Telomerase RNA (TERC) is subject to various modifications, yet the implications of these modifications for telomerase biology remain largely unexplored. In this study, we conducted a comprehensive mapping of N6-Methyladenosine (m6A) modifications within TERC RNA and elucidated their regulatory role in telomerase function. Our findings demonstrate that TERC undergoes methylation at adenosine residues A111 and A435 by METTL3. A deficiency in TERC m6A, which is also linked to various human telomerase disease-related mutations and deletions, significantly reduces telomerase activity and telomere length by disrupting the association between TERC and TERT. Mechanistically, YTHDC1 was identified as a scaffold facilitating the interaction between TERT and TERC, binding to TERT while recognizing m6A sites on TERC. Knockdown of YTHDC1 significantly diminished the interaction between TERT and TERC, thereby reducing telomerase activity and phenocopying the deficiency of METTL3. Furthermore, reconstituting wild-type YTHDC1 rescued telomere attrition, proliferation defects, and senescence in YTHDC1-knockdown alveolar epithelial cells, whereas truncated YTHDC1 (which retains m6A recognition but lacks TERT-binding capacity) failed to restore these phenotypes. Collectively, our work establishes m6A modification of TERC as a central regulator of telomerase function and reveals YTHDC1's scaffolding role in TERT-TERC assembly, shedding new light on the regulation of telomerase and related diseases.

Pramipexole (PPX), dopamine D2/D3 receptor agonist, exhibits neuroprotective effects in patients with restless legs syndrome and Parkinson's disease. The present study aimed to investigate the cellular and molecular mechanisms underlying PPX treatment in memory and cognitive impairments induced by sevoflurane anesthesia in the aged mice. Aged mice received 2-h daily exposures to 60% oxygen, either with or without 3% sevoflurane, for three consecutive days. For interventional evaluation, PPX was administered intraperitoneally 1 h before sevoflurane exposure on three consecutive days. After the behavioral tests, hippocampal tissues were harvested from the mice for histochemical and biochemical examinations. Ex vivo electrophysiology was used to assess the activity of pyramidal neurons, while in vivo electrophysiology to evaluate changes in neural oscillations in the hippocampus of mice. PPX attenuated glial cell activation, reversed the downregulation of parvalbumin (PV) expression, rescued the reduction in the activity of PV interneuron and the imbalance of local neuronal excitatory/inhibitory, reversed the reduction in gamma oscillations, and ameliorated the downregulation of dopamine D2 receptors in the hippocampus after repeated sevoflurane exposure in aged mice. Our findings suggest that PPX confers neuroprotection, at least in part, by attenuating neuroinflammation and PV interneuron dysfunction via upregulation of hippocampal dopamine D2 receptors, thereby ameliorating memory and cognitive deficits.

Aging involves morphological and functional changes across different organs, but how these changes are linked among the different organs remains to be elucidated. Here, we uncover a central role of platelets in systemic aging. In aged mice, the levels of platelet-secreted pro-inflammatory factors (PSPF) increased greatly in the serum and platelets, leading to a diffuse increase of platelet infiltration in the brain, liver, lung, kidney, and aortic root. The RNA-binding protein HuR/ELAVL1, a major regulator of RNA metabolism, promoted the production of PSPF in platelets. Platelet-specific deletion of HuR reduced the expression of PSPF in platelets, alleviated platelet infiltration in the brain, liver, lung, kidney, and aortic root, and delayed systemic aging. By using single-nucleus sequencing, platelet-specific HuR ablation was found to alleviate p53 and pro-inflammatory signaling pathways in liver, lung, and brain tissues in aged mice. Our findings highlight a role of platelets in coordinating aging traits across organs.

PURPOSE:To evaluate the effectiveness of an anesthesia recovery management model based on the ORTCC framework in patients undergoing painless gastrointestinal endoscopy. DESIGN:In August 2023, our hospital implemented an anesthesia recovery management model structured around the ORTCC model-comprising five components: Objective, Rules, Training, Check, and Culture. After a one-month pilot period, a total of 1000 patients were enrolled using a pre-post quasi-experimental design. METHODS:Five hundred patients who underwent endoscopy before the implementation formed the control group (conventional care), while 500 patients treated afterward constituted the intervention group, receiving care under the ORTCC-based recovery model. Recovery time, incidence of anesthesia-related complications, frequency of nursing adverse events, and patient satisfaction were measured and compared between groups. FINDINGS:Compared to the control group, the intervention group demonstrated significantly shorter anesthesia recovery times, lower rates of anesthesia-related complications and nursing adverse events, and higher levels of patient satisfaction (P < 0.05). CONCLUSIONS:The implementation of an ORTCC-based anesthesia recovery management model in patients undergoing painless gastrointestinal endoscopy was associated with enhanced recovery efficiency, reduced complication rates, and improved care safety. By integrating structured objectives, standardized rules and workflows, targeted training, performance assessment, and a culture of quality, this model provides a replicable framework for improving perioperative nursing outcomes and patient-centered care in ambulatory surgical settings.

