Application of baicalein in preparation of medicine for delaying rheumatoid arthritis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of baicalein in preparation of a medicine for delaying rheumatoid arthritis. The medicine in the application is a medicine for inhibiting the activity of the rheumatoid arthritis fibroblast-like synovial cells, and further, the medicine is a medicine for down-regulating the protein expression of NOX2 in the rheumatoid arthritis fibroblast-like synovial cells. In the application, the effective in-vivo medication concentration of the baicalein is 300-600 mg / kg, and the effective in-vitro medication concentration of the baicalein is 20-60 [mu] mol / L. According to the application, rheumatoid arthritis fibroblast-like synovial cells are taken as a research object, and baicalein is used for intervening cell viability and functions by down-regulating NOX2, so that the rheumatoid arthritis is relieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of baicalein in preparing a drug for delaying rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic autoimmune disease characterized by synovitis. According to WHO statistics, about 1% of the world's population suffers from RA. Its main pathological changes include synovitis and destruction of bone and cartilage in joints, the latter of which is the direct cause of progressive ankylosis, deformity, dysfunction and even disability of joints. RA-fibroblast-like synoviocytes (RA-FLS) play an important role in the occurrence and development of RA. On the one hand, RA-FLS can migrate to the surface of cartilage and erode cartilage and bone tissue by secreting proteolytic enzymes such as matrix metalloproteinases, leading to joint destruction; on the other hand, when RA-FLS migrate to bones, they can activate osteoclasts and enhance cartilage and bone erosion; in addition, the long-distance migration of activated RA-FLS may also participate in joint damage at different sites. Therefore, finding the key factors that regulate the proliferation, migration and invasion of RA-FLS and clarifying their specific molecular mechanisms of action have important guiding significance and translational value for the clinical discovery of new therapeutic targets and potential therapeutic drugs for RA.

[0003] At present, the treatment of RA is mainly to relieve pain, control inflammation, improve joint function and protect organs. The main measures include drug therapy, immune purification and surgical treatment, among which drug therapy is the most important and widely used method. Commonly used drugs in clinical practice include non-steroidal anti-inflammatory drugs (such as loxoprofen), glucocorticoid drugs (such as prednisone), immunosuppressants (such as methotrexate) and biological agents (such as ysaipu and adalimumab). However, the use of these drugs inevitably produces some side effects. Non-steroidal anti-inflammatory drugs can cause gastrointestinal discomfort and ulcers. Improper use of hormones can cause osteoporosis, opportunistic infections, etc. Immunosuppressants and biological agents can completely suppress immunity and easily cause infection in patients. Therefore, it is necessary to seek a safe and effective drug that can protect joint bones and cartilage while controlling inflammation.

[0004] According to the Yellow Emperor's Classic of Internal Medicine, RA is a "bi syndrome" caused by the three major evil spirits of wind, cold and dampness. Scutellaria baicalensis is a commonly used medicine in traditional Chinese medicine to treat rheumatic immune diseases. Baicalein is one of the effective monomers of Scutellaria baicalensis and has a wide range of pharmacological effects, including anti-inflammatory, anti-oxidant, anti-tumor, anti-bacterial, anti-viral, and anti-cardiovascular disease. Baicalein (CAS: 491-67-8, molecular formula C 15 H 10O5) is an extract of the traditional Chinese medicine Scutellaria baicalensis. Previous studies have shown that baicalein has a wide range of pharmacological effects, including anti-inflammatory, antioxidant, anti-tumor, antibacterial, antiviral, and anti-cardiovascular disease.

[0005] Therefore, developing a new baicalin for use in the preparation of drugs for delaying rheumatoid arthritis plays an important role. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies in the prior art and to provide an application of baicalin in the preparation of a drug for delaying rheumatoid arthritis, specifically in the preparation of a pharmaceutical composition for inhibiting the proliferation and activity of RA-FLS, providing a new idea and solution for the clinical treatment of RA.

