WO2021057599A1 - 吡非尼酮在制备防治类风湿性关节炎的药物中的应用 - Google Patents

吡非尼酮在制备防治类风湿性关节炎的药物中的应用 Download PDF

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WO2021057599A1
WO2021057599A1 PCT/CN2020/115952 CN2020115952W WO2021057599A1 WO 2021057599 A1 WO2021057599 A1 WO 2021057599A1 CN 2020115952 W CN2020115952 W CN 2020115952W WO 2021057599 A1 WO2021057599 A1 WO 2021057599A1
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pfd
cells
mmp
preventing
tnf
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French (fr)
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张鹏
甘东浩
成文翔
柯丽青
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the application relates to the technical field of medicine, in particular to the application of pirfenidone in the preparation of a medicine for preventing and treating rheumatoid arthritis, and a medicine for preventing and treating rheumatoid arthritis.
  • Rheumatoid Arthritis is a chronic systemic disease with unknown etiology, mainly synovial hyperplasia, inflammatory cell infiltration, pannus formation, cartilage and bone damage. It is characterized by multiple joints, symmetry, and aggressive joint inflammation of the hand and foot facet joints, often accompanied by involvement of extra-articular organs and positive serum rheumatoid factor, which can lead to joint deformities and loss of function. It is an autoimmune inflammatory disease. In the early stage of the disease, there are symptoms of joint swelling, heat, pain, and dysfunction. When it reaches the late stage, the joints may have different degrees of stiffness and deformity, accompanied by atrophy of bone and skeletal muscle, which is extremely disabling.
  • RA is a type of joint synovium that mainly affects joint cartilage, bone tissue, joint ligaments and muscle bonds, followed by a wide range of connective tissues such as serosa, heart, lung, and eyes. Inflammatory disease. In addition to joint disease, the systemic manifestations of RA include fever, fatigue, pericarditis, subcutaneous nodules, pleurisy, arteritis, and peripheral neuropathy.
  • the treatment of RA mainly uses hormones and anti-rheumatic drugs for anti-inflammatory and immunotherapy, but the existing treatment methods have relatively large adverse reactions and are difficult to cure.
  • the current RA treatment drugs have a single function, which cannot effectively prevent the progression of RA, and the side effects are relatively large. Therefore, looking for drugs with pleiotropic functions and small side effects is of great significance for the treatment of RA.
  • synovial cells are involved in almost all pathological processes of RA, including inflammation and bone destruction, and the aponeurosis is closely related to the progressive destruction of joints, research in recent years has mainly focused on treatment strategies for synovial inflammation and angiogenesis.
  • One of the purposes of the embodiments of the present application is to provide an application of pirfenidone (PFD) in the preparation of drugs for the prevention and treatment of RA, aiming to solve the single function of the current RA treatment drugs, which cannot effectively prevent the progression of RA, and has relatively side effects. Big problem.
  • PFD pirfenidone
  • the second purpose of the embodiments of the present application is to provide a medicine for preventing and treating rheumatoid arthritis.
  • the PFD can prevent and treat RA by inhibiting the angiogenesis of EA.hy926 cells.
  • the PFD can prevent and treat RA by inhibiting the JAK2/STAT3 pathway and the AKT pathway of EA.hy926 cells.
  • the PFD acts by inhibiting the secretion of matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) by EA.hy926 cells.
  • MMP-2 matrix metalloproteinase 2
  • MMP-9 matrix metalloproteinase 9
  • VEGF vascular endothelial growth factor
  • the PFD can prevent and treat RA by inhibiting the angiogenesis of EA.hy926 cells, and the effective concentration of the PFD is 10 ⁇ mol/L-100 ⁇ mol/L.
  • the PFD can prevent and treat RA by inhibiting the phosphorylation of synovial cells.
  • the PFD can prevent and treat RA by inhibiting STAT3 phosphorylation and AKT phosphorylation of human RA fibroblast-like synovial cells (MH7A cells).
  • the PFD plays a role in preventing and treating RA by reducing the increase in inflammatory factors, cartilage destruction factors, and angiogenesis-related factors secreted by MH7A induced by tumor necrosis factor- ⁇ (TNF- ⁇ ).
