Use of saikosaponin B2 in preparation of a medicine

Abstract

This invention provides the use of saikosaponin B2 in drug preparation, belonging to the field of natural medicine technology. This invention discovers that saikosaponin B2 has a significant inhibitory effect on NLRP3 inflammasome activation, and can be used to prepare NLRP3 inflammasome inhibitors, which can be used to prepare drugs for treating diseases related to NLRP3 inflammasomes (including gout and gouty arthritis), showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to the use of saikosaponin B2 in the preparation of NLRP3 inflammasome inhibitors and drugs for treating diseases related to NLRP3 inflammasomes. Background Technology

[0002] Hyperuricemia is defined as an abnormal fasting serum uric acid level exceeding 420 μmol / L on two separate occasions in adults, regardless of gender, under normal purine dietary conditions. Hyperuricemia caused by abnormal purine metabolism has become a prevalent chronic disease affecting the health of the Chinese population. The formation of monosodium urate crystals (MSU) from uric acid in joints and kidneys is a major cause of gout. However, gout and hyperuricemia are two different diseases, and their treatments are not interchangeable.

[0003] Currently, gout patients primarily lower serum uric acid levels through preventative treatments that inhibit uric acid synthesis (such as the xanthine oxidase inhibitor febuxostat) or promote uric acid excretion (such as benzbromarone, which inhibits uric acid transport by renal tubular URAT-1). First-line treatment for acute gout attacks involves colchicine or nonsteroidal anti-inflammatory drugs (NSAIDs), but these have limitations in efficacy and can cause significant side effects. Second-line treatment involves glucocorticoids, which can cause joint and bone damage. Currently, there is no universally accepted and effective treatment for gout. Therefore, developing safe, effective, cost-effective, and convenient novel anti-gout drugs is of great importance.

[0004] Bupleurum is the dried root of *Bupleurum chinense* DC. or *Bupleurum corzonerifolium* Willd., both belonging to the Apiaceae family. Its functions and indications include harmonizing the exterior and interior, soothing the liver, and raising yang. It is used for colds with fever, alternating chills and fever, chest and rib pain, irregular menstruation, uterine prolapse, and rectal prolapse. Annual production is 5000-7000 tons. Although approximately 87 Chinese herbal medicines with Bupleurum as a main ingredient are currently undergoing clinical research in China for diseases related to colds such as fever, depression, tumors, and sepsis, research on gout is very limited. If the components of Bupleurum can inhibit the inflammatory response of gout, it has broad application prospects in gout, the second largest metabolic disease in my country, and may fill the market gap for acute gout drugs. Summary of the Invention

[0005] The purpose of this invention is to provide the use of saikosaponin B2 in the preparation of NLRP3 inflammasome inhibitors and medicaments for treating diseases related to NLRP3 inflammasomes.

[0006] This invention provides the use of saponin B2 in the preparation of NLRP3 inflammasome inhibitors.

[0007] This invention also provides the use of saponin B2 in the preparation of medicaments for the prevention and / or treatment of diseases associated with the NLRP3 inflammasome.

[0008] Furthermore, the disease associated with the NLRP3 inflammasome is gout.

[0009] Furthermore, the gout mentioned is acute gout.

[0010] Furthermore, the disease associated with the NLRP3 inflammasome is gouty arthritis.

[0011] Furthermore, the gouty arthritis mentioned is acute gouty arthritis.

[0012] Furthermore, the drug is a formulation prepared by adding pharmaceutically acceptable excipients with saponin B2 as the active ingredient.

[0013] Furthermore, the formulation is an oral formulation or an injectable formulation.

[0014] The present invention has achieved the following beneficial effects:

[0015] 1. This invention discovers that the active ingredient of Bupleurum chinense, Saikosaponin B2 (SSB2), significantly inhibits the activation of the NLRP3 inflammasome. In experiments activating the NLRP3 inflammasome in the mouse macrophage cell line J774A.1, Saikosaponin B2 significantly inhibited the release of IL-1β, IC50... 50 The concentration was 3.544 μM. Further research revealed that saikosaponin B2 can target ASC protein, an important component of the NLRP3 inflammasome. The affinity of saikosaponin B2 for ASC was approximately 9.39 μM, as determined by biomembrane interference technology.

