I. Introduction

Obesity is a major risk factor for metabolic syndrome, type 2 diabetes, and cardiovascular disease, and its progression is tightly linked to structural and functional alterations within adipose tissue (Koenen et al. 2021). As adipose depots expand through hypertrophy and hyperplasia, the tissue requires coordinated vascular remodeling to maintain adequate oxygen and nutrient supply (Corvera et al. 2022). However, obesity disrupts this regulatory process, leading to pathological angiogenesis, increased vascular permeability, hypoxia, and chronic inflammation. These micro environmental changes further promote adipocyte dysfunction and systemic insulin resistance (Sun et al. 2011).

Macrophages play a central role in obesity-induced adipose tissue inflammation (Li et al. 2021). They undergo increased infiltration mediated through the CCL2-CCR2 chemokine axis, and phenotypic and functional reprogramming that enhances cytokine production, extracellular matrix remodeling, and angiogenic signaling. Both classically (M1) and alternatively activated (M2) macrophages expand in obese adipose tissue, creating a hyperactivated immune state that drives tissue remodeling rather than resolving inflammation (Boutens and Stienstra 2016). Thus, targeting macrophage activation and its downstream effects on vasculature may provide an effective strategy for restoring adipose tissue homeostasis (Weisberg et al. 2003;Chavakis et al. 2023).

Natural products have gained increasing attention as therapeutic agents due to their structural diversity and broad biological activities (Atanasov et al. 2021). Berberine, a plant-derived isoquinoline alkaloid, has been widely studied for its antimicrobial, anti-diabetic, and anti-inflammatory properties (Zhou et al. 2025;Yin et al. 2008;Wang et al. 2019). It regulates metabolic pathways, suppresses pro-inflammatory signaling, and modulates endothelial cell function (Rui et al. 2021;Kong et al. 2025). Additionally, it influences macrophage polarization and angiogenesis in various disease models (Liu et al. 2018;Zhu et al. 2019); however, its coordinated effects on immune and vascular remodeling within obese adipose tissue remain poorly understood. Moreover, whether berberine attenuates pathological angiogenesis indirectly through immune modulation or exerts direct vascular effects has not been fully clarified.

In this study, we investigated the effects of berberine on macrophage activation, vascular remodeling, and adipose tissue structure in a high-fat diet (HFD)-induced obesity model. By integrating morphological analysis, vascular permeability assays, and macrophage gene profiling, we aimed to determine whether berberine restores adipose tissue homeostasis through simultaneous regulation of immune and vascular remodeling.

II. Materials and methods

1. Animal experiments

Male C57BL/6 mice (8 weeks old) were purchased from Charles River Korea (Seoul, Republic of Korea). Mice were maintained under specific pathogen-free conditions at 22–26°C under a 12-h light/dark cycle with free access to food and water. All animal procedures were performed in accordance with the guidelines for the Care and Use of Laboratory Animals of the Korea Food Research Institute (KFRI) and approved by the Institutional Animal Care and Use Committee of KFRI (Approval No. KFRI-M-17056).

Mice were randomly assigned to three groups (n = 10 per group): (1) normal diet (ND), (2) high-fat diet (HFD), and (3) HFD plus berberine (HFD+BBR). The ND group received a standard chow diet containing 10% kcal from fat, whereas the HFD groups received a diet containing 60% kcal from fat for 8 weeks. Berberine was administered orally by gavage at a dose of 50 mg/kg throughout the experimental period. A previous study investigating the effects of berberine in HFD-induced obese mice was considered during the study design (Ye et al., 2016). Body weight and food intake were monitored weekly. Mice were sacrificed after 8 weeks, and epididymal adipose tissues were collected for further analyses.

2. Histopathological analysis

Epididymal adipose tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4-µm-thick slices. Tissue sections were stained with hematoxylin and eosin (H&E) for histological evaluation. For immunohistochemical analysis, sections were deparaffinized in xylene, rehydrated through graded ethanol, and blocked for 1 h. Slides were incubated overnight at 4°C with anti-ER-HR3 antibody (BMA, Augst, Switzerland). After washing, sections were treated with AEC substrate-chromogen solution (DakoCytomation, Glostrup, Denmark) to visualize immunoreactive signals.

