OBJECTIVE  To study the effects of Ro 64-6198, a selective nociceptin/orphanin FQ receptor (NOPR) agonist, on heroin self-administration and drug-seeking behavior in rats. METHODS  Rats were trained to self-administer heroin intravenously at a dose of 0.05 mg·kg^-1 under a fixed ratio 1 (FR1) reinforcement schedule. Heroin motivation was assessed using a progressive ratio (PR) schedule. Firstly, a stable heroin self-administered rat model was established before the effects of Ro 64-6198 on heroin rewarding under the FR1 schedule were observed. After three days of self-administration recovery training, the effects of Ro 64-6198 on heroin reward motivation were observed under the PR3-4 schedule. Following extinction, the reinstatement of heroin seeking induced by either conditioned cues or heroin priming was evaluated in rats withdrawn from self-administration. The expressions of cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in the ventral tegmental area (VTA) were analyzed using Western blotting, while the expression of the NOPR in neurons in the VTA was examined through immunofluorescence staining. RESULTS  Pretreatment with 3 mg·kg^-1 Ro 64-6198 significantly reduced active responses and heroin infusions during FR1 testing, as well as decreased breakpoints, indicating reduced motivation under the PR schedule. At a dose of 1 mg·kg^-1, Ro 64-6198 markedly attenuated the reinstatement of heroin-seeking behavior induced by conditioned cues or heroin priming. Furthermore, the administration of SB-612111, an NOPR antagonist, blocked the inhibitory effects of Ro 64-6198 on cue-induced heroin-seeking, although SB-612111 alone had no effect on heroin-seeking behavior. Ro 64-6198 treatment also suppressed the reduction of both phosphorylated CREB (p-CREB) and BDNF levels in the VTA and the decreased expression of NOPR and p-CREB in dopaminergic neurons of the VTA. CONCLUSION  These results demonstrate that Ro 64-6198 can mitigate heroin-seeking behavior through NOPR activation and CREB/BDNF pathway in the VTA. This study is expected to offer evidence for its potential as a clinical treatment for heroin addiction and relapse. 

OBJECTIVE  To investigate the protective effects of hydroxytyrosol hydroxybutyrate (HTHB) against cognitive impairment induced by acute sleep deprivation in mice and to explore the underlying mechanisms. METHODS  Male C57BL/6J mice were assigned to the following groups: normal control, normal groups administered with HTHB 30, 60 and 120 mg·kg^-1 or hydroxytyrosol (HT 19.25, 38.5 and 77 mg·kg^-1), a sleep deprivation (SD) model group, and SD groups co-administered with the same doses of HTHB or HT. Acute sleep deprivation was induced for 72-96 h using a rotarod apparatus in all groups except the normal control and normal drug-treated groups. Based on dose-response assessments in the Y-maze and novel object recognition tests, the effective doses (HTHB 60 mg·kg^-1 and HT 38.5 mg·kg^-1) were selected for subsequent evaluation in the two-choice visual discrimination task that was performed in a subset of groups: normal control, normal+HTHB 60 mg·kg^-1, normal+HT 38.5 mg·kg^-1, SD model, SD+HTHB 60 mg·kg^-1, and SD+HT 38.5 mg·kg^-1. Cognitive functions that were assessed included spatial working memory (Y-maze spontaneous alternation), object recognition memory (novel object recognition) and visual discrimination ability (two-choice visual discrimination task). Biochemically, levels of hippocampal reactive oxygen species (ROS) were quantified by ELISA while the ATP content was determined using a firefly luciferase-based assay. RESULTS  In non-sleep-deprived mice, neither HTHB nor HT administration significantly altered locomotor activity, spatial working memory, object recognition memory, or visual discrimination performance. Following sleep deprivation, the model group displayed significant cognitive deficits, including reduced spontaneous alternation rate, lower novel object recognition indexes, and decreased accuracy in the visual discrimination task at 48 h and 96 h. These impairments were accompanied by elevated hippocampal ROS levels and reduced ATP contents compared to the control group. Treatment with HT 38.5 and 77 mg·kg^-1 significantly attenuated the deficit in spontaneous alternation, but did not affect other parameters. In contrast, HTHB at 60 and 120 mg·kg^-1 produced broader restorative effects and significantly reversed impairment in both spontaneous alternation and novel object recognition. Furthermore, HTHB at 60 mg·kg^-1 significantly improved visual discrimination accuracy at 48 h and 96 h, while lowering hippocampal ROS levels and increasing ATP contents. CONCLUSION  HTHB effectively mitigates acute sleep deprivation-induced impairment in spatial working memory, object recognition memory, and visual discrimination in mice. This protection is likely mediated by the enhancement of mitochondrial antioxidant capacity and the restoration of energy metabolism.

