ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

AIM: To investigate the effect and potential mechanism of liraglutide(LIR)combined with neural stem cells(NSCs)in alleviating diabetic retinopathy(DR)in rats.METHODS:A DR rat model was established using a high-fat diet combined with a single intraperitoneal injection of streptozotocin(STZ). Rats in the control group were fed a normal diet and received an intraperitoneal injection of citric acid-sodium citrate buffer. After modeling, the rats were divided into the control group, DR group, DR+LIR group, DR+BMSCs group, DR+NSCs group, DR+BMSCs+LIR group, and DR+NSCs+LIR group. According to the grouping instructions, rats were given intraperitoneal injection of LIR at 200 μg/(kg·d)and intravitreal injection of bone marrow mesenchymal stem cells(BMSCs), or NSCs, or the combined therapy accordingly for 8 wk. Fasting blood glucose(FBG)and serum insulin levels, as well as retinal oxidative stress indexes and serum inflammatory indexes, were detected in each group. Retinopathy was evaluated by hematoxylin-eosin(HE)staining, retinal cell apoptosis was detected by TUNEL staining, SIRT1 expression in retinal tissues was detected by immunohistochemistry, and BDNF expression in retinal tissues was detected by immunofluorescence. The transcriptional levels of cZNF532, miR-29a-3p, SIRT1, TGF-β1, NRF2, Keap1, and BDNF in retinal tissues were detected by qRT-PCR, and the protein expression levels of SIRT1, TGF-β1, SMAD2/3, p-SMAD2/3, NRF2, Keap1, p65, and p-p65 in retinal tissues were detected by Western blot. RESULTS:Compared with the other groups of rats, the retinal morphology of rats in the DR+NSCs+LIR group was significantly improved, the levels of FBG, TNF-α, IL-1β, IL-6 and MDA were decreased(all P<0.05), the serum insulin level and the levels of SOD, CAT, and GSH-Px were increased(all P<0.05), the TUNEL-positive rate of retinal cells was reduced(all P<0.05). The expression levels of cZNF532, SIRT1, NRF2, and BDNF in retinal tissue were up-regulated(all P<0.05), whereas the expression levels of miR-29a-3p, TGF-β1, p-SMAD2/3, Keap1, and p-p65 were down-regulated(all P<0.05). CONCLUSION:LIR combined with NSCs alleviates DR by regulating the cZNF532-miR-29a-3p-SIRT1 network and its downstream TGF-β/SMAD, NF-κB, and Keap1/NRF2 signaling pathways.

Objective: To investigate the trends in incidence and mortality of lung cancer in Huangpu District, Shanghai Municipality from 2002 to 2019, so as to provide the evidence for formulating lung cancer prevention and control measures. Methods: Data of lung cancer incidence and mortality among residents in Huangpu District from 2002 to 2019 were collected through the Shanghai Cancer Registration and Reporting Management System. The crude incidence and mortality of lung cancer was calculated, and standardized by the data from the Chinese Fifth National Population Census in 2000 (Chinese-standardized rate) and the Segi's world standard population in 1960 (world-standardized rate). The trends in incidence and mortality of lung cancer among residents by age and gender were evaluated using annual percent change (APC). Results: A total of 12 965 cases of lung cancer were reported in Huangpu District from 2002 to 2019, and the crude incidence rate was 80.66/105, the Chinese-standardized incidence rate was 34.54/105, and the world-standardized incidence rate was 31.30/105, all showing upward trends (APC=4.588%, 2.933% and 3.247%, all P<0.05). A total of 10 102 deaths of lung cancer were reported, and the crude mortality rate was 62.30/105, showing an upward trend (APC=0.959%, P<0.05); the Chinese-standardized mortality was 25.93/105, and the world-standardized mortality was 22.05/105, both showing downward trends (APC=-1.282% and -1.263%, both P<0.05). The crude incidence and mortality rates of lung cancer in males were higher than those in females (101.39/105 vs. 60.52/105, 85.45/105 vs. 39.87/105, both P<0.05). The crude incidence and mortality rates of lung cancer showed upward trends with age (both P<0.05), reaching their peaks in the age groups of 80-<85 years (341.37/105) and 85 years or above (355.97/105), respectively. Conclusions The incidence of lung cancer showed an upward trend, while the mortality showed a downward trend in Huangpu District from 2002 to 2019. Elderly men were the high-risk group for lung cancer incidence and mortality.

