Journal: Nature Cardiovascular Research

Article Title: Angiopoietin–TIE2 feedforward circuit promotes PIK3CA-driven venous malformations

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: a , Volcano plot of 7,700 DEGs between Pik3ca-2 and vein clusters. Negative log 2 fold changes ( x axis) represent downregulated gene expression, while positive log 2 fold changes represent upregulated gene expression in the mutant. Differential gene expression was assessed using Seurat’s Wilcoxon rank-sum test. Significant upregulated and downregulated genes are marked in green ( P value < 0.00001, log 2 FC ≥ 1; y axis) and those shared with FOXO1 A3 downregulated genes (from c ) are highlighted in red. b , Whole-mount immunofluorescence of mouse ear skin showing ANGPT2 in EMCN + venous vessels (arrowheads), but not in vascular lesions in the Pik3ca H1047R mutant mouse (arrow). Single-channel images for ANGPT2 staining are shown on the right. Similar results were obtained from three mice in two independent experiments. c , ANGPT2 transcript levels in HUVECs transduced with AKT-resistant FOXO1 A3 and Ctrl, analyzed by RNA-seq at different time points. Data points represent individual biological replicates of ANGPT2 mRNA expression levels (in fold change), mean ± s.d. ( n = 3 (Ctrl) and n = 3 (FOXO1 A3 ) at each time point). *** P < 0.001, unpaired two-tailed Student’s t -test: P (16 h) = 0.0006, P (24 h) = 0.0005, P (32 h) = 0.0001. d , ChIP–seq, ATAC-seq and RNA-seq signals at the ANGPT2 genomic locus performed in FOXO1 A3 -expressing HUVECs. FOXO consensus motifs bound by FOXO1 are indicated in orange. Unbound FOXO motifs are shown in gray. Sequencing signals are represented as reads per kilobase million (RPKMs). e , f , Immunoblot analysis of immunoprecipitated TIE2 (top) or total cell lysates (TCL; below) from Ctrl HUVECs and HUVECs expressing PIK3CA H1047R ( e ) or FOXO1 A3 ( f ) using the indicated antibodies. AKT, S6, TIE2 and tubulin TCL western blots were used as sample loading controls. Cells were starved of serum and left untreated, or stimulated with ANGPT1 (50 ng ml -1 ( e ) or 200 ng ml -1 ( f )), in the presence or absence of the TIE2 inhibitor BAY-826. M r (K) indicates protein molecular weight marker (in kDa). IgG isotype control was used as a negative control. Data are representative of two independent experiments. g , Illustration of the TIE2–PI3K–FOXO pathways (left) and effects of FOXO1 (via FOXO1 A3 expression, middle) and PI3K activation (via PIK3CA H1047R expression, right) on their pathway effectors. Blue indicates reduced activity; red indicates increased activity. Scale bars, 50 μm ( a ).

Article Snippet: Rabbit anti-ANGPT1 antibody (Proteintech, 23302-1-AP) was used at a dilution of 1:100.

Techniques: Gene Expression, Mutagenesis, Immunofluorescence, Staining, Transduction, RNA Sequencing, Expressing, Two Tailed Test, ChIP-sequencing, Sequencing, Western Blot, Immunoprecipitation, Molecular Weight, Marker, Control, Negative Control, Activation Assay, Activity Assay

Journal: Nature Cardiovascular Research

Article Title: Angiopoietin–TIE2 feedforward circuit promotes PIK3CA-driven venous malformations

