NR8383 Cells Research Articles

Penehyclidine hydrochloride inhibits renal ischemia/reperfusion-induced acute lung injury by activating the Nrf2 pathway.

The nuclear factor (NF)-κB and NOD-like receptor protein 3 (NLRP3) pathways promote inflammatory signaling that injures the kidneys, whereas the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway promotes anti-inflammatory signaling that inhibits oxidative damage. Penehyclidine hydrochloride (PHC) inhibits NF-κB and activates Nrf2 signaling. We investigated whether PHC induces communication between the Nrf2 and NF-κB/NLRP3 pathways, thereby protecting against renal ischemia/reperfusion (rI/R)-induced lung inflammation. Rat alveolar macrophages (NR8383 cells) were stimulated for 24 h with PHC with or without brusatol (a Nrf2 antagonist), after which they were treated for 4 h with tert-butyl hydroperoxide (10 mM). PHC Nrf2-dependently alleviated tert-butyl hydroperoxide-induced reactive oxygen species production in alveolar macrophages. Additionally, wild-type and Nrf2−/− rats were each divided into four groups: (1) sham, (2) PHC (1 mg/kg), (3) rI/R and (4) rI/R + PHC (1 mg/kg). PHC markedly induced the Nrf2 and adenosine monophosphate-activated protein kinase pathways and suppressed rI/R-induced NF-κB and NLRP3 activation in the lungs. Nrf2 deficiency diminished the ability of PHC to ameliorate rI/R-induced histopathological alterations and reactive oxygen species release in the lungs; however, PHC inhibited NLRP3 signaling Nrf2-dependently, while it inhibited NF-κB signaling Nrf2-independently. Our findings demonstrate the beneficial effects of PHC on rI/R-induced lung inflammation.

Toxicity of TiO2 Nanoparticles: Validation of Alternative Models.

There are many studies concerning titanium dioxide (TiO2) nanoparticles (NP) toxicity. Nevertheless, there are few publications comparing in vitro and in vivo exposure, and even less comparing air–liquid interface exposure (ALI) with other in vitro and in vivo exposures. The identification and validation of common markers under different exposure conditions are relevant for the development of smart and quick nanotoxicity tests. In this work, cell viability was assessed in vitro by WST-1 and LDH assays after the exposure of NR8383 cells to TiO2 NP sample. To evaluate in vitro gene expression profile, NR8383 cells were exposed to TiO2 NP during 4 h at 3 cm2 of TiO2 NP/cm2 of cells or 19 μg/mL, in two settings—submerged cultures and ALI. For the in vivo study, Fischer 344 rats were exposed by inhalation to a nanostructured aerosol at a concentration of 10 mg/m3, 6 h/day, 5 days/week for 4 weeks. This was followed immediately by gene expression analysis. The results showed a low cytotoxic potential of TiO2 NP on NR8383 cells. Despite the absence of toxicity at the doses studied, the different exposures to TiO2 NP induce 18 common differentially expressed genes (DEG) which are involved in mitosis regulation, cell proliferation and apoptosis and inflammation transport of membrane proteins. Among these genes, we noticed the upregulation of Ccl4, Osm, Ccl7 and Bcl3 genes which could be suggested as early response biomarkers after exposure to TiO2 NP. On the other hand, the comparison of the three models helped us to validate the alternative ones, namely submerged and ALI approaches.

Inhibition of NLRP3 inflammasome activation on the inflammatory response of macrophage induced by silica dust

Objective: To investigate the effect of inhibiting the activation of NLRP3 inflammatory bodies on the inflammatory response of macrophages induced by silica (SiO(2)) dust. Methods: Rat alveolar macrophages (NR8383) cells were used to establish the cell model, which was divided into four groups: blank control group, dust exposure group (50 mg/L silica dust suspension) , NLRP3 inhibitor group (50 mg/L silica dust suspension and 20 μmol/L NLRP3 inhibitor) , luteolin group (50 mg/L silica dust suspension and 20 μmol/L luteolin) . Samples were collected at 12, 24 and 48 hours after culture. The secretion of inflammatory factors IL and TGF-β(1) were detected by ELISA. The levels of NLRP3 and Caspase-1 were detected by Western Blot. Results: Compared with the blank control group, the survival rates of NR8383 cells in the dust exposure group, NLRP3 inhibitor group and luteolin group were all decreased after 12, 24 and 48 hours of dust exposure, and the expression levels of IL-1β, IL-18, TGF-β1, NLRP3 and Caspase-1 were significantly increased (P<0.05) . Compared with the dust exposure group, NR8383 cells in NLRP3 inhibitor group and luteolin group had higher cell survival rates and lower levels of IL-1β, IL-18, TGF-β1, NLRP3 and Caspase-1 after 12, 24 and 48 hours of dust exposure (P<0.05) . Compared with the NLRP3 inhibitor group, the IL-1β level of NR8383 cells in luteolin group decreased significantly at 12, 24 and 48 h, the IL-18 and TGF-β1 levels decreased significantly at 48 h, the NLRP3 and Caspase-1 levels decreased significantly at 24 h (P<0.05) . Conclusion: Inhibition of NLRP3 inflammaasome activation can reduce the inflammatory response of macrophages induced by SiO(2) dust.

