Preparation method of dihydroquercetin self-assembled nanoparticles and application thereof in liver protection

Abstract

The application discloses a preparation method of dihydroquercetin self-assembled nanoparticles and application of the nanoparticles in liver protection, and the preparation method of the nanoparticles is as follows: betaine and dihydroquercetin are respectively dissolved in methanol; the methanol solutions of the betaine and the dihydroquercetin are mixed in a 1:1 molar ratio, and the pH is adjusted to 7.0-7.5 by using a sodium hydroxide solution; the mixture is added into preheated PBS under stirring, sealed and kept at 55-65 DEG C for 20-40 minutes; after being cooled to room temperature, the mixture is subjected to ultra-pure water dialysis by using a dialysis bag, and the BTNPs, i.e. dihydroquercetin self-assembled nanoparticles, are obtained after freeze-drying. The nanoparticles have good ALI treatment effect, high safety, can simultaneously solve the defects of low solubility and poor bioavailability of dihydroquercetin, and the defects of betaine, such as easy occurrence of gastrointestinal adverse reactions and existence of dose-dependent safety risks; meanwhile, the nanoparticles can also regulate intestinal flora disorder caused by alcohol and prevent endotoxemia.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing dihydroquercetin self-assembled nanoparticles and their application in liver protection. Background Technology

[0002] Excessive alcohol consumption is a leading cause of liver-related morbidity and mortality worldwide. Alcoholic liver disease (ALD) presents with a variety of clinicopathological manifestations, progressing from early simple fatty liver to acute alcoholic hepatitis (AH), liver fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. Among these, acute alcoholic liver injury (ALI) is a key driver of ALD progression, characterized by hepatocellular steatosis, inflammatory infiltration, and necrosis. If left untreated or recurrent, it can accelerate the development of alcoholic hepatitis, fibrosis, and ultimately cirrhosis.

[0003] The pathophysiological mechanism of acute liver injury (ALI) is complex, consisting of multiple components including oxidative stress, inflammatory response, and immune dysregulation. These components interact and collectively exacerbate ALI liver damage. Studies have confirmed that reactive oxygen species produced by alcohol metabolism directly damage hepatocytes, triggering an inflammatory cascade; in turn, inflammation further aggravates oxidative stress, creating a vicious cycle. Furthermore, alcohol intake disrupts the balance of the gut microbiota, leading to dysbiosis and impairing the integrity of the intestinal barrier. This dysbiosis and barrier damage mutually reinforce each other, increasing intestinal permeability, inducing endotoxemia, and consequently exacerbating ALI through the gut-liver axis.

[0004] Currently, clinical treatment of liver injury mainly relies on nutritional supplements and adjunctive drug therapy, which are often limited in effectiveness and accompanied by side effects. For example, desulfuron-methyl, naltrexone, and acampprosecte can reduce alcohol intake in patients with alcohol use disorder. Clometrazazole can inhibit CYP2E1 activity and may promote early recovery from ALD. However, some of these drugs have side effects; clometrazazole has sedative-hypnotic effects, and acampprosecte can cause diarrhea. Therefore, developing novel treatment strategies that can effectively and safely intervene in ALI is of significant clinical importance and urgent need.

[0005] Compared to Western medicine, natural bioactive compounds have advantages such as safety and fewer side effects, and are considered a promising intervention strategy. However, existing natural bioactive compounds have shortcomings such as low solubility and poor bioavailability, which limit their ability to exert their true therapeutic effects in vivo. Betaine (BET) is a natural compound with good hepatoprotective effects and good water solubility, but long-term oral administration can easily cause gastrointestinal adverse reactions and poses a dose-dependent safety risk, which limits its clinical application to some extent. Summary of the Invention

[0006] In view of the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide a method for preparing dihydroquercetin self-assembled nanoparticles. This method can simultaneously solve the disadvantages of low solubility and poor bioavailability of dihydroquercetin, as well as the defects of betaine, which is prone to gastrointestinal adverse reactions and has dose-dependent safety risks. At the same time, compared with the simple mixing or use of the two alone, it can significantly improve hepatic steatosis, cell apoptosis and inflammatory infiltration, and alleviate oxidative stress by inhibiting the activation of the IKK / IκBα / NF-κB signaling pathway, thereby improving acute alcoholic liver injury (ALI), with good therapeutic effect and high safety. In addition, the prepared nanoparticles can also regulate alcohol-induced intestinal flora dysbiosis and prevent endotoxemia.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing self-assembled dihydroquercetin nanoparticles includes the following steps: first, dissolving betaine and dihydroquercetin separately in methanol; then mixing the methanol solutions of betaine and dihydroquercetin at a 1:1 molar ratio and adjusting the pH to 7.0-7.5 with sodium hydroxide solution; then adding the mixture to preheated PBS under stirring, sealing and maintaining at 55-65℃ for 20-40 minutes; after cooling to room temperature, dialyzing the mixture with ultrapure water using a dialysis bag, and lyophilizing to obtain BTNPs, i.e., self-assembled dihydroquercetin nanoparticles.

[0008] As a preferred embodiment of the present invention, the nanoparticles have a uniform morphology, are regularly spherical, have an average diameter of 78.55 ± 12.99 nm, and the drug exists in an amorphous form.

[0009] As a preferred embodiment of the present invention, the amount of betaine is 35.145 mg, the amount of dihydroquercetin is 91.275 mg, and the betaine and dihydroquercetin are dissolved in 10 mL of methanol respectively; the mixture is added to preheated PBS with stirring, sealed and kept at 60°C for 30 minutes; the molecular weight cutoff of the dialysis bag is 3500 Da, and ultrapure water dialysis is performed for 24 hours.

[0010] The self-assembled dihydroquercetin nanoparticles prepared in this invention significantly reduced liver index and also significantly improved serum liver injury markers (ALT, AST, LDH), with effects comparable to the high-dose positive control SILY. They effectively reduced alcohol-induced pathological liver swelling and had a synergistic protective effect in maintaining hepatocyte membrane integrity and reducing direct toxicity and secondary inflammatory damage caused by alcohol. Therefore, they can be used in the preparation of drugs for treating acute alcoholic liver injury.

[0011] The self-assembled dihydroquercetin nanoparticles prepared in this invention can regulate intestinal flora disorder caused by alcohol, improve the diversity, richness and uniformity of the flora (significantly increasing the abundance of Firmicutes and decreasing the abundance of Bacteroidetes, thereby normalizing the F / B ratio), and therefore can be used in the preparation of drugs for treating or regulating intestinal flora disorder caused by alcohol.