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the inflammatory factors include interleukin 6 (IL-6), interleukin 8 (IL-8), and interleukin 1 ⁇ (IL-1 ⁇ ).
  • the cartilage destruction factors include matrix metalloproteinase 1 (MMP-1) and matrix metalloproteinase 3 (MMP-3).
  • the angiogenic factors include VEGF, matrix metalloproteinase 2 (MMP-2), and matrix metalloproteinase 9 (MMP-9).
  • the PFD reduces the inflammatory factors IL-6, IL-8, IL-1 ⁇ , MMP-1, MMP-3, MMP-2, MMP-9 and VEGF secreted by MH7A induced by TNF- ⁇ .
  • the increase in the prevention and treatment of RA plays a role.
  • the PFD reduces the inflammatory factors IL-6, IL-8, IL-1 ⁇ , MMP-1, MMP-3, MMP-2, MMP-9, and MMP-9 secreted by MH7A cells induced by TNF- ⁇ .
  • the increase of VEGF plays a role in preventing and treating RA, and the effective concentration of the PFD is 10 ⁇ mol/L-1 mmol/L.
  • the drug is a pharmaceutical composition made with the PFD as an active ingredient and a pharmaceutically acceptable auxiliary agent.
  • the pharmaceutical composition is prepared as a tablet, pill, capsule, granule, powder, liquid, emulsion, suspension, ointment, injection, or skin patch for oral or parenteral use.
  • a medicine for preventing and treating rheumatoid arthritis including PFD, and the chemical structural formula of the PFD is shown in the following formula 1, or is a salt having the structure shown in the following formula 1.
  • pharmaceutically acceptable adjuvants are also included.
  • the dosage form of the drug is one of tablets, pills, capsules, granules, powders, liquids, emulsions, suspensions, ointments, injections, and skin patches.
  • the auxiliary agent includes one or more of fillers, disintegrants, binders, emulsifiers, lubricants, glidants, flavoring agents, odorants, and coloring agents.
  • the beneficial effects of the application of the PFD provided in the examples of this application in the preparation of drugs for the prevention and treatment of RA are: the PFD can effectively inhibit the process of RA and inhibit the formation of EA.hy926 cells by inhibiting the inflammatory response and inhibiting the formation of vascular proliferation and other key pathological links.
  • the beneficial effect of a drug for preventing and treating rheumatoid arthritis is that the PFD is used as the active ingredient, and the PFD can effectively inhibit RA by inhibiting the inflammatory response and inhibiting the formation of pannus and other key pathological links. It inhibits the angiogenesis of EA.hy926 cells, and significantly reduces the expression of inflammatory factors, cartilage destruction factors and angiogenesis-related factors secreted by MH7A cells induced by TNF- ⁇ , thus having a significant inhibitory effect on RA.
  • Figure 1A is a visual diagram of the collagen-induced arthritis (CIA) rat's foot swelling after 21 days of continuous administration provided by Experimental Example 1;
  • CIA collagen-induced arthritis
  • Figure 1B is a broken line graph of the thickness (mm) of the rat's foot measured every 7 days after the successful modelling provided in Experimental Example 1;
  • Figure 2A is a visual diagram of hematoxylin and eosin staining of CIA rat knee joint section provided by Experimental Example 2;
  • Figure 2B is a quantitative diagram of knee synovial inflammation in CIA rats provided by Experimental Example 2;
  • Figure 3A is a visual diagram of toluidine blue staining of the knee joint section of CIA rat provided by Experimental Example 3;
  • Figure 3B is a quantitative diagram of cartilage destruction in the knee joint of a CIA rat provided by Experimental Example 3;
  • Figure 4A is an intuitive view of three-dimensional reconstruction of the knee joint of a CIA rat provided by Experimental Example 4;
  • Figure 4B is the imaging score of subchondral bone in the knee joint of CIA rat provided by Experimental Example 4;