[0016] 2. Saikosaponin B2 showed no significant toxicity to macrophages or hepatocytes. However, saikosaponin A, saikosaponin D, and saikosaponin B1 exhibited cytotoxicity against SK-Hep-1, HCCLM3, and J774A.1 macrophages at a concentration of 25 μM. Combined with data reported in the literature (Planta Medica, 1978, 34(2): 160-166), it can be seen that saikosaponin B2 showed lower hemolytic activity than saikosaponin A and saikosaponin D, indicating better safety.

[0017] 3. In the pharmacokinetic test, the results of this invention showed that when 20 mg / kg was administered orally, saikosaponin B2 reached its peak at 1.6 h, with a maximum concentration of 378 ng / ml, and decreased to half in about 4 h; however, according to the literature, when 20 mg / kg was administered orally, saikosaponin A was detected at 74±17 ng / ml at 30 min and disappeared after 2 h (Journal of Chromatography, 1984, 307(2): 488-492); after oral administration of saikosaponin A, it reached its peak at 15 min and decreased to half in 40 min (LifeSciences, 1986, 39(4): 297-301). The results of this invention indicate that saikosaponin B2, when administered intravenously (2 mg / kg), achieves a high maximum plasma concentration of 10512 ng / ml, and maintains a plasma concentration above 1000 ng / ml for the first 2 hours. However, according to literature reports, saikosaponin D, when administered intraperitoneally (5 mg / kg), reaches a maximum concentration of ~915 ng / ml after 4 hours (He Yu, Zhang Ruping, "Determination of Saikosaponin D Concentration in Rat Plasma by Solid Phase Extraction-High Performance Liquid Chromatography"). In other words, the pharmacokinetic properties of saikosaponin B2 in vivo are superior to those of saikosaponin A and saikosaponin D.

[0018] 4. In the experiment of an acute gouty arthritis model in mice, at the same dose of 40 mg / kg administered by gavage, 24 hours after administration, saikosaponin B2 relieved foot swelling better than saikosaponin A and saikosaponin D. Its inhibitory effect on IL-1β in swollen tissue was comparable to that of saikosaponin D and better than that of saikosaponin A.

[0019] In summary, this invention reveals that saikosaponin B2 significantly inhibits NLRP3 inflammasome activation and can be used to prepare NLRP3 inflammasome inhibitors for the treatment of diseases related to NLRP3 inflammasomes. Compared with saikosaponin A and saikosaponin D, saikosaponin B2 exhibits advantages in safety, in vivo pharmacokinetic properties, and therapeutic efficacy, demonstrating broad application prospects.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 This study screened saikosaponins for their inhibitory activity against the NLRP3 inflammasome. Figure A shows the chemical structures of each saikosaponin compound; Figure B shows the cytotoxic effects of saikosaponins on J774A.1 macrophages; Figure C shows the effect of saikosaponins on NLRP3 inflammasome activation-induced IL-1β expression; and Figure D shows the effect of saikosaponins on NLRP3 inflammasome activation-induced LDH release.

[0023] Figure 2 The effect of each group on the release of IL-1β under three different stimuli (ATP, Nigericin, and MSU stimulation from left to right) on mouse J774A.1 macrophages (Figure A) and mouse-derived primary macrophage BMDMs (Figure B) is shown. 1, 5, and 10 represent groups with saikosaponin B2 concentrations of 1, 5, and 10 μM, respectively.

[0024] Figure 3 The IC50 of saikosaponin B2 in inhibiting NLRP3 inflammasome activation in macrophages 50 (Figure A) and Western blotting results (Figure B).

[0025] Figure 4 Figure 1: Cell thermal displacement experiments of saikosaponin B2 under different temperature gradients (Figure A) and different concentration gradients of saikosaponin B2 (Figure B); Molecular docking analysis results (Figure C); Biomembrane interference experiment results (Figure D).

[0026] Figure 5 The cytotoxic effects of saikosaponins on hepatocytes SK-Hep1 (Figure A) and HCCLM3 (Figure B).