Dexmedetomidine (Dex), an α2-adrenergic receptor (α2-AR) agonist, is widely used for its antihyperalgesic effects in perioperative pain management, yet its underlying mechanisms remain largely undefined. Here, we identify a rapid, circuit-specific mechanism by which Dex reverses stress-induced hyperalgesia (SIH) in mice. Stress exposure strengthens excitatory drive from the orbitofrontal cortex (OFC) to the anterior insula cortex (AIC), shifting the excitatory/inhibitory balance toward excitation and increasing the intrinsic excitability of AIC glutamatergic neurons. Viral tracing and optogenetics reveal a direct OFC-AIC glutamatergic projection. Optogenetic or chemogenetic activation of this pathway in otherwise naïve mice potentiates glutamatergic synaptic transmission and is sufficient to induce hyperalgesia, phenotyping SIH. Dex rapidly suppresses these effects by engaging presynaptic α2-ARs on OFC terminals, thereby reducing transmitter release at OFC-AIC synapses. Consistently, chemogenetic silencing of AIC neurons or of the OFC-AIC projection alleviates hyperalgesia in SIH mice. These findings define the OFC-AIC glutamatergic circuit as a key substrate for stress-related pain amplification and uncover a rapid presynaptic α2-AR "brake" as the mechanism underlying Dex's antihyperalgesic action, highlighting a tractable therapeutic entry point for stress-exacerbated pain states.

Objective Postoperative delirium (POD) is common after cardiac surgery, linked to prolonged hospitalization and adverse outcomes. Despite its clinical impact, objective preoperative neuroimaging markers with robust POD predictive value remain elusive. This study explored whether preoperative regional homogeneity (ReHo) alterations correlate with POD and validated their predictive utility, with a focus on heart-brain interaction implications. Methods In this prospective observational study, resting-state functional MRI data were collected from patients undergoing valve replacement surgery with cardiopulmonary bypass preoperatively, as well as on postoperative days 7 and 30, alongside data from healthy controls. POD was assessed twice daily over 5 postoperative days using standardized delirium tools. Voxel-wise analyses detected group-level differences and longitudinal ReHo changes, and explored associations between preoperative ReHo alterations, cardiac function, and cognitive performance. Support vector machine (SVM) models incorporating preoperative ReHo features and age were constructed to evaluate their discriminative performance for POD. Results Of 43 recruited patients, 35 were included in final analyses, with 7 (20%) developing POD. Compared with non-POD patients, POD patients exhibited significantly reduced preoperative ReHo in the left hippocampus, precuneus, and rolandic operculum. Reduced hippocampal ReHo correlated with cardiac function, while rolandic operculum ReHo linked to episodic memory. SVM models incorporating preoperative ReHo features and age showed discriminative performance for distinguishing patients who developed POD from those who did not. Conclusions Preoperative ReHo alterations in specific brain regions reflect latent neural vulnerability to POD. These findings support the potential value of integrating ReHo features with age for preoperative risk stratification, and shed light on heart-brain interactions underlying POD susceptibility after cardiac surgery.

