Utilizing network pharmacology, molecular docking, and cellular experimental validation, this study delved into the therapeutic efficacy and underlying mechanisms of matrine in combating senescence. Databases were utilized to predict targets related to the anti-senescence effects of matrine, resulting in the identification of 81 intersecting targets for matrine in the treatment of senescence. A protein-protein interaction(PPI) network was constructed, and key targets were screened based on degree values. Gene Ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed on the key targets to elucidate the critical pathways involved in the anti-senescence effects of matrine. Molecular docking was conducted between matrine and key targets. A senescence model was established using human umbilical vein endothelial cells(HUVECs) induced with hydrogen peroxide(H_2O_2). Following treatment with varying concentrations of matrine(0.5, 1, and 2 mmol·L~(-1)), cell viability was assessed by using the CCK-8. SA-β-galactosidase staining was employed to observe the positive rate of senescent cells. Flow cytometry was utilized to measure the apoptosis rate. Real-time quantitative PCR(RT-PCR) was utilized to measure the mRNA expression of apoptosis-related cysteine peptidase 3(CASP3), albumin(ALB), glycogen synthase kinase 3β(GSK3B), CD44 molecule(CD44), and tumor necrosis factor-α(TNF-α). Western blot was performed to detect the protein expression of tumor protein p53(p53), cyclin-dependent kinase inhibitor 1A(p21), cyclin-dependent kinase inhibitor 2A(p16), and retinoblastoma tumor suppressor protein(pRb) in the senescence signaling pathway, p38 protein kinase(p38), c-Jun N-terminal kinase(JNK), and extracellular regulated protein kinases(ERK) in the mitogen-activated protein kinase(MAPK) pathway, and phosphatidylinositol 3-kinase(PI3K) and protein kinase B(Akt) in the PI3K/Akt signaling pathway. The experimental results revealed that matrine significantly increased the viability of HUVECs(P<0.05), decreased the positive rate of senescent cells and the apoptosis rate(P<0.05), and reduced the mRNA expression levels of CASP3, ALB, GSK3B, CD44, and TNF-α(P<0.05). It also inhibited the protein expression of p53, p21, p16 and pRb in the senescence signaling pathway(P<0.05), upregulated the protein expression of p-PI3K/PI3K and p-Akt/Akt(P<0.05), and downregulated the protein expression of p-p38/p38, p-JNK/JNK, and p-ERK/ERK(P<0.05). Collectively, these findings suggest that matrine exerts an inhibitory effect on HUVECs senescence, and its mechanism involves the modulation of the senescence signaling pathway, MAPK pathway, and PI3K/Akt signaling pathway to suppress cell apoptosis and inflammation.
Humans ; Matrines ; Quinolizines/chemistry* ; Alkaloids/chemistry* ; Human Umbilical Vein Endothelial Cells/cytology* ; Cellular Senescence/drug effects* ; Network Pharmacology ; Molecular Docking Simulation ; Signal Transduction/drug effects* ; Protein Interaction Maps/drug effects* ; Cell Survival/drug effects* ; Apoptosis/drug effects* ; Drugs, Chinese Herbal/pharmacology*

This study explored the active ingredients for anti-angiogenesis in Wenyujin Rhizoma Concisum. Ten sesquiterpenoids were isolated from Wenyujin Rhizoma Concisum by silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. According to the results of multiple spectroscopic methods and circular dichroism, they were identified as wenyujinlactam A(1),(4S,7S)11-hydroxycurdione(2), 8,9-seco-4β-hydroxy-1α,5βH-7(11)-guaen-8,10-olide(3), curcumadione(4), phaeocaulisin E(5), procurcumadiol(6), zedouronediol(7), epiprocurcumenol(8), gajutsulactone A(9), and(7Z)-1β,4α-dihydroxy-5α,8β(H)-eudesm-7(11)-en-8,12-olide(10). Compounds 1 and 2 were new sesquiterpenoids. Compounds 1, 6, 8, and 10 can inhibit human umbilical vein endothelial cells(HUVEC) proliferation with IC_(50) values of 38.83, 45.19, 32.12, and 37.80 μmol·L~(-1), respectively. Compounds 1 and 10 can inhibit HUVEC migration with IC_(50) values of 29.70 and 36.48 μmol·L~(-1), respectively.
Sesquiterpenes/isolation & purification* ; Humans ; Drugs, Chinese Herbal/isolation & purification* ; Rhizome/chemistry* ; Human Umbilical Vein Endothelial Cells/drug effects* ; Molecular Structure ; Cell Proliferation/drug effects*