[0012] As a preferred embodiment of the present invention, the dihydroquercetin self-assembled nanoparticles are used to improve the diversity, richness and uniformity of bacterial communities, increase the abundance of Firmicutes, and decrease the abundance of Bacteroidetes.

[0013] As a preferred embodiment of the present invention, the dihydroquercetin self-assembled nanoparticles are used to increase the abundance of beneficial bacteria, wherein the beneficial bacteria include Oscillospira .

[0014] The self-assembled dihydroquercetin nanoparticles prepared by this invention can improve endotoxemia by restoring intestinal flora homeostasis, and therefore can be used in the preparation of drugs for the treatment or prevention of endotoxemia.

[0015] Advantages and beneficial effects of the present invention: (1) The self-assembled dihydroquercetin nanoparticles provided by the present invention have a uniform spherical morphology and the drug exists in an amorphous form in the nano system, resulting in better solubility and effectively solving the shortcomings of low solubility and poor bioavailability of dihydroquercetin. In addition, low doses of BTNPs (40 mg / kg) can achieve similar efficacy to high doses of SILY (80 mg / kg), and compared with BET alone, mice showed good mental state and no gastrointestinal adverse reactions during administration. This result indicates that the nanoparticles have the advantages of lower dosage and higher safety, effectively solving the defects of betaine, which is prone to gastrointestinal adverse reactions and has dose-dependent safety risks.

[0016] (2) The antioxidant activity of the self-assembled dihydroquercetin nanoparticles provided by the present invention is significantly higher than that of the physical mixture of the two. After the self-assembly of betaine and dihydroquercetin, the antioxidant activity of TAX is effectively preserved, and the antagonistic effect that may exist when BET and TAX are physically mixed is avoided.

[0017] (3) The self-assembled dihydroquercetin nanoparticles provided by this invention can effectively inhibit the release of pro-inflammatory factors TNF-α and IL-1β, and restore IL-10 levels to a much higher level than either of the free drugs alone. This result indicates that the co-delivery of BET and TAX in the nanoparticles achieves a synergistic anti-inflammatory effect.

[0018] (4) The self-assembled dihydroquercetin nanoparticles provided by the present invention significantly reduced the liver index compared with BET or TAX alone. They also significantly improved serum liver injury markers (ALT, AST, LDH), and the effect was comparable to that of the high-dose positive control SILY. It can be seen that after self-assembly, BET and TAX can synergistically reduce alcohol-induced pathological liver swelling and have a synergistic protective effect in maintaining the integrity of hepatocyte membranes and reducing the direct toxicity and secondary inflammatory damage caused by alcohol.

[0019] (5) The self-assembled dihydroquercetin nanoparticles provided by the present invention significantly restored GSH levels and CAT activity compared with BET or TAX administration alone, and effectively reduced MDA content; the effect of low-dose BTNPs in restoring oxidative imbalance is similar to that of high-dose SILY, effectively disrupting the vicious cycle between oxidative stress and inflammation in alcoholic liver injury.

[0020] (6) The self-assembled dihydroquercetin nanoparticles provided by the present invention can significantly improve hepatic steatosis, apoptosis and inflammatory infiltration. They can effectively inhibit the TLR4 / MyD88 / NF-κB signaling pathway in macrophages, thereby effectively reducing the inflammatory cascade response triggered by LPS; thus achieving the purpose of improving acute alcoholic liver injury (ALI).

[0021] (7) The present invention used the MTT assay to evaluate the cytotoxicity of BET, TAX and BTNPs on RAW264.7 cells. High concentrations (80 μg / mL) of BET and TAX reduced cell viability to 90.2 ± 5.3% and 91.5 ± 4.8%, respectively, while BTNPs maintained viability at 96.7 ± 3.9%. At the highest concentration of 160 μg / mL, although BET, TAX and BTNPs all induced cytotoxicity, the cell viability of the BTNPs-treated group (83.1 ± 4.7%) was significantly higher than that of the free BET (74.6 ± 6.2%) and TAX (78.3 ± 5.5%) groups, further demonstrating that BTNPs formed after the self-assembly of betaine and dihydroquercetin have lower toxicity and higher safety to RAW264.7 cells.

[0022] (8) The self-assembled dihydroquercetin nanoparticles provided by the present invention can regulate the intestinal flora disorder caused by alcohol, improve the diversity, richness and uniformity of the flora (significantly increase the abundance of Firmicutes and reduce the abundance of Bacteroidetes, thereby normalizing the F / B ratio), and improve alcohol-induced endotoxemia by restoring the intestinal flora homeostasis.

[0023] (9) The dihydroquercetin self-assembled nanoparticles provided by this invention uniquely restore the Oscillospira Increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria. Attached Figure Description

[0024] Figure 1 Characterization analysis of BTNPs. (A) Representative TEM images of BTNPs. (B) Histogram of BTNP particle size distribution based on TEM analysis. (C) FTIR spectra of BET, TAX, and BTNPs. (D) XRD patterns of BET, TAX, and BTNPs. (E) Characterization analysis of BET, TAX, and BTNPs. 1 H NMR spectrum.

[0025] Figure 2 Molecular dynamics simulations of BTNPs. (A) RMSD curve at 100 ns. (B) Rg curve. (C) SASA curve. (D) Simulation snapshots at different time points. (E) Interaction energy distribution maps (LJ-SR and Coul-SR). (F) Schematic diagram of non-covalent interaction types.

[0026] Figure 3 To demonstrate how BTNPs inhibit LPS-induced macrophage inflammation via the TLR4 / MyD88 / NF-κB pathway. (A) Viability of RAW264.7 macrophages after treatment with the indicated concentrations of BET, TAX, or BTNPs for 24 hours, as determined by the MTT assay. (BD) Levels of TNF-α (B), IL-1β (C), and IL-10 (D) in the culture supernatant. Macrophages were pretreated with BET, TAX, or BTNPs (40 μg / mL) for 2 hours, followed by stimulation with LPS (4 μg / mL) for 22 hours. (E) Representative Western blot images of TLR4, MyD88, p-IκBα, IκBα, p-NF-κB, and NF-κB protein expression in treated cells. (FI) Quantitative analysis of TLR4 (F), MyD88 (G), p-IκBα / IκBα ratio (H), and p-NF-κB / NF-κB ratio (I). Data are expressed as mean ± standard deviation (n ≥ 3). Statistical significance: # p <0.05, ## p <0.01, ### p <0.001 vs. control group; *p < 0.05, ** p < 0.01, *** p<0.001 vs. LPS (model) group.

