ObjectiveTo integrate network pharmacology prediction with multi-level experimental verification methods， and to explore in depth the therapeutic efficacy and potential mechanism of luteolin in treating gout. MethodsDatabases were used to obtain potential pharmacodynamic targets of luteolin. Protein-protein interaction （PPI） network construction and network pharmacology analysis techniques were used to screen key core targets of luteolin in gout treatment. Further biological function enrichment analysis and signaling pathway analysis were performed on these targets. Molecular docking simulation was used to calculate the binding energy between luteolin and potential core targets， clarifying the strength of their interactions. In the in vivo experiment for hyperuricemia， 48 mice were randomly divided into a blank group， a model group， an allopurinol group （5 mg·kg^-1）， and low-dose （10 mg·kg^-1）， medium-dose （30 mg·kg^-1）， and high-dose （90 mg·kg^-1） luteolin groups. For the first three days， the blank and model groups were gavaged with an equal volume of normal saline， while the allopurinol group and luteolin groups were gavaged with corresponding drugs. From day 4 onwards， modeling was performed by intraperitoneal injection at 12：00 daily （normal saline for the blank group， and oxonic acid potassium-hypoxanthine mixture for other groups， with 300 mg·kg^-1 for each group）. Gavage intervention was administered at 18：00 daily （normal saline for the blank/model groups， and corresponding drugs for the treatment groups） until day 7. After sampling， levels of serum uric acid （UA）， alanine aminotransferase （ALT）， and aspartate aminotransferase （AST） were measured. Levels of xanthine oxidase （XO） in the liver and kidney， ATP-binding cassette transporter G2 （ABCG2） and malondialdehyde （MDA） in the kidney， and superoxide dismutase （SOD） in the liver were determined. Renal HE staining was also performed. In the pharmacodynamic study of gouty arthritis， 36 rats were randomly divided into a blank group， a model group， a colchicine group （0.315 mg·kg^-1）， and low-dose （7 mg·kg^-1）， medium-dose （21 mg·kg^-1）， and high-dose （63 mg·kg^-1） luteolin groups. The model was established by vertically injecting 100 µL of 25 g·L^-1 monosodium urate suspension into the posterior lateral aspect of the right ankle joint （the blank group was injected with an equal volume of normal saline）， with repeated injections every two days for reinforcement. From day 2 after modeling， daily gavage administration was performed （normal saline for the blank/model groups， and corresponding drugs for the treatment groups） for a total of 16 days. During the experiment， ankle swelling and pain threshold were measured regularly. After sampling， levels of serum tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β） were determined. Ankle joints were subjected to HE， Masson， and safranin O-fast green staining， and HE staining was also performed on ankle synovial tissue and various organs. Western blot was used to determine the expression levels of key proteins in gout-related signaling pathways. ResultsNetwork pharmacology analysis predicted that luteolin may regulate over 20 core targets， such as XO， ABCG2， nuclear factor erythroid 2-related factor 2 （Nrf2）， and SOD， through acting on signaling pathways including NF-κB， phosphoinositide 3-kinase/protein kinase B （PI3K/Akt）， and ABC transporters， thereby affecting uric acid metabolism and inflammatory responses. In the hyperuricemia model， compared with the blank group， the model group showed significantly increased serum UA level， liver and kidney XO activity， renal ABCG2 expression， and liver SOD activity （P<0.01）. Compared with the model group， the high-dose luteolin group significantly reduced serum UA level （P<0.01）， inhibited liver and kidney XO activity （P<0.01）， and significantly increased renal ABCG2 expression and liver SOD activity （P<0.01）， effectively alleviating renal oxidative stress damage and improving renal histopathological status. In the gouty arthritis model， compared with the blank group， the model group showed significant ankle swelling， decreased pain threshold， and significantly increased levels of IL-6， IL-1β， and TNF-α in serum and synovial tissue （P<0.01）. The high-dose luteolin group significantly reduced ankle swelling， prolonged hot plate pain threshold， effectively decreased the levels of the above inflammatory factors in serum and synovial tissue （P<0.01）， and significantly improved ankle pathological damage， showing good analgesic and anti-inflammatory effects. Western blot results further confirmed that luteolin significantly upregulated Nrf2 protein expression and downregulated XO and nucleotide-binding oligomerization domain （NOD）-like receptor protein 3 （NLRP3） expression in animals. ConclusionLuteolin can improve symptoms of hyperuricemia and gouty arthritis， and its potential mechanism may be related to inhibiting XO activity， increasing ABCG2 and SOD levels， and regulating Nrf2-mediated oxidative stress-related pathways.