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: a , PLA staining of activated TIE2 on ear skin paraffin sections from Pik3ca H1047R ;Vegfr1-CreER T2 and Cre − littermate Ctrl mice, detected using pTyr and TIE2 antibodies. DAPI marks cell nuclei. b , Quantification of PLA signals within PECAM1 + blood vessels. Data represent the number of PLA dots per μm 2 of PECAM1 + vessel area, mean ± s.d. ( n = 8 (Ctrl) and n = 26 ( Pik3ca H1047R ) vessels from four mice per genotype, unpaired two-tailed Student’s t -test, **** P = 0.0000054). c , Immunofluorescence of ear skin paraffin sections from Pik3ca H1047R ;Vegfr1-CreER T2 and Cre − littermate Ctrl mice using phospho-TIE2 antibodies. d , Phospho-TIE2 signal within EMCN + vessels, represented as corrected total cell fluorescence (CTFC) of EMCN + vessel area. Data points represent pTIE2 CTFC, mean ± s.d. ( n = 20 (Ctrl) and n = 31 ( Pik3ca H1047R ) vessels from two mice per genotype, unpaired two-tailed Student’s t -test, **** P = 0.000096; Extended Data Fig. ). e , Whole-mount immunofluorescence of ear skin from Pik3ca H1047R ;Vegfr1-CreER T2 and Cre − littermate Ctrl mice using αSMA antibodies 6 weeks after 4-OHT induction. f , Quantification of SMC coverage of veins and capillaries, shown as average percentage of EMCN + area, mean ± s.d. ( n = 4 (Ctrl) and n = 8 ( Pik3ca H1047R ) mice, unpaired two-tailed Student’s t -test, P (vein) = 0.173 (NS, not significant) and **** P (capillary) = 0.0000078). g , Whole-mount immunofluorescence of ANGPT1 + cells associated with veins and capillaries one week after 4-OHT induction. Proliferating cells (arrowheads) were labeled in mutant mice with EdU 16 h before analysis. Asterisks indicate vessel-detached ANGPT1 + cells. h , i , Violin plots showing gene expression of Angpt1 and selected marker genes of EC–SMC interaction ( h ) and heat map showing relative importance of two selected ligand–receptor pairs, generated using CellChat ( i ), in Ctrl and Pik3ca EC clusters from Fig. as well as in SMCs from the same dataset. Scale bars, 50 μm ( a and g ), 20 μm ( c ) and 100 μm ( e ).

Article Snippet: Rabbit anti-ANGPT1 antibody (Proteintech, 23302-1-AP) was used at a dilution of 1:100.

Techniques: Staining, Two Tailed Test, Immunofluorescence, Fluorescence, Labeling, Mutagenesis, Gene Expression, Marker, Generated

Journal: Nature Cardiovascular Research

Article Title: Angiopoietin–TIE2 feedforward circuit promotes PIK3CA-driven venous malformations

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: ( a ) Organ-wide average gene expression (normalized transcripts per million, nTPM), of ANGPT1 in different cell types, extracted from Human Protein Atlas scRNA-seq data. ANGPT1 expression in SMCs is highlighted in red and in BECs in blue. ( b ) Gene expression of ANGPT1 and selected mural cell markers in different skin cell types extracted from Human Protein Atlas scRNA-seq data. ( c ) Whole mount immunofluorescence of ANGPT1 + SMCs (αSMA) associated with veins (arrows) but not arteries (arrowhead) in the ear skin of a control mouse. Similar results were obtained from 3 mice in 2 independent experiments. Scale bars: 50 μm.

Article Snippet: Rabbit anti-ANGPT1 antibody (Proteintech, 23302-1-AP) was used at a dilution of 1:100.

Techniques: Gene Expression, Expressing, Immunofluorescence, Control

Journal: Nature Cardiovascular Research

Article Title: Angiopoietin–TIE2 feedforward circuit promotes PIK3CA-driven venous malformations

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: PI3K-AKT pathway regulates diverse cellular processes including protein translation (via TSC1/2-mTORC1) and gene transcription through direct (for example, inhibition of FOXO1) or indirect (for example, activation of β-catenin through inhibition of GSK3β) mechanisms. Examples of other AKT targets involved for example, in the regulation of cell survival (BAD) and vascular tone (eNOS) are indicated. FOXO1 inhibition-dependent PI3K signaling contributes to regulation of cellular metabolism, vascular growth and the acquisition of post-capillary venous phenotype. Activation of the PI3K pathway results in “feedforward” signaling to TIE2 by suppressing FOXO1-regulated expression of the autocrine TIE2 antagonist, ANGPT2. The resulting upstream activation of PI3K signaling is reinforced by an increase in coverage by smooth muscle cells (SMCs) that produce the TIE2 agonist, ANGPT1.

Article Snippet: Rabbit anti-ANGPT1 antibody (Proteintech, 23302-1-AP) was used at a dilution of 1:100.

