Application of astragaloside in preparation of HMGB1 inhibitor

Abstract

The invention provides an application of a natural compound astragaloside in preparation of an HMGB1 inhibitor. Proved by experiments on a protein level, a cell level and an animal level, astragaloside can be directly combined with HMGB1 to influence a protein space structure of the HMGB1 and inhibit HMGB1-induced inflammation, has a certain protection effect on drug-induced liver injury, can reduce the death rate of mice with septicopyemia induced by cecum ligation perforation (CLP), and can be used for preparing medicines for treating liver injury. Astragaloside is expected to become a medicine for preventing and treating HMGB1 induced diseases.

Description

Technical Field

[0001] This invention belongs to the field of natural medicines and relates to the application of astragaloside A in the preparation of HMGB1 inhibitors. Technical Background

[0002] High mobility group box-1 (HMGB1) is a non-histone chromosome-binding protein found in the nucleus of eukaryotic cells.

[0003] HMGB1, as an important biomarker and potential drug target, has been validated in various diseases, including neurodegenerative diseases, lung injury, and trauma / ischemia-reperfusion injury. HMGB1 resides in the nucleus of eukaryotic cells and participates in vital activities such as DNA replication, recombination, repair, and gene transcription regulation. When released extracellularly by activated immune cells and necrotic cells, HMGB1 primarily activates intracellular pathways such as NF-κB by binding to advanced glycation end products (RAGE) and Toll-like receptors (TLRs) on the cell membrane. It not only has a cascade amplification effect on its own secretion but also promotes the secretion of other inflammatory factors, vascular adhesion molecules, and chemokines, playing a crucial role in the occurrence, development, and maintenance of inflammatory responses. It is a core component of the inflammatory cytokine network in sepsis and related diseases. Furthermore, the key role of HMGB1 in drug-induced liver injury (DILI) has been confirmed in a hepatocyte-specific HMGB1 knockout mouse model of DILI, where HMGB1 antibodies or inhibitors significantly alleviate liver injury. Therefore, inhibiting HMGB1 activity and developing HMGB1 inhibitors is one of the important ways to prevent and treat HMGB1-induced diseases.

[0004] Currently, the development of HMGB1 inhibitors is being studied from four technical perspectives: 1. Receptor-type inhibitors, which negatively regulate the stimulatory activity of HMGB1 by binding to it, such as anti-HMGB1 antibodies; 2. Inhibitors that inhibit HMGB1 release, such as bicyclol; 3. Inhibitors that inhibit HMGB1 expression, such as berberine; 4. Inhibitors that inhibit HMGB1 nucleoplasmic translocation, such as emodin.

[0005] However, to date, there is only limited clinical evidence for therapeutics targeting extracellular HMGB1, and some promising candidates are still in preclinical or clinical development. However, existing natural products such as aspirin can interact with HMGB1, thereby interfering with its function and achieving therapeutic effects. Given the limited number of therapeutic drugs and the fact that potential drugs are still in clinical trials, identifying HMGB1 inhibitors from natural products is of great significance.

[0006] Astragalus is the dried root of *Astragalus mongholicus* or *Astragalus membranaceus*, belonging to the legume family. It possesses properties such as tonifying qi and strengthening the exterior, promoting tissue regeneration and healing, and diuresis. Astragalosides IV (ASI) is a tetracyclic triterpenoid compound isolated from Astragalus, exhibiting good anti-inflammatory and immunomodulatory activities. This study found that it directly binds to HMGB1, affecting its protein spatial structure and inhibiting HMGB1-induced inflammation. It also provides some protection against drug-induced liver injury and reduces mortality in mice with cecal ligation-perforation (CLP)-induced septicemia. Therefore, astragalosides IV can serve as an HMGB1 inhibitor and holds promise as a drug for preventing and treating HMGB1-induced diseases. Summary of the Invention

[0007] This invention relates to the application of astragaloside A in the preparation of HMGB1 inhibitors.

[0008] This invention relates to the application of astragaloside A in the preparation of drugs for treating sepsis.

