Application of daidzein in preparation of medicine for treating and / or preventing gouty arthritis

Abstract

The invention relates to the technical field of medicine preparation, in particular to application of daidzein in preparation of a medicine for treating and / or preventing gouty arthritis. Based on the activity of daidzein for resisting inflammation in vitro and relieving gouty arthritis in vivo and the binding effect of daidzein and daidzein probes on protein in inflammatory macrophages, research on the mechanism of daidzein for killing inflammatory macrophages and relieving gouty arthritis is developed. Direct acting target proteins of daidzein have been found in inflammatory macrophages, which are present in oxidative phosphorylation and tricarboxylic acid cycles. In inflammatory macrophages, it is proved that daidzein can improve the activity of succinate dehydrogenase (SDH), promote generation of reactive oxygen species (ROS) in the inflammatory macrophages and induce apoptosis of the inflammatory macrophages. A new thought is provided for research and development of new drugs for treating gouty arthritis and local administration of micro-injections.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to the application of soybean extract in the preparation of drugs for treating and / or preventing gouty arthritis. Background Art

[0002] Gout is the most common inflammatory arthritis with a high incidence rate. It can induce various serious complications and affect the physical and mental health of 410,000 people worldwide. In the past three decades, the number of gout patients has been increasing worldwide, but the management of gout is still not ideal. Its main cause is abnormal purine metabolism or obstructed uric acid excretion, which leads to an increase in serum uric acid concentration and exceeds the solubility (68μg / mL) to precipitate urate (Monosodiumurate, MSU) crystals. MSU is deposited in the synovium, cartilage and other locations of the joints, leading to the occurrence of an inflammatory cascade. The typical symptom of gouty arthritis is severe pain in the joints within just a few hours. The clinical treatment strategy for patients with this acute attack is often to use strong anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs, colchicine, glucocorticoids, IL-1β blockers, etc. However, due to adverse drug reactions and various other reasons, the use of these drugs has caused concerns. Despite this, when facing acute gouty arthritis, many gout patients must use these potentially harmful and need to be monitored at all times for treatment. Therefore, finding new safe, low-toxic drugs to relieve gouty arthritis has become an urgent problem to be solved. Summary of the invention

[0003] The object of the present invention is to provide application of soybean extract in preparing medicine for treating and / or preventing gouty arthritis.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides application of soybean extract in preparing medicine for treating and / or preventing gouty arthritis.

[0006] Preferably, the concentration of soy bean curd in the preparation of a drug for treating and / or preventing gouty arthritis is 10-500 μM.

[0007] Preferably, when soybean extract is used to prepare a drug for treating and / or preventing gouty arthritis, gouty arthritis is treated and / or prevented by promoting the activity of succinate dehydrogenase, intervening in the cellular oxidative phosphorylation process, and inducing inflammatory cell apoptosis.

[0008] Preferably, the dosage form of the drug includes one or more of injection, tablet, capsule and oral solution.

[0009] The present invention also provides the use of soybean extract in preparing a preparation for promoting the activity of succinate dehydrogenase.

[0010] Preferably, the concentration of daidzein in the preparation for promoting the activity of succinate dehydrogenase is 10 to 500 μM.

[0011] The present invention also provides the use of soybean extract in preparing a preparation for inducing inflammatory cell apoptosis.

[0012] Preferably, the concentration of soy bean extract in the preparation for inducing inflammatory cell apoptosis is 10-500 μM.

[0013] The present invention also provides the use of soybean isoflavones in the preparation of drugs for treating and / or preventing gouty arthritis. The soybean isoflavones of the present invention are mainly present in bean products, are abundant in content, and have multiple biological activities.

