The calcineurin-NFATc pathway modulates the lipid mediators in BAL fluid extracellular vesicles, thereby regulating microvascular endothelial cell barrier function.

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function.

Epigallocatechin gallate regulates the myeloid-specific transcription factor PU.1 in macrophages.

Our previous research demonstrated that PU.1 regulates expression of the genes involved in inflammation in macrophages. Selective knockdown of PU.1 in macrophages ameliorated LPS-induced acute lung injury (ALI) in bone marrow chimera mice. Inhibitors that block the transcriptional activity of PU.1 in macrophages have the potential to mitigate the pathophysiology of LPS-induced ALI. However, complete inactivation of PU.1 gene disrupts normal myelopoiesis. Although the green tea polyphenol Epigallocatechin gallate (EGCG) has been shown to regulate inflammatory genes in various cell types, it is not known if EGCG alters the transcriptional activity of PU.1 protein. Using Schrodinger Glide docking, we have identified that EGCG binds with PU.1 protein, altering its DNA-binding and self-dimerization activity. In silico analysis shows that EGCG forms Hydrogen bonds with Glutamic Acid 209, Leucine 250 in DNA binding and Lysine 196, Tryptophan 193, and Leucine 182 in the self-dimerization domain of the PU.1 protein. Experimental validation using mouse bone marrow-derived macrophages (BMDM) confirmed that EGCG inhibits both DNA binding by PU.1 and self-dimerization. Importantly, EGCG had no impact on expression of the total PU.1 protein levels but significantly reduced expression of various inflammatory genes and generation of ROS. In summary, we report that EGCG acts as an inhibitor of the PU.1 transcription factor in macrophages.

H3K4 Trimethylation Mediate Hyperhomocysteinemia Induced Neurodegeneration via Suppressing Histone Acetylation by ANP32A.

Homocysteine (Hcy) is an independent and serious risk factor for dementia, including Alzheimer's disease (AD), but the precise mechanisms are still poorly understood. In the current study, we observed that the permissive histone mark trimethyl histone H3 lysine 4 (H3K4me3) and its methyltransferase KMT2B were significantly elevated in hyperhomocysteinemia (HHcy) rats, with impairment of synaptic plasticity and cognitive function. Further research found that histone methylation inhibited synapse-associated protein expression, by suppressing histone acetylation. Inhibiting H3K4me3 by downregulating KMT2B could effectively restore Hcy-inhibited H3K14ace in N2a cells. Moreover, chromatin immunoprecipitation revealed that Hcy-induced H3K4me3 resulted in ANP32A mRNA and protein overexpression in the hippocampus, which was regulated by increased transcription Factor c-fos and inhibited histone acetylation and synapse-associated protein expression, and downregulating ANP32A could reverse these changes in Hcy-treated N2a cells. Additionally, the knockdown of KMT2B restored histone acetylation and synapse-associated proteins in Hcy-treated primary hippocampal neurons. These data have revealed a novel crosstalk mechanism between KMT2B-H3K4me3-ANP32A-H3K14ace, shedding light on its role in Hcy-related neurogenerative disorders.

Elamipretide(SS-31) Attenuates Idiopathic Pulmonary Fibrosis by Inhibiting the Nrf2-Dependent NLRP3 Inflammasome in Macrophages.

