Application of curcumin in regulating hyperuricemia and acute gouty arthritis and research method of curcumin

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of curcumin in regulating hyperuricemia and acute gouty arthritis and a research method of curcumin. Through cell culture, establishment of a hyperuricemia and acute gout model cell model, verification of combination of curcumin and NLRP3 and NEK7 through a cell thermal migration experiment CETSA and a research system of an in-vivo animal experiment, how the curcumin regulates hyperuricemia and acute gouty arthritis through an ROS-mediated NLRP3-NEK7 inflammasome signal channel is deeply revealed. And a new perspective is provided for understanding the pathogenesis and treatment of the disease. The research method focuses on an NLRP3-NEK7 inflammasome signal channel, and provides a new therapeutic target and a new drug research and development direction for treating hyperuricemia and acute gouty arthritis. Through deep research on the pathway, a more efficient and safer therapeutic drug is expected to be developed.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to an application of curcumin in regulating hyperuricemia and acute gouty arthritis and a research method thereof. Specifically, it relates to the application of curcumin in regulating hyperuricemia and acute gouty arthritis through the ROS-mediated NLRP3-NEK7 inflammasome signaling pathway. Background Art

[0002] Hyperuricemia is a metabolic disease mainly caused by excessive uric acid production and insufficient renal and intestinal uric acid excretion, which increases the uric acid level in the serum, causes chronic inflammatory damage to the kidneys and intestines, leads to glomerular arteriosclerosis and renal tubular damage, thereby inducing more insufficient uric acid excretion. The excessive accumulation of uric acid in the serum results in the deposition of sodium urate crystals in or around joints, stimulating the release of a large number of inflammatory mediators by local tissues to cause an acute inflammatory reaction, further causing damage to joint tissues and forming gout. For the treatment of gout and hyperuricemia, allopurinol and benzbromarone are common drugs for inhibiting uric acid production and increasing uric acid excretion, and colchicine, non-steroidal anti-inflammatory drugs and glucocorticoids are common drugs for inhibiting inflammation. However, these drugs have large side effects, all have hepatotoxicity and are excreted through the kidneys, which easily causes drug accumulation and leads to toxic reactions.

[0003] Curcumin is a bioactive polyphenol compound extracted from the rhizomes of Curcuma longa. Curcuma longa is widely used in life and medicine and is commonly used for coloring in Asian cooking. In vitro studies have found that curcumin has a variety of biological activities, including antioxidant, anti-inflammatory, inhibiting ferroptosis and ROS production. In animal experiments, curcumin not only shows the characteristics of anti-tumor immunity, but also can improve various inflammations, and has preventive and therapeutic effects on diabetes, systemic lupus erythematosus, as well as the liver, nervous system and cardiovascular system. However, it is not clear whether curcumin can regulate hyperuricemia and acute gouty arthritis by regulating NLRP3-NEK7. Therefore, it is necessary to explore the pharmacodynamics of curcumin affecting hyperuricemia and acute gouty arthritis and clarify its mechanism of action through in vitro and in vivo experiments.

[0004] At present, the relevant research on the effect of curcumin on hyperuricemia and acute gouty arthritis mainly focuses on the research of traditional Chinese medicine compositions and preparation methods. Chinese Patent CN112587543B discloses a complex with the function of preventing the attack of gouty arthritis, taking the complex of DNA tetrahedron and curcumin as the active ingredient, which can reduce the levels of reactive oxygen species (ROS) and nitric oxide (NO), and reduce the expression and secretion of inflammatory factors, showing a significant anti-inflammatory effect; Chinese Patent CN113274478A discloses a traditional Chinese medicine fermentation preparation for treating gout, which is made of four traditional Chinese medicines, namely Erythropalum scandens, Curcuma longa, Glycyrrhiza uralensis and Coix lacryma-jobi, in the form of powder packaging or made into any one of capsules, tablets and granules, which can effectively reduce blood uric acid, so as to achieve the effect of preventing or treating gout; Chinese Patent CN111000973A discloses a composition with the function of improving gout, including the following raw material components: 1-10 parts of soy peptide, 0.1-2 parts of Cynara scolymus leaf extract, 0.1-2 parts of curcumin, etc., which can play a synergistic effect in reducing uric acid level, anti-inflammatory and other effects, and improve the treatment effect of gout. At present, there are reports that curcumin has a therapeutic effect on hyperuricemia and acute gouty arthritis (Li, X et al. 2019, J. Cell. Biochem, 120(4), 6718-6728; Chen, Y. et al. 2019, Biomed Pharmacother, 118, 109195), but no in-depth mechanism research has been carried out.