[0027] Figure 6 Figure A shows the in vitro hemolysis results of saikosaponin B2, and Figure B shows the hemolysis rate and hemolysis status.

[0028] Figure 7 The results of an acute blood toxicity test of saikosaponin B2 administered by gavage to mice.

[0029] Figure 8 The results of an acute blood toxicity test of saikosaponin B2 administered by gavage to mice.

[0030] Figure 9 The results of an acute toxicity test on the major organs of mice after oral administration of saikosaponin B2.

[0031] Figure 10 The results of an acute toxicity test on the major organs of mice after oral administration of saikosaponin B2.

[0032] Figure 11Results of acute blood toxicity test of saikosaponin B2 administered intraperitoneally in mice.

[0033] Figure 12 Results of acute blood toxicity test of saikosaponin B2 administered intraperitoneally in mice.

[0034] Figure 13 Results of acute toxicity tests on major organs of mice after intraperitoneal injection of saikosaponin B2.

[0035] Figure 14 Results of acute toxicity tests on major organs of mice after intraperitoneal injection of saikosaponin B2.

[0036] Figure 15 The results show the blood concentrations of saikosaponin B2, saikosaponin D, and saikosaponin D.

[0037] Figure 16 Pharmacokinetic parameters of saikosaponin B2, saikosaponin D and saikosaponin D administered by gavage (Figure A) and intravenously (Figure B).

[0038] Figure 17 The oral bioavailability of saikosaponin B2, saikosaponin D and saikosaponin D was determined.

[0039] Figure 18 The results are from the liver microsomal stability experiment of saikosaponin B2.

[0040] Figure 19 The experimental data on the relief of MSU+C18-induced acute gouty arthritis by intraperitoneal injection of saikosaponin B2 are shown in Figure A, which shows the swelling of the mouse paw; Figure B shows the change curve of the mouse paw circumference at different time points; Figure C shows the detection results of IL-1β content in the swollen tissue of the mouse paw; and Figure D shows the expression of inflammation-related proteins in the swollen tissue of the mouse paw.

[0041] Figure 20 Experimental data on the effects of oral administration of saikosaponin B2 on the relief of MSU+C18-induced acute gouty arthritis. Figure A shows mouse paw swelling, Figure B shows the change curve of mouse paw circumference at different time points, Figure C shows the detection results of IL-1β content in mouse paw swelling tissue, and Figure D shows the expression of inflammation-related proteins in mouse paw swelling tissue.

[0042] Figure 21 Animal experimental data on the anti-acute gouty arthritis effects of oral administration of saikosaponin B2, saikosaponin A and saikosaponin D. Figure A shows the swelling of the mouse paw, Figure B shows the change curve of mouse paw circumference at different time points, and Figure C shows the detection results of IL-1β content in the swollen tissue of the mouse paw. Detailed Implementation

[0043] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0044] Example 1: Inhibitory effect of saikosaponin B2 on NLRP3 inflammasome

[0045] 1. Experimental Methods

[0046] This invention first evaluated nine saponin compounds isolated from Bupleurum chinense (structures shown below). Figure 1 (As shown in Figure A) Effects on the NLRP3 inflammasome. The experimental method is as follows:

[0047] (1) Cytotoxicity assay: Cells were seeded in 96-well plates overnight. Then, the cells were treated with different concentrations of different compounds and incubated for another 24 hours. After that, medium containing 10% CCK8 was added and incubated for 1-4 hours, and the absorbance was measured at 450 nm.

[0048] (2) Activation of the NLRP3 inflammasome: To activate the classic NLRP3 inflammasome, cells were first treated with LPS (1 μg / ml) for 3 hours, and then treated with different concentrations of compounds or the positive control VX-765 (0.5 μM) for 30 minutes. Afterward, a second stimulant was added (150 μg / ml MSU for 4 hours, 5 mM ATP for 30 minutes, or 10 μM Nigericin for 45 minutes).