[0027] Figure 4To alleviate alcohol-induced liver injury and oxidative stress in mice using BTNPs. (A) Schematic diagram of the timeline of the ALI model experiment. (B) Weekly body weight changes in different groups of mice during the 6-week experiment. (C) Liver index. (DF) Serum ALT(D), AST(E), and LDH(F) levels in each experimental group of mice. (GI) Liver GSH(G), CAT(H) activity, and MDA(I) content in each experimental group of mice. Data are expressed as mean ± standard deviation (n ≥ 3). Statistical significance: # p < 0.05, ## p <0.01, ### p<0.001 vs. control group; * p <0.05, ** p < 0.01 , *** p <0.001 vs. ethanol (model) group.

[0028] Figure 5 BTNPs were used to improve alcohol-induced liver histopathological damage and hepatocyte apoptosis in mice. (A) Gross appearance of liver tissue from different groups of mice and corresponding H&E-stained liver sections. (B) Representative immunofluorescence images of TNF-α, IL-1β, Bax and Bcl-2 staining and TUNEL apoptosis detection in liver sections.

[0029] Figure 6 To investigate the role of BTNPs in regulating gut microbiota composition and improving endotoxemia. (A) Serum LPS levels in mice. (B) Venn diagram of mouse gut microbiota based on OTUs. (C) Dilution curves of gut microbiota sequencing. (D) Abundance ranking curves of gut microbiota. (EG) Alpha diversity indices of gut microbiota: Chao1 index (E), Shannon index (F), and Simpson index (G). Data are presented as mean ± standard deviation (n≥3). Statistical significance: # p <0.05, ## p <0.01, ### p <0.001 vs. control group; * p <0.05,** p <0.01,*** p <0.001 vs. ethanol (model) group.

[0030] Figure 7To investigate the role of BTNPs in modulating gut microbiota composition and improving endotoxemia, β-diversity analysis of the gut microbiota was performed using PCoA (A) and NMDS (B). (C) Relative abundance of gut microbiota at the phylum level. (D) Relative abundance of the 10 most abundant bacterial genera. (E) Relative abundance of Firmicutes. (F) Relative abundance of Bacteroidetes. (G) Firmicutes / Bacteroidetes (F / B) ratio. (H) Relative abundance of *Vibrio*. Data are expressed as mean ± standard deviation (n≥3). Statistical significance: # p <0.05, ## p <0.01, ### p <0.001 vs. control group; * p <0.05, **p <0.01,*** p<0.001 vs. ethanol (model) group. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] 1. Materials and Methods 1.1. Materials Dihydroquercetin (purity 98.0%, batch number 111816-201102) was purchased from the National Institutes for Food and Drug Control, China. Betaine (LOT: JB251686, purity ≥98.0%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS) was purchased from Solarbio Biotechnology Co., Ltd. (Beijing, China). RAW264.7 cell line was purchased from Newgen Biotech (Wuxi, China). Lipopolysaccharide (LPS) from Escherichia coli O55:B5 was purchased from Sigma-Aldrich (Shanghai, China). 1.2. Preparation of self-assembled dihydroquercetin nanoparticles (BTNPs) Betaine (BET, 35.145 mg) and dihydroquercetin (TAX, 91.275 mg) were each dissolved in 10 mL of methanol. The methanol solutions of betaine and dihydroquercetin were mixed 1:1 molar and the pH was adjusted to 7.0–7.5 with sodium hydroxide solution (5 mg / mL). The mixture was slowly added to 10 mL of preheated PBS with stirring, sealed, and kept at 60 °C for 30 min. After cooling to room temperature, the mixture was dialyzed against ultrapure water for 24 h using a dialysis bag (MWCO 3500 Da). After lyophilization, BTNPs were obtained with a particle yield of 35.27%.

[0033] 1.3. Characterization of BTNPs 1.3.1. Transmission Electron Microscopy (TEM) The morphology of BTNPs was observed using TEM (JEM-1200 EX, Hitachi, Japan). Diluted samples were dropped onto a carbon-coated copper grid, negatively stained with 0.2% phosphotungstic acid, and imaged after drying overnight at room temperature.

[0034] 1.3.2. Fourier Transform Infrared Spectroscopy (FTIR) Lyophilized BET, TAX, and BTNPs samples were ground with KBr, compressed into tablets, and analyzed using an FTIR spectrophotometer (PerkinElmer, Norwalk, Connecticut, USA) at 400–4000 cm⁻¹. -1 The analysis is conducted within the specified range.

[0035] 1.3.3. X-ray diffraction analysis (XRD) XRD patterns were recorded on an X-ray diffractometer at a scan rate of 5° / min, a 2θ range of 5° to 60°, a voltage of 40 kV, and a current of 40 mA. Data were processed using Origin software (OriginLab, Massachusetts, USA).

[0036] 1.3.4. 1 H NMR analysis BET, TAX, and BTNPs samples were dissolved in DMSO-d6 and recorded at 600 MHz. 1 1H NMR spectrum (Rheinstein, Germany). Chemical shifts are reported in ppm.

[0037] 1.3.5. Molecular Dynamics Simulation The 3D structures of betaine (CID: 247) and dihydroquercetin (CID: 439533) were downloaded from the PubChem small molecule database. Molecular dynamics simulations were performed using GROMACS 2021 software with a GAFF2 force field. BET and TAX were placed in a water tank at a 1:1 molar ratio, and the water model was TIP3P. Energy minimization, 100 ps NVT, and 100 ps NPT pre-equilibrium were performed sequentially, followed by a 100 ns final simulation with a step size of 0.002 ps. The temperature was 298.15 K (V-rescale temperature control) and the pressure was 1.0 bar (Parrinello-Rahman pressure control). Long-range electrostatics were treated with PME, van der Waals interactions were corrected for dispersion, and periodic boundary conditions were used in all three directions. Trajectory analysis was performed using GROMACS built-in tools, visualization was performed using VMD, and plotting was performed using Matplotlib.

[0038] 1.4. Antioxidant Experiment 1.4.1. Scavenging ability against DPPH free radicals Take 2 mL of 0.2 mmol / L DPPH-ethanol solution and add 2 mL of BET, TAX, BTNPs, and a mixture of BET and TAX (molar ratio 1:1) solutions with a concentration of 0.05 mg / mL. Let stand in the dark for 30 min and measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging rate according to the following formula.

[0039]

[0040] In the formula, A1: absorbance of DPPH free radicals after reacting with the sample solution, A2: absorbance of the sample solution and anhydrous ethanol, and A0: absorbance of the mixture of DPPH free radicals and anhydrous ethanol.

