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.

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.

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.

S-allyl-l-cysteine attenuates bleomycin-induced pulmonary fibrosis and inflammation via AKT/NF-κB signaling pathway in mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by inflammation, multifocal fibrotic lesions and excessive collagen deposition with limited therapies. As a major bioactive compound in garlic, S-allyl-l-cysteine (SAC) is a neuroprotective drug candidate to prevent cognitive decline, however, its anti-pulmonary fibrotic activity remains unknown. Here, we investigated whether SAC could attenuate bleomycin (BLM)-induced pulmonary fibrosis and inflammation in mice. Our results showed that SAC dose-dependently reduced the infiltration of inflammatory cells, pulmonary lesions and collagen deposition in BLM treated mice with downregulated mRNA expression levels of fibrotic genes including alpha smooth muscle actin (α-SMA), fibronectin, collagen I and collagen III as well as the protein level of α-SMA. In addition, SAC could also reduce the mRNA expression of inflammatory mediators such as TNF-α and iNOS. Furthermore, higher phosphorylation of AKT and NF-κB p65 in IPF patient samples and murine samples was verified by immunohistochemistry while SAC could decrease the phosphorylation level of AKT and NF-κB p65 in mice stimulated with BLM. These findings, for the first time, indicate that SAC might mediate AKT/NF-κB signaling pathway to inhibit BLM-induced pulmonary fibrosis and support the potential role of SAC as an anti-pulmonary fibrosis agent.

AKT2 Regulates Pulmonary Inflammation and Fibrosis via Modulating Macrophage Activation.

Idiopathic pulmonary fibrosis (IPF) is a highly lethal pathological process that is characterized by inflammation, fibroblast accumulation, and excessive collagen deposition. Although AKT2-mediated signaling pathways modulate inflammatory responses, their role in IPF has not been defined. We report that AKT2 deficiency (Akt2-/-) protected against bleomycin-induced pulmonary fibrosis and inflammation. Adoptive transfer of wild-type macrophages or administration of IL-13 to Akt2-/- mice could restore pulmonary fibrosis. In response to IL-33 treatment, Akt2-/- macrophages displayed decreased production of IL-13 and TGF-ß1 and attenuated phosphorylation of FoxO3a compared with Akt2+/+ macrophages. Furthermore, the expression of IL-13 was increased by small interfering RNA knockdown of FoxO3a or in FoxO3a-deficient macrophages. By evaluating lung sections from pulmonary fibrosis patients, we found that the phosphorylation of AKT2 and FoxO3a was remarkably upregulated. Collectively, these results indicate that AKT2 modulates pulmonary fibrosis through inducing TGF-ß1 and IL-13 production by macrophages, and inhibition of AKT2 may be a potential strategy for treating IPF.

Lanatoside C protects mice against bleomycin-induced pulmonary fibrosis through suppression of fibroblast proliferation and differentiation.

It has been established that lanatoside C, a FDA-approved cardiac glycoside, reduces proliferation of cancer cell lines. The proliferation of fibroblasts is critical to the pathogenesis of pulmonary fibrosis (PF), a progressive and fatal fibrotic lung disease lacking effective treatment. In this study we have investigated the impact of lanatoside C on a bleomycin (BLM)-induced mouse model of PF and through the evaluation of fibroblast proliferation and activation in vitro. We evaluated explanted lung tissue by histological staining, western blot analysis, qRT-PCR and survival analysis, demonstrating that lanatoside C was able to protect mice against BLM-induced pulmonary fibrosis. The proliferation of cultured pulmonary fibroblasts isolated from BLM-induced PF mice was suppressed by lanatoside C, as hypothesized, through the induction of cell apoptosis and cell cycle arrest at the G2/M phase. The Akt signalling pathway was involved in this process. Interestingly, the production of α-SMA, fibronectin, and collagen I and III in response to TGF-ß1 in healthy mouse fibroblasts was suppressed following lanatoside C administration by inhibition of TGF-ß1/Smad signalling. In addition, TGF-ß1-induced migration in lung fibroblasts was also impeded after lanatoside C treatment. Together, our data revealed that lanatoside C alleviated BLM-induced pulmonary fibrosis in mice via attenuation of growth and differentiation of fibroblasts, suggesting that it has potential as a candidate therapy for PF patients.

Dehydrocostus Lactone Suppresses LPS-induced Acute Lung Injury and Macrophage Activation through NF-κB Signaling Pathway Mediated by p38 MAPK and Akt.

Acute lung injury (ALI) is a severe clinical disease marked by dysregulated inflammation response and has a high rate of morbidity and mortality. Macrophages, which play diverse roles in the inflammatory response, are becoming therapeutic targets in ALI. In this study we investigated the effects of dehydrocostus lactone (DHL), a natural sesquiterpene, on macrophage activation and LPS-induced ALI. The macrophage cell line RAW264.7 and primary lung macrophages were incubated with DHL (0, 3, 5, 10 and 30 µmol/L) for 0.5 h and then challenged with LPS (100 ng/mL) for up to 8 hours. C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) to induce acute lung injury (ALI) and then treated with a range of DHL doses intraperitoneally (5 to 20 mg/kg). The results showed that DHL inhibited LPS-induced production of proinflammatory mediators such as iNOS, NO, and cytokines including TNF-α, IL-6, IL-1ß, and IL-12 p35 by suppressing the activity of NF-κB via p38 MAPK/MK2 and Akt signaling pathway in macrophages. The in vivo results revealed that DHL significantly attenuated LPS-induced pathological injury and reduced cytokines expression in the lung. NF-κB, p38 MAPK/MK2 and Akt signaling molecules were also involved in the anti-inflammatory effect. Collectively, our findings suggested that DHL is a promising agent for alleviating LPS-induced ALI.

