Journal: CNS Neuroscience & Therapeutics

Article Title: ANGPT2 /Tie2 Enhances H3K18la ‐Mediated Macrophage M2 Polarization to Promote Endothelial Cell Proliferation in the Chronically Ischaemic Brain

doi: 10.1002/cns.70879

Figure Lengend Snippet: Results of flow cytometry, western blotting, and qPCR for determining the macrophage polarization status and the expression of proangiogenic factors. (A) Representative flow cytometry histogram showing the fluorescence intensity of 6 macrophage polarization markers. (B) Bar charts showing the MFIs of macrophage polarization markers ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (C) Representative western blot showing the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 (normalized to β‐Actin expression) in primary macrophages. (D) Densitometric analyses of the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (E) qPCR results showing the relative mRNA expression of iNOS , TNF‐α , IL‐6 , VEGFA , IGF1 , EGR1 , IL‐10 , and MMP9 in primary macrophages ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (F) Representative flow cytometry histogram showing the fluorescence intensity of macrophage polarization markers. (G) Bar charts showing the MFIs of macrophage polarization markers ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (H) Representative western blot showing the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 (normalized to β‐Actin expression) in TDMs. (I) Densitometric analyses of the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (J) qPCR results showing the relative mRNA expression of iNOS , TNF‐α , IL‐6 , VEGFA , IGF1 , EGR1 , IL‐10 , and MMP9 in TDMs ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). The error bars represent the ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. MFI, mean fluorescence intensity; TDMs, THP‐1‐derived macrophages; TEMs, Tie2‐expressing monocytes/macrophages; TNMs, Tie2‐negative monocytes/macrophages.

Article Snippet: In accordance with previous studies, primary TEMs and TNMs were treated with 100 ng/mL ANGPT2 protein (HY‐P7510; MedChemExpress, USA) for 3 h, while TDMs were exposed to the same concentration of ANGPT2 for 6 h.

Techniques: Flow Cytometry, Western Blot, Expressing, Fluorescence, Derivative Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: ANGPT2 /Tie2 Enhances H3K18la ‐Mediated Macrophage M2 Polarization to Promote Endothelial Cell Proliferation in the Chronically Ischaemic Brain

doi: 10.1002/cns.70879

Figure Lengend Snippet: Western blot and immunofluorescence staining showing the expression of relevant cytokines in the CIB of 2VO + EMS rats. (A) Representative images of triple immunofluorescence staining showing Tie2 + , CD11b + , and CD206 + cells in the CIB of each group. Bar = 50 μm. (B) Counts of M2 TEMs (Tie2 + /CD11b + /CD206 + ) in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (C) Representative western blot showing the relative expression of ANGPT2, CD206, and ARG1 (normalized to β‐Actin expression). (D) Densitometric analyses of the relative expression of ANGPT2, CD206, and ARG1 ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (E) Representative images of double immunofluorescence staining showing CD31 + and Ki67 + cells in the CIB of each group. Bar = 50 μm. (F) Column chart showing the counts of CD31 + cells in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (G) Column chart showing the proportion of Ki67‐positive ECs in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (H) Representative western blot showing the relative expression of CD31 (normalized to β‐Actin expression). (I) Densitometric analyses of the relative expression of CD31 ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (H) * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The error bars represent the ±SDs. CIB: Chronically ischaemic brain; TEMs, Tie2‐expressing monocytes/macrophages.

Article Snippet: In accordance with previous studies, primary TEMs and TNMs were treated with 100 ng/mL ANGPT2 protein (HY‐P7510; MedChemExpress, USA) for 3 h, while TDMs were exposed to the same concentration of ANGPT2 for 6 h.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Double Immunofluorescence Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: ANGPT2 /Tie2 Enhances H3K18la ‐Mediated Macrophage M2 Polarization to Promote Endothelial Cell Proliferation in the Chronically Ischaemic Brain

doi: 10.1002/cns.70879

Figure Lengend Snippet: Behavioral test, western blotting, and immunofluorescence results in 2VO + EMS rats 4‐week post‐modeling. (A) Quantitative analysis of the percentage of recognition preference for the novel object in each group rats in NORT test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (B) Representative image showing the swimming paths in each group rats during the MWM test. (C) Line chart showing the average escape latencies in each group rats during the MWM test ( n = 6; two‐way ANOVA and Tukey's multiple comparisons test; ***: TEMs vs. TEMs ANGPT2 , ****: TEMs ANGPT2 vs. TEMs ANGPT2+Oxamate , TEMs ANGPT2 vs. TEMs ANGPT2+C646 , TEMs ANGPT2 vs. TEMs ANGPT2+LPS ). (D) Quantitative analysis of the time spent in target quadrant in each group rats during the MWM test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (E) Quantitative analysis of the number of platform crossings in each group rats during the MWM test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (F) Quantitative analysis of the average swimming speed in each group rats during the MWM test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (G) Representative western blot showing the relative expression of CD31 (normalized to β‐Actin expression). (H) Densitometric analyses of the relative expression of CD31 ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (I) Representative images of double immunofluorescence staining showing CD31 + and Ki67 + cells in the CIB of each group. Bar = 50 μm. (J) Column chart showing the counts of CD31 + cells in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (K) Column chart showing the proportion of Ki67‐positive ECs in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). The error bars represent the ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 2VO, 2‐vessel occlusion; EMS, encephalomyosynangiosis; MWM, Morris water maze; NORT, novel object recognition test; TEMs, Tie2‐expressing monocytes/macrophages.

