Rutin targets AKT to inhibit ferroptosis in ventilator-induced lung injury.

Our previous research confirmed that rutin reduced ventilator-induced lung injury (VILI) in mice. Ferroptosis has been reported to participate in the pathogenic process of VILI. We will explore whether rutin inhibits ferroptosis to alleviate VILI. A mouse model of VILI was constructed with or without rutin pretreatment to perform a multiomics analysis. Hematoxylin-eosin (HE) staining and transmission electron microscopy were used to evaluate lung injury in VILI mice. Dihydroethidium (DHE) staining and the malondialdehyde (MDA) and superoxide dismutase (SOD) levels were detected. Molecular docking was performed to determine the binding affinity between rutin and ferroptosis-related proteins. Western blot analysis, real-time PCR (RT-PCR) and immunohistochemical (IHC) staining were conducted to detect the expression levels of GPX4, XCT, ACSL4, FTH1, AKT and p-AKT in lung tissues. Microscale thermophoresis (MST) was used to evaluate the binding between rutin and AKT1. Transcriptomic and proteomic analyses showed that ferroptosis may play a key role in VILI mice. Metabolomic analysis demonstrated that rutin may affect ferroptosis via the AKT pathway. Molecular docking analysis indicated that rutin may regulate the expression of ferroptosis-related proteins. Moreover, rutin upregulated GPX4 expression and downregulated the expression of XCT, ACSL4 and FTH1 in the lung tissues. Rutin also increased the ratio of p-AKT/AKT and p-AKT expression. MST analysis showed that rutin binds to AKT1. Rutin binds to AKT to activate the AKT signaling pathway, contributing to inhibit ferroptosis, thus preventing VILI in mice. Our study elucidated a possible novel strategy of involving the use of rutin for preventing VILI.

Electroacupuncture Attenuates Ventilator-Induced Lung Injury by Modulating the Nrf2/HO-1 Pathway.

INTRODUCTION: Ventilator-induced lung injury (VILI) is the most common complication associated with mechanical ventilation. Electroacupuncture (EA) has shown potent anti-inflammatory effects. This study aimed to investigate the effects of EA on VILI and explore the underlying mechanisms. METHODS: Male C57BL/6 mice were subjected to high tidal volume ventilation to induce VILI. Prior to mechanical ventilation, mice received treatment with EA, nonacupoint EA, or EA combined with zinc protoporphyrin. RESULTS: EA treatment significantly improved oxygenation, as indicated by increased PaO2 levels in VILI mice. Moreover, EA reduced lung injury score, lung wet/dry weight ratio, and protein concentration in bronchoalveolar lavage fluid. EA also decreased the expression of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, IL-18, chemokine keratinocyte chemoattractant, macrophage inflammatory protein 2, and malondialdehyde. Furthermore, EA increased the activities of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase in VILI mice. At the molecular level, EA upregulated the expression of Nrf2 (nucleus) and heme oxygenase -1, while down-regulating the expression of p-NF-κB p65, NLR Family Pyrin Domain Containing 3, Cleaved Caspase-1, and ASC in VILI mice. Notably, the effects of EA were reversed by zinc protoporphyrin treatment, nonacupoint EA did not affect the aforementioned indicators of VILI. CONCLUSIONS: EA alleviates VILI by inhibiting the NLR Family Pyrin Domain Containing three inflammasome through activation of the Nrf2/HO-1 pathway.

[Effects of electroacupuncture pretreatment on NLRP3 inflammasome in mice with ventilator-induced lung injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) pretreatment on inflammatory response in ven-tilator-induced lung injury (VILI) mice, so as to explore the underlying mechanism of EA pretreatment on prevention of VILI. METHODS: C57BL/6 mice were randomly divided into sham-operation group, model group, EA group and sham-acupoint group，with 8 mice in each group. The VILI model was established by ventilation with high tidal volume. Mice in the EA group and sham-acupoint group were given EA at "Zusanli" (ST36)and "Feishu"(BL13) or non-acupoints (located at 1-2 cm on both sides of the tail root of the proximal trunk) before mechanical ventilation, 30 min each time, once a day for 5 days. Arterial blood was collec-ted for blood gas analysis, the total protein content in bronchoalveolar lavage fluid (BALF) was detected by BCA method. The contents of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in BALF were detected by ELISA. Lung injury score was determined after HE staining. The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and Caspase-1 in lung tissue was detected by Western blot. RESULTS: Compared with the sham-operation group, the arterial partial pressure of oxygen and oxygenation index were decreased(P<0.05), the levels of total protein, IL-1ß and IL-18 in BALF, the W/D value and the pathological injury score of lung tissue and the protein expression levels of NLRP3, Caspase-1 and ASC were increased(P<0.05)in the model group. Following the interventions, the above mentioned increased or decreased indicators were reversed(P<0.05) in the EA group rather than in the sham-acupoint group. CONCLUSION: EA pretreatment of ST36 and BL13 can reduce the damage of lung tissue caused by mechanical ventilation, which may be related to its effect in reducing the expression of NLPR3 inflammasome related proteins, reducing the activation of inflammasome, and thereby reducing the inflammatory response.

