Application of rehmannia D in preparation of medicine for treating arthritis

Abstract

The invention discloses an application of rehmannia D in preparation of a medicine for treating arthritis. The invention provides and verifies that the rehmannia D can simultaneously inhibit cartilage degeneration and bone destruction and promote synovial fibroblast-like cell death so as to inhibit synovial hyperplasia for the first time, the three aspects are combined to effectively relieve the progress of rheumatoid arthritis, and a new natural medicine choice is provided for clinical treatment of arthritis.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, and more particularly to the application of rehmannia D in the preparation of drugs for treating arthritis. Background Technology

[0002] Arthritis is a general term for a group of joint inflammations, encompassing various types, the most common being osteoarthritis and rheumatoid arthritis (RA). Rheumatoid arthritis (RA), a chronic, systemic autoimmune disease, is fundamentally characterized by synovial inflammation: the immune system mistakenly attacks the synovial membrane of the joints, leading to its proliferation, congestion, edema, and ultimately, erosion of cartilage and bone. The pathological progression of RA is closely related to the abnormal activation of fibroblast-like synovial cells (FLSs). These cells acquire tumor-like invasiveness, proliferate excessively, and secrete inflammatory factors and matrix metalloproteinases, destroying joint structure.

[0003] Rehmaglutinin D is a natural iridoid compound, mainly isolated from the tuberous roots of the traditional Chinese medicine Rehmannia glutinosa. However, the pharmacological activity of rehmaglutinin D is currently unclear. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the application of rehmannia D in the preparation of related therapeutic drugs, based on the combined effects of inhibiting abnormal proliferation of synovial tissue, inhibiting cartilage degeneration, and inhibiting bone destruction, thereby alleviating the progression of rheumatoid arthritis.

[0005] Technical solution: The application of rehmannia D as described in this invention in the preparation of drugs for treating arthritis.

[0006] Preferably, the CAS number of the rehmannia D is 103744-84-9.

[0007] Preferably, the arthritis is rheumatoid arthritis.

[0008] Preferably, the application is in the preparation of a drug for inhibiting cartilage degeneration caused by arthritis.

[0009] Preferably, the application is in the preparation of a medicament for inhibiting bone destruction caused by arthritis.

[0010] Preferably, the application is in the preparation of a drug that inhibits abnormal proliferation of synovial tissue.

[0011] Preferably, the drug contains rehmannia D or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0013] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to propose and verify that rehmannia D can simultaneously inhibit cartilage degeneration and bone destruction, and promote the death of synovial fibroblasts, thereby inhibiting synovial hyperplasia. The three aspects work together to effectively alleviate the progression of rheumatoid arthritis, providing a new natural drug option for the clinical treatment of arthritis. Attached Figure Description

[0015] Figure 1 Representative images of foot morphology at the endpoint of the experiment in a mouse model of rheumatoid arthritis after treatment with rehmannia D; Figure 2 The graph shows the change in foot thickness in a mouse model of rheumatoid arthritis after treatment with rehmannia D. Figure 3 A graph showing the change in foot volume in a mouse model of rheumatoid arthritis after treatment with rehmannia D. Figure 4 Figure 1 shows the results of peripheral blood matrix metalloproteinase levels in a mouse model of rheumatoid arthritis after treatment with rehmannia D. Figure 5 The figure shows the toxicity test results of rehmannia D in a mouse model of rheumatoid arthritis after treatment. Figure 6 Representative H&E staining images of the tibia of a mouse model of rheumatoid arthritis after treatment with rehmannia D; Figure 7 Representative images of safranin O-fast green staining of the tibia of mice with rheumatoid arthritis model after treatment with rehmannia D; Figure 8 Representative images of the foot reconstructed by Micro-CT scan of a mouse model of rheumatoid arthritis after treatment with rehmannia D; Figure 9 The image shows the results of TNFα level detection in the synovial tissue of a mouse model of rheumatoid arthritis after treatment with rehmannia D. Figure 10 The image shows the results of LDH level detection in the synovial tissue of a mouse model of rheumatoid arthritis after treatment with rehmannia D. Figure 11The image shows the results of MH7A cell viability assay after treatment with rehmannia glutinosa D. Figure 12 Representative images of MH7A cells after scratch assay following treatment with rehmannia D; Figure 13 Figure 1 shows the flow cytometry results of apoptosis level in MH7A cells after treatment with rehmannia glutinosa D. Figure 14 Flow cytometry analysis of apoptosis levels in MH7A cells after treatment with rehmannia glutinosa D. Figure 15 Representative images for measuring the proliferation capacity of MH7A cells after treatment with rehmannia D; Figure 16 Graph showing the quantitative analysis of the proliferation capacity of MH7A cells after treatment with rehmannia glutinosa D; Figure 17 Figure 1 shows the flow cytometry results of mitochondrial membrane potential levels in MH7A cells after treatment with rehmannia glutinosa D. Figure 18 This is a quantitative analysis diagram of the mitochondrial membrane potential level of MH7A cells after treatment with rehmannia D by flow cytometry. Detailed Implementation