[0009] This invention relates to the application of astragaloside A in the preparation of drugs for drug-induced liver injury.

[0010] Preferably, the HMGB1 inhibitor further includes pharmaceutically acceptable excipients.

[0011] In another aspect of the present invention, a pharmaceutical composition for preventing and treating HMGB1-induced diseases is provided, wherein the pharmaceutical composition uses astragaloside A as the active ingredient.

[0012] Preferably, the dosage form of the composition includes patches, pastes, ointments, gels, films, poultices, tablets, oral liquids, injections, capsules, or granules.

[0013] The application of astragaloside A in the screening and preparation of anti-HMGB1 drugs; astragaloside A is derived from the following Astragalus membranaceus.

[0014] After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Then dry it until it is completely dry.

[0015] After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Spray the small bundles with wood ash and dry them thoroughly.

[0016] Fresh or dried astragalus root, sliced ​​and stir-fried, with wood ash added during the stir-frying process.

[0017] Fresh or dried Astragalus membranaceus is soaked in a wood ash solution and then dried.

[0018] The astragaloside A described in this invention directly binds to HMGB1, affecting its protein spatial structure, inhibiting HMGB1-induced inflammation, and exhibiting a certain protective effect against drug-induced liver injury. Furthermore, it can reduce the mortality rate of HMGB1-induced diseases. Therefore, astragaloside A shows potential application prospects in the prevention and treatment of HMGB1-induced diseases. The beneficial effects of this invention are:

[0019] This invention expands the application of astragaloside A in the prevention and treatment of HMGB1-induced diseases, enriching the existing range of HMGB1 inhibitors. Furthermore, astragaloside A used is a natural compound, abundant and readily available, inexpensive, easy to prepare, with fewer side effects, and high patient acceptance, making it suitable for widespread clinical use. Therefore, astragaloside A has promising development prospects in inhibiting HMGB1 activity. Attached Figure Description

[0020] Figure 1 Surface plasmon resonance spectroscopy: Real-time association and dissociation curves of astragaloside A (A) and HMGB1 binding.

[0021] Figure 2 Fluorescence spectra of HMGB1 and astragaloside A (AI). (HMGB1 concentration: 3 μM; astragaloside A (AI) concentrations: 0, 2.5, 5, 10, 20, 40, 60, 80, 100 μM; λex = 273 nm).

[0022] Figure 3 Synchronous fluorescence spectra of astragaloside A (AC)-HMGB1 (Δλ = 15 nm or Δλ = 60 nm) (HMGB1 concentration was 3 μM, and the astragaloside A concentrations of AI were 0, 2.5, 5, 10, 20, 40, 60, 80, 100 μM, λex = 273 nm).

[0023] Figure 4 Three-dimensional fluorescence spectra of HMGB1 (A) and HMGB1-astragaloside A (B) (HMGB1 concentration: 3 μM, astragaloside A concentration: 20 μM, 298 K).

[0024] Figure 5 The inhibitory effect of astragaloside A on HMGB1-induced NO release.

[0025] Figure 6 Effects of astragaloside A on L02 cell viability (8mM APAP stimulation of L02 cells to establish a liver injury model). Detailed Implementation

[0026] The present invention will be further described in detail below through specific embodiments.

[0027] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0029] Example 1. Detection experiment of direct binding between astragaloside A and HMGB1

[0030] The affinity between astragaloside A and HMGB1 was detected using a Biacore T200 instrument. Channels 1 and 3 of the CM5 chip were set as reference channels, and channels 2 and 4 were set as detection channels immobilized with HMGB1. The CM5 chip was activated with EDC / NHS solution for 7 min. HMGB1 protein was diluted to 100 μg / mL with 10 mM sodium acetate solution (pH = 5.0) and coupled to the second channel at a coupling amount of 4500 response units (RU) and a flow rate of 10 μL / min.

