Objective To investigate the relationship between triglyceride glucose body mass index(TyG-BMI),high density lipoprotein cholesterol to Apolipoprotein A ratio(HAR),glycemic risk index(GRI)and diabetic retinopathy(DR).Methods A total of 159 patients with type 2 diabetes mellitus(T2DM)complicated with DR(the DR group)were divided into the non-proliferative retinopathy group(NPDR group,66 cases)and the proliferative retinopathy group(PDR group,93 cases)according to DR international clinical grading criteria,and 159 T2DM patients without DR were selected as the control group.Clinical information and baseline laboratory test results were recorded,and TyG-BMI,HAR and GRI were calculated.Multivariate Logistic regression analysis was conducted to analyze the related factors of the incidence of DR,and receiver operating characteristic(ROC)curve was used to analyze the diagnostic value of TyG-BMI,HAR and GRI for DR.Results The duration of T2DM,the proportion of hypertension,diabetic nephropathy and diabetic foot,levels of HbA1c,homeostasis model insulin resistance index(HOMA-IR),total cholesterol(TC),C-reactive protein(CRP),TyG-BMI,HAR and GRI were higher in the DR group than those in the control group(P<0.05).TyG-BMI,HAR and GRI were higher in the PDR group than those in the NPDR group(P<0.05).Multivariate Logistic regression analysis showed that the longer course of T2DM disease[OR(95%CI):2.781(1.398-5.534)],high TyG-BMI[2.036(1.169-3.546)],high HAR[1.890(1.090-3.280)]and high GRI[1.836(1.065-3.167)]were risk factors for DR(P<0.05).ROC curve analysis results showed that,the area under the curve(AUC)of combined TyG-BMI,HAR and GRI diagnosis of DR was higher than that of single diagnosis[0.940(0.908-0.964),0.864(0.821-0.900),0.796(0.747-0.839)and 0.836(0.790-0.875),all P<0.05].Conclusion The increased TyG-BMI,HAR and GRI in T2DM patients is associated with the onset and severity of DR,and which can be used to assess the risk of DR.

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.

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.

Objective:To compare the dosimetric differences between a 3D-printed non-coplanar multi-channel applicator and traditional single-channel/co-planar multi-channel applicators in postoperative vaginal brachytherapy for early-stage endometrial cancer.Methods:CT scan data of 66 patients with stage I endometrial cancer, encompassing 100 3D brachytherapy CT imaging datasets, admitted to Department of Gynecologic Oncology of the Fourth Hospital of Hebei Medical University from December 2021 to June 2024 were retrospectively analyzed. Based on CT images and delineated structures, offline reconstructions of radiotherapy plans were performed for single-channel, coplanar multi-channel, and 3D-printed non-coplanar multi-channel applicators. These 3 radiotherapy plans were optimized, and the high-risk clinical target volume (HR-CTV) coverage (V 100 ≥90%) and doses to organs at risk (rectum, bladder) were compared. The prescription dose was standardized at 600 cGy, with constraints of rectal D 2 cm3 ≤420 cGy and bladder D 2 cm3 ≤480 cGy. Comparison among multiple groups was conducted by ANOVA. Bonferroni method was used to correct P-values for comparison between two groups. Results:When defined as HR-CTV D 90%≥600 cGy, bladder D 2 cm3 was (398.29±76.13)cGy and rectum D 2 cm3 was (402.10±49.77)cGy of the 3D-printed non-coplanar multi-channel group,which were significantly lower than those in the single-channel group [bladder D 2 cm3 (424.09±131.52) cGy, rectum D 2 cm3 (493.11±115.17) cGy] and coplanar group [bladder D 2 cm3 (461.28±134.84) cGy, rectum D 2 cm3 (508.75±119.02) cGy], respectively. When limiting bladder D 2 cm3≤480 cGy, rectal D 2 cm3 was (446.81±78.53 cGy) of the 3D-printed non-coplanar multi-channel group, which was significantly lower than those in the single-channel group [(589.71±153.91) cGy] and the coplanar group [(545.51±122.00) cGy], respectively. Meanwhile, HR-CTV V 100% (94.53%±3.42%) was higher than (91.19%±7.63%) in the coplanar group. When the rectal D 2 cm3 was ≤ 420 cGy, HR-CTV V 100% was (91.92%±4.04%) of the 3D-printed non-coplanar multi-channel group,which was significantly better than (79.23%±13.95%) in the single-channel group and (85.88%±6.86%) in the coplanar group, respectively. Conclusions:The 3D-printed non-coplanar multi-channel applicator significantly reduces bladder and rectal doses while enhancing target coverage, outperforming traditional single-channel and co-planar multi-channel applicators. This innovation provides an optimized solution for individualized precision radiotherapy.

Chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH) are both chronic progressive respiratory diseases that cannot be completely cured. COPD is characterized by irreversible airflow limitation, chronic airway inflammation, and gradual decline in lung function, whereas PH is characterized by pulmonary vasoconstriction, remodeling, and infiltration of inflammatory cells. These diseases have similar pathological features, such as vascular hyperplasia, arteriolar contraction, and inflammatory infiltration. Despite these well-documented observations, the exact mechanisms underlying the occurrence and development of COPD and PH remain unclear. Evidence that mitochondrial dynamics imbalance is one major factor in the development of COPD and PH. Mitochondrial dynamics is precisely regulated by mitochondrial fusion proteins and fission proteins. When mitochondrial dynamics equilibrium is disrupted, it causes mitochondrial and even cell morphological dysfunction. Mitochondrial dynamics participates in various pathological processes for heart and lung disease. Mitochondrial dynamics may be different in the early and late stages of COPD and PH. In the early stages of the disease, mitochondrial fusion increases, inhibiting fission, and thereby compensatorily increasing adenosine triphosphate (ATP) production. With the development of the disease, mitochondria decompensation causes excessive fission. Mitochondrial dynamics is involved in the development of COPD and PH in a spatiotemporal manner. Based on this understanding, treatment strategies for mitochondrial dynamics abnormalities may be different at different stages of COPD and PH disease. This article will provide new ideas for the potential treatment of related diseases.
Humans ; Mitochondrial Dynamics/physiology* ; Pulmonary Disease, Chronic Obstructive/metabolism* ; Hypertension, Pulmonary/metabolism* ; Mitochondria/metabolism* ; Animals

Andrographolide sulfonate (AS) is a sulfonated derivative of andrographolide extracted from Andrographis paniculata (Burm.f.) Nees, and has been approved for several decades in China. The present study aimed to investigate the novel therapeutic application and possible mechanisms of AS in the treatment of rheumatoid arthritis. Results indicated that administration of AS by injection or gavage significantly reduced the paw swelling, improved body weights, and attenuated pathological changes in joints of rats with adjuvant-induced arthritis. Additionally, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β in the serum and ankle joints were reduced. Bioinformatics analysis, along with the spleen index and measurements of IL-17 and IL-10 levels, suggested a potential relationship between AS and Th17 cells under arthritic conditions. In vitro, AS was shown to block Th17 cell differentiation, as evidenced by the reduced percentages of CD4⁺ IL-17A⁺ T cells and decreased expression levels of RORγt, IL-17A, IL-17F, IL-21, and IL-22, without affecting the cell viability and apoptosis. This effect was attributed to the limited glycolysis, as indicated by metabolomics analysis, reduced glucose uptake, and pH measurements. Further investigation revealed that AS might bind to hexokinase2 (HK2) to down-regulate the protein levels of HK2 but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or pyruvate kinase M2 (PKM2), and overexpression of HK2 reversed the inhibition of AS on Th17 cell differentiation. Furthermore, AS impaired the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signals in vivo and in vitro, which was abolished by the addition of lactate. In conclusion, AS significantly improved adjuvant-induced arthritis (AIA) in rats by inhibiting glycolysis-mediated activation of PI3K/AKT to restrain Th17 cell differentiation.
Animals ; Th17 Cells/immunology* ; Diterpenes/pharmacology* ; Arthritis, Rheumatoid/metabolism* ; Proto-Oncogene Proteins c-akt/immunology* ; Glycolysis/drug effects* ; Cell Differentiation/drug effects* ; Phosphatidylinositol 3-Kinases/genetics* ; Rats ; Male ; Rats, Sprague-Dawley ; Humans ; Andrographis paniculata/chemistry* ; Arthritis, Experimental/drug therapy* ; Interleukin-17/immunology* ; Signal Transduction/drug effects*

