Lipopolysaccharide-induced Acute Respiratory Distress Syndrome Research Articles

The Role of Pericytes in Lipopolysaccharide-Induced Murine Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a heterogeneous clinical syndrome that is most commonly triggered by infection-related inflammation. Lung pericytes can respond to infection and act as immune and proangiogenic cells; moreover, these cells can differentiate into myofibroblasts in non-resolving ARDS and contribute to the development of pulmonary fibrosis. Here, we aimed to characterize the role of lung cells which present characteristic of pericytes, such as peri-endothelial location and expression of panel of specific markers. To study their role in ARDS we used a murine model of lipopolysaccharide induced (LPS) resolving ARDS. We confirmed the development of ARDS after LPS instillation which was resolved 14 days after onset. Using immunofluorescence and flow cytometry we observed early expansion of NG2+PDGFRβ+ pericytes in murine lungs with loss of CD31+ PDGFRβ+ endothelial cells. These changes were accompanied by specific changes in lung structure and loss of vascular integrity. On day 14 after ARDS onset the composition of pericytes and endothelial cells returned to baseline values. LPS-induced ARDS activated NOTCH signaling in lung pericytes, the inhibition of which during LPS stimulation reduced the expression of its downstream target genes, pericyte markers and angiogenic factors. Together, lung pericytes in response to inflammatory injury activate NOTCH signaling that supports their maintenance and in turn can contribute to recovery of the microvascular endothelium.

Indole-3-carbinol attenuates lipopolysaccharide-induced acute respiratory distress syndrome through activation of AhR: role of CCR2+ monocyte activation and recruitment in the regulation of CXCR2+ neutrophils in the lungs.

Indole-3-carbinol (I3C) is found in cruciferous vegetables and used as a dietary supplement. It is known to act as a ligand for aryl hydrocarbon receptor (AhR). In the current study, we investigated the role of AhR and the ability of I3C to attenuate LPS-induced Acute Respiratory Distress Syndrome (ARDS). To that end, we induced ARDS in wild-type C57BL/6 mice, Ccr2gfp/gfp KI/KO mice (mice deficient in the CCR2 receptor), and LyZcreAhRfl/fl mice (mice deficient in the AhR on myeloid linage cells). Additionally, mice were treated with I3C (65 mg/kg) or vehicle to investigate its efficacy to treat ARDS. I3C decreased the neutrophils expressing CXCR2, a receptor associated with neutrophil recruitment in the lungs. In addition, LPS-exposed mice treated with I3C revealed downregulation of CCR2+ monocytes in the lungs and lowered CCL2 (MCP-1) protein levels in serum and bronchoalveolar lavage fluid. Loss of CCR2 on monocytes blocked the recruitment of CXCR2+ neutrophils and decreased the total number of immune cells in the lungs during ARDS. In addition, loss of the AhR on myeloid linage cells ablated I3C-mediated attenuation of CXCR2+ neutrophils and CCR2+ monocytes in the lungs from ARDS animals. Interestingly, scRNASeq showed that in macrophage/monocyte cell clusters of LPS-exposed mice, I3C reduced the expression of CXCL2 and CXCL3, which bind to CXCR2 and are involved in neutrophil recruitment to the disease site. These findings suggest that CCR2+ monocytes are involved in the migration and recruitment of CXCR2+ neutrophils during ARDS, and the AhR ligand, I3C, can suppress ARDS through the regulation of immune cell trafficking.

Blocking P2Y2 purinergic receptor prevents the development of lipopolysaccharide-induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality resulting from a direct or indirect injury of the lung. It is characterized by a rapid alveolar injury, lung inflammation with neutrophil accumulation, elevated permeability of the microvascular-barrier leading to an aggregation of protein-rich fluid in the lungs, followed by impaired oxygenation in the arteries and eventual respiratory failure. Very recently, we have shown an involvement of the Gq-coupled P2Y2 purinergic receptor (P2RY2) in allergic airway inflammation (AAI). In the current study, we aimed to elucidate the contribution of the P2RY2 in lipopolysaccharide (LPS)-induced ARDS mouse model. We found that the expression of P2ry2 in neutrophils, macrophages and lung tissue from animals with LPS-induced ARDS was strongly upregulated at mRNA level. In addition, ATP-neutralization by apyrase in vivo markedly attenuated inflammation and blocking of P2RY2 by non-selective antagonist suramin partially decreased inflammation. This was indicated by a reduction in the number of neutrophils, concentration of proinflammatory cytokines in the BALF, microvascular plasma leakage and reduced features of inflammation in histological analysis of the lung. P2RY2 blocking has also attenuated polymorphonuclear neutrophil (PMN) migration into the interstitium of the lungs in ARDS mouse model. Consistently, treatment of P2ry2 deficient mice with LPS lead to an amelioration of the inflammatory response showed by reduced number of neutrophils and concentrations of proinflammatory cytokines. In attempts to identify the cell type specific role of P2RY2, a series of experiments with conditional P2ry2 knockout animals were performed. We observed that P2ry2 expression in neutrophils, but not in the airway epithelial cells or CD4+ cells, was associated with the inflammatory features caused by ARDS. Altogether, our findings imply for the first time that increased endogenous ATP concentration via activation of P2RY2 is related to the pathogenesis of LPS-induced lung inflammation and may represent a potential therapeutic target for the treatment of ARDS and predictably assess new treatments in ARDS.