Utilising Magnetically Isolated Lysosomes for Direct Quantification of Intralysosomal Drug Concentrations by LC-MS/MS Analysis: An Investigatory Study With Imipramine

Lysosomes are acidic intracellular organelles that can extensively sequester basic lipophilic drugs due to pH and membrane partitioning, and therefore may significantly influence subcellular drug concentrations. Current in vitro methods for lysosomal drug sequestration evaluation typically lack the ability to accurately and sensitively quantify drug concentrations directly within the lysosome. In the current study, magnetic lysosomal isolation was used in the lysosome rich rat NR8383 cell line and combined with LC-MS/MS analysis to quantify intralysosomal concentrations and lysosomal partitioning (KpLysosome) values of imipramine. The purity of the isolated lysosomes was validated by enzymatic and electron microscopy analysis. Lysosomal imipramine accumulation was explored using 2 methods: addition of imipramine to cells followed by lysosomal isolation (Method 1), and direct addition of imipramine to isolated lysosomes (Method 2). This work highlighted that both experimental buffers and ATP influence intralysosomal drug concentrations, and non-specific drug binding and re-distribution limits the use of Method 1. Method 2 may benefit future lysosomal drug accumulation studies, as imipramine demonstrated high KpLysosome values (3500), comparable to in silico predictions. This study reports a novel method for the direct quantification of intralysosomal drug concentrations that has the ability to be adapted to other cell types.

MiR-124-3p helps to protect against acute respiratory distress syndrome by targeting p65.

Background: Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury that has a high mortality rate and leads to substantial healthcare costs. MicroRNA-124-3p (miR-124-3p) helps to suppress inflammation during a pulmonary injury. However, its mechanism of action is largely unknown, and its role in ARDS remains to be determined.Methods: Mice and NR8383 cells were exposed to lipopolysaccharides (LPS) to induce ARDS, and their miR-124-3p levels were determined. After a miRNA agomir was administrated to the mice, their pulmonary injuries were evaluated by H&E staining and assays for peripheral inflammatory cytokine levels. The direct interaction between miR-124-3p and p65 was predicted, and then confirmed by a luciferase activity assay. The role played by miRNA-124-3p in regulating p65 expression was further examined by transfection with its agomir, and its role in cell apoptosis was investigated by observing the effects of miRNA overexpression in vitro and in vivo.Results: After exposure to LPS, there was a consistent decrease in miR-124-3p expression in the lungs of mice and in NR8383 cells. After treatment with the miR-124-3p agomir, the degrees of pulmonary injury (e.g. alveolar hemorrhage and interstitial edema), and the increases in IL-1β, IL-6, and TNF-α levels induced by LPS were significantly attenuated. Overexpression of miR-124-3p in NC8383 cells and lung tissues significantly suppressed LPS-induced p65 expression and cell apoptosis.Conclusions: These results suggest that miR-124-3p directly targeted p65, and thereby decreased the levels of inflammation and pulmonary injury in a mouse model of ARDS.

Biomimetic Gold Nanoshell-Loaded Macrophage for Photothermal Biomedicine.