OBJECTIVE  To investigate the anti-fatigue effect of chicory polysaccharide (CP) on mice exposed to simulated hypobaric hypoxia. METHODS  Male C57BL/6J mice were randomly divided into the control group, model group, model+CP 150, 300 and 600 mg·kg^-1 groups. The control and model groups were given normal saline, while the CP groups were given drugs of different doses. After a 14 d pre-administration period, all the mice except the control group were exposed to a simulated altitude of 7 000 m in a hypobaric and hypoxic animal experimental chamber. After 7 d, a treadmill fatigue test was conducted to assess exercise endurance. The body weight and organ indexes were evaluated. The pathological changes in organs and tissues were observed via HE staining. The levels of fatigue-related and oxidative stress-related indicators were measured. The expression levels of phosphorylated AMP-activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and cytochrome c oxidase Ⅳ (COXⅣ) were determined using Western blotting analysis. RESULTS  Compared with model group, exercise endurance was significantly enhanced, body weight and organ indexes improved, and pathological damage to the lung, liver and skeletal muscle mitigated in the model+CP 600 mg·kg^-1 group. Compared with model group, the model+CP 600 mg·kg^-1 group had the contents of serum lactate and blood urea nitrogen reduced, but the contents of glycogen and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver and skeletal muscle were increased. The malondialdehyde content was lowered, but the expressions of p-AMPK, PGC-1α, and COXⅣ in skeletal muscle were significantly increased. CONCLUSION  CP can alleviate altitude-induced fatigue by reducing the metabolite accumulation, increasing glycogen storage, and lowering oxidative stress levels. The underlying mechanism may involve the activation of the AMPK/PGC-1α signaling pathway.

OBJECTIVE  To investigate the acute lung injury effects of pulmonary phospholipids and their phospholipase A2 (PLA2) decomposition products—lysophospholipids and fatty acids—on mice. METHODS  Mice were randomly assigned to the following groups: ① solvent control (PBS) and PLA2; ② solvent control and glycerol phospholipid groups: 1, 2-dioleoyl-sn-glycero-3-phosphoserine  (DOPS), 1, 2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine  (DPPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC); ③ solvent control and fatty acid groups: palmitic acid (PA), oleic acid; ④ solvent control and lysophospholipid groups: 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoserine (18:1 LysoPS), 1-stearoyl-sn-glycero-3-phosphoserine (18:0 LysoPS), 1-palmitoyl-sn-glycero-3-phosphoserine (16:0 LysoPS), 1-palmitoyl-sn-glycero-3-phosphoethanolamine (16:0 LysoPE), 1-palmitoyl-sn-glycero-3-phosphocholine (16:0 LysoPC); ⑤ solvent control, PLA2, DPPC, PA, 16:0 LysoPC, 16:0 LysoPS, and 18:1 LysoPS. Following anesthesia, mice were administered nebulized PBS in the solvent control group， 2.1 μg·kg^-1 PLA2 in PBS in the PLA2 group and 2.5 mg·kg^-1 of the corresponding substance in PBS in other experimental groups. For group ①, survival times were recorded and survival curves were plotted. At 1 h post-treatment, lung 