[0007] In order to solve the above technical problems, the present invention discloses an application of baicalin in preparing a drug for delaying rheumatoid arthritis;

[0008] Specifically, in some embodiments of the present invention, a rat rheumatoid arthritis model (CIA) was established, and the CIA rats were grouped and administrated. The dosage of baicalin was controlled for each group, and then the arthritis of the CIA rats and the ankle joint pathology of the CIA rats were evaluated and scored after continuous administration. This proved that baicalin can delay rheumatoid arthritis.

[0009] Preferably, the drug is a drug that inhibits the activity of rheumatoid arthritis fibroblast-like synoviocytes;

[0010] Specifically, in some embodiments of the present invention, by conducting immunohistochemical experiments on inflammatory factors in the synovial tissue of CIA rats and evaluating the vitality of the in vitro RA-FLS model, it was proved that the expression of inflammatory factors in the synovial tissue of CIA rats after the action of baicalin was reduced and baicalin could effectively inhibit the functions of RA-FLS, including cell viability, cell migration ability, and the ability to secrete pro-inflammatory factors.

[0011] More preferably, the drug for inhibiting the activity of rheumatoid arthritis fibroblast-like synoviocytes is a drug for down-regulating the protein expression of NOX2 in rheumatoid arthritis fibroblast-like synoviocytes.

[0012] Specifically, in some embodiments of the present invention, through cell thermal migration experiments and the effect of overexpression of NOX2 in RA-FLS on the effect of baicalein, it was proved that baicalein can significantly downregulate the protein expression of NOX2 in RA-FLS.

[0013] Wherein, the effective in vivo drug concentration of baicalein is 300-600 mg / kg;

[0014] Preferably, the effective in vivo concentration of baicalein is 300 mg / kg.

[0015] Among them, the effective in vitro drug concentration of baicalein is 20-60 μmol / L.

[0016] The present invention also discloses a drug for delaying rheumatoid arthritis and a composition thereof;

[0017] Wherein, the drug includes baicalin;

[0018] Wherein, the pharmaceutical composition comprises baicalein and other pharmaceutically acceptable components;

[0019] Preferably, the pharmaceutically acceptable components include pharmaceutically active ingredients and / or pharmaceutically acceptable excipients that can be used in combination pharmaceutically;

[0020] More preferably, the pharmaceutically acceptable excipients include one or a mixture of carriers, excipients, disintegrants or binders;

[0021] Further preferably, the carrier and excipient include one or a mixture of lactose hydrate, microcrystalline cellulose, mannitol, sodium citrate, calcium phosphate, glycine or starch;

[0022] Further preferably, the disintegrant comprises one or a mixture of crospovidone, copovidone, sodium starch glycolate, croscarboxymethyl cellulose sodium or a specific composite silicate;

[0023] Further preferably, the adhesive comprises one or a mixture of polyvinyl pyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sucrose, gelatin or gum arabic.

[0024] Beneficial effects: The present invention provides an application in the preparation of a drug for inhibiting rheumatoid arthritis. The present invention uses rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) as the research object, and uses baicalin to intervene in cell viability and function by downregulating NOX2 to achieve relief of rheumatoid arthritis. This mechanism is first proposed by the present invention. Experiments have shown that baicalin can significantly inhibit the proliferation, migration ability and secretion of inflammatory cytokines of RA-FLS by downregulating NOX2, thereby slowing down the disease progression of RA, effectively inhibiting the arthritis index, arthritis swelling degree and systemic arthritis pathological score of CIA rats, reducing the swelling volume of the secondary side paw of CIA rats, and significantly alleviating the ankle pathological manifestations of CIA rats. It can be used for clinical prevention / treatment of rheumatoid arthritis. The pharmaceutical composition capable of inhibiting rheumatoid arthritis prepared based on baicalin is safe and low-toxic, has a strong pharmacological effect, and has a clear therapeutic effect. The present invention provides a new source of drugs for the prevention, protection, treatment and research of rheumatoid arthritis, and is easy to promote and apply clinically, and can produce huge clinical application prospects and social benefits in a relatively short period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0026] Figure 1 The following are the scores for the pathological evaluation of arthritis in CIA rats given different doses of baicalein for 18 days in Example 1 of the present invention, where (a) is the body weight score, (b) is the whole body score, (c) is the arthritis index, (d) is the degree of joint swelling, and (e) is the secondary side paw volume score (mean ± SD; N = 6; # P<0.05Compared with normal rats, ## P<0.01Compared with normal rats, * P<0.05Compared with CIA rats, ** P < 0.01 compared with CIA rats);