  • 4C is a histogram of bone volume fraction from the subchondral bone of the knee joint of CIA rats to the distal 1.5mm range provided by Experimental Example 4;
  • Figure 5 is a diagram of the percentage of cell survival rate of MH7A cells 72h after PFD intervention by the CCK-8 method provided in Experimental Example 5;
  • Fig. 6A is a statistical diagram of IL-1 ⁇ expression level secreted by MH7A cells stimulated by PFD drug intervention by TNF- ⁇ using enzyme-linked immunosorbent assay (ELISA) method provided by Experimental Example 6;
  • ELISA enzyme-linked immunosorbent assay
  • Fig. 6B is a statistical graph of IL-6 expression levels secreted by MH7A cells stimulated by PFD drugs interfered with TNF- ⁇ by ELISA method provided in Experimental Example 6;
  • Fig. 6C is a statistical graph of IL-8 expression level secreted by MH7A cells stimulated by TNF- ⁇ by PFD drug intervention by ELISA method provided in Experimental Example 6;
  • Fig. 6D is a statistical diagram of VEGF expression secreted by MH7A cells stimulated by TNF- ⁇ by PFD drug intervention by ELISA method provided in Experimental Example 6;
  • Figure 7A is a western blot of PFD intervention TNF- ⁇ stimulated MH7A cells to express matrix metalloproteinase and vascular endothelial growth factor provided by Experimental Example 7;
  • Figure 7B is a semi-quantitative statistical diagram of western blot detection of PFD intervention by TNF- ⁇ to stimulate MMP-1 expression in MH7A cells provided by Experimental Example 7;
  • Figure 7C is a semi-quantitative statistical diagram of western blot detection of PFD intervention by TNF- ⁇ to stimulate MH7A cells to express MMP-3 provided by Experimental Example 7;
  • Fig. 7D is a semi-quantitative statistical diagram of western blot detection of PFD intervention by TNF- ⁇ to stimulate MH7A cells to express MMP-2 provided by Experimental Example 7;
  • Figure 7E is a semi-quantitative statistical diagram of western blot detection of PFD intervention by TNF- ⁇ to stimulate MH7A cells to express MMP-9 provided by Experimental Example 7;
  • Figure 7F is a semi-quantitative statistical diagram of western blot detection of PFD intervention in TNF- ⁇ stimulation of VEGF expression in MH7A cells provided by Experimental Example 7;
  • Fig. 8A is a statistical diagram of experimental results of using QRT-PCR method to detect the relative expression of IL-1 ⁇ at the RNA level in MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD intervention provided by Experimental Example 8;
  • Figure 8B is a statistical diagram of the experimental results of using QRT-PCR method to detect the relative expression of IL-6 at the RNA level of MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD provided by Experimental Example 8;
  • Fig. 8C is a statistical chart of experimental results of using QRT-PCR method to detect the relative expression of IL-8 at the RNA level in MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD, provided in Experimental Example 8;
  • Figure 8D is a statistical diagram of experimental results provided by Experimental Example 8 using QRT-PCR to detect the relative expression of VEGF at the RNA level in MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD;
  • Fig. 8E is a statistical diagram of experimental results provided by Experimental Example 8 using QRT-PCR method to detect the relative expression of MMP-1 at the RNA level in MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD;
  • Fig. 8F is a statistical diagram of the experimental results of using QRT-PCR method to detect the relative expression of MMP-3 at the RNA level of MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD provided by Experimental Example 8;
  • Fig. 8G is a statistical diagram of experimental results of using QRT-PCR method to detect the relative expression of MMP-2 at the RNA level in MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD, provided by Experimental Example 8.