Umbilical cord mesenchymal stem cells overexpressing CXCR7 facilitate treatment of ARDS-associated pulmonary fibrosis via inhibition of Notch/Jag1 mediated by the Wnt/β-catenin pathway

The therapeutic efficacy of umbilical cord mesenchymal stem cells (UCMSCs) in acute respiratory distress syndrome (ARDS) is mainly limited by the efficiency of homing of UCMSCs toward tissue damage. C-X-C chemokine receptor type 7 (CXCR7), which is involved in the mobilization of UCMSCs, is only expressed on the surface of a small proportion of UCMSCs. This study examined whether overexpression of CXCR7 in UCMSCs (UCMSCsOE-CXCR7) could improve their homing efficiency, and therefore, improve their effectiveness in fibrosis repair at the site of lung injury caused by ARDS. A lentiviral vector expressing CXCR7 was built and then transfect into UCMSCs. The impacts of CXCR7 expression of the proliferationand homing of UCMSCs were examined in a lipopolysaccharide-induced ARDS mouse model. The potential role and underlying mechanism of CXCR7 were examined by performing scratch assays, transwell assays, and immunoassays. The therapeutic dose and treatment time of UCMSCsOE-CXCR7 were directly proportional to their therapeutic effect on lung injury. In addition, overexpression of CXCR7 increased SDF-1-induced proliferation and migration of lung epithelial cells (Base-2b cells), and upregulation of CXCR7 inhibited α-SMA expression, suggesting that CXCR7 may have a role in alleviating pulmonary fibrosis caused by ARDS. Overexpression of CXCR7 in UCMSCs may improve their therapeutic effect of acute lung injury mouse, The mechanism of fibrosis repair by CXCR7 is inhibition of Jag1 via suppression of the Wnt/β-catenin pathway under the chemotaxis of SDF-1.

Protective Effect of Unfractionated Heparin on Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Neonatal Mice via the JAK2/STAT3 Signaling Pathway.

Neonatal acute respiratory distress syndrome (ARDS) is a clinical disorder characterized by excessive acute inflammatory response in lung parenchyma and has high morbidity and mortality. However, the therapeutic treatments are still lacking. The aim of this study is to evaluate the role of unfractionated heparin in neonatal ARDS and explore the underlying mechanism of its effects. To conduct the ARDS model, the mouse pups were treated by intraperitoneal injection of lipopolysaccharide (LPS) (10 mg/kg). For unfractionated heparin intervention group, C57BL/6 mouse pups received a single subcutaneous injection of unfractionated heparin (400 IU/kg) 30 minutes prior to LPS. The survival rate was recorded for each group. Histological analysis was used to evaluate lung injury. MPO (myeloperoxidase) concentration level in lung tissues and extracellular histones in serum were detected by enzyme linked immunosorbent assay (ELISA). A commercially available kit was used to detect inflammatory cytokine levels in serum. Real time quantitative polymerase chain reaction (qPCR) and western blot were used to detect the mRNA and protein in the JAK2/STAT3 signaling pathway, respectively. Intervention of unfractionated heparin significantly increased the survival rate of mouse pups with ARDS, restored lung architecture, inhibited neutrophil infiltration as evidenced by reduced MPO concentration, and attenuated the LPS-induced inflammatory responses, characterized by the down-regulation of proinflammatoy factors and up-regulation of anti-inflammatory factor when compared with the ARDS group. In addition, the concentration of extracellular histones, which have been proven to be mediated in the pathogenesis of ARDS, was diminished by unfractionated heparin. Moreover, the protein expressions of p-JAK2 (Y1007/1008) and p-STAT3 (Y705) in the ARDS group were remarkably up-regulated, which were reversed by unfractionated heparin. Unfractionated heparin protects LPS-induced ARDS via inhibiting JAK2/STAT3 pathway in neonatal mice, which might present a novel therapeutic target for ARDS of neonates.