Breast cancer is a kind of malignant tumor with a complex mechanism， and its morbidity and mortality are increasing year by year， which seriously threatens women's health. At present， the main clinical treatments are surgical resection， radiotherapy， chemotherapy， and drug therapy， but they are often accompanied by side effects and adverse reactions， which affect the therapeutic effect. Traditional Chinese medicine （TCM） has the advantages of multi-component and multi-target treatment in the fight against breast cancer. The wnt/β-catenin signaling pathway is one of the classic pathways in cancer research. Abnormally activated Wnt/β-catenin signaling pathway inhibits β-catenin degradation by blocking the formation of Axin/glycogen synthase kinase 3β/adenomatous polyposis coli complex， thus promoting β-catenin nuclear metastasis， and it binds to T cell transcription factor/lymphoenhancer factor-1 to initiate downstream target genes and further interfere with the proliferation， migration， and invasion of tumor cells to affect the tumor process. Previous studies have shown that TCM monomers and compounds can mediate the Wnt/β-catenin signaling pathway to inhibit the malignant phenotype of breast cancer cells， thus playing an anti-breast cancer role， and the biochemical process involved in the regulation of therapeutic drugs has not been systematically combed. By analyzing and collating Chinese and foreign literature at the present stage， this paper discussed the association mechanism between Wnt/β-catenin signaling pathway and breast cancer and analyzed the internal mechanism of TCM monomers and compounds in mediating Wnt/β-catenin signaling pathway to exert anti-breast cancer effect. The statistical results showed that the flavonoids， alkaloids， and terpenoids in TCM monomers could target the Wnt/β-catenin signaling pathway and block the further development of malignant phenotype of breast cancer cells. TCM compounds with functions of clearing heat and detoxifying， promoting blood circulation and removing blood stasis， and tonifying kidney and liver were commonly used to intervene in the Wnt/β-catenin signaling pathway to prevent breast cancer. Compared with the current inhibitors of Wnt/β-catenin signaling pathway， the application of TCM monomers and compounds is expected to bring low-toxicity and high-efficiency breast cancer treatment drugs to the clinical practice， and the existing results provide a reference for the subsequent screening， research， and development of TCM small-molecule compounds and TCM compounds against breast cancer.

Objective:To investigate the effect of natural plant compound puerarin(PUE)on expression of NLRP3 inflamma-some in macrophages and its effect in ulcerative colitis(UC)in mice.Methods:A mouse model of UC was established using dextran sulfate sodium(DSS).Mice received PUE via gavage for 7 consecutive days,body weight,disease activity index and colon length were measured.HE staining was performed to assess tissue pathological damage.Flow cytometry was used to determine the proportion of peripheral blood mononuclear cells in colonic lamina propria.Immunofluorescence was employed to assess the colocalization of NLRP3 inflammasome and macrophages.qRT-PCR was conducted to measure mRNA expression levels of pro-inflammatory cytokines and NLRP3-related genes in colonic tissues.Protein expression levels of ZO-1,Occludin,cleaved caspase-1 and IL-1β in colonic tissues were detected by Western blot.A cell model was established using lipopolysaccharide(LPS)and adenosine triphosphate(ATP).mRNA expression levels of genes related to NLRP3 inflammasome were detected by qRT-PCR.Western blot was used to detect effects of PUE on expression levels of proteins related to NLRP3 inflammasome and NF-κB signaling pathway.Results:PUE treatment signifi-cantly improved symptoms of DSS-induced UC in mice,including body weight,disease index,colon length and pathological damage.Following PUE intervention,infiltration of Ly6C+MHC Ⅱ-monocyte derived macrophages in the colonic lamina propria was reduced.Expression levels of pro-inflammatory cytokines and NLRP3 inflammasome related molecules in colonic tissues were decreased.PUE treatment increased expression levels of ZO-1 and Occludin in intestinal epithelial cells.In vitro experiments confirmed that PUE reduced expression levels of NLRP3 inflammasome-related molecules in macrophages induced by LPS combined with ATP,as well as protein expression level of p-NF-κB p65.Conclusion:PUE significantly alleviates the symptoms of UC by reducing intestinal tissue inflamma-tion and repairing the epithelial barrier.The mechanism may involve regulating NF-κB signaling pathway,thereby reducing the forma-tion and activation of NLRP3 inflammasome in macrophages.