[0041] 1.4.2. Scavenging ability against ABTS free radicals Prepare 2.45 mmol·L -1 K2S2O7 solution and 7 mmol·L -1 Mix equal volumes of ABTS solution thoroughly and let stand in the dark for 12 hours to form stable ABTS. + Free radical mother liquor. ABTS was prepared by controlling the measured value of the mother liquor to 0.70 ± 0.02 at a wavelength of 734 nm through appropriate dilution. + Diluent. Take 1 mL of a 0.05 mg / mL solution of BET, TAX, BTNPs, and a mixture of BET and TAX (molar ratio 1:1) and dilute with ABTS. +After reacting 4 mL of the diluent in the dark for 10 min, the absorbance was measured at a wavelength of 734 nm. Using the VC test solution as a positive control, the ABTS free radical scavenging rate was calculated according to the following formula.

[0042]

[0043] In the formula, A i : Absorbance of the sample solution after reaction with ABTS+, A j A0: Absorbance of distilled water and sample solution; A0: Absorbance of distilled water and ABTS. + Absorbance.

[0044] 1.5. Cell Experiments 1.5.1. Cytotoxicity assay RAW264.7 cells were seeded in 96-well plates (1 × 10⁻⁶). 4 Cells / well were treated with BET, TAX, or BTNPs at concentrations of 5, 10, 20, 40, 80, and 160 μg / mL for 24 hours. MTT solution (5 mg / mL) was added, and after incubation for 4 hours, formazan crystals were dissolved in DMSO. Absorbance was measured at 490 nm. A concentration of 40 μg / mL was selected for subsequent experiments.

[0045] 1.5.2. ELISA kit detection RAW264.7 cells were seeded in culture dishes (1 × 10⁻⁶). 5 Cells / well). After reaching 80% confluence, cells were treated with BET, TAX, or BTNPs (40 μg / mL) for 24 hours, followed by stimulation with LPS (4 μg / mL) for 22 hours. The culture supernatant was collected, and the levels of TNF-α, IL-1β, and IL-10 were measured using an ELISA kit according to the manufacturer's instructions.

[0046] 1.5.3. Western Blot Analysis Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA kit. Equal volumes of protein were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk, and incubated overnight at 4°C with a primary antibody, followed by incubation with an HRP-conjugated secondary antibody. The membrane was developed using ECL and quantified using ImageJ software.

[0047] 1.6. Animal Experiments 1.6.1. Animals Male ICR mice (6-8 weeks old, 25 ± 3 g) were purchased from Changchun Yiyis Animal Science Co., Ltd., China. All animal experimental procedures were approved by the Animal Ethics Committee of Jilin Agricultural University (ethics number 202504002).

[0048] 1.6.2. Experimental Design Mice were housed under standard conditions (22 ± 2 ℃, 60 ± 5% humidity, 12-hour light / dark cycle) with free access to food and water. After one week of acclimatization, mice were randomly divided into six groups (n=12): control group, model group, BET group (40 mg / kg), TAX group (40 mg / kg), BTNPs group (40 mg / kg), and positive control silymarin (SILY) group (80 mg / kg). From week 2 to week 7, the control and model groups were orally administered 0.5% sodium carboxymethyl cellulose solution, while the other groups were orally administered the corresponding drug (dissolved in 0.5% sodium carboxymethyl cellulose solution) daily. In week 7, two hours after the last administration, mice were fasted but allowed free access to water. Except for the control group, all mice were administered 56% alcohol (6 mL / kg) twice by gavage at 6-hour intervals. Six hours after the second alcohol gavage, blood was collected through the orbital sinus, and the mice were euthanized by cervical dislocation. Serum and liver tissue were collected for analysis.

[0049] 1.6.3. Biochemical Analysis Serum ALT, AST, and LDH levels, as well as liver GSH, CAT, and MDA levels, were measured using a commercial kit (Nanjing Jiancheng). Liver tissue was homogenized in cold physiological saline (1:9, w / v), centrifuged, and the supernatant was collected. Blood samples were centrifuged at 3500 × g for 10 minutes at 4°C to obtain serum.

[0050] 1.6.4. Histological Analysis Liver tissue was fixed in 10% formalin, embedded in paraffin, and cut into 5-micrometer-thick sections. The sections were stained with H&E and observed under an optical microscope (Bio-Rad, Hercules, USA).

[0051] 1.6.5. Immunofluorescence and TUNEL staining For immunofluorescence, sections were incubated overnight at 4°C with primary antibodies, followed by incubation at 37°C with secondary antibodies for 30 minutes. Cell nuclei were counterstained with DAPI. For TUNEL staining, sections were treated with 20 μg / mL proteinase K at 37°C for 15 minutes, then incubated with TUNEL reaction solution at 37°C in the dark for 1 hour. After DAPI staining, apoptotic cells were observed under a fluorescence microscope.

[0052] 1.6.6. 16S rRNA gene sequencing Microbial DNA was extracted from mouse intestinal contents. The V3-V4 region of the 16S rRNA gene was amplified using primers 338F and 806R and sequenced on the Illumina NovaSeq platform. ASV clustering, α and β diversity, and taxonomic annotation were performed using QIIME2.

[0053] 1.7. Statistical Analysis Data are expressed as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA, followed by Tukey's post-hoc test or Student's t-test. A p-value < 0.05 was considered statistically significant. Image and data analysis were performed using ImageJ, GraphPad Prism 8.0, and Origin 2018 software. Charts were compiled using Adobe Photoshop 2025 and Adobe Illustrator 2024.

[0054] 2. Results 2.1. Characterization Analysis of BTNPs 2.1.1 TEM Analysis Transmission electron microscopy (TEM) analysis of BTNPs showed that the obtained nanoparticles had a uniform spherical morphology with an average diameter of 78.55 ± 12.99 nm. Figure 1 AB).

[0055] 2.1.2 FTIR Analysis Fourier transform infrared spectroscopy (FTIR) is used to study the intermolecular interactions within BTNPs. Figure 1 C). Several significant changes were observed compared to the spectra of free BET and TAX. BET showed changes at 1620 cm⁻¹. -1 The characteristic carboxylate ion (-COO) at the location - The peak completely disappears in the BTNP spectrum, indicating its involvement in strong intermolecular interactions. Meanwhile, the carbonyl (C=O) stretching peak of TAX disappears from 1640 cm⁻¹. -1 The shift to lower wavenumbers and broadening indicate that the carbonyl group is involved in hydrogen bonding. Furthermore, at 3200–3500 cm⁻¹... -1 The broad hydroxyl (OH) absorption bands in the region are significantly enhanced and broadened in the BTNP spectra, indicating the formation of an extensive hydrogen bond network. Overall, these spectral changes suggest the formation of intermolecular hydrogen bonds, primarily between the carboxyl groups of BET and the phenolic hydroxyl groups of TAX. This interaction is considered a key driving force for molecular self-assembly and contributes to the stability of the nanoparticles.