Objective To construct an in vitro research model for studying human liver regeneration based on human pluripotent stem cells(hPSCs)-derived hepatocyte-like organoid(HLO).Methods The hPSCs-derived HLO was obtained by inducing differentiation and the regeneration model after liver injury was constructed by adding acetaminophen(APAP)at fixed time points in HLO culture conditions to simulate acute liver injury.Subse-quently,HLO with catenin/cadherin-associated protein beta 1(CTNNB1)knockout,a key gene regulating liver re-generation,was constructed using CRISPR/Cas9 gene editing technology,and regeneration experiments with APAP injury were performed.HLO as a model for liver regeneration studies was further evaluated by morphological observation,RT-qPCR,Western blot and pathological analysis.Results Morphology evidence as well as expres-sion of marker genes showed that hPSCs-derived HLO was able to initiate a post-injury regeneration response after APAP treatment.CTNNB1-deficient HLO showed delayed recovery in dimension and down-regulated or delayed ex-pression of related genes during post-injury regeneration as compared to control HLO.Conclusions A HLO-based hPSCs-derived human liver regeneration model is successfully constructed,which can be used for gene function studies during liver regeneration.

Objective To investigate alteration of blood routine parameters,blood gas analysis profile,coagulation function,and inflammatory factors during exposure to high-altitude hypoxic environments.Methods Rats were raised in a hypobaric oxygen chamber to simulate the altitude of 5 500 meters.The animals were divided into groups with exposure duration of 0(control),1,3,5,7,14,and 28 days.Arterial blood gas was measured using a blood gas analyzer.The routine blood test was performed by an automatic five-differential animal hematology analyzer.The coagulation function was measured by a fully automatic coagulation analyzer.The level of plasma D-dimer(DD),erythropoietin(EPO),interleukin-10(IL-10),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and C-reactive protein(CRP)was detected by ELISA method.The protein expression of IL-10,IL-6,and TNF-α in lung tissues of the animals was detected by Western blot.Results As compare to control group,the arterial partial pressure of ox-ygen(PaO2)decreased at different durations of hypoxia exposure.The PaO2 in the group exposed to hypoxia for 7 d was the lowest(P＜0.05).The red blood cell count(RBC),hemoglobin(HGB),hematocrit(HCT),mean cor-puscular volume(MCV),mean corpuscular hemoglobin concentration(MCHC)and mean platelet volume(MPV)were all higher than control group(P＜0.05).Erythropoietin(EPO),prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT),fibrinogen(FIB)and D-dimer(DD)all gradually increased(P＜0.05).The white blood cell count(WBC)and platelet count(PLT),as well as the plasma inflammatory fac-tors including interleukin-10(IL-10),interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),C-reactive protein(CRP),and the expression of inflammatory factors in the lung tissue including IL-10,IL-6 and TNF-α pro-teins all showed a curve of increasing at beginning and then followed by a slow decrease with the prolongation of the hypoxia time(P＜0.05).Conclusions In high-altitude hypoxic environment,the blood circulation undergoes dy-namic evolution of functional remodeling with higher risk of inflammatory response.As the hypoxia time prolongs,the animals adapt the environment and the level of inflammatory cytokines gradually decline but remains at a level which is still higher than that of control animals.

Objective This study aims to investigate the current status and potential profiles of emotional inhibition in stoma patients with colorectal cancer,to analyze the differences and influencing factors,and thereby provide a basis for the development of intervention strategies.Methods A convenience sample of 348 colorectal cancer patients with ostomies was recruited from colorectal surgery departments and ostomy outpatient clinics of a tertiary A hospital in Anhui Province.Data were collected using the General Information Questionnaire,Emotional Inhibition Scale,Chinese Perceived Stress Scale,10 item Conner Davidson Resilience Scale,and Family Care Index Questionnaire.Latent profile analysis was performed using Mplus 8.3,while univariate analysis and multivariate logistic regression analysis were conducted using SPSS 26.0.Results Valid questionnaires were collected from 336 participants(response rate 96.6％).Colorectal cancer patients with ostomies demonstrated a mean emotional inhibition score of 30.84±10.49.Latent profile analysis identified 3 distinct emotional inhibition patterns:high inhibition-suppressive type(31.25％),moderate inhibition-adaptive type(39.88％),and low inhibition-excessive type(28.87％).Gender,residential location,household income per capita,ostomy self-care proficiency,perceived stress levels,psychological resilience scores and family functioning status emerged as significant determinants of emotional inhibition heterogeneity among colorectal cancer patients with ostomies across distinct latent profiles(P＜0.05).Conclusion Colorectal cancer patients with ostomies exhibit significant heterogeneity in emotional inhibition profiles.Clinicians should conduct early identification of these latent classes through standardized assessments and develop tailored interventions to improve emotional inhibition outcomes in this clinical population.

