原代内皮细胞专用培养基

Astragaloside IV (ASIV) promotes the proliferation of key cells, endothelial progenitor cells (EPCs), during the wound healing process, while exosomes and hydrogels are ideal drug delivery carriers. This study aims to explore the mechanism of action of the “ROS-responsive hydrogel-engineered EPCs-targeted exosomes” composite ASIV delivery system (PF–PEG@ASIV-EXO) in diabetic wound healing. Surface markers of EPCs and PF–PEG@ASIV-EXO were detected separately. The degradation rate of PF–PEG@ASIV-EXO was assessed after coculturing with human dermal fibroblasts (HDF), immortalized human epidermal cells (HaCAT), and human EPCs, and the biocompatibility of EPCs and PF–PEG@ASIV-EXO was evaluated through exosome release and uptake. The effects of PF–PEG@ASIV-EXO on the viability, angiogenesis, ferroptosis, and mitochondria of high-glucose-treated EPCs (HS-EPCs) were investigated. A diabetic wound rat model was established, and the effects of PF–PEG@ASIV-EXO on diabetic wounds were evaluated through HE and Masson staining, as well as levels of VWF, CD31, and ferroptosis in the skin. EPCs were successfully isolated, and PF–PEG@ASIV-EXO was successfully constructed. PF–PEG@ASIV-EXO exhibited a high degradation rate within EPCs, and both EPCs and PF–PEG@ASIV-EXO showed good biocompatibility. PF–PEG@ASIV-EXO promoted the vitality and angiogenesis of EPCs, inhibited ferroptosis, and mitigated mitochondrial damage. Following treatment with PF–PEG@ASIV-EXO, the healing of diabetic rat skin accelerated, accompanied by elevated expression of VWF and CD31, and reduced ferroptosis levels. PF–PEG@ASIV-EXO hydrogel inhibits ferroptosis, promotes angiogenesis, and thereby accelerates the healing of diabetic wounds.

Background Bone fracture healing is a multifaceted process that involves different stages and intercellular interactions. In this study, we aimed to investigate the effect of Taohong Siwu decoction (TSD) on bone fracture healing and the underlying mechanisms. Methods First, a mouse model of femur fracture was constructed, and TSD intervention was administered for durations of 7, 14, and 21 days. Following this, immunofluorescence (IF) was employed to evaluate the expression of CD90 (a marker for mesenchymal stem cells [MSCs]), endomucin (Emcn), and CD31. We also treated MSCs with normal serum and 10% TSD-containing serum to investigate the effects of TSD. Molecular docking was applied to verify the binding of active compounds in TSD to pVon Hippel–Lindau (VHL). Additionally, MSCs were treated with paeoniflorin and 2-methoxyestradiol (2-ME2) to explore the effects of paeoniflorin. Subsequently, mouse aortic endothelial cells were extracted and identified. Furthermore, normally cultured MSCs were cocultured with endothelial cells. MSCs were exposed to control serum, 10% TSD-containing serum, and a combination of 10% TSD-containing serum with 2-ME2. Finally, we administered a combination of 2-ME2 over 21 days to evaluate its effects on the fractured mice. Results TSD significantly influenced H-type angiogenesis during the healing process of fractured mice. Compared to the sham group, the model group exhibited lower levels of Emcn, CD90, hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF), while there was an increase in pVHL expression. After 7, 14, and 21 days of TSD intervention, the levels of Emcn, CD90, HIF-1α, VEGF, and pVHL gradually increased, whereas HIF-1α expression decreased. In vitro experiments revealed that TSD enhanced the proliferation and migration of MSCs while inhibiting the ubiquitination of pVHL/HIF-1α. Moreover, ferulic acid, amygdalin, hydroxysafflor yellow A, and paeoniflorin demonstrated a strong affinity for binding with pVHL. Notably, paeoniflorin promoted the proliferation and migration of MSCs through the pVHL/HIF-1α pathway to promote angiogenesis. Furthermore, TSD was found to enhance endothelial angiogenesis in MSCs. In summary, TSD affects H-type angiogenesis and MSCs homing during the healing process of fractured mice through the HIF-1α axis. Conclusions TSD regulated MSC-mediated H-type angiogenesis to accelerate fracture healing through VHL/HIF-1α ubiquitination.