[0014] The present invention is based on the activity of soymilk in in vitro anti-inflammatory and in vivo relief of gouty arthritis, as well as the binding effect of soymilk and soymilk probe on proteins in inflammatory macrophages, to carry out research on the mechanism of soymilk killing inflammatory macrophages and relieving gouty arthritis. The direct target protein of soymilk was found in inflammatory macrophages, which exists in oxidative phosphorylation and tricarboxylic acid cycle. The importance of this protein for the activity of soymilk was confirmed by RNA interference experiments. It was confirmed in inflammatory macrophages that soymilk can increase the activity of succinate dehydrogenase (SDH), promote the production of reactive oxygen species ROS in inflammatory macrophages and induce apoptosis of inflammatory macrophages. The treatment method of local injection of soymilk into the ankle joint can relieve gouty arthritis in mice. These provide new ideas for the research and development of new drugs for the treatment of gouty arthritis and local administration with microinjections. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The anti-inflammatory effect of soymilk in vitro and the in vivo relief of gouty arthritis in mice (a shows the effect of soymilk on the level of TNF-α produced by inflammatory RAW264.7 cells, b shows the effect of soymilk on the survival rate of RAW264.7 cells, c shows the effect of soymilk on the degree of ankle swelling in mice, d shows the effect of soymilk on the temperature of the soles of mice, the swelling rate of the ankles and the swelling rate of the soles of the feet, and e shows the results of H&E staining of the ankle joint sections of mice).

[0016] Figure 2 This is the study of the direct acting proteins of soymilk in anti-inflammatory and promoting apoptosis of inflammatory macrophages, and the verification of the direct acting target proteins, carried out with the help of soymilk probe (a is the soymilk probe, b is the principle of fluorescence imaging, c is the schematic diagram of the competitive binding process between the soymilk probe and soymilk, d is the KEGG pathway of the soymilk target, and e is the effect of RNA interference on soymilk killing inflammatory macrophages).

[0017] Figure 3 The effect of soy sauce on SDH activity of inflammatory macrophages.

[0018] Figure 4 This is an investigation of the effect of soy sauce on promoting ROS production and apoptosis of inflammatory macrophages (a is the effect of soy sauce on ROS production of inflammatory macrophages, b is the effect of soy sauce on the degree of apoptosis of inflammatory macrophages).

[0019] Figure 5 To verify in vivo the importance of SDH for soy sauce to relieve gouty arthritis in mice (a is a visual diagram showing that soy sauce can relieve ankle swelling in mice, b is the effect of soy sauce on the sole temperature, ankle swelling rate and instep swelling rate of mice, c is the result of H&E staining of mouse ankle joint sections). DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] The soybean extract in the examples of the present invention was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0022] Example 1

[0023] Experimental study on the anti-inflammatory effect of soybean extract in vitro and the relief of gouty arthritis in vivo

[0024] (1) In vitro anti-inflammatory assay on inflammatory macrophages

[0025] Soybean extract has an inhibitory effect on the inflammatory RAW264.7 cells induced by sodium urate (MSU). The specific implementation scheme is:

[0026] RAW264.7 cells (from China Center for Type Culture Collection, Wuhan, China) were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific). 10,000 cells were plated in a 96-well plate, 100 μL per well; after the cells adhered, they were converted to serum-free medium containing soymilk (0 μM, 12.5 μM, 25 μM, 50 μM, 100 μM and 200 μM) and MSU (final concentration of 200 μg / mL) for 24 hours; after incubation, 20 μL of the culture medium was aspirated from each well and the level of inflammatory factor TNF-α in the culture medium was detected using an ELISA kit (Beijing Sizhengbai Biotechnology Co., Ltd.).

[0027] The results showed that soy extract could significantly reduce the increase in the level of inflammation in RAW264.7 cells induced by MSU ( Figure 1 a), killing inflammatory macrophages ( Figure 1 b), therefore, soybean extract is an effective small molecule anti-inflammatory compound in vitro.

[0028] (2) In vitro gouty arthritis relief experiment

[0029] SPF Kunming male mice (n=60) were randomly divided into blank control group, model group, positive drug group, and soymilk low-, medium-, and high-dose groups.