[0056] 2.1.3 XRD Analysis X-ray diffraction (XRD) analysis is used to assess the physical state of BTNPs. Figure 1 D). Both free BET and free TAX exhibit sharp, intense diffraction peaks characteristic of the crystalline state. In contrast, the diffraction patterns of BTNPs show significantly reduced intensity and broadened peaks, indicating a significant decrease in the crystallinity of both components. This transition to an amorphous state suggests that the intermolecular forces driving self-assembly disrupt the native lattices of BET and TAX. This amorphous dispersion is typically associated with increased dissolution rates and improved bioavailability.

[0057] 2.1.4 1 H NMR analysis Proton nuclear magnetic resonance (NMR) 1 ¹H NMR spectroscopy was used to further elucidate the self-assembly mechanism of BTNPs. Figure 1 E). The spectra of BTNPs contain the main proton signals of BET and TAX, confirming the successful binding of these two components in the nanoparticle structure. Notably, the spectra of BTNPs exhibit characteristics distinct from the simple superposition of precursor signals. In free BET, the methyl proton (–N) + (CH3)3 appears at δ 3.26 ppm, while the methylene proton (–CH2–) appears at δ 3.89 ppm. In BTNP spectra, these signals shift to δ 3.16 ppm and δ 3.93 ppm, respectively, resulting in a broadening of the distance between the two characteristic peaks. This unique chemical shift pattern may be attributed to changes in the local environment of these hydrogen atoms, due to intermolecular interactions involving adjacent carboxyl groups.

[0058] Meanwhile, the proton signals attributable to the TAX framework remained detectable, but underwent significant changes. Specifically, the multiple dispersed phenolic hydroxyl proton peaks (chemical shift range δ8.91–11.85 ppm) observed in free TAX were significantly reduced in the BTNP spectra. This spectral change suggests that hydrogen bonds may have formed between the phenolic hydroxyl groups of TAX and the carboxyl groups of betaine (BET) during nanoparticle self-assembly, thereby altering the NMR signals of the corresponding protons. Significant changes were also observed in the aromatic region of TAX. For example, the aromatic proton doublets at δ5.85 and 5.80 ppm in free TAX merged and shifted, appearing as a triplet at δ5.92 ppm (J=3.3 Hz) and a doublet at δ5.88 ppm in the BTNP spectra. Furthermore, the patterns of alicyclic C-ring protons (e.g., δ 5.70, 4.92, 4.45 ppm in free TAX) were altered in the nanoparticle spectra (e.g., δ 4.97, 4.49 ppm), indicating a change in the conformation or electronic environment of the TAX core after complexation. The emergence of new resonances in the alicyclic region (δ 0.85–2.29 ppm) further confirms the formation of a novel supramolecular entity with a unique chemical environment.

[0059] comprehensive 1 ¹H NMR analysis revealed clear intermolecular interactions between BET and TAX during self-assembly. In BET, the protons of the methyl and methylene groups shifted, and the peak spacing increased. In TAX, the proton signal of the phenolic hydroxyl group weakened or disappeared, and the protons in the aromatic and alicyclic regions shifted, resulting in changes in peak shape. Furthermore, a new resonance peak appeared in the aliphatic region. These changes collectively demonstrate that BET and TAX formed BTNPs through non-covalent interactions such as hydrogen bonding.

[0060] 2.1.5 Molecular Dynamics Simulation Analysis A 100 ns all-atom molecular dynamics simulation was performed on BET and TAX (molar ratio 1:1) in aqueous solution to analyze the molecular mechanism of BTNP self-assembly at the molecular level. Root mean square deviation (RMSD) analysis showed that the structural fluctuations of the simulated BTNP system gradually decreased and stabilized within the first 60 ns. Thereafter, the RMSD stabilized at approximately 2.947 nm and maintained small oscillations, indicating that the assembly had reached a stable state. Figure 2 A). The radius of gyration (Rg) of BTNPs shows a continuous decreasing trend, from approximately 2.95 nm initially to approximately 1.45 nm at the end. Figure 2 (B) indicates that BTNPs gradually curled and packed into more compact aggregates from a relatively loose state. The solvent accessible surface area (SASA) increased from 195.674 nm in the initial simulation. 2 It sharply decreased to 52.928 nm at the end. 2 The decrease reached 73% ( Figure 2C indicates that intermolecular aggregation occurred to reduce the exposed solvent and surface area. This significant interfacial shielding effect is corroborated by the decrease in Rg, indicating a densification process in the assembly. Snapshots at different times allow for a direct examination and analysis of the system's dynamic behavior. Figure 2 D): At 0 ns, BET and TAX molecules are randomly dispersed; at 20 ns, local aggregation begins; at 40–60 ns, loose clusters form; at 80–100 ns, they eventually evolve into a spherical, dense nanoaggregate, which is consistent with the uniform spherical nanoparticles observed by TEM. Figure 1 A) The results are consistent.

[0061] Interaction energy analysis of the stability and binding strength between BET and TAX ( Figure 2 E). Throughout the equilibrium phase, the average van der Waals contribution (LJ-SR) was -1363.4 kJ / mol, and the average electrostatic Coulomb contribution (Coul-SR) was -1159.8 kJ / mol; at the simulation endpoint, they reached -1432.0 kJ / mol and -1423.9 kJ / mol, respectively. The absolute values ​​of the van der Waals energy and the electrostatic energy are almost equivalent, indicating that the self-assembly of BET and TAX is a synergistic process dominated by both hydrophobic / dispersion forces and electrostatic attraction.

[0062] Further analysis of the types of effects ( Figure 2 F) shows that there are various non-covalent interactions between BET and TAX, including π-π stacking, hydrogen bonding, C-H bonding, electrostatic interactions, and π-σ interactions. Among these, hydrogen bonding mainly occurs between the carboxylate group of BET and the phenolic hydroxyl group of TAX, which is related to the disappearance of the carboxylate peak in the FTIR ( Figure 1 C) and a significant decrease in the phenolic hydroxyl proton signal in ¹H NMR ( Figure 1 E) Complete agreement. π-π stacking and cation-π interaction (between the quaternary ammonium group of betaine and the aromatic ring of TAX) are manifestations of van der Waals forces.

