Ginsenoside Rg_2(GRg2) is a triterpenoid compound found in Panax notoginseng. This study explored its effects and mechanisms on diabetic retinopathy and angiogenesis. The study employed endothelial cell models induced by glucose or vascular endothelial growth factor(VEGF), the chorioallantoic membrane(CAM) model, the oxygen-induced retinopathy(OIR) mouse model, and the db/db mouse model to evaluate the therapeutic effects of GRg2 on diabetic retinopathy and angiogenesis. Transwell assays and endothelial tube formation experiments were conducted to assess cell migration and tube formation, while vascular area measurements were applied to detect angiogenesis. The impact of GRg2 on the retinal structure and function of db/db mice was evaluated through retinal thickness and electroretinogram(ERG) analyses. The study investigated the mechanisms of GRg2 by analyzing the activation of Yes-associated protein(YAP) and Toll-like receptors(TLRs) pathways. The results indicated that GRg2 significantly reduced cell migration numbers and tube formation lengths in vitro. In the CAM model, GRg2 exhibited a dose-dependent decrease in the vascular area ratio. In the OIR model, GRg2 notably decreased the avascular and neovascular areas, ameliorating retinal structural disarray. In the db/db mouse model, GRg2 increased the total retinal thickness and enhanced the amplitudes of the a-wave, b-wave, and oscillatory potentials(OPs) in the ERG, improving retinal structural disarray. Transcriptomic analysis revealed that the TLR signaling pathway was significantly down-regulated following YAP knockdown, with PCR results consistent with the transcriptome sequencing findings. Concurrently, GRg2 downregulated the expression of Toll-like receptor 4(TLR4), TNF receptor-associated factor 6(TRAF6), and nuclear factor-kappaB(NF-κB) proteins in high-glucose-induced endothelial cells. Collectively, GRg2 inhibits cell migration and tube formation and significantly reduces angiogenesis in CAM and OIR models, improving retinal structure and function in db/db mice, with its pharmacological mechanism likely involving the down-regulation of YAP expression.
Animals ; Ginsenosides/pharmacology* ; Diabetic Retinopathy/physiopathology* ; Mice ; YAP-Signaling Proteins ; Humans ; Male ; Signal Transduction/drug effects* ; Cell Movement/drug effects* ; Adaptor Proteins, Signal Transducing/genetics* ; Mice, Inbred C57BL ; Neovascularization, Pathologic/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Panax notoginseng/chemistry* ; Endothelial Cells/metabolism* ; Transcription Factors/genetics* ; Angiogenesis

This article investigated the effect and mechanism of salidroside(SAL) on the expression of adhesion molecules in oxidized low-density lipoprotein(oxLDL)-induced mouse aortic endothelial cell(MAEC). The oxLDL-induced endothelial cell injury model was constructed, and the safe concentration and action time of SAL were screened. The cells were divided into control group, oxLDL group, low and high concentration groups of SAL, and ferrostatin-1(Fer-1) group. The cell viability was detected by CCK-8 assay; lactate dehydrogenase(LDH) leakage was measured by colorimetry; the expression of intercellular adhesion molecule 1(ICAM-1) and recombinant vascular cell adhesion molecule 1(VCAM-1) were detected by immunofluorescence; Fe~(2+),glutathione(GSH),malondialdehyde(MDA),and 4-hydroxynonenal(4-HNE) levels were detected by kit method; reactive oxygen species(ROS) was detected by DCFH-DA probe; the levels of glutathione peroxidase 4(GPX4),silent mating type information regulation 2 homolog 1(SIRT1), and nuclear factor erythroid 2-related factor 2(Nrf2) were determined by using Western blot. The inhibitors of Nrf2 and SIRT1 were used, and endothelial cell were divided into control group, oxLDL group, SAL group, ML385 group(Nrf2 inhibitor), and EX527 group(SIRT1 inhibitor). The ultrastructure of mitochondria was observed by electron microscope; mitochondrial membrane potential(MMP) was detected by flowcytometry; the expressions of SIRT1,Nrf2,solute carrier family 7 member 11(SLC7A11),GPX4,ferroportin 1(FPN1),ferritin heavy chain 1(FTH1),ICAM-1, and VCAM-1 were detected by Western blot. The results showed that similar to Fer-1,low and high concentrations of SAL could improve cell viability, inhibit LDH release and the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cells(P<0.05 or P<0.01). It was related to increase in GSH level, decrease in Fe~(2+),ROS,MDA, and 4-HNE level, and up-regulation of SIRT1,Nrf2, and GPX4 expression to inhibit ferroptosis(P<0.05 or P<0.01). The intervention effect of high concentration SAL was the most significant. ML385 and EX527 could partially offset the protection of SAL on mitochondrial structure and MMP and reverse the ability of SAL to up-regulate the expression of SIRT1,Nrf2,SLC7A11,GPX4,FPN1, and FTH1 and down-regulate the expression of ICAM-1 and VCAM-1(P<0.05 or P<0.01).To sum up, SAL could reduce the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cell, which may relate to activation of SLC7A11/GPX4 antioxidant signaling pathway mediated by SITR1/Nrf2, up-regulation of FPN1 and FTH1 expression, and inhibition of ferroptosis.
Sirtuin 1/genetics* ; Animals ; Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; NF-E2-Related Factor 2/genetics* ; Mice ; Endothelial Cells/cytology* ; Glucosides/pharmacology* ; Phenols/pharmacology* ; Cell Adhesion Molecules/genetics* ; Reactive Oxygen Species/metabolism* ; Intercellular Adhesion Molecule-1/genetics* ; Vascular Cell Adhesion Molecule-1/genetics* ; Cell Survival/drug effects*

