Application of DHA-RXR-PPAR signal pathway in promotion of post-HIFU postoperative recovery of hysteromyoma

Abstract

The invention discloses an application of a DHA-RXR-PPAR signal channel in promotion of postoperative recovery of hysteromyoma after HIFU (high intensity focused ultrasound). Clinical sample metabolic spectrum analysis and in-vitro cell function experiments are combined, and research proves that the differential metabolite DHA drives macrophage metabolism reprogramming through an RXR-PPAR signal axis, so that the macrophage is promoted to be transformed into an anti-inflammatory and phagocytosis-promoting repair phenotype, the absorption and repair process of the HIFU postoperative hysteromyoma is accelerated, and the treatment effect of the HIFU postoperative hysteromyoma is improved. A new intervention target spot (RXR-PPAR gamma axis) and a new potential medicine are provided for HIFU (high intensity focused ultrasound) postoperative rehabilitation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of a DHA-RXR-PPAR signaling pathway in promoting postoperative recovery after HIFU treatment for uterine fibroids. Background Technology

[0002] Uterine fibroids are the most common benign pelvic tumors in women. The incidence of leiomyomas in perimenopausal women is as high as 70%. Uterine fibroids are considered a typical fibrotic disease. Although the etiology is not fully understood, one model suggests that fibroid formation may be a result of excessive wound healing, an abnormal healing process driven by dysregulation of the inflammatory process.

[0003] High-intensity focused ultrasound (HIFU) is a non-invasive local treatment technique. Its principle is to concentrate externally emitted ultrasound energy onto the lesion, causing irreversible coagulative necrosis and inactivating the tumor tissue through thermal, mechanical, and cavitation effects. HIFU provides a safer and more effective treatment option for patients with uterine fibroids, featuring non-invasiveness, no scarring, repeatable treatment, no radiation, precise one-time full-coverage thermal ablation, few complications, and short treatment time. Furthermore, it has no significant impact on ovarian endocrine function, and the clinical application of HIFU tumor ablation is becoming increasingly widespread.

[0004] Studies have shown that HIFU ablation of tumors produces a significant immune inflammatory response related to the ablation process, manifested as the infiltration of immune cells around the ablated lesion. Research has also monitored significant metabolic changes in cortisol, endorphins, and prostaglandins after HIFU, suggesting that HIFU treatment affects inflammation and homeostasis. The inflammatory response after ablation is a dynamic process; as necrotic tissue is cleared and repaired, the inflammatory response gradually weakens and stabilizes, laying a good foundation for the body's full recovery.

[0005] Following HIFU ablation, necrotic tissue can induce the infiltration and activation of numerous immune cells, particularly macrophages. Macrophages, as key members of the innate immune system, possess high plasticity and can polarize into either the pro-inflammatory M1 subpopulation or the anti-inflammatory and repair-promoting M2 subpopulation under different signal stimuli. Studies have shown that endogenous signals released from necrotic tissue can induce macrophage activation, polarization, and metabolic remodeling. In particular, M2 macrophages play a crucial role in tissue repair, inflammation suppression, and lesion clearance, and their function depends on enhanced fatty acid oxidation and mitochondrial metabolism.

[0006] Metabolic reprogramming, a key mechanism for macrophage phenotypic transformation, has received widespread attention in recent years. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, include three isoforms: PPARα, PPARβ / δ, and PPARγ. They regulate transcription by forming heterodimers with retinoid X receptors (RXR) and binding to PPAR response elements (PPREs) of target genes. PPARγ is an important transcription factor regulating fatty acid metabolism and oxidative phosphorylation, promoting M2 polarization. RXR (retinoic acid X receptor), as its heterodimer binding partner, works with PPARγ to regulate the expression of downstream target genes, forming the RXR-PPAR signaling axis. Previous studies have confirmed that activation of this pathway not only enhances fatty acid uptake and oxidation but also drives the shift from glycolysis to aerobic metabolism, thereby inducing macrophage transformation to the M2 phenotype. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned problems by providing an application of the DHA-RXR-PPAR signaling pathway in promoting postoperative recovery after HIFU treatment for uterine fibroids.

[0008] To achieve its objective, the present invention employs the following technical solution:

[0009] The first aspect of the present invention provides the application of the DHA-RXR-PPAR signaling pathway as a target in screening drugs that promote postoperative recovery after HIFU for uterine fibroids.

[0010] In the above-mentioned application technology solution, the drug upregulates the DHA level of subjects after HIFU treatment for uterine fibroids.

[0011] In the above-mentioned application technology, the drug upregulates the expression levels of PPARγ and RXRα in macrophages, promotes their phagocytic function, and promotes the shrinkage of uterine fibroids and postoperative recovery.

[0012] In the above-mentioned application technology, DHA mediates the regulation of macrophage energy metabolism through the RXR-PPAR signaling pathway, thereby affecting its functional state.

[0013] Furthermore, DHA drives macrophage metabolic reprogramming through the RXR-PPAR signaling axis, thereby promoting their transformation into an anti-inflammatory, pro-phagocytic repair phenotype and accelerating the absorption and repair process of uterine fibroids after HIFU surgery.