[0027] Figure 2 The ankle pathological evaluation and scoring of CIA rats given different doses of baicalein for 18 days in Example 2 of the present invention, wherein (a) is H&E staining of ankle joint tissue sections, (b) is ankle joint pathological score, and (c) is micro-CT detection (mean±SD; N=6; 、* P<0.05, ** P < 0.01 compared with CIA rats);

[0028] Figure 3The expression of proinflammatory cytokines IL-1β, TNF-α and IFN-β in synovial tissue of CIA rats after administration of different doses of baicalein for 18 days in Example 3 of the present invention;

[0029] Figure 4 The viability and function of MH7A (RA-FLS) cells stimulated by TNFα in Example 4 of the present invention, wherein (a) is the effect of baicalin on the proliferation of RA-FLS, (b) is the effect of baicalin on the migration ability of RA-FLS cells, and (c) is the effect of baicalin on the production of verification factors by RA-FLS (mean ± SD; N = 3; ## P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 compared with the untreated control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the TNF-α stimulated model group);

[0030] Figure 5 The effect of baicalin on NOX2 protein in MH7A (RA-FLS) stimulated by TNFα in Example 5 of the present invention, wherein (a) is the effect of baicalin on the thermal stability of NOX2 protein, (mean±SD; N=3; * P<0.05 compared with the DMSO control group); (b) Effect of baicalein on NOX2 protein expression (mean±SD; N=3; ## P<0.01, ### P<0.001, ### P < 0.0001 compared with the untreated control group; ** P<0.01, *** P<0.001, **** P < 0.0001 compared with the TNF-α stimulation model group);

[0031] Figure 6 The effect of NOX2 expression on the activity of MH7A (RA-FLS) stimulated by TNFα with baicalin in Example 6 of the present invention, including an untreated wild-type MH7A cell (FLS) control group, a TNFα-stimulated wild-type MH7A cell (RA-FLS) model group, a baicalin intervention group of RA-FLS, and a TNFα-stimulated MH7A cell overexpressing NOX2 (RA-FLS+NOX2 OE ) model group, and RA-FLS+NOX2 OE Baicalein intervention group, where (a) is the expression of migration proteins in each cell, and (b) is the secretion capacity of inflammatory factors in each group of cells (mean±SD; N=3; * P<0.05,** P<0.01, RA-FLS baicalein intervention group and RA-FLS+NOX2 OE compared with the baicalin intervention group). DETAILED DESCRIPTION

[0032] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0033] Example 1: Pathological evaluation and scoring of arthritis in CIA rats

[0034] 1. Establish CIA rat model by subcutaneous injection of a mixture of type II collagen and an equal amount of complete Freund's adjuvant (CFA)

[0035] S1. Preparation of collagen emulsion: Complete Freund's Adjuvant (20022, Chondrex) and Bovine Type II Collagen Solution (7023, Chondrex) were mixed in equal volumes on ice, and a 1 mL injection needle was used to repeatedly blow until a white emulsion was formed, which remained in water for 1 hour.

[0036] S2. Establishment of CIA model: On the first day, 0.1 mL of the above emulsion was injected at multiple points at the base of the rat's tail and the right sole of the foot. On the seventh day, 0.1 mL of the above emulsion was injected at multiple points at the base of the rat's tail again. On the 14th day, the model was established.