This study elucidates the mechanism of Rhodiolae Crenulatae Radix et Rhizoma(RCRR) in protecting brain microvascular endothelial cells from oxygen-glucose deprivation(OGD) injury and reveals the modern pharmacological mechanism of RCRR's traditional use in nourishing Qi and promoting blood circulation to protect endothelial cells. The scratch assay was employed to assess the migratory capacity of endothelial cells. Immunofluorescence and Western blot techniques were employed to assess the protein expression of tight junction proteins zonula occludens-1(ZO-1), occludin, claudin-5, and proteins of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/glycogen synthase kinase-3beta(GSK3β) pathway. The results demonstrated that 63 bioactive components and 125 potential core targets of RCRR were identified from the ETCM, TCMBank, and SwissTargetPrediction databases, as well as from the literature. A total of 1 708 brain microvascular endothelial cell-related targets were identified from the GeneCards and OMIM databases, and 52 targets were obtained by intersecting drug components with cell targets. The protein-protein interaction(PPI) network analysis revealed that AKT1, epidermal growth factor receptor(EGFR), matrix metalloproteinase 9(MMP9), estrogen receptor 1(ESR1), proto-oncogene tyrosine-protein kinase(SRC), peroxisome proliferator-activated receptor gamma(PPARG), GSK3β, and matrix metalloproteinase 2(MMP2) were considered hub genes. The KEGG enrichment analysis identified the PI3K/AKT pathway as the primary signaling pathway. Cell experiments demonstrated that RCRR-containing serum could enhance the migratory capacity of brain microvascular endothelial cells and the expression of tight junction proteins following OGD injury, which may be associated with the downregulation of the PI3K/AKT/GSK3β pathway. This study elucidates the pharmacological mechanism of RCRR in protecting brain microvascular endothelial cells through network pharmacology, characterized by multiple components and targets. These findings were validated through in vitro experiments and provide important ideas and references for further research into the molecular mechanisms of RCRR in protecting brain microvascular endothelial cells.
Endothelial Cells/cytology* ; Glycogen Synthase Kinase 3 beta/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Phosphatidylinositol 3-Kinases/genetics* ; Signal Transduction/drug effects* ; Brain/metabolism* ; Humans ; Animals ; Rhizome/chemistry* ; Microvessels/metabolism* ; Brain Ischemia/drug therapy*