[0014] A second aspect of the invention provides the use of a promoter of the DHA-RXR-PPAR signaling pathway in the preparation of a product for promoting postoperative recovery after HIFU treatment for uterine fibroids.

[0015] The application involves driving the metabolic reprogramming of macrophages after HIFU treatment for uterine fibroids through promoters of the DHA-RXR-PPAR signaling pathway, thereby promoting their transformation into an anti-inflammatory, pro-phagocytic repair phenotype and accelerating the absorption and repair process of uterine fibroids after HIFU treatment.

[0016] The promoter of the DHA-RXR-PPAR signaling pathway is any one of the following:

[0017] (1) A promoter of DHA;

[0018] (2) RXR promoter;

[0019] (3) PPAR promoters;

[0020] (4) Promoters of the RXR-PPAR signaling pathway.

[0021] A third aspect of the invention provides the use of docosahexaenoic acid in the preparation of a medicament for promoting postoperative recovery after HIFU treatment for uterine fibroids.

[0022] A fourth aspect of the present invention provides the use of biomaterials containing docosahexaenoic acid (DHA) or substances that promote DHA expression in subjects in the preparation of a medicament for promoting postoperative recovery after HIFU treatment for uterine fibroids.

[0023] The beneficial effects of this invention are:

[0024] This invention, through the integration of clinical cohort analysis and molecular and cell biology experiments, systematically elucidates the crucial role and molecular mechanism of the differentially expressed metabolite docosahexaenoic acid (DHA) in the repair process of uterine fibroids after ultrasound ablation (HIFU). By comparing the metabolomics profiles of serum from patients with good and slow repair after HIFU, we discovered for the first time that DHA is one of the most significantly different metabolites, and its level is significantly positively correlated with the rate of fibroid absorption after surgery. This clinical clue suggests that DHA may not only be a simple nutritional indicator, but also a key signaling molecule that actively regulates the tissue repair process.

[0025] To verify the above hypothesis, we constructed a human macrophage model in vitro and conducted a series of loss-of-function experiments. The study confirmed that the differential metabolite DHA drives macrophage metabolic reprogramming through the RXR-PPAR signaling axis, thereby promoting its transformation into an anti-inflammatory, pro-phagocytic repair phenotype and accelerating the absorption and repair process of uterine fibroids after HIFU.

[0026] This invention not only reveals a novel mechanism by which DHA regulates macrophage function, but more importantly, it connects clinical metabolomics phenomena with precise molecular pathways and well-defined pathophysiological outcomes, providing new biomarkers (serum DHA) and potential intervention targets (RXR-PPARγ axis) for post-HIFU rehabilitation. This lays a solid theoretical foundation for developing adjuvant therapy strategies based on nutritional metabolic regulation to improve tissue repair quality after HIFU and other minimally invasive surgeries.

[0027] This invention provides new targets and potential new drugs for screening drugs that promote postoperative recovery after HIFU treatment for uterine fibroids. Attached Figure Description

[0028] Figure 1 The study showed metabolic changes at three time points before and after HIFU ablation, and preliminarily screened out DHA, a metabolite significantly associated with fibroid shrinkage.

[0029] Figure 2 The results showed: A. The effect of metabolite concentration gradients on macrophage proliferation was detected using the CCK-8 assay; B. Changes in the morphology of M0 giant cells (HE staining); C. Phagocytic function was assessed by neutral red phagocytosis assay and acid phosphatase activity assay.

[0030] Figure 3 The results of detecting the surface expression of CD206, IL-10, and TNF-α after DHA treatment.

[0031] Figure 4 The results show the fatty acid oxidation assay after DHA / GLA intervention in macrophages.

[0032] Figure 5 The results show the detection results of CPT1A, the rate-limiting enzyme of fatty acid β-oxidation, and PFKFB3, a key enzyme of glycolysis, in macrophages after DHA treatment.

[0033] Figure 6 This is a heatmap for metabolomics analysis.

[0034] Figure 7 The study showed that 89 metabolites were significantly altered in extracellular, intracellular, and cell residue samples after DHA intervention.

[0035] Figure 8 The string diagram shows the KEGG metabolic pathway associated with metabolites that were significantly altered after DHA treatment (p < 0.05).

[0036] Figure 9 The differentially expressed genes were shown to be primarily associated with cholesterol binding, lipid transport activity, and cytokine receptor binding.

[0037] Figure 10 The KEGG pathway enrichment analysis revealed significantly enriched differentially expressed genes in the pathway.

[0038] Figure 11 The study shows that DHA treatment induces a characteristic correlation pattern (A) between fatty acid-related metabolites and key transcriptional regulators of the PPAR signaling pathway, and constructs a metabolite-gene correlation network (B).

[0039] Figure 12 The results showed that DHA treatment significantly upregulated the mRNA levels of PPARγ and RXRα.

[0040] Figure 13 The results of immunofluorescence localization assay were used to detect the expression levels of PPARγ and RXRα in DHA-treated macrophages.

[0041] Figure 14 The optimal IC50 concentration of the inhibitors was shown for detecting changes in macrophage proliferation under different concentration gradients of RXR-α (UVI 3003) and PPAR-γ (GW 9662) inhibitors using the CCK-8 assay.