[0037] 2. Drug administration in CIA rats

[0038] On the 14th day, after the rat model was successfully induced, the rats were divided into groups for drug administration. The positive drug methotrexate (MTX) was prepared with 0.5% sodium carboxymethylcellulose (CMC-Na) at a dose of 0.5 mg / kg, administered by gavage once every three days; baicalein was mixed with 0.5% CMC-Na solution at doses of 300 mg / kg and 600 mg / kg, respectively, administered by gavage once a day. The experiment was terminated after 18 consecutive days of administration.

[0039] 3. Pathological evaluation and scoring of arthritis in CIA rats

[0040] S1. Body weight: The body weight of Wistar rats was measured and recorded every 3 days.

[0041] S2. Systemic score: The maximum score for systemic joint inflammation in CIA rats is 8 points. Starting from the first inflammation, the score was observed and scored every 3 days. The scoring criteria are shown in Table 1 below:

[0042] Table 1 Systemic joint inflammation criteria (full score: 8 points)

[0043]

[0044]

[0045] S3. Arthritis index and joint swelling score: From the first injection of the induction adjuvant, the pathological changes of inflammation in the rats' limb joints were observed every 3 days, and the number and degree of swelling of the rats' joints were recorded and scored. The scoring criteria are shown in Tables 2 and 3 below:

[0046] Table 2 Scoring criteria for joint swelling (full score: 24 points)

[0047]

[0048] Table 3 Arthritis index scoring criteria (full score: 16 points)

[0049]

[0050] S4. Measurement of the volume of the paw on the secondary side: The volume of the non-inflammation side paw of each rat was measured and recorded before inflammation. Starting from the 7th day of immunization-induced inflammation, the volume of the paw on the secondary side was measured (once every 3 days).

[0051] Swelling of the secondary paw (ΔmL): ΔmL = volume of the paw after inflammation - volume of the paw before inflammation

[0052] The results are as follows Figure 1 As shown, compared with the normal control group, the body weight of CIA rats decreased significantly due to acute pathology on day 14, and then slowly recovered with the stabilization and self-healing of the disease. After treatment with MTX and baicalein, the body weight recovered compared with the CIA group; compared with the normal control group, the systemic pathology score of CIA rats was increased, and MTX and baicalein treatment could reduce the systemic pathology score of CIA rats; compared with normal rats, the arthritis index and joint swelling of CIA rats were increased, and MTX and baicalein treatment could reduce them; compared with the CIA group, the paw volume of the MTX and baicalein treatment groups was significantly decreased.

[0053] Example 2: Pathological evaluation and scoring of ankle joints in CIA rats

[0054] 1. Hematoxylin-eosin (H&E) staining

[0055] S1. Take the ankle joint of the rat, remove the surface fur and other materials as much as possible, fix it with 10% formaldehyde for 24 hours, decalcify it with decalcifying solution until the bone softens and the needle of the syringe can gently pierce the bone, embed and slice it.

[0056] S2. Dewaxing of sections: The sections were sequentially placed in xylene I, xylene II and xylene III for 5 min each, anhydrous ethanol for 5 min × 2 times, 95%, 90%, and 70% ethanol for 5 min each, and then rinsed in distilled water for 10 min.

[0057] S3. Hematoxylin staining: Hematoxylin staining, rinse with tap water.

[0058] S4. Eosin staining of cytoplasm: staining for 5 minutes.

[0059] S5. Dehydration and sealing: Immerse the sections in 70%, 95%, 95%, anhydrous ethanol and anhydrous ethanol for 5 min each, and xylene for 5 min × 2 times, dry naturally, and seal with neutral gum.

[0060] S6. Microscope observation and analysis.