This study aims to explore the mechanism of Buyang Huanwu Decoction(BHD) in promoting angiogenesis after oxygen-glucose deprivation/reoxygenation(OGD/R) of mouse brain microvascular endothelial cell line(brain-derived Endothelial cells.3, bEnd.3) based on the caveolin-1(Cav1)/Yes-associated protein 1(YAP1)/hypoxia-inducible factor-1α(HIF-1α) signaling pathway. Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to analyze the blood components of BHD. The cell counting kit-8(CCK-8) method was used to detect the optimal intervention concentration of drug-containing serum of BHD after OGD/R injury of bEnd.3. The lentiviral transfection method was used to construct a Cav1 silent stable strain, and Western blot and polymerase chain reaction(PCR) methods were used to verify the silencing efficiency. The control bEnd.3 cells were divided into a normal group(sh-NC control group), an OGD/R model + blank serum group(sh-NC OGD/R group), and an OGD/R model + drug-containing serum group(sh-NC BHD group). Cav1 silent cells were divided into an OGD/R model + blank serum group(sh-Cav1 OGD/R group) and an OGD/R model + drug-containing serum group(sh-Cav1 BHD group). The cell survival rate was detected by the CCK-8 method. The cell migration ability was detected by a cell migration assay. The lumen formation ability was detected by an angiogenesis assay. The apoptosis rate was detected by flow cytometry, and the expression of YAP1/HIF-1α signaling pathway-related proteins in each group was detected by Western blot. Finally, co-immunoprecipitation was used to verify the interaction between YAP1 and HIF-1α. The results showed astragaloside Ⅳ, formononetin, ferulic acid, and albiflorin in BHD can all enter the blood. The drug-containing serum of BHD at a mass fraction of 10% may be the optimal intervention concentration for OGD/R-induced injury of bEnd.3 cells. Compared with the sh-NC control group, the sh-NC OGD/R group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, significantly increased cell apoptotic rate, significantly lowered phosphorylation level of YAP1 at S127 site, and significantly elevated nuclear displacement level of YAP1 and protein expression of HIF-1α, vascular endothelial growth factor(VEGF), and vascular endothelial growth factor receptor 2(VEGFR2). Compared with the same type of OGD/R group, the sh-NC BHD group and sh-Cav1 BHD group had significantly increased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly decreased cell apoptotic rate, a further decreased phosphorylation level of YAP1 at S127 site, and significantly increased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC OGD/R group, the sh-Cav1 OGD/R group exhibited significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC BHD group, the sh-Cav1 BHD group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at the S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. YAP1 protein was present in the protein complex precipitated by the HIF-1α antibody, and HIF-1α protein was also present in the protein complex precipitated by the YAP1 antibody. The results confirmed that the drug-containing serum of BHD can increase the activity of YAP1/HIF-1α pathway in bEnd.3 cells damaged by OGD/R through Cav1 and promote angiogenesis in vitro.
Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Signal Transduction/drug effects* ; Glucose/metabolism* ; Caveolin 1/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; YAP-Signaling Proteins ; Oxygen/metabolism* ; Endothelial Cells/metabolism* ; Cell Line ; Adaptor Proteins, Signal Transducing/genetics* ; Neovascularization, Physiologic/drug effects* ; Cell Hypoxia/drug effects* ; Angiogenesis

Retinopathy of prematurity (ROP) is a vision-threatening disorder that leads to pathological growth of the retinal vasculature due to hypoxia. Here, we investigated the potential effects of alamandine, a novel heptapeptide in the renin-angiotensin system (RAS), on hypoxia-induced retinal neovascularization and its underlying mechanisms. In vivo, the C57BL/6J mice with oxygen-induced retinopathy (OIR) were injected intravitreally with alamandine (1.0 μmol/kg per eye). In vitro, human retinal microvascular endothelial cells (HRMECs) were utilized to investigate the effects of alamandine (10 μg/mL) on proliferation, apoptosis, migration, and tubular formation under vascular endothelial growth factor (VEGF) stimulation. Single-cell RNA sequencing (scRNA-seq) matrix data from the Gene Expression Omnibus (GEO) database and RAS-related genes from the Molecular Signatures Database (MSigDB) were sourced for subsequent analyses. By integrating scRNA-seq data across multiple species, we identified that RAS-associated endothelial cell populations were highly related to retinal neovascularization. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a significant decrease in alamandine levels in both the serum and retina of OIR mice compared to those in the control group. Next, alamandine ameliorated hypoxia-induced retinal pathological neovascularization and physiologic revascularization in OIR mice. In vitro, alamandine effectively mitigated VEGF-induced proliferation, scratch wound healing, and tube formation of HRMECs primarily by inhibiting the hypoxia-inducible factor-1α (HIF-1α)/VEGF pathway. Further, coincubation with D-Pro⁷ (Mas-related G protein-coupled receptor D (MrgD) antagonist) hindered the beneficial impacts of alamandine on hypoxia-induced pathological angiogenesis both in vivo and in vitro. Our findings suggested that alamandine could mitigate retinal neovascularization by targeting the MrgD-mediated HIF-1α/VEGF pathway, providing a potential therapeutic agent for OIR prevention and treatment.
Animals ; Retinal Neovascularization/prevention & control* ; Mice, Inbred C57BL ; Vascular Endothelial Growth Factor A/metabolism* ; Humans ; Mice ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Oligopeptides/therapeutic use* ; Signal Transduction/drug effects* ; Cell Proliferation/drug effects* ; Endothelial Cells/drug effects* ; Retinopathy of Prematurity/drug therapy* ; Apoptosis/drug effects* ; Cell Movement/drug effects* ; Renin-Angiotensin System/drug effects* ; Cells, Cultured