OBJECTIVE: To explore the mechanism of antibiotic delivery system targeting bacterial biofilm with linezolid (LZD) based on ε-poly- L-lysine (ε-PLL) and cyclodextrin (CD) (ε-PLL-CD-LZD), aiming to enhance antibiotic bioavailability, effectively penetrate and disrupt biofilm structures, and thereby improve the treatment of bone and joint infections. METHODS: ε-PLL-CD-LZD was synthesized via chemical methods. The grafting rate of CD was characterized using nuclear magnetic resonance. In vitro biocompatibility was evaluated through live/dead cell staining after co-culturing with mouse embryonic osteoblast precursor cells (MC3T3-E1), human umbilical vein endothelial cells, and mouse embryonic fibroblast cells (3T3-L1). The biofilm-enrichment capacity of ε-PLL-CD-LZD was assessed using Staphylococcus aureus biofilms through enrichment studies. Its biofilm eradication efficacy was investigated via minimum inhibitory concentration (MIC) determination, scanning electron microscopy, and live/dead bacterial staining. A bone and joint infection model in male Sprague-Dawley rats was established to validate the antibacterial effects of ε-PLL-CD-LZD. RESULTS: In ε-PLL-CD-LZD, the average grafting rate of CD reached 9.88%. The cell viability exceeded 90% after co-culturing with three types cells. The strong biofilm enrichment capability was observed with a MIC of 2 mg/L. Scanning electron microscopy observations revealed the effective disruption of biofilm structure, indicating potent biofilm eradication capacity. In vivo rat experiments demonstrated that ε-PLL-CD-LZD significantly reduced bacterial load and infection positivity rate at the lesion site ( P<0.05). CONCLUSION The ε-PLL-CD antibiotic delivery system provides a treatment strategy for bone and joint infections with high clinical translational significance. By effectively enhancing antibiotic bioavailability, penetrating, and disrupting biofilms, it demonstrated significant anti-infection effects in animal models.
Biofilms/drug effects* ; Animals ; Anti-Bacterial Agents/pharmacology* ; Polylysine/chemistry* ; Cyclodextrins/administration & dosage* ; Humans ; Linezolid/pharmacology* ; Staphylococcus aureus/physiology* ; Rats, Sprague-Dawley ; Mice ; Rats ; Male ; Drug Delivery Systems ; Staphylococcal Infections/drug therapy* ; Microbial Sensitivity Tests ; Human Umbilical Vein Endothelial Cells ; Osteoblasts/cytology*