Fusu mixture alleviates inflammatory reactions in lipopolysaccharide-induced acute respiratory distress syndrome mice via regulation of surfactant-associated protein C, aquaporin 5, and Notch1.

Acute respiratory distress syndrome (ARDS) is a common life-threatening critical illness with high mortality. Fusu mixture (FSM) can improve the mechanical ventilation in ARDS patients. However, the detailed pharmacological mechanisms and active substances of FSM are still unclear. This study aimed to explore the potential pharmacological mechanisms of FSM for treating ARDS and its chemical compositions. A lipopolysaccharide (LPS)-induced ARDS mouse model was established, and the mice subsequently received FSM (50 mg/kg) orally for 5 days. Then, the blood samples and lung tissues were collected. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum, and histopathology examinations were applied to analyze the inflammatory response of lung tissues in ARDS mice. In addition, protein expressions of aquaporin 5 (AQP-5), surfactant-associated protein C (SP-C), and Notch1 were detected by western blot assays and immunohistochemical (IHC) examination. In addition, the chemical compositions of FSM were analyzed by high performance liquid chromatography (HPLC), using standard reference agents. After LPS induction, the serum levels of IL-6 and TNF-α in ARDS mice were significantly increased (P<0.01, vs. Control), and FSM significantly reduced these 2 pro-inflammatory cytokines (IL-6 and TNF-α) compared to the model mice (P<0.01). Histopathology examinations showed FSM significantly attenuated the inflammatory responses in lung tissues. Furthermore, after FSM treatment, the SP-C and AQP-5 were significantly increased, compared to the Model mice (P<0.01), and FSM also up-regulated the Notch1 expressions in lung tissues of ARDS mice (P<0.001, vs. Model). Collectively, it is suggested that FSM alleviates inflammatory reactions and promotes the proliferation of alveolar epithelial cells in LPS-induced ARDS mice via regulation of SP-C, AQP-5, and Notch1 in lung tissues.

Alveolar macrophage-derived secretory autophagosomes exacerbate inflammation in lipopolysaccharide-induced acute respiratory distress syndrome

Background: Acute respiratory distress syndrome (ARDS) has a high incidence among patients in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) contribute to ARDS-associated lung injury; however, the mechanisms underlying the inflammatory process remain unclear. Purpose: This study investigated the effects and the mechanisms through which AM-derived secretory autophagosomes (SAPs) contribute to ARDS-associated lung injury. Methods: SAPs were isolated from cell culture supernatants of AMs treated or untreated with lipopolysaccharide (LPS) and intratracheally injected to determine whether SAP exacerbated lung injury in mice with ARDS. The SAP proteome was analysed using mass spectrometry. Lung fibroblasts were treated with SAPs and collected for transcriptome sequencing. A transwell assay was conducted to study the chemotactic activity of fibroblasts induced by SAPs. Results: We found that AMs contributed to ARDS-associated lung injury by releasing a novel type of pro-inflammatory vesicles named SAPs, which were characterised as double-membraned vesicles approximately 200 nm in diameter and light-chain-3 expression. Proteomic analysis of SAPs and gene ontology (GO) enrichment analysis revealed that LPS-SAPs contain several differentially expressed proteins involved in effecting an inflammatory response, including interleukin (IL)-1β. We confirmed this result by quantifying IL-1β in SAPs using ELISA. Moreover, administering SAPs from LPS-stimulated macrophages to mice with ARDS exacerbated lung injury, whereas IL-1 receptor antagonist (IL-1RA) pre-treatment significantly ameliorated SAP-induced lung injury. In addition, we conducted transcriptome sequencing of lung fibroblasts treated with SAPs to explore the effect of SAPs on fibroblasts in ARDS, which demonstrated that the expression of several chemokines (CXCL2, CXCL15, and so forth) significantly increased with SAP stimulation. Furthermore, the transwell assay indicated that SAPs could enhance the chemotactic activity of fibroblasts, suggesting that SAPs may exacerbate inflammation in ARDS by promoting neutrophil or monocyte recruitment. Conclusion: This is the first study to show that AM-derived SAPs contribute significantly to ARDS by secreting pro-inflammatory cytokines and promoting inflammatory cell recruitment by activating fibroblasts, which may be beneficial for therapeutic development of ARDS. National Natural Science Foundation of China (Grant numbers 81870066, 81670074, 81930058, 82270083 and 81971888), Six Talent Peaks Project in Jiangsu Province(Grant numbers TD-SWYY-003), and Social Development Specifc Projects of Jiangsu Province [Grant number BE2020786]. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel tree shrew model of lipopolysaccharide-induced acute respiratory distress syndrome