Images were obtained using a Nikon Eclipse 80i light microscope (Nikon Instruments Inc., Melville, NY, USA). ER-HR3-positive macrophages were quantified in 10 randomly selected fields at ×400 magnification using ImageJ software (NIH, Bethesda, MD, USA).

3. Whole mount fluorescent immunohistochemistry

Whole-mount adipose tissues were incubated for 1 h at room temperature in blocking solution containing 5% donkey serum (Jackson ImmunoResearch Laboratories Inc.) in PBST (0.3% Triton X-100 in PBS). Tissues were then incubated overnight at 4°C with the following primary antibodies: hamster anti-mouse CD31 monoclonal antibody (1:1000; Millipore) for blood vessels, anti-mouse F4/80 antibody (1:1000; eBioscience) for macrophages, and anti-rabbit LYVE-1 antibody (1:1000; AngioBio) for lymphatic structures.

After washing with PBST, tissues were incubated for 1 h at room temperature with the corresponding secondary antibodies: Cy3-conjugated anti-hamster antibody, Cy5-conjugated anti-rat antibody, or Cy5-conjugated anti-rabbit antibody (all diluted 1:2000; Jackson ImmunoResearch Laboratories).

For fluorescent staining, tissues were additionally incubated with BODIPY 493/503 (1 µg/mL; Invitrogen, Carlsbad, CA, USA) to visualize neutral lipids and DAPI (1 µg/mL; Invitrogen) to stain nuclei. Fluorescent images were acquired using a confocal fluorescence microscope.

4. Quantitative real-time PCR

Total RNA was isolated from epididymal adipose tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Quantitative real-time PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.

Relative gene expression levels were normalized to GAPDH. The expression of macrophage-related inflammatory markers, including TNF-α, IL-1β, CD11c, CCL2, arginase-1, CD206, Fizz1, and TGF-β1, was subsequently analyzed.

5. Enzyme-linked immunosorbent assay (ELISA)

MCP-1 protein levels in epididymal adipose tissue were measured using a DuoSet sandwich ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instructions.

6. Vascular permeability analysis

Vascular permeability was evaluated using Evans blue dye (30 mg/kg; Sigma-Aldrich). A 1% Evans blue solution in 0.9% NaCl was intravenously injected into the tail vein (40 µL per 20 g body weight). After 30 min, epididymal adipose tissues were harvested and photographed. Tissues were incubated in formamide at 55°C for 18 h, followed by centrifugation at 12,000 × g for 20 min. Absorbance of the supernatant was measured at 620 nm to quantify Evans blue extravasation.

7. Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. Differences were considered statistically significant at P < 0.05.

III. Results and Discussion

1. Berberine attenuates adipocyte hypertrophy and macrophage infiltration in HFD-induced obese mice

To evaluate the effects of berberine on obesity-associated adipose tissue remodeling, histological and immunohistochemical analyses were performed using epididymal adipose tissue from HFD-fed mice. H&E staining revealed marked adipocyte hypertrophy in the HFD group compared with the normal diet (ND) group, whereas berberine treatment significantly reduced adipocyte enlargement (Figure 1A). Quantitative analysis showed that adipocyte diameter increased approximately six-fold in HFD-fed mice and was markedly reduced by berberine treatment (Figure 1B).

HFD feeding also significantly increased ER-HR3-positive macrophage infiltration in epididymal adipose tissue, whereas berberine treatment reduced macrophage accumulation to near-control levels (Figure 1C, 1D). In addition, berberine partially attenuated HFD-induced body weight gain during the 8-week experimental period (Figure 1E). These findings suggest that berberine alleviates obesity-associated adipose tissue remodeling by suppressing adipocyte hypertrophy and inflammatory macrophage infiltration.

2. Berberine attenuates obesity-associated vascular remodeling in epididymal adipose tissue

Whole-mount immunostaining revealed that HFD feeding markedly increased adipocyte size and PECAM-1-positive vascular structures in epididymal adipose tissue. Adipocytes in HFD-fed mice displayed pronounced hypertrophy compared with that in the ND group, whereas berberine treatment significantly reduced adipocyte diameter (Figure 2A, 2B).

