The aryl hydrocarbon receptor (AhR) plays a crucial role in regulating many physiological processes. Activating the AhR-CYP1A1 axis has emerged as a novel therapeutic strategy against various inflammatory diseases. Here, a practical in situ cell-based fluorometric assay was constructed to screen AhR-CYP1A1 axis modulators, via functional sensing of CYP1A1 activities in live cells. Firstly, a cell-permeable, isoform-specific enzyme-activable fluorogenic substrate for CYP1A1 was rationally constructed for in-situ visualizing the dynamic changes of CYP1A1 function in living systems, which was subsequently used for discovering the efficacious modulators of the AhR-CYP1A1 axis. Following screening of a compound library, LAC-7 was identified as an efficacious activator of the AhR-CYP1A1 axis, which dose-dependently up-regulated the expression levels of both CYP1A1 and AhR in multiple cell lines. LAC-7 also suppressed macrophage M1 polarization and reduced the levels of inflammatory factors in LPS-induced bone marrow-derived macrophages. Animal tests showed that LAC-7 could significantly mitigate DSS-induced ulcerative colitis and LPS-induced acute lung injury in mice, and markedly reduced the levels of multiple inflammatory factors. Collectively, an optimized fluorometric cell-based assay was devised for in situ functional imaging of CYP1A1 activities in living systems, which strongly facilitated the discovery of efficacious modulators of the AhR-CYP1A1 axis as novel anti-inflammatory agents.

Objective To investigate the characteristics of thickness changes in peripapillary retinal nerve fiber layer(pRNFL)and identify related risk factors in patients with Moyamoya disease(MMD).Methods A retrospective study was conducted on 150 MMD patients(150 eyes)aged 6-65 years admitted to the Neurosurgery Department of the Fifth Medical Center,Chinese PLA General Hospital from May 2016 to December 2023(observation group),and 150 age-matched healthy volunteers(150 eyes)from the hospital's ophthalmology outpatient department(control group).Both groups were subdivided into pediatric(≤18 years),young adult(18-40 years),and middle-aged(40-65 years)subgroups.The pRNFL thickness in four quadrants was measured by optical coherence tomography(OCT):superior(pRNFL-Sup),inferior(pRNFL-Inf),nasal(pRNFL-Nas),temporal(pRNFL-Tmp),and average thickness(pRNFL-Avg).General clinical data and pRNFL thickness were compared between two groups.Univariate and multivariate logistic regression analyses were performed to identify risk factors for pRNFL thinning in MMD patients.The cohort was randomly divided into training(n=210)and validation(n=90)sets at a 7:3 ratio.A predictive model for pRNFL thinning in MMD patients was constructed based on logistic regression results.Model performance was evaluated using the area under the receiver operating characteristic curve(AUC),and clinical utility was assessed via decision curve analysis.Results Compared with control group,MMD patients exhibited significantly reduced pRNFL-Avg,pRNFL-Sup,pRNFL-Tmp,and pRNFL-Inf thickness(P<0.05 or P<0.001),while pRNFL-Nas showed no significant difference(P>0.05).In the pediatric subgroup,pRNFL-Avg and pRNFL-Inf were thinner(P<0.05).In the young adult subgroup,pRNFL-Avg and pRNFL-Sup were reduced(P<0.001 or P<0.05).In the middle-aged subgroup,pRNFL-Avg,pRNFL-Sup,pRNFL-Inf,and pRNFL-Tmp were all thinner(P<0.05 or P<0.001).Multivariate logistic regression identified visual field defects(OR=15.28,95%CI 2.95-79.10),disease duration(OR=1.11,95%CI 1.05-1.18),and the number of involved cerebral vessels(OR=1.49,95%CI 1.01-2.22)as independent risk factors for pRNFL thinning.The predictive model achieved AUC of 0.94(95%CI 0.91-0.97)and 0.95(95%CI 0.91-0.99)in the training and validation sets,respectively.Decision curve analysis confirmed the model's favorable clinical net benefit.Conclusion Thinning of pRNFL was observed in Moyamoya disease patients with visual field defects,disease duration,and cerebral vascular involvement identified as independent risk factors for pRNFL atrophy.