Techniques: Inhibition, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

doi: 10.1371/journal.pone.0165371

Figure Lengend Snippet: (A) Internalization of AT 1 and ACE in the presence of 4 nM 3 H-Ang-II. Data are shown as mean from three independent experiments, each performed in duplicate. (B) CHO-ACE and CHO-AT 1 cells present the same relative protein level of each respective receptor. (Values are mean ± S.E.M, *p<0.05, n = 63 individual experiments). (C) Representative confocal images of internalized Ang-II-FITC (1 μM) in CHO-ACE and CHO-AT 1 cells after 30 seconds of Ang-II stimulation. DAPI (blue) and Wheat Germ Agglutinin (red), scale bar = 10 μm. On the right, quantification of internalized Ang-II-FITC is presented. Values are mean ± S.E.M, *p<0.05, n = 6. (D) Representative confocal images of unstimulated CHO-ACE and CHO-AT1 cells, labeled for DAPI and WGA. (E) Immunolocalization of ACE after stimulation with Ang-II (1μM), for the indicated times. ACE is shown in green, actin filaments in red, and nucleus in blue (DAPI). Right panel represents a 3D reconstruction of CHO-ACE cell after 15 minutes of incubation with Ang-II (1μM). Scale bar = 10μm. (F) Western blotting of nuclear and non-nuclear protein fractions from CHO-ACE cells, before (control) and after Ang-II (1 μM) stimulation for the indicated times. Histone-3 and GAPDH were used to shown the purification of nuclear and non-nuclear protein fractions, respectively. (G) Densitometry analysis of the western blot. Values are mean ± S.E.M., n = 3 (*** p<0.01).

Article Snippet: Fluo-4/AM, DAPI, secondary antibodies conjugated to Alexa-488, Alexa-633, Angiotensin-II FITC conjugate and Lipofectamine® 2000 were purchased from Life Technologies (New York, USA); rabbit IgG secondary antibody was purchased from Sigma-Aldrich, (St. Louis, USA).

Techniques: Labeling, Incubation, Western Blot, Control, Purification

Journal: PLoS ONE

Article Title: Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

doi: 10.1371/journal.pone.0165371

Figure Lengend Snippet: (A) Western immunoblotting to confirm the silencing of PLC isoforms. Densitometric analysis are shown on (B) for PLCβ3 and (C) for PLCγ1. (D) Quantitative representation of intracellular [Ca 2+ ] in CHO-ACE cells transfected with siRNA-PLCβ3 and PLCγ1 using Lipofectamine and stimulated with Ang-II (1μM). (E) Line scanning of Ca 2+ signal in CHO-ACE. Ang-II promotes Ca 2+ increase with greater intensity in the nuclear region. (F) Time course of Ca 2+ signaling in the nucleus (red traces) and cytosol (blue traces). (G) Quantification of fluorescence intensity signal in the nucleus and cytosol. Values are mean ± S.E.M., (* p<0.01), 45 cells, n = 3 individual experiments.

Article Snippet: Fluo-4/AM, DAPI, secondary antibodies conjugated to Alexa-488, Alexa-633, Angiotensin-II FITC conjugate and Lipofectamine® 2000 were purchased from Life Technologies (New York, USA); rabbit IgG secondary antibody was purchased from Sigma-Aldrich, (St. Louis, USA).

Techniques: Western Blot, Transfection, Fluorescence

Journal: PLoS ONE

Article Title: Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

doi: 10.1371/journal.pone.0165371

Figure Lengend Snippet: (A) Cell growth assay of CHO-ACE cells 12, 24 and 48 hours after stimulation with Ang-II (1μM), triplicate in 3 individual experiments. (B) Western blot to confirm the silencing of clathrin (upper panel) and densitometry analysis (bottom panel). Mean ± S.E.M., n = 5 (* p<0.05). (C) BrDU incorporation is decreased in CHO-ACE cells transfected with siRNA-Cla (Clathrin) and stimulated with Ang-II (1μM). Mean ± S.E.M., n = 6 (* p<0.05).

Article Snippet: Fluo-4/AM, DAPI, secondary antibodies conjugated to Alexa-488, Alexa-633, Angiotensin-II FITC conjugate and Lipofectamine® 2000 were purchased from Life Technologies (New York, USA); rabbit IgG secondary antibody was purchased from Sigma-Aldrich, (St. Louis, USA).

Techniques: Growth Assay, Western Blot, BrdU Incorporation Assay, Transfection

Journal: PLoS ONE

Article Title: Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

doi: 10.1371/journal.pone.0165371

Figure Lengend Snippet: (A) Real-Time PCR analysis for expression of ACE in melan-a and TM-5 cells. (B) ACE activity measured by cleavage of Abz-FRK(Dnp)P-OH in melan-a and TM-5 cells. For A and B, mean ± S.E.M, n = 6 (**p<0.01). (C-D) BrDU uptake assay 24 hours after stimulation with Ang-II (1μM) in the presence of lisinopril (1μM), showing inhibition of cell proliferation in TM-5 cells (D) but not in melan-a cells (C). Mean ± S.E.M., n = 6. (*p<0.05).