HFD feeding also induced a substantial expansion of PECAM-1-positive endothelial structures, indicative of obesity-associated pathological angiogenesis. Berberine markedly attenuated this increase (Figure 2A, 2C). LYVE-1-positive cells were detected in all groups; however, their morphology and distribution were unchanged after berberine treatment and consistent with LYVE-1-expressing macrophages but not lymphatic endothelial cells (Figure 2A, 2D).

3. Berberine suppresses macrophage activation in obese adipose tissue

Whole-mount immunofluorescence staining revealed a marked increase in F4/80-positive macrophages in epididymal adipose tissue from HFD-fed mice compared with the ND group (Figure 3A, 3B). These macrophages were predominantly localized around PECAM-1-positive vascular structures, indicating close association between macrophage accumulation and obesity-associated vascular remodeling. Berberine treatment markedly reduced F4/80-positive macrophage accumulation in obese adipose tissue.

4. Berberine suppresses macrophage-associated inflammatory responses in obese adipose tissue

To investigate the effects of berberine on obesity-associated inflammatory responses, macrophage-related cytokines and marker genes were analyzed in epididymal adipose tissue. ELISA analysis demonstrated that MCP-1 protein levels were markedly elevated in HFD-fed mice compared with the ND group, whereas berberine treatment significantly reduced MCP-1 production (Figure 4A). Consistently, HFD feeding significantly increased the expression of M1-associated inflammatory markers, including TNF-α, IL-1β, CD11c, and CCL2, all of which were markedly suppressed by berberine treatment (Figure 4B–F).

In addition to M1-associated genes, HFD feeding also upregulated M2-associated macrophage markers, including arginase-1, Fizz1, CD206, and TGF-β1 (Figure 4G–J). Berberine treatment broadly reduced the expression of these markers, indicating that berberine suppresses obesity-associated macrophage hyperactivation rather than inducing a simple M1-to-M2 polarization shift. These findings suggest that berberine attenuates adipose tissue inflammation through broad regulation of macrophage-associated inflammatory responses.

5. Berberine decreases vascular permeability in obese adipose tissue

Evans blue dye extravasation revealed marked vascular leakage in the epididymal adipose tissue of HFD-fed mice, indicating obesity-associated endothelial dysfunction (Figure 5A, 5B). Berberine treatment significantly reduced Evans blue accumulation, suggesting improved vascular barrier integrity and endothelial stabilization under obese conditions.

Notably, the reduction in vascular leakage was accompanied by decreased PECAM-1-positive vascular structures, indicating that berberine suppresses pathological vascular remodeling associated with obesity-induced adipose tissue inflammation. These findings suggest that berberine exerts protective effects on adipose tissue vasculature by attenuating both endothelial hyperpermeability and abnormal vascular expansion.

In this study, we demonstrated that berberine mitigates obesity-induced adipose tissue inflammation by suppressing excessive macrophage activation and stabilizing pathological vascular remodeling. HFD feeding led to pronounced adipocyte hypertrophy, increased PECAM-1-positive vasculature, and robust macrophage infiltration in epididymal adipose tissue. Berberine effectively reversed these changes, indicating its role in attenuating obesity-associated tissue remodeling. Notably, it reduced the expression of both M1-related cytokines (e.g., TNF-α, IL-1β, and CCL2) and M2 markers (e.g., arginase-1, CD206, and Fizz1). These findings suggest that berberine broadly suppresses macrophage-associated inflammatory responses in obese adipose tissue.