Cancer-related pain and anxiety significantly negate the quality of life in patients. Oligodendrocyte precursor cells (OPCs) were reported to involve in engulf synapses and remodel neural circuits. This study aimed to elucidate the role of OPCs-mediated phagocytosis of GABAergic synapses in a mouse model of bone cancer pain. Male C3H mice were utilized to establish a model of bone cancer pain. In vivo fiber photometry was used to monitor the activity of GABAergic neurons, and chemogenetic techniques were applied to modulate neuronal excitation. The phagocytosis of GABAergic synapses by OPCs was visualized via immunofluorescence-based 3D reconstruction and immunoelectron microscopy. Interventions targeting lipocalin-2 (LCN2) and its receptor SLC22A17 were carried out with adeno-associated virus (AAV), siRNA, and pharmacological tools. On the 21st postoperative day, mice with bone cancer displayed significant pain and anxiety-like behaviors. Tumor-bearing mice exhibited a compensatory increase in calcium activity among GABAergic neurons within the anterior cingulate cortex (ACC). Compared with sham-operated mice, OPC phagocytosis of GABAergic synapses was higher in the tumor-bearing mice than controls. Concurrently, a pronounced upregulation of LCN2 expression was observed in the tumor group. Administration of LCN2-neutralizing antibodies or AAV-mediated intervention markedly alleviated pain and anxiety-related behaviors in mice with bone cancer. Moreover, the LCN2 receptor SLC22A17 expression was significantly increased. Targeted inhibition of SLC22A17 induced cytoskeletal remodeling and decreased their phagocytic capacity of OPCs. Collectively, LCN2/SLC22A17 signal was involved in OPCs-mediated phagocytosis of GABAergic synapses and contributed to cancer pain and anxiety development.

Astrocytic activation is critically involved in the development and maintenance of bone cancer pain (BCP). Recent studies have shown that astrocytes participate in synaptic remodeling through synaptic phagocytosis, whereas neuronal cell adhesion molecule (NRCAM) restricts the extension of perisynaptic astrocytic processes and exerts an anti-phagocytic effect. However, whether astrocyte-mediated synaptic phagocytosis contributes to BCP remains to be explored. Hence, this study investigated the role of astrocytic NRCAM deficiency in synaptic remodeling and central sensitization in BCP. A mouse model of BCP was established by intrafemoral inoculation of fibrosarcoma cells. Pain-related behaviors were evaluated by spontaneous pain assessment and mechanical withdrawal threshold testing. Bone destruction and tumor infiltration were examined by hematoxylin and eosin staining. Synaptic alterations and astrocytic activation were assessed by western blotting and immunofluorescence. Astrocyte-mediated synaptic engulfment was analyzed using immunofluorescence, three-dimensional reconstruction, magnetic-activated cell sorting, and Golgi-Cox staining. Moreover, fluorocitrate was used to inhibit the astrocytic activation. Astrocytic NRCAM was overexpressed via adeno-associated virus (AAV)-mediated gene delivery. Fibrosarcoma cell inoculation induced persistent spontaneous pain and mechanical hyperalgesia in C3H/HeN mice, accompanied by trabecular bone destruction and tumor infiltration. In the spinal cord, expression of the excitatory synaptic markers VGLUT1 and PSD95 was increased, whereas expression of the inhibitory synaptic markers VGAT and Gephyrin was decreased, together with marked astrocytic activation. Multiple complementary assays consistently showed that astrocytes engulfed both excitatory and inhibitory synapses, with a clear preference for GABAergic synapses. Pharmacological inhibition of astrocytic activation with fluorocitrate alleviated pain behaviors and improved synaptic remodeling. NRCAM expression was significantly downregulated in tumor-bearing mice. Restoration of astrocytic NRCAM expression by AAV markedly reduced astrocyte-mediated engulfment towards GABAergic synapses, had minimal effect on excitatory synapses, and significantly attenuated BCP. Reactive astrocytes preferentially phagocytose GABAergic synapses in BCP, thereby contributing to synaptic imbalance and central sensitization. This process is associated with downregulation of astrocytic NRCAM. Restoring astrocytic NRCAM alleviates BCP by suppressing excessive astrocyte-mediated phagocytosis of GABAergic synapses. These findings identify astrocytic NRCAM-dependent synaptic phagocytosis as an unrecognized mechanism underlying BCP and as a potential therapeutic target.