JAK2/STAT1-mediated HMGB1 translocation increases inflammation and cell death in a ventilator-induced lung injury model.

Janus kinase 2/signal transducer and activators of transcription 1 (JAK2/STAT1) signaling is a common pathway that contributes to numerous inflammatory disorders, including different forms of acute lung injury (ALI). However, the role of JAK2/STAT1 in ventilator-induced lung injury (VILI) and its underlying mechanism remain unclear. In this study, using lipopolysaccharide (LPS) inhalation plus mechanical ventilation as VILI mouse model, we found that the administration of JAK2 inhibitor AZD1480 markedly attenuated lung destruction, diminished protein leakage, and inhibited cytokine release. In addition, when mouse macrophage-like RAW 264.7 cells were exposed to LPS and cyclic stretch (CS), AZD1480 prevented cell autophagy, reduced apoptosis, and suppressed lactate dehydrogenase release by downregulating JAK2/STAT1 phosphorylation levels and inducing HMGB1 translocation from the nucleus to the cytoplasm. Furthermore, HMGB1 and STAT1 knockdown attenuated LPS+CS-induced autophagy and apoptosis in RAW 264.7 cells. In conclusion, these findings reveal the connection between the JAK2/STAT1 pathway and HMGB1 translocation in mediating lung inflammation and cell death in VILI, suggesting that these molecules may serve as novel therapeutic targets for VILI.

Alpha 1-antitrypsin for treating ventilator-associated lung injury in acute respiratory distress syndrome rats.

Purpose: Mechanical ventilation (MV) is an essential life support tool for patients with acute respiratory distress syndrome (ARDS). However, MV for ARDS can result in ventilator-induced lung injury (VILI). This study aimed to assess whether alpha 1-antitrypsin (AAT) can reduce VILI in ARDS rats. Materials and Methods: Rats were randomly divided into five groups: the sham (S) group, MV (V) group, lipopolysaccharide (LPS) (L) group, MV/LPS (VL) group and MV/AAT (VA) group. Rats in the S group were anesthetized. The rats in the L group received LPS but not ventilation, the rats in the V group received only MV, and the rats in the VL and VA groups received LPS and MV. Additionally, the rats in the VA group were treated with AAT, and the other rats were injected with saline. The PaO2/FiO2 ratio and the wet/dry weight were assessed. The total protein and neutrophil elastase concentrations and the neutrophil and macrophage counts in bronchoalveolar lavage fluid (BALF) were evaluated. Proinflammatory factors in BALF and ICAM-1 and MIP-2 in serum were also tested. Furthermore, the oxidative stress response was detected, and histological injury and apoptosis were evaluated. Results: All the rats in the V, L and VL groups had significant lung injury, with the VL group exhibiting the most severe injury. Compared with the findings in the VL group, AAT significantly upregulated the PaO2/FiO2 ratio but decreased the wet/dry weight ratio and protein levels in BALF. AAT also reduced proinflammatory cytokine levels and inflammatory cell counts in BALF. Lung tissue injury and cell apoptosis were mitigated by AAT. Conclusions: AAT ameliorated VILI in ARDS rats. The protection conferred by AAT may be associated with the anti-inflammatory, antioxidative stress response and anti-apoptotic effects of AAT.

Protective effects of fucoidan against hyperoxic lung injury via the ERK signaling pathway.

