Application of cynanchum otoN in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of cynanchum otoN in preparation of medicine for treating rheumatoid arthritis. The invention discloses application of cynanchum otoN in preparation of a medicine for treating rheumatoid arthritis. The cynanchum otoN has an antagonistic effect on EP4, can be prepared into an antagonist of a prostaglandin receptor EP4, and can also effectively relieve rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of ginsenoside otoN in the preparation of a medicine for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease characterized by symmetrical joint synovitis, which is manifested by repeated attacks of joint synovitis and the formation of synovial pannus, leading to erosion and destruction of the affected joint cartilage and subchondral bone, and eventually forming different deformities in the affected joints, resulting in a series of joint dysfunctions.

[0003] Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (GC) and anti-rheumatic drugs (DMARDs) are used in clinical RA treatment. However, in the first-line treatment, all three have adverse reactions that are difficult to ignore. In the case of excessive or overtime use of NSAIDs, leukopenia, thrombocytopenia, and gastrointestinal lesions such as stomach pain, gastric ulcers, and even ulcer bleeding, liver damage, and kidney damage may occur. GC and DMARD may be toxic and have potential serious adverse reactions, such as liver damage, renal failure, and increased susceptibility to infection. Since the treatment of RA is a long-term process, long-term adverse drug reactions bring a serious burden to patients, and finding new drugs and new treatment methods has become a new goal for the treatment of RA.

[0004] Cyclooxygenases (COXs) are rate-limiting enzymes in metabolic pathways. COX has three isoenzymes, called COX-1, COX-2, and COX-3. COX-2 is an inducible enzyme whose expression is enhanced in cases of tissue damage, inflammation, etc. PGE2 is the main mediator of inflammatory responses triggered by COX activity, and can bind to and activate four GPCRs, namely EP1, EP2, EP3, and EP4. As a member of the GPCRs family, EP4's ligand can activate the ERK1 / 2 signaling pathway to regulate a wide range of physiological and pathological functions. After ERK1 / 2 is activated, it phosphorylates itself, then transfers to the nucleus to affect the activity of various transcription factors, and ultimately regulates cell metabolism and functional expression, thereby affecting the specific biological effects of cells.

[0005] Qingyang ginseng is the dried root of Cynanchum otophyllum, a plant of the genus Cynanchum, which is widely distributed in the southwestern provinces of my country, and is mainly produced in Dali, Lijiang and other western Yunnan regions in Yunnan. It is often used to treat rheumatism and bone pain in ethnic minority areas. However, there is no specific research on the treatment of rheumatism and bone pain with Qingyang ginseng in the prior art. Based on this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide the use of ginsenoside otoN in the preparation of a medicine for treating rheumatoid arthritis.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides the use of ginsenoside otoN in the preparation of a drug for treating rheumatoid arthritis. The structural formula of the ginsenoside otoN is as follows:

[0009]

[0010] The present invention also provides a medicine for treating rheumatoid arthritis, comprising ginsenoside otoN and one or more pharmaceutically acceptable excipients.

[0011] Preferably, the dosage form of the drug includes capsules, powders, tablets, granules, oral liquids or injections.

[0012] The present invention also provides the use of ginsenoside otoN in the preparation of a prostaglandin receptor EP4 antagonist, and the structural formula of the ginsenoside otoN is as follows:

[0013]

[0014] The present invention also provides a prostaglandin receptor EP4 antagonist, comprising ginsenoside otoN and one or more pharmaceutically acceptable excipients.