Objective To investigate the effects of ROCK-siRNA transfection on endothelial cell senescence and endothelial microparticles (EMPs) induced by angiotensin II (Ang II). Methods Human umbilical vein endothelial cells (HUVECs) were treated with Ang II (1.0 μmo/L) to induce cellular senescence models, followed by transfection with ROCK-siRNA. The cells were divided into four groups: control group, model group, negative transfection control group (Ang II combined with NC-siRNA), and ROCK-siRNA transfection group (Ang II combined with ROCK-siRNA). Cellular senescence was assessed by SA-β-Gal staining. EMP levels in cell supernatants and intracellular reactive oxygen species (ROS) levels were assessed using flow cytometry. The expression levels of silenced information regulator 1(SIRT1) and p53 protein in each group were analyzed by Western blotting. Results Following ROCK-siRNA transfection, the number of senescent cells induced by Ang II was significantly reduced, accompanied by decreased CD31⁺ EMP levels and suppressed intracellular ROS levels. Meanwhile, the expression levels of SIRT1 were up-regulated, while the expression levels of p53 were down-regulated. Conclusion Silencing ROCK expression suppresses EMP release, reduces ROS generation, regulates the expression of SIRT1 and p53, and ultimately attenuates Ang II-induced endothelial cell senescence.
Humans ; Angiotensin II/pharmacology* ; Cellular Senescence/genetics* ; Human Umbilical Vein Endothelial Cells/cytology* ; RNA, Small Interfering/metabolism* ; Reactive Oxygen Species/metabolism* ; Sirtuin 1/genetics* ; Transfection ; Tumor Suppressor Protein p53/genetics* ; Cell-Derived Microparticles/drug effects* ; rho-Associated Kinases/metabolism* ; Endothelial Cells/metabolism* ; Cells, Cultured

OBJECTIVES: To investigate the role and mechanism of fibroblast growth factor (FGF) 19 in inflammation-induced injury of vascular endothelial cells caused by high glucose (HG). METHODS: Human umbilical vein endothelial cells (HUVECs) were randomly divided into four groups: control, HG, FGF19, and HG+FGF19 (n=3 each). The effect of different concentrations of glucose and/or FGF19 on HUVEC viability was assessed using the CCK8 assay. Flow cytometry was utilized to examine the impact of FGF19 on HUVEC apoptosis. Levels of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), total superoxide dismutase (T-SOD), and malondialdehyde (MDA) were measured by ELISA. Real-time quantitative PCR and Western blotting were used to determine the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), nuclear factor erythroid 2 related factor 2 (Nrf2), and heme oxygenase-1 (HO-1). Cells were further divided into control, siRNA-Nrf2 (siNrf2), HG, HG+FGF19, HG+FGF19+negative control, and HG+FGF19+siNrf2 groups (n=3 each) to observe the effect of FGF19 on oxidative stress injury in HUVECs induced by high glucose after silencing the Nrf2 gene. RESULTS: Compared to the control group, the HG group exhibited increased apoptosis rate, increased IL-6, iNOS and MDA levels, and increased VEGF mRNA and protein expression, along with decreased T-SOD activity and decreased mRNA and protein expression of Nrf2 and HO-1 (P<0.05). Compared to the HG group, the HG+FGF19 group showed reduced apoptosis rate, decreased IL-6, iNOS and MDA levels, and decreased VEGF mRNA and protein expression, with increased T-SOD activity and increased Nrf2 and HO-1 mRNA and protein expression (P<0.05). Compared to the HG+FGF19+negative control group, the HG+FGF19+siNrf2 group had decreased T-SOD activity and increased MDA levels (P<0.05). CONCLUSIONS FGF19 can alleviate inflammation-induced injury in vascular endothelial cells caused by HG, potentially through the Nrf2/HO-1 signaling pathway.
Humans ; NF-E2-Related Factor 2/genetics* ; Signal Transduction ; Human Umbilical Vein Endothelial Cells/drug effects* ; Fibroblast Growth Factors/pharmacology* ; Heme Oxygenase-1/physiology* ; Apoptosis/drug effects* ; Glucose ; Inflammation ; Interleukin-6/analysis* ; Vascular Endothelial Growth Factor A/genetics* ; Nitric Oxide Synthase Type II/analysis* ; Cells, Cultured