[0042] Figure 15 The results show changes in macrophage phagocytic capacity as determined by the neutral erythropoietin assay and ACP acid phosphatase assay.

[0043] Figure 16 The results of ELISA detection of changes in inflammatory factors (IL-6, IL-10, TNF-α) secreted by macrophages. Detailed Implementation

[0044] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0046] Example 1: Clinical sample testing after HIFU treatment for uterine fibroids

[0047] This study recruited eligible healthy volunteers and uterine fibroid (UF) patients who underwent high-intensity focused ultrasound (HIFU) treatment at the Chongqing HIFU Hospital between April 2022 and February 2025. All participants signed informed consent forms before enrollment. The inclusion criteria were as follows: age 23-45 years, confirmed diagnosis, scheduled HIFU ablation for uterine fibroids, and voluntary participation after providing informed consent. Participants diagnosed with malignant lesions, severe comorbidities (such as cardiovascular disease, liver or kidney dysfunction), or pregnancy were excluded.

[0048] This study has been approved by the Ethics Committee of HIFU Hospital in Chongqing, China (approved protocol number: 2022006) and the Chinese Clinical Trial Registry (ChiCTR-IOR-16007700), and complies with the relevant provisions of the Declaration of Helsinki.

[0049] 1. Clinical sample collection

[0050] Plasma samples were collected from patients with uterine fibroids at the following time points: 1 day before high-intensity focused ultrasound (HIFU) treatment (UF), 1 day after treatment (HIFU-1D), 3 months after treatment (HIFU-3M), and 6 months after treatment (HIFU-6M). A professional nurse collected 10 mL of peripheral blood from each participant using two blood collection tubes containing heparin sodium anticoagulant (EDTA). Within 2 hours of collection, plasma was prepared by centrifugation at 3000 rpm for 10 minutes at room temperature and quickly transferred to a -80°C freezer for long-term storage. Approximately 5 mL of random midstream urine was collected and placed in a sterile urine collection tube, stored in a 4°C incubator, and delivered to the laboratory within 1 hour. The urine was centrifuged at 10000 g for 10 minutes at 4°C, and the supernatant was aliquoted into 1.5 mL cryovials, sealed, and then stored at -80°C. Hair samples were collected from the back of the patient's head. They were cut approximately 0.5 cm from the scalp using sterilized medical scissors, wrapped in medical foil, placed in a sealed bag, and stored at -20°C for subsequent analysis.

[0051] 2. MRI Data Collection

[0052] MRI examinations were performed using a 1.5T MRI system (United Imaging Healthcare Co., Ltd., Shanghai, China). Image acquisition time points were: 1 day before high-intensity focused ultrasound (HIFU) treatment (UF), 1 day after treatment (HIFU-1D), 3 months after treatment (HIFU-3M), and 6 months after treatment (HIFU-6M). Typical scanning parameters were: T2-weighted imaging with TR 4692 ms / TE 75 ms / slice thickness 5 mm / slice spacing 1 mm; T1-weighted imaging with TR 196 ms / TE 8.18 ms / slice thickness 6 mm / slice spacing 1.2 mm; and enhanced T1-weighted imaging with TR 4 ms / TE 2 ms / slice thickness 5 mm / slice spacing 1.2 mm. The volume of the fibroid was calculated by automatically delineating the fibroid layers and non-perfused areas using the MicroSea-HIFU integrated 3D imaging system (Chongqing MicroSea Software Development Co., Ltd., Chongqing, China). The preoperative fibroid volume (FV) and the volume at six months after HIFU (FV6M) were measured using T2-weighted imaging (T2WI), and the fibroid volume reduction rate (FVS) was calculated using the formula (FV-FV6M) / FV.

[0053] 3. Metabolite extraction and MCF derivatization

[0054] Frozen samples were thawed on ice. 150 μL of each sample was thoroughly mixed with 254 μL of pre-cooled methanol / sodium hydroxide solution (1:1), containing 0.3 μM internal standards 2,3,3,3-d4-alanine, DL-phenylalanine (cyclic d5), and DL-tyrosine-3,3-d2. After vortexing for 30 seconds, the mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was transferred to clean glass tubes for derivatization. The derivatization process was performed in steps: 34 μL of pyridine and 20 μL of methyl chloroform (MCF) were added to each tube sequentially, and the mixture was vortexed for 30 seconds. Then, 20 μL of MCF was added and the mixture was vortexed again. Immediately afterwards, 400 μL of chloroform was added to extract the derivatized metabolites, and the mixture was vortexed for 10 seconds. The organic phase was then separated by adding 400 μL of 50 mM sodium bicarbonate solution, followed by vortexing and centrifugation at 2000 rpm for 10 minutes at 4 °C. The upper aqueous phase and interfacial layer were carefully removed, and the remaining chloroform phase was dried with 5 mg of anhydrous sodium sulfate. Finally, 150 μL of the dehydrated organic phase was transferred to an automated sample vial (Agilent, USA) for gas chromatography-mass spectrometry analysis.