[0015] Preferably, the dosage form of the prostaglandin receptor EP4 antagonist includes capsules, powders, tablets, granules, oral solutions or injections.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides the use of ginsenoside otoN, which can effectively inhibit the activity of prostaglandin receptor EP4. Therefore, it can be used to prepare an antagonist of prostaglandin receptor EP4; ginsenoside otoN can inhibit the activity of cyclooxygenase-2 (COX-2) by inhibiting the activity of prostaglandin receptor EP4, thereby effectively treating rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the molecular docking of ginsenoside otoN and EP4, where A is the binding active pocket of ginsenoside otoN and EP4 (3D), and B is the hydrogen bond interaction between ginsenoside otoN and EP4 residues;

[0019] Figure 2Schematic diagram of using the EP4 receptor antagonist CJ-42794 to verify the EP4 overexpression model in Example 2, wherein A is the Western Blot band, B is the band quantification result, wherein the sample for NC detection is sterile water, - represents the antagonist CJ-42794 of PGE2 / EP4 receptor without adding the agonist, and + represents the antagonist CJ-42794 of PGE2 / EP4 receptor with adding the agonist;

[0020] Figure 3 The figure is a schematic diagram of the phosphorylation level and expression amount of ERK1 / 2 downstream of EP4 after treating cells with ginsenoside otoN in Example 2; A is a Western Blot result diagram, B is a WB grayscale scan value bar graph, wherein the sample detected by NC is sterile water, - represents the antagonist of PGE2 / EP4 receptor CJ-42794 without adding the agonist, and + represents the antagonist of PGE2 / EP4 receptor CJ-42794 with adding the agonist;

[0021] Figure 4 The Western Blot results in Example 3, wherein A is the Western Blot band and B is the band quantification result;

[0022] Figure 5 This is the test result of the thickness of the mouse hind limb paw;

[0023] Figure 6 This is the test result of bare joint thickness of mouse hind limb paw;

[0024] Figure 7 This is the test result of the width of the mouse hind limb paw;

[0025] Figure 8 is the average grip strength of the mouse hind paws. DETAILED DESCRIPTION

[0026] The present invention provides the use of ginsenoside otoN in the preparation of a drug for treating rheumatoid arthritis. The structural formula of the ginsenoside otoN is as follows:

[0027]

[0028] The present invention also provides a medicine for treating rheumatoid arthritis, comprising ginsenoside otoN and one or more pharmaceutically acceptable excipients.

[0029] In the present invention, the dosage form of the drug includes capsules, powders, tablets, granules, oral liquids or injections.

[0030] The present invention also provides the use of ginsenoside otoN in the preparation of a prostaglandin receptor EP4 antagonist, and the structural formula of the ginsenoside otoN is as follows:

[0031]

[0032] The present invention also provides a prostaglandin receptor EP4 antagonist, comprising ginsenoside otoN and one or more pharmaceutically acceptable excipients.

[0033] In the present invention, the dosage form of the prostaglandin receptor EP4 antagonist includes capsules, powders, tablets, granules, oral solutions or injections.

[0034] 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.

[0035] Example 1

[0036] Molecular docking method was used to predict the binding of Qingyang ginsenoside otoN to EP4 receptor:

[0037] 1. Preparation of Ligand Small Molecule

[0038] (1) Search the PubChemCID number of the target blood component in the online database Drugbank, and download and save its sdf format structure in the online database PubChem;

[0039] (2) Establish a small molecule library in the software MOE (Molecular Operating Environment), import all small molecules, wash and 3D the small molecule library, and obtain the optimized ligand (i.e., ginsenoside otoN);

[0040] 2. Protein Receptor Preparation

[0041] (1) Download and save the 3D structure of the target receptor protein (i.e., EP4 receptor) in the online database RCSB PDB (RCSB Protein DataBank);

[0042] (2) Import the structure of the target receptor protein into the software MOE, remove water molecules and organic solvents, and retain only the protein and the original ligand;

[0043] 3. Molecular docking

[0044] (1) The ligand optimized in step 1 and the protein in step 2 were docked. The active pocket was selected as LigandAtoms. 30 conformations were generated and 3 docking results were output as references. The other parameters remained default.

[0045] (2) Waiting for docking: The evaluation criteria first look at S, which is the docking binding energy score (unit: kcal / mol). The smaller the value (usually a negative number), the lowest energy conformation. The tighter the small molecule in this conformation binds to the ligand.