  • Fig. 8H is a statistical diagram of experimental results of using QRT-PCR method to detect the relative expression of MMP-9 at the RNA level in MH7A cells stimulated by TNF- ⁇ with different concentrations of PFD, provided by Experimental Example 8;
  • Figure 9A is a western blot of PFD intervention TNF- ⁇ stimulated MH7A cells to express JAK2, STAT3, AKT, p65 total protein and phosphorylated protein provided by Experimental Example 9;
  • Figure 9B is a semi-quantitative statistical diagram of western blot detection of PFD intervention in TNF- ⁇ stimulated MH7A cells to express JAK2 phosphorylated protein relative to total protein level provided by Experimental Example 9;
  • Figure 9C is a semi-quantitative statistical diagram of western blot detection of PFD intervention in TNF- ⁇ stimulated MH7A cells to express STAT3 phosphorylated protein relative to total protein level provided by Experimental Example 9;
  • Figure 9D is a semi-quantitative statistical diagram of western blot detection of PFD intervention in TNF- ⁇ stimulation of MH7A cells to express AKT phosphorylated protein relative to total protein level provided by Experimental Example 9;
  • Figure 9E is a semi-quantitative statistical diagram of western blot detection of PFD intervention in TNF- ⁇ stimulated MH7A cells to express p65 phosphorylated protein relative to total protein level provided by Experimental Example 9;
  • Figure 10 is a diagram of the percentage of cell survival rate of human umbilical vein cell fusion cells (EA.hy926) 72h after the use of CCK-8 method to detect PFD intervention provided by Experimental Example 10;
  • Figure 11A is an experimental visual diagram of the effect of PFD provided in Experimental Example 11 on the formation of EA.hy926 cell matrigel into a tube;
  • Fig. 11B is a quantitative statistical diagram of the tube network area of EA.hy926 cell matrigel provided by Experimental Example 11;
  • Fig. 11C is a quantitative statistical diagram of PFD on the branch length of EA.hy926 cell matrix glue tube provided by Experimental Example 11;
  • FIG. 12A is a visual diagram of the scratch test used in experimental example 12 to detect the effect of different concentrations of PFD on the recovery of the scratch width of EA.hy926 cells;
  • Figure 12B is a statistical diagram of the relative migration distance of EA.hy926 cells with different concentrations of PFD provided in Experimental Example 12;
  • Figure 13A is a diagram of experimental results provided by Experimental Example 13 using the transwell experiment to detect the effect of different concentrations of PFD on the penetration of EA.hy926 cells;
  • Figure 13B is a statistical diagram of the relative number of penetrating membranes of EA.hy926 cells with different concentrations of PFD provided by Experimental Example 13;
  • Figure 14A is a western blot of PFD intervention EA.hy926 cells expressing matrix metalloproteinase and VEGF provided by Experimental Example 14;
  • Fig. 14B is a semi-quantitative statistical chart of western blot detection of PFD intervening VEGF expression in EA.hy926 cells provided by Experimental Example 14;
  • Figure 14C is a semi-quantitative statistical diagram of western blot detection of PFD intervention in EA.hy926 cell expression MMP-2 provided by Experimental Example 14;
  • Figure 14D is a semi-quantitative statistical diagram of western blot detection of PFD intervention in EA.hy926 cells expressing MMP-9 provided by Experimental Example 14;
  • Figure 15A is a western blot of PFD intervention EA.hy926 cells expressing JAK2, STAT3, AKT total protein and phosphorylated protein provided by Experimental Example 15;
  • Figure 15B is a semi-quantitative statistical diagram of western blot detection of PFD intervention in EA.hy926 cells expressing JAK2 phosphorylated protein relative to total protein level provided by Experimental Example 15;
  • Fig. 15C is a semi-quantitative statistical diagram of western blot detection of PFD intervention in EA.hy926 cells expressing STAT3 phosphorylated protein relative to total protein level provided by Experimental Example 15;
  • Fig. 15D is a semi-quantitative statistical diagram of western blot detection of PFD intervention in EA.hy926 cells expressing AKT phosphorylated protein relative to total protein level provided by Experimental Example 15.
  • M is the abbreviation of the concentration unit "mol/L", for example, ⁇ M means " ⁇ mol/L”.
  • PFD was originally used to treat inflammatory diseases, and later found to have a good effect on fibrotic diseases such as idiopathic pulmonary fibrosis and renal fibrosis, with fewer side effects. Due to its extensive anti-inflammatory, anti-fibrotic and anti-oxidant effects, new indications are constantly being developed. PFD can effectively inhibit the proliferation of fibroblast-like synovial cells in the body, reduce local inflammatory cell infiltration, and reduce collagen deposition. In addition, it can also regulate wound healing and inhibit endothelial cell angiogenesis. In view of this,
  • the first aspect of the embodiments of the present application provides an application of PFD in the preparation of drugs for preventing and treating RA.
  • the chemical structural formula of PFD is shown in the following formula 1, or is a salt of the structure shown in the following formula 1.
