bovine serum albumin, 25 nmol/L TPL, and 50 nmol/L TPL, respectively. Flow cytometry was used to detect the effect of PMN-MDSCs on T-cell proliferation, and RT-qPCR was used to measure iNOS and ARG-1 mRNA expression. RESULTS: Compared with the NC group, the LPS group showed marked lung pathology with significantly increased histopathological scores and wet-to-dry ratios (both P<0.001). TW treatment significantly alleviated lung injury and reduced both indices compared with the LPS group (both P<0.05). Cytokine levels were significantly decreased in the LPS+TW group compared with the LPS group (all P<0.001). The proportions of MDSCs in CD45⁺ cells from spleen, bone marrow, peripheral blood, and lung, as well as PMN-MDSCs from spleen, peripheral blood, and lung, were significantly reduced in the LPS+TW group compared with the LPS group (all P<0.05), accompanied by reduced ROS levels in lung tissues (P<0.001). iNOS and ARG-1 mRNA expression in lung tissues was significantly lower in the LPS+TW group than in the LPS group (both P<0.001). In vitro, compared with the TPL-L group, the TPL-H group showed significantly increased CD3⁺ T-cell proliferation (P<0.001), and decreased iNOS and ARG-1 mRNA expression (all P<0.05). CONCLUSIONS TW alleviates the progression of LPS-induced ALI in mice, potentially by reducing the proportion of MDSCs in lung tissues and attenuating the immunosuppressive function of PMN-MDSCs.
Animals ; Acute Lung Injury/chemically induced* ; Myeloid-Derived Suppressor Cells/cytology* ; Tripterygium/chemistry* ; Mice, Inbred C57BL ; Mice ; Cell Differentiation/drug effects* ; Male ; Lipopolysaccharides ; Nitric Oxide Synthase Type II/genetics* ; Cytokines/metabolism* ; Reactive Oxygen Species/metabolism* ; Diterpenes/pharmacology* ; Epoxy Compounds ; Phenanthrenes

Acute lung injury (ALI) is a significant complication of sepsis, characterized by high morbidity, mortality, and poor prognosis. Neutrophils, as critical intrinsic immune cells in the lung, play a fundamental role in the development and progression of ALI. During ALI, neutrophils generate neutrophil extracellular traps (NETs), and excessive NETs can intensify inflammatory injury. Research indicates that Taohe Chengqi decoction (THCQD) can ameliorate sepsis-induced lung inflammation and modulate immune function. This study aimed to investigate the mechanisms by which THCQD improves ALI and its relationship with NETs in sepsis patients, seeking to provide novel perspectives and interventions for clinical treatment. The findings demonstrate that THCQD enhanced survival rates and reduced lung injury in the cecum ligation and puncture (CLP)-induced ALI mouse model. Furthermore, THCQD diminished neutrophil and macrophage infiltration, inflammatory responses, and the production of pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α). Notably, subsequent experiments confirmed that THCQD inhibits NET formation both in vivo and in vitro. Moreover, THCQD significantly decreased the expression of peptidyl arginine deiminase 4 (PAD4) protein, and molecular docking predicted that certain active compounds in THCQD could bind tightly to PAD4. PAD4 overexpression partially reversed THCQD's inhibitory effects on PAD4. These findings strongly indicate that THCQD mitigates CLP-induced ALI by inhibiting PAD4-mediated NETs.
Extracellular Traps/immunology* ; Acute Lung Injury/immunology* ; Animals ; Sepsis/immunology* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Neutrophils/immunology* ; Male ; Protein-Arginine Deiminase Type 4/genetics* ; Mice, Inbred C57BL ; Humans ; Disease Models, Animal ; Cytokines/metabolism*