Article Snippet: Fluo-4/AM, DAPI, secondary antibodies conjugated to Alexa-488, Alexa-633, Angiotensin-II FITC conjugate and Lipofectamine® 2000 were purchased from Life Technologies (New York, USA); rabbit IgG secondary antibody was purchased from Sigma-Aldrich, (St. Louis, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

doi: 10.1371/journal.pone.0165371

Figure Lengend Snippet: (A) Western bot (upper panel) to confirm the silencing of ACE and densitometry analysis (bottom panel). Mean ± S.E.M, n = 8. (***p<0.01 compared to respective columns). (B) BrDU uptake in Tm5 cells silenced for ACE and stimulated for 24 hours with Ang-II (1μM), showing a decrease in BrDU incorporation in the absence of ACE. Mean ± S.E.M., n = 12 (*p<0.05; ns = non-significant).

Article Snippet: Fluo-4/AM, DAPI, secondary antibodies conjugated to Alexa-488, Alexa-633, Angiotensin-II FITC conjugate and Lipofectamine® 2000 were purchased from Life Technologies (New York, USA); rabbit IgG secondary antibody was purchased from Sigma-Aldrich, (St. Louis, USA).

Techniques: Western Blot, BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

doi: 10.1371/journal.pone.0165371

Figure Lengend Snippet: (A-B) Wound healing assay using TM-5 cells stimulated with Ang-II (1μM). C) Representative confocal images of TM-5 cells double-labeled with vinculin (green) and phalloidin (red). Scale bar = 10 μm. (D-E) Quantification of the focal adhesion formation. Mean ± S.E.M., n = 6 (* p<0.05).

Article Snippet: Fluo-4/AM, DAPI, secondary antibodies conjugated to Alexa-488, Alexa-633, Angiotensin-II FITC conjugate and Lipofectamine® 2000 were purchased from Life Technologies (New York, USA); rabbit IgG secondary antibody was purchased from Sigma-Aldrich, (St. Louis, USA).

Techniques: Wound Healing Assay, Labeling

Journal: bioRxiv

Article Title: Chronic kidney disease promotes anxiety susceptibility through an angiotensin II–central amygdala axis

doi: 10.64898/2026.08.16.744184

Figure Lengend Snippet: (A) Serum Ang II levels in Control, CKD, SUS and CKD-SUS mice (n=10 per group).(B-C) Quantification and representative images of fluorescence accumulation in the CeA (n = 5; scale bars: left 100 μm and right 50 μm).(D) Representative immunofluorescence images of AT1R (red) and c-Fos (white) expression in CeA sections, with DAPI nuclear staining (blue) (scale bar: 50 μm).(E) Representative co-localization curve corresponding to the region indicated by the green arrow (pointing) in panel (D).Data are presented as mean ± SEM. Statistics used one-way analysis of variance followed by Bonferroni post hoc tests. *p < 0.05, **p < 0.01.

Article Snippet: Serum and CSF Ang II concentrations were measured using a Mouse Angiotensin II ELISA Kit (RayBiotech, Cat. No. EIA-ANGII, Guangzhou, China).

Techniques: Control, Fluorescence, Immunofluorescence, Expressing, Staining

Journal: bioRxiv

Article Title: Chronic kidney disease promotes anxiety susceptibility through an angiotensin II–central amygdala axis

doi: 10.64898/2026.08.16.744184

Figure Lengend Snippet: (A) Cerebrospinal fluid (CSF) Ang II levels. Due to limited CSF volume, each data point represents pooled samples from 3–5 mice (detection limit: 0.1 pg/mL).(B) Representative immunofluorescence images of Ang II in the central amygdala (CeA) (scale bars: 100 μm and 50 μm).(C) Quantification of Ang II fluorescence-positive area in the CeA (n = 4).(D) Representative images of viral transduction and AT1R expression in the CeA (n = 3). Green: virus; red: AT1R; blue: DAPI (scale bar: 100 μm).(E) Quantification of AT1R expression in the CeA after viral transduction (n = 3).Data are presented as mean ± SEM. Statistics used two-tailed unpaired t tests.“ns” indicates no significant difference, **p < 0.01.

Article Snippet: Serum and CSF Ang II concentrations were measured using a Mouse Angiotensin II ELISA Kit (RayBiotech, Cat. No. EIA-ANGII, Guangzhou, China).