[0049] (3) IL-1β detection: Cell culture supernatant or mouse tissue supernatant was collected for the detection of mouse IL-1β. The experimental procedure was described in the manufacturer's instructions as follows: Add the required strips to 300 μl of 1× washing buffer and let stand for 30 seconds. Prepare diluted standards and samples. Add 100 μL of standard to the standard wells, 100 μL of standard diluent or culture medium to the blank wells, and 100 μL of sample to the sample wells. Seal the membrane and incubate on a shaker at room temperature for 1.5 hours. Discard the liquid and then wash 6 times with 1× washing buffer. Add 100 μl of detection antibody (1:100 dilution) to each well. Seal the membrane and incubate at room temperature for 30 minutes. After incubation, wash 6 times with washing buffer. Add 100 μl of horseradish peroxidase-labeled streptavidin diluted 1:100. Seal the membrane and incubate at room temperature for 30 minutes, then wash 6 times. Add 100 μL of a 1:100 diluted signal enhancer to each well. Seal the membrane and incubate precisely at room temperature for 15 minutes. Wash 6 times. Add 100 μL of a 1:100 diluted horseradish peroxidase-labeled streptavidin again and incubate for 15 minutes. Wash 6 times. Add 100 μL of chromogenic substrate and incubate at room temperature in the dark for 30 minutes. Finally, add 100 μL of stop solution. Detect OD values ​​at 450 nm and 630 nm wavelengths within 30 minutes.

[0050] (4) LDH detection: J774A.1 cells were seeded in 24-well plates and inflammasomes were activated overnight according to the method in (2). One well was selected as the "sample maximum enzyme activity control well". 1 h before sample collection, 10% volume of LDH release reagent was added to the culture medium and the cells were returned to the cell culture incubator for further culture. After stimulation, the supernatant was collected and centrifuged at 2000 rpm for 5 min at room temperature. The supernatant was collected. 120 μl of the supernatant from each well was added to a new 96-well plate, and 60 μl of LDH detection working solution (20 μl each of lactate solution, 1×INT solution, and enzyme solution) was added to each well. The cells were incubated at room temperature in the dark for 30 min, and the absorbance was measured at 490 nm using a microplate reader. The calculation formula is as follows: Cytotoxicity or mortality (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100.

[0051] 2. Experimental Results

[0052] The results showed that the main components isolated from Bupleurum chinense, saponins A, D, and B1, had significant cytotoxic effects on J774A.1 macrophages. Figure 1 B).

[0053] Excluding cytotoxic effects, saikosaponin B2 significantly inhibited NLRP3 inflammasome activation-induced IL-1β expression. Figure 1 C). Saikosaponin B2 can also effectively inhibit LDH release induced by NLRP3 inflammasome activation (an indicator of pyroptosis). Figure 1 D).

[0054] Saikosaponin B2, in J774A.1 and BMDM macrophages, inhibited IL-1β release in a concentration-dependent manner under three different stimuli of the NLRP3 inflammasome. Figure 2 In J774A.1 cells, NLRP3 inflammasome is activated, and saikosaponin B2 inhibits the IC50 of IL-1β release. 50 3.544 μM ( Figure 3 Western blotting results showed that saikosaponin B2 could also significantly inhibit the secretion of mature IL-1β and p20, but had no effect on the expression of pro-IL-1β and pro-caspase-1. Figure 3 ).

[0055] The above results indicate that saikosaponin B2 has a significant inhibitory effect on NLRP3 inflammasome activation and can be used to prepare NLRP3 inflammasome inhibitors for the preparation of drugs to treat diseases related to NLRP3 inflammasomes.

[0056] Example 2: Mechanism study of bufotoxin B2 inhibiting NLRP3 inflammasome activation

[0057] 1. Experimental Methods

[0058] (1) Cell thermal displacement assay: When J774A.1 cells reached 90% confluence in the culture dish, the cells were collected with PBS and then subjected to three freeze-thaw cycles in liquid nitrogen and a 37°C water bath. After centrifugation of the cell lysate, the supernatant was collected and divided into a dimethyl sulfoxide (DMSO) group and a saikosaponin B2 (100 μM) group. All samples were incubated at room temperature for 30 minutes, then at a specific temperature (48–58°C) for 3 minutes, and then placed on ice for 3 minutes. Finally, the samples were centrifuged again, and the supernatant was collected for Western blot analysis.

