Background: High-mobility group box protein 1 (HMGB1) is a chromatin-binding protein involved in arthritis, ischemia, sepsis, atherosclerosis, neurodegenerative disorders, meningitis, and cancer. HMGB1 exhibits dual roles in cancer, acting as either a tumor suppressor or oncoprotein depending on context. Objective: This research aimed to elucidate HMGB1’s functional significance in cutaneous squamous cell carcinoma (cSCC). Methods: We overexpressed HMGB1 in cSCC cell lines using recombinant adenovirus and examined its effects on cell proliferation, colony formation, and cell migration. Results: Immunohistochemical analysis revealed elevated HMGB1 expression levels in cSCC tissue relative to normal epidermis. To assess the influence of HMGB1, we employed recombinant adenoviruses expressing HMGB1 to transduce SCC cell lines (SCC12 and SCC13). Enhanced HMGB1 expression significantly promoted cellular proliferation and colony formation capacity.Notably, HMGB1 overexpression elevated the levels of proliferation regulators, including P63, SOX2, CDK4 and CDK6. Furthermore, HMGB1 overexpression substantially enhanced tumor invasiveness, accompanied by upregulation of epithelial-mesenchymal transition (EMT) biomarkers. Mechanistically, overexpression of HMGB1 enhanced transforming growth factor-β signaling by increasing phosphorylation of SMAD2/3, the key mediators of EMT. Conclusion These data imply that HMGB1 acts as a tumor-promoting factor in cSCC.

[摘 要] 目的：探究着丝粒蛋白M（CENPM）在脑胶质瘤中的表达特征、临床预后价值及其对肿瘤恶性生物学行为的调控机制，为脑胶质瘤的精准治疗提供潜在靶点。方法：基于中国胶质瘤基因组图谱（CGGA）和癌症基因组图谱（TCGA）数据库分析CENPM在胶质瘤中的表达及其与患者临床病理特征和预后的相关性。通过基因本体论（GO）分析、京都基因与基因组百科全书（KEGG）富集分析和单细胞转录组分析探索CENPM的生物学功能和作用机制。WB法检测CENPM在胶质瘤细胞（LN-18、LN-229、U-138MG、U-251MG）和正常胶质细胞（HEB）中的表达；构建CENPM敲低细胞后，通过CCK-8、集落形成、Transwell和划痕实验评估恶性表型改变。结果：CENPM在WHO高级别胶质瘤中呈高表达（P < 0.05），与肿瘤恶性程度正相关。高表达组患者总体生存期显著短于低表达组（P < 0.01），Cox回归证实CENPM是影响胶质瘤患者预后的独立危险因素（P < 0.05）。功能富集分析结果显示CENPM相关基因主要富集于细胞周期调控、PI3K-Akt通路和免疫相关过程。单细胞分析结果显示CENPM主要在CD8⁺ T细胞高表达，并通过PTN-PTPRZ1/NCL配受体调控细胞通信。体外实验证实CENPM在胶质瘤细胞表达高于正常胶质细胞（LN-18：P < 0.01，LN-299：P < 0.05）；敲低CENPM显著抑制迁移（P < 0.05），但增强集落形成，提示其在肿瘤进展中的双重调控作用。结论：CENPM作为胶质瘤独立预后危险因子，通过调控细胞周期、PTN通路和免疫微环境驱动肿瘤进展，其差异化调控机制（抑制迁移、促进增殖）具有潜在的临床转化价值，可作为分子分型和靶向治疗候选标志物。