[0030] Blank control group (n=10): 20 μL of normal saline was injected into the ankle joint of mice as the blank control group.

[0031] Model group (n = 10): Weigh an appropriate amount of MSU crystal powder and place it in a 15mL centrifuge tube for high-pressure steam sterilization, then add physiological saline in a biological safety cabinet to resuspend it to a concentration of 30 mg / mL, seal it with a sealing film, and mix it ultrasonically to obtain an MSU suspension. After the SPF-grade KM mice were anesthetized by an isoflurane gas anesthesia machine, the ankle joints of the mice were disinfected with 75% alcohol, and the MSU suspension was drawn with an insulin syringe. The ankle joint cavity was pierced at a 45° angle to the ankle of the mouse. After a clear sense of emptiness was felt, 20 μL of MSU suspension was injected into the ankle joint of the mouse, and the needle was slowly withdrawn to prevent the suspension from leaking out, and the mouse in vitro gouty arthritis model was established.

[0032] Positive drug group (n=10): Colchicine (0.65 mg / kg) was used as the positive drug and was administered by oral gavage 1 h before modeling. Afterwards, an in vitro gouty arthritis model of mice was established in the same manner as the model group.

[0033] Soybean extract low-dose group (n=10), medium-dose group (n=10), and high-dose group (n=10): Soybean extract was dissolved in a 30 mg / mL MSU suspension prepared with normal saline, and three concentrations of 125 μM, 250 μM, and 500 μM were prepared as the low, medium, and high dose groups, respectively, and injected into the ankle joint cavity of mice together with the MSU suspension. Each mouse was injected with 20 μL, and soybean extract intervention was carried out while the model was being established.

[0034] All animal experiments complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985).

[0035] The results showed that this treatment with soy extract could reduce the overall redness and swelling of the feet of mice with gouty arthritis ( Figure 1c), and can effectively reduce the temperature of the mouse's soles and reduce the swelling of the ankles and feet ( Figure 1 d); H&E staining results of the ankle joints of mice showed that there was no macrophage infiltration in the ankle joint cavity of the control group mice, and the synovial tissue was normal. There was a large number of macrophage infiltration in the ankle joint cavity of the model group mice, and the synovial tissue also had inflammatory infiltration, and the soy bean extract group had a significant improvement effect, especially in the high-dose group (500μM), the macrophage infiltration in the ankle joint cavity of the mice was significantly reduced, and the synovial inflammation disappeared ( Figure 1 e).

[0036] Example 2

[0037] Soybean extract binds to succinate dehydrogenase A (SDHA) protein, and SDHA is associated with the activity of soybean extract in killing inflammatory macrophages

[0038] (1) Experimental principle:

[0039] Based on the design strategy of small molecule probes and the structural characteristics of soybean protein, the research group designed and synthesized a soybean protein probe with a photocrosslinking group diaziridine and a Click chemical reaction group alkyne. Figure 2 a). The soy protein probe is used to compete with the soy protein binding protein. The protein that can be competed with represents the target protein of soy protein. It can be seen that soy protein can compete with the soy protein probe in a concentration-dependent manner and weaken the fluorescence displayed in the gel ( Figure 2 b) When the protein bound by the daidzein probe is competed with daidzein, it is enriched again. Compared with using the daidzein probe alone, the abundance of some proteins is reduced. These proteins are the target proteins of daidzein ( Figure 2 c).