Objective Dipeptidyl peptidase-4 (DPP4)-targeted therapy is widely employed in the therapy of pulmonary diseases, but the role of its H3K4me1 modification in pulmonary arterial hypertension (PAH) disease remains unknown. This study aims to investigate the function of histone methyltransferase SET domain containing 7 (Set7)-mediated monomethylation of histone 3 lysine 4 (H3K4me1) modification of DPP4 in PAH, with the goal of providing new insights for the broader application of DPP4-targeted therapies. Methods The PAH mouse model was constructed and intervened with overexpression (oe) or knockdown (sh) of DPP4, sh-Set7, oe-NADPH oxidase 4 (NOX4) or sh-Set7 + erastin. Human pulmonary arterial endothelial cells were induced by hypoxia and treated with sh-DPP4, erastin, oe-DPP4, sh-Set7, oe-NOX4, oe-Set7 or oe-Set7 + ferrostatin-1. The enrichment of H3K4me1 level in the DPP4 promoter region was analyzed by ChIP and dual-luciferase assay. Pulmonary vascular remodeling, fibrosis, and endothelial injury were observed by echocardiography, HE, MASSON, and α-SMA staining. Ferroptosis markers and protein expression were measured using biochemical assay kits, RT-qPCR, WB, immunofluorescence, and transmission electron microscopy. Results Silencing DPP4 alleviates pulmonary vascular remodeling, fibrosis, and endothelial injury in PAH mice, reduces cardiac fibrosis and pulmonary inflammation, while improving mitochondrial damage in the lungs and downregulating the level of ferroptosis-related proteins. ChIP assays confirmed increased enrichment of H3K4me1 in the DPP4 promoter region in both hypoxia-induced endothelial cells and lung tissues of PAH mice. Overexpression of Set7 resulted in elevated H3K4me1 enrichment in the DPP4 promoter region and increased NOX4 protein expression. Ferrostatin-1 inhibited the promotion of oe-Set7 in hypoxia-induced endothelial cell injury. Silencing Set7 mitigated hypoxia-induced endothelial cell injury, ferroptosis, and inflammatory responses by downregulating DPP4/NOX4. Erastin reversed the treatment effect of sh-Set7 in PAH mice. Furthermore, Set7 knockdown ameliorated PAH in mice by suppressing DPP4/NOX4-mediated ferroptosis. Conclusion The H3K4me1 modification of DPP4 is upregulated in PAH, a process regulated by Set7. Silencing Set7 alleviates PAH by suppressing ferroptosis through the DPP4/NOX4 signaling pathway, offering a novel gene therapy approach for this disease.