The purpose of this study was to investigate the effect of photothermal treatment (PTT) with gold nanoshell (ANS) using a macrophage-mediated delivery system in a head and neck squamous cell carcinoma (HNSCC) cell line. To achieve this, ANS-loaded rat macrophages (ANS-MAs) were prepared via the coculture method with ANS. The human HNSCC (FaDu cell) and macrophage (rat macrophage; NR8383 cell) hybrid spheroid models were generated by the centrifugation method to determine the possibility of using ANS-MAs as a cancer therapy. These ANS-MAs were set into the tumor and macrophage hybrid spheroid model to measure PTT efficacy. Kinetic analysis of the spheroid growth pattern revealed that this PTT process caused a decreasing pattern in the volume of the hybrid model containing ANS-MAs (p < 0.001). Comparison with empty macrophages showed harmony between ANS and laser irradiation for the generation of PTT. An annexin V/dead cell marker assay indicated that the PTT-treated hybrid model induced increasing apoptosis and dead cells. Further studies on the toxicity of ANS-MAs are needed to reveal whether it can be considered biocompatible. In summary, the ANS was prepared with a macrophage as the delivery method and protective carrier. The ANS was successfully localized to the macrophages, and their photoabsorption property was stationary. This strategy showed significant growth inhibition of the tumor and macrophage spheroid model under NIR laser irradiation. In vivo toxicology results suggest that ANS-MA is a promising candidate for a biocompatible strategy to overcome the limitations of fabricated nanomaterials. This ANS-MA delivery and PTT strategy may potentially lead to improvements in the quality of life of patients with HNSCC by providing a biocompatible, minimally invasive modality for cancer treatment.

Trichostatin A exerts anti-inflammation functions in LPS-induced acute lung injury model through inhibiting TNF-α and upregulating micorRNA-146a expression.

Acute lung disease is characterized by inflammation. This research aimed to investigate effect of trichostatin A (TSA) on microRNA-146a (miR-146a) and tumor necrosis factor α (TNF-α) in lipopolysaccharide (LPS)-induced alveolar macrophage injury model. Rat alveolar macrophage, NR8383, was cultured and induced using LPS to establish acute lung injury model in vitro level. Cell Counting Kit-8 (CCK-8) assay was used to determine cell viability of NR8383 cells. TSA was administrated to LPS-induced NR8383 cells. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay was utilized to evaluate TNF-α and miR-146a mRNA expression in LPS and/or TSA treated NR8383 cells. Enzyme-link immunosorbent assay (ELISA) was used to examine TNF-α levels. This study selected 1 ng/ml and 10 ng/ml TSA as the optimal concentrations for treating NR8383 cells. LPS-induced acute lung injury model was successfully established. TSA administration significantly enhanced accounts of LPS-stimulated NR8383 cells. LPS induction significantly increased miR-146a mRNA expression in NR8383 cells compared to NR8383 cells (p<0.05). TSA administration significantly reduced the levels of TNF-α in LPS-induced NR8383 cells compared to those in LPS-induced NR8383 cells (p<0.05). TSA administration significantly enhanced miR-146a expression in LPS-induced NR8383 cells compared to that in LPS-induced NR8383 cells (p<0.05). TSA administration exerted anti-inflammation functions in LPS-induced acute lung injury model in vitro, which might be triggered by inhibiting TNF-α molecule and upregulating miR-146a expression. The present data hint that TSA could be considered as a potential therapeutic agent for treating acute lung injury.

Understanding PM2.5‐Induced Oxidative Stress In Alveolar Macrophages

In situations of oxidative stress, the antioxidant processes of any eukaryotic organism can eventually become overwhelmed. Exposure to ambient particulate matter 2.5 (PM2.5), composed of transition metals and organic compounds 2.5μm or less in diameter, has been shown to cause oxidative stress in alveolar macrophages (AMs). This can lead to various adverse health effects, including cardiovascular and pulmonary diseases. In this study, we examine the capacity of ambient PM2.5 samples collected during periods of peak air pollution in Fresno and Claremont, CA to trigger ROS production in AM cells. These PM2.5 samples were characterized for their chemical content using chromatography and mass spectrometry. A DCF‐fluorescence based microplate assay was used to estimate the potential of the PM2.5 samples to trigger ROS production in NR8383, rat AM cells. Two types of quinones (1,2‐Naphtaquinone and 1,4‐Naphtaquinone) and two transition metals (Cu2+ and Fe2+) were associated with samples that had a high capacity to induce ROS generation in NR8383. While establishing a cytotoxic link between PM2.5 constituents and ROS, we also seek to understand the underlying biological mechanisms through which PM2.5 interacts with AM cells to produce excessive ROS. Previous observations suggest that the upregulation of NADPH Oxidase (NOX1), an enzyme that enhances ROS production, may contribute to cellular oxidative stress in macrophages. In this study, NOX1’s potential role in a PM2.5‐ induced oxidative stress pathway is investigated in NR8383 cells incubated with concentrations of iron and copper relevant with those found in the collected PM2.5 samples. The change in NOX1 expression was quantified using fluorescence‐based western blotting, with zymosan, a well‐known inducer of oxidative stress in macrophages, used as positive control. Our preliminary results indicate that 4‐hour treatments with increasing relevant iron concentrations triggered both an increase of ROS and NOX1 protein levels in NR8383. Our current experiments consist of testing the potential of relevant concentrations of other chemical constituents of PM2.5 suspected of triggering increased ROS production and NOX1 expression (these include naphtaquinones, phenanthraquinones, and copper). Understanding which chemical components of PM2.5 are able to induce significant amounts of ROS will allow us to understand how pollution is pernicious and advocate for the global reduction of PM2.5 emission.Support or Funding InformationThis work is supported by Fresno State ASI, the Division of Research and Graduate Studies, and Fresno State’s CSU‐LSAMP program funded by NSF under grant #HRD‐1826490.