[0005] More importantly, there is a lack of a systematic research method in the prior art. Summary of the Invention

[0006] On the one hand, the present invention provides the use of curcumin in the preparation of a drug for preventing or treating hyperuricemia and acute gouty arthritis.

[0007] In the present invention, curcumin treats hyperuricemia and acute gouty arthritis by inhibiting the ROS-mediated NLRP3-NEK7 inflammasome signaling pathway.

[0008] The curcumin is the curcumin produced by Shanghai Yuanye Bio-Technology Co., Ltd., with the product number B20614.

[0009] Another invention is a research method for curcumin to regulate hyperuricemia and acute gouty arthritis through the ROS-mediated NLRP3-NEK7 inflammasome signaling pathway, including the following steps:

[0010] S1: In vitro cytological experiments, specifically including the following steps:

[0011] S1.1: Cell culture;

[0012] HK-2 cells were cultured in DMEM / F12 medium, and RAW264.7 cells (from iCell Bioscience Inc, Shanghai, China) were cultured in DMEM medium. Both were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and cultured in a humid environment at 37 °C and 5% CO2.

[0013] S1.2: Establishment of cell models for hyperuricemia and acute gout;

[0014] HK-2 cells were stimulated with 200 μM MSU for 24 hours to serve as a hyperuricemia cell model. RAW264.7 cells were stimulated with 10 μg / mL LPS for 4 h, and then 200 μM MSU was added and stimulated for another 20 h to serve as a gout cell model. The cultured HK-2 and RAW264.7 cells were respectively divided into a control group, a model group, a curcumin group (40 μM), and an NAC group (5 mM). Curcumin and the ROS scavenger NAC were added 1 h before model establishment.

[0015] The cells were seeded in a 96-well cell culture plate at a density of 5×10 3 cells / well. Then, 20 μL (5 μg / mL) of MTT solution was added to each well after drug administration, and incubated for 4 hours. Then, the medium was removed, 150 μL of dimethyl sulfoxide was added to each well, and the absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader to calculate cell viability.

[0016] S1.3: Cellular thermal shift assay (CETSA) to verify the binding of curcumin to NLRP3 and NEK7;

[0017] RAW264.7 gout model cells were collected using pre-cooled PBS containing protease inhibitors, and frozen and thawed five times in liquid nitrogen. Centrifuged at 12,000 rpm for 15 minutes at 4 °C to obtain the cell lysate supernatant. Then, an equal amount of the supernatant was treated with DMSO or 100 μM curcumin, and co-incubated on ice for 2 hours. After curcumin bound to the protein, it was treated at multiple temperatures from 37 - 67 °C for 3 minutes, and further centrifuged to obtain the supernatant. The protein levels of NLRP3 and NEK7 bound to curcumin were detected by Western blot.

[0018] The study was conducted by establishing a hyperuricemia model in HK-2 renal tubular epithelial cells and a gout inflammation model in RAW264.7 macrophages.

[0019] The interaction between curcumin and NLRP3-NEK7 was further studied, and theoretical calculations were performed through molecular docking. The results showed that there was a binding pocket at the binding interface of NLRP3 and NEK7, where curcumin could bind to the two proteins, and the binding energy of molecular docking was -7.0 kcal / mol.