Objective To investigate the differential expression genes(DEGs)related to angiogenesis in rosacea(RA)by utilizing bioinformatics analysis in order to screen the key genes and verify their mRNA expression levels.Methods The gene microarray dataset GSE65914 was retrieved from the Gene Expression Omnibus(GEO)repository.Analyzed by R programming,the dataset was refined to identify DEGs related to RA,and then cross-referenced with angiogenesis-related genes from the GeneCards database to get a subset specific to RA angiogenesis.The process of identifying key genes was augmented by employing protein-protein interaction(PPI)network analysis and Cytoscape-based computational algorithms.The mRNA expression levels of the aforementioned pivotal genes were detected by real-time fluorescent quantitative reverse transcription PCR(RT-qPCR).Results A total of 947 RA-associated DEGs were identified from GEO dataset,and then 202 genes related to RA angiogenesis were further delineated.PPI network analysis and Cytoscape algorithm finally identified 3 key genes,that is,CXCL8,IL-1B,and STAT1.The results of RT-qPCR showed that the mRNA expression levels of MIP-2,GCP-2,IL-1B and STAT1 in RA lesions were significantly higher than those in normal controls(P<0.05).Conclusion With aid of bioinformatics analysis,our study has screened and validated key genes associated with angiogenesis in RA,namely CXCL8,IL-1B,and STAT1,which providing a theoretical basis for elucidating the potential mechanisms underlying RA-induced angiogenesis and developing targeted therapeutic strategies.

This research investigated the impact of Ganoderma lucidum polysaccharides(GLP) on hepatoblastoma HepG2 and Huh6 cell models, as well as KM mouse model with in situ transplanted tumors, so as to provide a theoretical basis for the clinical application of GLP. Cell viability was assessed through the CCK-8 assay, whereas cell proliferation was evaluated by using the BeyoClick~(TM)EdU-488 test. Cell apoptosis was visualized via Hochest 33258 staining, and autophagy was detected through Mrfp-GFP-LC3 dual fluorescence staining. An in situ tumor transplantation model was created by using HepG2 cells in mice, and mice were treated with normal saline and GLP of 100, 200, and 300 mg·kg~(-1) for tumor count calculation and size assessment. Hematoxylin-eosin(HE) staining was used to observe pathological changes in tumor tissue and vital organs(liver, kidney, lung, spleen, and heart). Western blot analysis was conducted to measure the protein expressions of tumor protein P53(P53), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-caspase-3, Beclin-1, autophagy related protein-5(Atg-5), microtubule-associated protein-light chain-3Ⅰ(LC3Ⅰ)/LC3Ⅱ, autophagy adapter protein 62(P62), protein kinase B(Akt), p-Akt, mammalian target of rapamycin(mTOR), and p-mTOR. The in vitro experiment revealed that compared with the control group, after GLP treatment, tumor cell viability decreased significantly; apoptosis rate increased in a dose-dependent manner, and autophagic flux was inhibited. The in vivo experiments showed that compared with the model group, mice treated with GLP exhibited significantly fewer and smaller tumors. Western blot results showed that compared with the control group or model group, levels of P53, Bax, cleaved-caspase-3, Beclin-1, Atg-5, and LC3-Ⅱ/LC3-Ⅰ were significantly increased after GLP treatment, and the levels of Bcl-2, P62, p-Akt/Akt, and p-mTOR/mTOR were significantly decreased. These outcomes suggest that GLP promotes apoptosis and autophagy in hepatoblastoma cells by regulating the Akt/mTOR pathway.
Animals ; Humans ; Autophagy/drug effects* ; Reishi/chemistry* ; Mice ; Apoptosis/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Liver Neoplasms/genetics* ; Hepatoblastoma/genetics* ; Polysaccharides/pharmacology* ; Cell Line, Tumor ; Signal Transduction/drug effects* ; Male ; Cell Proliferation/drug effects* ; Hep G2 Cells

The efficacy mechanism of the alcoholic extract of Gnaphalium affine was investigated by observing its influence on oxidative stress and intestinal flora in rats modeled for chronic obstructive pulmonary disease(COPD). UPLC-MS was used to evaluate the quality of the alcoholic extract of G. affine, and 72 rats were randomly divided into six groups, with COPD models established in five groups by cigarette smoke combined with airway drip lipopolysaccharide, and the rats were given the positive drug of Danlong Oral Solution, as well as low-, medium-, and high-doses alcoholic extract of G. affine, respectively. After two weeks of continuous gastric gavage, the body weights and general morphology observations were performed; HE staining and Masson staining were used to verify the effects of the alcoholic extract of G. affine on alveolar inflammation and collagen deposition area in COPD rats; the oxidative stress indexes CAT and GSH in serum and SOD and MDA in lung tissue of the rats were measured, and the mRNA expression of HO-1, Nrf2, and NQO1 were determined by qRT-PCR. The protein expressions of HO-1, Nrf2, and NQO1 were determined by the Western blot method, and the mechanism by which the alcoholic extract of G. affine affected oxidative stress in COPD rats was explored. Finally, the influence of G. affine on the changes in intestinal flora caused by COPD was studied by 16S rRNA sequencing. The results showed that a total of 121 chemical components were identified by UPLC-MS, including 70 positive and 51 negative ion modes. In animal experiments, it was found that the alcoholic extracts of G. affine were able to reduce the percentage of collagen deposition, affect the oxidative stress indexes such as CAT, GSH, SOD, MDA, as well as the mRNA and protein expression of Nrf2, HO-1, and NQO1. The 16S rRNA sequencing results showed an increase in the level of Lactobacillales and a decrease in the level of Desulfovibrio and Desulfovibrionales, suggesting that the alcoholic extracts of G. affine could reverse the changes in intestinal flora caused by COPD. In conclusion, the alcoholic extracts of G. affine may exert anti-COPD effects by affecting the oxidative stress pathway and modulating the changes in intestinal flora.
Animals ; Oxidative Stress/drug effects* ; Pulmonary Disease, Chronic Obstructive/genetics* ; Rats ; Male ; Gastrointestinal Microbiome/drug effects* ; Rats, Sprague-Dawley ; Drugs, Chinese Herbal/administration & dosage* ; NF-E2-Related Factor 2/metabolism* ; Humans ; Lung/metabolism*