[0061] 2. H&E pathological scoring of ankle joints in CIA rats

[0062] H&E staining was used to observe the pathology of the ankle joints of rats, and the scores were based on the observed pathological lesions. Table 4 shows the H&E pathological scores of the ankle joints of CIA rats:

[0063] Table 4 Ankle joint pathology scoring table

[0064]

[0065] The score of 0 was no pathological or injury symptoms.

[0066] 3. Micro CT examination of ankle joints in CIA rats

[0067] S1. Ankle joint removal: After killing CIA rats, the ankle joint tissue on the secondary side was removed, the fur, muscle tissue and meridians were removed, and fixed in formalin for 24 hours.

[0068] S2. CT scan: The fixed ankle joint is scanned with CT scan.

[0069] The results are as follows Figure 2 As shown in the figure, compared with normal rats, the synovium in the secondary ankle joint of CIA rats was abnormally proliferated and a large number of pannus were formed, synovial cells accompanied by immune cells infiltrated the bone layer, and bone destruction, etc., and these symptoms were significantly relieved after MTX and baicalein treatment. The micro CT images of the ankle joints of CIA rats clearly showed that the articular surface of the ankle joints was irregular and had destructive lesions, and MTX and baicalein treatment could significantly alleviate these pathological changes.

[0070] Example 3: Immunohistochemical evaluation of the expression of inflammatory factors IL-1β, TNF-α and INF-α in the synovial tissue of CIA rats

[0071] S1. Dewaxing: The tissue sections were placed on a rack and dewaxed in an electric blast drying oven at 80°C for 60 min. The sections were cooled naturally at room temperature. The sections were then placed in xylene for 5 min × 3 times, dewaxed in gradient ethanol (100%, 95% and 70% ethanol for 3 min each × 2 times), rinsed in distilled water for 3 min, and rinsed in PBS solution for 5 min.

[0072] S2. Antigen repair: Preheat in a microwave oven. Transfer the slices to the antigen repair buffer and perform antigen repair by microwave radiation in a microwave oven (power is 800w×3min, 200w×5min, 800w×10s, 200w×5min, respectively). Remove the slices, cool them naturally to room temperature, and then rinse them in distilled water and PBS solution for 5min each.

[0073] S3. Permeabilization: permeabilize with 0.3% Triton X-100 solution for 30 min, rinse with PBS solution for 5 min x 3 times.

[0074] S4. Blocking: Place the slices in a humidified box, draw circles around the tissue with a grease pen, add endogenous peroxidase blocker, incubate for 30 minutes, rinse with PBS for 5 minutes × 2 times. Take out the slices and place them in a humidified box, add nonspecific staining blocker, incubate at room temperature for 1 hour, and remove the blocking solution.

[0075] S5. Incubate with primary antibody: add primary antibody and incubate overnight at 4°C. The next day, rinse with PBS solution for 5 min × 3 times.

[0076] The primary antibody information is as follows:

[0077] IFN Beta Polyclonal antibody (use ratio 1:200, Proteintech, 27506-1-AP);

[0078] IL-1Beta Polyclonal antibody (ratio 1:200, Proteintech, 16806-1-AP);

[0079] TNF Alpha Monoclonal antibody (ratio 1:200, Proteintech, 60291-1-Ig);

[0080] S6. Incubate with secondary antibody: add biotin-labeled goat anti-mouse / rabbit, incubate at room temperature for 1 hour, and rinse with PBS for 5 minutes × 3 times.

[0081] S7. Diaminobenzidine (DAB) and hematoxylin staining: Incubate streptavidin peroxidase for 30 minutes at room temperature in the dark, rinse with PBS solution. Add DAB staining solution for 5 minutes, then rinse in distilled water. Stain with hematoxylin for a certain period of time (depending on the tissue), then rinse with tap water. Place in gradient ethanol (70%-95%-100%) for 3 minutes each, and in xylene for 5 minutes × 3 times.

[0082] S8. Seal the slides: Seal the slides with neutral gum and air dry them in a fume hood. Take photos under a microscope.