[0020] S2: In vivo animal experiment, establishment of hyperuricemia and acute gout mouse model: mice were intraperitoneally injected with 250 mg / kg potassium oxonate once a day for 12 days. On the tenth day, 0.02 mL of 25 mg / mL sodium urate was injected into the right ankle joint of the mice with a microsyringe to prepare a hyperuricemia and acute gout mouse model. Normal saline was injected intraperitoneally or injected into the right ankle joint as a control during model preparation.

[0021] The present invention verifies that the therapeutic effect is definite and significant. Pharmacological experiments have found that curcumin regulates hyperuricemia and acute gouty arthritis through the ROS-mediated NLRP3-NEK7 inflammasome signaling pathway. Therefore, curcumin can be used to prevent or treat hyperuricemia and acute gouty arthritis, and has good development prospects.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. Innovative mechanism of action: This technical solution proposes for the first time that curcumin regulates hyperuricemia and acute gouty arthritis through the ROS-mediated NLRP3-NEK7 inflammasome signaling pathway, providing a new target and theoretical basis for the treatment of the disease, and is expected to develop new and highly effective therapeutic drugs.

[0024] 2. Highly effective anti-inflammatory effect: By directly acting on the NLRP3-NEK7 inflammasome, curcumin can specifically block the activation of the inflammasome and reduce the release of pro-inflammatory factors such as IL-1β, thereby effectively alleviating the inflammatory symptoms of acute gouty arthritis and reducing the pain of patients.

[0025] 3. High safety: As a natural product, curcumin has good biocompatibility and low toxicity. It is relatively safe for long-term use and is expected to reduce the side effects of traditional therapeutic drugs and improve patient compliance with treatment.

[0026] 4. Multi-target regulation: In addition to directly acting on the NLRP3-NEK7 inflammasome, curcumin may also work synergistically through other mechanisms (such as anti-oxidation, regulation of uric acid metabolism, etc.) to achieve multi-target regulation and improve the therapeutic effect.

[0027] 5. Reliable experimental model: This technical solution combines in vitro cytological experiments and in vivo animal experiments to comprehensively verify the therapeutic effect of curcumin on hyperuricemia and acute gouty arthritis. The experimental model is reliable and the results are persuasive, laying a solid foundation for subsequent clinical research.

[0028] 6. Reveal the mechanism of action of curcumin in treating hyperuricemia and acute gouty arthritis: Through this research method, it is possible to deeply reveal how curcumin regulates hyperuricemia and acute gouty arthritis through the ROS-mediated NLRP3-NEK7 inflammasome signaling pathway, providing a new perspective for understanding the pathogenesis and treatment of this disease.

[0029] 7. Verify the therapeutic effect of curcumin on hyperuricemia and acute gouty arthritis: Verify the therapeutic effect of curcumin on hyperuricemia and acute gouty arthritis through in vitro and in vivo experiments, providing experimental evidence for curcumin as a drug for treating this disease.

[0030] 8. Find new therapeutic targets and drug R & D directions: This research method focuses on the NLRP3-NEK7 inflammasome signaling pathway, providing new therapeutic targets and drug R & D directions for treating hyperuricemia and acute gouty arthritis. By deeply studying this pathway, it is expected to develop more efficient and safe therapeutic drugs.

[0031] 9. Evaluate the safety and side effects of curcumin: In in vivo animal experiments, the toxic effects and side effects of curcumin on mice can be observed to evaluate its safety. This helps to provide safety guarantees for the clinical application of curcumin and reduce potential risks and hazards. Description of the drawings

[0032] Figure 1 It is the swelling degree and inflammation of the ankle joint of mice with hyperuricemia and gout relieved by curcumin. (A) is the trend of ankle joint swelling degree, and (B-C) are the levels of TNF-α and IL-1β in the ankle joint. Compared with the control group:

[0033] ##p < 0.01, compared with the model group *p < 0.05, **p < 0.01;

[0034] Figure 2 It is curcumin reducing the blood uric acid level of mice with hyperuricemia and gout. (A) is the uric acid content in the serum, (B) is the activity of xanthine oxidase in the liver, (C) is the expression of ABCG2 in the kidney measured by Western blotting, and (C) is the result of relative quantitative analysis of proteins using Image J software. Compared with the control group: #p < 0.05, ##p < 0.01, compared with the model group **p < 0.01;