Techniques: Immunofluorescence, Fluorescence, Transduction, Expressing, Virus, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC effectively improves AngII-induced cell dysfunction and HF due to TAC. (A) Relative cell viability of different groups of H9c2 cells compared to the control group, and NT-proBNP content per mg of total cellular protein; (B) Gross images of rat hearts from each group for direct comparison of heart size, and comparison of heart weight to tibia length ratio in each group; (C) Parasternal long-axis M-mode echocardiographic images of the left ventricle in rats from each group, and quantitative comparison of LVEF; (D) Comparison of serum NT-proBNP levels in rats from each group; (F) Microscopic images of Masson’s trichrome-stained myocardium in rats from each group (×50, ×100, ×200), where the blue-stained areas represent collagen deposition, indicating myocardial fibrosis; (E) Quantification of myocardial fibrosis as indicated by Masson’s trichrome staining, expressed as the percentage of fibrotic area. p ns ≥ 0.05, p * < 0.05, p ** < 0.01, p *** < 0.001, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; AngII, Angiotensin II; HF, Heart failure; F, Ferrostatin-1; E, Erastin; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; LVEF, Left Ventricular Ejection Fraction; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction.

Article Snippet: Astragalus mongholicus Bunge [Fabaceae; Astragali radix] (Batch No. 2301001, Wan Zhen Chinese Herbal Pieces Factory, Bozhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), S. miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma] (Batch No. 20230202-01, Guizhou Tongde Pharmaceutical Co., Ltd., Guizhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), Angiotensin II (AngII) (S25704, Yuanye, China), Ferrostatin-1 (Fer-1) (S7243, Selleck, United States), Brequinar (BQR) (HY108325, MedChemExpress), Erastin (Era) (S7242, Selleck, United States), inhibitor of ferroptosis suppressor protein 1 (iFSP1) (MFCD01572665, Macklin, China), dimethyl sulfoxide (DMSO) (D8371, Solarbio, China).

Techniques: Control, Comparison, Staining

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC effectively inhibits ferroptosis in HF. (A) Levels of antioxidants GSH and CoQ10H2 in H9c2 cells of each group; (B) Extent of lipid peroxidation (MDA content) in H9c2 cells of each group; (C) Levels of Fe 2+ in H9c2 cells of each group; (D) Flow cytometry plots showing red and green fluorescence intensities after JC-1 staining of H9c2 cells from each group, and quantitative analysis of the proportion of green fluorescence signal; (E) GSH levels in myocardial tissue homogenates and serum CoQ10H2 levels in SD rats from each group; (F) MDA content in myocardial tissue homogenates of SD rats from each group; (G) Levels of Fe 2+ in myocardial tissues of SD rats from each group; (H) TEM images of myocardial tissue from each group, with indicating one of the mitochondria in the image. p ns ≥ 0.05, p * < 0.05, p ** < 0.01, p *** < 0.001, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; AngII, Angiotensin II; F, Ferrostatin-1; E, Erastin; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; GSH, Reduced Glutathione; CoQ10H2, Reduced Coenzyme Q10; MDA, Malondialdehyde; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Astragalus mongholicus Bunge [Fabaceae; Astragali radix] (Batch No. 2301001, Wan Zhen Chinese Herbal Pieces Factory, Bozhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), S. miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma] (Batch No. 20230202-01, Guizhou Tongde Pharmaceutical Co., Ltd., Guizhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), Angiotensin II (AngII) (S25704, Yuanye, China), Ferrostatin-1 (Fer-1) (S7243, Selleck, United States), Brequinar (BQR) (HY108325, MedChemExpress), Erastin (Era) (S7242, Selleck, United States), inhibitor of ferroptosis suppressor protein 1 (iFSP1) (MFCD01572665, Macklin, China), dimethyl sulfoxide (DMSO) (D8371, Solarbio, China).

Techniques: Flow Cytometry, Fluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC ameliorates H9c2 cell dysfunction by inhibiting ferroptosis through the elevation of GPX4 levels. (A) Western blotting analysis of GPX4 levels in H9c2 cells from various groups to evaluate the effect of AngII and various doses of HDC on GPX4 content. (B–F) All panels represent the conditions of H9c2 cells under HDC intervention, assessing the effects of GPX4 inhibition and non-inhibition in varying FSP1 and DHODH states. (B–E) Measurements in H9c2 cells include GSH levels, MDA content, Fe 2+ levels, cell viability, and NT-proBNP levels. (F) Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the proportion of green fluorescence signal in flow cytometry. p * < 0.05, p ** < 0.01, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; GPX4, Glutathione Peroxidase 4; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; GSH, Reduced Glutathione; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; E, Erastin; B, Brequinar; i, Inhibitor of FSP1; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Astragalus mongholicus Bunge [Fabaceae; Astragali radix] (Batch No. 2301001, Wan Zhen Chinese Herbal Pieces Factory, Bozhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), S. miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma] (Batch No. 20230202-01, Guizhou Tongde Pharmaceutical Co., Ltd., Guizhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), Angiotensin II (AngII) (S25704, Yuanye, China), Ferrostatin-1 (Fer-1) (S7243, Selleck, United States), Brequinar (BQR) (HY108325, MedChemExpress), Erastin (Era) (S7242, Selleck, United States), inhibitor of ferroptosis suppressor protein 1 (iFSP1) (MFCD01572665, Macklin, China), dimethyl sulfoxide (DMSO) (D8371, Solarbio, China).