[0016] The technical solution of the present invention will be further described below.

[0017] Example 1: In vivo efficacy verification of rehmaglutin D (REHD) in the treatment of rheumatoid arthritis (RA) 1. Construction of animal models of rheumatoid arthritis and drug treatment Eight-week-old female DBA / 1 mice weighing 16-18 g were purchased from the Experimental Animal Center of Nantong University and randomly divided into a normal group, a RA group, and a RA+REHD group, with 10 mice in each group.

[0018] On day 0, mice in the RA and RA+REHD groups were injected intradermally with 0.1 mL of the first immunization solution at the base of the tail. This solution contained an equal volume of complete Freund's adjuvant (CFA, MedChemExpress LLC., catalog number HY-153808) and 2 mg / mL type II collagen (purchased from MedChemExpress LLC., catalog number HY-NP003). Mice in the normal group were injected with an equal volume of 0.01 M glacial acetic acid at the same site. On day 14, mice in the RA and RA+REHD groups were injected with 0.1 mL of the second immunization solution at the same site. This solution contained an equal volume of incomplete Freund's adjuvant (IFA, MedChemExpress LLC., catalog number HY-153808A) and 2 mg / mL type II collagen. Mice in the normal group were injected with an equal volume of 0.01 M glacial acetic acid at the same site.

[0019] Treatment began on day 15, with the drug administered by gavage once daily. Mice in the RA+REHD group were given 20 mg / kg of REHD (purchased from MedChemExpress LLC., catalog number HY-N8586), while the normal group and RA group were given an equal volume of saline. Treatment continued until day 36.

[0020] 2. Monitoring of mouse paw condition 2.1 Foot Shape After the experiment, images of each mouse's paws were collected.

[0021] The results are as follows Figure 1 As shown, compared with normal mice, RA mice had significantly swollen paws, while the swelling of the toes was significantly relieved after REHD treatment.

[0022] 2.2 Changes in the sole of the foot Starting from day 1, the thickness of the mouse's foot sole was measured every 7 days using vernier calipers and the data were statistically analyzed.

[0023] The results are as follows Figure 2 As shown, from day 1 after modeling to the time of drug administration, the thickness of the paw edema in RA mice increased significantly, reaching 6 mm; the thickness of the paw edema in REHD-treated mice decreased significantly with increasing treatment time, showing a significant difference from RA mice, which preliminarily suggests that REHD can be used as a potential treatment for RA.

[0024] Starting from day 1, the foot volume of each mouse was measured every 7 days using a toe volume measuring instrument (purchased from Jiangsu Saiangs Biotechnology Co., Ltd., model SA701).

[0025] The results are as follows Figure 3 As shown, the paw volume of normal mice is 0.1 mL and hardly changes over time; while in RA mice, the paw volume is 0.2 mL on day 1 and reaches 0.42 mL on day 15, which further indicates that the RA model was successfully established; after REHD treatment, the paw volume of mice gradually decreased, and the volume dropped to 0.2 mL on day 36, which was significantly different from that of the RA group.