tissues from groups ①②③④ were collected, photographed to obtain white light images, and subjected to HE staining to assess histopathological changes and pathological scoring.  At 2 hours post-treatment, pulmonary blood flow in group ⑤ was assessed using laser speckle contrast imaging, arterial blood gas was analyzed with a blood gas analyzer, and lung function was evaluated using whole-body plethysmography. At 6 hours post-treatment, blood cells from group ⑤ were analyzed using an automated hematology analyzer. RESULTS  Compared with the solvent control group, severe pathological changes were observed in lung tissues of the PLA2 group, accompanied by extensive inflammatory infiltration and interstitial thickening, with all mice succumbing within 240 min. In mice treated with glycerol phospholipids, alveolar structures remained clear, alveolar walls were intact and continuous, and alveolar spaces were translucent, with only occasional minor inflammatory cell infiltration in the septa. No significant pathological alterations were detected in the fatty acid groups. Minor inflammatory cell infiltration was seen in the 16:0 LysoPE and 16:0 LysoPC groups. However, such pathological changes as patchy hemorrhage, alveolar interstitial edema, increased alveolar wall thickness, and elevated neutrophil counts were observed in the 18:1 LysoPS, 18:0 LysoPS, and 16:0 LysoPS groups. Pathological scores based on HE staining were significantly increased in the 16:0 LysoPS and 18:1 LysoPS groups compared with the solvent control. The percentage of the lung tissue injury area was also markedly higher in the 16:0 LysoPS group. A significant decrease in the mean fluorescence intensity of blood flow was observed in the 16:0 LysoPS group. Arterial partial pressure of oxygen (PO₂) was significantly reduced in the PLA2 group, while arterial partial pressure of carbon dioxide (PCO₂) was markedly elevated in the 16:0 LysoPS and 18:1 LysoPS groups. Lung function tests revealed that the 16:0 LysoPS group exhibited significant increases in expiratory time, end-expiratory pressure, and enhanced pause, in contrast to significant decreases in tidal volume, expired volume, and minute volume. The 18:1 LysoPS group also exhibited a significant decline in minute volume. No significant changes in inflammatory cell concentrations were detected in blood, with the exception of neutrophils in the 16:0 LysoPS group, which showed a significant but physiologically normal increase. CONCLUSION  Pulmonary phospholipids and their PLA2-derived fatty acid metabolites do not induce severe lung injury in mice while the lysophospholipid metabolites, particularly lysophosphatidylserine, are found to cause significant lung injury.

OBJECTIVE  To investigate the role and mechanisms of the soluble guanylate cyclase (sGC) stimulator sGC003 in improving high altitude pulmonary edema (HAPE) in mice. METHODS  Mice were randomly assigned to a normal control group, model group, model+dexamethasone 4 mg·kg^-1 group (before modeling, intragastric administration of saline was performed once daily for 6 d, followed by intragastric administration of dexamethasone 4 mg·kg^-1 on days 7 and 8), model+riociguat 10 mg·kg^-1 group (before modeling, intragastric administration once a day for 7 d), and model+sGC003 5 and 10 mg·kg^-1 groups (before modeling, intragastric administration once a day for 7 d). All groups except the normal control group received intratracheal instillation of lipopolysaccharide at a dose of 4 mg·kg^-1 1 h after drug administration on day 7, followed by placement in a hypoxic environment to establish the HAPE model. After 24 h of modeling, the expiratory time, end-inspiratory pause, enhanced pause, and breathing frequency were measured, Lung tissue morphology was examined using HE staining, and lung tissue edema was assessed by determining the wet to dry weight ratio (W/D). The level of interleukin-1β (IL-1β) was determined using immunofluorescence staining. The phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) in lung tissue was analyzed by Western blotting. Additionally, levels of sGC, hypoxia inducible factor-1α (HIF-1α), cyclic guanosine monophosphate (cGMP), IL-6, and IL-1β in serum were quantified using ELISA. RESULTS  Compared with the normal control group, the model group had obvious pulmonary edema, and the lung W/D, IL-1β levels, expiratory time, end-inspiratory pause, enhanced pause, as well as serum levels of IL-1β, HIF-1α and IL-6 were significantly increased. Concurrently, the frequency of breathing and serum levels of sGC and cGMP were significantly decreased. Compared with model group, the expiratory time, end-inspiratory pause, enhanced pause, lung W/D and IL-1β levels, and serum levels of IL-1β, HIF-1α and IL-6 were significantly decreased in the model+sGC003 10 mg·kg-1 group; while the frequency of breathing, serum sGC and cGMP levels, phosphorylation level of VASP in lung tissues were significantly increased. CONCLUSION  sGC003 can improve lung function, suppress pulmonary inflammation, and mitigate pulmonary edema in HAPE mice by activating the sGC/cGMP pathway.