Techniques: Western Blot, Inhibition, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC ameliorates H9c2 cell dysfunction by increasing FSP1 levels to inhibit ferroptosis. (A) Western blotting analysis of FSP1 levels in H9c2 cells from various groups to assess the effects of AngII and different doses of HDC on FSP1 content. (B–F) These panels represent the HDC-treated cells, examining the effects of FSP1 inhibition and non-inhibition under varying GPX4 and DHODH states. (B–E) Measurements in H9c2 cells include CoQ10H2 levels, MDA levels, Fe 2+ content, cell viability, and NT-proBNP levels. (F) Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the green fluorescence signal proportion in flow cytometry charts. p ** < 0.01, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; FSP1, Ferroptosis Suppressor Protein 1; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; DHODH, Dihydroorotate Dehydrogenase; i, Inhibitor of FSP1; B, Brequinar; E, Erastin; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Astragalus mongholicus Bunge [Fabaceae; Astragali radix] (Batch No. 2301001, Wan Zhen Chinese Herbal Pieces Factory, Bozhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), S. miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma] (Batch No. 20230202-01, Guizhou Tongde Pharmaceutical Co., Ltd., Guizhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), Angiotensin II (AngII) (S25704, Yuanye, China), Ferrostatin-1 (Fer-1) (S7243, Selleck, United States), Brequinar (BQR) (HY108325, MedChemExpress), Erastin (Era) (S7242, Selleck, United States), inhibitor of ferroptosis suppressor protein 1 (iFSP1) (MFCD01572665, Macklin, China), dimethyl sulfoxide (DMSO) (D8371, Solarbio, China).

Techniques: Western Blot, Inhibition, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC ameliorates H9c2 cell dysfunction by inhibiting ferroptosis through the elevation of DHODH levels. (A) "Western blotting analysis of DHODH levels in H9c2 cells from various groups, assessing the effects of AngII and different doses of HDC on DHODH content. (B–F) Experiments conducted on cells treated with HDC, evaluating the impact of inhibiting or not inhibiting DHODH under different GPX4 and iFSP1 states. (B–E) Measurements in H9c2 cells include CoQ10H2 levels, MDA levels, Fe 2+ content, cell viability, and NT-proBNP levels. Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the green fluorescence signal proportion in flow cytometry charts. p ns ≥ 0.05, p * < 0.05, p ** < 0.01, p *** < 0.001, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; DHODH, Dihydroorotate Dehydrogenase; WB, Western blotting; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; B, Brequinar; i, Inhibitor of FSP1; E, Erastin; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Astragalus mongholicus Bunge [Fabaceae; Astragali radix] (Batch No. 2301001, Wan Zhen Chinese Herbal Pieces Factory, Bozhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), S. miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma] (Batch No. 20230202-01, Guizhou Tongde Pharmaceutical Co., Ltd., Guizhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), Angiotensin II (AngII) (S25704, Yuanye, China), Ferrostatin-1 (Fer-1) (S7243, Selleck, United States), Brequinar (BQR) (HY108325, MedChemExpress), Erastin (Era) (S7242, Selleck, United States), inhibitor of ferroptosis suppressor protein 1 (iFSP1) (MFCD01572665, Macklin, China), dimethyl sulfoxide (DMSO) (D8371, Solarbio, China).