[0035] Figure 3Curcumin alleviates renal insufficiency and kidney inflammation in hyperuricemia and gout mice. (A) is the creatinine content in serum, (B) is the blood urea nitrogen content in serum, (C) is the level of TNF-α in the kidney, and (C-D) is the level of IL-1β in the kidney. All data are expressed as mean ± standard error. Compared with the control group: ##p < 0.01, compared with the model group: *p < 0.05, **p < 0.01;

[0036] Figure 4 The protective effect of curcumin on oxidative stress. (A) is the protective effect of curcumin on the viability of MSU-induced HK-2 cells, (B) is the protective effect of curcumin on the viability of LPS+MSU-induced RAW264.7 cells, (C-F) is the accumulation of ROS in HK-2 cells, and (G-J) is the accumulation of ROS in RAW264.7 cells. Compared with the control group: ##p < 0.01, compared with the model group: **p < 0.01;

[0037] Figure 5 The effect of curcumin on NLRP3-mediated pyroptosis. (A) is the expression of NLRP3, NEK7, ASC, Cleaved-CASPASE1, Cleaved-IL-1β, GSDMD, and ABCG2 in HK-2 cells determined by Western blotting, and (B) is the expression of NLRP3, NEK7, ASC, Cleaved-CASPASE1, Cleaved-IL-1β, and GSDMD in RAW264.7 cells determined by Western blotting;

[0038] Figure 6 Curcumin intervenes in the binding of NLRP3 and NEK7. (A) is the molecular docking simulation of the binding of curcumin to NLRP3 and NEK7, (B) is the target binding site at the interface of the docking pocket, (C) is the immunoblot analysis of the binding between curcumin and NLRP3, NEK7 in gout model cells at different temperatures (37°C, 45°C, and 53°C) by CETSA, (D) is the immunoblot analysis of the binding between curcumin and NLRP3, NEK7 in gout model cells at multiple temperatures by CETSA, and (E-F) is the quantitative analysis of NLRP3 and NEK7 and the display in the form of a melting curve graph. Detailed implementation mode

[0039] In the following, the present invention will be described in detail through examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0041] Materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0042] Example 1 In Vivo Experiment on the Activity of Curcumin in Treating Hyperuricemia and Acute Gout

[0043] 1.1 Preparation of Hyperuricemia and Acute Gout Model Mice and Group Administration

[0044] Male Kunming mice (7 - 9 weeks old, weighing 22 ± 2 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (License No.: SCXK(Liao)2022 - 0001) and adaptively fed for 1 week under the conditions of 22 ± 2°C, relative humidity of 55%, and a 12 - hour light / dark cycle. All animal experiments were approved by the Biomedical Ethics Committee of Dalian University of Technology.

[0045] To establish a hyperuricemia and acute gout model in mice, the mice were intraperitoneally injected with 250 mg / kg potassium oxonate once a day for 12 days. On the tenth day, 0.02 mL of 25 mg / mL sodium urate (MSU) was injected into the right ankle joint of the mice with a micro syringe to prepare a hyperuricemia and acute gout mouse model. During model preparation, intraperitoneal injection with normal saline or injection into the right ankle joint was used as a control.

[0046] Fifty - six KM mice were randomly divided into 7 groups, with 8 mice in each group, namely the control group, the model group, the low - dose curcumin group (50 mg / kg), the medium - dose curcumin group (100 mg / kg), the high - dose curcumin group (200 mg / kg), the allopurinol group (10 mg / kg), and the colchicine group (1 mg / kg). The corresponding dose of curcumin was dissolved in corn oil and administered by gavage (0.1 mL / 20 g body weight) once a day for 12 days to investigate the effect of curcumin on hyperuricemia and acute gout. Mice were gavaged with an equal volume of corn oil as a blank administration control, and allopurinol and colchicine were used as positive drug controls.