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal lung disease with a limited therapeutic strategy. Mitochondrial oxidative stress in macrophages is directly linked to IPF. Elamipretide(SS-31) is a mitochondrion-targeted peptide that has been shown to be safe and beneficial for multiple diseases. However, whether SS-31 alleviates IPF is unclear. In the present study, we used a bleomycin (BLM)-induced mouse model followed by SS-31 injection every other day to investigate its role in IPF and explore the possible mechanism. Our results showed that SS-31 treatment significantly suppressed BLM-induced pulmonary fibrosis and inflammation, with improved histological change, and decreased extracellular matrix deposition and inflammatory cytokines release. Impressively, the expression percentage of IL-1ß and IL-18 was downregulated to lower than half with SS-31 treatment. Mechanistically, SS-31 inhibited IL-33- or lipopolysaccharide(LPS)/IL-4-induced production of IL-1ß and IL-18 in macrophages by suppressing NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammasome activation. Nuclear factor erythroid 2-related factor 2(Nrf2) was dramatically upregulated along with improved mitochondrial function after SS-31 treatment in activated macrophages and BLM-induced mice. Conversely, there was no significant change after SS-31 treatment in Nrf2-/- mice and macrophages. These findings indicated that SS-31 protected against pulmonary fibrosis and inflammation by inhibiting the Nrf2-mediated NLRP3 inflammasome in macrophages. Our data provide initial evidence for the therapeutic efficacy of SS-31 in IPF.

Aerobic exercise attenuates autophagy-lysosomal flux deficits by ADRB2/ß2-adrenergic receptor-mediated V-ATPase assembly factor VMA21 signaling in APP-PSEN1/PS1 mice.

Growing evidence suggests that macroautophagy/autophagy-lysosomal pathway deficits contribute to the accumulation of amyloid-ß (Aß) in Alzheimer disease (AD). Aerobic exercise (AE) has long been investigated as an approach to delay and treat AD, although the exact role and mechanism are not well known. Here, we revealed that AE could reverse autophagy-lysosomal deficits via activation of ADRB2/ß2-adrenergic receptor, leading to significant attenuation of amyloid-ß pathology in APP-PSEN1/PS1 mice. Molecular mechanism research found that AE could reverse autophagy deficits by upregulating the AMP-activated protein kinase (AMPK)-MTOR (mechanistic target of rapamycin kinase) signaling pathway. Moreover, AE could reverse V-ATPase function by upregulating VMA21 levels. Inhibition of ADRB2 by propranolol (antagonist, 30 µM) blocked AE-attenuated Aß pathology and cognitive deficits by inhibiting autophagy-lysosomal flux. AE may mitigate AD via many pathways, while ADRB2-VMA21-V-ATPase could improve cognition by enhancing the clearance of Aß through the autophagy-lysosomal pathway, which also revealed a novel theoretical basis for AE attenuating pathological progression and cognitive deficits in AD.

Fraxetin alleviates BLM-induced idiopathic pulmonary fibrosis by inhibiting NCOA4-mediated epithelial cell ferroptosis.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a debilitating lung condition with few available treatments. The early driver of wound repair that contributes to IPF has been extensively identified as repetitive alveolar epithelial damage. According to recent reports, IPF is linked to ferroptosis, a unique type of cell death characterized by a fatal buildup of iron and lipid peroxidation. OBJECTIVE AND METHOD: There is little information on epithelial cells that induce pulmonary fibrosis by going through ferroptosis. In this study, we used bleomycin (BLM) to examine the impact of ferroptosis on IPF in mouse lung epithelial cells (MLE-12). RESULTS: We discovered that BLM increases ferroptosis in MLE-12. Additionally, we found that NCOA4 is overexpressed and plays a key role in the ferroptosis of epithelial cells throughout the IPF process. Using Molecular docking, we found that Fraxetin, a natural component extracted from Fraxinus rhynchophylla, formed a stable binding to NCOA4. In vitro investigations showed that Fraxetin administration greatly decreased ferroptosis and NCOA4 expression, which in turn lowered the release of inflammatory cytokines. CONCLUSION: Fraxetin treatment significantly alleviated BLM-induced lung inflammation and fibrosis. Our findings imply that fraxetin possesses inhibitory roles in ferroptosis and can be a potential drug against IPF.

Orexin A alleviates LPS-induced acute lung injury by inhibiting macrophage activation through JNK-mediated autophagy.