[0063] Molecular dynamics simulations, from a microscopic perspective, explain that BET and TAX can spontaneously assemble into dense spherical nanoparticles. The driving force for this assembly comes from the synergistic contribution of hydrogen bonding, electrostatic interactions, and hydrophobic / π-stacking, providing a clear molecular explanation for the structural stability of BTNPs.

[0064] In summary, multiple characterization methods consistently demonstrate that BTNPs are self-assembled nanoparticles synergistically driven by BET and TAX through hydrogen bonding, electrostatic interactions, and hydrophobic interactions. TEM shows that they are uniform spherical; FTIR and... 1H NMR confirmed the formation of hydrogen bonds between the BET carboxylate group and the TAX phenolic hydroxyl group, and the changes in proton chemical shift and peak shape reflected intermolecular interactions. XRD showed that the drug transformed from a crystalline state to an amorphous state, which is beneficial to improving dissolution. Molecular dynamics simulations further verified the densification process of the assembly and the synergistic contribution of van der Waals forces and electrostatic forces from a microscopic perspective. These results lay the physicochemical foundation for BTNPs as a stable and uniform nano-formulation.

[0065] 2.2. Antioxidant Activity Analysis of BTNPs Table 1 Antioxidant activity of BTNPs Antioxidant type BET TAX BTNPs A mixture of BET and TAX (molar ratio 1:1) DPPH removal rate % 17.28 76.82 73.49 62.94 ABTS clearance rate % 25.71 85.53 78.43 68.77 In the DPPH and ABTS free radical scavenging experiments, the antioxidant activities of all tested samples showed a consistent trend: TAX > BTNPs > BET / TAX physical mixture > BET. BET exhibited almost no direct antioxidant activity, while TAX demonstrated the strongest free radical scavenging ability. The antioxidant activity of BTNPs was slightly lower than that of free TAX, but significantly higher than that of the physical mixture, indicating that the nanodelivery system can effectively retain the antioxidant activity of TAX and avoid the potential antagonistic effects that may exist when BET and TAX are physically mixed.

[0066] 2.3. Solubility of BTNPs Table 2 Solubility of BTNPs Temperature (°C) BET (mg / 10mL) TAX (mg / 10mL) BTNPs (mg / 10mL) 25（℃） Very soluble 8.94 23.68 50（℃） Very soluble 57.71 219.46 80（℃） Very soluble 146.65 656.39 100（℃） Very soluble 278.34 1442.63 Note: Very soluble means that 1g of solute can dissolve in less than 1ml of solvent.

[0067] 2.4. BTNPs inhibit LPS-induced macrophage inflammation In the early stages of alcoholic liver injury, LPS translocation is a key event triggering liver inflammation. LPS activates hepatic macrophages via TLR4, initiating the downstream myeloid differentiation factor 88 (MyD88) and NF-κB signaling cascade. This leads to the massive release of pro-inflammatory cytokines, a central process in the progression from ALI to a chronic inflammatory state. Therefore, targeting the TLR4 / NF-κB pathway in macrophages is a crucial strategy for treating ALI. In this invention, the effectiveness of BTNPs in inhibiting LPS-induced inflammation was first evaluated, and their potential mechanisms in RAW264.7 macrophages were investigated.

[0068] First, the cytotoxicity of BET, TAX, and BTNPs to RAW264.7 cells was evaluated using the MTT assay. Figure 3As shown in Figure A, no significant cytotoxicity was observed in any treatment at concentrations up to 40 μg / mL. Notably, BTNPs at concentrations of 20 and 40 μg / mL even showed slight proliferative effects (p<0.05). When the concentration was increased to 80 μg / mL, free BET and TAX reduced cell viability to 90.2 ± 5.3% and 91.5 ± 4.8%, respectively, while BTNPs maintained viability at 96.7 ± 3.9%. At the highest concentration of 160 μg / mL, all formulations induced cytotoxicity, reducing viability to 74.6 ± 6.2% (BET), 78.3 ± 5.5% (TAX), and 83.1 ± 4.7% (BTNPs). Based on these results, 40 μg / mL was selected as the safe concentration for subsequent anti-inflammatory experiments.

[0069] Next, the effects of BTNPs on LPS-induced cytokine release were evaluated. LPS stimulation strongly promoted the release of key pro-inflammatory cytokines such as TNF-α and IL-1β, while inhibiting the anti-inflammatory cytokine IL-10, reflecting the characteristic inflammatory imbalance in ALI. Figure 3 As shown in BD, compared with the control group, LPS stimulation significantly increased the secretion of TNF-α and IL-1β and significantly inhibited the secretion of IL-10 (p<0.001). Pretreatment with free BET or TAX alone partially reversed this imbalance. However, BTNPs exhibited excellent modulatory capabilities, inhibiting the release of TNF-α and IL-1β and restoring IL-10 levels to a much higher level than either of the free drugs alone. This result strongly suggests that the co-delivery of BET and TAX in nanoparticles achieves a synergistic anti-inflammatory effect.

[0070] Western blot analysis showed that LPS stimulation significantly activated the TLR4 / MyD88 / NF-κB signaling pathway in RAW264.7 cells. Compared with the control group, the expression levels of TLR4 and MyD88 proteins in the model group were significantly increased (P<0.01 or P<0.001), and the phosphorylation ratio of p-IκBα / IκBα to p-NF-κB / NF-κB was also sharply increased (P<0.001). Figure 3 E). Using BET or TAX alone partially alleviated the aforementioned pathway changes. Compared to the model group, the levels of TLR4 and MyD88 proteins decreased in both the BET and TAX groups (P<0.05 or P<0.01), but the phosphorylation ratios of p-IκBα / IκBα and p-NF-κB / NF-κB remained high (P<0.05 or P<0.01). Figure 3FH). Notably, the therapeutic effect of BTNPs exceeded the inhibitory effect of single drugs. After BTNP intervention, the expression of TLR4 and MyD88 proteins, as well as the phosphorylation ratios of both, were significantly inhibited, returning to levels close to those of the control group (P<0.01 or P<0.001), indicating that it almost reversed LPS-induced activation of this pathway. Figure 3 FH).

[0071] Based on the above data analysis, BTNPs can effectively inhibit the TLR4 / MyD88 / NF-κB signaling pathway in macrophages, thereby effectively alleviating the LPS-triggered inflammatory cascade response.