Effect of lncRNA-AC013472.3 on LPS-stimulated secretion of tumor necrosis factor-α in NR8383 rat alveolar macrophages

Objective: To investigate the effect of long non-coding RNA-AC013472.3 on lipopolysaccharide (LPS)-stimulated secretion of tumor necrosis factor (TNF)-α in NR8383 rat alveolar macrophages. Methods: Silencing and overexpression models of lncRNA-AC013472.3 were established with NR8383 rat alveolar macrophages as the experimental subjects. The silencing models were divided into three groups: random nonsense negative small interfering RNA sequence (si-con) group (si-con group, si-con transfected NR8383 cells), LPS+si-con group (10 μg/L LPS was used to treat si-con transfected NR8383 cells for 24 h), and siRNA group (siRNA transfected NR8383 cells), and LPS+siRNA group (10 μg/L LPS was used to treat siRNA transfected NR8383 cells for 24 h). The overexpression models were divided into the empty plasmid (p-con) group (p-con transfected NR8383 cells), LPS+p-con group (10 μg/L LPS was used to treat p-con transfected NR8383 cells for 24 h), lncRNA overexpression plasmid (plncRNA) group (plncRNA transfected NR8383 cells), and the LPS+plncRNA group (10 μg/L LPS was used to treat plncRNA transfected NR8383 cells for 24 h). The mRNA levels of TNF-α in each group were examined by quantitative real-time PCR (qPCR). The protein levels of tumor necrosis factor receptor-related factor-6 (TRAF-6) and phosphorylated nuclear factor-κB (NF-κB) p65 were examined by Western blot. Results: In the silencing model, the mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+si-con group were significantly higher than those in the si-con group (2.040±0.195 vs 1.048±0.207, 0.473±0.022 vs 0.293±0.076 and 0.469±0.062 vs 0.252±0.038)(all P<0.05). The mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+siRNA group were significantly higher than those in the siRNA group (4.158±0.119 vs 1.028±0.019, 0.700±0.104 vs 0.231±0.023 and 0.771±0.095 vs 0.258±0.050)(all P<0.05). The relative expression levels of all indexes in the LPS+siRNA group were significantly higher than those in the LPS+si-con group (all P<0.05). In the overexpression model, the mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+p-con group were significantly higher than those in the p-con group (1.961±0.169 vs 0.999±0.143, 0.533±0.047 vs 0.247±0.020 and 0.565±0.108 vs 0.276±0.048) (all P<0.05). The mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+plncRNA group were significantly higher than those in the plncRNA group (1.322±0.110 vs 1.043±0.093, 0.347±0.035 vs 0.232±0.023 and 0.405±0.072 vs 0.268±0.031) (all P<0.05). The relative expression of all indexes in the LPS+plncRNA group were significantly lower than that in the LPS+p-con group (all P<0.05). Conclusion: LncRNA-AC013472.3 may inhibit the activation of NF-κB signaling pathway, thereby inhibiting the LPS-stimulated secretion of TNF-α in NR8383 rat alveolar macrophages.

PAI-1 Regulation of TGF-β1-induced Alveolar Type II Cell Senescence, SASP Secretion, and SASP-mediated Activation of Alveolar Macrophages.

