Background: Influenza A virus (IAV) is a negative-sense RNA virus of the Orthomyxoviridae family and is the etiological agent of a highly contagious acute respiratory disease that can lead to acute lung injury. Objective: To elucidate the molecular mechanisms of IAV infection, an integrative research approach combining gene expression profiling, multinetwork analysis, and in vivo experimental validations was employed. Methods: First, a series of network-based analyses were performed, including protein-protein interaction network construction, weighted gene co-expression network analysis, and subsequent gene set enrichment analysis, to identify the major underlying mechanisms of IAV infection. Following gene expression analysis, core targets, both direct and indirect regulators, were screened. An IAV (H1N1) strain A/PR/8/34-induced acute lung injury mouse model was constructed for in vivo validations. Batch one included two groups to evaluate findings from the multi-network analysis: Mock (n = 10; 5 males and 5 females) and IAV (n = 10; 5 males and 5 females). Batch two included three groups to assess the role of toll-like receptor 3 (TLR3) in IAV infection: Mock (n = 6; 3 males and 3 females), IAV (n = 6; 3 males and 3 females), and TLR3 inhibitor (n = 6; 3 males and 3 females). Body weight was measured on days 0, 3, and 5 after infection. On day 5, lung tissues were collected to assess viral load and histopathological changes. Key targets were examined using enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining, both in sera and lung tissues. Results: IAV infection was significantly associated with dysregulation of the immune-inflammation system, such as the LTR, nucle-otide-binding oligomerization domain-(NOD) like receptor, retinoic acid-inducible gene I-like receptor, and nuclear factor kappa-B signaling pathways. Gene set enrichment analysis further indicated that the TLR and necroptosis signaling pathways played crucial roles in the progression of IAV infection (TLR signaling pathway normalized enrichment score = 2.3941, P = 1.00 × 10 ⁻¹⁰; necroptosis normalized enrichment score = 1.9421, P = 6.21 × 10 ⁻⁷). Among the core targets, TLR3 and mixed lineage kinase domain-like protein (MLKL) may regulate gene expression at the transcriptional level (all P < 0.05). In vivo validation using an IAV (PR8) infected acute lung injury mouse model demonstrated increased viral load and lung index, alveolar structural damage, and inflammatory cell infiltration. Immunofluorescence staining exhibited large gaps in Lamin B1 staining and breaches in Emerin signals following IAV-PR8 infection. Expression levels of TLR3, p-receptor-interacting serine/threonine-protein kinase 3 (RIPK3)/RIPK3, and p-mixed lineage kinase domain-like protein (MLKL)/MLKL proteins in lung tissues, as well as proinflammatory factors and mediators in sera, were significantly elevated after IAV infection. Moreover, enhanced neutrophil infiltration (myeloperoxidase) and citrullinated histone H3 (a neutrophil extracellular trap-specific marker), both established indicators of neutrophil extracellular trap formation, were observed. Notably, treatment with a TLR3 inhibitor significantly ameliorated IAV-induced acute lung injury by regulating necroptosis-related targets. Conclusion: Our study provides network-based in vivo evidence that TLR3-receptor-interacting serine/threonine-protein kinase 3-MLKL-mediated necroptosis may underlie IAV-induced acute lung injury and could serve as a potential therapeutic target in severe influenza cases.

ObjectiveHepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. The main risk factors associated with HCC include viral hepatitis (B and/or C), alcohol abuse, and metabolic dysfunction-associated steatotic liver disease (MASLD), which progressively advance to liver fibrosis, cirrhosis, and ultimately evolve into HCC. Surgical resection represents the most effective treatment for HCC, while recent advances in immunotherapy, including immune checkpoint inhibitors and adoptive cell therapies, have provided improved treatment prospects for patients with unresectable HCC. However, the complex metabolic heterogeneity of HCC limits the therapeutic efficacy. Metabolic intermediates acyl-CoA not only provide energy and substrates for numerous biochemical reactions but also serve as donors for protein lysine acylation, a major class of post-translational modification (PTM). Therefore, a deeper understanding of the molecular mechanisms underlying protein lysine acylation and hepatocarcinogenesis is urgently needed. MethodsThe levels of protein lysine acylation and silence information regulator 5 (SIRT5) expression levels in clinical HCC samples were analyzed by Western blot. Quantitative malonylome and succinylome of HCC samples were analyzed by antibody-based affinity enrichment coupled with tandem mass spectrometry. The proliferation of HCC cells was analyzed with Cell Counting Kit-8 (CCK-8) assays, the apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining coupled with flow cytometry, and the ability of cells to migrate was assayed by Transwell assays. The enzymatic activity of glutathione S-transferase Mu 1 (GSTM1) was quantified. Transgenic mice with hepatic overexpression of SIRT5 were constructed using CRISPR-Cas9, and primary hepatocarcinogenesis was induced by administration of diethylnitrosamine. ResultsWestern blot analysis indicated that the expression level of SIRT5 was elevated in clinical samples from HCC patients, and the levels of lysine malonylation, glutarylation, and succinylation were significantly reduced in HCC tissues. Knockout of SIRT5 in MHCC-97H and MHCC-97L hepatoma cells suppressed cell proliferation, and increased the percentage of apoptotic cells significantly. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially malonylome and succinylome of HCC samples revealed significant enrichment in two major classes of biological processes: core energy metabolism (e.g., glycolysis/gluconeogenesis, tricarboxylic acid metabolic process, fatty acid beta oxidation) and detoxification and oxidative stress response (e.g., response to toxic substance, chemical carcinogenesis, reactive oxygen species (ROS)). SIRT5 removes malonylation from lysine residues in GSTM1 and restores its detoxification activity, which is crucial for the survival of hepatocytes under stressed conditions. More importantly, in vivo experiment indicated that hepatic-specific overexpression of SIRT5 in mice accelerated diethylnitrosamine-induced liver fibrosis and hepatocarcinogenesis, indicating the critical role of SIRT5 in HCC progression. ConclusionThis study highlights the previously unrecognized SIRT5-GSTM1 axis as a key regulator in hepatocarcinogenesis, and suggests a potential target for the treatment of patients with HCC.

Bladder cancer is a common malignant tumor in the urinary system.At present,the main treatment methods include surgery and drug therapy.Although surgical treatment has shown remarkable effects,the prognosis and quality of life of patients still need to be improved.In recent years,immunotherapy has developed rapidly,with programmed cell death-1(PD-1)/programmed cell death-ligand-1(PD-L1)emerging as particularly prominent targets.In order to deeply understand the potential of PD-1/PD-L1 inhibitors in the treatment of bladder cancer and provide new ideas for the treatment of bladder cancer,this study reviewed the research progress of PD-1/PD-L1 inhibitors in the field of bladder cancer.

We reported the clinical,imaging,and treatment process of a patient with severe cranial penetrating injury caused by a nail from a nail gun that accidentally shot into the eye and penetrated into the skull while working.The patient was admitted to the Qingpu Branch of Zhongshan Hospital,Fudan University after being injured by the nail gun.The patient was admitted to the hospital 1 hour after injury with coma.After admission,the foreign body was completely removed and part of the hematoma was cleared through emergency surgery.After surgery,there were high-risk complications such as cerebral edema peak,intracranial hypertension,intracranial infection,cerebrospinal fluid leakage,and intracranial pseudoaneurysm.The patient eventually regained consciousness and was transferred to rehabilitation treatment.