High oxygen mechanical ventilation is widely used to treat various lung diseases; however, it may result in hyperoxia, which induces inflammation and lung injury. Fucoidan is an extract of the seaweed Fucus vesiculosus, which has previously been reported to exert effects against diabetic nephropathy. The present study is the first, to the best of our knowledge, to investigate the protective effects of fucoidan against hyperoxic lung injury. Balb/c mice were ventilated with 100% oxygen, with or without the atomization inhalation of fucoidan, for 36 h. Hyperoxia reduced the body weight and increased the relative lung weight of the mice. In addition, cell quantity and differentiation were determined using a hemocytometer, hyperoxia increased the total number of cells, and the number of macrophages, neutrophils and lymphocytes in the bronchoalveolar lavage fluid. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) demonstrated that hyperoxia also increased the mRNA expression levels of cluster of differentiation (CD)68, F4/80, CD64 and CD19 in lung tissue, and induced lung morphological alterations. Furthermore, western blotting assay demonstrated that hyperoxia increased the expression levels of interleukin (IL)­1, IL­6 and tumor necrosis factor (TNF)­α, and the phosphorylation of extracellular signal­regulated kinase (ERK)1/2. Conversely, hyperoxia­induced inflammation and morphological alterations were significantly attenuated in the mice treated with fucoidan. Atomization inhalation of fucoidan also reduced the hyperoxia­induced expression of IL­1, IL­6 and TNF­α, and the phosphorylation of ERK1/2. These findings suggested that fucoidan may attenuate hyperoxic lung injury via the ERK1/2 signaling pathway.

BML-111, a lipoxin receptor agonist, attenuates ventilator-induced lung injury in rats.

Mechanical ventilation can cause structural and functional disturbances in the lung termed ventilator-induced lung injury (VILI). The aim of this study was to evaluate whether BML-111, a lipoxin receptor agonist, could attenuate VILI. Following induction of anesthesia and tracheostomy, Sprague-Dawley rats were ventilated with low tidal volume (6 mL/kg) or high tidal volume (20 mL/kg, HVT) for 4 h. Some rats subjected to HVT ventilation received BML-111 or vehicle (saline) by intraperitoneal injection. Some rats subjected to HVT and BML-111(1 mg/kg) received BOC-2 (a FPR2/ALX antagonist) intraperitoneally 30 min before BML-111. Sham rats were tracheotomized without ventilation. Treatment with BML-111 attenuated VILI, as evidenced by improved oxygenation and reduced histological injury compared with HVT-induced lung injury. BML-111 decreased indices of inflammation such as interleukin 1ß, interleukin 6, tumor necrosis factor α, and bronchoalveolar lavage neutrophil infiltration. Administration with BML-111 suppressed the decrement of the nuclear factor κB (NF-κB) inhibitor IκB-α, diminished NF-κB activation, and reduced activation of mitogen-activated protein kinase in VILI. This study indicates that BML-111 attenuated VILI via a NF-κB and mitogen-activated protein kinase dependent mechanism. BML-111 may be a promising strategy for alleviation of VILI in patients subjected to mechanical ventilation.

Activation of nicotinic cholinergic receptors prevents ventilator-induced lung injury in rats.

Respiratory distress syndrome is responsible for 40 to 60 percent mortality. An over mortality of about 10 percent could result from additional lung injury and inflammation due to the life-support mechanical ventilation, which stretches the lung. It has been recently demonstrated, in vitro, that pharmacological activation of the alpha 7 nicotinic receptors (α7-nAChR) could down regulate intracellular mediators involved in lung cell inflammatory response to stretch. Our aim was to test in vivo the protective effect of the pharmacological activation of the α7-nAChR against ventilator-induced lung injury (VILI). Anesthetized rats were ventilated for two hours with a high stretch ventilation mode delivering a stroke volume large enough to generate 25-cmH(2)O airway pressure, and randomly assigned to four groups: pretreated with parenteral injection of saline or specific agonist of the α7-nAChR (PNU-282987), or submitted to bilateral vagus nerve electrostimulation while pre-treated or not with the α7-nAChR antagonist methyllycaconitine (MLA). Controls ventilated with a conventional stroke volume of 10 mL/kg gave reference data. Physiological indices (compliance of the respiratory system, lung weight, blood oxygenation, arterial blood pressure) and lung contents of inflammatory mediators (IL-6 measured by ELISA, substance P assessed using HPLC) were severely impaired after two hours of high stretch ventilation (sham group). Vagal stimulation was able to maintain the respiratory parameters close to those obtained in Controls and reduced lung inflammation except when associated to nicotinic receptor blockade (MLA), suggesting the involvement of α7-nAChR in vagally-mediated protection against VILI. Pharmacological pre-treatment with PNU-282987 strongly decreased lung injury and lung IL-6 and substance P contents, and nearly abolished the increase in plasmatic IL-6 levels. Pathological examination of the lungs confirmed the physiological differences observed between the groups. In conclusion, these data suggest that the stimulation of α7-nAChR is able to attenuate VILI in rats.