Article Snippet: In accordance with previous studies, primary TEMs and TNMs were treated with 100 ng/mL ANGPT2 protein (HY‐P7510; MedChemExpress, USA) for 3 h, while TDMs were exposed to the same concentration of ANGPT2 for 6 h.

Techniques: Western Blot, Immunofluorescence, Expressing, Double Immunofluorescence Staining

Journal: Cell Death & Disease

Article Title: Decreased ZNF750 promotes angiogenesis in a paracrine manner via activating DANCR/miR-4707-3p/FOXC2 axis in esophageal squamous cell carcinoma

doi: 10.1038/s41419-020-2492-2

Figure Lengend Snippet: a PCR array in ZNF750 knockdown cells using the kit of Cancer Pathway Finder PCR Array. b , c qRT-PCR showed the expression of FOXC2 in ZNF750 knockdown cells ( b ) and ZNF750-wt cells ( c ). GAPDH was performed as a loading control. Statistical analysis was performed with a two-sided t test. d Representative images of tube formation ( upper ), migration ( middle ) and invasion( lower ) of HUVEC. e The conditioned medium from FOXC2 knockdown in ZNF750 knockdown cells reversed the effect on tube formation ( left ), migration ( middle ) and invasion( right ) of HUVEC. f Angiogenic biomarkers ANGPT2 , FLT1 , CCL2 and FGF2 were detected by qRT-PCR in ZNF750 knockdown cells and FOXC2 knockdown in ZNF750 knockdown cells. Statistical analysis was performed with one-way ANOVA. ** p < 0.01, *** p < 0.0001.

Article Snippet: The antibodies used in this experiment are shown as follows: Ki-67 (Zhongshan, China, ZM-0166), ZNF750 (Sigma, USA, HPA021573), FOXC2 (Proteintech, USA, 23066-1-AP), CD31(Abcam, USA, ab134168), FLT1 (Proteintech, USA, 13687-1-AP), ANGPT2 (Proteintech, USA, 24613-1-AP).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Control, Migration

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: AJ attenuates microglial activation and neuroinflammation in the PVN while modulating key RAAS components. (a) Representative immunofluorescence images (left) and quantification (right) of Iba1 (red) in the hypothalamic Paraventricular Nucleus (PVN). Nuclei are stained with DAPI (blue). AJ treatment significantly reduced Iba1 fluorescence intensity and density, indicating suppression of microglial activation. Scale bar, 50 μ m. (b) ELISA quantification of proinflammatory cytokines (TNF-α, IL-6, IL-1β) in PVN tissue lysates. (c) Serum levels of RAAS components (ACE, Renin [PRN], Angiotensin II [AngII], Aldosterone [ALD]). AJ treatment restored the RAAS balance towards the WKY control phenotype. Data are mean ± S.D. ( n = 5 per group). Δ P < 0.05 vs. Control; * P < 0.05 vs. Model (One-way ANOVA with Tukey’s post hoc test).

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: AJ alleviates oxidative stress and inflammation in AngII-stimulated BV-2 microglia. (a) Representative fluorescence images of Dihydroethidium (DHE, red) staining for intracellular ROS. (b) Representative images of MitoSOX Red staining for mitochondrial superoxide. Nuclei are counterstained with Hoechst (blue). Scale bars, 50 μ m. (c) Quantification of DHE fluorescence intensity. (d) Quantification of MitoSOX fluorescence intensity. (e) Intracellular SOD activity and MDA levels measured by ELISA. (f) Western blot analysis (left) and densitometric quantification (right) of antioxidant (NQO1) and oxidative (Nox1, Nox4) enzymes. Pre-treatment with AJ (6.25 and 12.5 μ g/mL) significantly reversed AngII-induced oxidative stress. Data are mean ± S.D. ( n = 3 independent experiments). Δ P < 0.05 vs. Control; * P < 0.05 vs. AngII Model.

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Fluorescence, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: AJ reduces inflammatory cytokine levels in AngII-stimulated BV-2 microglia. ELISA quantification of proinflammatory cytokines (a) TNF-α, (b) IL-6, and (c) IL-1β in the culture supernatants of BV-2 microglia. Cells were stimulated with Angiotensin II (100 n M) in the presence or absence of AJ (6.25, 12.5 μ g/mL) or Minocycline. AJ treatment resulted in a dose-dependent reduction of all measured cytokines. Data are mean ± S.D. ( n = 3). Δ P < 0.05 vs. Control; * P < 0.05 vs. AngII Model.