Crosstalk between the central nervous system and immune system by the neuroendocrine and autonomic nervous systems is critical during the inflammatory response. Exposure to endotoxin alters the activity of hypothalamic homeostatic systems, resulting in changed transmitter release within the brain. This study investigated the effects and cellular molecular mechanisms of neurogenic and exogenous orexin-A (OXA) in LPS-induced acute lung injury (ALI). We found the production of OXA in the hypothalamus and lungs was both decreased following LPS infection. LPS-induced lung injury including the destruction of the structure, inflammatory cell infiltration, and pro-inflammatory cytokines generation was aggravated in mice in which orexin neurons were lesioned with the neurotoxin orexin-saporin (orexin-SAP). Administration of exogenous OXA greatly improved lung pathology and reduced inflammatory response. Orexin receptors were found in cultured mouse bone marrow-derived macrophages (BMDMs) and lung macrophages (LMs), adoptive transfer of OXA-treated macrophages showed alleviative lung injury compared to adoptive transfer of macrophages without OXA treatment. Mechanistically, it is the induction of autophagy via JNK activation that is responsible for OXA to suppress macrophage-derived pro-inflammatory cytokine production. These findings highlight the importance of neuro-immune crosstalk and indicate that OXA may be a potential therapeutic agent in the treatment of ALI.

NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages.

Idiopathic pulmonary fibrosis (IPF) is a progressive and highly lethal inflammatory interstitial lung disease characterized by aberrant extracellular matrix deposition. Macrophage activation by cytokines released from repetitively injured alveolar epithelial cells regulates the inflammatory response, tissue remodeling, and fibrosis throughout various phases of IPF. Our previous studies demonstrate that nuclear factor of activated T cells cytoplasmic member 3 (NFATc3) regulates a wide array of macrophage genes during acute lung injury pathogenesis. However, the role of NFATc3 in IPF pathophysiology has not been previously reported. In the current study, we demonstrate that expression of NFATc3 is elevated in lung tissues and pulmonary macrophages in mice subjected to bleomycin (BLM)-induced pulmonary fibrosis and IPF patients. Remarkably, NFATc3 deficiency (NFATc3+/-) was protective in bleomycin (BLM)-induced lung injury and fibrosis. Adoptive transfer of NFATc3+/+ macrophages to NFATc3+/- mice restored susceptibility to BLM-induced pulmonary fibrosis. Furthermore, in vitro treatment with IL-33 or conditioned medium from BLM-treated epithelial cells increased production of CCL2 and CXCL2 in macrophages from NFATc3+/+ but not NFATc3+/- mice. CXCL2 promoter-pGL3 Luciferase reporter vector showed accentuated reporter activity when co-transfected with the NFATc3 expression vector. More importantly, exogenous administration of recombinant CXCL2 into NFATc3+/- mice increased fibrotic markers and exacerbated IPF phenotype in BLM treated mice. Collectively, our data demonstrate, for the first time, that NFATc3 regulates pulmonary fibrosis by regulating CCL2 and CXCL2 gene expression in macrophages.

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo.

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is accompanied by the activation of inflammatory cells such as macrophages and neutrophils, as well as the release of inflammatory factors including TNF-α, IL-1ß, and IL-6. Early inflammation caused by activated pulmonary interstitial macrophages (IMs) in response to IL-33 stimulation is known to play a vital role in the pathological process of IPF. This protocol describes the adoptive transfer of IMs stimulated by IL-33 into the lungs of mice to study IPF development. It involves the isolation and culture of primary IMs from host mouse lungs, followed by the adoptive transfer of stimulated IMs into the alveoli of bleomycin (BLM)-induced IPF recipient mice (which have been previously depleted of alveolar macrophages by treatment with clodronate liposomes), and the pathological evaluation of those mice. The representative results show that the adoptive transfer of IL-33-stimulated macrophages aggravates pulmonary fibrosis in mice, suggesting that the establishment of the macrophage adoptive transfer experiment is a good technical means to study IPF pathology.