[0040] (2) Experimental process:

[0041] When the cell confluence in the culture dish reached 75%, inflammatory RAW264.7 cells were collected after treatment with 200μg / mL MSU. Protein quantification was performed using a BCA kit, and the protein was quantified to 2mg / mL, divided into 6 groups, 1mL / group. Among them, 3 groups were competitive groups and 3 groups were non-competitive groups. Dimethyl sulfoxide DMSO (40μL) was added to the non-competitive group, and 20mM soy bean extract (40μL) was added to the competitive group to react in a constant temperature mixer (25℃, speed 800, 30min). After the reaction, it was separated instantly, vortexed and separated instantly. Then 5mM soy bean extract probe (40μL) was added to each group to react in a constant temperature mixer (25℃, speed 800, 60min). After the reaction, it was separated instantly, vortexed and separated instantly. This process was protected from light throughout. The reacted protein was transferred to a 24-well plate on ice and irradiated under a 365nm UV lamp for 10 minutes. After UV curing, the protein solution was transferred back to a new EP tube. 100μL Click mixture (the composition and ratio of the mixture were tris(2-chloroethyl) phosphate (TCEP): tetramethylbenzidine (TBTA): copper sulfate = 1:3:1) was added to each sample tube, and 10μL 20mM azide biotin solution was added to react. The reaction was kept away from light. Protein precipitation was generated after the reaction. 600μL of pre-cooled methanol was added to the precipitate, ultrasonicated on ice, centrifuged, and the supernatant was discarded. Repeat three times. Next, 1.2% sodium dodecyl sulfate SDS was added to the precipitate, heated in a metal bath to dissolve the protein precipitate, centrifuged, and the supernatant was retained. The above protein solution was added to the centrifuge tube containing Beads, and the centrifuge tube was fixed on a rotating mixer for incubation. After the incubation was completed, the Beads were washed with PBS, water and TEAB, centrifuged, and the supernatant was discarded. Finally, use 100mM TEAB to transfer the beads to a 1.5mL centrifuge tube, transfer multiple times, centrifuge, and discard the supernatant. Add 6M urea and 1M dithiothreitol (DTT) to different groups of centrifuge tubes and react in a constant temperature mixer. Next, add 1M iodoacetamide (IAA) to each sample to close the open disulfide bonds. After the reaction is completed, add 100mM TEAB to the sample, centrifuge, discard the supernatant, and repeat this process twice. Add 200μL 1M urea, 2μL 100mM calcium chloride, and 6μL trypsin to each sample. Prepare the solution and add the premix, shake the reaction, centrifuge, and transfer the supernatant to a 1.5mL centrifuge tube. Add 12μL of differently labeled 4% formaldehyde on ice (L group: CH2O; H group: 13CD2O). Then add 12μL 0.6M sodium cyanoborohydride (L group: NaBH3CN, H group: NaBD3CN) on ice, mix well, centrifuge, and react in a constant temperature mixer (29℃, speed 900, 1h). After the dimethylation reaction is completed, add 48μL 1% ammonia water to each sample to terminate the dimethylation. After the reaction is completed, add 24μL 5% formic acid to neutralize the excess ammonia water. Mix the light, medium and heavy into a new EP tube, vortex, centrifuge, and spin dry in a vacuum centrifugal concentrator (45℃, 1400rpm, 4h). Perform three biological replicates to ensure the reliability of the data. The prepared samples are stored in a -80℃ refrigerator.

[0042] The samples were desalted and then analyzed by LC-MS / MSF: After the samples were taken out of the -80°C freezer, they were centrifuged at high speed (15000rpm, 5min) and dissolved in 20μL of 0.1% formic acid-water solution, vortexed to dissolve, left to dissolve for 5min, and centrifuged (15000rpm, 15min, 4°C). The supernatant was removed and transferred to a dedicated mass spectrometer loading tube. After ultrasonic degassing for 15min, 2μL was loaded onto the Ultimate 3000LC tandem Q-Exactive PlusOrbitrap mass spectrometer for LC-MS / MS analysis. In brief, the flow rate through the column was set to 0.3μL / min, and the applied distal spray voltage was set to 2.8kV. MS2 data collection was performed using a full scan (350-1800MW), followed by a data-dependent MS2 scan of the 20 most abundant ions by enabling dynamic exclusion.