[摘 要] 目的：通过多组学整合分析，解析肠道菌群在胆管癌（CCA）发生发展中的潜在作用机制并识别相关关键基因。方法：基于SRA数据库的16S rRNA测序数据，比较CCA患者与健康对照者的肠道菌群组成；采用孟德尔随机化（MR）分析评估菌群与CCA风险的遗传关联。通过gutMGene和GeneCards数据库获取相关代谢物与基因，进行代谢和功能富集分析。整合GEO单细胞转录组数据（GSE213452），解析肿瘤微环境的细胞组成，重点关注T细胞亚群及其功能状态，并结合TCGA-CHOL数据集验证关键候选基因的表达差异。结果：与健康对照组相比，CCA患者肠道菌群组成发生显著改变，变形菌门下菌群异常富集（LDA > 4）。MR分析进一步证实，肠杆菌目与肠杆菌科的遗传易感性均与CCA风险呈正向关联。代谢通路富集分析提示，菌群相关代谢物主要参与嘌呤代谢及糖酵解/糖异生等通路；功能富集分析显示，相关基因显著富集于NOD样受体、IL-17、Toll样受体及NF-κB信号通路等炎症免疫通路。单细胞转录组分析结果显示，CCA组织中肿瘤细胞比例显著升高（P < 0.05），T细胞比例由20.7%增至39.2%；拟时序分析结果表明，MKI67⁺ T细胞处于分化末期并呈高增殖特征，其差异基因与菌群相关基因存在交集，其中SERPINA1和IFNG表达在肿瘤免疫微环境中显著变化（P < 0.001），可能发挥核心调控作用。TCGA-CHOL数据集验证显示，SERPINA1在CCA肿瘤组织中显著下调（P < 0.001），而IFNG在肿瘤与正常组织间无显著差异（P > 0.05）。结论：肠道菌群失衡（尤其是肠杆菌科异常增殖）可能通过代谢-免疫调控网络促进CCA进展，T细胞功能变化与关键基因（SERPINA1和IFNG）在MKI67+ T细胞中的差异化表达模式密切相关。

The core pathogenesis of attention deficit hyperactivity disorder （ADHD） lies in deficiency， stasis and stagnation. Deficiency arises from kidney essence depletion and spleen dysfunction in transportation and transformation， leading to inadequate nourishment of the marrow sea. Stasis caused by qi deficiency leads to obstruction in channels and collaterals， resulting in obstructed marrow transport. Stagnation is associated with the excess of the five minds transforming into fire， which scorches the brain orifices and leads to loss of control over marrow utilisation. Based on this， a "supplementation-unblocking-regulation" therapeutic approach is proposed. For deficiency， the focus is on supplementing kidney and fortifying spleen， and replenishing the marrow sea. For stasis， the priority is to unblock and open the orifices， and clear the marrow channels. For stagnation， the core is to clear fire and contain the mind， regulate and restore vital activity. In clinical practice， it is necessary to identify the primary and secondary pathogenic mechanisms and apply dynamic， combined treatment， integrating Chinese herbal medicine， acupuncture， and guiding exercises throughout the process， aiming to provide a reference for the diagnosis and treatment of ADHD with traditional Chinese medical.

BackgroundMonitoring the blood concentrations of antipsychotic drugs and their metabolites can guide the adjustment of clinical treatment plans， improving therapeutic efficacy while reducing adverse effects. However， there is currently a lack of a method that can accurately and efficiently quantitatively detect multiple antipsychotic drugs and their metabolites. ObjectiveTo establish a ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method for the simultaneous identification and quantitation of 12 antipsychotic drugs and their main metabolites in human serum. MethodsUsing UPLC-MS/MS technology， protein precipitation method was employed for sample pretreatment. An Agela Technologies Durashell C₈ chromatographic column （50 mm×3.0 mm， 5 μm） was selected for chromatographic separation with gradient elution. The flow rate was 0.4 mL/min， and the total analysis time was 5 minutes. The column temperature was 40℃. The mass spectrometry detection was carried out in the multiple reaction monitoring （MRM） mode， and the isotope internal standard method was used for quantification. ResultsThe relative standard deviation （RSD） of the internal standard normalization matrix effect factor for 12 antipsychotic drugs and their main metabolites at low and high quality concentrations was all less than 15%. The extraction recovery rate was 85% to 115%. They showed good linear relationships within their respective standard curve ranges （r>0.995）. At low， medium， and high quality concentrations， the accuracy was 85.24% to 114.71%， and the RSD of intra-batch and inter-batch precision was all ≤14.15%， with good stability. ConclusionAll the analytical performance indicators of this method meet the verification requirements， providing an analytical means for the quantitative detection of antipsychotic drugs and their main metabolites in human serum. ［Funded by The Third Batch of Science and Technology Projects in Chaozhou City in 2023 （number， 202303GY02）］