AIM:To investigate the immunosuppressive effects of M2-like tumor-associated macrophages(M2-TAMs)on CD8+T cells within the tumor microenvironment of esophageal cancer.METHODS:Multiplex fluores-cence immunohistochemistry was used to analyze the distribution of immune cells in esophageal cancer tissues.An in vitro co-culture system was established,and flow cytometry along with Calcein-AM/PI staining was employed to assess the im-pact of M2-TAMs on CD8+T cell function.The GEPIA database was utilized to evaluate the prognostic significance of PDCD1 expression in esophageal cancer patients and to analyze the correlations between gene expressions.Immunohisto-chemistry(IHC)was performed to detect the expression of TGF-β1 in esophageal cancer tissues.Flow cytometry and en-zyme-linked immunosorbent assay(ELISA)were used to measure PD-1,IFN-γ and TNF-α expression in CD8+T cells fol-lowing treatment with a TGF-β1 inhibitor.RESULTS:Compared with early-stage(stage I)esophageal cancer patients,the patients with advanced disease(stages Ⅱ to Ⅳ)exhibited dynamic changes in the infiltration of CD4+T cells,CD8+T cells,Tregs,and M2-TAMs within tumor tissues,with significant correlations observed among these cell populations(P<0.05).The distribution of M2-TAMs and Tregs was positively correlated with poor prognosis(P<0.05),while that of CD8+T cells was negatively correlated(P<0.05).In contrast,CD4+T cell infiltration showed no significant association with clinical outcomes(P>0.05).Co-culture of CD8+T cells with M2-TAMs resulted in significant downregulation of CD107a,granzyme B,IFN-γ and TNF-α expression(P<0.01).Additionally,M2-TAM-treated CD8+T cells co-cultured with esophageal cancer cells led to reduced apoptosis of cancer cells.High expression of PDCD1 was significantly associated with poor prognosis(P<0.05),and significant correlations were observed between CD8A and PDCD1 expression,as well as between TGF-β1 and CD274 gene expression(P<0.01).TGF-β1 was also significantly associated with CD163+macro-phage infiltration and the progression of esophageal cancer(P<0.05).Treatment with a TGF-β1 inhibitor in the M2-TAM and CD8+T cell co-culture system significantly down-regulated PD-1 expression and increased the secretion of IFN-γ and TNF-α(P<0.01).CONCLUSION:The TGF-β1 derived from M2-TAMs inhibits the antitumor activity of CD8+T cells in the esophageal cancer microenvironment,suggesting potential therapeutic targets for overcoming immunosuppression in esophageal cancer.

Objective·To identify different comorbidity types resulting from the spontaneous association of psychopathological symptoms among male drug addicts,explore the relationship between latent types and drug addiction,and examine the mediating role of psychological distress.Methods·Four hundred and fifty male drug addicts,recruited by Yunnan First Compulsory Isolation Drug Rehabilitation Center according to the 2017 revised Drug Addiction Identification Method by the Ministry of Public Security,were enrolled as participants.The Symptom Checklist-90(SCL-90),Craving Automated Scale for Substances(CAS-S),and Psychache Scale(PAS)were used to assess the severity of psychopathological symptoms,drug addiction,and psychological distress,respectively.Latent class analysis was employed to identify different types of psychopathological comorbidity that may exist in the participants,and the types were named based on the analysis results.Variance analysis was used to test differences in the severity of drug addiction and psychological distress among different latent types,and a mediation model was established to explore the mediating role of psychological distress between different latent types and drug addiction.Results·Latent class analysis identified three types of psychopathology:high,medium,and low levels,with decreasing positive rates and numbers of comorbidity in each type based on the ten psychopathological symptoms included in the SCL-90.There were significant differences in the severity of psychological distress and drug addiction among the three types of participants.Psychopathological symptoms positively predicted the severity of psychological distress and drug addiction.Psychological distress completely mediated the relationship between medium levels of psychopathology and drug addiction,while partially mediating the relationship between high levels of psychopathology and drug addiction.Conclusion·There are three types of psychopathological comorbidity among male drug addicts:high,medium,and low.Psychological distress plays a complete mediating role between medium levels of psychopathology and drug addiction,and a partial mediating role between high levels of psychopathology and drug addiction.