Objective:To explore the influencing factors of evacuation associated pulmonary edema (WIPE) in patients with mechanical ventilation of cardiogenic respiratory failure, and to build a risk warning model based on independent influencing factors.Methods:A total of 220 patients with cardiogenic respiratory failure who were treated and received mechanical ventilation in Chengbei Campus of Hangzhou First People′s Hospital from April 2021 to December 2023 were retrospectively selected by cross-sectional investigation method, and were divided into WIPE group (34 cases) and non WIPE group (186 cases) according to whether the patients had WIPE or not. Clinical data of the patients were analyzed using the hospital electronic medical record system. The influencing factors of WIPE were determined by univariate analysis and multivariate Logistic regression analysis, and the risk early warning model was constructed based on regression analysis. The corresponding nomogram was drawn by R language software, and the predictive efficiency of the model was tested by receiver operating characteristic curve and calibration curve.Results:WIPE group included 18 males and 16 females, aged (65.12±9.28) years. Non WIPE group included 107 males and 79 females, aged (60.25±8.40) years. Multivariate Logistic regression analysis showed that age ( OR=1.072), smoking history ( OR=3.412), acute physiology and chronic health evaluationⅡ( OR=1.184), cardiac function classification ( OR=4.043), shallow rapid breathing index ( OR=1.100), mechanical ventilation time ( OR=1.540), hypertension ( OR=4.903), left ventricular diastolic dysfunction ( OR=5.151) and chronic obstructive pulmonary disease ( OR= 5.536) were independent influencing factors (all P < 0.05). The area under the curve of the risk early warning model constructed based on the above 9 independent influencing factors was 0.938, and the sensitivity and specificity corresponding to the optimal cutoff value of 0.620 were 0.971 and 0.801, respectively, indicating good differentiation ability. The calibration curve results show that the average absolute error was 0.020, the calibration curve fits the ideal curve, and the model calibration performance was good. Conclusions:WIPE in patients with cardiogenic respiratory failure induced by mechanical ventilation is affected by cardiac function status, mechanical ventilation parameters and other factors. The risk early warning model based on the above 9 independent influencing factors has good predictive efficacy, and can provide reference for clinical prevention of WIPE.

Objective:To investigate the correlation between oral cleanliness and secondary Pulmonary infection in patients with severe chronic obstructive pulmonary disease (COPD) in mechanical ventilation, and to investigate the predictive effect of oral cleanliness on the risk of secondary pulmonary infection.Methods:Using the cross-sectional survey method, the purposeful sampling method was adopted to select 216 patients with severe COPD who were hospitalized in Hangzhou First People′s Hospital from June 2020 to December 2023 and received mechanical ventilation. The oral cleanliness index and general clinical data of patients at admission were collected using the hospital electronic medical record system. The independent influencing factors of secondary lung infection were analyzed by univariate analysis and multivariate Logisitic regression. The predictive value of oral cleanliness index on secondary lung infection was analyzed by patient operating characteristic (ROC) curve.Results:216 patients with severe COPD who underwent mechanical ventilation were included.Patients aged 37-84 (66.81 ± 8.98) years were included, including 125 males and 91 females.Among them, 89 cases developed secondary pulmonary infection, with an infection rate of 41.20%.Univariate analysis and multivariate Logistic regression analysis showed that, Beck Oral Rating Scale (BOAS) score ( OR = 1.371), visual simulation score of oral odor ( OR = 1.405), gum index ( OR = 3.508), plaque index ( OR = 14.357), smoking history ( OR = 6.772), duration of disease ( OR = 1.391), COPD assessment test score ( OR = 1.269) and mechanical ventilation time ( OR = 1.302) were independent factors for secondary pulmonary infection (all P<0.05). ROC curve analysis showed that oral cleanliness index combined with infection prediction was effective (the area under the ROC curve was 0.833) . Conclusions:Oral cleanliness was closely related to secondary pulmonary infection in patients with severe COPD with mechanical ventilation. BOAS score, visual simulation score of oral odor, gingival index and plaque index could predict secondary pulmonary infection independently, and combined test could predict secondary pulmonary infection.

Objective:To investigate the pharmacological mechanism of Compound Xuanju Capsule in the treatment of erectile dysfunction(ED)by using network pharmacology and molecular docking technology.Methods:The active ingredients and targets of Compound Xuanju Capsule were screened using Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform(TCMSP).TTD,OMIM,DrugBank and GeneCards databases were used to obtain genes related to ED,and the union of the results was taken as the disease genes of ED.The common target of drug and disease was taken as the potential target of Compound Xuanju Capsule in ED,and the drug-disease interaction network was constructed by using Cytoscape software.The protein-protein interaction(PPI)network was constructed by using String database,which was then imported into Cytoscape to identify the key target.Based on the drug-disease intersection genes,GO and KEGG enrichment analyses were performed to predict the relevant signaling pathways and molecular mechanisms of Compound Xuanju Capsule for the treatment of ED.Autodock software was used to perform molecular docking between the active ingredients and the core targets.Results:Forty chemical components of Compound Xuanju Capsule were screened,and 239 predicted targets were obtained.A total of 1 907 ED-related genes were screened,and 97 common targets were identified between Compound Xuanju Capsule and ED,among which the core targets included EGFR,ESR1,HIF1A,PTGS2,and STAT3.The signaling pathways obtained by KEGG enrichment analysis included calcium signaling pathway,HIF-1 signaling pathway,PI3K-Akt signaling pathway,cGMP-PKG signaling pathway,relaxin signaling pathway,Serotonergic synapse signaling pathway.The molecular docking results showed that there were molecular binding sites between the key active ingredients and the core targets with strong binding activity.Conclusion:Compound Xuanju Capsule may treat ED through multi-target pathways such as anti-inflamnmato-ry and improving cellular oxidative stress.