OBJECTIVE  To investigate the impact of lysosomal associated membrane protein 2b (Lamp2b) modification on the mucosal immune efficacy of engineered exosome-based vaccines. METHODS  In vitro experiments: The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein. Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions, flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells, and immunofluorescence staining was performed to determine their membrane localization. Exosomes were isolated via ultracentrifugation, and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis. Western blotting was applied to confirm exosomal marker proteins [cluster of differentiation 9 (CD9), CD63, ALG-2-interacting protein X (Alix), and Golgi marker GM130] and Lamp2b-RBD expression. In vivo experiments: ① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle (LNP) group, and administered intratracheally for mucosal immunization. Pulmonary retention was assessed by immunofluorescence staining. ② Female BALB/c mice were divided into three groups: placebo group (PBS group), Lamp2b-RBD-Exo intratracheal administration group, and Lamp2b-RBD-Exo intramuscular injection group (im). Immunizations were performed on days 0 and 14, and on days 7 and 21. The titers of RBD-specific immunoglobulin G (IgG) in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS  In vitro experiments: Lamp2b-RBD-positive cells accounted for 71.16%. Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls, with Lamp2b-RBD proteins localized on the cell membrane. Purified engineered exosomes displayed regular morphology, expressed CD9, CD63, and Alix but not GM130, had an average diameter of approximately 124 nm, and carried 3 009 pg of RBD protein per 1×109 exosomes. In vivo experiments: At 4 h after administration, fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups. At 24 h, the fluorescence signal in the LNPs group shifted to the liver, while in the Lamp2b-RBD-Exo group, the fluorescence expanded from the trachea to the bronchioles and lung tissue, showing significantly better distribution and retention capacity than the LNPs group. Seven days after immunization, both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo (im) groups induced RBD-specific IgG antibody titers. At 21 days after immunization, Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response, with serum IgG titers reaching 1:8 100 and bronchoalveolar lavage fluid (BALF) IgA titers reaching 1:300. No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo (im) group. CONCLUSION  Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes, thereby inducing potent antigen-specific mucosal immune responses. 

Excessive activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome drives neuronal injury in neurological disorders. The NLRP3 inflammasome complex comprises NLRP3, apoptosis-associated speck - like protein containing a CARD (ASC) and cysteine aspartic acid specific protease-1 (Caspase-1). Upon activation through canonical or non-canonical pathways, this complex triggers the release of IL-1β/IL-18 and induces pyroptosis. Clinical evidence has pointed to the co-localization of ASC with amyloid β-protein (Aβ) plaques in the brains of Alzheimer disease (AD) patients, specific upregulation of NLRP3 in monocytes of multiple sclerosis patients, and a positive correlation between serum NLRP3 levels and infarct volume in stroke patients, suggesting that NLRP3 inflammasome can be used as a critical therapeutic target. This review summarizes the pathological roles of the NLRP3 inflammasome and recent advances in candidate therapeutics. The targeting strategies include direct inhibitors, upstream regulators and downstream blockers. However, clinical translation of small-molecule inhibitors faces challenges due to the blood-brain barrier, inadequate disease modeling and off-target toxicity concerns. Emerging solutions include nanocarrier systems, AI-assisted drug design and novel target exploration. Future research should use stem cell-derived organoid models and molecular subtyping approaches to advance precision medicine and facilitate clinical translation.

Adenosine, as a purine nucleoside widely present in living organisms, plays an important role in such physiological and pathological processes as vasodilation, inflammation regulation, fibrosis, and viral infection. Research has found that in pulmonary hypertension, adenosine acts as a vasodilator through multiple targets. In diseases such as mycoplasma pneumonia, asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis, adenosine primarily mediates the progression of inflammation through interactions with adenosine A2A and A2B receptors, thus delivering a dual regulatory effect. This suggests that modulating adenosine receptors is a potential key target for the treatment of pulmonary diseases. Adenosine derivatives—such as acyclovir monophosphate, which has antiviral effects, cyclic adenosine monophosphate, which can alleviate airway narrowing, and N-6-methyladenosine, which is involved in regulating cilia homeostasis and anti-pulmonary fibrosis—are closely related to the occurrence and development of pulmonary diseases. Targeting the adenosine A2A receptor or antagonizing the adenosine A2B receptor can precisely intervene in specific pulmonary diseases. Combination medications, regulation of adenosine metabolic pathways, and gene editing technologies promise new treatments. This article reviews the role of adenosine, its receptors, and derivatives in the development of pulmonary diseases in the hopes of providing evidence for related treatment.