  • PFD effectively inhibits the process of RA by inhibiting key pathological links such as inflammatory response and inhibiting vascular proliferation.
  • PFD can prevent and treat RA by inhibiting the angiogenesis of EA.hy926 cells.
  • the effect of preventing and treating RA can be achieved.
  • PFD can prevent and treat RA by inhibiting JAK2/STAT3 pathway and AKT pathway in EA.hy926 cells.
  • PFD can prevent and treat RA by inhibiting the secretion of MMP-2, MMP-9 and VEGF from EA.hy926 cells.
  • PFD can prevent and treat RA by inhibiting the angiogenesis of EA.hy926 cells, and the effective concentration of PFD is 10 ⁇ mol/L-100 ⁇ mol/L.
  • the effective concentration of PFD is 10 ⁇ mol/L, 20 ⁇ mol/L, 30 ⁇ mol/L, 40 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 80 ⁇ mol/L, 100 ⁇ mol/L, etc. Concentration, but not limited to this.
  • PFD can prevent and treat RA by inhibiting the phosphorylation of synovial cells.
  • PFD inhibits STAT3 phosphorylation and AKT phosphorylation of MH7A, significantly reduces the increase in inflammatory factors and angiogenesis, and plays a role in preventing and treating RA.
  • PFD plays a role in preventing and treating RA by reducing the increase in TNF- ⁇ -induced inflammatory factors, cartilage destruction factors, and angiogenesis-related factors secreted by MH7A.
  • PFD can reduce the swelling of RA joints by inhibiting inflammatory factors, reduce the destruction of articular cartilage by inhibiting cartilage destruction factors, prevent and treat RA, and inhibit the formation of joint synovial pannus by reducing angiogenesis-related factors, and play a role in preventing and treating RA. effect. In the end, through the triple action, it plays an effective role in preventing RA.
  • the causes of inflammation include IL-6, IL-8, and IL-1 ⁇ .
  • PFD inhibits TNF- ⁇ -induced MH7A cells to secrete inflammatory factors IL-6, IL-8, IL-1 ⁇ , reduces joint swelling, and plays a role in preventing and treating RA.
  • cartilage destruction factors include MMP-1 and MMP-3.
  • PFD inhibits the secretion of MMP-1 and MMP-3 by MH7A cells induced by TNF- ⁇ , reduces the destruction of articular cartilage, and plays a role in preventing and treating RA.
  • angiogenesis-related factors include VEGF, MMP-2, and MMP-9.
  • PFD can prevent and treat RA by inhibiting the secretion of VEGF, MMP-2 and MMP-9 in MH7A cells induced by TNF- ⁇ , inhibiting the formation of pannus in the joint synovium.
  • PFD reduces the increase in IL-6, IL-8, IL-1 ⁇ , MMP-1, MMP-3, MMP-2, MMP-9, and VEGF secreted by MH7A cells induced by TNF- ⁇ Play the role of prevention and treatment of RA.
  • PFD reduces the increase in IL-6, IL-8, IL-1 ⁇ , MMP-1, MMP-3, MMP-2, MMP-9, and VEGF secreted by MH7A cells induced by TNF- ⁇ Play the role of prevention and treatment of RA, and the effect of PFD in prevention and treatment of RA is concentration-dependent.
  • the effective concentration of PFD is 10 ⁇ mol/L-1 mmol/L.
  • the effective concentration of PFD is 10 ⁇ mol/L, 20 ⁇ mol/L, 50 ⁇ mol/L, 80 ⁇ mol/L, 100 ⁇ mol/L, 200 ⁇ mol/L, 250 ⁇ mol/L, 500 ⁇ mol/L, 800 ⁇ mol/L, 1 mmol/L, etc. Concentration, but not limited to this.
  • the drug when PFD is used in the preparation of a drug for preventing and treating RA, the drug is a pharmaceutical composition made with PFD as an active ingredient and a pharmaceutically acceptable auxiliary agent. That is to say, it can be understood as a drug for preventing and treating RA, including active ingredients and academically acceptable adjuvants, where the active ingredient is PFD.
  • the dosage form of the pharmaceutical composition can be flexibly set according to the mode of administration, and can be used as tablets, pills, capsules, granules, powders, and liquids. , Emulsion, suspension, ointment, injection, skin patch for oral or non-oral drug preparation.