Objective To investigate the relationships between adolescents'body mass index(BMI),blood pressure(BP),and myopia and provide a scientific basis for the formulation of myopia prevention and control measures.Methods Adolescent students who par-ticipated in physical fitness surveillance in Chenghua District,Chengdu,from 2014 to 2023 were selected as the study population.A cross-sectional study was conducted using multiple logistic regression,adjusting for age,sex,BP,and pulse as covariates.A Cox model was employed to examine the relationship between obesity and myopia.Additionally,for the 2023 dataset,characterized by the largest sample size and most complete data,a mediation analysis was performed with mean BP as the mediator.Results Annual cross-sectional analysis from 2014 to 2023 revealed a significant positive association between obesity and myopia,with OR and 95％CI of 1.16(1.10-1.22),1.15(1.08-1.21),1.19(1.12-1.26),1.11(1.04-1.18),1.10(1.05-1.15),1.10(1.05-1.16),1.07(1.03-1.12),1.10(1.05-1.15),1.12(1.07-1.17),and 1.11(1.06-1.15),respectively.Similarly,cohort analysis showed a significant positive association between obesity and myopia(HR=1.09,95％CI:1.01-1.19).Mediation analysis indicated that BP accounted for 21.2％of the total effect of obesity on myopia.Conclusion Obesity facilitates the onset of myopia,and weight reduction can help decrease the likelihood of latter.Furthermore,BP mediates the impact of obesity on myopia.Implementing BP-lowering interventions in obese populations may aid in the prevention of myopia.

Objective:To learn about the current situation of organizational management and inter-departmental coordination and provide a basis for optimization the national joint prevention and control strategy of endemic fluorosis and arsenicosis.Methods:The staff engaged in prevention and control of endemic fluorosis and arsenicosis at the provincial, municipal, county, township, and village levels were selected as the investigation subjects. An online questionnaire survey was conducted to collect relevant information on organizational management and departmental coordination. SAS 9.4 software was used for data statistical analysis.Results:A total of 3 107 valid questionnaires were collected, covering 25 provinces, distributed in 6 regions including Northeast China, North China, East China, Central China, Northwest China, and Southwest China. Totally 92.52% (1 088/1 176) of the respondents believed that a leading group for prevention and control of endemic diseases had been established in their localities, there were statistically significant differences among different regions (χ 2 = 17.18, P = 0.004). However, the highest proportion of those who believed that no leading group had been established was in the Southwest China (14.09%, 21/149). Totally 83.97% (906/1 079) of the respondents believed that the coordination role of the leading group for endemic disease prevention and control was very good or relatively good. The proportion of survey respondents who believed that the local water resources department had a good/relatively good main responsibility in implementation of water improvement measures in drinking-water-borne fluorosis and arsenic poisoning areas, as well as in management of fluoride and arsenic reduction water improvement projects, were 90.51% (2 203/2 434) and 89.37% (2 143/2 398), respectively. The differences between different regions were statistically significant (χ 2 = 70.90, 57.40, P < 0.001). The highest proportion of general/poor cases was believed to be in the southwest region [25.14% (46/183), 24.58% (44/179)]. Totally 71.37% (187/262) of the respondents believed that the supply and distribution of low-fluorine brick tea in tea-drinking-borne endemic fluorosis areas were very good or good. Totally 90.55% (1 447/1 598) of the respondents believed that local medical insurance departments had included skeletal fluorosis patients who were covered by medical insurance. Totally 90.71% (1 474/1 625) of the respondents believed that social assistance departments had included eligible patients with skeletal fluorosis in the scope of social assistance. There were significant differences in the inclusion rate among different regions (χ 2 = 50.45, 46.22, P < 0.001). North China [18.99% (30/158), 21.43% (33/154)] and Southwest China [18.64% (33/177), 15.22% (28/184)] were the two regions with the highest percentage of respondents who believed that the above two were not included. Totally 83.19% (1 425/1 713) of the respondents believed that the local designated hospital for treatment of skeletal fluorosis had been established, there were statistically significant differences among different regions (χ 2 = 31.54, P < 0.001). North China (26.40%, 47/178) and Northwest China (24.56%, 42/171) had the highest proportion of those who believed that there were no designated treatment hospitals for skeletal fluorosis. Totally 83.58% (1 502/1 797) of the respondents believed that the utilization of medical insurance and other policy assistance was very good or good by skeletal fluorosis patients. In Northeast China (30.34%, 27/89), North China (28.41%, 50/176), Southwest China (24.00%, 48/200), and Northwest China (21.43%, 39/182), the proportion of those who believed that the utilization was average and poor were significantly lower than those in East China (11.57%, 96/180) and Central China (10.94%, 35/320, Pcorrect < 0.05). Totally 92.96% (2 747/2 955) of the respondents believed that the cooperation degree of education departments in school monitoring and health education was very good or good, and there were significant differences between different regions (χ 2 = 26.11, P < 0.001), and the highest proportion of respondents who believed that the degree of cooperation was average and poor was in Southwest China (12.63%, 37/293). Conclusions:Except for the East China and Central China, there are different degrees of problems in the organization management and/or departmental coordination and cooperation between departments of endemic fluorosis and arsenicosis prevention and control, especially in the Southwest region. All regions should raise awareness of risk prevention and control, strengthen joint prevention and control, and integrate medical and prevention mechanisms, and consolidate and improve the achievements of endemic fluorosis and arsenicosis prevention and control.