[0031] Finally, unreacted active sites on the CM5 chip were blocked with ethanolamine (pH = 8.5) for 7 min. The compounds were dissolved and diluted in PBS containing 5% DMSO to test concentrations (3.1 μM, 6.2 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM), and then injected into the Biacore T200 detection system at a rate of 30 μL / min. The binding and dissociation times of the two ligands with HMGB1 protein were set to 60 s. Data were analyzed using Biacore T200 evaluation software, and the affinity constants with HMGB1 were calculated using a 1:1 steady-state affinity model.

[0032] Results: See attached. Figure 1 and Table 1

[0033] Real-time association and dissociation curves showed that astragaloside A interacts with HMGB1, exhibiting rapid association and dissociation rates. The dissociation constant (KD) was calculated to be 2.77 × 10⁻⁶ using steady-state fitting. -5 M. As attached Figure 1 As shown, there may be a transient and weak interaction between astragaloside A and the target protein HMGB1, which is consistent with the mechanism of action of traditional Chinese medicine.

[0034] Table 1 Affinity constants of astragaloside A and HMGB1

[0035]

[0036] Example 2. Experiment on the effect of astragaloside A on the spatial structure of HMGB1

[0037] Fluorescence spectroscopy can be used to detect intermolecular interactions and their effects on protein spatial conformation by observing changes in excitation and emission spectra and the fluorescence intensity of proteins with and without ligands. In this work, astragaloside-HMGB1 complexes with different ligand concentrations were prepared, with a protein solution without small molecules serving as a control. The final concentration of HMGB1 was fixed at 3.0 μM, and the final concentrations of astragaloside ranged from 2.5 to 100 μM. Steady-state fluorescence spectra were recorded in the wavelength range of 290–400 nm, with an excitation wavelength of 273 nm and a path length of 0.1 cm. The slit widths for excitation and emission were set to 10 nm, and the scan rate was 1200 nm / min. Each concentration was prepared and measured in triplicate.

[0038] The differences in excitation and emission wavelengths (Δλ = λem - λex) for the synchronous fluorescence spectra of tyrosine (Tyr) and tryptophan (Trp) were set to 15 nm and 60 nm, respectively. At 298 K, the Tyr and Trp spectra of HMGB1 were detected in the wavelength ranges of 250–350 nm and 240–350 nm, respectively. The three-dimensional fluorescence spectrum at 298 K was scanned using an optical path length of 0.1 cm. The final concentration of HMGB1 used for synchronous and three-dimensional fluorescence was 3.0 μM, and the final concentration of astragaloside A was 20.0 μM for both.

[0039] The results are attached. Figure 2 Appendix Figure 3 Appendix Figure 4 and Table 2

[0040] To investigate the effects of interactions on the conformation of HMGB1 and its surrounding amino acid microenvironment, steady-state fluorescence spectra were scanned using an excitation wavelength of 273 nm and an emission wavelength of 290–400 nm. The results showed that the maximum emission wavelength of HMGB1 was approximately 330 nm, indicating that the Trp residues were surrounded by a hydrophobic environment. Astragaloside IV did not emit fluorescence in the 290–400 nm wavelength range and did not interfere with the fluorescence spectrum of the HMGB1 protein. In the presence of astragaloside IV, the fluorescence intensity of HMGB1 gradually decreased in a concentration-dependent manner, indicating that astragaloside IV binds to HMGB1. Furthermore, the addition of astragaloside IV caused a slight redshift of the maximum absorption wavelength of HMGB1 (see attached image). Figure 2 The above results demonstrate that astragaloside A can directly bind to HMGB1, thereby affecting the microenvironment of its amino acid residues.

[0041] In the appendix Figure 3The study also recorded the synchronous fluorescence spectra of HMGB1 in the presence and absence of astragaloside. When Δλ = 15 nm, it refers to the fluorescence spectrum of Tyr residues; when Δλ = 60 nm, it reflects the fluorescence characteristics of Trp residues. Changes in the maximum emission wavelength of HMGB1 protein can reveal the polarity / hydrophobicity around the chromogenic group and the spatial conformation of HMGB1. A red shift in the maximum emission wavelength of HMGB1 indicates an increased polarity of the amino acid microenvironment. With increasing astragaloside concentration, the fluorescence intensity of Trp and Tyr exhibited a regular quenching. Specifically, astragaloside was more effective at quenching the fluorescence of Trp residues. However, no significant red or blue shift in the maximum emission wavelength indicated that the binding of astragaloside to HMGB1 had little effect on the polarity and hydrophobicity of the microenvironment.