[0055] 4. Gas chromatography-mass spectrometry analysis

[0056] All derivatized metabolites were analyzed using a BD-1701 capillary column (30 m × 250 μm × 0.25 μm, Agilent Technologies, USA) and an Agilent Intuvo 9000-5977B gas chromatograph-mass spectrometer (USA). The electron impact ionization (EI) energy was set to 70 eV. The injection port temperature was set to 300 °C, and a pulsed splitless injection mode was used. Helium was used as the carrier gas at a constant flow rate of 0.8 mL / min. The temperatures of the guard and auxiliary chips were maintained at 300 °C. The quadrupole temperature was set to 230 °C, and the ion source temperature was set to 150 °C. Mass spectrometry data were acquired in full scan mode, with a mass number range of 38–550 and a scan rate of 2.9 scans / second. To avoid solvent interference, data acquisition was initiated 4.5 minutes after injection. After each run, the gas chromatographic column was backflushed at 300 °C for 4 minutes to remove residual compounds.

[0057] Raw gas chromatography-mass spectrometry (GC-MS) data were processed using Agilent MassHunter software for peak detection and retention time alignment. Metabolite deconvolution and identification were performed using the Automated Mass Spectrometry Deconvolution and Identification System (AMDIS). For unidentified metabolites, provisional identification results were further confirmed using the NIST mass spectrometry database. Metabolite quantification was based on the peak height of the most abundant fragment ions, and annotation was performed using a MassOmics script implemented in R. Data standardization was performed using internal standards and median centering based on quality control (QC) samples to correct for batch effects. The final dataset (including retention time, peak area, and ionic intensity) was exported for statistical analysis.

[0058] Partial least squares discriminant analysis (PLS-DA) was used for multivariate statistical analysis to visualize clustering and between-group separation. Student's t-test with false discovery rate (FDR) correction was applied to identify significantly different metabolites. Principal component analysis (PCA) was performed using MetaboAnalyst 3.0 software to compare metabolomic profiles among different groups. Metabolites were identified using enrichment analysis based on the KEGG pathway database. Hierarchical clustering and heatmaps were used to visualize metabolic patterns. Longitudinal variations in metabolite levels were assessed using repeated measures ANOVA. All visualizations (including heatmaps, line graphs, and chord plots) were generated using R packages such as ggplot2 and GOplot.

[0059] 5. Results

[0060] To investigate the changes in metabolic status over time after HIFU ablation of uterine fibroids, we conducted a longitudinal analysis of plasma metabolite changes at different time points post-procedure in patients with uterine fibroids from different efficacy groups. The plasma metabolite change curves at three time points—1 day before surgery, 1 day after surgery, and 3 months post-surgery—are presented, including only metabolites with p-values ​​less than 0.05. The results showed significant differences between the treatment-effective group (n=6) and the treatment-ineffective group (n=6) in amino acid and fatty acid metabolites, involving a total of 12 metabolites, including α-ketoglutarate, asparagine, N-acetylglycine, L-hydroxyproline, aspartic acid, glutamate, gamma-linolenic acid (GLA), isoleucine, leucine, eicosapentaenoic acid (EPA), pentadecanoic acid, and valine. The changing trends of these metabolites suggest that they may play an important role in the post-HIFU treatment efficacy.

[0061] By analyzing metabolic changes at three time points before and after HIFU ablation, DHA (docosahexaenoic acid) was preliminarily screened out from the differentially changed metabolites that showed significant changes and were significantly associated with fibroid shrinkage. Figure 1 DHA levels were significantly higher in the effective treatment group than in the ineffective treatment group at all three time points. Patients with higher DHA levels had a higher rate of uterine fibroid shrinkage. Preoperative DHA levels were higher in the effective treatment group than in the ineffective treatment group.

[0062] Furthermore, preoperative DHA levels were higher in the treatment-significant group than in the treatment-ineffective group, suggesting a potential role of DHA in preoperative immune status and metabolic activity. As an omega-3 polyunsaturated fatty acid, DHA has significant anti-inflammatory effects, reducing chronic inflammation by regulating the immune system and optimizing macrophage function. The higher preoperative DHA levels in the treatment-significant group may indicate that they already possessed strong immunomodulatory capabilities and a favorable anti-inflammatory microenvironment before treatment, thereby promoting tissue repair and therapeutic efficacy after HIFU. Higher preoperative DHA levels may help improve the immune microenvironment, making the patient's immune system more adaptable to the immune response and repair processes required during treatment, thus improving the success rate and efficacy of HIFU treatment. In contrast, the treatment-ineffective group may lack this metabolic advantage, leading to limited immune and repair processes after HIFU, ultimately affecting the treatment outcome.

[0063] Example 2, Cell Experiment

[0064] This embodiment includes preliminary functional tests (qPCR, neutral red, BODIPY staining) of macrophages under GLA and DHA intervention in terms of polarization phenotype, phagocytic capacity and lipid droplet accumulation; as well as RXR and PPAR inhibitor intervention experiments.

[0065] 1. Preparation and treatment of THP-1-M0 macrophages

[0066] THP-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2. To induce macrophage differentiation, logarithmically growing THP-1 monocytes were treated with PMA (100 ng / mL) for 48 hours. After differentiation, non-adherent cells were gently removed by aspiration, and adherent cells were washed three times with PBS. After induction with PMA, THP-1 monocytes changed from suspension growth to adherent growth, with further increase in volume, loose cytoplasm, enlarged nuclei, numerous organelles, and a few protrusions around the cell membrane.