IntroductionAcute respiratory distress syndrome (ARDS) is a leading cause of respiratory failure, with substantial attributable morbidity and mortality. The small animal models that are currently used for ARDS do not fully manifest all of the pathological hallmarks of human patients, which hampers both the studies of disease mechanism and drug development. ObjectivesTo examine whether the phenotypic changes of primate-like tree shrews in response to a one-hit lipopolysaccharides (LPS) injury resemble human ARDS features. MethodsLPS was administered to tree shrews through intratracheal instillation; then, the animals underwent CT or PET/CT imaging to examine the changes in the structure and function of the whole lung. The lung histology was analyzed by H&E staining and immunohistochemical staining of inflammatory cells. ResultsResults demonstrated that tree shrews exhibited an average survival time of 3–5 days after LPS insult, as well as an obvious symptom of dyspnea before death. The ratios of PaO2 to FiO2 (P/F ratio) were close to those of moderate ARDS in humans. CT imaging showed that the scope of the lung injury in tree shrews after LPS treatment were extensive. PET/CT imaging with 18F-FDG displayed an obvious inflammatory infiltration. Histological analysis detected the formation of a hyaline membrane, which is usually present in human ARDS. ConclusionThis study established a lung injury model with a primate-like small animal model and confirmed that they have similar features to human ARDS, which might provide a valuable tool for translational research.

Fusu mixture alleviates inflammatory reactions in lipopolysaccharide-induced acute respiratory distress syndrome mice via regulation of surfactant-associated protein C, aquaporin 5, and Notch1

Song Zhang, Yan Yue, Jing Liu, Lijia Zhi, Li Zhang, Kaichen Zhang, Peng Ding, Peiyang Gao, Kunlan Long

MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a prevalent illness in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) assist in ARDS lung injury and the inflammatory process through mechanisms that are unclear. This study investigated the role of AM-derived secretory autophagosomes (SAPs) in lung injury and microRNA (MiR)-199a-3p-regulated inflammation associated with ARDS in vitro and in a murine model. The ARDS model in mouse was established by intratracheal LPS lipopolysaccharide (LPS) injection. The agomirs or antagomirs of MiR-199a-3p were injected into the caudal vein to figure out whether MiR-199a-3p could influence ARDS inflammation and lung injury, whereas the mimics or inhibitors of MiR-199a-3p, siRNA of Rab8a, or PAK4 inhibitor were transfected or applied to RAW264.7 cells to evaluate the mechanism of SAP release. Culture supernatants of RAW264.7 cells treated with LPS or bronchoalveolar lavage fluid from mice were collected for the isolation of SAPs. We found that MiR-199a-3p was over-expressed in the lungs of ARDS mice. The MiR-199a-3p antagomir alleviated, whereas the MiR-199a-3p agomir exacerbated LPS-induced inflammation in mice by promoting AM-derived SAP secretion. In addition, MiR-199a-3p over-expression exacerbated LPS-induced ARDS via activating Rab8a, and Rab8a silencing significantly suppressed the promoting influence of the MiR-199a-3p mimic on SAP secretion. Furthermore, MiR-199a-3p mimic activated Rab8a by directly inhibiting PAK4 expression. The novel finding of this study is that MiR-199a-3p participated in the regulation of SAP secretion and the inflammatory process via targeting of PAK4/Rab8a, and is a potential therapeutic candidate for ARDS treatment.

Protective effect of Lindera aggregata on lipopolysaccharide-induced mice acute respiratory distress syndrome by regulating p38MAPK/ERK pathway