[0083] The results are as follows Figure 3 As shown in the figure, the expressions of IL-1β, TNF-α, and INF-α in the synovial tissue of CIA rats were reduced after treatment with MTX or baicalein.

[0084] Example 4: Evaluation of the activity and function of RA-FLS

[0085] 1. Establishment of in vitro RA-FLS model

[0086] Human rheumatoid arthritis fibroblast synoviocytes MH7A were purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai. Culture conditions: 20% fetal bovine serum (BSA) + MEM containing NEAA (Punosai, PM150410) + 100U / mL penicillin and 100mg / mL streptomycin (Chenxin Pharmaceutical Co., Ltd., Jining, China), cultured in a 5% CO2, 37°C incubator. In the in vitro study, MH7A cells were stimulated with 50ng / mL TNF-α for 24h to simulate RA-FLS cells.

[0087] 2. CCK8 assay to detect cell viability

[0088] Cell viability was detected by CCK-8 method. Dehydrogenase can reduce WST-8 to generate orange-yellow formazan, and the absorbance (OD) value detected at a wavelength of 450nm can reflect the relative cell activity level.

[0089] Operation process:

[0090] S1. Collect cells: wash cells with PBS (containing EDTA), digest with trypsin, resuspend in culture medium, centrifuge at 1000 rpm for 3 min at room temperature, and discard the culture medium.

[0091] S2. Spotting: Resuspend cells, count, dilute cells, and spot in groups (control group and TNF-α induced group): 100 μL cells are evenly cultured in a 96-well plate, 5,000 cells per well, and cultured in an incubator for 24 hours (if medication is required, a certain concentration of NAC is given after 24 hours and culture is continued for 24 hours).

[0092] S3. Measure OD value: Aspirate the culture medium, add the prepared CCK-8 solution (the volume ratio of MEM culture medium: CCK-8 stock solution = 100:10), incubate at 37°C, and measure the OD value at 450 nM using an ELISA reader.

[0093] 3. Transwell chamber cell migration assay

[0094] The cell migration ability was detected using 8μm Transwell chamber. The operation process is as follows:

[0095] S1. Collect cells: collect MH7A cells and resuspend them;

[0096] S2. Cell inoculation: prepare a 12-well plate, add 1 mL of 20% fetal bovine serum culture medium to each well, and place it in the corresponding chamber. Add a certain amount of the above resuspended cells to each chamber, and after adhering to the wall for 12 hours, treat the cells (stimulation induction, drug addition, etc.);

[0097] S3. Fixation and staining: fix with 4% paraformaldehyde for 30 min, wash three times with PBS; stain with 1% crystal violet for 10 min.

[0098] S4. Rinse off excess crystal violet with tap water, let dry naturally, and take photos.

[0099] 4. Western blot assay to detect protein levels of inflammatory cytokines in RA-FLS

[0100] S1. Protein extraction: Wash cells twice with PBS. Lyse cells with pre-cooled RIPA protein lysis buffer (containing 0.1mM PMSF) and resuspend. Lyse on ice for 30min; gently flick the bottom of the EP tube every 5min to mix the settled cells with the lysis buffer. Centrifuge at 4℃, 12000rpm for 15min, transfer the supernatant to a new 1.5mL EP tube. Quantify protein by BCA, add loading buffer, denature in a metal bath at 95℃ for 10min, and store at -20℃.

[0101] S2. Gel preparation and electrophoresis: Prepare gel electrophoresis (Sodium laurylsulfonate-polyacrylamide gel electrophoresis, SDS-PAGE) of corresponding concentration, with sample protein at 40 μg / well; run the concentrated gel at 70V until a red band appears on the marker, and adjust the voltage to 160V until the end.

[0102] S3. Transfer: prepare separation gel, filter paper, NC membrane (nitrocellulose filter membrane) and pre-cooled full wet transfer buffer, stack them in a specific order in the transfer clip, put them into the transfer tank, fill it with pre-cooled full wet buffer, put it on ice, and transfer at 250mA for 2h.