Protostemonine effectively attenuates lipopolysaccharide-induced acute lung injury in mice.

Protostemonine (PSN) is the main anti-inflammatory alkaloid extracted from the roots of Stemona sessilifolia (known as "Baibu" in traditional Chinese medicine). Here, we reported the inhibitory effects of PSN on lipopolysaccharide (LPS)-induced macrophage activation in vitro and LPS-induced acute lung injury in mice. Macrophage cell line RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs) were treated with PSN (1, 3, 10, 30 and 100 µmol/L) for 0.5 h and then challenged with LPS (0.1 µg/mL) for 24 h. Pretreatment with PSN significantly inhibited LPS-induced phosphorylation of MAPKs and AKT, iNOS expression and NO production in the macrophages. C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) to induce acute lung injury (ALI). The mice were subsequently treated with PSN (10 mg/kg, ip) at 4 and 24 h after LPS challenge. PSN administration significantly attenuated LPS-induced inflammatory cell infiltration, reduced pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) production and eliminated LPS-mediated lung edema. Furthermore, PSN administration significantly inhibited LPS-induced pulmonary MPO activity. Meanwhile, LPS-induced phosphorylation of p38 MAPK, iNOS expression and NO production in the lungs were also suppressed. The results demonstrate that PSN effectively attenuates LPS-induced inflammatory responses in vitro and in vivo; the beneficial effects are associated with the decreased phosphorylation of MAPK and AKT and the reduced expression of pro-inflammatory mediators, such as iNOS, NO and cytokines. These data suggest that PSN may be a potential therapeutic agent in the treatment of ALI.

LYRM03, an ubenimex derivative, attenuates LPS-induced acute lung injury in mice by suppressing the TLR4 signaling pathway.

Toll-like receptor 4 (TLR4)-mediated signaling plays a critical role in sepsis-induced acute lung injury (ALI). LYRM03 (3-amino-2-hydroxy-4-phenyl-valyl-isoleucine) is a novel derivative of ubenimex, a widely used antineoplastic medicine. We previously found that LYRM03 has anti-inflammatory effects in cecal ligation puncture mouse model. In this study we determined whether LYRM03 attenuated LPS-induced ALI in mice. LPS-induced ALI mouse model was established by challenging the mice with intratracheal injection of LPS (5 mg/kg), which was subsequently treated with LYRM03 (10 mg/kg, ip). LYRM03 administration significantly alleviated LPS-induced lung edema, inflammatory cell (neutrophils and macrophages) infiltration and myeloperoxidase (MPO) activity, decreased pro-inflammatory and chemotactic cytokine (TNF-α, IL-6, IL-1ß, MIP-2) generation and reduced iNOS and COX-2 expression in the lung tissues. In cultured mouse alveolar macrophages in vitro, pretreatment with LYRM03 (100 µmol/L) suppressed LPS-induced macrophage activation by reducing Myd88 expression, increasing IκB stability and inhibiting p38 phosphorylation. These results suggest that LYRM03 effectively attenuates LPS-induced ALI by inhibiting the expression of pro-inflammatory mediators and Myd88-dependent TLR4 signaling pathways in alveolar macrophages. LYRM03 may serve as a potential treatment for sepsis-mediated lung injuries.

PTPA activates protein phosphatase-2A through reducing its phosphorylation at tyrosine-307 with upregulation of protein tyrosine phosphatase 1B.

Protein phosphatase-2A (PP2A), an important phosphatase in dephosphorylating tau and preserving synapse, is significantly suppressed in Alzheimer's disease (AD), but the mechanism is not well understood. Here, we studied whether phosphotyrosyl phosphatase activator (PTPA) could activate PP2A by reducing its inhibitory phosphorylation at tyrosine 307 (P-PP2AC). We found that overexpression of PTPA activated PP2A by decreasing the level of P-PP2AC with reduced phosphorylation of tau, while knockdown of PTPA inhibited PP2A by increasing the level of P-PP2AC with enhanced tau phosphorylation. We also observed that expression of PTPA could upregulate the protein and mRNA levels of protein tyrosine phosphatase 1B (PTP1B) and simultaneous downregulation of PTP1B eliminated PTPA-induced PP2A activation. Importantly, we also found that the protein level of PTPA is downregulated in the brains of AD patients, and the AD transgenic mouse models with expression of mutant human amyloid precursor protein (hAPP) or the longest human tau (htau), respectively. Our data indicate that PTPA may activate PP2A through activating PTP1B and thus reducing the level of P-PP2AC, therefore upregulation of PTPA may represent a potential strategy in rescuing PP2A and arresting tau pathology in AD.

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.

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.

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.

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.

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.

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.

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.

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.

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.