4-OI ameliorates bleomycin-induced pulmonary fibrosis by activating Nrf2 and suppressing macrophage-mediated epithelial-mesenchymal transition.

OBJECTIVES: Pulmonary fibrosis (PF) is a chronic and refractory interstitial lung disease with limited therapeutic options. 4-octyl itaconate (4-OI), a cell-permeable derivative of itaconate, has been shown to have anti-oxidative and anti-inflammatory properties. However, the effect and the underlying mechanism of 4-OI on PF are still unknown. METHODS: WT or Nrf2 knockout (Nrf2-/-) mice were intratracheally injected with bleomycin (BLM) to establish PF model and then treated with 4-OI. The mechanism study was performed by using RAW264.7 cells, primary macrophages, and conditional medium-cultured MLE-12 cells. RESULTS: 4-OI significantly alleviated BLM-induced PF and EMT process. Mechanism studies have found that 4-OI can not only directly inhibit EMT process, but also can reduce the production of TGF-ß1 by restraining macrophage M2 polarization, which in turn inhibits EMT process. Moreover, the effect of 4-OI on PF and EMT depends on Nrf2. CONCLUSION: 4-OI ameliorates BLM-induced PF in an Nrf2-dependent manner, and its role in alleviating PF is partly due to the direct inhibition on EMT, and partly through indirect inhibition of M2-mediated EMT. These findings suggested that 4-OI has great clinical potential to develop as a new anti-fibrotic agent for PF therapy.

NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophage.

Idiopathic pulmonary fibrosis (IPF) is a progressive and highly lethal inflammatory interstitial lung disease characterized by aberrant extracellular matrix deposition. Macrophage activation by cytokines released from repetitively injured alveolar epithelial cells regulates the inflammatory response, tissue remodeling, and fibrosis throughout various phases of IPF. Our previous studies demonstrate that nuclear factor of activated T cells cytoplasmic member 3 (NFATc3) regulates a wide array of macrophage genes during acute lung injury pathogenesis. However, the role of NFATc3 in IPF pathophysiology has not been previously reported. In the current study, we demonstrate that expression of NFATc3 is elevated in lung tissues and pulmonary macrophages in mice subjected to bleomycin (BLM)-induced pulmonary fibrosis and IPF patients. Remarkably, NFATc3 deficiency (NFATc3+/-) was protective in bleomycin (BLM)-induced lung injury and fibrosis. Adoptive transfer of NFATc3+/+ macrophages to NFATc3+/- mice restored susceptibility to BLM-induced pulmonary fibrosis. Furthermore, in vitro treatment with IL-33 or conditioned medium from BLM-treated epithelial cells increased production of CCL2 and CXCL2 in macrophages from NFATc3+/+ but not NFATc3+/- mice. CXCL2 promoter-pGL3 Luciferase reporter vector showed accentuated reporter activity when co-transfected with the NFATc3 expression vector. More importantly, exogenous administration of recombinant CXCL2 into NFATc3+/- mice increased fibrotic markers and exacerbated IPF phenotype in BLM treated mice. Collectively, our data demonstrate, for the first time, that NFATc3 regulates pulmonary fibrosis by regulating CCL2 and CXCL2 gene expression in macrophages.

Minocycline Attenuates Sevoflurane-Induced Postoperative Cognitive Dysfunction in Aged Mice by Suppressing Hippocampal Apoptosis and the Notch Signaling Pathway-Mediated Neuroinflammation.