[0103] S4. Blocking: Incubate with 5% skimmed milk powder blocking solution at room temperature for 1 hour.

[0104] S5. Antibody incubation: wash the membrane, dilute the antibody with primary antibody diluent, incubate the corresponding bands at 4°C overnight; at the end of incubation, wash the membrane, incubate the secondary antibody at room temperature for 1 hour, and wash the membrane with PBST.

[0105] The primary antibody information is as follows:

[0106] IFN Beta Polyclonal antibody (use ratio 1:2000, Proteintech, 27506-1-AP);

[0107] IL-1Beta Polyclonal antibody (ratio 1:2000, Proteintech, 16806-1-AP);

[0108] Beta Actin Polyclonal antibody (ratio 1:10000, Proteintech, 20536-1-AP);

[0109] S6. Prepare ECL color developing solution and expose.

[0110] The results are as follows Figure 4 As shown, TNF-α-stimulated MH7A cells (inflammatory synovial cells RA-FLS) were treated with 20, 40 and 60 μM baicalin for 24 h, and cell viability was detected by CCK-8. The results showed that baicalin significantly inhibited the cell viability of MH7A cells stimulated by TNF-α. The effect of baicalin on the migration ability of RA-FLS cells was studied by Transwell chamber experiment. TNF-α stimulation increased the number of MH7A cells passing through the chamber pore, and 20, 40 and 60 μM baicalin significantly inhibited the number of cells passing through the chamber after 24 h. Baicalin can significantly downregulate the protein level of inflammatory cytokines in TNF-α-induced MH7A cells, that is, the ability to secrete proinflammatory cytokines. Therefore, baicalin can effectively inhibit the functions of RA-FLS, including cell viability, cell migration ability, and the ability to secrete proinflammatory factors.

[0111] Example 5: Effect of baicalin on NOX2 protein in RA-FLS

[0112] 1. Cell thermal migration assay (CETSA) to detect the effect of baicalin on the thermal stability of NOX2 protein

[0113] CETSA is a method for detecting drug molecules binding to target proteins. After binding to drug molecules, the target protein structure becomes stable, and protein degradation slows down under gradient heating.

[0114] S1. Cell collection: MH7A cells were treated with 60 μM baicalein for 24 h and then the cells were collected. The cells were resuspended in 1 mL of pre-cooled PBS containing protease inhibitors and evenly divided into 8 EP (eppendorf) tubes.

[0115] S2. Gradient heating: Place the EP tube on the PCR instrument and set the gradient temperature: 37℃, 40℃, 45℃, 48℃, 50℃, 52℃, 55℃, 60℃, 65℃, and heat for 3 minutes.

[0116] S3. Protein extraction: After heating, take out the sample, freeze and thaw repeatedly three times in liquid nitrogen-refrigerated storage. After completion, transfer the sample to a 1.5mL EP tube and centrifuge at 12000rpm and 4℃ for 10min. Aspirate the supernatant, add the corresponding proportion of Loading buffer, heat denature, and store at -20℃.

[0117] S4. Detect target protein by Western blot.

[0118] 2. Western blot assay to detect NOX2 protein levels in RA-FLS (same method as in Example 4)

[0119] The results are as follows Figure 5 As shown in the figure, CETSA experiments proved that after the action of baicalin, the degradation rate of NOX2 protein in TNF-α-stimulated MH7A cells (RA-FLS) was significantly lower than that in the DMSO group as the temperature increased, indicating that NOX2 protein under the action of baicalin was more stable under heat incubation, suggesting that baicalin may interact with NOX2. The results of Western blot experiments showed that the protein expression of NOX2 in RA-FLS was significantly downregulated by 20, 40 and 60 μM baicalin treatment of TNF-α-stimulated MH7A cells (RA-FLS) for 24 hours.