OBJECTIVES: To investigate the role and mechanism of copper overload-mediated endoplasmic reticulum stress (ERS) in vascular endothelial injury in Kawasaki disease (KD). METHODS: Four-week-old male C57BL/6 mice were randomly divided into four groups: control, KD, KD plus copper chelator tetrathiomolybdate (TTM), and KD plus ERS inhibitor AMG PERK 44 (AMG) (n=20 per group). A KD mouse model was established using Candida albicans extract. Human umbilical vein endothelial cells (HUVECs) were divided into control (intervention with healthy children's serum), KD (intervention with KD patients' serum), and KD+TTM (intervention with KD patients' serum plus 20 µmol/L TTM). Copper deposition in mouse heart tissue was assessed using rubeanic acid staining. Vascular pathological changes were observed using hematoxylin-eosin staining and measurement of abdominal aortic diameter and area. ERS activation was detected by transmission electron microscopy and immunofluorescence. HUVEC viability, apoptosis, and functional changes were evaluated using CCK8, flow cytometry, cell scratch assay, and angiogenesis experiments. ERS marker protein expression levels were measured by Western blot. RESULTS: Compared to the KD group, the KD+TTM and KD+AMG groups showed reduced copper deposition in the vascular wall, decreased swelling of coronary endothelial cells and endoplasmic reticulum, reduced inflammatory cell infiltration, and less abdominal aortic lesion expansion. The abdominal aortic diameter and area, and the fluorescence intensity of ERS marker proteins (GRP78 and CHOP) were significantly lower (P<0.05). Compared to the KD group, the KD+TTM group exhibited increased cell viability, tube number, and scratch healing rate, along with decreased apoptosis rate and expression of ERS marker proteins (GRP78, CHOP, ATF6, and p-PERK) (P<0.05). CONCLUSIONS Copper overload aggravates vascular endothelial injury in KD by activating the ERS pathway. TTM can exert protective effects on the endothelium by regulating copper metabolism and inhibiting the ERS pathway.
Endoplasmic Reticulum Stress ; Copper/toxicity* ; Male ; Mucocutaneous Lymph Node Syndrome/metabolism* ; Animals ; Humans ; Endoplasmic Reticulum Chaperone BiP ; Mice, Inbred C57BL ; Mice ; Human Umbilical Vein Endothelial Cells ; Apoptosis ; Endothelium, Vascular/injuries*

OBJECTIVE: To explore and verify the effect and potential mechanism of Brucea javanica Seed Oil Emulsion Injection (YDZI) and Shengmai Injection (SMI) on peripheral microcirculation dysfunction in treatment of gastric cancer (GC). METHODS: The potential mechanisms of YDZI and SMI were explored through network pharmacology and verified by cellular and clinical experiments. Human microvascular endothelial cells (HMECs) were cultured for quantitative real-time polymerase chain reaction, Western blot analysis, and human umbilical vein endothelial cells (HUVECs) were cultured for tube formation assay. Twenty healthy volunteers and 97 patients with GC were enrolled. Patients were divided into surgical resection, surgical resection with chemotherapy, and surgical resection with chemotherapy combining YDZI and SMI groups. Forearm skin blood perfusion was measured and recorded by laser speckle contrast imaging coupled with post-occlusive reactive hyperemia. Cutaneous vascular conductance and microvascular reactivity parameters were calculated and compared across the groups. RESULTS: After network pharmacology analysis, 4 ingredients, 82 active compounds, and 92 related genes in YDZI and SMI were screened out. β-Sitosterol, an active ingredient and intersection compound of YDZI and SMI, upregulated the expression of vascular endothelial growth factor A (VEGFA) and prostaglandin-endoperoxide synthase 2 (PTGS2, P<0.01), downregulated the expression of caspase 9 (CASP9) and estrogen receptor 1 (ESR1, P<0.01) in HMECs under oxaliplatin stimulation, and promoted tube formation through VEGFA. Chemotherapy significantly impaired the microvascular reactivity in GC patients, whereas YDZI and SMI ameliorated this injury (P<0.05 or P<0.01). CONCLUSIONS YDZI and SMI ameliorated peripheral microvascular reactivity in GC patients. β-Sitosterol may improve peripheral microcirculation by regulating VEGFA, PTGS2, ESR1, and CASP9.
Humans ; Microcirculation/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Stomach Neoplasms/physiopathology* ; Emulsions ; Male ; Plant Oils/administration & dosage* ; Brucea/chemistry* ; Middle Aged ; Female ; Drug Combinations ; Human Umbilical Vein Endothelial Cells/metabolism* ; Seeds/chemistry* ; Injections ; Vascular Endothelial Growth Factor A/metabolism* ; Aged ; Network Pharmacology

OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

Bone repair remains an important target in tissue engineering, making the development of bioactive scaffolds for effective bone defect repair a critical objective. In this study, β-tricalcium phosphate (β-TCP) scaffolds incorporated with processed pyritum decoction (PPD) were fabricated using three-dimensional (3D) printing-assisted freeze-casting. The produced composite scaffolds were evaluated for their mechanical strength, physicochemical properties, biocompatibility, in vitro pro-angiogenic activity, and in vivo efficacy in repairing rabbit femoral defects. They not only demonstrated excellent physicochemical properties, enhanced mechanical strength, and good biosafety but also significantly promoted the proliferation, migration, and aggregation of pro-angiogenic human umbilical vein endothelial cells (HUVECs). In vivo studies revealed that all scaffold groups facilitated osteogenesis at the bone defect site, with the β-TCP scaffolds loaded with PPD markedly enhancing the expression of neurogenic locus Notch homolog protein 1 (Notch1), vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and osteopontin (OPN). Overall, the scaffolds developed in this study exhibited strong angiogenic and osteogenic capabilities both in vitro and in vivo. The incorporation of PPD notably promoted the angiogenic-osteogenic coupling, thereby accelerating bone repair, which suggests that PPD is a promising material for bone repair and that the PPD/β-TCP scaffolds hold great potential as a bone graft alternative.
Calcium Phosphates/chemistry* ; Animals ; Bone Regeneration ; Rabbits ; Tissue Scaffolds ; Printing, Three-Dimensional ; Humans ; Human Umbilical Vein Endothelial Cells ; Neovascularization, Physiologic ; Osteogenesis ; Tissue Engineering/methods* ; Biomimetic Materials ; Cell Proliferation ; Angiogenesis

OBJECTIVES: To investigate the antioxidative mechanism of snake venom-derived protein C activator (PCA) in mitigating vascular endothelial cell injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 1.0 g/L D-glucose and exposed to hypoxia (1% O₂) for 6 h followed by reoxygenation for 2 h to establish a cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The cell model was treated with 2 μg/mL PCA alone or in combination with 2-ME2 (a HIF-1α inhibitor) or DMOG (a HIF-1α stabilizer), and intracellular production of reactive oxygen species (ROS) and protein expression levels of HIF-1α, BNIP3, and Beclin-1 were detected using DCFH-DA fluorescence probe, flow cytometry, and Western blotting. The OGD/R cell model was transfected with a BNIP3-specific siRNA or a scrambled control sequence prior to PCA treatment, and the changes in protein expressions of HIF-1α, BNIP3 and Beclin-1 and intracellular ROS production were examined. RESULTS: In the OGD/R cell model, PCA treatment significantly upregulated HIF-1α, BNIP3 and Beclin-1 expressions and reduced ROS production. The effects of PCA were obviously attenuated by co-treatment with 2-ME2 but augmented by treatment with DMOG (a HIF-1α stabilizer). In the cell model with BNIP3 knockdown, PCA treatment increased BNIP3 expression and decreased ROS production without causing significant changes in HIF-1α expression. Compared with HUVECs with PCA treatment only, the cells with BNIP3 knockdown prior to PCA treatment showed significantly lower Beclin-1 expression and higher ROS levels. CONCLUSIONS Snake venom PCA alleviates OGD/R-induced endothelial cell injury by upregulating HIF-1α/BNIP3 signaling to suppress ROS generation, suggesting its potential as a therapeutic agent against oxidative stress in vascular pathologies.
Humans ; Reactive Oxygen Species/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Human Umbilical Vein Endothelial Cells/drug effects* ; Membrane Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Up-Regulation ; Cell Hypoxia ; Cells, Cultured ; Snake Venoms/chemistry* ; Beclin-1