OBJECTIVES: Investigating the protective effect of naringenin (NAR) on the osteogenic potential of human periodontal ligament stem cells (hPDLSCs) under oxidative stress and its related mechanisms. METHODS: The oxidative damage model of hPDLSCs was established using hydrogen peroxide (H₂O₂) andthe hPDLSCs were treated with different concentrations of NAR and 0.5 μmol/L forkhead box protein O-1 (FOXO1) inhibitor AS1842856. After that, the cell counting kit-8 (CCK8) was used to determine the optimal concentrations of H₂O₂ and NAR. The alkaline phosphatase (ALP) staining and real time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR) were employed to assess the expression of ALP, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) in hPDLSCs of each group. The enzyme-linked immunosorbent assay (ELISA) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining were utilized to evaluate the expression of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in hPDLSCs. Meanwhile, qRT-PCR and western blot were used to detect the expression levels of FOXO1 and β-catenin, both are pathway related genes and proteins. RESULTS: H₂O₂ exposure led to an increase in oxidative damage in hPDLSCs, characterized by a rise in intracellular ROS levels and increased expression of MDA and LDH (P<0.05). At the same time, the osteogenic differentiation ability of hPDLSCs decreased, as evidenced by lighter ALP staining and reduced expression levels of osteogenic differentiation-related genes ALP, RUNX2 and OCN (P<0.05). Co-treatment with NAR alleviated the oxidative damage in hPDLSCs, enhanced their antioxidant capacity, and restored their osteogenic ability. The FOXO1 inhibitor AS1842856 downregulated the expression of β-catenin (P<0.05) and significantly diminished both the antioxidant effect of NAR and its ability to restore osteogenesis (P<0.05). CONCLUSIONS NAR can enhance the antioxidant capacity of hPDLSCs by activating the FOXO1/β-catenin signaling pathway within hPDLSCs, thereby mitigating oxidative stress damage and alleviating the loss of osteogenic capacity.
Humans ; Oxidative Stress/drug effects* ; Periodontal Ligament/cytology* ; Hydrogen Peroxide ; Forkhead Box Protein O1/metabolism* ; Stem Cells/cytology* ; Flavanones/pharmacology* ; beta Catenin/metabolism* ; Osteogenesis/drug effects* ; Signal Transduction ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Alkaline Phosphatase/metabolism* ; Osteocalcin/metabolism* ; Cells, Cultured ; Cell Differentiation/drug effects*

Objective To investigate biomarkers for sepsis-induced lung injury based on results of proteomics combined with transcriptomics analyses.Methods A total of 70 patients with sepsis and 70 patients with sepsis-induced lung injury were included as research objects.These patients were di-vided into experimental group and validation group.The experimental group included 10 patients with sepsis and 10 patients with sepsis-induced lung injury.Proteomics was used to analyze differentially expressed proteins in plasma,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed.The dataset GSE10474 of sepsis-induced lung injury was downloaded from the Gene Expression Omnibus(GEO),and GEO2R online database was used to analyze differential transcriptomics data for sepsis-induced lung injury.A Venn diagram was used online to analyze common differentially expressed genes related to sepsis-induced lung injury in pro-teomics and transcriptomics.The validation group included 60 patients with sepsis(sepsis group)and 60 patients with sepsis-induced lung injury(sepsis-induced lung injury group).Enzyme-linked immunosorbent assay(ELISA)was used to detect and compare the differences in protein expression level in peripheral blood between the sepsis group and the sepsis-induced lung injury group.Receiv-er operating characteristic(ROC)curve was used to analyze the clinical value of differential protein expression level in distinguishing sepsis and sepsis-induced lung injury.Results Proteomics results confirmed the presence of 239 significantly differentially expressed proteins in the plasma of patients with sepsis and sepsis-induced lung injury.Compared with patients with sepsis,there were 96 sig-nificantly upregulated proteins and 143 significantly downregulated proteins in patients with sepsis-induced lung injury.The results of GO enrichment analysis of differentially expressed proteins in-cluded cytoplasm,microtubule binding,adenosine triphosphate(ATP)binding,defense response to virus,and immune response.The results of KEGG enrichment analysis included metabolic path-ways,interleukin-17(IL-17)signaling pathway,and phosphatidylinositol-3-kinase-protein kinase B(PI3K-Akt)signaling pathway.In the GSE10474 dataset,compared with patients with sepsis,there were 77 significantly upregulated genes and 142 significantly downregulated genes in patients with sepsis-induced lung injury.The Venn diagram results showed that there were 6 common differential-ly expressed genes in proteomics and transcriptomics,namely BTNL8,FCGR2B,TAK1,KCNC1,TREM1,and SEC31A.Compared with the sepsis group,the levels of TAK1 and TREM1 proteins in the peripheral blood of the sepsis-induced lung injury group were significantly increased(P＜0.01).ROC curve showed that the areas under the curve(AUC)for the expression levels of TAK1 and TREM1 proteins in serum to distinguish sepsis and sepsis-induced lung injury were 0.925 and 0.785 respectively;when the cut-off value for TAK1 was 71.28 pg/mL,the sensitivity and specific-ity were 94.45％and 97.89％respectively;when the cut-off value for TREM1 was 58.22 mg/mL,the sensitivity and specificity were 83.43％and 82.19％respectively.Conclusion Proteomics and transcriptomics results confirm that the activation of TAK1,TREM1 and multiple inflammatory signa-ling pathways may play important roles in the progression of sepsis-induced lung injury.

