Journal:

Article Title: Targeting the Tie2/Tek Receptor in Astrocytomas

doi:

Figure Lengend Snippet: Expression profile of Tie2 in human GBM specimens and explant xenografts. A: Immunohistochemical analysis of normal brain (NB), low-grade astrocytoma (LGA), and GBM for Tie2 demonstrates increasing Tie2 expression in the ECs with increasing grade of astrocytoma (arrowheads). B: Immunohistochemical characterization of human GBM explants grown as subcutaneous xenografts in NODSCID mice. Similar to human GBM specimens, the explants were GFAP-positive [GFAP (glial fibrillary acidic protein), which is an intermediate filament present in glial cells and used to characterize the astrocytic cell type origin of GBM tumors]. The explants expressed high levels of Tie2 on the vessel EC, high levels of Ang1 in the astrocytoma cells and Ang2 on the vessel ECs. C: Tie2 anti-phosphotyrosine immunoblot assay demonstrating increased Tie2 phosphorylation in both operative GBM specimens and tumor explants grown in NODSCID mice compared to control normal brain.

Article Snippet: Immunohistochemical analysis was performed on paraffin-embedded sections with antibodies to: Ki-67 (polyclonal rabbit no. A0047, 1:400; DAKO, Carpinteria, CA); Factor VIII (polyclonal rabbit no. A0082, 1:2500; DAKO); GFAP (polyclonal rabbit, 1:3000; DAKO); Ang1, Ang2, and Tie2 (polyclonal goat, 1:200,1:400; Santa Cruz); Tie2 (polyclonal rabbit, 1:400; Santa Cruz).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with scrambled-siRNA (sc-siRNA) or CHFR-siRNA. At 72 h post-transfection, cells stimulated with rAng1 (400 ng/ml) for different time intervals were used for IB analysis to determine phosphorylation of Akt1 at S473 and T308 ( n = 3 independent experiments). b Control HMEC and CHFR knockdown HMEC stimulated with rAng1 as above were stained with phospho-473-Akt1 specific antibody. c Control HMEC and CHFR knockdown HMEC were transfected with the Akt1 biosensor plasmid and then stimulated with rAng1 as above to assess live cell Akt1 activity by measuring the FRET ratio ( p <0.0001) ( left panel shows basal Akt activity; right panel shows Akt activity in response to rAng1 challenge). d-f Control HMEC and CHFR-siRNA treated HMEC stimulated with rAng1 were used for IB analysis to determine tyrosine phosphorylation Tie2 ( d ), phosphorylation of GSK3β at S9 and phosphorylation of β-catenin ( e ), ( n = 3 independent experiments). a, d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Mouse mAb against β-actin (catalog #A5441; IB, 1:2000) was from Sigma-Aldrich Inc. Rabbit pAb against GFP (catalog #50430-2-AP; IB, 1:2000), rabbit pAb against CHFR (catalog #12169-1-AP; IB, 1:1000), rabbit pAb against Angiopoietin-2 (catalog #24613-1-AP; IB, 1:1000), rabbit pAb against β-catenin (catalog #61067-2-AP; IB, 1:1000), and rabbit pAb against mCherry (catalog #26765-1-AP; IB, 1:1000) were from Proteintech.

Techniques: Transfection, Phospho-proteomics, Control, Knockdown, Staining, Plasmid Preparation, Activity Assay

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