Background Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer (LC) cases, making it the primary cause of cancer-related mortality worldwide. T-box transcription factor 5 (TBX5) is an important regulator of embryonic and organ development and plays a key role in cancer development. Here, our objective was to investigate the involvement of TBX5 in ferroptosis within LC cells and the underlying mechanisms. Methods First, TBX5 expression was examined in human LC cells. Next, overexpression of TBX5 and Yes1-associated transcriptional regulator (YAP1) and knockdown of TEA domain 1 (TEAD1) were performed in A549 and NCI-H1703 cells. The proliferation ability of A549 and NCI-H1703 cells, GSH, MDA, ROS, and Fe 2+ levels were measured. Co-immunoprecipitation (Co-IP) was performed to verify whether TBX5 protein could bind YAP1. Then TBX5, YAP1, TEAD1, GPX4, p53, FTH1, SLC7A11 and PTGS2 protein levels were assessed. Finally, we verified the effect of TBX5 on ferroptosis in LC cells in vivo . Results TBX5 expression was down-regulated in LC cells, especially in A549 and NCI-H1703 cells. Overexpression of TBX5 significantly decreased proliferation ability of A549 and NCI-H1703 cells, downregulated GPX4 and GSH levels, and upregulated MDA, ROS, and Fe 2+ levels. Co-IP verified that TBX5 protein could bind YAP1. Moreover, oe-YAP1 promoted proliferation ability of A549 and NCI-H1703 cells transfected with Lv-TBX5, upregulated GPX4 and GSH levels and downregulated MDA, ROS, and Fe 2+ levels. Additionally, oe-YAP1 promoted FTH1 and SLC7A11 levels and inhibited p53 and PTGS2 levels in A549 and NCI-H1703 cells transfected with Lv-TBX5. However, transfection with si-TEAD1 further reversed these effects. In vivo experiments further validated that TBX5 promoted ferroptosis in LC cells. Conclusions TBX5 inhibited the activation of YAP1-TEAD1 pathway to promote ferroptosis in LC cells.

Background Heart failure (HF) is a clinical syndrome with a high risk. Our previous research showed a regulatory relationship between Sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor α (PPARA) and nuclear receptor co-repressor 1 (NCOR1). This study aimed to investigate the regulatory mechanism of SIRT1/PPARA/NCOR1 axis in HF. Methods HF models in vitro were established by doxorubicin (DOX)-induced AC16 and human cardiac microvascular endothelial cell (HCMEC) lines. The contents of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), interleukin-1β (IL-1β), and IL-18 were detected using enzyme-linked immunosorbent assay. Then, we assessed the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and adenosine triphosphate (ATP). Moreover, the relationship between SIRT1 and PPARA was detected using the co-immunoprecipitation (Co-IP) analysis. The connection between PPARA and NCOR1 was analyzed using chromatin immunoprecipitation (ChIP). Results Overexpression of SIRT1 or PPARA could reduce apoptosis in DOX-induced AC16 and HCMEC cells, the levels of IL-1β, IL-18, ANP, BNP, ROS and MDA, while increasing the levels of SOD and ATP. In addition, overexpression of PPARA could increase the viability of DOX-induced cells and the levels of myosin heavy chain 6 (Myh6) and Myh7. Co-IP showed that SIRT1 interacted with PPARA. Silencing PPARA could reverse the effect of SIRT1 overexpression on DOX-induced AC16 and HCMEC cells. ChIP assay demonstrated that PPARA could bind to the promoter region of NCOR1 . Silencing NCOR1 could reverse the effect of PPARA overexpression on DOX-induced AC16 and HCMEC cells. Conclusions This study revealed that PPARA could mediate SIRT1 to promote NCOR1 expression and thus protect damaged heart cells. The finding provided an important reference for the treatment of HF.