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: Effects of AJ on core targets that regulate oxidative stress in BV-2 microglia. (a) Relative mRNA expression levels of STAT3 , JUN , ROCK2 , and CREB1 assessed by qRT-PCR. (b) Western blot analysis (left) and quantification (right) of protein expression for JUN, ROCK2, and CREB1. GAPDH was used as a loading control. AngII stimulation upregulated ROCK2 and JUN while downregulating CREB1 . AJ treatment significantly reversed these expression patterns in a dose-dependent manner but did not significantly alter STAT3 expression, suggesting specificity for the ROCK2-JUN/CREB1 axis. Data are mean ± S.D. ( n = 3). Δ P < 0.05 vs. Control; * P < 0.05 vs. AngII Model; # P < 0.05 comparing AJ doses.

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: AJ inhibits the activation of the RhoA/ROCK2 signaling pathway in AngII-induced BV-2 microglia. (a) Relative mRNA expression of ROCK2. (b) Western blot analysis of ROCK2 protein levels. (c) Activity assays for RhoA (GTP-bound fraction) and ROCK kinase activity. AngII significantly increased RhoA/ROCK2 activity. This activation was suppressed by AJ and the specific ROCK inhibitor Y-27632. Crucially, co-treatment with the ROCK agonist Lysophosphatidic Acid (LPA) abolished the inhibitory effect of AJ, restoring pathological signaling levels. Data are mean ± S.D. ( n = 3). Δ P < 0.05 vs. Control; * P < 0.05 vs. AngII; # P < 0.05 vs. AJ + AngII.

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Activity Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: The antioxidant efficacy of AJ is dependent on the suppression of the RhoA/ROCK2 axis. (a) Relative mRNA expression of NQO1 , Nox1 , and Nox4 . (b) Representative DHE staining images. (c) Representative MitoSOX staining images. (d) Quantification of DHE fluorescence. (e) Quantification of MitoSOX fluorescence. (f) Intracellular SOD activity and MDA levels. The protective effects of AJ on oxidative stress parameters (reduced ROS/MDA/Nox, increased SOD/NQO1) were significantly negated by the addition of the ROCK agonist LPA, confirming that AJ exerts its antioxidant effects via ROCK2 inhibition. Data are mean ± S.D. ( n = 3). Δ P < 0.05 vs. Control; * P < 0.05 vs. AngII Model.

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Expressing, Staining, Fluorescence, Activity Assay, Inhibition, Control

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: AJ attenuates microglial neuroinflammation via the regulation of ROCK2 signaling. ELISA quantification of (a) TNF-α, (b) IL-6, and (c) IL-1β levels in BV-2 culture supernatants. The anti-inflammatory effect of AJ (reduction in cytokine release) was significantly reversed by co-incubation with the ROCK agonist LPA, mirroring the oxidative stress results. This confirms that the anti-neuroinflammatory mechanism of Shinflavanone (AJ) is dependent on the blockade of the RhoA/ROCK2 pathway. Data are mean ± S.D. ( n = 3). Δ P < 0.05 vs. Control; * P < 0.05 vs. AngII Model.

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Frontiers in Pharmacology

Article Title: Anjiang formula inhibits PVN microglial activation and lowers blood pressure by targeting RhoA/ROCK2 pathway: a retrospective clinical and experimental study

doi: 10.3389/fphar.2026.1795297

Figure Lengend Snippet: AJ (Shinflavanone) rescues PVN microglia from AngII-induced oxidative storm via the RhoA/ROCK2 switch. Schematic representation of the proposed molecular mechanism. Under hypertensive conditions, Angiotensin II (AngII) binds to the AT1 receptor, activating the RhoA/ROCK2 signaling cascade. Active ROCK2 promotes the assembly of NADPH oxidases (NOX1/NOX4), leading to a surge in Reactive Oxygen Species (ROS). Concurrently, ROCK2 activation modulates nuclear transcription factors (upregulation of JUN, downregulation of CREB1), suppressing endogenous antioxidant defenses (NQO1/SOD). This “Oxidative-Inflammatory” cycle drives microglial activation and sympathetic outflow. Shinflavanone (the active component of AJ) permeates the blood-brain barrier and physically locks ROCK2 in an inactive state, thereby breaking the feed-forward loop, restoring redox homeostasis, and lowering blood pressure.

Article Snippet: ELISA kits for ACE (E-EL-R2401), REN (E-EL-R3075), AngII (E-EL-R0125), ALD (E-EL-0070), TNF-α (E-EL-R2856, E-EL-M3063), IL-6 (E-EL-R0015, E-EL-M0044), and IL-1β (E-EL-R0012, E-EL-M0037) were obtained from Elabscience Biotechnology Inc. (Wuhan, China).

Techniques: Activation Assay