Objective To explore the regulatory effects of cytokines interleukin(IL)-1β,IL-6,tumor necrosis factor alpha(TNF-ɑ),gamma interferon(IFN-γ),granulocyte colony-stimulating factor(G-CSF),and granulocyte-macrophage colony-stimulating factor(GM-CSF)on spontaneous pyroptosis in neutrophils.Methods Neutrophils isolated from mouse bone marrow by density-gradient centrifugation were cultured in vitro for 20 h with or without 10,50 or 100 ng/mL IL-1β,IL-6,IFN-γ,G-CSF or GM-CSF,or for 12 h with or without 1,10 or 50 ng/mL TNF-α.After incubation,cells were stained with annexin Ⅴ(AV)/propidium iodide(PI),and the proportions and absolute number of neutrophils undergoing different forms of cell death were determined by fluorescence microscopy combined with manual counting.Pyroptotic neutrophils were identified by cell morphology in conjunction with AV/PI staining.Flow cytometry with counting beads was employed to measure the proportions and number of AV/PI-stained Ly6g⁺neutrophils in different forms of cell death.Western blotting was employed to assess the cleavage and activation levels of cysteinyl aspartate-specific proteinase-3(caspase-3)and gasdermin E(GSDME).Results Treatment with IL-1β or IL-6 had no significant effect on the proportion or number of neutrophils undergoing spontaneous pyroptosis.After 12 h of treatment with TNF-α at 1,10,and 50 ng/mL,the proportions of pyroptotic neutrophils were(14.79±0.45)%,(19.99±3.02)%,and(20.66±1.99)%,respectively,higher than that[(10.22±1.12)%]in the untreated control(P=0.024,P<0.001,and P<0.001,respectively).Treatment with 10,50,and 100 ng/mL IFN-γ for 20 h reduced the proportion of pyroptotic neutrophils from(17.43±1.88)%to 12.00%(all P<0.001).G-CSF at 10,50,and 100 ng/mL reduced the proportion of pyroptotic cells to around 6.00%and greatly inhibited the cleavage of both caspase-3 and GSDME.After 20 h of treatment with 10,50,and 100 ng/mL GM-CSF,the proportions of pyroptotic neutrophils decreased to(7.52±0.53)%,(5.27±2.30)%,and(0.64±1.11)%,respectively.Conclusions Neither IL-1β nor IL-6 affects GSDME-mediated spontaneous pyroptosis in neutrophils.TNF-ɑ induces spontaneous pyroptosis in neutrophils,whereas IFN-γ,G-CSF,and GM-CSF demonstrate inhibitory effects.
Pyroptosis/drug effects* ; Animals ; Neutrophils/cytology* ; Mice ; Cytokines/pharmacology* ; Interleukin-1beta/pharmacology* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology* ; Cells, Cultured ; Granulocyte Colony-Stimulating Factor/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Interferon-gamma/pharmacology* ; Interleukin-6/pharmacology*

OBJECTIVE: To observe the protective effect and mechanism of hydroxyl safflower yellow A (HSYA) from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with oxygen-glucose deprivation reperfusion (OGD/R) to simulate the ischemia reperfusion model, and cell counting kit-8 was used to detect the protective effect of different concentrations (1.25-160 µ mol/L) of HSYA on HUVECs after OGD/R. HSYA 80 µ mol/L was used for follow-up experiments. The contents of inflammatory cytokines interleukin (IL)-18, IL-1 β, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α) and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay. The protein expressions of toll-like receptor, NOD-like receptor containing pyrin domain 3 (NLRP3), gasdermin D (GSDMD) and GSDMD-N-terminal domain (GSDMD-N) before and after administration were detected by Western blot. NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt (CRID3 sodium salt, also known as MCC950) and agonist were added, and the changes of NLRP3, cysteine-aspartic acid protease 1 (Caspase-1), GSDMD and GSDMD-N protein expressions were detected by Western blot. RESULTS: HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18, IL-1 β, MCP-1, TNF-α and IL-6 (P<0.01 or P<0.05). At the same time, by inhibiting NLRP3/Caspase-1/GSDMD pathway, HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3, Caspase-1, GSDMD and GSDMD-N proteins (P<0.01). CONCLUSIONS The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis.
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Chalcone/analogs & derivatives* ; Quinones/pharmacology* ; Pyroptosis/drug effects* ; Caspase 1/metabolism* ; Glucose ; Phosphate-Binding Proteins/metabolism* ; Signal Transduction/drug effects* ; Intracellular Signaling Peptides and Proteins/metabolism* ; Oxygen/metabolism* ; Cytokines/metabolism* ; Gasdermins