[0026] 3. Measurement of MMP levels in peripheral blood After the experiment, peripheral blood was collected from mice. Solarbio erythrocyte lysis buffer (product number R1010) was added at a volume ratio of 1:3, and the cells were lysed on ice for 15 min. Then, an equal volume of pre-cooled physiological saline was added and thoroughly mixed. The mixture was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The levels of MMP1, MMP3, MMP9, and MMP13 in the supernatant were detected using the following assay kits: Ellete mouse matrix metalloproteinase 1 (MMP-1) enzyme-linked immunosorbent assay kit (product number E-EL-M0779), Ellete mouse matrix metalloproteinase 3 (MMP-3) enzyme-linked immunosorbent assay kit (product number E-EL-M0626), Ellete mouse matrix metalloproteinase 9 (MMP-9) enzyme-linked immunosorbent assay kit (product number E-EL-M3052), and Ellete mouse matrix metalloproteinase 13 (MMP-13) enzyme-linked immunosorbent assay kit (product number E-EL-M0076).

[0027] The results are as follows Figure 4 As shown, for MMP1, the concentration in normal mice was 506.7 pg / mL, in RA mice it was 1076.1 pg / mL, and in mice treated with REHD it was 730.6 pg / mL; for MMP3, the concentration in normal mice was 406.5 pg / mL, in RA mice it was 910.7 pg / mL, and in mice treated with REHD it was 600.4 pg / mL; for MMP9, the concentration in normal mice was 713.2 pg / mL, in RA mice it was 1500.3 pg / mL, and in mice treated with REHD it was 906.8 pg / mL; for MMP13, the concentration in normal mice was 221.1 pg / mL, in RA mice it was 760.4 pg / mL, and in mice treated with REHD it was 470.7 pg / mL. pg / mL; the above results preliminarily indicate that REHD can effectively reduce the MMP content in RA mice, thereby effectively alleviating the symptoms of RA.

[0028] 4. Biological toxicity assays for REHD treatment After the experiment, liver and kidney tissues were collected from each group of mice. The collected tissues were then supplemented with BeyoLysis at a ratio of 10 mg of tissue to 100 μL. TMBuffer A for Metabolic Assay: After homogenization in an ice bath, centrifuge at 12000 ×g for 5 min at 4°C and collect the supernatant. For liver-derived supernatant, use the Elletet mouse aspartate aminotransferase (AST) enzyme-linked immunosorbent assay kit (catalog number E-EL-M0160) and the Elletet alanine aminotransferase (ALT / GPT) colorimetric assay kit (catalog number E-BC-K235-M). For kidney-derived supernatant, use the Elletet creatinine (Cr) colorimetric assay kit (catalog number E-BC-K188-M) and the Elletet urea (BUN) colorimetric assay kit (catalog number E-BC-K183-M).

[0029] The results are as follows Figure 5 As shown, there were no significant differences in the levels of the above indicators among the three groups of mice, indicating that the REHD dose in the experiment did not cause biological toxicity.

[0030] 5. Histopathological analysis 5.1 H&E staining After the experiment, tibias of mice in each group were collected and fixed in 4% paraformaldehyde solution for 48 h. Then, they were decalcified in Beyotime EDTA decalcification solution (C0167-500ml), with fresh solution changed daily until the tissue softened. After rinsing with running water for 24 h to remove acid, the tissue was dehydrated in a gradient, embedded in paraffin, and cut into 4 μm sections. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (H&E), mounted with neutral resin, and observed and images were acquired under a microscope.

[0031] 5.2 Safranin O-Fast Green Staining The preliminary steps were the same as those for H&E staining. In the staining part, the tibia was stained with Beyotime Safranin O-Fix Green Cartilage Staining Kit (Catalog No. C0621S). After mounting with neutral resin, the tibia was observed and images were acquired under a microscope.