Background:Bone cancer pain (BCP) frequently develops following cancerous lesions in the bone, severely affecting patients' daily activities and long-term quality of life. However, effective therapeutic options remain limited. Therefore, it is essential to explore its underlying mechanisms to identify new therapeutic targets. Methods:A mouse model of BCP was established to investigate pain development using the von Frey test. Immunofluorescence staining and Western blotting were performed to detect neuroinflammation and astrocyte activation. Transcriptome sequencing and targeted metabolomics were conducted to identify significantly altered genes and pathways in BCP mice, and the results were validated by Western blotting, enzyme-linked immunosorbent assay, and immunofluorescence staining. Small interfering RNA was used to examine the relationship between Arpc1b and ALDOA using the aforementioned methods. Results:Neuroinflammation in the spinal cord of BCP mice was accompanied by activation of C3-positive reactive astrocytes. Sequencing analysis revealed that Arpc1b and glycolysis were associated with C3-positive astrocyte activation. Further experiments demonstrated that BCP-induced neuroinflammation downregulated Arpc1b and increased the expression of glycolytic enzymes in spinal cord astrocytes. Additionally, increased soluble ALDOA appeared to contribute to BCP-induced glycolysis in astrocytes, while Arpc1b-mediated cytoskeletal remodeling increased free ALDOA levels. Conclusions:The significant reduction of Arpc1b in C3-positive astrocytes within the spinal cord of BCP mice mediates cytoskeletal remodeling, causing dissociation of ALDOA from F-actin. This process promotes excessive glycolysis in astrocytes and persistent neuroinflammation, which may underlie BCP.

Obesity markedly exacerbates nociceptive sensitivity and substantially compromises the quality of life of affected people. Astrocytes orchestrate metabolic regulation and homeostatic maintenance in the central nervous system. Notably, fatty acid binding protein 7 (FABP7) is highly expressed in astrocytes that governs intracellular fatty acid uptake and transport. While systemic hyperlipidemia is pathognomonic of obesity, the mechanistic contribution of FABP7 in astrocytes to obesity-associated pain pathophysiology remains poorly characterized. The present study established a model of high-fat diet (HFD)-induced obesity combined with a standardized hind paw surgical incision paradigm, aiming to unveil the role of astrocytic FABP7 in HFD-induced chronic pain. Furthermore, an in vitro high-fat environment was induced by palmitic acid (PA),aiming to investigate the molecular mechanisms by which primary astrocytes were activated into the A1 phenotype to mediate neuroinflammation. FABP7 was overexpressed in the spinal dorsal of HFD mice. The activation of A1-type astrocytes and neuroinflammation cascades involving elevated iNOS eventually resulted in mechanical allodynia in HFD mice. Pharmacological inhibition of FABP7 via an intraperitoneal administration of SBFI-26 (20 mg/kg) significantly attenuated the paw withdrawal mechanical threshold and inhibited the A1-type astrocytes activation. PA-induced high-fat conditions promoted lipid droplet accumulation and upregulated FABP7 in astrocytes. Pharmacological inhibition of FABP7 using SBFI-26 (100 μmol/L) significantly suppressed the neurotoxic C3-positive A1 astrocyte phenotype, reduced intracellular lipid droplet accumulation, thereby inhibiting the activation of A1-type astrocytes and alleviating neuroinflammation. Overall, FABP7-mediated astrocytic reprogramming was a critical nexus bridging obesity with chronic pain. A1-astrocyte polarization initiated neuroinflammatory amplification, forming a feedforward loop perpetuating central sensitization. Our findings are expected to offer a viable target for metabolic pain management.

Patients admitted for surgery commonly experience preoperative anxiety. Previous studies have shown that preoperative anxiety often delays recovery from postoperative pain or even aggravates pain. Therefore, it is necessary to explore the mechanisms by which anxiety prolongs chronic postoperative pain. A single prolonged stress (SPS) rat model was constructed to investigate the effects of anxiety and depression using behavioral tests. Changes in the levels of tight junction proteins in the cerebral striatum (CPu) of the rats were assessed by western blotting 1 to 21 days after the operation. The level of inflammation was detected using western blotting and enzyme-linked immunosorbent assay (ELISA). Glucose metabolism levels and changes in related signaling pathways in microglia were assessed using western blotting, immunofluorescence, ELISA, and flow cytometry. The effects of S-ketamine treatment on the rats were also determined using the above methods. Preoperative SPS aggravated acute pain after plantar incision in rats and significantly prolonged the postoperative pain recovery time. The incised SPS rats began to show significant blood-brain-barrier (BBB) damage on the third day after surgery. Simultaneously, SPS caused neuroinflammation and microglial activation in the CPu after plantar incision. CPu microglia participated in neuroinflammation by undergoing glucose metabolic reprogramming mediated by the mTOR-p70S6K-4EBP1 pathway. Preoperative administration of a single dose of S-ketamine was an effective analgesic, as it inhibited SPS-induced postoperative inflammation. S-ketamine partially corrected SPS-induced abnormal glycolysis in striatal microglia through the mTOR-p70S6K-4EBP1 pathway. S-ketamine effectively relieved postoperative chronic pain caused by preoperative anxiety by correcting glucose metabolic reprogramming in CPu microglia.