[0047] 1.2 Detection of Biochemical Indexes

[0048] The circumference of the right ankle of the mice was measured at 0, 4, 12, 24, and 48 h after MSU injection. The degree of ankle edema was expressed as a percentage based on 0 h. The opposite side bulge of the joint capsule was considered the standard for successful injection. Blood and organ samples were collected at 48 h for further analysis.

[0049] The xanthine oxidase activity in the liver, and the contents of uric acid, creatinine, and urea in the serum were detected using kits.

[0050] The right ankle joint tissues and kidneys of mice were collected and added to pre-cooled normal saline at a ratio of 1:10 (g / mL). After homogenizing and grinding on ice with a tissue grinder, they were centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant samples were collected to detect the levels of TNF-α and IL-1β.

[0051] The mouse kidneys were homogenized in RIPA lysis buffer containing 1 mM PMSF and 1 μg / mL serine protease inhibitor, and then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected, and the concentration of the protein extract was detected using a BCA protein assay kit. The same amount of 20 μg protein samples were separated by SDS-PAGE gel and then transferred to a PVDF membrane. After blocking with 5% skim milk powder, the membrane was incubated overnight at 4°C with antibodies against ABCG2 or β-actin (primary antibody concentration 1:1000). Then, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. Finally, the protein bands were visualized by chemiluminescent reagent, and the images were captured using a ChemiDoc TM Touch imaging system (Bio-Rad, USA).

[0052] We first evaluated the ankle swelling in mice. As Figure 1 shown in A, compared with the control group, the model group (28.5 ± 4.1%) showed obvious ankle swelling, and the degree of swelling at 48 hours was significantly higher than that of the control group (2.5 ± 2.3%) (p < 0.01). Curcumin treatment alleviated this swelling, and the effect of the high-dose curcumin group was comparable to that of the positive control drug colchicine. In addition, as Figure 1 shown in B and 1C, the levels of inflammatory cytokines TNF-α and IL-1β in the ankle joint tissues of the model group were significantly increased (74.0 ± 8.1 ng / L, p < 0.01; 128.6 ± 13.4 ng / L, p < 0.01), while those in the control group were 36.9 ± 4.2 ng / L and 35.0 ± 6.7 ng / L. High-dose curcumin significantly reduced the levels of TNF-α (p < 0.01) and IL-1β (p < 0.05). These results indicate that curcumin can effectively alleviate ankle joint inflammation in mice with acute gout model.

[0053] We measured the serum uric acid levels of the model mice. As Figure 2As shown, compared with the control group (33.5 ± 2.3 mg / L), the serum uric acid level in the model group was significantly increased (59.7 ± 2.4 mg / L, p < 0.01), confirming the successful establishment of a hyperuricemia with acute gout mouse model. High-dose curcumin treatment significantly reduced the serum uric acid level (p < 0.01). Xanthine oxidase (XOD) is the key enzyme that catalyzes the conversion of xanthine and hypoxanthine to uric acid. Compared with the control group (4.95 ± 0.37 U / gprot), the XOD activity in the liver of mice in the model group was significantly increased (8.96 ± 0.62 U / gprot, p < 0.01). However, in the high-dose curcumin treatment group, the XOD activity was significantly decreased (p < 0.01), indicating that curcumin may reduce the serum uric acid level by inhibiting uric acid production. The regulation of uric acid homeostasis depends to a large extent on the activity of uric acid transporters. In the model group, compared with the control group, the expression of ABCG2 protein in the kidney was significantly decreased; while in the curcumin treatment group, the expression level of ABCG2 protein was comparable to that of the control group. These results suggest that curcumin may promote uric acid excretion by regulating the expression of uric acid transporters.

[0054] Hyperuricemia is known to cause kidney damage. To evaluate the protective effect of curcumin on kidney damage, we measured the levels of creatinine and urea nitrogen in the serum of mice. As Figure 3 shown in Figures 3A and 3B, compared with the control group (70.0 ± 21.2 μM, 3.0 ± 0.4 mM), the serum creatinine (230.3 ± 16.3 μM, p < 0.01) and urea nitrogen (6.8 ± 0.4 mM, p < 0.01) in mice in the model group were significantly increased. Curcumin treatment significantly reduced the serum creatinine (p < 0.01) and urea nitrogen levels (p < 0.05), indicating that curcumin may relieve kidney damage caused by hyperuricemia. Next, we measured the levels of pro-inflammatory cytokines TNF-α and IL-1β in the kidney tissues of mice. As Figure 3 shown in Figures 3C and 3D, compared with the control group (65.4 ± 10.3 ng / L, 75.3 ± 27.3 ng / L), the levels of TNF-α (155.9 ± 3.4 ng / L, p < 0.01) and IL-1β (245.9 ± 65.9 ng / L, p < 0.01) in mice in the model group were significantly increased. High-dose curcumin treatment significantly reduced these levels, with significant decreases in both TNF-α (p < 0.01) and IL-1β (p < 0.05). This shows that curcumin relieves joint swelling and inflammation in the hyperuricemia and acute gout model mice

[0055] Example 2 Experiment on the treatment of hyperuricemia and acute gout by curcumin in vitro through the ROS-mediated NLRP3-NEK7 inflammasome signaling pathway

[0056] 2.1 Cell culture, model preparation, and grouping for drug administration

[0057] HK-2 cells were cultured in DMEM / F12 medium, while RAW264.7 cells (from iCell Bioscience Inc, Shanghai, China) were cultured in DMEM medium. Both were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and cultured in a humid environment at 37 °C and 5% CO2.

[0058] HK-2 cells were stimulated with 200 μM MSU for 24 hours to establish a hyperuricemia cell model. RAW264.7 cells were stimulated with 10 μg / mL LPS for 4 h, and then 200 μM MSU was added for another 20 h to establish a gout cell model. The cultured HK-2 and RAW264.7 cells were divided into control group, model group, curcumin group (40 μM), and NAC group (5 mM). Curcumin and the ROS scavenger NAC were added 1 h before model establishment.

[0059] The cells were seeded into 96-well cell culture plates at a density of 5×10 3 cells / well. Then, 20 μL (5 μg / mL) of MTT solution was added to each well after drug administration, and the cells were incubated for 4 hours. Then, the culture medium was removed, and 150 μL of dimethyl sulfoxide was added to each well. The absorbance at 490 nm was measured using a microplate reader to calculate cell viability.

[0060] 2.2 Intracellular ROS detection

[0061] The cell culture medium was removed, and DCFH-DA probe (10 μM) diluted with serum-free medium was added. The cells were incubated for 20 min, washed three times with serum-free cell culture medium, and observed under a laser confocal microscope.

[0062] 2.3 Cell protein extraction and Western blot analysis

[0063] RAW264.7 or HK-2 cells were homogenized in RIPA lysis buffer containing 1 mM PMSF and 1 μg / mL aprotinin, and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected, and the protein expressions of NLRP3, NEK7, ASC, Caspase-1, GSDMD, IL-1β, and ABCG2 were detected using Western blot.

[0064] 2.4 Molecular docking

[0065] Obtain the protein crystal structure of the core target from the PDB database, import it into autodocktools, adjust the parameters of the receptor protein for dehydrogenation and dehydration, and convert it into a pdbqt file. Obtain the three-dimensional structure of curcumin from the PubChem database, save it in the "SDF" format, import it into the PyMol software to set parameters for dehydration and hydrogenation treatment, and convert it into a pdb format file. Then import it into the AutoDock Tools software for docking, and evaluate the final docking results according to the binding energy and the number of binding sites. Finally, use the PyMol software for visualization processing.