OBJECTIVES: To investigate the effect of Didang Decoction-medicated serum on autophagy in high glucose (HG)-induced rat glomerular endothelial cells (RGECs) and explore the pathway that mediates its effect. METHODS: Primary RGECs were isolated and cultured using sequential sieving combined with collagenase digestion, followed by identification using immunofluorescence assay for factor VIII. High glucose medium was used to induce RGECs to simulate a diabetic environment, and the effects of Didang Decoction-medicated serum and 3-MA (an autophagy inhibitor), either alone or in combination, on autophagy of HG-exposed cells were evaluated by observing autophagic vacuoles using monodansylcadaverine (MDC) staining. RT-qPCR and Western blotting were employed to measure mRNA and protein expression levels of Beclin-1, p62, LC3B, p-PI3K, p-Akt, and p-mTOR. RESULTS: Compared with the control cells, the HG-exposed RGECs showed significantly reduced autophagic fluorescence intensity, decreased Beclin-1 mRNA expression, increased p62 mRNA expression, downregulated Beclin-1 protein and LC3-II/I ratio, and upregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. Didang Decoction-medicated serum significantly enhanced autophagic fluorescence intensity in HG-exposed cells, increased Beclin-1 mRNA expression, decreased p62 mRNA expression, upregulated Beclin-1 protein, and downregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. CONCLUSIONS Didang Decoction-medicated serum enhances autophagy in HG-exposed RGECs by regulating the PI3K/Akt/mTOR signaling pathway, which sheds light on a new therapeutic strategy for diabetic nephropathy.
Animals ; Autophagy/drug effects* ; Signal Transduction/drug effects* ; Rats ; TOR Serine-Threonine Kinases/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Endothelial Cells/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glucose ; Cells, Cultured ; Kidney Glomerulus/cytology* ; Rats, Sprague-Dawley

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

This study aims to explore the synergistic effects of the main components in salvianolic acids for Injection(SAFI) on key pathological events in cerebral ischemia, elucidating the pharmacological characteristics of SAFI in neuroprotection. Two major pathological gene modules related to endothelial injury and neuroinflammation in cerebral ischemia were mined from single-cell data. According to the topological distance calculated in network medicine, potential synergistic component combinations of SAFI were screened out. The results showed that the combination of caffeic acid and salvianolic acid B scored the highest in addressing both endothelial injury and neuroinflammation, demonstrating potential synergistic effects. The cell experiments confirmed that the combination of these two components at a ratio of 1∶1 significantly protected brain microvascular endothelial cells(bEnd.3) from oxygen-glucose deprivation/reoxygenation(OGD/R)-induced reperfusion injury and effectively suppressed lipopolysaccharide(LPS)-induced neuroinflammatory responses in microglial cells(BV-2). This study provides a new method for uncovering synergistic effects among active components in traditional Chinese medicine(TCM) and offers novel insights into the multi-component, multi-target acting mechanisms of TCM.
Brain Ischemia/metabolism* ; Neuroprotective Agents/pharmacology* ; Animals ; Drugs, Chinese Herbal/administration & dosage* ; Benzofurans/pharmacology* ; Mice ; Drug Synergism ; Caffeic Acids/pharmacology* ; Polyphenols/pharmacology* ; Humans ; Alkenes/pharmacology* ; Endothelial Cells/drug effects* ; Depsides

OBJECTIVE: To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. METHODS: Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modelling, the rats were randomly divided into 4 groups ( n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL), group B received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL)+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. RESULTS: All groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences ( P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences ( P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNAs compared to the other 3 groups ( P<0.05). CONCLUSION ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
Animals ; Rats, Sprague-Dawley ; Skin/pathology* ; Rats ; Vascular Endothelial Growth Factor A/genetics* ; Basic Helix-Loop-Helix Transcription Factors/genetics* ; Signal Transduction ; Flavonoids/pharmacology* ; Adipose Tissue/cytology* ; Stem Cells/cytology* ; Receptors, Notch/metabolism* ; Radiation Injuries, Experimental/metabolism* ; Wound Healing/drug effects* ; Male

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