A single-center retrospective study was designed to investigate the risk factors associated with delayed discharge from the Anesthesia Intensive Care Unit (AICU). This retrospective study involved patients admitted in the AICU from January 2017 to December 2022. Risk factors for the delayed discharge from the AICU were analyzed by the binary multivariate logistic regression analysis. Nomogram was constructed to predict the risk of delayed discharge from AICU. The performance of the nomogram was assessed using the receiver operating characteristic curve and calibration curve. A decision curve analysis was also performed to determine the net benefit threshold of prediction. A total of 14,338 patients admitted in the AICU were retrospectively recruited, involving 9,271 males and 5,067 females. The incidence of delayed discharge from the AICU in the cohort was 1.54

Perineurial network (PNN) is a special extracellular matrix structure in the central nervous system, and its alterations are associated with the pain hypersensitivity. Recent studies have suggested a potential interaction between abnormal activation of spinal microglia and PNN. This study investigates whether S-ketamine mitigates neuropathic pain via inhibiting degradation of PNNs by spinal microglia. C57BL/6 mice were utilized for CCI modeling to induce neuropathic pain. Subsequent to modeling, we assessed the expression changes of spinal microglia, PNN and inflammatory factors. Microglia colocalization with PNN was evaluated via 3D reconstruction to quantify spatial overlap. Minocycline was administered to target microglia. S-ketamine was subsequently administered to CCI mice, and its effects on pain behavior, microglial activation, and PNN were investigated. Microglia-PNN colocalization was evaluated via 3D reconstruction to quantify spatial overlap. CCI mice exhibited significant neuropathic pain, accompanied by increased microglia-mediated phagocytosis of PNN. Minocycline and S-ketamine treatment of CCI mice led to improved pain thresholds, suppression of neuroinflammation, and reduction in microglia-mediated phagocytosis of PNN. Increased microglial phagocytosis leading to PNNs degradation in the spinal dorsal horn plays a critical role in neuropathic pain pathogenesis. The analgesic effects of S-ketamine may be attributed to its modulation of this mechanism.

Cancer pain, a frequent complication in patients with cancer, adversely affects quality of life and survival rates. Microglia promote nociceptive information transmission by modulating myelin integrity during pain perception. However, the specific mechanisms by which microglia regulate myelin in the context of cancer pain remain poorly understood. In this study, we developed a bone cancer pain model to examine the interactions among microglia, myelin, and oligodendrocyte precursor cells and their roles in cancer pain. Our study found that mice with bone cancer pain had oligodendrocyte differentiation defects and myelin loss, and that promoting myelination did not relieve pain. In addition, we observed that reactive microglia and inflammatory cytokines increased and microglia phagocytosed myelin in mice with bone cancer pain. Inhibition of microglia not only alleviated pain behaviors in mice with bone cancer but also mitigated myelin phagocytosis and the proliferation of oligodendrocyte precursor cells. Our study suggests that microglia-mediated myelin loss and oligodendrocyte precursor cell proliferation may be one of the pathological mechanisms underlying pain in mice with bone cancer.

Isoflurane, a widely used anesthetic, has raised concerns due to its potential neurotoxic effects, including oxidative stress and astrocytic gap junction (GJ) dysfunction. This study investigates whether ω-3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can mitigate these effects by activating the Wnt/β-catenin signaling pathway. Using primary astrocytes, we found that long-term isoflurane exposure uncoupled GJ-Cx43, increased reactive oxygen species (ROS), and inhibited Wnt/β-catenin signaling. ω-3 PUFA treatment restored GJ-Cx43 coupling, reduced ROS levels, and partially reactivated the Wnt/β-catenin pathway. These findings suggest that ω-3 PUFAs protect against isoflurane-induced neurotoxicity by enhancing GJ-Cx43 coupling and reducing oxidative stress, offering a potential therapeutic strategy for perioperative neurocognitive disorders."