[0067] DHA significantly improved cell viability at a concentration of 100 µM (p < 0.0001), but showed inhibitory effects at higher concentrations (120 µM and 140 µM). Therefore, the optimal concentration was set at 100 µM, and this concentration was used for subsequent interventions. Well-growing THP-1-M0 cells were cultured at 2 × 10⁻⁶ cells / year. 4 / wells were seeded into 96-well plates, cultured until adherent and stable, and then subjected to DHA metabolite intervention treatment.

[0068] The proliferation of macrophages under different concentration gradients of RXR-α (UVI 3003, CAS No. 847239-17-2) and PPAR-γ (GW 9662, CAS No. 22978-25-2) inhibitors was detected by CCK-8 assay. The optimal concentration IC50 of the inhibitors was calculated. The inhibitors UVI 3003 and PPAR-γ were applied to macrophages at concentrations of 2.279 μM and 48.42 μM, respectively.

[0069] 2. DHA plays an important role in promoting macrophage proliferation and phagocytosis.

[0070] (1) Cell viability detection

[0071] THP-1-M0 cells were seeded into 96-well microplates (flat-bottomed, TC-treated) (2 × 10⁻⁶ cells). 4 Cells per well were placed at 37°C and 5% CO2 and exposed to different concentrations of differential metabolites or solvent controls (PBS) for 24 hours. Cell viability was then measured using the CCK-8 cell viability assay kit (BIOAGRIO, USA) according to the manufacturer's instructions. Each treatment condition was performed in five replicate wells, and each well was independently repeated three times.

[0072] (2) Detection of macrophage phagocytic capacity

[0073] THP-1-M0 macrophages were used at a rate of 5 × 10⁻⁶ 4 Cells were seeded at a density of 96-well plates (flat-bottomed, TC-treated) and co-cultured at 37°C and 5% CO2 for 24 hours with either a single differential metabolite or a PBS control. After treatment, 100 μL of 0.1% Neutral Red solution (Shanghai Aladdin Reagent Co., Ltd.) was added to each well, and the cells were incubated for 30 minutes. Cells were then washed three times with PBS, followed by lysis of each well with 100 μL of a 1:1 mixture of ethanol and glacial acetic acid, and incubated for 4 hours. Absorbance was measured at 540 nm using a microplate reader.

[0074] result( Figure 2The results showed that: A. DHA significantly improved cell viability at a concentration of 100 µM (p < 0.0001), but inhibited it at higher concentrations (120 µM and 140 µM). B. Changes in cell morphology can serve as an important indicator of the activation or inhibition of macrophage function. Microscopic images revealed significant changes in cell morphology in the experimental group compared to the control group: after intervention, cells exhibited stronger extensibility, higher differentiation, and a significant increase in protrusions, indicating that the intervention promoted cell differentiation. C. Macrophages can absorb liquids or other substances from the surrounding medium through pinocytosis, which is essential for metabolism. Neutral red is a dye that can be taken up by intracellular lysosomes, and changes in its uptake can reflect changes in cellular pinocytosis capacity. Compared to the control group, DHA (p < 0.001) significantly improved the phagocytic capacity of cells. Acid phosphatase (ACP) is a marker hydrolase in macrophage lysosomes, and its activity is closely related to the phagocytic function of macrophages. The enzyme activity of macrophages stimulated by differential metabolites was detected using an acid phosphatase (ACP) ELISA kit.

[0075] 3. Polarization state detection

[0076] After DHA treatment, the surface expression of CD206, IL-10, and TNF-α (M2 markers) was detected, and it was observed that DHA induced macrophage polarization towards the M2 anti-inflammatory phenotype. Figure 3 ).

[0077] 4. Fatty acid oxidation detection

[0078] THP-1-M0 macrophages were seeded into 35 mm glass-bottom confocal culture dishes and allowed to adhere. After treatment with different metabolites for 24 hours, the culture medium was removed, and the cells were gently washed three times with PBS for 5 minutes each time. They were then fixed with 4% paraformaldehyde (Servicebio, China) at room temperature for 15-20 minutes. After fixation, the cells were washed three times with PBS and incubated for 30 minutes at room temperature in the dark with 5 μM BODIPY™ 493 / 503 (MedChemExpress, USA). Excess dye was removed by washing three times with PBS again. Finally, the cells were mounted with DAPI-containing anti-fading mounting medium (Bristol-Myers Squibb, China), and lipid droplet aggregation was observed using a laser scanning confocal microscope (Nikon, Japan). Quantitative analysis of the lipid droplets was performed using ImageJ software.

[0079] After DHA / GLA intervention in macrophages, BODIPY staining revealed a decrease in extracellular lipid droplets and an increase in fatty acid consumption. Figure 4 ).

[0080] DHA treatment increased the expression of CPT1A, the rate-limiting enzyme for fatty acid β-oxidation in macrophages, enhancing fatty acid metabolism and oxidation; simultaneously, inhibition of PFKFB3, a key enzyme in glycolysis, was observed, suggesting macrophage metabolic reprogramming. Figure 5 ).