Senescence of alveolar type II (ATII) cells, progenitors of the alveolar epithelium, is a pathological feature and contributes importantly to the pathogenesis of idiopathic pulmonary fibrosis. Despite recognition of the importance of ATII cell senescence in idiopathic pulmonary fibrosis pathogenesis, how ATII cell senescence is regulated and how senescent ATII cells contribute to lung fibrogenesis remain unclear. In this study, we show that TGF-β1 (transforming growth factor-β1), a most ubiquitous and potent profibrotic cytokine, induces plasminogen activator inhibitor-1 (PAI-1), a cell senescence and fibrosis mediator, and p16 as well as senescence, but not apoptosis, in primary mouse ATII cells. We also found that senescent ATII cells secrete various cytokines and chemokines, including IL-4 and IL-13, which stimulate the expression of genes associated with a profibrotic phenotype in alveolar macrophages. Similar responses were also observed in TGF-β1-treated rat ATII (L2) and rat macrophage NR8383 cells. Deletion of PAI-1 or inhibition of PAI-1 activity with a small molecule PAI-1 inhibitor, however, blocks TGF-β1-induced senescence as well as a senescence-associated secretory phenotype in ATII and L2 cells and, consequently, the stimulatory effects of the conditioned medium from senescent ATII/L2 cells on macrophages. Moreover, we show that silencing p16 ameliorates PAI-1 protein-induced ATII cell senescence and secretion of profibrotic mediators. Our data suggest that PAI-1 mediates TGF-β1-induced ATII cell senescence and secretion of profibrotic mediators through inducing p16, and they also suggest that senescent ATII cells contribute to lung fibrogenesis in part by activating alveolar macrophages through secreting profibrotic and proinflammatory mediators.

Genes expression profiling of alveolar macrophages exposed to non-functionalized, anionic and cationic multi-walled carbon nanotubes shows three different mechanisms of toxicity

Functionalized multi-walled carbon nanotubes (MWCNT) have become the focus of increased research interest, particularly in their application as tools in different areas, such as the biomedical field. Despite the benefits associated with functionalization of MWCNT, particularly in overcoming issues relating to solubility, several studies have demonstrated that these functionalized nanoparticles display different toxicity profiles. For this study, we aim to compare NR8383 cells responses to three well-characterized MWCNT with varying functional groups. This study employed cytotoxicity assays, transcriptomics and proteomics to assess their toxicity using NR8383 rat alveolar macrophages as an in vitro model. The study findings indicated that all MWCNT altered ribosomal protein translation, cytoskeleton arrangement and induced pro-inflammatory response. Only functionalized MWCNT alter mTOR signaling pathway in conjunction with increased Lamtor gene expression. Furthermore, the type of functionalization was also important, with cationic MWCNT activating the transcription factor EB and inducing autophagy while the anionic MWCNT altering eukaryotic translation initiation factor 4 (EIF4) and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) signaling pathway as well as upregulation Tlr2 gene expression. This study proposes that MWCNT toxicity mechanisms are functionalization dependent and provides evidence that inflammatory response is a key event of carbon nanotubes toxicity.

Silica dust exposure induces autophagy in alveolar macrophages through switching Beclin1 affinity from Bcl-2 to PIK3C3.

Increased deposition of silica dust in pulmonary interstitial tissues leads to silicosis, in which autophagy plays a defensive role in silica dust-associated stress response and cell death. Our previous studies revealed that silica dust exposure contributed to autophagy in pulmonary macrophages in vivo, while the specific regulatory mechanism is still unclear. This study aimed to figure out the regulatory mechanism as well as the role of autophagy in the pathogenesis of experimental silicosis. We used 3-methyladenine (3-MA) and ABT-737 to suppress the expression of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) and B cell leukemia/lymphoma 2 (Bcl-2), two critical initiators of autophagy, and detected and evaluated the autophagy in NR8383 cells with or without silica dust exposure. We found that exposure of silica dust increased autophagy in NR8383 cells and elevated the expression of Beclin1 and PIK3C3, but it reduced the expression of Bcl-2. The relationship among Beclin1, PIK3C3, and Bcl-2 were then investigated using immunoprecipitation analysis, and we found that suppression of PIK3C3 and/or Bcl-2 using 3-MA and/or ABT-737 could alter the autophagy induced by silica dust in NR8383 cells, and the complexes of Beclin1/PIK3C3 and Beclin1/Bcl-2 were both downregulated, which may be that inhibition of PIK3C3 and Bcl-2 altered the affinity of Beclin1 with PIK3C3 and Bcl-2 and lead to the silence of PIK3C3 signaling. These findings indicate that silica dust exposure induces autophagy via changing the connectivity of Beclin1 from Bcl-2 to PIK3C3.