  • PFD can effectively inhibit the process of RA and inhibit the angiogenesis of EA.hy926 cells by inhibiting the inflammatory response and inhibiting the formation of pannus and other key pathological links.
  • PFD can effectively inhibit the process of RA and inhibit the angiogenesis of EA.hy926 cells by inhibiting the inflammatory response and inhibiting the formation of pannus and other key pathological links.
  • PFD can effectively reduce the expression of inflammatory factors, cartilage destruction factors and angiogenesis-related factors secreted by MH7A cells induced by TNF- ⁇ , thus having a significant inhibitory effect on RA.
  • the embodiments of the present application provide a medicine for preventing and treating rheumatoid arthritis, including PFD, and the chemical structural formula of PFD is shown in the following formula 1, or is a salt having the structure shown in the following formula 1.
  • the PFD shown in Formula 1 above functions as an active ingredient for preventing and treating rheumatoid arthritis.
  • the PFD inhibits the phosphorylation of STAT3 and AKT, significantly reduces downstream inflammatory signaling pathways and vascular pathways, and jointly plays the role of preventing and treating RA.
  • PFD inhibits the secretion of MMP-2, MMP-9 and VEGF by EA.hy926 cells, inhibits angiogenesis, and plays a role in preventing and treating RA;
  • PFD inhibits the secretion of MH7A cells induced by TNF- ⁇ Inflammation factors IL-6, IL-8, IL-1 ⁇ , cartilage destruction factors MMP-1, MMP-3, and angiogenesis-related factors VEGF, MMP-2, MMP-9, slow down the swelling of RA joints and reduce the destruction of articular cartilage, Inhibit the formation of synovial pannus in joints, and play the role of preventing and treating RA together.
  • the drug for the prevention and treatment of rheumatoid arthritis in addition to the PFD or the salt of the PFD, also includes a pharmaceutically acceptable adjuvant to facilitate its preparation to meet various requirements.
  • the dosage form of the drug route in addition to the PFD or the salt of the PFD, also includes a pharmaceutically acceptable adjuvant to facilitate its preparation to meet various requirements.
  • the dosage form of the drug is one of tablets, pills, capsules, granules, powders, liquids, emulsions, suspensions, ointments, injections, and skin patches.
  • the auxiliary agent includes one or more of fillers, disintegrants, binders, emulsifiers, lubricants, glidants, flavors, odorants, and colorants, But it is not limited to this.
  • the beneficial effect of a medicine for preventing and treating rheumatoid arthritis is that the PFD is used as the active ingredient, and the PFD is effective by inhibiting the synovial inflammation and inhibiting the formation of pannus. Inhibit the process of RA, significantly inhibit the angiogenesis pathway of EA.hy926 cells, and significantly reduce the expression of inflammatory factors, cartilage destruction factors and angiogenesis-related factors secreted by MH7A cells induced by TNF- ⁇ , thus having a significant inhibitory effect on RA .
  • 24 male Lewis rats (8 weeks old) were raised in a pathogen-free environment.
  • the 24 rats were randomly divided into 4 groups (6 rats/group): the normal group (Normal, the first group) and 3 CIA model groups (the second to the fourth group).
  • the second group of mice were given intragastric carboxymethylcellulose sodium suspension at 10 ml/kg/day (CIA), and the third group of mice were given methotrexate as a positive control drug, 0.1 mg/kg /3 days intraperitoneal injection (CIA-MTX), the fourth group of rats were given intragastric PFD at 500 mg/kg/day (CIA-PFD). After 21 days of continuous administration, the rats were euthanized and the lower limbs were taken. After confirming that the model was successfully established, the thickness of the hind paw of the rat was measured every 7 days.
  • Figure 1A is a visual photograph of the foot swelling of a CIA rat after 21 days of continuous administration (the abscissa indicates the group);
  • Figure 1B is provided by Experimental Example 1: Rat-made After the modeling is successful, the line graph of the thickness of the rat's foot is measured every 7 days (where the abscissa represents the time of starting the modeling, and the ordinate represents the thickness of the foot (mm)). It can be seen from the figure that compared with the normal group, the feet of CIA rats were significantly swollen, and the swelling of the feet was reduced after methotrexate (MTX) and PFD treatment (P ⁇ 0.05).