OBJECTIVES: To report the development, validation, and findings of the Multi-dimensional Attention Rating Scale (MARS), a self-report tool crafted to evaluate six-dimension attention levels. METHODS: The MARS was developed based on Classical Test Theory (CTT). Totally 202 highly educated healthy adult participants were recruited for reliability and validity tests. Reliability was measured using Cronbach's alpha and test-retest reliability. Structural validity was explored using principal component analysis. Criterion validity was analyzed by correlating MARS scores with the Toronto Hospital Alertness Test (THAT), the Attentional Control Scale (ACS), and the Attention Network Test (ANT). RESULTS: The MARS comprises 12 items spanning six distinct dimensions of attention: focused attention, sustained attention, shifting attention, selective attention, divided attention, and response inhibition.As assessed by six experts, the content validation index (CVI) was 0.95, the Cronbach's alpha for the MARS was 0.78, and the test-retest reliability was 0.81. Four factors were identified (cumulative variance contribution rate 68.79%). The total score of MARS was correlated positively with THAT (r = 0.60, P < 0.01) and ACS (r = 0.78, P < 0.01) and negatively with ANT's reaction time for alerting (r = -0.31, P = 0.049). CONCLUSIONS The MARS can reliably and validly assess six-dimension attention levels in real-world settings and is expected to be a new tool for assessing multi-dimensional attention impairments in different mental disorders.
Humans ; Adult ; Male ; Attention/physiology* ; Female ; Middle Aged ; Reproducibility of Results ; Young Adult ; Psychometrics

BACKGROUND:Bone morphogenetic protein 2 is vital in embryonic development,bone formation,and regeneration,but its high-dose application is linked to cancer.Bone morphogenetic protein 2 osteogenic peptide L20 reduces adverse effects like cancer and boosts bone tissue regeneration.OBJECTIVE:To graft bone morphogenetic protein 2 active peptide segments onto mesopores and surfaces through a peptide mimicry strategy inspired by oysters,and explore its impact on osteogenic properties of tissue-engineered bone.METHODS:(1)Mesoporous bioactive glass was synthesized using a template method.Bone morphogenetic protein 2 osteogenic peptide L20 was loaded onto mesoporous bioactive glass using a one-step synthesis method to characterize the morphology and in vitro sustained release properties of mesoporous active glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20.(2)Bone marrow mesenchymal stem cells were isolated and extracted from SD rats.After two generations,they were co-cultured with PBS(blank group),mesoporous bioactive glass nanoparticles(control group),and mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20(experimental group).Cell live/dead fluorescence staining and CCK-8 assay were used to detect cytotoxicity and cell proliferation.Scanning electron microscopy was used to observe cell adhesion.After osteogenic induction and differentiation,alkaline phosphatase staining,Alizarin red S staining,and osteogenesis-related gene expression were detected.(3)Fifteen SD rats were selected to establish bilateral femoral condyle defect models and divided into three groups using a random number table method:the blank group(n=5)was not implanted with any material;the control group(n=5)was implanted with mesoporous bioactive glass nanoparticles,and the experimental group(n=5)was implanted with mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20.Eight weeks after surgery,femoral Micro-CT scanning and tissue morphology observation were performed.RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20 were spherical and monodisperse particles.Transmission electron microscopy showed their porous structure with an average particle size of(268.10±0.58)nm,which could release L20 in vitro.(2)Mesoporous bioglass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20 were non-cytotoxic and could promote the proliferation and adhesion of bone marrow mesenchymal stem cells.Compared with the blank group and the control group,the alkaline phosphatase activity and extracellular matrix mineralization capacity of the experimental group were increased(P<0.05),and the mRNA expression levels of alkaline phosphatase,Runx2,and osteocalcin were increased(P<0.05).(3)The results of femoral Micro-CT scanning showed that compared with the blank group and the control group,the new bone mass and bone density of the experimental group were increased(P<0.05).The results of hematoxylin-eosin and Masson staining showed that compared with the blank group and the control group,the new bone formation and collagen fibers of the experimental group were increased.(4)These findings indicate that mesoporous bioactive glass loaded with bone morphogenetic protein 2 active peptide L20 exhibits excellent biocompatibility and in vitro and in vivo osteogenic properties,promoting regeneration and repair of SD rat femoral condyle defects.