Acotiamide attenuates central urocortin 2‐induced intestinal inflammatory responses, and urocortin 2 treatment reduces TNF‐α productions in LPS‐stimulated macrophage cell lines

To determine whether central and in vitro administration of urocortin 2 (Ucn 2) affected intestinal inflammatory responses in LPS-stimulated rat models and macrophage cell lines and acotiamide modified mucosal inflammation in this model. Rats were divided into four groups. LPS-stimulated group (n=4); LPS- and urocortin 2-treated group (n=4); LPS- and acotiamide-treated group (n=4); and LPS-, urocortin 2-, and acotiamide-treated group (n=4). CD68-, CCR2-, and corticotropin-releasing hormone receptor type 2 (CRHR2)-positive cells were assessed by immunostaining. Myeloperoxidase (MPO) activity was measured. TNF-α, IL-6, and IL-4 levels were measured by ELISA method. Gastric emptying and small intestinal transit time were determined using Evans blue. Central administration of Ucn 2 significantly aggravated infiltrations of CD68- and CCR2-positive cells in the intestinal mucosa of LPS-stimulated rat models compared to those in LPS treatment alone. Interestingly, acotiamide treatment significantly reduced the migrations of both CD68- and CCR2-positive cells in the jejunum of central Ucn 2-treated LPS-stimulated rat models. Acotiamide significantly reduced the expression levels of IkB-α phosphorylation in LPS- and MCP-1-stimulated NR8383 cells. Central administration of Ucn 2 significantly delayed gastric emptying. In contrast, Ucn 2 stimulation significantly reduced TNF-α and IL-6 productions in LPS-stimulated NR8383 cells and astressin B reversed the inhibition of TNF-α production in stimulated NR8383 cells. Acotiamide (30μmol/L) significantly reduced TNF-α and IL-6 productions in LPS- and MCP-1-stimulated NR8383 cells. Central and in vitro treatments of Ucn 2 affected intestinal inflammatory responses, respectively, and acotiamide improved them.

Macrophage escape by cholesterol-polyoxyethylene sorbitol oleate micelles for pulmonary delivery.

Aim: Micelles are one of the most promising nanoplatforms for drug delivery, and here, cholesterol-conjugated polyoxyethylene sorbitol oleate (CPSO) micelles have been fabricated for the pulmonary delivery of paclitaxel (PTX). Materials & methods: PTX-CPSO micelles were prepared by a dialysis-ultrasonic method, and a single-factor experiment with a Box-Behnken designwas conducted to optimize the formulation. Furthermore, intracellular and phagocytosis escape studies of the optimized formulation were performed on A549 and NR8383 cells. Results: The optimal micelles exhibited satisfactory encapsulation efficiency (78.48±2.36%) and drug loading (17.06±1.71%). In vitro studies showed enhanced CPSO micelle A549 cellular uptake and their ability to escape macrophages. Conclusion: PTX-CPSO micelles could be a promising system for pulmonary targeting by intravenous administration.

Siglec1 enhances inflammation through miR-1260-dependent degradation of IκBα in COPD

It has been documented that sialic acid-binding Ig-like lectin 1 (Siglec1) is a cell surface protein with a variety of functions in the immune system. In the present study, we evaluated whether Siglec1 plays a role in chronic obstructive pulmonary disease (COPD). Results show that the expression of Siglec1 was increased in the lung of COPD rats, and that Siglec1 overexpression greatly enhanced the expression of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6 in cigarette smoke extract (CSE)-treated NR8383 cells, a rat lung-derived macrophage cell line. Notably, the proinflammatory effect of Siglec1 was totally inhibited by overexpression of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α (IκBα). Importantly, Siglec1 overexpression increased miR-1260, which then degraded IκBα through its 3′ untranslated region (3′UTR). Further study demonstrated that miR-1260 inhibitor attenuated inflammation in CSE-induced rat COPD lung and in CSE-treated NR8383 cells. Finally, the inhibitory effect of miR-1260 on inflammation was totally lost when IκBα was inhibited. In summary, the present study demonstrated that Siglec1 exerts its proinflammatory effects through increasing miR-1260, leading to decreased expression of IκBα.

Soluble PTX3 of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Attenuates Hyperoxic Lung Injury by Activating Macrophage Polarization in Neonatal Rat Model.