  • MTX methotrexate
  • PFD treatment P ⁇ 0.05
  • the right knee joint was fixed with 4% paraformaldehyde, decalcified with 10% EDTA for 40 days, and then subjected to routine dehydration, paraffin embedding, and 5- ⁇ m sectioning. Then the sections were stained with hematoxylin and eosin (H&E): paraffin sections of the knee joint were conventionally deparaffinized and rehydrated with xylene and gradient alcohol, and then stained with hematoxylin staining solution for 7 minutes. After washing the excess staining solution, use it again Yin Hong was stained for 1 minute, then dehydrated with gradient alcohol, transparent with xylene, then mounted with neutral gum, and finally the slices were photographed and analyzed.
  • H&E hematoxylin and eosin
  • Figure 2A is an intuitive diagram of hematoxylin and eosin staining of CIA rat knee joint section provided by Experimental Example 2 (the abscissa indicates the group; * indicates P ⁇ 0.05); 2B is a quantitative map of knee synovial inflammation in CIA rats provided by Experimental Example 2 (wherein, the abscissa represents the group, and the ordinate represents the knee synovial inflammation score). It can be seen from the figure that compared with the normal group, CIA rats have obvious synovial hyperplasia and inflammatory cell infiltration, while PFD can significantly reduce knee synovial hyperplasia and inflammatory cell infiltration (P ⁇ 0.05).
  • Toluidine blue (TB) staining After the rats in Example 1 were euthanized, paraffin sections of the knee joints were dewaxed and rehydrated with xylene and gradient alcohol, and then stained with toluidine blue solution for 10 minutes to wash away the excess dye solution , Gradient alcohol dehydration, transparent xylene, neutral gum mounts, observe and take pictures under a microscope and analyze the degree of articular cartilage erosion.
  • Figure 3A is a visual image of toluidine blue staining of the knee joint section of CIA rats provided in Experimental Example 3 (the abscissa indicates the group; * indicates P ⁇ 0.05);
  • Figure 3B is the experiment Example 3 provides a quantitative map of cartilage destruction in the knee joint of CIA rats (where the abscissa represents the group, and the ordinate represents the cartilage destruction score). It can be seen from the figure that compared with the normal group, the articular cartilage of CIA rats is destroyed and the gap is narrowed, while PFD can significantly reduce the destruction of knee cartilage (P ⁇ 0.05).
  • Microcomputer tomography After the rats were euthanized, the knee joint was taken, and the right knee joint was fixed with 4% paraformaldehyde for 1 day, and the bone tissue of the knee joint was scanned by Micro-CT (SkyScan1176).
  • the scanning parameters are: the x-ray tube voltage is 65kV, the tube current is 385 ⁇ A, and the pixel size is 12.63 ⁇ m.
  • the scanned images were reconstructed in three dimensions and evaluated and analyzed.
  • Figure 4A is an intuitive three-dimensional reconstruction of the knee joint of a CIA rat provided by Experimental Example 4 (the abscissa indicates the group; * indicates P ⁇ 0.05);
  • Figure 4B is the experimental example 4 provided CIA rat knee joint subchondral bone imaging score (where the abscissa represents the group, the ordinate represents the bone destruction imaging score);
  • Figure 4C is the CIA rat knee joint subchondral bone to the experimental example 4 Bone volume fraction histogram in the distal 1.5mm range (where the abscissa represents the group and the ordinate represents the bone volume fraction).
  • the CIA rat has erosion of subchondral bone, joint destruction, BV/TV value and bone mass decrease, while PFD can significantly reduce knee joint subchondral bone damage and increase BV/TV ratio , Increase bone mass (P ⁇ 0.05).
  • the CCK-8 method is used to detect the activity of PFD on MH7A.
  • Fig. 5 is the percentage graph of cell survival rate of MH7A cells 72h after PFD intervention by the CCK-8 method provided in Experimental Example 5.