[0046] 4. Visual analysis of docking results

[0047] The results were visualized in 3D mode using PyMOL and in 2D mode using MOE. Figure 1 As shown;

[0048] from Figure 1 It can be seen that Qingyang ginsenoside otoN can bind to the EP4 receptor with a binding energy of -8.4 kcal / mol.

[0049] Example 2

[0050] Western Blot technology was used to verify the antagonistic effect of ginsenoside otoN on EP4 based on the ERK1 / 2 signaling pathway:

[0051] 1. Cell Culture

[0052] (1) Preparation: Use ultraviolet light to sterilize the biosafety cabinet and necessary supplies for 30 min to ensure the cleanliness and sterility of the operation area; turn on a constant temperature water bath (37°C) and preheat the culture medium, 1× PBS buffer, and 0.25% trypsin to ensure that the temperature matches the in vivo environment and provide a suitable growth environment for the cells;

[0053] (2) Washing: Slowly add 2 mL of 1× PBS buffer along the wall of the culture dish, and aspirate and discard after rinsing the cell surface. Repeat twice;

[0054] (3) Digestion: Add 0.5 mL of 0.25% trypsin to digest the cells, place them in an incubator and allow to digest. When the cells become like quicksand, use complete medium to terminate the digestion, mix the cells, and obtain a cell suspension.

[0055] (4) Centrifugation: Gently blow the cell suspension to make it evenly distributed, transfer it to a clean and sterilized 1.5 mL EP tube, and centrifuge at 800 rpm for 3 min. After centrifugation, remove the supernatant and retain the cell pellet;

[0056] (5) Culture: The cell pellet was inoculated into a 100 mm dish, 5 mL of DMEM medium containing 10% FBS was added, and the dish was placed in a cell culture incubator at 37°C and 5% CO2 for cell culture until the cell density reached 75%;

[0057] 2. Cell Transfection

[0058] (1) Preparation: Preheat Opti-MEM medium and PBS in a 37°C water bath; remove cells from the cell culture incubator, use a pipette to remove the old medium and discard it, wash the cells with preheated PBS, add 5 mL of preheated Opti-MEM medium to the cells, place in a 37°C, 5% CO2 cell culture incubator, and culture for 1 h to obtain cells to be transfected;

[0059] (2) Prepare solution A: 200 μL Opti-MEM medium + 18 μL lipofectamine2000 / well, incubate at 37°C for 5 min; solution B: 200 μL Opti-MEM medium + 9 μL plasmid (pcDNA5 / FRT / TO-VSV-GluR5-EP4) (purchased from Beijing Qingke Biotechnology Co., Ltd.), incubate at 37°C for 5 min; mix solution A and solution B, gently pipette and continue incubation at 37°C for 20 min; obtain a mixed solution;

[0060] (3) adding the cells to be transfected into the mixed solution and placing them in a 37°C constant temperature incubator for transfection. After 6 hours of transfection, replacing the medium with fresh DMEM containing 10% FBS, and transfecting for another 12 hours to obtain transfected cells;

[0061] 3. Cell Plating

[0062] (1) washing, digesting, and centrifuging the transfected cells [same as steps 1(2) to (4) of Example 2] to obtain treated cells, wherein during the digestion process, the amount of trypsin added is increased to 1 mL;

[0063] (2) Open the cell well plate in a biological cabinet, soak each well with poly-L-lysine and aspirate it, and place it in the cabinet to air dry;

[0064] (3) Add 1 mL of 10% FBS DMEM medium to the treated cells, mix well by pipetting, and then add an equal amount evenly to the cell wells coated with poly-lysine, and shake the wells in a cross shape;

[0065] (4) 4 h after plating, the cells were starved by replacing the medium with 600 μL of DMEM medium without FBS;

[0066] 4. Cell Protein Extraction

[0067] (1) Use DMEM medium without FBS to prepare a concentration of 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11, 10 -12 mol / L (M) EP4 receptor antagonist CJ-42794 (CJ) and ginsenoside otoN solution, after starving the cells for 12 h, replace the DMEM medium without FBS with 600 μL of the solution and incubate for 40 min;