Objective To establish an epirubicin (EPI)-resistant murine triple-negative breast cancer (TNBC) (4T1/EPI) cell line and evaluate its biological characteristics and drug resistance. Methods The EPI-resistant cell line 4T1/EPI was developed through intermittent induction with gradually increasing EPI concentrations in vitro. Morphological changes were observed under an inverted microscope. Drug resistance index (MTT assay), cell doubling time (CCK-8 assay), and migration ability (wound healing assay) were evaluated. Western blot was used to detect the expression of drug resistance-related proteins. Transcriptome sequencing and KEGG pathway enrichment analysis were performed to identify the pathways and targets involved in EPI resistance, followed by experimental validation. Results The 4T1 cells eventually grew normally in a medium containing 100 ng/mL EPI, confirming the establishment of the 4T1/EPI resistant cell line. After stable resistance was acquired, morphological alterations were observed. Compared with their parental 4T1 cells, 4T1/EPI cells showed significantly prolonged doubling time (P<0.01) and enhanced migration ability (P<0.05). Expression levels of drug resistance-related proteins MDR1, MRP1 (P<0.01), and ABCG2 (P<0.05) were elevated in 4T1/EPI cells. In vivo models also demonstrated significant EPI resistance in 4T1/EPI tumors in terms of tumor weight and volume. Transcriptome sequencing highlighted the involvement of the PI3K/Akt signaling pathway and ABC transporter pathway. Validation experiments showed the upregulation of Erbb3, Egfr, PI3K, and Akt (P<0.05) and significant downregulation of Fgfr1 (P<0.01) in 4T1/EPI cells. Conclusion The EPI-resistant TNBC cell line 4T1/EPI was successfully established, exhibiting significant resistance in vitro and in vivo. The mechanism may involve the EPI-induced upregulation of Egfr and Erbb3, activating the PI3K/Akt pathway and subsequently enhancing ABC transporter expression.

OBJECTIVE To explore a synergistic medication strategy integrating stage-specific modifications of Bushen huoxue decoction with hormone replacement therapy （HRT） for premature ovarian insufficiency （POI）. METHODS The connotation of Academician Tong Xiaolin’s “state-target differentiation and treatment” theory and the disease stage characteristics of POI were summarized. The latent stage， subclinical stage， clinical stage and exhaustion stage of POI were classified into corresponding core pathogenesis “states” and key objective indicator “targets”. Taking Bushen huoxue decoction as the basic prescription， its staged modification scheme and its sequential combination with HRT were analyzed. RESULTS & CONCLUSIONS The four stages of POI correspond to four pathogenic states respectively： kidney yin deficiency， kidney deficiency and blood stasis， heart-kidney disharmony， and deficiency of both yin and yang. Follicle-stimulating hormone （FSH）， anti-Müllerian hormone （AMH）， antral follicle count （AFC） and Kupperman score were taken as the evaluation targets of staging. Combined with the “state-target” characteristics of each stage， a medication principle based on Bushen huoxue decoction with syndrome differentiation modification and stage-adjusted administration was established： traditional Chinese medicine dominates in the latent stage， a sequential collaborative regimen is adopted in the subclinical stage， HRT is dominant supplemented by traditional Chinese medicine in the clinical stage， and integrated traditional Chinese and Western medicine is applied to consolidate the root cause in the exhaustion stage.

Background Amid global climate change, extreme environmental events are occurring more frequently, and it is imperative to investigate the impacts of combined exposure to fine particluate matter (PM_2.5) and cold spells (CS) on population mortality. Objective To analyze the association between sequential extreme PM_2.5-cold spell (EP-CS) events and non-accidental mortality among residents in Zigong City from 2016 to 2021. Methods Using time-series study design, meteorological data in Zigong were collected from the Zigong Meteorological Bureau for the period from January 1, 2016 to December 31, 2021, while daily non-accidental mortality data were obtained from the mortality surveillance system of the Zigong Center for Disease Control and Prevention. We adopted the percentile method to define extreme PM_2.5 events and cold spells. We analyzed the risk effect of EP-CS events on non-accidental mortality among residents in this city and explored the potential amplification of damage resulting from different patterns of consecutive extreme events by using distributed lag nonlinear model (DLNM). We also conducted stratified analyses based on age, gender, education level, and marital status. Results The EP-CS events demonstrated a significant impact on non-accidental mortality among the local residents, exhibiting a certain lagged effect. The effects on the overall residents lasted from lag0 (RR=1.030, 95%CI: 1.013, 1.048) to lag14 (RR=1.035, 95%CI: 1.019, 1.052). Notably, the effects were more pronounced among females, individuals aged 65 years and above, and those who were never married, divorced, or widowed. Different patterns of EP-CS events all associated with adverse effects, the health impact of EP-CS events was significantly greater than that of individual PM_2.5 pollution or CS events. The analysis of lag effects across different event patterns revealed that the overall effect of EP-CS events with shorter intervals (0–7 d) had a stronger effect compared to EP-CS with longer intervals (8–14 d), and the RR values of lag14 were 1.034 (95%CI: 1.015, 1.054) and 1.017 (95%CI: 1.007, 1.027), suggesting that the damaging effect of compound events occurring in the short term was more significant. Conclusion All sequential extreme EP-CS events have an impact on non-accidental mortality among residents in this city, with compound events demonstrating a stronger effect. Females, individuals aged ≥65 years, and those who were never married, divorced, or widowed are more sensitive to EP-CS events.