[0042] Three-dimensional fluorescence spectroscopy comprehensively presents information on excitation, emission spectra, and fluorescence intensity, and is often used to explore the influence of small molecules on the spatial conformation of proteins.

[0043] As attached Figure 4 As shown, the three-dimensional fluorescence of HMGB1 contains four characteristic peaks. Peak a is the Rayleigh scattering peak (λex = λem), and peak b is the second-order scattering peak (2λex = λem). Peak 1 (λex = 280 nm, λem = 340 nm) reflects the spectral characteristics of Trp and Tyr and changes in the microenvironment. Peak 2 (λex = 230 nm, λem = 340 nm) originates from changes in the polypeptide chain backbone of n-π* transition proteins, representing changes in the protein's secondary structure. By observing the changes in the four characteristic peaks in the presence of two ligands, their interaction characteristics with HMGB1 were studied. (See attached image for details.) Figure 4 In the fluorescence spectrum, peak 1 showed a 12.25% decrease in fluorescence intensity, indicating that the addition of astragaloside A affected the polarity of the Trp microenvironment. Peak 2 showed a 13.20% decrease in fluorescence intensity, indicating a change in the secondary structure of HMGB1. The three-dimensional fluorescence results were consistent with the steady-state fluorescence spectroscopy results, further confirming that astragaloside A can induce conformational changes in HMGB1 through direct binding.

[0044] Table 2. Three-dimensional fluorescence spectral data of the interaction between astragaloside A and HMGB1.

[0045]

[0046] Example 3. Astragaloside A inhibits HMGB1-induced inflammation.

[0047] RAW 264.7 cells (1.0 × 10⁵ cells / well) were seeded in 96-well plates and cultured at 37°C for 12 hours in an incubator containing 5% CO₂. Astragaloside A (100, 50, 25, 12.5, 6.25, 3.125 μM) and HMGB1 (1 μg / mL) were co-incubated in vitro at 4°C for 1 hour, and then administered to the cells as the treatment group.

[0048] For the model group, 100 μL of DMEM medium containing 1 μg / mL HMGB1 was added. Free serum DMEM served as a blank control. Incubation continued at 37°C for 24 hours in an incubator containing 5% CO2. 100 μL of culture supernatant from each well was transferred to a new 96-well plate, followed by the addition of 50 μL of Griess reagent A and 50 μL of Griess reagent B. The mixture was thoroughly shaken and mixed, and the OD value at 540 nm was measured using a microplate reader (Tecan Trading AG, Switzerland).

[0049] The concentration of NO in the cell culture supernatant could be calculated using a NaNO2 standard curve. Cell viability was assessed using the standard MTT assay. The supernatant was discarded, and 100 μL of MTT (0.5 mg / mL) was added. After incubation at 37°C for 2 hours, the reaction was terminated by adding 150 μL of LDMSO. The amount of MTT formazan product was recorded by measuring the absorbance at 570 nm (test wavelength) and 630 nm (reference wavelength) using a microplate reader.

[0050] Results: See attached. Figure 5

[0051] Astragaloside A showed that it effectively downregulated the pro-inflammatory function of HMGB1 (see attached image). Figure 5 In the absence of HMGB1 in RAW264.7, no toxicity was observed and it significantly affected NO production.