Experimental teaching is an important component of the course Medical Immunology,which requires the use of experimental animals and the execution of experimental animals by medical students.By applying the carbon dioxide euthanasia system,it can effectively reduce the pain of experimental animals,ensure their welfare,and meet ethical requirements.It can also improve the efficiency of experimental teaching in Medical Immunology,reduce environmental pollution,and promote medical students to estab-lish scientific values and worldviews that pay attention to experimental animals and respect life,which is conducive to becoming future medical service talents.In the experimental teaching of Medical Immunology,the appropriate application of carbon dioxide euthanasia system combined with effective ideological and political construction of the curriculum can further implement the Party's educational policy of cultivating morality and talents,and lay a good foundation for cultivating medical talents with comprehensive knowledge,high skills and excellent quality.

Bladder cancer is a common malignant tumor in the urinary system.At present,the main treatment methods include surgery and drug therapy.Although surgical treatment has shown remarkable effects,the prognosis and quality of life of patients still need to be improved.In recent years,immunotherapy has developed rapidly,with programmed cell death-1(PD-1)/programmed cell death-ligand-1(PD-L1)emerging as particularly prominent targets.In order to deeply understand the potential of PD-1/PD-L1 inhibitors in the treatment of bladder cancer and provide new ideas for the treatment of bladder cancer,this study reviewed the research progress of PD-1/PD-L1 inhibitors in the field of bladder cancer.

Objective:To evaluate the effect of electroacupuncture (EA) on ionotropic purinergic receptor 4 (P2X4R)/nuclear factor-kappa B (NF-κB) signaling pathway during spinal cord injury (SCI) in rats.Methods:Thirty-six clean-grade healthy adult female Sprague-Dawley rats, weighing 210-250 g, were divided into 3 groups ( n=12 each) using a random number table method: sham surgery group (S group), SCI group, and SCI+ EA treatment group (SCI+ EA group). The SCI model was established by the Allen′s method in anesthetized animals. In group S, only the spinous processes and vertebral laminae were resected, but the spinal cord was not injured. On the 7th day after developing the model, EA of Jiaji, Dazhui, and Mingmen lasting 30 min was performed once a day for 7 consecutive days, with a depth of 2 mm, intensity of 12-15 mV, frequency of 2 Hz, in SCI+ EA group. The mechanical paw withdraw threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before developing the model and 3, 7, 14, 21 and 28 days after developing the model, and the motor function was assessed using the Basso-Beattie-Bresnahan (BBB) score. The recovery of motor function was assessed using footprint analysis at 28 days after developing the model. After the final behavioral testing, the rats were sacrificed, and spinal cord tissues were harvested to observe the pathological changes of the spinal cord tissues using hematoxylin-eosin staining, to detect the expression of P2X4R and phosphorylated NF-κB p65 (p-NF-κB p65) (by immunohistochemical analysis and Western blot) and to determine contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay). Results:Compared with the baseline measured at 1 day before developing the model, the MWT and BBB scores were significantly decreased and the TWL was shortened at each time point after developing the model in SCI group and SCI+ EA group ( P<0.05). Compared with S group, the MWT and BBB scores were significantly decreased and the TWL was shortened at each time point after developing the model, the expression of P2X4R and p-NF-κB p65 in spinal cord tissues was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI group ( P<0.05). Compared with SCI group, the MWT and BBB scores were significantly increased and the TWL was prolonged at 14, 21 and 28 days after developing the model, the expression of P2X4R and p-NF-κB p65 in spinal cord tissues was down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased ( P<0.05), and the pathological damage of spinal cord tissues was alleviated and footprints were reduced in SCI+ EA group. Conclusions:The mechanism by which EA alleviates SCI may be related to the inhibition of the activation of the P2X4R/NF-κB signaling pathway and the reduction in the inflammatory response in rats.