ObjectiveTo observe the effects of Yiqi Huoxue Jiedu formula（YHJF） on immune cell exhaustion in the spleen of septic mice and to explore and validate its potential intervention targets. MethodsMice were randomly divided into the sham-operated， model， low-dose YHJF（4.1 g·kg^-1）， and high-dose YHJF（8.2 g·kg^-1） groups. Except for the sham-operated group， a cecal ligation and puncture（CLP） procedure was performed to establish a mouse sepsis model. The treatment groups received oral administration of the corresponding doses， while the sham-operated and model groups received an equal volume of physiological saline. After the intervention， the 7-day survival rate of each group was recorded， and spleen samples were collected 72 h post-intervention， and the spleen index was calculated. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate（dUTP） nick end labeling（TUNEL） staining was used to detect apoptosis in spleen cells. Enzyme-linked immunosorbent assay（ELISA） was performed to measure the levels of interleukin（IL）-4 and IL-10 in the serum. Transcriptomics and metabolomics were used to screen for differentially expressed genes（DEGs） and differential metabolites in the spleen， followed by bioinformatics analysis to identify key targets. Real-time quantitative polymerase chain reaction（Real-time PCR）， flow cytometry， and multiplex immunofluorescence were used to verify the expressions of key genes and proteins. ResultsThe high-dose YHJF group significantly improved the 7-day survival rate of septic mice（P0.05）. Compared with the sham-operated group， the model group showed a significant increase in apoptosis of spleen cells and a decrease in the spleen index at 72 h post-modeling， with markedly elevated peripheral serum IL-4 and IL-10 levels（P0.01）. Compared with the model group， the high-dose YHJF group showed a reduction in apoptosis of spleen cells， an increase in the spleen index， and a significant decrease in peripheral serum IL-4 and IL-10 levels（P0.05）. Spleen transcriptomics identified 255 DEGs between groups， potentially serving as intervention targets for YHJF. Gene Ontology（GO） enrichment analysis revealed that DEGs were mainly involved in biological processes such as natural killer（NK） cell-mediated positive immune regulation， cell killing， cytokine production， positive regulation of innate immune cells， and interferon production. Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis showed that DEGs were mainly involved in cytokine-cytokine receptor interactions， viral protein interactions with cytokines and cytokine receptors， chemokine signaling pathway， and nuclear transcription factor-κB（NF-κB） signaling pathway. Protein-protein interaction（PPI） network analysis identified CD160， granzyme B（GZMB）， and chemokine ligand 4（CCL4） as key targets for YHJF in treating sepsis. Metabolomics identified 46 differential metabolites that were significantly reversed by YHJF intervention， and combined transcriptomics and metabolomics analysis identified 17 differential metabolites closely related to CD160. Pathway enrichment revealed that these metabolites were mainly involved in glycerophospholipid metabolism， arachidonic acid metabolism， glycosylphosphatidylinositol（GPI） anchor biosynthesis， linoleic acid metabolism， and α-linolenic acid metabolism pathways. Verification results showed that， compared with the sham-operated group， the model group exhibited significantly elevated CD160 mRNA expression level in the spleen， along with markedly decreased CCL4 and GZMB mRNA expression， and had a significant increase in CD160 expression on the surface of natural killer T（NKT） cells in the spleen（P0.01）. Compared with the model group， the high-dose YHJF group had a significant decrease in CD160 mRNA expression in the spleen， a significant increase in CCL4 and GZMB mRNA expressions. Further flow cytometry and immunofluorescence revealed that compared with the sham-operated group， CD160 expression on the surface of splenic NKT cells in the model group was significantly increased（P0.01）， while high-dose YHJF intervention significantly reduced CD160 expression（P0.01）. ConclusionYHJF may alleviate NKT cell exhaustion in sepsis by downregulating the expression of the negative co-stimulatory molecule CD160， and this regulatory effect is closely related to fatty acid metabolism pathways. This study provides new insights and targets for further exploration of strengthening vital Qi and detoxifying strategy to improve immune cell exhaustion in acute deficiency syndrome of sepsis.