[0032] The results are as follows Figure 6 , 7 As shown, REHD effectively alleviated intra-articular inflammation, cartilage degeneration, and bone resorption in the RA model.

[0033] 6. Foot CT scan Mouse paw tissue collected after the experiment was scanned and analyzed using a micro-CT scanner (Micro-CT, purchased from Pingsheng Medical Technology, model VNC-102) at 55 kV and 144 μA current, with a resolution of 9 μm. Three-dimensional reconstruction was performed after scanning to study changes in microstructure.

[0034] The results are as follows Figure 8As shown, RA mice exhibit bone destruction compared to normal mice, and REHD can alleviate the degree of this bone destruction. Therefore, REHD could be considered a potential drug for the treatment of RA.

[0035] 7. Detection of TNFα and lactate dehydrogenase (LDH) levels in synovial tissue After the experiment, synovial tissue from the knee joint cavity of mice was collected, and an equal volume of pre-cooled physiological saline was added to fully lyse and homogenize the tissue. After centrifugation at 12,000 rpm for 15 min, the supernatant was collected, and the levels of TNFα and LDH in the supernatant were detected using the Elite Mouse Tumor Necrosis Factor α (TNFα) Enzyme-Linked Immunosorbent Assay Kit (Catalog No. E-EL-M3063) and the Elite Lactate Dehydrogenase Cytotoxicity Assay Kit (Catalog No. E-BC-K771-M).

[0036] TNFα detection results are as follows Figure 9 As shown, the TNFα content in normal mice is 73 pg / mgprot, while it is 195 pg / mgprot in RA mice. After REHD treatment, the TNFα level decreased to 106 pg / mgprot, indicating that REHD can significantly reduce the level of the pro-inflammatory cytokine TNFα in RA patients, thereby alleviating RA symptoms.

[0037] LDH test results are as follows Figure 10 As shown, the content in normal mice was 840 U / mgprot, while in RA mice it was 927 U / mgprot. After REHD treatment, it increased to 2550 U / mgprot. This experimental result indicates that REHD can induce a strong cytotoxic effect, significantly inhibit the activity of synovial fibroblasts, and alleviate the symptoms of RA.

[0038] Example 2: In vitro efficacy verification of rehmannia D in the treatment of renal fibrosis 1. Cell viability assay after REHD treatment Human arthritis fibroblasts MH7A (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., product number CL-0747) were administered at a rate of 1×10⁻⁶. 3 Cells were seeded at a density of 1‰ in 96-well plates. After cell adhesion, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or containing a final concentration of 200 ng / mL TNFα (purchased from MedChemExpress LLC., catalog number HY-P7058), or containing a final concentration of 200 ng / mL TNFα and 0, 0.625, 1.25, 2.5, 5, 10, 15, or 20 µM REHD.

[0039] After 24 h of treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the relative level of cell viability was calculated.

[0040] The results are as follows Figure 11 As shown, compared with the DMSO treatment group, the TNFα treatment group showed a 4.2-fold increase in MH7A cell viability. With REHD treatment, TNFα-induced MH7A cell viability significantly decreased in a dose-dependent manner. Furthermore, when REHD reached 10 µM, MH7A cell viability was no longer significantly affected by REHD concentration, so the REHD concentration selected in the later stages was 10 µM.

[0041] 2. Cell scratch assay after REHD treatment MH7A cells were grown at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a specific density in 6-well plates. After cell adhesion, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or a final concentration of 200 ng / mL TNFα, or a final concentration of 200 ng / mL TNFα and 10 µM REHD. After 24 h of treatment, cells were scratched in each well using a 10 µL pipette tip, washed with PBS to remove detached cells, and then cultured in DMEM medium containing 1% FBS for another 24 h. After culture, the healing of cell scratches in each group was observed under an optical microscope and images were acquired.

[0042] The results are as follows Figure 12 As shown, TNFα treatment enhanced the scratch healing ability of cells, while REHD treatment significantly inhibited the migration and healing ability of MH7A cells.