Postoperative cognitive dysfunction (POCD), an important postoperative neurological complication, is very common and has an elevated incidence in elderly patients. Sevoflurane, an inhaled anesthetic, has been demonstrated to be associated with POCD in both clinical and animal studies. However, how to prevent POCD remains unclear. Minocycline, a commonly used antibiotic can cross the blood-brain barrier and exert an inhibitory effect on inflammation in the central nervous system. The present work aimed to examine the protective effect and mechanism of minocycline on sevoflurane-induced POCD in aged mice. We found that 3% sevoflurane administered 2 h a day for 3 consecutive days led to cognitive impairment in aged animals. Further investigation revealed that sevoflurane impaired synapse plasticity by causing apoptosis and neuroinflammation and thus induced cognitive dysfunction. However, minocycline pretreatment (50 mg/kg, i.p, 1 h prior to sevoflurane exposure) significantly attenuated learning and memory impairments associated with sevoflurane in aged animals by suppressing apoptosis and neuroinflammation. Moreover, a mechanistic analysis showed that minocycline suppressed sevoflurane-triggered neuroinflammation by inhibiting Notch signaling. Similar results were also obtained in vitro. Collectively, these findings suggested minocycline may be an effective drug for the prevention of sevoflurane-induced POCD in elderly patients.

Activation of ß2-adrenergic Receptor Ameliorates Amyloid-ß-induced Mitophagy Defects and Tau Pathology in Mice.

Defective mitophagy and mitochondrial dysfunction have been linked to aging and Alzheimer's disease (AD). ß2-Adrenergic receptor (ADRB2) is critical for mitochondrial and cognitive function. However, researchers have not clearly determined whether ADRB2 activation ameliorates defective mitophagy and cognitive deficits in individuals with AD. Here, we observed that the activation of ADRB2 by clenbuterol (Clen, ADRB2 agonist, 2 mg/kg/day) ameliorated amyloid-ß-induced (Aß1-42 bilateral intracerebral infusion, 2 µl, 5 µg/µl) memory deficits. Activation of ADRB2 also attenuated Aß-induced mitochondrial dysfunction, as revealed by increased ATP levels, mitochondrial membrane potential (MMP/Δψm) and complex I activity. Further studies revealed that ADRB2 activation restored mitophagy deficits, as revealed by the increased light chain 3 (LC3)-II/LC3-I ratio, Atg5 levels, and Atg7 levels and decreased p62 levels, along with the upregulation of PTEN-induced putative kinase 1 (PINK1), Parkin and NAD+ levels. Activation of ADRB2 rescued Aß-induced oxidative stress and neuronal death. ADRB2 activation also attenuated Aß-induced tau hyperphosphorylation by regulating glycogen synthase kinase-3ß expression in the hippocampus. Finally, we established that Clen improved mitophagy and attenuated mitochondrial dysfunction, and tau pathology in mice by activating the ADRB2/Akt/PINK1 signaling pathway. Conversely, the inhibition of ADRB2 by propranolol (ßAR antagonist, 10 µM) blocked the Clen-mediated improvements in pathological changes in N2a cells. The results from the present study indicate that ADRB2 activation may be a therapeutic strategy for AD.

Investigation into the anti-airway inflammatory role of the PI3Kγ inhibitor JN-PK1: An in vitro and in vivo study.

Phosphatidylinositol 3-kinase gamma (PI3Kγ) has been proven to be a potential target for the treatment of inflammatory diseases of the airway; however, there are few reports of selective PI3Kγ inhibitors being used in the field of airway inflammation thus far. Herein, a study employing in vitro and in vivo methodologies was carried out to assess the anti-airway inflammatory effects of JN-PK1, a selective PI3Kγ inhibitor. In RAW264.7 macrophages, JN-PK1 inhibited PI3Kγ-dependent, cellular C5a-induced AKT Ser473 phosphorylation in a concentration- and time-dependent manner and had no significant effect on cell viability.Furthermore, JN-PK1 significantly suppressed LPS-induced, proinflammatory cytokine expression and nitric oxide production through inhibition of the PI3K signaling pathway in RAW264.7 cells. Then, a murine asthma model was established to evaluate the anti-airway inflammation effect of JN-PK1. BALB/c mice were sensitized and challenged with ovalbumin (OVA) to develop an inflammatory response, fibrosis formation, and other airway changes similar to the symptomatology of asthma in humans. Oral administration of JN-PK1 remarkably attenuated OVA-induced asthma in association with the inhibition of the PI3K signaling pathway. That is to say, the oral administration significantly inhibited increases in inflammatory cell counts and reduced T-helper type 2 cytokine production in bronchoalveolar lavage fluid. Pulmonary histological studies showed that oral administration of JN-PK1 not only reduced the infiltration of inflammatory cells but also retarded airway inflammation and fibration. Taken together, JN-PK1 could be developed as a promising candidate for inflammation therapy, and our findings support some potential for therapeutic inhibition of PI3Kγ to treat inflammatory airway diseases.