[0120] The primary antibody information used in Western blot experiments is as follows:

[0121] NOX2 Polyclonal antibody (ratio 1:2000, Proteintech, 19013-1-AP);

[0122] NOX4 Polyclonal antibody (ratio 1:2000, Proteintech, 14347-1-AP);

[0123] Beta Actin Polyclonal antibody (ratio 1:10000, Proteintech, 20536-1-AP);

[0124] Example 6: Effect of NOX2 expression in RA-FLS on the effect of baicalin

[0125] NOX2 plasmid was overexpressed in TNF-α stimulated MH7A cells (RA-FLS), and then the protein levels of cell migration-related proteins and inflammatory factors in RA-FLS were detected by Western blot experiment (the method was the same as that shown in Example 4).

[0126] The primary antibody information used in Western blot experiments is as follows:

[0127] MMP3 Polyclonal antibody (use ratio 1:1000, Proteintech, 17873-1-AP);

[0128] MMP1 Polyclonal antibody (ratio 1:2000, Proteintech, 10371-2-AP);

[0129] MMP9 (N-terminal) Polyclonal antibody (ratio 1:500, Proteintech, 10375-2-AP);

[0130] NOX2 Polyclonal antibody (ratio 1:2000, Proteintech, 19013-1-AP);

[0131] IFN Beta Polyclonal antibody (use ratio 1:2000, Proteintech, 27506-1-AP);

[0132] IL-1Beta Polyclonal antibody (ratio 1:2000, Proteintech, 16806-1-AP);

[0133] Beta Actin Polyclonal antibody (ratio 1:10000, Proteintech, 20536-1-AP);

[0134] The results are as follows Figure 6 As shown in the results, NOX2 overexpression could reverse the down-regulation of baicalein on the cell migration-related proteins MMP1, MMP3 and MMP9, as well as IL-1β and IFN proteins in RA-FLS, suggesting that NOX2 is a key molecule in baicalein's inhibition of RA-FLS function.

[0135] The present invention provides an application of baicalein in the preparation of a drug for delaying rheumatoid arthritis. There are many methods and approaches to implement the technical solution. 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 considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. The use of baicalin in the preparation of a drug for delaying rheumatoid arthritis, wherein: The drug for delaying rheumatoid arthritis is a drug for inhibiting the activity of rheumatoid arthritis fibroblast-like synoviocytes.

2. The use according to claim 1, characterized in that: The drug for inhibiting the activity of rheumatoid arthritis fibroblast-like synoviocytes is a drug for down-regulating the protein expression of NOX2 in rheumatoid arthritis fibroblast-like synoviocytes.

3. The use according to claim 1, characterized in that: The effective in vivo drug concentration of baicalein is 300-600 mg / kg.

4. The use according to claim 1, characterized in that: The effective in vitro drug concentration of baicalein is 20-60 μmol / L.

5. A drug for delaying rheumatoid arthritis and its composition, characterized in that: The medicine comprises baicalein; and the pharmaceutical composition comprises baicalein and other pharmaceutically acceptable components.

6. The medicine and composition thereof according to claim 5, characterized in that: The pharmaceutically acceptable components include pharmaceutically active ingredients and / or pharmaceutically acceptable excipients that can be used in combination pharmaceutically.

7. The medicine and composition thereof according to claim 6, characterized in that: The pharmaceutically acceptable excipients include one or a mixture of carriers, excipients, disintegrants or binders.

8. The medicine and composition thereof according to claim 7, characterized in that: The carrier and excipient include one or a mixture of lactose hydrate, microcrystalline cellulose, mannitol, sodium citrate, calcium phosphate, glycine or starch.

9. The medicine and composition thereof according to claim 7, characterized in that: The disintegrant comprises one or a mixture of cross-linked polyvinylpyrrolidone, copolyvinylpyrrolidone, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose or a specific composite silicate.

10. The medicine and composition thereof according to claim 7, characterized in that: The adhesive comprises one or a mixture of polyvinyl pyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sucrose, gelatin or gum arabic.

Images