To explore the protective effect of Lindera aggregata on acute respiratory distress syndrome (ARDS) induced by lipopolysaccharide (LPS) in mice and its possible mechanism. Forty C57BL/6 mice were randomly divided into sham operation group, ARDS model group, low-dose Lindera aggregata (L-LA) group and high-dose Lindera aggregata (H-LA) group, with 10 mice in each group. ARDS model was established by injecting 5 mg/kg LPS through the trachea. The L-LA group and H-LA group were orally administrated 1 g/kg and 5 g/kg of the Lindera aggregate extract once a day, respectively, while the ARDS model group was given the same volume of normal saline, the sham group received no treatment. The Lindera aggregata was preadministered for 3 days before modeling, and continued for 2 days after modeling, then the animals were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The pathological changes of lung tissue in each group of mice were observed under the microscope and the wet/dry weight ratio (W/D) of the lung were measured. Enzyme linked immunosorbent assay (ELISA) was used to examine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in mice serum and BALF, and flow cytometry was used to detect the expression rate of CD40 on the surface of BALF macrophages. The phosphorylation levels of p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) proteins in lung tissue were measured by Western blotting. Lung histopathology under light microscope showed that the damage of alveolar structure, thickening of alveolar septum and infiltration of inflammatory cells in the H-LA group were less severe than those in the ARDS model group, while the pathological characteristics of ARDS in the L-LA group were not significantly different from those in the ARDS model group. Compared with the sham operation group, the lung W/D ratio, TNF-α and IL-6 protein contents in serum and BALF, BALF macrophage CD40 expression rate and lung tissue p38 and ERK1/2 protein phosphorylation levels were significantly increased in ARDS model group. The W/D ratio, the concentrations of TNF-α and IL-6 in serum and BALF, the expression rate of CD40 in BALF macrophages, and the phosphorylation levels of p38 and ERK1/2 protein in lung tissue in the L-LA group were not significantly different from those in the ARDS model group. The above indexes in the H-LA group were significantly lower than those in the ARDS model group and the L-LA group [W/D ratio: 5.70±0.19 vs. 6.20±0.31, 6.01±0.17; serum TNF-α (ng/L): 83.63±15.04 vs. 111.75±18.45, 108.12±13.98; serum IL-6 (ng/L): 111.38±8.75 vs. 244.13±26.85, 227.50±9.37; BALF TNF-α (ng/L): 36.25±2.82 vs. 51.13±5.44, 47.50±5.78; BALF IL-6 (ng/L): 35.63±2.20 vs. 49.63±4.90, 46.38±3.50; CD40 expression rate (%): 23.28±2.45 vs. 30.32±2.40, 28.17±1.98; p-p38/p38: 0.50±0.04 vs. 0.74±0.07, 0.69±0.04; p-ERK1/2/ERK1/2: 0.47±0.07 vs. 0.72±0.07, 0.68±0.05; all P < 0.01]. Lindera aggregata can inhibit LPS-induced lung inflammation and alleviate lung injury in ARDS mice. The mechanism may be related to the inhibition of the activation of p38 mitogen activated protein kinase/ERK (p38MAPK/ERK) signaling pathway.

Selexipag Improves Lipopolysaccharide-Induced ARDS on C57BL/6 Mice by Modulating the cAMP/PKA and cAMP/Epac1 Signaling Pathways.

Selexipag, a long-acting and selective prostacyclin (PGI2) IP receptor agonist, has in aged rats with stroke revealed effects of inhibiting inflammation, ameliorating damage to the blood-brain barrier, and alleviating oxidative stress. However, in the case of acute respiratory distress syndrome (ARDS) characterized by diffuse alveolar damage and lung capillary endothelial injury, its effects yet remain unknown. In this study, we investigated effects of the prophylaxis by Selexipag on a mouse model of ARDS established by the lipopolysaccharide (LPS) challenge and potential mechanism. Compared to the LPS-challenged mice, the LPS-challenged mice with the prophylaxis by 0.5 or 1 mg/kg of Selexipag exhibited significantly alleviated lung histological manifestations, reduced protein leakage, decreased levels of interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1), diminished expressions of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) mRNA, noticeably increased expressions of zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) protein, escalated lung cAMP levels, and raised levels of lung relative phosphorylated-protein kinase A catalytic subunit (p-PKA C) at Thr197 and exchange protein activated by cAMP 1 (Epac1) protein. These results suggest that, through suppressing inflammation and reducing vascular endothelial damage, Selexipag can effectively ameliorate the LPS-induced ARDS on mice. The lung cAMP and its downstream signaling modules, PKA and Epac1, possibly constitute the main regulative molecular mechanism. Selexipag appears to hold promise to become a new potential therapeutic option for ARDS.

SRC3 deficiency exacerbates lipopolysaccharide-induced acute respiratory distress syndrome in mice

Acute respiratory distress syndrome (ARDS) is a severe disease. Inflammation is the key element implicated in ARDS. Steroid receptor coactivator 3 (SRC3), a coactivator protein for transcription, is involved in regulation of inflammatory response. Here we explored the potential roles of SRC3 in ARDS. We utilized the SRC3 deficient (SRC3-/-) mice and established the lipopolysaccharides (LPS)-induced ARDS model. The mortality, lung injury, leucocytes infiltration and inflammatory cytokine production were compared between wild type (WT) and SRC3-/- mice. The NF-κB activation in lung of WT and SRC3-/- mice was measured. After LPS treatment, SRC3-/- mice had higher mortality and more severe lung damage than WT mice. LPS-treated SRC3-/- mice had more leucocytes infiltration and upregulated inflammatory cytokine production. LPS-treated SRC3-/- mice had elevated NF-κB activation. SRC3-/- mice had exacerbated ARDS in LPS-treated mice.