[0043] (3) Experimental results

[0044] The results of the experiment showed that there were 73 direct target proteins of soymilk (at least 2 out of 3 biological replicates, with an average light / heavy ratio > 1.5). Through biological pathway analysis, it was found that the direct target of soymilk was related to the energy metabolism pathway ( Figure 2 d); The proteins involved include SDHA (Q8K2B3), MDH2 (P08249), IDH3α (Q9D6R2), and OSCP (Q9DB20).

[0045] Example 3

[0046] Soybean extract binds to SDHA and activates SDH

[0047] (1) RNA interference experiment

[0048] siRNA was designed and synthesized by Shanghai Genema Gene, and the sequences are shown in SEQ ID NO.1-26 and Table 1.

[0049] Table 1 siRNA sequences

[0050]

[0051] The cells were plated in 96-well plates, and cell transfection was performed after the cells adhered to the wall. First, the culture medium in the 96-well plate was replaced with a culture medium without FBS and PS, 100 μL / well. The NC group and different siRNA powders were dissolved in DEPC water to prepare a 15 μM solution, and the transfection reagents required for different groups were prepared according to the method in Table 2. First, each group was divided into two tubes, one Lipofectamine3000 tube and one siRNA / NC tube. After preparing the two tubes, the two were mixed. After gently blowing and standing for 15 minutes, the siRNA / NC was fully entered into the liposomes. Except for the model control group, the corresponding 10 μL / well mixed transfection reagent was added to the other groups, and gently shaken to make it evenly distributed, and then the 96-well plate was transferred to the cell culture incubator for 8 hours. After transfection, the medium in the 96-well plate was aspirated and replaced with 100 μL of FBS-free medium (containing 200 μg / mL MSU) with or without 50 μM soy bean extract for each group. The culture was continued for 24 h and then CCK-8 assay was performed. The results are shown in Figure 2 As shown in e.

[0052] Table 2 Reagent dosage required for cell transfection in each group

[0053]

[0054] (2) SDH activity detection

[0055] The cells were divided into a model group, a SDHI group, and a soymilk group (12.5, 25, 50, and 100 μM). Serum-free culture medium containing 200 μg / mL MSU was added to the model group, and serum-free culture medium containing 50 μM fluopyram and 200 μg / mL MSU was added to the SDHI group. After 24 hours, the original culture medium was discarded, and the cells were washed twice with PBS. The cells were scraped into a 1.5 mL EP tube with a cell scraper and centrifuged (4°C, 3000 rpm, 5 min), and the supernatant was discarded. Lysate was added to the cells of different groups, and the protein was extracted and quantified to 10 mg / mL. Fluopyram and soymilk of different concentrations were added to the corresponding groups, and the reaction was carried out in a constant temperature shaking mixer (25°C, 800 rpm, 60 min). After the reaction, the SDH activity of different groups was detected according to the instructions of the succinate dehydrogenase activity detection kit. The results are as follows. Figure 3 shown.

[0056] The results showed that SDHA is essential for the activity of daidzein ( Figure 2 d), and soybean extract can activate SDH ( Figure 3 ).

[0057] Example 4

[0058] Soybean extract promotes the production of reactive oxygen species (ROS) and apoptosis of inflammatory cells in RAW264.7 cells

[0059] (1) Detection of ROS levels: RAW264.7 cells were plated in glass-bottomed culture dishes at a density of 300,000 cells / dish. After the cells adhered to the wall, the original culture medium was aspirated and 200 μg / mL MSU serum-free culture medium containing different concentrations of soy bean extract was added to the well plate. After 24 hours, the cells were stained according to the instructions of the reactive oxygen species detection kit, and the intensity of ROS fluorescence (green) was observed under a research-grade inverted microscope.