[0068] (2) After incubation, the EP4 receptor agonist PGE2 was added for activation for 5 min; then an appropriate amount of an equal volume of strong RIPA lysis buffer containing a protease inhibitor cocktail and sodium orthovanadate was added to each well, and the cells were lysed on ice for 20 min to obtain lysed cells;

[0069] (3) Transfer the lysed cells to a 1.5 mL EP tube, centrifuge at 16,000 rpm for 15 min, discard the precipitate, and keep the supernatant;

[0070] (4) Add 1 / 4 volume of 5× SDS Loading Buffer to the supernatant and shake to mix to obtain the protein sample;

[0071] (5) Western Blot was used to detect protein samples. The results are as follows Figures 2-3 As shown;

[0072] from Figures 2-3 It can be seen that VSV tag expression and EP4 were successfully overexpressed; the phosphorylation of ERK was antagonized by the EP4 receptor antagonist CJ-42794 in a dose-dependent manner, proving that the model was successfully established; the phosphorylation of ERK was antagonized by ginsenoside otoN in a dose-dependent manner, proving that ginsenoside otoN has an antagonistic effect on EP4.

[0073] Example 3

[0074] Western Blot technology was used to verify the protective effect of ginsenoside otoN on EP4 based on the SIP method:

[0075] According to the experimental principle of SIP, the binding of drugs to proteins can protect proteins from denaturation by organic solvents. The protective effect of drugs on protein receptors will decrease as the concentration of organic solvents increases. When the effective concentration reaches the limit of drug protection, protein receptors almost disappear.

[0076] 1. Cell culture and transfection, the same as the cell culture and transfection steps in Example 2;

[0077] 2. Cell sampling:

[0078] (1) After transfection, discard the original culture medium and wash the transfected cells twice with pre-cooled 1× PBS buffer to obtain washed transfected cells;

[0079] (2) Add 1 mL of cell lysis buffer (lysis buffer formula: 200 μL Cell Lysis Buffer (10×) + 1800 μL ddH2O + 20 μL PMSF Solution + 20 μL protein inhibitor cocktail) to the washed transfected cells, shake and lyse for 20 min to obtain lysed cells;

[0080] (3) Place the lysed cells into a 1.5 mL EP tube, centrifuge at 16,000 rpm for 15 min, discard the precipitate, and keep the supernatant;

[0081] (4) The supernatant was transferred and evenly divided into two clean 1.5 mL EP tubes, 460 μL in each tube, and the agonist PGE2 (PGE2) and ginsenoside otoN (otoN) were added respectively; sterile water was used as the blank control (NC), and dimethyl sulfoxide (DMSO) was added to the sterile water to ensure that the DMSO concentration in the PGE2 group, otoN group, and NC group was consistent, and the mixture was incubated on a shaker at 25°C for 40 min to obtain the incubated samples;

[0082] (5) Take 80 μL of the incubated sample and mix it with 20 μL of sterile water to prepare a mixed solution of 0% AEA; take 80 μL of the incubated sample, 5 μL of AEA organic solvent mixture (acetone: anhydrous ethanol: glacial acetic acid 1:1:2) and 15 μL of sterile water to prepare a mixed solution of 5% AEA; take 80 μL of the incubated sample, 10 μL of AEA organic solvent mixture and 10 μL of sterile water to prepare a mixed solution of 10% AEA; take 80 μL of the incubated sample and 20 μL of AEA organic solvent mixture to prepare a mixed solution of 20% AEA;

[0083] (6) The mixture was equilibrated at 25°C and 60 rpm for 20 min, then centrifuged at 4°C and 16,000 rpm for 20 min, and the supernatant was collected;

[0084] (7) Add 1 / 4 volume of 5× SDS Loading Buffer to the supernatant and shake to mix to obtain the protein sample;

[0085] (8) Western Blot was used to detect protein samples. The results are as follows Figure 4 As shown;

[0086] from Figure 4 It can be seen that under the damage of organic reagents, Qingyang ginsenoside otoN can bind to EP4 to inhibit protein denaturation.