[0059] (2) Molecular docking: The crystal structure of ASC [PDB ID: 2KN6] was obtained from the Protein Data Bank. Chem 3D was used to minimize the energy of saikosaponin B2 to obtain the most stable conformation, and molecular docking was performed using Autodock. First, water molecules and ligands were removed from the ASC crystal structure, and then hydrogen atoms were added. To better predict the interaction between saikosaponin B2 and ASC, 100 docking operations were performed, and the result with the lowest binding free energy was selected. Finally, the structures of saikosaponin B2 and ASC were derived, and image processing was performed using PyMol.

[0060] (3) Biological membrane interference experiment

[0061] The binding affinity of PYD protein to compound B2 was determined using the GatorPrime biomembrane interferometry system (Gator Bio, Palo Alto, California, USA). All procedures were performed at 30°C and 1000 rpm. A simplified procedure was as follows: the SAS sensor (Gator Bio) was immersed in a PYD protein solution for 10 minutes to load the protein, followed by immersion in PBS buffer (containing 2% DMSO) with different concentrations of B2. A set of parallel sensors incubated with unloaded buffer was used as a background control. All data were processed using Gator Bio data analysis software, and global fitting was performed based on multiple curves generated from serial dilutions of the compound. The equilibrium dissociation constant (KD) was calculated as the ratio of the dissociation rate constant (Koff) to the binding rate constant (Kon).

[0062] 2. Experimental Results

[0063] Cellular thermal displacement analysis (CETSA) and molecular docking results showed that incubation with saikosaponin B2 increased the thermostability of ASC protein, and that saikosaponin B2 interacted with multiple sites of the PYD domain of ASC protein, including Asn-71 and Arg-74. Furthermore, biomembrane interference experiments demonstrated that saikosaponin B2 can directly interact with the PYD domain of ASC protein, with an affinity of approximately 9.39 μM. Figure 4 ).

[0064] The above results suggest that saikosaponin B2 may exert its inhibitory effect on NLRP3 inflammasome activation by targeting ASC.

[0065] Example 3: Cytotoxicity of Saikosaponin B2

[0066] 1. Experimental Methods

[0067] This invention simultaneously investigated the cytotoxic effects of saikosaponins on two hepatocellular carcinoma cell lines, SK-Hep1 and HCCLM3. Cells were seeded in 96-well plates overnight. Then, the cells were treated with different compounds at the same concentration and incubated for another 24 hours. Afterward, medium containing 10% CCK8 was added, and the cells were incubated for 1–4 hours, with absorbance measured at 450 nm.

[0068] 2. Experimental Results

[0069] The results showed that after 24 h of treatment with the compounds, saikosaponin A, saikosaponin D, and saikosaponin B1 exhibited cytotoxicity against SK-Hep-1 and HCCLM3 cells at a concentration of 25 μM, while saikosaponin B2 showed no cytotoxicity against hepatocytes. Figure 5 ).

[0070] The above results indicate that saikosaponin B2 has excellent safety.

[0071] Example 4: In vitro hemolysis experiment of saikosaponin B2

[0072] 1. Experimental Methods

[0073] After collecting blood from mouse eyeballs, the blood was washed three times with 0.9% saline solution (9 times the volume of blood), centrifuged at 150g for 5 minutes each time, and red blood cells were collected. The red blood cells were then diluted with 0.9% saline solution at a ratio of 1:9 and divided into eight equal portions, each 1 mL. A concentration gradient of saikosaponin B2 was set at 6.25, 12.5, 25, 50, 100, and 200 μM. An equal volume of DMSO was added to the negative control group, and 0.1% (v / v) Tween 20 was added to the positive control group. After mixing, the mixture was incubated at 37℃ for 4 hours, and the absorbance was measured at 545 nm. The hemolysis rate was calculated using the formula: Hemolysis rate = (Experimental group - Negative group) / (Positive group - Negative group) x 100%.

[0074] 2. Experimental Results

[0075] In vitro hemolysis experiments showed that saikosaponin B2 had low hemolytic activity, with an EC50 value of 0.5%. 50 The concentration was 74.40 μM, which is much higher than the effective concentration in vivo. Figure 6 ).