ObjectiveTo observe the effects of Yiqi Huoxue Jiedu formula（YHJF） on immune cell exhaustion in the spleen of septic mice and to explore and validate its potential intervention targets. MethodsMice were randomly divided into the sham-operated， model， low-dose YHJF（4.1 g·kg^-1）， and high-dose YHJF（8.2 g·kg^-1） groups. Except for the sham-operated group， a cecal ligation and puncture（CLP） procedure was performed to establish a mouse sepsis model. The treatment groups received oral administration of the corresponding doses， while the sham-operated and model groups received an equal volume of physiological saline. After the intervention， the 7-day survival rate of each group was recorded， and spleen samples were collected 72 h post-intervention， and the spleen index was calculated. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate（dUTP） nick end labeling（TUNEL） staining was used to detect apoptosis in spleen cells. Enzyme-linked immunosorbent assay（ELISA） was performed to measure the levels of interleukin（IL）-4 and IL-10 in the serum. Transcriptomics and metabolomics were used to screen for differentially expressed genes（DEGs） and differential metabolites in the spleen， followed by bioinformatics analysis to identify key targets. Real-time quantitative polymerase chain reaction（Real-time PCR）， flow cytometry， and multiplex immunofluorescence were used to verify the expressions of key genes and proteins. ResultsThe high-dose YHJF group significantly improved the 7-day survival rate of septic mice（P0.05）. Compared with the sham-operated group， the model group showed a significant increase in apoptosis of spleen cells and a decrease in the spleen index at 72 h post-modeling， with markedly elevated peripheral serum IL-4 and IL-10 levels（P0.01）. Compared with the model group， the high-dose YHJF group showed a reduction in apoptosis of spleen cells， an increase in the spleen index， and a significant decrease in peripheral serum IL-4 and IL-10 levels（P0.05）. Spleen transcriptomics identified 255 DEGs between groups， potentially serving as intervention targets for YHJF. Gene Ontology（GO） enrichment analysis revealed that DEGs were mainly involved in biological processes such as natural killer（NK） cell-mediated positive immune regulation， cell killing， cytokine production， positive regulation of innate immune cells， and interferon production. Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis showed that DEGs were mainly involved in cytokine-cytokine receptor interactions， viral protein interactions with cytokines and cytokine receptors， chemokine signaling pathway， and nuclear transcription factor-κB（NF-κB） signaling pathway. Protein-protein interaction（PPI） network analysis identified CD160， granzyme B（GZMB）， and chemokine ligand 4（CCL4） as key targets for YHJF in treating sepsis. Metabolomics identified 46 differential metabolites that were significantly reversed by YHJF intervention， and combined transcriptomics and metabolomics analysis identified 17 differential metabolites closely related to CD160. Pathway enrichment revealed that these metabolites were mainly involved in glycerophospholipid metabolism， arachidonic acid metabolism， glycosylphosphatidylinositol（GPI） anchor biosynthesis， linoleic acid metabolism， and α-linolenic acid metabolism pathways. Verification results showed that， compared with the sham-operated group， the model group exhibited significantly elevated CD160 mRNA expression level in the spleen， along with markedly decreased CCL4 and GZMB mRNA expression， and had a significant increase in CD160 expression on the surface of natural killer T（NKT） cells in the spleen（P0.01）. Compared with the model group， the high-dose YHJF group had a significant decrease in CD160 mRNA expression in the spleen， a significant increase in CCL4 and GZMB mRNA expressions. Further flow cytometry and immunofluorescence revealed that compared with the sham-operated group， CD160 expression on the surface of splenic NKT cells in the model group was significantly increased（P0.01）， while high-dose YHJF intervention significantly reduced CD160 expression（P0.01）. ConclusionYHJF may alleviate NKT cell exhaustion in sepsis by downregulating the expression of the negative co-stimulatory molecule CD160， and this regulatory effect is closely related to fatty acid metabolism pathways. This study provides new insights and targets for further exploration of strengthening vital Qi and detoxifying strategy to improve immune cell exhaustion in acute deficiency syndrome of sepsis.