[0066] 2.5 Cell thermal migration experiment

[0067] Collect RAW264.7 gout model cells using pre-cooled PBS containing protease inhibitors, and perform five freeze-thaw cycles in liquid nitrogen. Centrifuge at 12,000 rpm for 15 minutes at 4°C to obtain the cell lysate supernatant. Then, treat equal amounts of the supernatant with DMSO or 100 μM curcumin, co-incubate on ice for 2 hours. After curcumin binds to the protein, treat it at multiple temperatures from 37 - 67°C for 3 minutes, further centrifuge to obtain the supernatant, and detect the protein levels of NLRP3 and NEK7 bound to curcumin by Western blot.

[0068] The research was carried out by establishing a hyperuricemia model in HK-2 renal tubular epithelial cells and a gout inflammation model in RAW264.7 macrophages. As Figure 4 shown in Figures 4A and 4B, in the two cell lines of HK-2 and RAW264.7, compared with the control group, the cell viability in the model group decreased significantly (35.5 ± 1.2%, p < 0.01; 60.1 ± 1.3%, p < 0.01), while curcumin dose-dependently inhibited cell death in the range of 10 - 80 μM. As Figure 3 shown in Figures 4C - 4J, using the ROS scavenger NAC as a control, the effect of curcumin on oxidative stress was explored. Compared with the control group, the ROS fluorescence intensity in the two cell model groups increased significantly, while compared with the model group, curcumin (40 μM) treatment significantly reduced the ROS fluorescence intensity in the cells, inhibited oxidative stress, and its effect was similar to that of the ROS scavenger NAC treatment.

[0069] The abnormal accumulation of ROS can activate the NLRP3 inflammasome, and the activation of the NLRP3 inflammasome will cause pyroptosis. Therefore, we further explored the intervention effect of curcumin on the pyroptosis pathway mediated by the NLRP3 inflammasome. As Figure 5As shown in Figures 5A and 5B, the WB result analysis showed that, compared with the control group, the expression levels of NEK7, NLRP3, ASC, caspase1, GADMD, and cleaved-IL-β in the two cell models were all increased, and pyroptosis occurred. Curcumin (40 μM) reversed these phenomena, showing a similar effect to the ROS scavenger NAC. Meanwhile, in the HK-2 cell model, both curcumin and NAC showed the effect of restoring the expression of ABCG2 protein, further confirming that curcumin in the model mice had the effect of restoring uric acid excretion and reducing blood uric acid. Thus, curcumin has an inhibitory effect on the release of NEK7 and IL-1β, and the activation of Caspase-1 and GSDMD, and inhibits pyroptosis in the process of hyperuricemia and gout inflammation from the ROS / NEK7-NLRP3 pathway.

[0070] We further investigated the interaction between curcumin and NLRP3-NEK7. As Figure 6 shown in Figures 6A and 6B, theoretical calculations were performed by molecular docking. The results showed that there was a binding pocket at the binding interface of NLRP3 and NEK7, and curcumin could bind to the two proteins at this site, and the binding energy of molecular docking was -7.0 kcal / mol. Further, based on the principle of protein thermal stability, we used the Cellular Thermal Shift Assay (CETSA) to evaluate the binding effect between curcumin and the target proteins NEK7 and NLRP3. As Figure 6 shown in Figures 6C and 6D, the CETSA experimental results showed that, compared with the drug-free control group, when the target proteins NEK7 or NLRP3 bound to curcumin, the thermal stability increased. At the same temperature, the amount of undegraded NEK7 or NLRP3 protein increased, and the melting curve of the complex protein shifted to the right. It indicated that curcumin might specifically interfere with the binding of NLRP3 and NEK7, thereby inhibiting the assembly and activation of the NLRP3 inflammasome.