Sevoflurane inhibits histone acetylation and contributes to cognitive dysfunction by enhancing the expression of ANP32A in aging mice.

Postoperative cognitive dysfunction (POCD) is a major clinical complication after surgery under general anesthesia, particularly in elderly patients, but the mechanisms remain unclear. We recently found that the anaesthetization of aging C57BL/6 J mice (14-16 months) with sevoflurane (3%, two hours each day for three consecutive days) can induce cognitive dysfunction and synaptic plasticity deficits. Further studies demonstrated that sevoflurane induced ANP32A (acidic leucine-rich nuclear phosphoprotein-32A) overexpression by stimulating C/EBPß (CCAAT/enhancer binding protein-ß), which could suppress histone acetylation at H3K18, H3K14, H4K5, and H4K12 and decrease the binding of H3K18 and H3K14 to the promoters of GluN2B and GluN2A, respectively. These results suggest that sevoflurane can inhibit histone acetylation and contribute to cognitive dysfunction by enhancing the expression of ANP32A in aging mice. Our study provides new insights into aging-associated POCD and potential molecular markers for protection.

Role of IL-17 family cytokines in the progression of IPF from inflammation to fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic interstitial lung disease with no established treatment and is characterized by progressive scarring of the lung tissue and an irreversible decline in lung function. Chronic inflammation has been demonstrated to be the pathological basis of fibrosis. Emerging studies have revealed that most interleukin-17 (IL-17) isoforms are essential for the mediation of acute and chronic inflammation via innate and adaptive immunity. Overexpression or aberrant expression of IL-17 cytokines contributes to various pathological outcomes, including the initiation and exacerbation of IPF. Here, we aim to provide an overview of IL-17 family members in the pathogenesis of IPF.

Dehydrocostus Lactone Attenuates Methicillin-Resistant Staphylococcus aureus-Induced Inflammation and Acute Lung Injury via Modulating Macrophage Polarization.

Dehydrocostus lactone (DHL), a natural sesquiterpene lactone isolated from the traditional Chinese herbs Saussurea lappa and Inula helenium L., has important anti-inflammatory properties used for treating colitis, fibrosis, and Gram-negative bacteria-induced acute lung injury (ALI). However, the effects of DHL on Gram-positive bacteria-induced macrophage activation and ALI remains unclear. In this study, we found that DHL inhibited the phosphorylation of p38 MAPK, the degradation of IκBα, and the activation and nuclear translocation of NF-κB p65, but enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of Nrf2 and HO-1 in lipoteichoic acid (LTA)-stimulated RAW264.7 cells and primary bone-marrow-derived macrophages (BMDMs). Given the critical role of the p38 MAPK/NF-κB and AMPK/Nrf2 signaling pathways in the balance of M1/M2 macrophage polarization and inflammation, we speculated that DHL would also have an effect on macrophage polarization. Further studies verified that DHL promoted M2 macrophage polarization and reduced M1 polarization, then resulted in a decreased inflammatory response. An in vivo study also revealed that DHL exhibited anti-inflammatory effects and ameliorated methicillin-resistant Staphylococcus aureus (MRSA)-induced ALI. In addition, DHL treatment significantly inhibited the p38 MAPK/NF-κB pathway and activated AMPK/Nrf2 signaling, leading to accelerated switching of macrophages from M1 to M2 in the MRSA-induced murine ALI model. Collectively, these data demonstrated that DHL can promote macrophage polarization to an anti-inflammatory M2 phenotype via interfering in p38 MAPK/NF-κB signaling, as well as activating the AMPK/Nrf2 pathway in vitro and in vivo. Our results suggested that DHL might be a novel candidate for treating inflammatory diseases caused by Gram-positive bacteria.