[0052] Example 4. Experiment on the reduction of drug-induced liver injury by astragaloside A

[0053] Logarithmic growth phase L02 cells (2×10⁶ cells / mL) were seeded at 2 mL per well in 6-well cell culture plates and cultured overnight at 37°C in a 5% CO₂ incubator. After cell attachment, the 6-well plates were removed, and the supernatant was aspirated. Control, model, and drug-treated (100 μM astragaloside A) groups were set up. 1 mL of serum-free DMEM medium was added to each well in the control and model groups. 1 mL of serum-free DMEM medium containing different concentrations of the test compound was added to each well in the drug-treated group. Each group was divided into triplicates. The cells were cultured for another 1 h. 10 μL of APAP solution (final concentration 8 mM) was added to each well in the model and drug-treated groups, and the cells were cultured for another 36 h.

[0054] Cell viability was assessed using the standard MTT assay. The supernatant was discarded, and 100 μL of MTT (0.5 mg / mL) was added. After incubation at 37°C for 2 hours, the reaction was terminated by adding 150 μL of LDMSO. The amount of MTT formazan product was recorded by measuring the absorbance at 570 nm (test wavelength) and 630 nm (reference wavelength) using a microplate reader. (See attached image.) Figure 6 .

[0055] Results: Cell viability was significantly reduced in the model group (52.4%), almost unchanged in the blank group (99.8%), and in the drug-treated group (62.2%), demonstrating that astragaloside A can protect against drug-induced liver injury.

[0056] Example 5. Experiment on the improvement of survival rate of mice with cecal ligation-perforation (CLP) induced sepsis by astragaloside A.

[0057] Male Kunming mice weighing 18–30 g, SPF grade, were randomly divided into four groups of 10 mice each after 3 days of feeding: a sham-operated group, a model group, and an astragaloside A group. Except for the sham-operated group, the other mice underwent cecal ligation and perforation to induce sepsis. In the sham-operated group, the cecum was only exposed and sutured, without ligation or perforation.

[0058] The astragaloside A group was administered 40 mg / kg by gavage 2 hours before anesthesia for modeling. The sham-operated group and the model group were administered the same volume of distilled water by gavage. The administration was continued for 5 days. The condition of the mice was observed daily, and the time of death was recorded until the 10th day after modeling.

[0059] Results: No mice in the sham-operated group died within 10 days; mice in the model group died after day 2, with a survival rate of 20% after 10 days of modeling; mice in the astragaloside A group died on day 3 after modeling, with a survival rate of 60%. These results demonstrate that astragaloside A can reduce the mortality rate of mice with sepsis.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of astragaloside A in the preparation of HMGB1 inhibitors.

2. As described in claim 1, characterized in that, The application is a drug for preventing and treating HMGB1-induced diseases.

3. As described in claim 1, characterized in that, The application described is the use of astragaloside A in the preparation of drugs for treating sepsis.

4. As described in claim 1, characterized in that, The application described is the use of astragaloside A in the preparation of drugs for drug-induced liver injury.

5. A pharmaceutical composition for preventing and treating HMGB1-induced diseases, characterized in that, The pharmaceutical composition described herein uses astragaloside A as the active ingredient and further contains pharmaceutically acceptable excipients.

6. As described in claim 4, characterized in that, The dosage forms of the composition include patches, pastes, ointments, gels, films, poultices, tablets, oral liquids, injections, capsules, or granules.

7. Application of astragaloside A in the screening and preparation of anti-HMGB1 drugs. Astragaloside A is derived from the following Astragalus membranaceus. After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Then dry it until it is completely dry.

8. Application of astragaloside A in the screening and preparation of anti-HMGB1 drugs. Astragaloside A is derived from the following Astragalus membranaceus. After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Spray the small bundles with wood ash and dry them thoroughly.

9. Application of astragaloside A in the screening and preparation of anti-HMGB1 drugs. Astragaloside A is derived from the following Astragalus membranaceus. Fresh or dried astragalus root, sliced ​​and stir-fried, with wood ash added during the stir-frying process.

10. Application of astragaloside A in the screening and preparation of anti-HMGB1 drugs. Astragaloside A is derived from the following Astragalus membranaceus. Fresh or dried Astragalus membranaceus is soaked in a wood ash solution and then dried.

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