Introduction: The proprotein convertase subtilisin/kexin type 9/lectin-like oxidized low-density lipoprotein receptor-1 (PCSK9/LOX-1) axis plays a crucial role in regulating vascular endothelial cell function, but its specific involvement in type 2 diabetes mellitus (T2DM) remains unclear. This study aims to explore the potential mechanism of the PCSK9/LOX-1 axis in high-glucose (HG)-induced vascular endothelial cell dysfunction. Material and methods: Peripheral blood samples were collected from T2DM patients to analyse the correlation between PCSK9 and blood lipid levels. Human microvascular endothelial cells (HMEC-1) exposed to high glucose concentration were used as a model of diabetic= angiopathy (DA). Levels of PCSK9, reactive oxygen species (ROS), malonodialdehyde (MDA), interleukins (IL): IL-6, IL-1β, superoxide dismutase (SOD), and tumour necrosis factor alpha (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA) and biochemical methods. Additionally, intracellular total cholesterol (TC) and cholesterol ester (CE) levels were detected using enzyme chemistry. Expression of PCSK9 and LOX-1 was assessed through real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. Results: Compared to the normal group, PCSK9 levels were significantly elevated in T2DM patients. Furthermore, PCSK9 levels were found to be positively correlated with body mass index (BMI), waistline, triglyceride (TG), cholesterol, low-density lipoprotein cholesterol (LDL-C), glycated hemoglobin (HbA lc ), and intracardiac fat pad levels in T2DM patients. HG exposure led to reduced activity of HMEC-1 cells, along with increased levels of apoptosis, oxidative stress, and inflammation, all of which were counteracted by si-PCSK9. The inhibitory effects of si-PCSK9 on LOX-1 expression, as well as TC and CE contents in HMEC-1 cells induced by HG, were observed. Moreover, intervention with oe-LOX-1 reversed the effects of si-PCSK9 in HG-induced HMEC-1 cells. Conclusion: Silencing of PCSK9 inhibited HG-induced inflammation, oxidative stress, and lipid metabolic dysfunction in HMEC-1 cells via LOX-1.

This study proposes to investigate the therapeutic efficacy and mechanism of combining tibial transverse transport (TTT) with platelet-rich plasma (PRP) for diabetic foot ulcer (DFU). The diabetic rabbit model was constructed with Streptozotocin, which was intervened with TTT and PRP. PRP injection combined with TTT significantly promoted vascularisation and enhanced CD31, VEGFA, and VEGFR2 expressions compared to traditional TTT. However, the VEGFR2 inhibitor suppressed these phenomena. In the in vitro injury model, PRP reversed the diminished human umbilical vein endothelial cells (HUVECs) function and vascularisation caused by high-glucose damage. Additionally, PRP reduced inflammation and oxidative stress (approximately 47% ROS level) and enhanced VEGFA and VEGFR2 expression in HUVECs. However, the knockdown of VEGFR2 reversed the effect of PRP. In conclusion, TTT combined with intraosseous flap injection of PRP sustained-release microspheres activated the VEGFA/VEGFR2 pathway to promote microcirculatory reconstruction in DFU. These findings may provide new potential therapeutic strategies for DFU.

Ginsenoside Rb1 ameliorates renal fibrosis, yet its effects on myocardial fibrosis (MF) remain unclear. In this study, we aimed to explore the role of ginsenoside Rb1 in chronic heart failure (CHF) and MF. To explore the correlation between endothelial-mesenchymal transition (EndMT) in endothelial cells and IGFBP2 expression in M1 macrophages, M1 macrophages were polarized and co-cultured with myocardial microvascular endothelial cells (MMVECs). IGFBP2 levels in the macrophages and levels of endothelial-specific markers and EndMT-related indexes in MMVECs were measured. Additionally, we treated the macrophages with ginsenoside Rb1. The CHF mice model was established using transverse aortic constriction (TAC) and then treated with ginsenoside Rb1. The effects of Rb1 on cardiac function, MF, and cardiomyocyte hypertrophy in CHF mice were assessed. We observed the successful differentiation of M1 macrophages using in vitro experiments. M1 macrophages co-cultured with MMVECs demonstrated the ability to enhance the EndMT effect in MMVECs, as evidenced by elevated levels of IGFBP2 in the macrophages and a reduction in the viability of MMVECs. This decrease in cell viability was mitigated following the knockdown of IGFBP2. Rb1 treatment significantly suppressed the expression of IGFBP2 and inhibited the occurrence of the EndMT in MMVECs. The in vivo experiment findings showed that ginsenoside Rb1 notably enhanced cardiac function, attenuated cardiomyocyte hypertrophy, and alleviated MF in CHF mice. Furthermore, ginsenoside Rb1 inhibited M1 macrophage polarization, reduced IGFBP2 expression in the myocardium, and suppressed the EndMT effect of MMVECs in mice. Ginsenoside Rb1 alleviated MF in mice with CHF by inhibiting M1 macrophage IGFBP2-mediated EndMT.