[0071] In this study, it was found that curcumin at a concentration of 200 mg / kg significantly down-regulated the activity of xanthine oxidase in the liver of model mice, alleviated the increase of uric acid, creatinine and urea nitrogen in the serum, and reduced joint swelling. At the same time, curcumin also significantly increased the expression of uric acid transporter ABCG2 in the kidney. Curcumin treatment significantly reduced the intracellular ROS level induced by LPS-MSU and the protein levels of NEK7, NLRP3, Caspase-1, GSDMD and IL-1β, thereby inhibiting pyroptosis mediated by the NLRP3 inflammasome signaling pathway. To further explore the inhibitory mechanism of curcumin on NLRP3 inflammasome activation, this study predicted the binding of curcumin to NEK7 and NLRP3 using molecular docking method, and detected the binding of curcumin to NEK7 and NLRP3 using CESTA technology respectively. The protein bound to the drug degraded less at different temperatures. The CESTA experimental results of this study showed that curcumin might inhibit the assembly and activation of NLRP3 inflammasome by directly targeting and interfering with the binding of NEK7 to NLRP3 in a protein-binding manner, thereby improving or alleviating hyperuricemia and gout injury.

[0072] The above is the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be thought of by those skilled in the art within the technical scope disclosed by the present invention without creative labor should be covered by the protection scope of the present invention.

Claims

1. An application of curcumin, characterized in that, Use in the preparation of drugs for preventing and treating hyperuricemia and acute gouty arthritis.

2. A research method for curcumin to regulate hyperuricemia and acute gouty arthritis, characterized in that, It includes the following steps: S1: In vitro cytological experiments; S1.1: Cell culture; S1.2: Establishment of cell models for hyperuricemia and acute gout models; S1.3: Verification of the binding of curcumin to NLRP3 and NEK7 by cell thermal shift assay CETSA; S2: In vivo animal experiment verification.

3. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 2, wherein S1.1: The cells used for cell culture are HK-2 cells and RAW264.7 cells.

4. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 3, characterized in that, S1.1: The steps of cell culture include: HK-2 cells are cultured in DMEM / F12 medium, and RAW264.7 cells are cultured in DMEM medium. Both are supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and cultured in a humid environment at 37°C and 5% CO2.

5. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 2, characterized in that, S1.2: The methods for establishing cell models for hyperuricemia and acute gout models include: Stimulate HK-2 cells with MSU as the hyperuricemia cell model, stimulate RAW264.7 cells with LPS, and then add MSU for further stimulation as the gout cell model. The cultured HK-2 and RAW264.7 cells are respectively divided into a control group, a model group, a curcumin group, and an NAC group. Curcumin and the ROS scavenger NAC are added 1 h before model establishment; Inoculate the cells into a 96-well cell plate, add MTT solution to each well after drug administration, incubate for 4 hours, then remove the medium, add dimethyl sulfoxide to each well, and calculate the cell viability.

6. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 2, wherein S1.3: The methods for verifying the binding of curcumin to NLRP3 and NEK7 by cell thermal shift assay CETSA include: detecting the protein levels of NLRP3 and NEK7 bound by curcumin by Western blot method, and conducting research by establishing a hyperuricemia model in HK-2 renal tubular epithelial cells and a gout inflammation model in RAW264.7 macrophages.

7. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 6, characterized in that, The Western blot method includes: collecting RAW264.7 gout model cells with pre-cooled PBS containing protease inhibitors, and performing freeze-thaw cycles in liquid nitrogen, centrifuging at 12,000 rpm for 15 minutes at 4°C to obtain cell lysate supernatant; treating equal amounts of the supernatant with DMSO or 100 μM curcumin, co-incubating on ice for 2 hours, and after curcumin binds to the protein, treating it at multiple temperatures from 37 - 67°C for 3 minutes, and further centrifuging to obtain the supernatant.

8. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 2, characterized in that, The interaction between curcumin and NLRP3-NEK7 was theoretically calculated by molecular docking.

9. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 2, characterized in that, S2: The methods for in vivo animal experiment verification include: constructing a mouse model of hyperuricemia and acute gout: mice are intraperitoneally injected with 250 mg / kg potassium oxonate once a day for 12 days. On the 10th day, 0.02 mL of 25 mg / mL sodium urate is injected into the right ankle joint of the mice with a microsyringe to prepare a mouse model of hyperuricemia and acute gout.

10. The research method for curcumin to regulate hyperuricemia and acute gouty arthritis according to claim 9, characterized in that, During model construction, intraperitoneal injection with normal saline or injection into the right ankle joint is used as a control.

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