Objective:To observe the effects of moxibustion at Shenshu(BL23)and Zusanli(ST36)on Toll-like receptor 4(TLR4)-myeloid differentiation factor 88(Myd88)signaling pathway-mediated inflammatory factors in the synovial tissue of ankle joints of rats with rheumatoid arthritis(RA),and to explore the molecular and biological mechanisms underlying the anti-inflammatory and analgesic effects.Methods:A total of 24 male Sprague-Dawley(SD)rats were randomly divided into a normal group,a model group,and a moxibustion group,with 8 rats in each group.The RA model was established with exposure to wind,cold,and damp environmental factors,along with Freund's complete adjuvant.After three days of modeling,mild moxibustion was applied to bilateral Shenshu(BL23)and Zusanli(ST36)in the moxibustion group using moxa sticks of 0.9 cm in diameter for 30 min each time,once a day for 14 d.Structural changes in the synovial tissue and cells were then observed using hematoxylin-eosin staining and transmission electron microscopy,while immunohistochemistry analysis was used to detect tumor necrosis factor(TNF)-α,interleukin(IL)-17,IL-1β,and IL-6 levels.Moreover,the protein expression levels of Myd88,TLR4,and transient potential receptor vanilloid type 1(TRPV1)in the synovial tissue were detected using Western blotting,while their mRNA expression levels were detected using reverse transcription-polymerase chain reaction.Finally,the levels of IL-1β,IL-2,IL-6,IL-17A,and TNF-α in rat serum were detected using enzyme-linked immunosorbent assay.Results:Compared to the normal group,the model group exhibited notable pathological synovial tissue damage,along with significantly higher IL-1β,IL-6,and TNF-α levels(P<0.01)and a slightly higher IL-17 content(P>0.05).Furthermore,the Myd88,TLR4,and TRPV1 protein and mRNA expression levels and serum IL-1β,IL-2,IL-6,IL-17A,and TNF-α levels were all significantly higher in the model group than in the normal group(P<0.01).Compared to the model group,the moxibustion group exhibited a lower degree of synovial tissue pathological damage,along with significantly lower IL-1β,IL-6,and TNF-α levels(P<0.05 or P<0.01)and a lower IL-17 content without statistical significance(P>0.05).Moreover,the Myd88,TLR4,and TRPV1 protein and mRNA expression levels,and serum IL-1β,IL-2,IL-6,IL-17A,and TNF-α levels were all significantly lower in the moxibustion group than in the model group(P<0.01 or P<0.05).Conclusion:Mild moxibustion at Shenshu(BL23)and Zusanli(ST36)can effectively inhibit TLR4-MyD88 signaling pathway-mediated inflammatory factor expression in the synovial tissue of ankle joints of RA rats.Furthermore,the effect of moxibustion on synovial tissue inflammation in RA rats may be attributed to TRPV1 channel activation.

Geraniin, a polyphenol derived from the fruit peel of Nephelium lappaceum L., has been shown to possess anti-inflammatory and antioxidant properties in the cardiovascular system. The present study explored whether geraniin could protect against an isoproterenol (ISO)-induced cardiac hypertrophy model. Mice in the ISO group received an intraperitoneal injection of ISO (5 mg/kg) once daily for 9 days, and the administration group were injected with ISO after 5 days of treatment with geraniin or spironolactone. Potential therapeutic effects and related mechanisms analysed by anatomical coefficients, histopathology, blood biochemical indices, reverse transcription-PCR and immunoblotting. Geraniin decreased the cardiac pathologic remodeling and myocardial fibrosis induced by ISO, as evidenced by the modifications to anatomical coefficients, as well as the reduction in collagen I/III á1mRNA and protein expression and cross-sectional area in hypertrophic cardiac tissue. In addition, geraniin treatment reduced ISO-induced increase in the mRNA and protein expression levels of interleukin (IL)-6, IL-1β and tumor necrosis factor-α, whereas ISO-induced IL-10 showed the opposite behaviour in hypertrophic cardiac tissue.Further analysis showed that geraniin partially reversed the ISO-induced increase in malondialdehyde and nitric oxide, and the ISO-induced decrease in glutathione, superoxide dismutase and glutathione. Furthermore, it suppressed the ISO-induced cellular apoptosis of hypertrophic cardiac tissue, as evidenced by the decrease in Bcell lymphoma-2 (Bcl-2)-associated X/caspase-3/caspase-9 expression, increase in Bcl-2 expression, and decrease in TdT-mediated dUTP nick-end labeling-positive cells.These findings suggest that geraniin can attenuate ISO-induced cardiac hypertrophy by inhibiting inflammation, oxidative stress and cellular apoptosis.