[0060] (2) Flow cytometry to detect cell apoptosis: RAW264.7 cells were divided into blank control group, FITC single staining group, PI single staining group, negative control group, model group, and 25μM / 50μM / 100μM soy bean extract group. The blank control group, FITC single staining group, and PI single staining group were all MSU modeled and treated with apoptosis inducers. After the cells adhered to the wall, the cells in different groups were treated with MSU and soy bean extract. After 24 hours, the cells were collected for cell staining. First, 100μL of Annexin V buffer was added to different groups. After the cells were blown evenly, FITC-Annexin V and PI were added according to the grouping needs. The cells were incubated at room temperature in the dark for 15 minutes. During this period, the EP tube was shaken regularly to make the dye solution uniform. Finally, an appropriate amount of Annexin V buffer was added for flow cytometry detection.

[0061] According to the results, as the concentration of soy bean extract increases, the level of ROS in inflammatory RAW264.7 cells increases significantly ( Figure 4 a), and the degree of early and late apoptosis of cells increased ( Figure 4 b).

[0062] Example 5

[0063] SDH is the key protein for soybean extract to relieve gouty arthritis

[0064] The effect of soymilk on relieving gouty arthritis by activating SDH was verified at the animal level. After the mice were modeled with MSU-induced gouty arthritis, soymilk and flupyraclostrobin (SDHI, Shanghai Taoshu Biotechnology Co., Ltd.) were used for intervention. After the treatment, the swelling degree of the ankle and foot surface of the mice and the temperature of the soles were measured every 2 mice.

[0065] The results showed that soymilk can relieve ankle swelling in mice, but SDHI can inhibit the effect of soymilk ( Figure 5 a), Similarly, soy extract can reduce ankle swelling and paw temperature, as well as reduce macrophage infiltration in the ankle joints of mice, but these effects are inhibited by SDHI ( Figure 5 b, Figure 5 c), indicating the importance of soymilk in activating SDH in its role in alleviating gouty arthritis.

[0066] As can be seen from the above examples, the present invention provides the use of soymilk in the preparation of drugs for treating and / or preventing gouty arthritis. The present invention is based on the activity of soymilk in in vitro anti-inflammatory and in vivo relief of gouty arthritis, as well as the binding effect of soymilk and soymilk probe on proteins in inflammatory macrophages, to carry out the study of the mechanism of soymilk killing inflammatory macrophages and relieving gouty arthritis. The direct target protein of soymilk was found in inflammatory macrophages, which is present in oxidative phosphorylation and tricarboxylic acid cycle. The importance of this protein for the activity of soymilk was confirmed by RNA interference experiments. It was confirmed in inflammatory macrophages that soymilk can increase the activity of succinate dehydrogenase (SDH), promote the production of reactive oxygen ROS in inflammatory macrophages and induce apoptosis of inflammatory macrophages. The treatment method of local injection of soymilk into the ankle joint can relieve gouty arthritis in mice. These provide new ideas for the research and development of new drugs for the treatment of gouty arthritis and local administration preparations with microneedles.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of soybean extract in the preparation of medicines for treating and / or preventing gouty arthritis.

2. The use according to claim 1, characterized in that: The concentration of soybean extract in preparing the medicine for treating and / or preventing gouty arthritis is 10-500 μM.

3. The use according to claim 1 or 2, characterized in that: When soybean extract is used to prepare a drug for treating and / or preventing gouty arthritis, gouty arthritis is treated and / or prevented by promoting the activity of succinate dehydrogenase, intervening in the cellular oxidative phosphorylation process, and inducing inflammatory cell apoptosis.

4. The use according to claim 3, characterized in that: The dosage form of the drug includes one or more of injection, tablet, capsule and oral solution.

5. Application of soybean extract in the preparation of preparations for promoting the activity of succinate dehydrogenase.

6. The use according to claim 4, characterized in that: The concentration of daidzein in the preparation for promoting the activity of succinate dehydrogenase is 10 to 500 μM.

7. The application of soybean extract in the preparation of preparations for inducing apoptosis of inflammatory cells.

8. The use according to claim 7, characterized in that: The concentration of soy bean extract in the preparation for inducing apoptosis of inflammatory cells is 10 to 500 μM.

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