[0043] 3. Determination of apoptosis levels after REHD treatment MH7A cells were grown at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After cell adhesion, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or containing a final concentration of 200 ng / mL TNFα, or containing a final concentration of 200 ng / mL TNFα and 10 µM REHD.

[0044] Cells were collected 24 h after treatment and stained using the Annexin V-FITC / PI apoptosis detection kit (catalog number A211-01). The stained cells were analyzed by flow cytometry to quantify the level of apoptosis.

[0045] The results are as follows Figure 13 , 14As shown, the apoptosis rate of cells in the DMSO group was 9.4%, the apoptosis rate of cells in the TNFα group was 8.2%, while the apoptosis rate of cells after REHD treatment increased to 31.3%, indicating that REHD can effectively induce apoptosis of MH7A cells, thereby alleviating the symptoms of RA.

[0046] 4. Determination of cell proliferation capacity after REHD treatment MH7A cells were grown at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After cell adhesion, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or containing a final concentration of 200 ng / mL TNFα, or containing a final concentration of 200 ng / mL TNFα and 10 µM REHD.

[0047] After 24 h of treatment, 20 μL of EdU reagent (purchased from Novizan, catalog number A413-01) was added to each well, and the cells were incubated at 37 °C for 2 h. After fixation with 4% paraformaldehyde solution at room temperature for 15 min, the cells were permeated with Beyotime immunostaining permeabilization solution (catalog number P0096) at room temperature for 20 min. Then, Click reaction solution (purchased from Novizan, catalog number A413-01) was added, and the cells were incubated in the dark for 30 min. The cells were then mounted with Beyotime anti-fluorescence quenching mounting solution (catalog number P0131). The cells were observed and images were acquired using a fluorescence microscope, and the percentage of red fluorescent cells in each group was analyzed.

[0048] The results are as follows Figure 15 , 16 As shown, compared with the DMSO group, the proportion of EdU-positive cells in the TNFα group did not change significantly, but after REHD treatment, the proportion of EdU-positive cells decreased to 18%, indicating that REHD can significantly inhibit the proliferation of MH7A cells.

[0049] 5. Measurement of mitochondrial membrane potential after REHD treatment MH7A cells were grown at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After cell adhesion, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or containing a final concentration of 200 ng / mL TNFα, or containing a final concentration of 200 ng / mL TNFα and 10 µM REHD.

[0050] Cells were collected 24 h after treatment, washed with pre-cooled PBS, and incubated at 37°C for 30 min in the dark with PBS buffer containing 10 µg / mL JC-1 (purchased from MedChemExpress LLC., catalog number HY-15534). Fluorescence signals were detected by flow cytometry. The excitation wavelength for JC-1 monomer (green fluorescence) was set to 488 nm, and the emission wavelengths were detected at 525 / 50 nm. The emission wavelengths for JC-1 multimer (red fluorescence) were detected at 590 / 40 nm.

[0051] The results are as follows Figure 17 , 18 As shown, in the DMSO group, the mitochondrial membrane potential was high (JC-1 multimer / JC-1 monomer ratio was 5.3), while in the TNFα group, there was no significant change in mitochondrial membrane potential (JC-1 multimer / JC-1 monomer ratio was 5.0). However, after REHD treatment, the mitochondrial membrane potential was significantly reduced (JC-1 multimer / JC-1 monomer ratio was 1.3). This indicates that REHD can significantly induce mitochondrial dysfunction in MH7A cells, thereby inhibiting their cell activity and ultimately alleviating the symptoms of RA.

Claims

1. The application of rehmannia D in the preparation of an arthritis treatment drug.

2. The application according to claim 1, characterized in that, The CAS number of the rehmannia D is 103744-84-9.

3. The application according to claim 1, characterized in that, The arthritis mentioned is rheumatoid arthritis.

4. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit cartilage degeneration caused by arthritis.

5. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit bone destruction caused by arthritis.

6. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit abnormal proliferation of synovial tissue.

7. The application according to claim 1, characterized in that, The drug contains rehmannia D or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

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