[0081] 5. Metabolomics analysis

[0082] Extraction and derivatization of intracellular, extracellular and biomass metabolites from macrophages.

[0083] THP-1-M0 macrophages were seeded in 60 mm culture dishes and treated with different metabolites or PBS (as a control) for 24 hours. After treatment, 2 mL of supernatant was collected from each dish for extracellular metabolite analysis. Cell metabolism was terminated by liquid nitrogen freezing, and cells were extracted with pre-chilled methanol / sodium hydroxide solution (1:1) containing 0.3 µM internal standard 2,3,3,3-d4-alanine. Samples were subjected to three freeze-thaw cycles (-80°C and room temperature), followed by centrifugation at 15,000 × g for 15 min at 4°C. The supernatant containing intracellular metabolites was collected for subsequent chemical derivatization. The biomass precipitate was resuspended in 200 µL of 1 M sodium hydroxide solution and incubated at 95°C for 1 hour. After cooling to room temperature, 200 µL of methanol was added, and the mixture was centrifuged at 15,000 × g for 15 min at 4°C. The supernatant was collected for subsequent derivatization.

[0084] For derivatization, 400 µL of sample was mixed sequentially with 400 µL of 1 M NaOH, 400 µL of methanol, and 4 µL of internal standard. After shaking for 30 seconds, 68 µL of pyridine and 30 µL of methylchloroform were added in two portions, shaking for 30 seconds after each addition. Then, 200 µL of chloroform was added and vortexed for 10 seconds. Next, 800 µL of 50 mM sodium bicarbonate solution was added, and the mixture was centrifuged at 2000 rpm for 10 minutes at 4 °C. The upper aqueous phase and the medium phase were discarded. The lower chloroform layer was dried with anhydrous sodium sulfate, and 150 µL was analyzed by gas chromatography-mass spectrometry.

[0085] Gas chromatography-mass spectrometry (GC-MS): All derivatized metabolites were analyzed using a BD-1701 capillary column (30 m × 250 μm × 0.25 μm, Agilent Technologies, USA) and an Agilent Intuvo 9000-5977B GC-MS system (USA). The electron impact ionization (EI) energy was set to 70 eV. The injection port temperature was set to 300℃, and a pulsed splitless injection mode was used. Helium was used as the carrier gas at a constant flow rate of 0.8 mL / min. The temperatures of the guard and auxiliary chips were maintained at 300℃. The quadrupole temperature was set to 230℃, and the ion source temperature was set to 150℃. Mass spectrometry data were acquired in full scan mode, with a mass number range of 38–550 and a scan rate of 2.9 scans / second. To avoid solvent interference, data acquisition was initiated 4.5 minutes after injection. After each run, the gas chromatography column was backflushed at 300°C for 4 minutes to remove residual compounds.

[0086] Raw gas chromatography-mass spectrometry (GC-MS) data were processed using Agilent MassHunter software for peak detection and retention time alignment. Metabolite deconvolution and identification were performed using the Automated Mass Spectrometry Deconvolution and Identification System (AMDIS). For unidentified metabolites, provisional identification results were further confirmed using the NIST mass spectrometry database. Metabolite quantification was based on the peak height of the most abundant fragment ions, and annotation was performed using a MassOmics script implemented in R. Data standardization was performed using internal standards and median centering based on quality control (QC) samples to correct for batch effects. The final dataset (including retention time, peak area, and ionic intensity) was exported for statistical analysis.

[0087] Partial least squares discriminant analysis (PLS-DA) was used for multivariate statistical analysis to visualize clustering and between-group separation. Student's t-test with false discovery rate (FDR) correction was applied to identify significantly different metabolites. Principal component analysis (PCA) was performed using MetaboAnalyst 3.0 software to compare metabolomic profiles among different groups. Metabolites were identified using enrichment analysis based on the KEGG pathway database. Hierarchical clustering and heatmaps were used to visualize metabolic patterns. Longitudinal variations in metabolite levels were assessed using repeated measures ANOVA. All visualizations (including heatmaps, line graphs, and chord plots) were generated using R packages such as ggplot2 and GOplot.

[0088] PLS-DA results showed significant separation between the CON and DHA groups in the metabolomic profiles of intracellular, extracellular, and cellular biomass samples. Venn diagrams revealed 8, 63, and 12 unique metabolites in intracellular, extracellular, and cellular biomass samples, respectively. In addition, 5 differentially expressed common metabolites were identified (p < 0.05). Heatmaps and volcano plots showed (…). Figure 6 and Figure 7 Following DHA intervention, 89 metabolites showed significant alterations in extracellular, intracellular, and cell residue samples (p < 0.05); extracellular fatty acids were upregulated across a large area, and CA cycle intermediates increased; DHA intervention may promote enhanced TCA cycle and fatty acid metabolism. Enrichment analysis based on the KEGG metabolic network showed that DHA intervention remodeled the macrophage metabolic network: TCA cycle was upregulated, fatty acid synthesis metabolism was upregulated, and pantothenic acid and CoA synthesis increased.