[0072] 2.5. Effects of BTNPs on mouse condition and liver injury 2.5.1. Effects of BTNPs on mouse condition During the administration period, the mice were observed daily for their mental state, behavior, fur luster, and fecal characteristics. Results showed that, compared to the control group, mice given BTNPs and TAX exhibited good mental state, normal activity, and smooth, clean fur, without any abnormal behaviors such as restlessness, lethargy, or huddling. Regarding gastrointestinal reactions, the mice produced well-formed, normal-textured feces, without diarrhea, loose stools, bloody stools, or abdominal distension. In contrast, a small number of mice given BET showed lethargy but normal activity, and a few had rough fur, with occasional diarrhea and loose stools.

[0073] To investigate whether BTNPs could improve the gastrointestinal adverse reactions of BET, this invention administered BET to 10 mice simultaneously at a dose of 15 mg / kg for 6 weeks. It was found that regardless of whether the dose was 15 mg / kg or 40 mg / kg, long-term administration of BET resulted in varying degrees of diarrhea and loose stools in some mice. These results indicate that at the experimental dose, BTNPs had no significant adverse effects on the overall condition of the mice, demonstrated good safety, did not cause significant irritation to the gastrointestinal tract, and could improve the gastrointestinal adverse reactions induced by BET.

[0074] 2.5.2. Effects of BTNPs on acute liver injury in mice To evaluate the in vivo hepatoprotective effect of BTNPs, this invention established a mouse model of acute alcoholic liver injury (ALI). Figure 4 A). During the six-week treatment period, the body weight of mice in all groups increased steadily, with no significant difference between groups. Figure 4 B). Alcohol exposure led to a significant increase in liver index in the model group compared to the control group (P<0.001), indicating hepatomegaly, a typical pathological feature of ALI (amyotrophic lateral sclerosis). Figure 4C). BET or TAX administration alone only resulted in a slight decrease in liver indices (P < 0.05), with limited improvement. In contrast, BTNPs treatment significantly reduced liver indices (P < 0.01), with effects comparable to the positive control SILY. These results suggest that BTNPs can synergistically alleviate alcohol-induced pathological liver swelling.

[0075] Consistent with changes in liver indices, BTNPs also significantly improved serum markers of liver injury. In the model group mice, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels all increased sharply. Figure 4 DF, p The levels were <0.001, confirming severe impairment of hepatocyte membrane integrity and parenchymal damage. Treatment with free BET or TAX partially reversed the elevation of these enzymes. In contrast, BTNPs treatment more effectively suppressed alcohol-induced elevations of ALT, AST, and LDH (p<0.001 or p<0.001). <0.01). The efficacy of BTNPs was comparable to that of SILY and significantly superior to the corresponding monotherapy. These results indicate that BTNPs have a synergistic protective effect in maintaining hepatocyte membrane integrity and reducing direct alcohol toxicity and secondary inflammatory damage.

[0076] Oxidative stress is a core pathological component of ALI, forming a vicious cycle with inflammation to jointly drive hepatocellular damage. Therefore, this invention assesses key indicators of hepatic redox status. For example... Figure 4 As shown by GI, compared with the control group, the liver's antioxidant defense system in the model group was severely impaired: the levels of reduced glutathione (GSH) and catalase (CAT) activity were significantly reduced (p < 0.05). <0.001), while the level of malondialdehyde (MDA), an end product of lipid peroxidation, was significantly increased (p <0.001). <0.001). This clearly indicates a severe state of alcohol-induced oxidative damage. Free BET or TAX treatment has a mitigating effect on these oxidative stress markers. However, BTNPs showed the most effective improvement, more significantly restoring GSH levels and CAT activity, and effectively reducing MDA content (p < 0.001). <0.001). BTNPs were similar to SILY in restoring oxidative imbalance and superior to both drugs alone. This result indicates that BTNPs effectively disrupt the vicious cycle between oxidative stress and inflammation in alcoholic liver injury, which is crucial for their synergistic hepatoprotective effect.

[0077] 2.6. BTNPs ameliorate alcohol-induced liver histopathological damage and hepatocyte apoptosis in mice. 2.6.1. Macroscopic and Histopathological Observations Macroscopic and histopathological examinations of the liver provide the most direct evidence of damage. For example... Figure 5 As shown in Figure A, the livers of the control group were a healthy, deep red color with a smooth surface. In contrast, the livers of the model group were pale white with obvious white granular spots on the surface, suggesting steatosis and focal inflammation. The livers of the groups treated with free BET or TAX showed some improvement in morphology. In contrast, the livers of mice treated with BTNPs or SILY recovered to near-normal morphology, and were very similar to the control group in both color and surface texture.

[0078] H&E staining further revealed histopathological changes at the microscopic level. Figure 5 A). The control group showed well-structured hepatic cords with centrally located nuclei and no signs of inflammation or steatosis. Alcohol exposure leads to severe liver injury characterized by extensive hepatocellular steatosis, disruption of hepatic cord structure, and irregular hepatocyte arrangement. Treatment with free BET or TAX alone significantly reduced the area of ​​necrosis. However, BTNPs treatment showed the most significant protective effect: markedly reduced liver injury, decreased necrosis, orderly arrangement of hepatic cords, and normal radiating of hepatocytes from the central vein.

[0079] 2.6.2. Improvement of hepatocyte apoptosis TUNEL staining was performed to directly assess hepatocyte apoptosis in ALI mice. Figure 5 B). The liver tissue of the model group had abundant TUNEL-positive cell nuclei and a significantly increased apoptosis index (p). <0.001). All treatment groups reduced the number of TUNEL-positive cells and decreased the apoptosis index. Although the free BET and TAX groups showed a moderate reduction (p <0.001). <0.05), but the BTNPs group showed a significantly strong anti-apoptotic effect (p ...). <0.001). TUNEL-positive cells were extremely sparse, and the apoptosis index was significantly lower than that of the model group and each single drug group, recovering to a level comparable to that of SILY and the control group.

[0080] In summary, these histopathological findings consistently demonstrate that acute alcohol exposure induces hepatic steatosis, inflammatory infiltration, and apoptosis. BTNPs most effectively reversed these pathological processes, exhibiting significantly superior protective effects compared to single-component administration. This nanosystem not only alleviates liver inflammation by effectively inhibiting the expression of pro-inflammatory factors TNF-α and IL-1β, but also inhibits hepatocyte apoptosis at its source by restoring the Bax / Bcl-2 balance, thereby achieving optimal preservation of liver tissue structure and function. These results are highly consistent with data on oxidative stress and inflammatory cytokines, collectively confirming the remarkable synergistic therapeutic effect of BTNPs against acute alcoholic liver injury.

[0081] 2.7 BTNPs regulate gut microbiota and improve alcohol-induced endotoxemia by restoring gut microbiota homeostasis. Alcohol intake disrupts the integrity of the intestinal barrier, inducing gut microbiota dysbiosis and leading to the translocation of gut-derived endotoxins (LPS) into the portal circulation. LPS entering the portal vein can induce endotoxemia. This invention evaluates the effects of BTNPs on endotoxemia and the composition and structure of the gut microbiota.