Pre-treatment with morphine prevents lipopolysaccharide-induced acute respiratory distress syndrome in rats via activation of opioid receptors

PurposeAcute respiratory distress syndrome (ARDS), a severe medical condition, is among the major causes of death in critically ill patients. Morphine is used as a therapeutic agent against severe pain. The mechanisms of its reactions over ARDS are not fully understood. The aim of this study was to assess the mechanism of morphine in rats with ARDS. MethodsRats were injected with lipopolysaccharide to induce ARDS, and some rats were pre-treated with graded doses of morphine in the lateral ventricles to assess survival and non-infected mortality. Immunohistochemical and HE staining were performed to measure MPO and CD68 activity in the lungs and lung injury. ELISA was conducted to detect the inflammatory factor levels in the plasma and BALF. Co-labeling of μ-opioid receptor (MOR) and c-Fos was observed in the brain tissues. MOR-positive cells in brain tissues were evaluated using immunohistochemistry. The effect of MOR antagonists on ARDS was examined in rats by pre-injection of naloxone or methylnaltrexone. The expression of MyD88, TLR4, and NF-κB was lastly assessed. ResultsDose-independent improvement was observed in respiratory capacity and lung injury in ARDS rats after morphine pre-injection, along with reduced inflammatory factors in the plasma and BALF. MOR-positive cells were elevated after morphine, which occurred within the ventral part of the gigantocellular reticular nucleus (GiV). Naloxone and methylnaltrexone blocked the effects of morphine via central and peripheral MOR. Morphine activated TLR pathway in a MyD88-dependent manner. ConclusionMorphine activates MOR within the GiV and the TLR pathway to attenuate ARDS in rats.

Anti-inflammatory effects of a SERP 30 polysaccharide from the residue of Sarcandra glabra against lipopolysaccharide-induced acute respiratory distress syndrome in mice

Ethnopharmacological relevanceSarcandra glabra (Thunb.) Nakai, a valuable dietetic Chinese herb, is still widely used today. Multiple ingredients of S. glabra with a variety of activities such as anti-inflammatory, antiviral, and antitumor were studied. However, the Sarcandra glabra (Thunb.) Nakai polysaccharide hasn’t been reported for its anti-inflammatory effect. Aim of the studyIn this study, the anti-inflammatory activity of Sarcandra glabra (Thunb.) Nakai polysaccharide was assessed in LPS-induced ARDS mice. Materials and methodsA polysaccharide coded as SERP 30 was obtained by water extraction, alcohol precipitation, and gel filtration. After the physicochemical properties determination and structural characterization, LPS induced-mice ARDS model was used to evaluate the anti-inflammatory and associated antioxidant activities of SERP 30. H&E staining was used to observe the seriousness of lung injury in mice. The ELISA method was used to measure the expression of inflammatory factors (TNF-α and IL-6) in the serum of the mice. The TBA method and the WST-1 method were used to evaluate the oxidative stress injury. Immunohistochemistry was used to distinguish the expression of metalloproteinase-9 (MMP-9), heparinase (HPA), syndecan-1, and decorin in ARDS-mice lung tissue. Western blotting was used to confirm the expression of related proteins in mouse lung tissue. ResultsSERP 30 had a potential role in improving lung damage, reducing inflammation, and preventing oxidative stress. Moreover, SERP 30 significantly attenuated the damage to the endothelial glycocalyx and maintained the integrity of the glycocalyx. The western blotting result implied that the main anti-inflammatory mechanism is directed towards NF-κB and MAPK signaling pathways with inhibiting the activation of associated proteins. ConclusionThis research provides a theoretical basis for treating ARDS by using a byproduct from food resource.

A Probe into the Intervention Mechanism of Yiqi Huayu Jiedu Decoction on TLR4/NLRP3 Signal Pathway in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome (ARDS) Rats.