[0089] 6. Transcriptomics analysis

[0090] To further elucidate how docosahexaenoic acid (DHA) regulates metabolic pathways in polarized macrophages, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Notably, extracellular metabolites involved in gluconeogenesis, the tricarboxylic acid cycle, valine / leucine / isoleucine degradation, steroid hormone biosynthesis, and unsaturated fatty acid biosynthesis were significantly upregulated, indicating enhanced metabolic flux and increased availability of bioenergy precursors. In the intracellular component, we observed significant downregulation of pathways related to arachidonic acid metabolism, adipocyte lipolysis regulation, and necrosis / apoptosis, which may indicate a weakening of the inflammatory cascade. Consistent with this, pathways related to the glucagon signaling pathway, the tricarboxylic acid cycle, and carbon metabolism also showed an enrichment trend in the biomass component. KEGG metabolic pathways associated with significantly altered metabolites (p < 0.05) after DHA treatment were visualized using string plots. Figure 8 Analysis revealed the strongest associations between the tricarboxylic acid cycle, alanine, aspartate, and glutamate metabolism, carbon metabolism, ABC transporters, and unsaturated fatty acid biosynthesis pathways and differentially metabolites. In energy metabolism, key intermediates of glycolysis / gluconeogenesis (such as pyruvate and lactate) underwent significant changes; while the tricarboxylic acid cycle showed synergistic changes in multiple metabolites, including citrate, fumarate, pyruvate, and succinate. Furthermore, unsaturated fatty acid biosynthesis pathways were significantly altered, particularly involving arachidonic acid, linoleic acid, and octadecanoic acid. Overall, DHA treatment significantly activated multiple pathways in macrophages related to energy metabolism and lipid biosynthesis.

[0091] Transcriptome sequencing revealed changes in gene expression. A total of 1,939 differentially expressed genes (DEGs; fold change > 1.5, P-adj < 0.01) were identified, of which 755 were upregulated and 1,184 were downregulated. To visualize the most significant expression changes, the top 50 differentially expressed genes (DEGs) by P-value were selected for heatmap display. The heatmap showed strong homogeneity within each group, with a clear separation between the control group and the DHA-treated group. Notably, a group of genes, including ATP9A (phospholipid transporter ATPase 9A), NFATC2 (activated T cell nuclear factor 2), and TYMP (thymidine phosphorylase), were significantly upregulated after DHA exposure, while another group of genes (such as SAMD11 (protein 11 containing the sterile α motif domain), FAM124B (a member of sequence-similar family 124B), and DXDC1 (protein 1 containing the dioxin-inducible dialkylphenyl dioxygenase domain)) were significantly downregulated. These coordinated expression changes reveal robust transcriptional reprogramming induced by DHA in macrophages. To elucidate the biological significance of these differentially expressed genes, gene ontology (GO) and KEGG pathway enrichment analyses were performed.

[0092] In the category of biological processes, differentially expressed genes are mainly enriched in pathways related to checkpoint signaling of mitotic spindle assembly, macrophage activation involved in immune responses, phagocytosis, the phagocytic process, and the positive regulation of phagocytosis. Figure 9 In the cellular component category, differentially expressed genes were significantly enriched in structures such as foot processes, folded membranes, and cell processes defined by the plasma membrane, including the cytoplasm. These structures are closely related to macrophage migration and phagocytosis. In the molecular function category, differentially expressed genes were mainly associated with cholesterol binding, lipid transport activity, and cytokine receptor binding. Figure 9 Furthermore, KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in the chemokine signaling pathway, MAPK signaling pathway, actin cytoskeleton regulation, cytokine-cytokine receptor interaction, and PPAR signaling pathway. Figure 10 ).

[0093] Multi-omics analysis revealed that DHA, a key differential metabolite after HIFU, may mediate the regulation of macrophage energy metabolism through the PPAR signaling pathway, thereby affecting their functional status.

[0094] To elucidate the molecular mechanism by which DHA induces functional reprogramming in THP-1-derived M0 macrophages, this study employed an integrated analysis of transcriptomic and metabolomic datasets. Differentially expressed metabolites (P < 0.05) and the top ten upregulated genes (FC > 1.5, P < 0.05) were screened for correlation analysis. By calculating the Pearson correlation coefficient between metabolites and genes, significantly associated metabolite-gene pairs (|r| > 0.8, P < 0.05) were identified. Figure 11 As shown in Figure A, DHA treatment induces a characteristic correlation pattern between fatty acid-related metabolites and key transcriptional regulators of the PPAR signaling pathway. Notably, pyruvate dehydrogenase kinase 4 (PDK4), a classic downstream effector of PPAR activation, shows a strong positive correlation with various long-chain polyunsaturated fatty acids, including DHA (r = 0.935), conjugated linoleic acid (r = 0.877), arachidonic acid (r = 0.821), and myristic acid and palmitic acid (r > 0.82).