Our previous studies have demonstrated that neutrophils play a key role in septic organ injury partly through the excessive formation of neutrophil extracellular traps (NETs) and that exosomes participate in the regulation of NET formation during sepsis. Therefore, this study aimed to determine whether neutrophil-derived exosomes promote the formation of NETs and induce multiple organ dysfunction during sepsis. Initially, polymorphonuclear neutrophil (PMN)-derived exosomes following in vitro stimulation with PBS or LPS (1 μg/mL) for 6 h. In vivo, PMN-derived exosomes were intravenously administered to wild-type C57BL/6 mice. Then, histopathological injury and NET formation in multiple organs were evaluated. In vitro, PMN-derived exosomes were cocultured with PMNs freshly isolated from healthy volunteers, and subsequently, NET formation and activation of associated molecular pathways were detected. Administration of LPS-stimulated PMN-derived exosomes in mice significantly enhanced NET formation, resulting in multi-organ inflammation and tissue injury. In vitro coculture experiments also demonstrated that exosomes from LPS-stimulated PMNs promote ROS-dependent NET formation. Proteomic analysis revealed enrichment of matrix metalloproteinase 9 (MMP9) expression in exosomes from LPS-stimulated PMNs, and further mechanistic investigations showed that exosomal MMP9 induced NET formation through the p38 MAPK pathway. Clinical data analysis suggests a close association between sepsis severity/prognosis and plasma-derived exosomal MMP9 expression levels. PMN-derived exosomes facilitate the excessive formation of NETs in sepsis, leading to the subsequent development of multiple organ dysfunction. This discovery reveals a novel role for PMN-derived exosomes in the pathogenesis of sepsis-related multiple organ dysfunction and suggests their potential as prognostic indicators for this condition.

BackgroundEarly life stress (ELS) causes functional gastrointestinal issues linked to visceral hyperalgesia. Activation of spinal neurons and glial cells is key to the development and persistence of visceral hyperalgesia. Our previous research has shown that EphrinB2/EphB2 signaling in the spinal cord facilitates this hyperalgesia through neuron and glial cell activation. Gallic acid (GA), a natural compound with recognized anti-inflammatory and analgesic effects, may attenuate visceral hyperalgesia. This study investigates whether GA mitigates visceral hyperalgesia induced by ELS in mice via inhibiting EphrinB2/EphB2-mediated activation of neurons and glial cells.MethodsWe employed a maternal separation (MS)-induced ELS model and recorded abdominal withdrawal reflex (AWR) scores following colorectal distension (CRD) in adult mice. Molecular docking analysis was used to evaluate the binding stability of GA with the EphrinB2-EphB2 complex or EphrinB2 alone. After CRD, we assessed EphrinB2 and EphB2 expression, glial and neuronal activation, and synaptic plasticity in the spinal cord of MS mice, with or without GA treatment. C-fos levels were measured via immunohistochemistry, and protein expression was quantified by Western blotting. EphrinB2/EphB2 co-expression with neurons or glial cells was examined by double-labeling, and a 3D reconstruction confirmed cell type-specific expression.ResultsMolecular docking confirmed that GA binds stably to EphrinB2-EphB2 complex or EphrinB2 alone. In adult MS mice, CRD stimulation induced pain behaviors, accompanied by substantial activation of spinal neurons and glial cells, as well as upregulation of synaptic N-methyl-D-aspartate receptors (NMDARs). EphrinB2 and EphB2 were localized within spinal astrocytes, microglia, and neurons. Furthermore, exogenous EphrinB2 induced the activation of glial cells and neurons, NMDARs phosphorylation, and visceral hypersensitivity in naive mice. Intraperitoneal injection of GA can alleviate the above conditions.ConclusionsOur findings suggest that spinal EphrinB2/EphB2 signaling is crucial in the development of maternal separation-induced visceral hyperalgesia. GA may alleviate hyperalgesia by inhibiting the EphrinB2/EphB2 signaling pathway, thereby modulating nociceptive processing by MS.

Persistent postoperative pain (PPP) is associated with neuroinflammation and excitatory/inhibitory(E/I) imbalance in the spinal cord. Notably, trained immunity enhances the immune reactivity of microglia to secondary stimuli, exacerbating neuroinflammation and synaptic engulfment of microglia. Here, we investigated whether preoperative anxiety stress promotes trained immunity in microglia and how this phenomenon influences microglia-mediated synaptic engulfment. Given the role of glycolysis in trained immunity, we focused on microglial glucose metabolism. Rutin inhibits microglial glycolysis and reduces neuroinflammation, prompting further investigation into its therapeutic potential for PPP. Preoperative anxiety was modeled in male Sprague-Dawley (SD) rats using single prolonged stress (SPS). We found that SPS aggravated and prolonged incision pain in SD rats. Mechanistically, SPS promoted trained immunity in microglia via mammalian target of rapamycin (mTOR)/hypoxia-inducible factor-1α (HIF-1α) signaling. This amplified inflammatory responses to surgical stimuli and enhanced microglial engulfment of inhibitory synapses. Rutin inhibited microglial activation and inhibitory synaptic engulfment via mTOR/HIF-1α signaling, relieving SPS-induced PPP. These findings suggest preoperative anxiety induces trained immunity in microglia, amplifying neuroinflammation and E/I imbalance in the spinal cord after surgery. Rutin attenuates this process by suppressing mTOR/HIF-1α-driven glycolysis, thereby alleviating PPP.