This study aimed to investigate the role of mesenchymal homeobox 2 (MEOX2) on breast cancer cell metastasis and its underlying mechanism. Overexpression of MEOX2 in human lymphatic endothelial cell (HLEC) lines was established to assess the adhesion and transendothelial migration of MCF7 and MDA-MB-231 cells to the HLEC cells. After being treated with the oxidative stress inducer H 2 O 2 and the antioxidant N-acetylcysteine (NAC), cell viability, reactive oxygen species (ROS) levels, adhesion, and transendothelial migration of MCF7 and MDA-MB-231 cells to HLEC cells were detected. Tumor volume changes were observed in the xenograft model. The expression of C-X-C chemokine receptor type 4 (CXCR4), C–C chemokine receptor type 7 (CCR7), MEOX2, and G protein signal transduction regulator 5 (RGS5) in tumor tissues and ROS levels were detected. MEOX2 was lowly expressed in breast cancer tissues. Upregulated MEOX2 inhibited the proliferation of lymphatic endothelial cells and the adhesion and transendothelial migration of MCF7 and MDA-MB-231 cells to HLEC cells. After MCF7 and MDA-MB-231 cells were treated with oxidative stress inducer H 2 O 2 , ROS levels increased, and cell viability and MEOX2 expression decreased. After NAC or overexpressed MEOX2 treatment, MEOX2 expression increased, ROS and RGS5 levels, adhesion, and transendothelial migration ability decreased in HLEC cells. Overexpression of MEOX2 resulted in smaller tumor volume, lower ROS levels, and lower CXCR4 and CCR7 expression levels. MEOX2 and RGS5 are pivotal in regulating breast cancer metastasis, offering valuable insights into potential therapeutic strategies for breast cancer metastasis.

5-Methoxytryptophan (5-MTP), a candidate biomarker for chronic kidney disease (CKD), has an undefined role in cerebrovascular pathophysiology. To investigate this, we employed a folic acid (FA)–induced CKD to simulate cerebrovascular complications in vivo. Additionally, in vitro models of cerebral ischemia and cerebrovascular endothelial cell injury were established. 5-MTP was administered to rats and cells, along with nuclear factor-κB (NF-κB) expression. The pathological characteristics of kidney and brain tissue were observed by histological staining. Cell proliferation was assessed using the Cell Counting Kit 8, while tube formation and migration were examined using tube formation and wound healing assays. Cell apoptosis was detected using both TdT-mediated dUTP-biotin nick end labeling and flow cytometry. Levels of renal injury markers, blood biomarkers of cerebrovascular disease, and inflammatory cytokines were measured using biochemical assays. Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expression, respectively. Key findings revealed that FA successfully induced CKD in rats, which subsequently exacerbated cerebrovascular dysfunction. 5-MTP reduced the levels of proteinuria, N-acetyl-beta- d -glucosaminidase, nephrin, endothelin-1, von Willebrand factor, and thrombomodulin; improved the degree of renal fibrosis and structural damage to the brain tissue; and inhibited cell apoptosis in rats. In vitro, 5-MTP promoted cell proliferation, tube formation, migration, and the upregulation of B-cell lymphoma-2 and caspase-3 expression. This treatment also led to an increase in interleukin (IL)-10 levels while suppressing cell apoptosis, Bcl-2-associated X protein (Bax), and cleaved caspase-3 expression. Furthermore, it reduced the IL-6 and tumor necrosis factor-alpha levels. NF-κB overexpression reversed the effects of 5-MTP in vitro and in vivo. Our results demonstrate that 5-MTP ameliorated CKD-induced cerebrovascular injury through the NF-κB pathway, indicating its potential as an innovative and efficacious therapeutic target for CKD-induced cerebrovascular dysfunction.