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

Figures

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  FIGURE 1. Clinical efficacy of Anjiang Formula (AJ) and improvement in autonomic phenotypes. (a) Detailed CONSORT flow diagram illustrating the screening, randomization, and allocation of elderly hypertensive patients (N = 85) into the AJ treatment group (n = 43) and the Lifestyle Control group (n = 42). (b) Raincloud plots depicting the distribution and probability density of the change in Systolic Blood Pressure (ΔSBP) from baseline to week 8. The box plots indicate the median and interquartile range (IQR). AJ treatment resulted in a significantly greater mean reduction in SBP compared to control (Mean difference: 10.2 mmHg; P < 0.001). (c) Waterfall plot of individual therapeutic responses. Each bar represents a single patient. The dashed line indicates the responder cutoff (>10 mmHg reduction). The responder rate was significantly higher in the AJ group (93%) compared to the control group (62%). (d) Forest plot of subgroup analyses stratified by age, sex, baseline BP severity, diabetes status, and BMI. Data are presented as mean difference with 95% Confidence Intervals (CI). P-values for interaction indicate consistency of the treatment effect across all subgroups. Statistical…

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  FIGURE 2. AJ improves vascular endothelial function and arterial stiffness in hypertensive patients. (a) Flow chart summarizing the imaging sub-cohort (N = 85). (b–e) Longitudinal changes in vascular and cardiac parameters from baseline to week 8. Paired dot plots show individual participant trajectories; bold lines represent the group means. (b) Flow-Mediated Dilation (FMD) significantly improved in the AJ group (+1.3% net difference; P < 0.01). (c) Pulse Wave Velocity (PWV), a marker of arterial stiffness, decreased significantly with AJ treatment (−125 cm/s net difference; P < 0.05). (d) Carotid Intima-Media Thickness (cIMT) and (e) Cardiac indices (Left Ventricular Mass Index [LVMI] and E/e' ratio) showed non-significant trends toward improvement (P > 0.05). (f) Intraclass Correlation Coefficients (ICC) table confirming high reproducibility for the blinded re-read of imaging metrics. (g) Representative ultrasound images for FMD, PWV, cIMT, and echocardiography at baseline and week 8. Data are presented as mean ± S.D. P-values were calculated using Linear Mixed-Effects Models.

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  FIGURE 3. AJ reprograms the systemic inflammatory-oxidative axis and suppresses ROCK2 activity. (A-D) Longitudinal analysis of serum biomarkers measured at baseline and week 8. Gray lines indicate individual patient data; colored lines indicate group means with 95% CIs. (A) Serum Interleukin-6 (IL-6) levels decreased significantly in the AJ group compared to control (β GT = −0.90 pg/mL; P = 0.0003). (B) Serum 8-iso-PGF2α, a marker of lipid peroxidation, was significantly reduced by AJ treatment (β GT = −45 pg/mL; P = 0.008). (C) Serum Superoxide Dismutase (SOD) activity increased, indicating restored antioxidant capacity (β GT = +19 U/mL; P = 0.012). (D) Peripheral Blood Mononuclear Cell (PBMC) ROCK2 protein levels were significantly suppressed in the AJ group (β GT = −0.26 ng/mL; P = 0.035). β GT represents the Group × Time interaction coefficient derived from Linear Mixed-Effects Models.

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  FIGURE 4. Systemic inflammatory dynamics predict autonomic recovery and therapeutic success. (a) Partial correlation matrix (heatmap) visualizing the associations between changes in systemic biomarkers (Δ) and changes in physiological parameters. Red indicates positive correlation; blue indicates negative correlation. Numbers represent Spearman’s rho coefficients. ΔIL-6 is significantly correlated with ΔSBP-CV (Coefficient of Variation). (b) Standardized regression coefficients showing independent predictors of improved Blood Pressure Variability (ΔSBP-CV) and Heart Rate Variability (ΔSDNN). Reductions in IL-6 and 8-iso-PGF2α are strong predictors of autonomic stabilization. (c) Scatter plots with linear regression lines highlighting the key associations: ΔIL-6 vs. ΔSBP-CV (R 2 = 0.20, p < 0.01) and ΔIL-6 vs. ΔSDNN (R 2 = 0.15, p < 0.05). (d) Multivariable logistic regression forest plot identifying predictors of achieving “Blood Pressure Control” (<140/90 mmHg) at Week 8. AJ treatment, ΔIL-6 reduction, and ΔROCK inhibition are significant independent predictors. Data are Odds Ratios (OR) with 95% CIs.

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  FIGURE 5. Shinflavanone directly engages the ROCK2 kinase domain: Structural basis and biophysical kinetics. (a) Molecular docking simulation showing the predicted binding mode of Shinflavanone (yellow sticks) within the ATP-binding pocket of the ROCK2 kinase domain (PDB: 4WZG). Key hydrogen bond interactions with residues Met172, Asp185, and Lys121 are indicated by yellow dashed lines. (b) Surface Plasmon Resonance (SPR) sensorgrams demonstrating the binding kinetics of Shinflavanone to immobilized recombinant ROCK2. Curves represent varying concentrations (3.125–50 μM). The calculated equilibrium dissociation constant (K D) is 20.0 nM (c) Cellular Thermal Shift Assay (CETSA) validating target engagement in intact cells. Western blots (top) and melting curves (bottom) show that Shinflavanone (10 μM) increases the thermal stability of endogenous ROCK2 (Tm shift = +5.2 °C) compared to DMSO control. (d) Microscale Thermophoresis (MST) analysis confirming the binding affinity in solution (K D = 22.5 ± 3.1 nM). Data in c and d are mean ± S.D. of n = 3 independent experiments.

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  FIGURE 6. AJ reduces blood pressure and ameliorates pathological changes in the aorta and heart. (a) Time-course measurements of Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), and Mean Arterial Pressure (MAP) in Spontaneously Hypertensive Rats (SHRs) over 10 weeks of treatment. Groups: Control (WKY), Model (SHR + vehicle), AJ-L (8.1 g/kg), AJ-H (16.2 g/kg), and Minocycline (positive control). AJ treatment dose-dependently lowered BP compared to the Model group ( Δ P < 0.05 vs. Control; *P < 0.05 vs. Model). (b) Representative Hematoxylin and Eosin (H&E) staining of thoracic aorta sections showing reduced wall thickening in AJ-treated groups. Scale bar, 50 μm. (c) Representative H&E staining of left ventricular myocardium showing attenuated cardiomyocyte hypertrophy and disarray. Scale bar, 100 μm. Data in a are mean ± S.D. (n = 5 rats per group). Statistical significance determined by repeated measures ANOVA.

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  FIGURE 7. 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 μm. (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).

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  FIGURE 8. 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 μm. (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 μg/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.

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  FIGURE 9. 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 nM) in the presence or absence of AJ (6.25, 12.5 μg/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.

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  FIGURE 10. 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.

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  FIGURE 11. 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.

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  FIGURE 12. 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.