Objective:To investigate epithelial cell heterogeneity in ovarian cancer(OC)and its impact on the tumor microenvironment(TME)by constructing OC epithelial subtypes using publicly available single-cell RNA sequencing(scRNA-seq)data and bioinformatics approaches.Methods:Eight cell types and gene expression matrices were identified from the GSE118828 OC scRNA-seq dataset,and a deconvolution approach was used to estimate cellular composition in bulk RNA sequencing(bulk RNA-seq)data.OC samples were stratified into high-epithelial(C1)and low-epithelial(C2)subtypes based on epithelial cell abundance.Differences in overall survival(OS)and immune microenvironment features between patients with C1 and C2 subtypes were analyzed.Differentially expressed genes(DEGs)between C1 and C2 subtypes,as well as between early-stage(stageⅠ～Ⅱ)and late-stage(stageⅢ-Ⅳ)OC groups were identified to screen epithelial-related genes associated with OC progression,and their influence on the TME was analyzed.Protein expression of the epithelial-related genes in OC progression was validated using data from the Human Protein Atlas(HPA)database.Results:Patients with the C1 subtype exhibited significantly better OS compared to those with C2 subtype(P<0.05).The C1 and C2 subtypes demonstrated distinct immune microenvironment infiltration profiles.Infiltration levels of T helper(Th)cells,M1 macrophages,and activated dendritic cells(DCs)were significantly higher in the C1 subtype(all P<0.05).In late-stage OC patients with C1 subtype,the expression of HAS1,DAPL1,and ADH1B was significantly upregulated(all P<0.05).Furthermore,DAPL1 expression was positively correlated with the degree of macrophage and regulatory T cells(Treg)infiltration in OC(P<0.05).HPA database analysis confirmed that DAPL1 protein expression was significantly upregulated in OC tissues.Conclusion:The high-epithelial subtype of ovarian cancer may exert anti-tumor effects through extensive infiltration of T helper(Th)cells,M1 macrophages,and activated dendritic cells within the tumor microenvironment.Conversely,upregulation of the epithelial-related gene DAPL1 may induce the infiltration of macrophages and Treg,thereby promoting OC progression.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.