OBJECTIVE: To investigate the inhibitory effect of Celastrus orbiculatus extracts (COE) on the proliferation of lymphoma cells and the immune regulation ability on inflammation and thrombophilia in vivo. METHODS: The 38B9 lymphoma cells were treated with COE (160 µ g/mL) and CTX (25 µ mol/L). The apoptosis rate and cell cycle of each group were detected by flow cytometry. The secretion of inflammatory factors, including interleukin (IL)-6, IL-10, and tumor necrosis factor α (TNF-α), in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). In vivo, BALB/c mice were subcutaneously injected with 38B9 lymphoma cells to establish lymphoma model. COE (3 mg·kg^-1·d^-1) and CTX (40 mg·kg^-1·d^-1) were administered to the model mice, respectively. The expression of plasma inflammatory factors (IL-6, IL-10 and TNF-α) and thrombus indexes, including D-dimer (D-D), von Willebrand factor (vWF) and tissue factor (TF), were detected by ELISA before tumor bearing (1 d), after tumor formation (14 d) and after intervention (21 d). PicoGreen dsDNA was used to detect the level of serum neutrophil extracellular traps (NETs). Flow cytometry was used to detect the expression of platelet activation marker calcium-dependent lectin-like receptor 2 (CLEC-2). The tumor growth and survival of mice were recorded. RESULTS: The 38B9 lymphoma cells were apoptotic after the intervention of COE and CTX. The ratio of G₂-M phase cells decreased in COE intervented cells compared with the control cells (P<0.05), and S phase cells decreased in CTX intervented cells (P<0.05). Also, the secretion level of IL-6 was significantly reduced after COE or CTX intervention (P<0.05), and IL-10 was significantly increased (P<0.05). Furthermore, the tumor mass was reduced, and the median survival time was longer in COE and CTX intervented tumor-bearing mice than in non-intervented mice. The significantly lower levels of TNF-α, IL-6, NETs, TF, DD and CLEC-2, as well as higher IL-10 were observed in COE and CTX treatment mice in comparision with the control mice (P<0.05). CONCLUSION COE has a mild and stable anti-tumor effect, which can reduce the secretion of inflammatory factors by lymphoma cells and regulate thrombophilic state caused by tumor inflammatory microenvironment.
Animals ; Plant Extracts/pharmacology* ; Mice, Inbred BALB C ; Thrombosis/drug therapy* ; Celastrus/chemistry* ; Cell Line, Tumor ; Lymphoma, B-Cell/pathology* ; Apoptosis/drug effects* ; Inflammation/pathology* ; Cell Proliferation/drug effects* ; Mice ; Cell Cycle/drug effects* ; Male ; Cytokines/metabolism* ; Inflammation Mediators/metabolism*

OBJECTIVE: To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism. METHODS: THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot. RESULTS: 1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway. CONCLUSION Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.
Humans ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/genetics* ; Macrophages/metabolism* ; Cytokines/metabolism* ; Lipopolysaccharides/pharmacology* ; NF-kappa B