Long-term or repeated anesthesia has been shown to potentially contribute to cognitive dysfunction. Existing animal studies have demonstrated that long-term anesthesia using inhaled anesthetics, such as isoflurane, may impact cognitive performance via mechanisms involving neuroinflammation, synaptic plasticity, and dysregulation of protein degradation. However, the molecular mechanisms underlying isoflurane-induced cognitive dysfunction remain inadequately understood. Here, through bioinformatics analyses, we have established the association between ubiquitin ligase Cullin-1 and cognitive impairment, and innovatively explored the molecular mechanism of Cullin-1 in anesthesia-related cognitive dysfunction via protein degradation regulation. By Y-maze and fear conditioning experiments, we observed that long-term isoflurane-anesthesia (1.5 % isoflurane for 6 h) impaired cognitive performance, and increased expression of Cullin-1 in mice. Injection of siRNA targeting Cullin-1 into lateral ventricle effectively mitigated cognitive impairments. In vitro cell experiments, we found that overexpression of Cullin-1 promote the degradation of PHLPP1 via the ubiquitin-proteasome system. In mice, co-injection of PHLPP1 siRNA prevented the therapeutic effects of Cullin-1 siRNA on long-term anesthesia induced cognitive dysfunction indicating that Cullin-1 siRNA ameliorates cognitive impairments through the stabilization of PHLPP1. Our studies provided a novel theoretical foundation and potential intervention strategies for prevention and mitigation of anesthesia-related cognitive impairment in future clinical management.

IntroductionSepsis-associated acute kidney injury (SA-AKI) is a highly lethal condition with a rapid onset, and effective treatments are lacking because the molecular pathogenesis remains unclear. Tubular epithelial cells (TECs) have increasingly been recognized as driving forces in the progression of kidney diseases, partly through the release of extracellular vesicles (EVs) carrying proinflammatory cargos. However, the role of TEC-derived EVs on neutrophil extracellular traps (NETs) formation, which is an established feature of sepsis, and SA-AKI remains unclear.MethodsEVs isolated from phosphate buffer saline (PBS)/lipopolysaccharide (LPS)-treated TECs were injected intravenously into C57BL/6J wild type mice to determine whether TECs-derived EVs can directly induce NETs formation and kidney injury. Proteomics and single-cell RNA sequencing analysis were used to screen the key molecules that mediate the effects of TECs-derived EVs. EVs secretion from TECs and serum amyloid A1 (SAA1) expression in TECs were specifically inhibited via adeno-associated virus (AAVs). Finally, the association between SAA1 level in plasma EVs and clinical features of septic patients was determined.ResultsThis study demonstrated that EVs secreted from LPS-stimulated TECs exacerbated AKI by promoting NETs formation. Specifically blocking EVs secretion from TECs via AAVs reduced NETs formation and alleviated LPS-induced AKI. Bioinformatics analysis suggested that LPS increased SAA1 expression in TECs, and then released extracellularly through EVs. Further mechanistic studies revealed that SAA1 packaged in TECs-derived EVs was responsible for NETs formation and AKI via activation of the TLR4/p38 MAPK signaling pathway in neutrophils. Specifically inhibiting SAA1 upregulation in TECs via AAVs also reduced NETs formation and alleviated LPS-induced AKI. Interestingly, modulating EVs release from TECs or SAA1 expression in TECs also alleviated remote lung injury induced by LPS, indicated that TECs-derived EVs may participate in kidney‒lung crosstalk during sepsis. Furthermore, plasma TECs-derived EVs proportion and SAA1 expression in plasma EVs may be promising prognostic indexes for SA-AKI patients.DiscussionHere, we explored a new mode of TECs-neutrophils crosstalk mediated by EVs during SA-AKI, and strategies to modify TECs-derived EVs and the cargo SAA1 could be a new avenue for developing therapeutics against SA-AKI.