[0076] Example 5: Acute toxicity test of saikosaponin B2

[0077] 1. Experimental Methods

[0078] Animal experiments were approved by the Animal Experiment Ethics Committee of the Chengdu Institute of Biology, Chinese Academy of Sciences. Kunming mice were randomly divided into a blank group (blank solvent), a low-dose treatment group (saikosaponin B2 100 mg / kg), and a high-dose treatment group (saikosaponin B2 600 mg / kg), with 5 males and 5 females in each group. Administration was by gavage. Additionally, Kunming mice were randomly divided into a blank group (blank solvent) and a treatment group (saikosaponin B2 200 mg / kg), with 5 males and 5 females in each group. Administration was by intraperitoneal injection. Mice were closely monitored after administration and sacrificed 14 days later. Blood was collected for routine blood tests and other blood biochemical analysis. The hearts, kidneys, lungs, spleens, and livers of the mice were fixed and stained with hematoxylin and eosin (HE) to observe pathological changes in each organ.

[0079] 2. Experimental Results

[0080] Acute toxicity tests showed that saikosaponin B2 had no significant toxicity to blood and major organs when administered by gavage and intraperitoneal injection. Figures 7-14 ).

[0081] Example 6: Pharmacokinetic Test of Saikosaponin B2

[0082] 1. Experimental Methods

[0083] This invention investigates the pharmacokinetic properties of saikosaponin B2, saikosaponin D (PSD), and saikosaponin D (SGD). The structures of saikosaponin D (PSD) and saikosaponin D (SGD) are as follows:

[0084]

[0085] The specific experimental method is as follows:

[0086] SD rats (200±20g) were randomly divided into six groups and administered the drugs via gavage (SSB2 20mg / kg, PSD 10mg / kg, and SGD 40mg / kg) and intravenous injection (SSB2 2mg / kg, PSD 2mg / kg, and SGD 3mg / kg), respectively. Animals were fasted overnight (10-14 hours) before administration. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 12 hours after administration. Blood samples were centrifuged within 1 hour of collection to separate serum and plasma. Ethanol was added to the plasma for protein precipitation, and 100 ng / mL α-sulfobutylurea (IS) was added as an internal standard. The plasma mixture was homogenized and centrifuged, and the supernatant was collected and transferred to a 96-well plate. 2 µL of the supernatant was analyzed by LC-MS / MS. The standard parameter set includes the area under the curve (AUC(0-t) and AUC(0-∞)), elimination half-life (T). 1 / 2), maximum plasma concentration (Cmax), and time to reach maximum plasma concentration (Tmax), etc.

[0087] 2. Experimental Results

[0088] Blood drug concentration results showed that the highest blood drug concentrations of the three compounds were obtained at the 5-minute blood sampling point after intravenous injection, and the time to peak concentration was significantly prolonged after gavage, with Tmax of 1.6 h, 1.33 h, and 3.00 h, respectively. Figure 15 The pharmacokinetic parameters showed that the Tmax and t1 / 2 of both saikosaponin B2 and SGD were increased after oral administration compared to intravenous injection, indicating that absorption and elimination were significantly slower during oral administration, and the circulation time of the drugs in the body was prolonged after oral administration. Figure 16 From the oral bioavailability results, the oral bioavailability of saikosaponin B2, PSD, and SGD in rats increased sequentially, at 0.90%, 2.18%, and 8.56%, respectively. The oral bioavailability of saikosaponin B2 was relatively low, consistent with the poor oral absorption characteristics of other saikosaponin compounds. Figure 17 The results of the liver microsomal assay for saikosaponin B2 showed that, compared with the Ketanserin positive control, saikosaponin B2 had better stability in liver microsomes, with Eh(%) <30% indicating stability. Figure 18 ).