This study aims to explore the neuroprotective effect of bilobalide(BB) and the mechanisms such as inhibiting inflammatory response in macrophage/microglia, promoting neurotrophic factor secretion, and interfering with the activation and differentiation of peripheral CD4~+ T cells. BB of different concentration(12.5, 25, 50, 100 μg·mL~(-1)) was used to treat the RAW264.7 and BV2 cells for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and cell counting kit-8(CCK-8) were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment. Lipopolysaccharide(LPS) was applied to elicit inflammation in RAW264.7 and BV2 cells, mouse bone marrow-derived macrophages(BMDMs), and primary microglia, respectively. The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA). Spleen monocytes of C57BL/6 female mice(7-8 weeks old) were isolated, and CD4~+ T cells were separated by magnetic beads under sterile conditions. Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs. The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4~+ T cells. In addition, PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed. The cytoto-xicity was determined by lactate dehydrogenase(LDH) assay. The result showed that BB with the concentration of 12.5-100 μg·mL~(-1) had no toxicity to RAW264.7 and BV2 cells, and had no significant effect on the activity of cell model with low inflammation. The 50 μg·mL~(-1) BB was selected for further experiment, and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines. The experiment on CD4~+ T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activation and differentiation of CD4~+ T cells, while the conditioned medium of the experimental group with BB intervention reduced the activation and differentiation of CD4~+ T cells. In addition, BB also enhanced the release of neurotrophic factors from BMDMs and primary microglia. The conditioned medium after BB intervention can significantly reduce the death of PC12 neurons, inhibit neuronal damage, and protect neurons. To sum up, BB plays a neuroprotective role by inhibiting macrophage and microglia-mediated inflammatory response and promoting neurotrophic factors.
Female ; Rats ; Mice ; Animals ; Bilobalides/pharmacology* ; Neuroprotection ; Lipopolysaccharides/toxicity* ; Culture Media, Conditioned/pharmacology* ; Mice, Inbred C57BL ; Macrophages/metabolism* ; Microglia ; Cytokines/metabolism* ; Nerve Growth Factors/pharmacology* ; Inflammation/metabolism*

Platelets not only have hemostatic function, but can also directly or indirectly recognize pathogenic microorganisms and the signals they produce to capture and destroy them through membrane receptors. They can collaborate with various components of the body's immune system by releasing of intraplatelet particulate matter, cytokines and chemokines to perform bactericidal functions. And it can also play a bactericidal role by swallowing pathogens, releasing antimicrobial proteins and chemokines and activating and enhancing other specialized anti-inflammatory cells bactericidal effect, such as leukocytes and so on. However, the bacteriostatic composition and bacteriostatic mechanism of platelets remain unclear, so attention should be paid to the immune mechanism and bacteriostatic effect of platelets.
Blood Platelets ; Anti-Bacterial Agents/pharmacology* ; Cytokines ; Leukocytes ; Particulate Matter

Tumors in which the microenvironment is characterized by lack of immune cell infiltration are referred as "cold tumors" and typically exhibit low responsiveness to immune therapy. Targeting the factors contributing to "cold tumors" formation and converting them into "hot tumors" is a novel strategy for improving the efficacy of immunotherapy. Adenosine, a hydrolysis product of ATP, accumulates with a significantly higher concentration in the tumor microenvironments compared with normal tissue and exerts inhibitory effects on tumor-specific adaptive immunity. Tumor cells, dendritic cells, macrophages, and T cells express abundant adenosine receptors on their surfaces. The binding of adenosine to these receptors initiates downstream signaling pathways that suppress tumor antigen presentation and immune cell activation, consequently dampening adaptive immune responses against tumors. Adenosine down-regulates the expression of major histocompatibility complex Ⅱ and co-stimulatory factors on dendritic cells and macrophages, thereby inhibiting antigen presentation to T cells. Adenosine also inhibits ligand-receptor binding and transmembrane signaling on T cells, concomitantly suppressing the secretion of anti-tumor cytokines and impairing T cell activation. Furthermore, adenosine hinders effector T cell trafficking to tumor sites and infiltration by inhibiting chemokine secretion and KCa3.1 channels. Additionally, adenosine promotes the secretion of immunosuppressive cytokines, increases immune checkpoint protein expression, and enhances the activity of immunosuppressive cells, collectively curbing cytotoxic T cell-mediated tumor cell killing. Given the immunosuppressive role of adenosine in adaptive antitumor immunity, several inhibitors targeting adenosine generation or adenosine receptor blockade are currently in preclinical or clinical development with the aim of enhancing the effectiveness of immunotherapies. This review provides an overview of the inhibitory effects of adenosine on adaptive antitumor immunity, elucidate the molecular mechanisms involved, and summarizes the latest advances in application of adenosine inhibition strategies for antitumor immunotherapy.
Humans ; Adenosine/pharmacology* ; T-Lymphocytes ; Adaptive Immunity ; Cytokines ; Neoplasms/therapy* ; Tumor Microenvironment