[0095] Furthermore, succinate, an intermediate in the tricarboxylic acid cycle, showed a stronger correlation with PDK4 (r = 0.979). Besides PDK4, various fatty acids also showed broad positive correlations with other transcriptional targets, including ST6-N-acetylgalactosamine-α-2,6-sialyltransferase 3 (ST6GALNAC3), glycoprotein 5 (GPC5), spinin 1 (SPON1), neural cell adhesion molecule (NRCAM), CD247 (CD247), and cell adhesion molecule 2 (CADM2). These genes are mainly involved in membrane organization, intercellular interactions, cytoskeleton regulation, and immune receptor signaling, suggesting that DHA-responsive lipid metabolites may drive co-transcriptional remodeling. Conversely, non-lipid metabolites such as tryptophan, glycine, indole, and decane showed negative correlations with these genes (r < -0.82).

[0096] To visualize the overall architecture of these associations, a metabolite-gene correlation network was constructed. Figure 11 (B) This network reveals that DHA and succinate constitute the most highly metabolic hub, forming strong positive correlation edges (|r| > 0.82) with PDK4, GRAM domain-containing protein 1C (GRAMD1C), and other transcriptional nodes. Combining the heatmap-derived correlations with the network-defined DHA hub indicates that DHA remodels macrophage transcriptional programs through the PDK4-related PPAR signaling pathway.

[0097] 7. Research on the mechanism of DHA regulation of macrophage metabolism

[0098] To investigate the mechanism by which DHA regulates macrophage metabolism, the expression of PPARγ and RXRα, core pathways of fatty acid oxidation, was first examined. Results ( Figure 12The results showed that DHA treatment significantly upregulated the mRNA levels of PPARγ and RXRα.

[0099] Immunofluorescence localization assays further confirmed these findings, and the results ( Figure 13 The results showed that the expression levels of PPARγ and RXRα were significantly increased in macrophages treated with DHA, and the co-localization of the two was significantly enhanced.

[0100] The proliferation of macrophages under different concentration gradients of RXR-α (UVI 3003) and PPAR-γ (GW 9662) inhibitors was detected by CCK-8 assay. The optimal IC50 concentration of the inhibitors was calculated. When UVI 3003 and PPAR-γ inhibitors were applied to macrophages at concentrations of 2.279 μM and 48.42 μM, respectively... Figure 14 ).

[0101] Changes in macrophage phagocytic capacity were detected by neutral erythropoietin assay and ACP acid phosphatase assay. The addition of RXR-α (UVI 3003) and PPAR-γ (GW 9662) inhibitors significantly suppressed macrophage phagocytic capacity, but this phenomenon was reversed upon the addition of DHA. Figure 15 ).

[0102] Changes in macrophage secretion of inflammatory factors (IL-6, IL-10, TNF-α) were detected by ELISA. DHA treatment increased anti-inflammatory factors and decreased pro-inflammatory factors; these changes were reversed upon the addition of RXR-α (UVI 3003) and PPAR-γ (GW 9662) inhibitors. Figure 16 ).

[0103] This study, through a complete in vitro mechanism experimental system, strongly demonstrates that DHA activates the PPARγ / RXR heterodimer, transcribedly upregulates target genes such as PDK4, thereby driving metabolic reprogramming in macrophages and ultimately enhancing their phagocytic capacity. The elucidation of this signaling axis provides a solid theoretical foundation for immunometabolic intervention strategies targeting DHA and other Omega-3 fatty acids or PPARγ / RXR.

Claims

1. Application of the DHA-RXR-PPAR signaling pathway as a target in screening drugs that promote postoperative recovery after HIFU for uterine fibroids.

2. The application according to claim 1, characterized in that: The drug upregulated DHA levels in subjects who underwent HIFU treatment for uterine fibroids.

3. The application according to claim 1, characterized in that: The drug upregulates the expression levels of PPARγ and RXRα in macrophages, promoting their phagocytic function, and thus promoting the shrinkage of uterine fibroids and postoperative recovery.

4. The application according to claim 1, characterized in that: DHA mediates the regulation of energy metabolism in macrophages through the RXR-PPAR signaling pathway, thereby affecting their functional state.

5. The application according to claim 1, characterized in that: DHA drives macrophage metabolic reprogramming through the RXR-PPAR signaling axis, thereby promoting their transformation into an anti-inflammatory, pro-phagocytic repair phenotype and accelerating the absorption and repair process of uterine fibroids after HIFU surgery.

6. Application of DHA-RXR-PPAR signaling pathway promoters in the preparation of products for promoting postoperative recovery after HIFU treatment for uterine fibroids.

7. The application according to claim 6, characterized in that: The application involves driving the metabolic reprogramming of macrophages after HIFU treatment for uterine fibroids through promoters of the DHA-RXR-PPAR signaling pathway, thereby promoting their transformation into an anti-inflammatory, pro-phagocytic repair phenotype and accelerating the absorption and repair process of uterine fibroids after HIFU treatment.

8. The application according to claim 6, characterized in that: The promoter of the DHA-RXR-PPAR signaling pathway is any one of the following: (1) A promoter of DHA; (2) RXR promoter; (3) PPAR promoters; (4) Promoters of the RXR-PPAR signaling pathway.

9. Application of docosahexaenoic acid in the preparation of drugs for promoting postoperative recovery after HIFU treatment for uterine fibroids.

10. The use of biomaterials containing docosahexaenoic acid (DHA) or substances that promote DHA expression in subjects in the preparation of drugs for promoting postoperative recovery after HIFU treatment for uterine fibroids.

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