Objective In this paper,the ethnic patent medicines(EPMs)in the Chinese Pharmacopeia,National Basic Medical Insurance,Employment Injury Insurance and Maternity Insurance Medicine List,and National Essential Medicine List(re-ferred to as"one pharmacopeia and two lists")were counted and analyzed.Methods The EPMs in"one pharmacopeia and two lists"were summarized,and the information of manufacturers,medicinal materials used in EPMs,and their current quality standards were collated,and their indicative function,adverse reactions,contraindications,and precautions were analyzed.The modern research level of the above EPMs was analyzed by searching relevant literature on CNKI and PubMed.Results A total of 142 EPMs in"one pharmacopeia and two lists"were included,and 68 of the EPMs had exclusive manufacturers and 98.75％of the medicinal materials had provincial-level or higher quality standards.There were issues such as the expression of indicative function in the instruction being mixed between medical terms,91.55％ of the EPMs lacking sufficient information on adverse re-actions,contraindications,and precautions,and modern basic research was weak.Conclusions In the development of EPMs,it is important to improve information on adverse reactions,contraindications,and precautions in the instructions.In expressing indic-ative function,it is necessary to avoid mixing medical terms,which is more conducive to the clinical promotion of EPMs.

AIM:To investigate the regulatory mechanism of silent information regulator 6(Sirt6)on nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway and ferroptosis in skeletal muscle aging in-duced by D-galactose(D-Gal)in mice.METHODS:A D-Gal-induced mouse aging model was established and randomly divided into control and D-Gal groups.In vitro,D-Gal-treated C2C12 mouse myoblasts were treated with ferroptosis ago-nist erastin(Era)and inhibitor ferrostatin-1(Fer-1),Sirt6 agonist MDL-800 and inhibitor OSS-128167,and Nrf2 siRNA.Mouse body weight and forelimb relative grip strength were monitored.RT-qPCR and Western blot were used to measure the expression of Sirt6,Nrf2,HO-1,P53,P21,P16,muscle ring finger protein 1,muscle atrophy F-box,solute carrier family 7 member 11,and glutathione peroxidase 4 in gastrocnemius muscle and myoblasts.Hematoxylin-eosin staining was performed to examine muscle fiber diameter.Levels of reactive oxygen species(ROS),mitochondrial ROS,mitochon-drial membrane potential,senescence-associated β-galactosidase activity,glutathione,lipid peroxidation,and Fe2? con-centration were measured in myoblasts and myotubes.Immunofluorescence staining was used to detect myosin heavy chain(MyHC)expression in myotubes.RESULTS:Mice in the D-Gal group exhibited significant reductions in body weight and forelimb grip strength(P<0.01),upregulation of aging and muscle atrophy markers,and decreased mRNA and pro-tein levels of ferroptosis markers and Sirt6(P<0.01).Additionally,gastrocnemius muscle fiber diameter significantly de-creased(P<0.01).In D-Gal-treated myoblasts and myotubes,aging and muscle atrophy markers were elevated(P<0.01),MyHC expression was reduced,and protein levels of ferroptosis-related markers,Sirt6,Nrf2,and HO-1 were de-creased(P<0.05 or P<0.01).Fer-1 pre-treatment alleviated these changes(P<0.05 or P<0.01).MDL-800 significantly improved D-Gal-induced aging and muscle atrophy in myoblasts and myotubes,while increasing the expression of ferropto-sis-related proteins(P<0.05 or P<0.01).However,the addition of Erastin abolished the beneficial effects of MDL-800(P<0.05 or P<0.01).Following Nrf2 siRNA transfection,the ability of MDL-800 to improve ferroptosis and the quality of myotube formation was significantly diminished(P<0.05 or P<0.01).CONCLUSION:Sirt6 inhibits ferroptosis in myo-blasts through the Nrf2/HO-1 signaling pathway,thereby alleviating age-related changes in myoblasts and the decline in myotube formation quality,which is beneficial for improving skeletal muscle aging.