Techniques: Western Blot, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: Comparative analysis of the effectiveness of GPX4, FSP1, and DHODH in mediating HDC’s improvement of H9c2 cell dysfunction. In this figure, H9c2 cells are divided into three groups: the AngII + H-S + i + E group, primarily mediated by DHODH for HDC’s protective effect; the AngII + H-S + B + E group, primarily mediated by FSP1 for HDC’s protective effect; and the AngII + H-S + B + i group, primarily mediated by GPX4 for HDC’s protective effect. (A–F) Measurements in these three groups of H9c2 cells include GSH levels, CoQ10H2 levels, MDA levels, Fe 2+ content, cell viability, and NT-proBNP levels. (G) Flow cytometry images post JC-1 staining of the H9c2 cells and the quantitative analysis of the proportion of green fluorescence signal. p ns ≥0.05, p *<0.05, p **<0.01, p ****<0.0001. represents an individual sample data point. GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; AngII, Angiotensin II; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; i (iFSP1), Inhibitor of FSP1; E, Erastin; B, Brequinar; GSH, Reduced Glutathione; CoQ10H2, Reduced Coenzyme Q10; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Astragalus mongholicus Bunge [Fabaceae; Astragali radix] (Batch No. 2301001, Wan Zhen Chinese Herbal Pieces Factory, Bozhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), S. miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma] (Batch No. 20230202-01, Guizhou Tongde Pharmaceutical Co., Ltd., Guizhou, China; procured via Jiangsu Province Academy of Traditional Chinese Medicine), Angiotensin II (AngII) (S25704, Yuanye, China), Ferrostatin-1 (Fer-1) (S7243, Selleck, United States), Brequinar (BQR) (HY108325, MedChemExpress), Erastin (Era) (S7242, Selleck, United States), inhibitor of ferroptosis suppressor protein 1 (iFSP1) (MFCD01572665, Macklin, China), dimethyl sulfoxide (DMSO) (D8371, Solarbio, China).

Techniques: Flow Cytometry, Staining, Fluorescence

Journal: International journal of molecular medicine

Article Title: Attenuation of exogenous angiotensin II stress-induced damage and apoptosis in human vascular endothelial cells via microRNA-155 expression.

doi: 10.3892/ijmm.2012.1182

Figure Lengend Snippet: Figure 2. Ang II affects HUVEC proliferation in vitro and miR-155 attenuates that effect. (A) MTT proliferation assay was used to show that exogenous Ang II inhibited HUVEC proliferation in a dose-dependent manner, with a half maximal inhibitory concentration of 68.94 ng/µl. **P<0.01 vs. HUVEC-PBS group; #P>0.05 vs. HUVEC-PBS group; n=3. (B) qRT-PCR showed that mRNA expression of the apoptosis inhibitor Bcl-2 was markedly lower in the HUVEC-Ang II group (Ang II) than in the HUVEC-PBS group (PBS) and the blank control (BC). mRNA expression of the apoptosis-promoting factors Bax, cytochrome c, caspases-9 and -3 was markedly higher in the HUVEC-Ang II group (Ang II) than in the PBS and the BC groups. However, after transfection with miR-155, mRNA expression of Bcl-2 in the miR-155-transfected group (A+miR155) was markedly elevated; Bax, cytochrome c, caspases-9 and -3 were markedly decreased in this group, but not in the miR-155 mutant-transfected group (A+miR-mut). *P<0.05 vs. BC; **P<0.01 vs. BC; #P>0.05 vs. BC; n=3. (C) Western blotting showed that Bcl-2 protein expression was significantly reduced, but Bax, active caspase-9 and active caspase-3 were significantly increased in the HUVEC-Ang II group (Ang II) compared with the PBS and the BC groups. Bcl-2 protein expression was significantly increased in the miR‑155-transfected group (A+miR155) compared with the HUVEC-Ang II group (Ang II) and the miR-155 mutant-transfected group (A+miR-mut), and Bax, active caspase-9 and active caspase-3 in this group were markedly decreased. *P<0.05 vs. BC; **P<0.01 vs. BC; #P>0.05 vs. BC; n=3.

Article Snippet: The cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking, the cells were incubated first with rabbit anti-human Angiotensin II polyclonal antibody (1:200;) and rabbit anti-human ERK1/2 or pERK1/2 polyclonal antibody (1:200; were from Santa Cruz Biotechnology, Inc.) overnight at 4 ̊C, and then with FITC- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Abcam, Cambridge, UK) and 5 mg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

Techniques: In Vitro, Proliferation Assay, Concentration Assay, Quantitative RT-PCR, Expressing, Control, Transfection, Mutagenesis, Western Blot

Journal: International journal of molecular medicine

Article Title: Attenuation of exogenous angiotensin II stress-induced damage and apoptosis in human vascular endothelial cells via microRNA-155 expression.

doi: 10.3892/ijmm.2012.1182

Figure Lengend Snippet: Figure 4. Mitochondrial membrane potential (ΔΨm) of HUVECs was determined by JC-1 staining. ΔΨm changes occurred in the HUVEC-Ang II group (Ang II) and the miR-155 mutant-transfected group (A+miR-mut), suggesting that exogenous Ang II can damage the mitochondria of HUVECs. However, miR-155 attenuated the exogenous Ang II-induced mitochondrial damage in miR-155-transfected HUVECs (A+miR155). *P<0.05 vs. BC group; **P<0.01 vs. BC; #P>0.05 vs. BC; n=3.