[0082] 2.7.1 Improvement of endotoxemia First, serum LPS levels were measured, a direct indicator of intestinal barrier function and systemic endotoxemia. Results showed that the serum LPS level in the alcohol-fed model group (535.68 ± 10.99 ng / L) was significantly higher than that in the control group (406.42 ± 6.23 ng / L) (p<0.001), confirming that alcohol successfully induced endotoxemia. Figure 6 A). Administration of free BET (517.69 ± 10.21 ng / L) or TAX (516.44 ± 8.05 ng / L) only slightly reduced LPS levels. In contrast, BTNPs treatment showed a stronger modulatory effect, significantly reducing LPS levels to 484.47 ± 13.19 ng / L (p<0.01), close to the normal range. This finding suggests that BTNPs are superior to monotherapy in reducing endotoxin levels.

[0083] 2.7.2 Analysis of the overall structure and diversity of gut microbiota To gain a deeper understanding of the global impact of BTNPs on the gut microbiota, 16S rRNA gene sequencing was performed on the cecal contents of mice. Venn diagram analysis showed that alcohol intake significantly reduced the number of unique species in the gut microbiota (model group unique OTU number: 10,134), while BTNP intervention restored it to 12,184, indicating that this nanotherapy can reverse alcohol-induced loss of species richness. Figure 6 B). Both the dilution curve and the abundance ranking curve tend to flatten out, confirming that the sequencing depth is sufficient to cover the microbial diversity in the sample. Figure 6 C, D).

[0084] Further utilize the alpha diversity index to assess the overall ecological health of the microbial community. For example... Figure 6As shown in E–G, alcohol intake severely impaired the richness and evenness of the gut microbiota, significantly reducing the Chao1, Shannon, and Simpson indices (p<0.01 or p<0.001). Free BET or TAX had limited effects in improving these indices. Notably, BTNPs treatment effectively reversed the decline in diversity, restoring these indices to near-control levels. The efficacy of BTNPs was comparable to that of SILY and significantly superior to any single drug (e.g., Shannon index, BTNPs vs. model group, p<0.05). This indicates that BTNPs can synergistically restore the ecological stability of the gut microbiota.

[0085] β-diversity analyses (PCoA and NMDS) provided more visual evidence at the community structure level. In ordination plots, the microbial community structure of the model group formed clusters that were clearly separated from the control group. Conversely, the BTNPs treatment group and the SILY group clustered more tightly with the control group. Figure 7 (A, B). This clearly demonstrates that BTNPs can restore the overall microbial community structure damaged by alcohol to a healthy state.

[0086] 2.7.3 Specific changes in gut microbiota at the phylum and genus levels Further analysis revealed specific changes in the composition of the microbial community at the phylum and genus levels. At the phylum level, the model group exhibited characteristic dysbiosis: the relative abundance of Firmicutes significantly decreased from 42.88% to 29.92%, while Bacteroidetes increased from 52.42% to 64.69%, resulting in a substantial decrease in the crucial Firmicutes / Bacteroidetes (F / B) ratio. Figure 7 C, E–G, p<0.001). BTNPs treatment effectively reversed this imbalance, significantly increasing Firmicutes abundance (to 61.27%) and decreasing Bacteroidetes abundance (to 30.92%), thereby normalizing the F / B ratio. Its improvement was significantly superior to monotherapy.

[0087] At the genus level, this invention focuses on key bacterial genera beneficial to intestinal barrier function and anti-inflammation. Alcohol intake significantly reduces probiotic levels. Lactobacillus and related to metabolic health Oscillospira abundance ( Figure 7 D, H). BTNP intervention uniquely restored the abundance of these beneficial bacteria (e.g., observed). Oscillospira (a significant increase).

[0088] Based on the above data analysis, this invention provides multidimensional evidence, from endotoxemia to gut microbiota, demonstrating that BTNPs can remodel alcohol-induced gut microbiota dysbiosis. Their effects include restoring α-diversity, correcting overall β-diversity structure, improving phylum-level compositional imbalances, and specifically enriching beneficial genera. This broad and synergistic microbiota regulation helps alleviate endotoxemia.

[0089] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing self-assembled dihydroquercetin nanoparticles, characterized in that, The method includes the following steps: first, dissolve betaine and dihydroquercetin separately in methanol; then mix the methanol solutions of betaine and dihydroquercetin at a 1:1 molar ratio and adjust the pH to 7.0-7.5 with sodium hydroxide solution; then add the mixture to preheated PBS with stirring, seal and keep at 55-65℃ for 20-40 minutes; after cooling to room temperature, dialyze the mixture with ultrapure water using a dialysis bag, and freeze-dry to obtain BTNPs, i.e., self-assembled dihydroquercetin nanoparticles.

2. The method for preparing dihydroquercetin self-assembled nanoparticles according to claim 1, characterized in that, The nanoparticles have a uniform morphology, are regular spherical, and have an average diameter of 78.55 ± 12.99 nm, and the drug exists in an amorphous form.

3. The method for preparing dihydroquercetin self-assembled nanoparticles according to claim 1, characterized in that, The amount of betaine used was 35.145 mg, and the amount of dihydroquercetin used was 91.275 mg. The betaine and dihydroquercetin were dissolved in 10 mL of methanol respectively. The mixture was added to preheated PBS with stirring, sealed and kept at 60°C for 30 minutes. The molecular weight cutoff of the dialysis bag was 3500 Da, and ultrapure water was used for dialysis for 24 hours.

4. The use of the dihydroquercetin self-assembled nanoparticles prepared according to claim 1 or 3 in the preparation of a medicament for treating acute alcoholic liver injury.

5. The use of the dihydroquercetin self-assembled nanoparticles prepared according to claim 1 or 3 in the preparation of medicaments for treating or regulating intestinal flora imbalance caused by alcohol.

6. The application according to claim 5, characterized in that, The dihydroquercetin self-assembled nanoparticles are used to improve the diversity, richness, and uniformity of bacterial communities, increase the abundance of Firmicutes, and decrease the abundance of Bacteroidetes.

7. The application according to claim 5, characterized in that, The dihydroquercetin self-assembled nanoparticles are used to increase the abundance of beneficial bacteria, which include... Oscillospira .

8. The use of the dihydroquercetin self-assembled nanoparticles obtained according to claim 2 or 3 in the preparation of drugs for the treatment or prevention of endotoxemia.

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