[0087] Example 4

[0088] Establish a rheumatoid arthritis model in C57BL / 6 mice to study the therapeutic effect of ginsenoside otoN on rheumatoid arthritis:

[0089] Thirty-six 7-week-old mice with mature immune systems (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were randomly divided into six groups: normal (NC) group, model (Model) group, methotrexate 4 mg / kg (MTX) group, ginsenoside otoN 18 mg / kg (otoN-18) group, ginsenoside otoN 16 mg / kg (otoN-16) group, ginsenoside otoN 20 mg / kg (otoN-21) group, 6mg / kg (otoN-6) group; after the mice were adaptively fed for 7 days, 0.1mL of agent (an emulsion containing 2.5mg / mL Mycobacterium tuberculosis mixed with collagen and complete adjuvant) was injected into the tail of the mice in the Model group, MTX group, otoN-18 group, otoN-16 group, and otoN-6 group for the first immunization; 21d after the first immunization, 0.1mL of agent (an emulsion containing 2.5mg / mL Mycobacterium tuberculosis mixed with collagen and complete adjuvant) was injected again for booster immunization to complete the rheumatoid arthritis model; 35d after the first immunization, the mice in the MTX group, The mice in the otoN-18, otoN-16, and otoN-6 groups were intraperitoneally injected with 4 mg / kg methotrexate, 18 mg / kg otoN, 16 mg / kg otoN, and 6 mg / kg otoN. The Model group was injected with 0.1 mL of normal saline (0.9% NaCl, sterile) at the same site and time. The NC group was injected with 0.1 mL of normal saline (0.9% NaCl, sterile) at the same site and time. The paw thickness, bare joint thickness, and palm width of the mice were measured every day from the start of drug administration. The paw pulling force was measured on the 40th day. The measurement results are shown in the table below. Figures 5 to 8 shown.

[0090] from Figures 5 to 7 It can be seen that after the rheumatoid arthritis model was established, the rat's hind paws showed obvious swelling; after the injection of Qingyang ginsenoside otoN, the thickness of the rat's paws, the thickness of the bare joints, and the width of the palms were significantly reduced, indicating that Qingyang ginsenoside otoN can effectively improve the swelling of the rat's paws;

[0091] from Figure 8 It can be seen that after the injection of Qingyang ginsenoside otoN, the pulling force of the mouse paw was weakened. It can be seen that Qingyang ginsenoside otoN can effectively improve the pulling force of the mouse paw.

[0092] According to the above embodiments, the present invention provides the use of ginsenoside otoN in the preparation of drugs for treating rheumatoid arthritis. Ginsenoside otoN has an antagonistic effect on EP4, can be prepared into an antagonist of prostaglandin receptor EP4, and can also effectively relieve rheumatoid arthritis.

[0093] 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 ginsenoside otoN in the preparation of a drug for treating rheumatoid arthritis, wherein the structural formula of ginsenoside otoN is as follows:

2. A drug for treating rheumatoid arthritis, characterized in that: The invention comprises ginsenoside otoN and one or more pharmaceutically acceptable excipients.

3. The drug according to claim 2, characterized in that The dosage form of the drug includes capsule, powder, tablet, granule, oral solution or injection solution.

4. The use of ginsenoside otoN in the preparation of prostaglandin receptor EP4 antagonist, wherein the structural formula of ginsenoside otoN is as follows:

5. A prostaglandin receptor EP4 antagonist, characterized in that The invention comprises ginsenoside otoN and one or more pharmaceutically acceptable excipients.

6. The prostaglandin receptor EP4 antagonist according to claim 5, characterized in that The dosage form of the prostaglandin receptor EP4 antagonist includes capsules, powders, tablets, granules, oral solutions or injections.

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