Objective:To explore the influence of Stat1 on Foxp3 expression and production of Treg.Methods:C57BL/6 mice were used and separated into normal control group and Stat1 specific inhibitor Fludarabine(Flud)treatment group.Ratio of CD4+Foxp3+Treg and expression of Foxp3 in spleen,lymph nodes and peripheral blood of mice in each group were detected by flow cy-tometry.Human Stat1 overexpression plasmid was constructed and transfected into human breast cancer MCF-7 cells,and expression changes of Foxp3 was detected by RT-qPCR and Western blot.Results:Compared with mice in normal control group,proportion of Treg and expression of Foxp3 in lymph nodes and peripheral blood of mice in Flud treatment group were increased,while Stat1 overex-pression resulted in decreased Foxp3 mRNA and protein expression in MCF-7 cells.Conclusion:Stat1 inhibits expression of Foxp3 and production of Tregs.

BACKGROUND:Bone morphogenetic protein 2 is vital in embryonic development,bone formation,and regeneration,but its high-dose application is linked to cancer.Bone morphogenetic protein 2 osteogenic peptide L20 reduces adverse effects like cancer and boosts bone tissue regeneration.OBJECTIVE:To graft bone morphogenetic protein 2 active peptide segments onto mesopores and surfaces through a peptide mimicry strategy inspired by oysters,and explore its impact on osteogenic properties of tissue-engineered bone.METHODS:(1)Mesoporous bioactive glass was synthesized using a template method.Bone morphogenetic protein 2 osteogenic peptide L20 was loaded onto mesoporous bioactive glass using a one-step synthesis method to characterize the morphology and in vitro sustained release properties of mesoporous active glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20.(2)Bone marrow mesenchymal stem cells were isolated and extracted from SD rats.After two generations,they were co-cultured with PBS(blank group),mesoporous bioactive glass nanoparticles(control group),and mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20(experimental group).Cell live/dead fluorescence staining and CCK-8 assay were used to detect cytotoxicity and cell proliferation.Scanning electron microscopy was used to observe cell adhesion.After osteogenic induction and differentiation,alkaline phosphatase staining,Alizarin red S staining,and osteogenesis-related gene expression were detected.(3)Fifteen SD rats were selected to establish bilateral femoral condyle defect models and divided into three groups using a random number table method:the blank group(n=5)was not implanted with any material;the control group(n=5)was implanted with mesoporous bioactive glass nanoparticles,and the experimental group(n=5)was implanted with mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20.Eight weeks after surgery,femoral Micro-CT scanning and tissue morphology observation were performed.RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20 were spherical and monodisperse particles.Transmission electron microscopy showed their porous structure with an average particle size of(268.10±0.58)nm,which could release L20 in vitro.(2)Mesoporous bioglass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20 were non-cytotoxic and could promote the proliferation and adhesion of bone marrow mesenchymal stem cells.Compared with the blank group and the control group,the alkaline phosphatase activity and extracellular matrix mineralization capacity of the experimental group were increased(P<0.05),and the mRNA expression levels of alkaline phosphatase,Runx2,and osteocalcin were increased(P<0.05).(3)The results of femoral Micro-CT scanning showed that compared with the blank group and the control group,the new bone mass and bone density of the experimental group were increased(P<0.05).The results of hematoxylin-eosin and Masson staining showed that compared with the blank group and the control group,the new bone formation and collagen fibers of the experimental group were increased.(4)These findings indicate that mesoporous bioactive glass loaded with bone morphogenetic protein 2 active peptide L20 exhibits excellent biocompatibility and in vitro and in vivo osteogenic properties,promoting regeneration and repair of SD rat femoral condyle defects.

Objective:To explore the influence of Stat1 on Foxp3 expression and production of Treg.Methods:C57BL/6 mice were used and separated into normal control group and Stat1 specific inhibitor Fludarabine(Flud)treatment group.Ratio of CD4+Foxp3+Treg and expression of Foxp3 in spleen,lymph nodes and peripheral blood of mice in each group were detected by flow cy-tometry.Human Stat1 overexpression plasmid was constructed and transfected into human breast cancer MCF-7 cells,and expression changes of Foxp3 was detected by RT-qPCR and Western blot.Results:Compared with mice in normal control group,proportion of Treg and expression of Foxp3 in lymph nodes and peripheral blood of mice in Flud treatment group were increased,while Stat1 overex-pression resulted in decreased Foxp3 mRNA and protein expression in MCF-7 cells.Conclusion:Stat1 inhibits expression of Foxp3 and production of Tregs.