[0089] Example 7: The therapeutic effect of saikosaponin B2 on acute gouty arthritis

[0090] 1. Experimental Methods

[0091] Animal experiments were approved by the Animal Experiment Ethics Committee of the Chengdu Institute of Biology, Chinese Academy of Sciences. Six-week-old Kunming mice were randomly divided into four groups: a control group, a model group (MSU+C18), a positive control group (colchicine), a low-dose administration group (saikosaponin B2 5 mg / kg), a medium-dose administration group (saikosaponin B2 10 mg / kg), and a high-dose administration group (saikosaponin B2 20 mg / kg), with six mice in each group (half male and half female). The mice were administered the drugs via intraperitoneal injection. Additionally, six-week-old Kunming mice were randomly divided into four groups: a control group, a model group (MSU+C18), a positive control group (colchicine), a low-dose administration group (saikosaponin B2 20 mg / kg), a medium-dose administration group (saikosaponin B2 40 mg / kg), and a high-dose administration group (saikosaponin B2 60 mg / kg), with six mice in each group (half male and half female). The mice were administered the drugs via gavage. Mice were housed under standard feeding conditions and induced with acute gouty arthritis by injecting MSU / C18:0 free fatty acids (300 μg / 200 μM) into the left hind paw 30 minutes after intraperitoneal injection or gavage. Swelling of the left hind paw was measured at 6, 12, and 24 hours post-modeling, and the pain threshold was measured using Von Frey fibers. After euthanasia, the swollen areas of the left hind paw were collected for ELISA and Western blot analysis.

[0092] To further compare the therapeutic effects of saikosaponin B2, saikosaponin A, and saikosaponin D on acute gouty arthritis, saikosaponin B2 (40 mg / kg), saikosaponin A (40 mg / kg), and saikosaponin D (40 mg / kg) were administered by gavage according to the above method, and the therapeutic effects were compared.

[0093] 2. Experimental Results

[0094] The results show that ( Figure 19 Following intraperitoneal injection of MSU and C18, mice exhibited significant paw swelling with increased temperature at the swollen site, accompanied by an increase in IL-1β release, indicating successful establishment of an acute gouty arthritis model. Intraperitoneal injection of saikosaponin B2 alleviated paw swelling, decreased surface temperature at the swollen site, and significantly reduced IL-1β release in a concentration-gradient manner. Western blotting results showed that saikosaponin B2 inhibited the protein expression of caspase-1 splice and mature IL-1β in swollen tissues of mice, but had no significant effect on the expression of caspase-1 precursor and IL-1β precursor proteins. Furthermore, gavage administration of saikosaponin B2 significantly improved joint swelling and pain sensation in the mouse acute gouty arthritis model, inhibited IL-1β release from swollen tissues, and reduced the expression of mature IL-1β and p20 proteins. Figure 20This indicates that saikosaponin B2 can be effectively treated with intraperitoneal injection or oral administration for acute gouty arthritis.

[0095] Further based on Figure 21 It can be seen that, at the same oral dose of 40 mg / kg, 24 hours after administration, saikosaponin B2 was superior to saikosaponin A and saikosaponin D in relieving foot swelling. Its inhibitory effect on IL-1β in swollen tissue was comparable to that of saikosaponin D, but superior to that of saikosaponin A. This indicates that saikosaponin B2 is significantly more effective than saikosaponin A and saikosaponin D in treating acute gouty arthritis.

[0096] In summary, this invention reveals that saikosaponin B2 significantly inhibits NLRP3 inflammasome activation and can be used to prepare NLRP3 inflammasome inhibitors for the treatment of diseases related to NLRP3 inflammasomes. Compared with saikosaponin A and saikosaponin D, saikosaponin B2 exhibits advantages in safety, in vivo pharmacokinetic properties, and therapeutic efficacy, demonstrating broad application prospects.

Claims

1. Use of saikosaponin B2 in the preparation of a medicament for the prevention and / or treatment of gout or gouty arthritis, wherein the gout is acute gout and the gouty arthritis is acute gouty arthritis; wherein the medicament is capable of inhibiting NLRP3 inflammasome activation and is capable of improving joint swelling.

2. The use according to claim 1, characterized in that, The drug is a preparation made by adding pharmaceutically acceptable excipients to saikosaponin B2 as the active ingredient.

3. The use according to claim 2, characterized in that, The preparation is an oral preparation or an injectable preparation.

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