Therapeutic treatment of various inflammation-related diseases using mesenchymal stem cells (MSCs) has increased in recent years because of the paracrine action of these cells but shows several limitations. First, MSC-based therapies exhibit varying efficacies; thus, biomarkers should be determined to identify who may benefit from these candidate therapeutic agents. Second, the mechanism underlying the therapeutic effects is poorly understood. To evaluate the effects of human umbilical cord blood-derived MSCs (UCB-MSCs) on macrophages, the macrophage cell line NR8383 stimulated with lipopolysaccharide (LPS) was cocultured by UCB-MSCs. We found that UCB-MSCs mediated changes in macrophage polarization towards M2 from M1 macrophages. To identify the paracrine action underlying the anti-inflammation effect of UCB-MSCs, the secretion of UCB-MSCs exposed to LPS-stimulated NR8383 cells was tested using a biotin label-based 507 antibody array. Among the secreted proteins, we selected pentraxin-related protein PTX3/tumor necrosis factor-inducible gene 14 protein (PTX3) to investigate its association with UCB-MSCs in macrophage polarization. We found that human PTX3 was secreted from UCB-MSCs under inflammation condition and reinforced the M2 macrophage marker via the Dectin-1 receptor by activating MSK1/2 phosphorylation signaling in NR8383 cells. Accordingly, knockdown of PTX3 in UCB-MSCs significantly attenuated their therapeutic effects in a neonatal hyperoxic lung injury resulting in reduced survival, lung alveolarization, M2 marker expression, Dectin-1 levels, anti-inflammatory cytokines, and improved M1 marker expression and inflammatory cytokines compared to control MSC-injected rats. UCB-MSCs show therapeutic potential by controlling macrophage polarization. Interestingly, higher PTX3 levels in UCB-MSCs induced greater improvement in the therapeutic effects than lower PTX3 levels. Collectively, PTX3 is a potential marker with critical paracrine effects for predicting the therapeutic potential of MSC therapy in inflammatory diseases; quality control assessments using PTX3 may be useful for improving the therapeutic effects of UCB-MSCs.

Formulation development and in-vitro evaluation of montelukast sodium pressurized metered dose inhaler

Montelukast is used as alternative treatment for asthma. However, some adverse drug reactions have been reported for the oral dosage form of montelukast. To counter this issue, the drug was formulated into a pressurized metered-dose inhaler (pMDI), which delivered montelukast directly to the lower regions of the lungs. Montelukast sodium, mixed with either HFA 134a propellant or a mixed propellant (HFA-134a and HFA227), was formulated into a pMDI with other excipients. The canister was prepared using a pressurized filling method, followed by disk sampling to assess content per spray and content uniformity. The effects of ethanol and polyethylene glycol (PEG) 400 on the fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD) of the formulation were evaluated. In vitro cytotoxicity of the developed montelukast pMDIs was tested in lung airway cell lines. Their effects on the levels of inflammatory nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), generated from NR8383 alveolar macrophage (AM) cells, were also evaluated. All selected formulations had a FPF >46%, whereas the formulation containing 20% v/v ethanol and 1% v/v PEG 400 showed the highest FPF. The MMADs of all formulations were within 1–5 μm. The montelukast pMDI formulations were found to be non-toxic to lung airway cell lines and did not induce inflammatory NO, TNF-α, or IL-1β production in NR8383 cells.

Kappa Opioid Receptor on Pulmonary Macrophages and Immune Function.

BackgroundRespiratory failure significantly increases mortality in critically ill patients. While opioids are often used during the perioperative period and in critically ill situations, little is known about how opioids are involved in pulmonary immune function and the inflammatory response. There is currently no clear information on the role of the kappa opioid receptor (KOR) in pulmonary inflammation. Here we evaluate whether KORs are involved in the modulation of lung macrophages by the use of selective KOR agonists in lipopolysaccharide (LPS) activated alveolar macrophages.MethodThe inflammatory response in rat NR8383 macrophages was induced by stimulation with LPS (100 ng/ml) at different time-points. The effects of the KOR agonists Salvinorin A (SA) and U50488 on inflammatory factors such as nitrite, TNF-α, IL-1β, iNOS and COX-2 were investigated. Nor-binaltorphimine, a selective KOR antagonist, was used to investigate the specific role of KOR.ResultsStimulation of NR8383 cells with LPS (100 ng/ml) significantly increased the level of TNF-α at 1h, 2h and 6h compared to un-stimulated cells. SA attenuated the inflammatory response by reducing the levels of TNF-α and IL-1β after LPS treatment. SA co-treatment reduced the elevated levels of NO induced by LPS and also alleviated the over-expression of iNOS and COX-2 within 2 hours after LPS activation, and such effects can be partially blocked by KOR antagonist, nor-binaltorphimine. Similar results from U50488 were observed.ConclusionOur results indicate that KORs may play a critical role in the modulation of the pulmonary inflammatory process by their activation in macrophages. Selective KOR agonists exert their anti-inflammatory effects acutely on lung macrophages, within 1–2 hours of LPS-stimulated inflammation in vitro.