Background This study discusses the anti-inflammatory mechanism of Yiqi Huayu Jiedu decoction (YQHYJD) and studies the intervening effect of YQHYJD on the inflammatory cytokines in acute respiratory distress syndrome (ARDS) rats by inhibiting the TLR4/NLRP3 signal pathway. The aim of the probe is to provide evidence to support the identification of therapeutic targets in Chinese medicine treatment, which broadens the alternatives for the treatment of ARDS. Method A lipopolysaccharide (LPS)-induced ARDS model group is established on rats by tail vein injection. A medicine group is established on ARDS rats by prophylactic administration using YQHYJD. Materials are collected, and tests are conducted according to experimental processes. Result The rats in the medicine group gained weight compared with those in the ARDS model group. Pathological sections from the medicine group indicated improved condition in terms of pulmonary and interstitial edema in the lung tissues of rats compared with that from the ARDS model group. The percentage of neutrophil of the medicine group was significantly brought down compared with that of the ARDS model group (P < 0.001). Enzyme-linked immunosorbent assay (ELISA) was used to detect the changes in the level of inflammatory cytokines. It was observed that the levels of IL-1β and IL-18 in serum of the medicine group significantly decreased (P < 0.001 and P < 0.01), the contents of TLR4 and NLRP3 in bronchoalveolar lavage fluid (BALF) of the medicine group decreased, and the contents of TLR4 and NLRP3 in lung tissue homogenate of the medicine group significantly decreased (P < 0.05, P < 0.001, P < 0.01, and P < 0.05). In further mass spectrum identification of the proteins from the same animal groups, it was observed that the expressions of inflammatory proteins TNFRSF1, LBP, and NOS2 of the medicine group were reduced. The differences were statistically significant. Conclusions The pharmacological action of YQHYJD's anti-inflammatory mechanism is closely associated with the regulation of inflammatory cytokines TLR4, NLRP3, IL-1β, IL-18, TNFRSF1, LBP, and NOS2 on the TLR4/NLRP3 signal pathway.

Meso-Dihydroguaiaretic Acid Ameliorates Acute Respiratory Distress Syndrome through Inhibiting Neutrophilic Inflammation and Scavenging Free Radical.

The pathogenesis of acute respiratory distress syndrome (ARDS) is very complex. Patients with ARDS still suffer high mortality rates. Infiltration and activation of neutrophils in lungs are critical pathogenic factors in ARDS. In this study, we demonstrate that meso-dihydroguaiaretic acid (MDGA), a natural lignan, inhibits inflammatory responses in human neutrophils and ameliorates ARDS in mice. MDGA inhibited superoxide anion generation and elastase release in various G-protein coupled receptor agonists-induced human neutrophils. However, MDGA did not alter superoxide anion generation and elastase activity in cell-free systems. These results suggest that the anti-inflammatory effects of MDGA are mediated by regulating cellular signals in human neutrophils. In consistent with this, MDGA suppressed phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase in activated human neutrophils. Moreover, MDGA inhibited CD11b expression and adhesion in activated human neutrophils. Interestingly, MDGA reduced reactive oxygen species (ROS) generation but not superoxide anion generation in protein kinase C (PKC) activator-induced human neutrophils, suggesting that MDGA may also have ROS scavenging ability. Indeed, MDGA showed strong free radical scavenging activity in cell-free assays. Significantly, MDGA suppressed PKC-induced neutrophil extracellular trap formation. Additionally, treatment of MDGA attenuated neutrophil infiltration and lung damage on lipopolysaccharide-induced ARDS in mice. In conclusion, our results demonstrate that MDGA has anti-neutrophilic inflammatory effects and free-radical scavenging activity. We also suggest that MDGA has potential to serve as a lead for developing new therapeutics to treat ARDS.

Ferulic acid regulates miR-17/PTEN axis to inhibit LPS-induced pulmonary microvascular endothelial cells apoptosis through activation of PI3K/Akt pathway.

Acute lung injury (ALI) is mainly mediated by the damage of pulmonary microvascular endothelial cells (PMVECs). LPS is one of the pathogenic factors leading to microcirculatory abnormalities of ALI. Ferulic acid (FA) exhibits therapeutic effects against various diseases. During lipopolysaccharide-induced acute respiratory distress syndrome, FA, when given beforehand, could depress inflammation and oxidative stress. However, the concrete role and underlying mechanism of FA in ALI have not been well characterized. Ten μg/mL Lipopolysaccharide (LPS) was used to treat rat PMVECs for 24 hr. qRT-PCR was used to detect the level of miR-17 and phosphatase and tensin homolog deleted on chromosome ten (PTEN). Western blot was used to analyze the associated proteins in the PI3K/Akt pathway, and the apoptosis-related proteins. Flow cytometric analysis was performed to detect the apoptosis of PMVECs. MTT assay was constructed to detect the cell viability. Luciferase assay was conducted to detect the target gene of miR-17 and PTEN. A cell model for in vitro studying the role of FA in ALI was established using PMVECs. Our data demonstrate that FA up-regulates miR-17 and declines apoptosis induced by LPS. FA inhibits apoptosis mediated by up-regulating miR-17. Furthermore, we found miR-17 targeted PTEN negatively. FA inhibits cleaved caspase-3 and Bax expression through the PI3K/Akt pathway mediated by up-regulating miR-17. Over-expression of PTEN could contribute to the similar expression trend of the PI3K/Akt signal pathway protein compared to miR-17 inhibitor transfected cells. FA inhibits PMVECs apoptosis induced by LPS via miR-17/PTEN to further regulate the activation of the PI3K/Akt pathway in ALI. We anticipate that our data will provoke additional studies for ALI clinical therapy.