Dehydrocostus lactone inhibits BLM-induced pulmonary fibrosis and inflammation in mice via the JNK and p38 MAPK-mediated NF-κB signaling pathways.

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible inflammatory disease with a high mortality rate and limited therapeutic options. This study explored the potential role and mechanisms of Dehydrocostus lactone (DHL) in the inflammatory and fibrotic responses in a bleomycin (BLM) induced model. Treatment with DHL significantly reduced pathological injury and fibrosis, the secretion of BLM-induced pro-fibrotic mediators TGF-ß and α-SMA, and components of the extracellular matrix (fibronectin). Additionally, in the early stages of inflammation, DHL administration inhibited the infiltration of inflammatory cells and downregulated the expression of TGF-ß, TNF-α, and IL-6, indicating that DHL treatment effectively alleviated BLM-induced pulmonary fibrosis and inflammation in a dose-dependent manner. Furthermore, BLM induced the production of IL-33 in vivo, which initiated and progressed pulmonary fibrosis by activating macrophages and enhancing the production of IL-13 and TGF-ß. In contrast, a significant decrease in the expression of IL-33 after DHL treatment in vitro showed that DHL strongly reduced IL-13 and TGF-ß. Regarding the mechanism, BLM-induced phosphorylation of JNK, p38 MAPK, and NF-κB were significantly reduced after DHL treatment, which further led to the down-regulation of IL-33 expression, thereby decreasing IL-13 and TGF-ß. Collectively, our data suggested that DHL could exert its anti-fibrosis effect via inhibiting the early inflammatory response by downregulating the JNK/p38 MAPK-mediated NF-κB signaling pathway to suppress macrophage activation. Therefore, DHL has therapeutic potential for pulmonary fibrosis.

PolyADP-Ribosylation of NFATc3 and NF-κB Transcription Factors Modulate Macrophage Inflammatory Gene Expression in LPS-Induced Acute Lung Injury.

Pulmonary macrophages play a critical role in the recognition of pathogens, initiation of host defense via inflammation, clearance of pathogens from the airways, and resolution of inflammation. Recently, we have shown a pivotal role for the nuclear factor of activated T-cell cytoplasmic member 3 (NFATc3) transcription factor in modulating pulmonary macrophage function in LPS-induced acute lung injury (ALI) pathogenesis. Although the NFATc proteins are activated primarily by calcineurin-dependent dephosphorylation, here we show that LPS induces posttranslational modification of NFATc3 by polyADP-ribose polymerase 1 (PARP-1)-mediated polyADP-ribosylation. ADP-ribosylated NFATc3 showed increased binding to iNOS and TNFα promoter DNA, thereby increasing downstream gene expression. Inhibitors of PARP-1 decreased LPS-induced NFATc3 ribosylation, target gene promoter binding, and gene expression. LPS increased NFAT luciferase reporter activity in lung macrophages and lung tissue that was inhibited by pretreatment with PARP-1 inhibitors. More importantly, pretreatment of mice with the PARP-1 inhibitor olaparib markedly decreased LPS-induced cytokines, protein extravasation in bronchoalveolar fluid, lung wet-to-dry ratios, and myeloperoxidase activity. Furthermore, PARP-1 inhibitors decreased NF-кB luciferase reporter activity and LPS-induced ALI in NF-кB reporter mice. Thus, our study demonstrates that inhibiting NFATc3 and NF-кB polyADP-ribosylation with PARP-1 inhibitors prevented LPS-induced ALI pathogenesis.