Network pharmacology based research into the effect and mechanism of Xijiao Dihuang decoction against sepsis

Sepsis is a critical illness that contributes a high mortality, while Xijiao Dihuang decoction (XJDHT) has been used in treatment against sepsis for many years by clinical doctors.Clinical studies confirmed a good efficacy of XJDHT against sepsis. The aim of this study is to observe the efficacy of XJDHT in sepsis model rats and macrophages activated by LPS, and to verify the underlying mechanisms.The key components of XJDHT and its targets against sepsis were analyzed and selected by network pharmacology. The potential mechanisms that XJDHT regulates the progress of sepsis were verified in sepsis rats and NR8383 cell lines. XJDHT at a dose of 25 mg/kg was administrated to rats which endured cecal ligation and perforation (CLP). After MTT assay, XJDHT at a dose of 4 mg/mL was selected to treat NR8383 cell lines activated by LPS. In vivo experiment, the survival of the rats was assessed. The content of cytokine in serum were assessed by Enzyme-linked immunosorbent assays (ELISA). Contents of cytokine and key molecules in relative signaling pathway were assessed by immunohistochemical method. The pathway protein expressions were detected by Western blotting. In vitro experiment, immunofluorescence was used to assess the content of cytokine and signaling pathway.A total of 42 targets of XJDHT against sepsis were identified by network pharmacology. After eliminating overlapping compounds and proteins, there were 8 compounds in XJDHT that associating with the 42 sepsis-related targets. NF-κB and HIF-1α signaling pathway were recognized to play important role for XJDHT against sepsis. XJDHT improved survival rate in the XJDHT group compared with the model group. The contents of IL-6 increased in the model group compared with the control group with ELISA and immunohistochemistry, while XJDHT reduced the content of IL-6. The expressions of p65 and HIF-1α reduced significantly in the XJDHT group compared with the model group. In vitro study, the content of IL-6 elevated significantly after LPS stimulation, while XJDHT reduced this increase. Furthermore, expressions of protein of p65 and HIF-1α decreased significantly compared with the LPS group.To conclude, our study demonstrated that XJDHT at a dose of 25 g/kg is capable of improving the survival of sepsis via regulating the NF-κB and HIF-1α signaling pathway.

Electroacupuncture ameliorates cardiopulmonary bypass induced apoptosis in lung via ROS/Nrf2/NLRP3 inflammasome pathway

AimsElectroacupuncture (EAc) has a pulmonary protective effect during cardiopulmonary bypass (CPB), but its molecular mechanisms including inflammasome activation signaling pathways remains unclear. Materials and methodsMale Sprague Dawley rats were divided into control, CPB + EAc and CPB groups. Lung injury model was developed by CPB treatment and EAc (2/100 Hz) was carried out before CPB in the CPB + EAc group. Lung tissues were collected at two time points (0.5 h; 2 h) to determine cytokines release by ELISA kits, and protein expressions by Western blot. Serum collected at two time points (0.5 h; 2 h) from CPB and CPB + EAc treated groups were used in NR8383 cells to confirm the effect of EAc. Key findingsCPB significantly increased the inflammatory mediators, histological damage and expression of inflammasome related protein and apoptosis, when compared with control group. The level of tumor necrosis factor-α(TNF-α), interleukin (IL)-18 and IL-1β in the CPB + EAc treated group was significantly decreased along with histological changes compared to CPB. Moreover, EAc inhibited the activation of Nod like receptor protein-3 (NLRP3) inflammasome complex, caspase-8 and activated NF-E2-related factor 2 (p-Nrf2). In addition, serum from the CPB + EAc group prevented CPB induced activation of inflammasome and related mediators, reducing ROS generation and apoptosis in NR8383 macrophages. SignificanceThese findings indicate that EAc had a critical anti-apoptotic role by suppression of ROS/Nrf2/NLRP3 inflammasome pathway. EAc might be a possible therapeutic treatment for CPB-induced acute lung injury.