Article Snippet: The cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking, the cells were incubated first with rabbit anti-human Angiotensin II polyclonal antibody (1:200;) and rabbit anti-human ERK1/2 or pERK1/2 polyclonal antibody (1:200; were from Santa Cruz Biotechnology, Inc.) overnight at 4 ̊C, and then with FITC- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Abcam, Cambridge, UK) and 5 mg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

Techniques: Membrane, Staining, Mutagenesis, Transfection

Journal: International journal of molecular medicine

Article Title: Attenuation of exogenous angiotensin II stress-induced damage and apoptosis in human vascular endothelial cells via microRNA-155 expression.

doi: 10.3892/ijmm.2012.1182

Figure Lengend Snippet: Figure 3. Flow cytometry was used to determine whether Ang II influenced the cell cycle in HUVECs and whether miR-155 could affect this. The HUVEC-Ang II group (Ang II) and the miR-155 mutant-transfected group (A+miR-mut) were arrested in the G0/G1 phase of the cell cycle and the percentage of cells in the S phase was significantly decreased. By contrast, the cell cycle in the miR-155-transfected group (A+miR155) had more ameliorated.

Article Snippet: The cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking, the cells were incubated first with rabbit anti-human Angiotensin II polyclonal antibody (1:200;) and rabbit anti-human ERK1/2 or pERK1/2 polyclonal antibody (1:200; were from Santa Cruz Biotechnology, Inc.) overnight at 4 ̊C, and then with FITC- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Abcam, Cambridge, UK) and 5 mg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

Techniques: Flow Cytometry, Mutagenesis, Transfection

Journal: International journal of molecular medicine

Article Title: Attenuation of exogenous angiotensin II stress-induced damage and apoptosis in human vascular endothelial cells via microRNA-155 expression.

doi: 10.3892/ijmm.2012.1182

Figure Lengend Snippet: Figure 5. miR-155 reduced the activation and phosphorylation of Ang II-induced ERK1/2. (A) Co-immunoprecipitation western blotting showed no obvious difference in the levels of normal ERK1/2 and phospho-ERK1/2 expression between the blank control (BC) and the HUVEC-PBS group (PBS). However, when exogenous Ang II was added to the culture medium, phospho-ERK1/2 levels were significantly higher in the HUVEC-Ang II group (Ang II) and the miR-155 mutant-transfected group (A+miR-mut) than in the miR-155-transfected group (A+miR155), the BC and the PBS groups. *P<0.05 vs. BC; **P<0.01 vs. BC; #P>0.05 vs. BC; n=3. (B) Immunofluorescence staining showed that ERK1/2 and phospho-ERK1/2 protein expression decreased in miR-155-transfected cells (A+miR155) compared with the HUVEC-Ang II group (Ang II) and miR-155 mutant-transfected cells (A+miR-mut) after exogenous Ang II stimulation.

Article Snippet: The cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking, the cells were incubated first with rabbit anti-human Angiotensin II polyclonal antibody (1:200;) and rabbit anti-human ERK1/2 or pERK1/2 polyclonal antibody (1:200; were from Santa Cruz Biotechnology, Inc.) overnight at 4 ̊C, and then with FITC- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Abcam, Cambridge, UK) and 5 mg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

Techniques: Activation Assay, Phospho-proteomics, Immunoprecipitation, Western Blot, Expressing, Control, Mutagenesis, Transfection, Immunofluorescence, Staining

Journal: International journal of molecular medicine

Article Title: Attenuation of exogenous angiotensin II stress-induced damage and apoptosis in human vascular endothelial cells via microRNA-155 expression.

doi: 10.3892/ijmm.2012.1182

Figure Lengend Snippet: Figure 6. miR-155 maintains the migration and capillary tubule formation abilities of HUVECs. HUVECs formed capillary tubules in migration gels under normal conditions, but migration and capillary tubule formation were markedly reduced when the cell culture medium contained exogenous Ang II. After transfection with miR-155, these abilities were maintained in the presence of Ang II. **P<0.01 vs. BC; #P>0.05 vs. BC; n=6.

Article Snippet: The cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking, the cells were incubated first with rabbit anti-human Angiotensin II polyclonal antibody (1:200;) and rabbit anti-human ERK1/2 or pERK1/2 polyclonal antibody (1:200; were from Santa Cruz Biotechnology, Inc.) overnight at 4 ̊C, and then with FITC- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Abcam, Cambridge, UK) and 5 mg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

Techniques: Migration, Cell Culture, Transfection