Berberine also significantly reduced vascular permeability and PECAM-1-positive vascular structures. Obesity induces pathological angiogenesis in adipose tissue, driven in part by macrophage-derived factors, such as VEGF and MMPs. Thus, the reduction in vascular density and leakage observed in this study may reflect an indirect anti-angiogenic effect mediated through the suppression of macrophage-derived vascular signals. The causal relationship between macrophage suppression and improved endothelial integrity remains to be fully clarified. In obese adipose tissue, macrophage-derived inflammatory mediators can promote endothelial activation, vascular leakage, and pathological vascular remodeling (Koenen et al., 2021;Li et al., 2021). Therefore, the reduction in macrophage accumulation and inflammatory marker expression observed after berberine treatment may indirectly contribute to improved vascular integrity by attenuating macrophage–endothelial inflammatory crosstalk. In addition, previous studies have reported that berberine suppresses macrophage inflammatory signaling through AMPK- and NF-κB-related pathways (Jeong et al., 2009;Liu at al., 2018) and can directly improve endothelial function in vascular disease models (Rui et al., 2021). Thus, the vascular protective effects observed in the present study may reflect both reduced macrophage-associated inflammation and direct modulation of endothelial responses by berberine. Furthermore, LYVE-1-positive cells observed in adipose tissue displayed morphology consistent with LYVE-1-expressing macrophages rather than lymphatic endothelial cells. LYVE-1-positive macrophages contribute to extracellular matrix remodeling, debris clearance, lipid handling, and local tissue homeostasis. Therefore, their limited response to berberine warrants further investigation.

Interestingly, berberine reduced the expression of both M1- and M2-associated macrophage markers in adipose tissue. This pattern may not simply indicate a shift in macrophage polarization but rather reflect a reduction in macrophage infiltration and/or an overall attenuation of macrophage activation. Consistent with this possibility, previous studies have demonstrated that berberine suppresses inflammatory signaling pathways in macrophages, including AMPK-, SIRT1-, and NF-κB-mediated signaling, thereby reducing the production of pro-inflammatory mediators (Jeong et al., 2009). Therefore, the observed decrease in both M1- and M2-associated markers may represent a broader suppression of obesity-associated macrophage activation within adipose tissue.

Previous studies have shown that berberine exerts anti-inflammatory and metabolic regulatory effects through multiple signaling pathways. In addition to suppressing NF-κB-mediated inflammatory responses, berberine activates AMPK, a key regulator of cellular energy homeostasis, thereby improving insulin sensitivity and metabolic dysfunction in obesity. Berberine has also been reported to modulate MAPK signaling pathways and regulate the production of inflammatory cytokines in macrophages and other metabolic tissues (Jeong et al., 2009;Zhou et al., 2025;Liu et al., 2018). These pleiotropic actions may contribute to the reductions in macrophage accumulation and adipose tissue remodeling observed in the present study. Although these pathways were not directly examined in the present study, they may contribute to the beneficial effects of berberine on macrophage-associated inflammation and adipose tissue remodeling.

Importantly, stabilization of the adipose vasculature may contribute to improved adipose tissue function. Excessive vascular remodeling and vascular leakage are closely associated with chronic inflammation and adipose tissue dysfunction during obesity. Therefore, the reduction in vascular density and permeability observed following berberine treatment may reflect an improvement in obesity-associated vascular abnormalities. However, the functional consequences of these vascular changes require further investigation. Previous studies have reported that berberine can either inhibit or promote angiogenesis depending on the pathological context. Its anti-angiogenic effects, mediated through inhibition of VEGF/VEGFR2 signaling, have been described in tumor and metabolic disease models. Conversely, berberine enhances angiogenesis and tissue repair in ischemic models through AMPK activation and M2-like macrophage polarization. These findings suggest that berberine attenuates obesity-associated vascular remodeling in adipose tissue. Taken together, the present findings support a beneficial role of berberine in improving obesity-associated alterations in adipose tissue vasculature.

Although food intake tended to be lower in the HF+BBR group, no significant difference was observed between the HF and HF+BBR groups. Therefore, the beneficial effects of berberine on adipose tissue remodeling are unlikely to be explained solely by reduced food intake. Nevertheless, the contribution of body weight changes to the observed improvements cannot be completely excluded. Further studies will be required to distinguish the direct effects of berberine on adipose tissue remodeling from secondary metabolic consequences associated with reduced weight gain.

IV. Summary and Conclusion

In conclusion, our findings show that berberine alleviates adipose tissue inflammation by reducing pathological vascular remodeling and broadly suppressing macrophage activation rather than inducing a simple polarization shift. These results highlight the therapeutic potential of berberine in obesity and metabolic dysfunction, and support its further investigation in vascular and immune-mediated diseases.