N-Acetylcysteine in Mechanically Ventilated Rats with Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome: The Effect of Intravenous Dose on Oxidative Damage and Inflammation.

Treatment of acute respiratory distress syndrome (ARDS) is challenging due to its multifactorial aetiology. The benefit of antioxidant therapy was not consistently demonstrated by previous studies. We evaluated the effect of two different doses of intravenous (i.v.) N-acetylcysteine (NAC) on oxidative stress, inflammation and lung functions in the animal model of severe LPS-induced lung injury requiring mechanical ventilation. Adult Wistar rats with LPS (500 μg/kg; 2.2 mL/kg) were treated with i.v. NAC 10 mg/kg (NAC10) or 20 mg/kg (NAC20). Controls received saline. Lung functions, lung oedema, total white blood cell (WBC) count and neutrophils count in blood and bronchoalveolar lavage fluid, and tissue damage in homogenized lung were evaluated. NAC significantly improved ventilatory parameters and oxygenation, reduced lung oedema, WBC migration and alleviated oxidative stress and inflammation. NAC20 in comparison to NAC10 was more effective in reduction of oxidative damage of lipids and proteins, and inflammation almost to the baseline. In conclusion, LPS-instilled and mechanically ventilated rats may be a suitable model of ARDS to test the treatment effects at organ, systemic, cellular and molecular levels. The results together with literary data support the potential of NAC in ARDS.

Astrocytes Downregulate Inflammation in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome: Applicability to COVID-19.

Background: An acute respiratory distress syndrome (ARDS) is caused by the increased amounts of pro-inflammatory cytokines and neutrophil-mediated tissue injury. To date, there is no effective treatment for the ARDS available, while the need for one is growing due to the most severe complications of the current coronavirus disease-2019 (COVID-19) pandemic. The human astrocytes (AstroRx) have shown immunomodulatory properties in the central nervous system (CNS). This study aimed to evaluate the capacity of astrocytes to decrease lung inflammation and to be applied as a treatment therapy in ARDS.Methods: First, we assessed the ability of clinical-grade AstroRx to suppress T-cell proliferation in a mixed lymphocyte reaction test. Next, we tested the therapeutical potential of AstroRx cells in a lipopolysaccharide (LPS)-based ARDS mouse model by injecting AstroRx intravenously (i.v). We determined the degree of lung injury by using a severity scoring scale of 0–2, based on the American Thoracic Society. The scoring measured the presence of neutrophils, fibrin deposits, and the thickening of alveolar walls. The state of inflammation was further assessed by quantifying the immune-cell infiltration to the bronchoalveolar lavage fluid (BALF) and by the presence of proinflammatory cytokines and chemokines in the BALF and serum.Results: We detected that AstroRx cells were capable to suppress T-cell proliferation in vitro after exposure to the mitogen concanavalin A (ConA). In vivo, AstroRx cells were able to lower the degree of lung injury in LPS-treated animals compared with the sham injected animals (P = 0.039). In this study, 30% of AstroRx treated mice showed no lung lesions (responder mice), these mice presented a steady number of eosinophils, T cells, and neutrophils comparable with the level of naïve control mice. The inflammatory cytokines and chemokines, such as TNFα, IL1b, IL-6, and CXCL1, were also kept in check in responder AstroRx-treated mice and were not upregulated as in the sham-injected mice (P < 0.05). As a result, the LPS-treated ARDS mice had a higher survival rate when they were treated with AstroRx.Conclusions: Our results demonstrate that the immunosuppressive activity of AstroRx cells support the application of AstroRx cells as a cell therapy treatment for ARDS. The immunoregulatory activity may also be a part of the mechanism of action of AstroRx reported in the amyotrophic lateral sclerosis (ALS) neurodegenerative disease.