OBJECTIVES: Osteoarthritis (OA) and sarcopenia are significant health concerns in the elderly, substantially impacting their daily activities and quality of life. However, the relationship between them remains poorly understood. This study aims to uncover common biomarkers and pathways associated with both OA and sarcopenia. METHODS: Gene expression profiles related to OA and sarcopenia were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between disease and control groups were identified using R software. Common DEGs were extracted via Venn diagram analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to identify biological processes and pathways associated with shared DEGs. Protein-protein interaction (PPI) networks were constructed, and candidate hub genes were ranked using the maximal clique centrality (MCC) algorithm. Further validation of hub gene expression was performed using 2 independent datasets. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive value of key genes for OA and sarcopenia. Mouse models of OA and sarcopenia were established. Hematoxylin-eosin and Safranin O/Fast Green staining were used to validate the OA model. The sarcopenia model was validated via rotarod testing and quadriceps muscle mass measurement. Real-time reverse transcription PCR (real-time RT-PCR) was employed to assess the mRNA expression levels of candidate key genes in both models. Gene set enrichment analysis (GSEA) was conducted to identify pathways associated with the selected shared key genes in both diseases. RESULTS: A total of 89 common DEGs were identified in the gene expression profiles of OA and sarcopenia, including 76 upregulated and 13 downregulated genes. These 89 DEGs were significantly enriched in protein digestion and absorption, the PI3K-Akt signaling pathway, and extracellular matrix-receptor interaction. PPI network analysis and MCC algorithm analysis of the 89 common DEGs identified the top 17 candidate hub genes. Based on the differential expression analysis of these 17 candidate hub genes in the validation datasets, AEBP1 and COL8A2 were ultimately selected as the common key genes for both diseases, both of which showed a significant upregulation trend in the disease groups (all P<0.05). The value of area under the curve (AUC) for AEBP1 and COL8A2 in the OA and sarcopenia datasets were all greater than 0.7, indicating that both genes have potential value in predicting OA and sarcopenia. Real-time RT-PCR results showed that the mRNA expression levels of AEBP1 and COL8A2 were significantly upregulated in the disease groups (all P<0.05), consistent with the results observed in the bioinformatics analysis. GSEA revealed that AEBP1 and COL8A2 were closely related to extracellular matrix-receptor interaction, ribosome, and oxidative phosphorylation in OA and sarcopenia. CONCLUSIONS AEBP1 and COL8A2 have the potential to serve as common biomarkers for OA and sarcopenia. The extracellular matrix-receptor interaction pathway may represent a potential target for the prevention and treatment of both OA and sarcopenia.
Sarcopenia/genetics* ; Osteoarthritis/genetics* ; Computational Biology/methods* ; Humans ; Protein Interaction Maps/genetics* ; Animals ; Mice ; Gene Expression Profiling ; Gene Ontology ; Transcriptome ; Male ; Signal Transduction/genetics* ; Gene Regulatory Networks

OBJECTIVES: Osteoarthritis (OA) is one of the most common chronic degenerative diseases, with chondrocyte apoptosis and extracellular matrix (ECM) degradation as the major pathological changes. The mechanical stimulation can attenuate chondrocyte apoptosis and promote ECM synthesis, but the underlying molecular mechanisms remain unclear. This study aims to investigate the role of primary cilia (PC) in mediating the effects of mechanical stimulation on OA progression. METHODS: In vivo, conditional knockout mice lacking intraflagellar transport 88 (IFT88^flox/flox IFT88 knockout; i.e., primary cilia-deficient mice) were generated, with wild-type mice as controls. OA models were established via anterior cruciate ligament transection combined with destabilization of the medial meniscus, followed by treadmill exercise intervention. OA progression was evaluated by hematoxylin-eosin staining, safranin O-fast green staining, and immunohistochemistry; apoptosis was assessed by TUNEL staining; and limb function by rotarod testing. In vitro, primary articular chondrocytes were isolated from mice and transfected with lentiviral vectors to suppress IFT88 expression, thereby constructing a primary cilia-deficient cell model. Interleukin-1β (IL-1β) was used to induce an inflammatory environment, while cyclic tensile strain (CTS) was applied via a cell stretcher to mimic mechanical loading on chondrocytes. Immunofluorescence and Western blotting were used to detect the protein expression levels of type II collagen α1 chain (COL2A1), primary cilia, IFT88, and caspase-12; reverse transcription polymerase chain reaction was performed to assess COL2A1 mRNA levels; and flow cytometry was used to evaluate apoptosis. RESULTS: In vivo, treadmill exercise significantly reduced Osteoarthritis Research Society International (OARSI) scores and apoptotic cell rates, and improved balance ability in wild-type OA mice, whereas IFT88-deficient OA mice showed no significant improvement. In vitro, CTS inhibited IL-1β-induced ECM degradation and apoptosis in primary chondrocytes; however, this protective effect was abolished in cells with suppressed primary cilia expression. CONCLUSIONS Mechanical stimulation delays OA progression by mediating signal transduction through primary cilia, thereby inhibiting cartilage degeneration and chondrocyte apoptosis.
Animals ; Chondrocytes/cytology* ; Apoptosis/physiology* ; Mice ; Cilia/metabolism* ; Osteoarthritis/pathology* ; Extracellular Matrix/metabolism* ; Mice, Knockout ; Disease Progression ; Interleukin-1beta ; Male ; Cells, Cultured