  • the abscissa indicates the PFD concentration of different groups, and the ordinate indicates the cell survival rate of each group relative to the blank group; * indicates P ⁇ 0.05). It can be seen from the figure that in the concentration range of 100 ⁇ mol/L-1mmol/L, PFD has no obvious inhibition on the proliferation activity of MH7A cells, while PFD at a concentration of 2mmol/L obviously inhibits the proliferation activity of MH7A cells. Therefore, 100 ⁇ mol/L-1mmol is selected /L concentration of PFD for follow-up experiments.
  • One line represents different groups of TNF- ⁇ added at a concentration of 20ng/ml (-: no addition, +: addition); the second line of abscissa represents the PFD concentration of different groups; the ordinate represents the concentration of cytokine expression in the culture medium;* Means P ⁇ 0.05, ** means P ⁇ 0.01):
  • Figure 6A is the expression of IL-1 ⁇ ;
  • Figure 6B is the expression of IL-6;
  • Figure 6C is the expression of IL-8;
  • Figure 6D is the expression of VEGF.
  • the experimental results show that PFD can significantly reduce the increase of IL-6, IL-8, IL-1 ⁇ and other inflammatory factors secreted by MH7A cells induced by TNF- ⁇ , and significantly reduce the expression of VEGF secreted by MH7A cells induced by TNF- ⁇ Abnormally elevated and concentration-dependent, that is, within the safe concentration range of 100 ⁇ mol/L-1mmol/L, the higher the concentration, the stronger the inhibitory effect.
  • anti-MMP-1 CST, Cat.no.54376S
  • anti-MMP-2 CST, Cat.no.40994S
  • anti-MMP-3 Abcam, Cat.no.ab53015
  • anti-MMP-9 Proteintech, Cat.no.10375-2-AP
  • anti-VEGF Abcam, Cat.no.ab46154
  • Fig. 7A is the western blot of PFD intervention TNF- ⁇ stimulated MH7A cells to express matrix metalloproteinase and VEGF provided in Experimental Example 7.
  • the first row of the abscissa represents the addition of 20ng/ml TNF- ⁇ in different groups (-: no addition, +: addition); the second row of the abscissa represents the PFD concentration of different groups; the ordinate represents each display The target protein corresponding to the color band);
  • FIGS. 7F are the western blots provided in Experimental Example 7 to detect PFD intervention in TNF- ⁇ to stimulate the expression of matrix metalloproteinases and angiogenic factors in MH7A cells
  • Semi-quantitative statistical graph (wherein the first row of the abscissa represents the addition of TNF- ⁇ at a concentration of 20ng/ml in different groups (-: no addition, +: addition); the second row of the abscissa represents the PFD concentration of different groups; the ordinate Indicates the target protein expression ratio of each group relative to the blank group; * means P ⁇ 0.05, ** means P ⁇ 0.01), where Figure 7B is the relative expression statistics of MMP-1; Figure 7C is the relative expression statistics of MMP-3 Figure; Figure 7D is a statistical diagram of the relative expression of MMP-2; Figure 7E is a statistical diagram of the relative expression of MMP-9; Figure 7F is a statistical diagram of the relative expression of VEGF.
  • TNF- ⁇ stimulated MH7A cells to increase the expression of IL1, IL6, IL8, VEGF, MMP-1, MMP-2, MMP-3 and MMP-9, while PFD significantly and dose-dependently reduced the above The expression level of cytokines.
  • Figure 8 is a statistical diagram of the experimental results of using QRT-PCR method to detect the cytokine expression at the RNA level of MH7A cells stimulated by PFD with TNF- ⁇ provided by Experimental Example 8 (wherein the first row of the abscissa indicates that different groups add 20ng /ml concentration of TNF- ⁇ (-: no addition, +: addition); the second line of the abscissa indicates the PFD concentration of different groups; the ordinate indicates the expression of cytokine in the culture medium relative to the blank group; * indicates P ⁇ 0.05, **Indicating P ⁇ 0.01): Figure 8A is the relative expression of IL-1 ⁇ ; Figure 8B is the relative expression of IL-6; Figure 8C is the relative expression of IL-8; Figure 8D is the relative expression of VEGF Figure 8E is a statistical diagram of relative expression of MMP-1; Figure 8F is a statistical diagram of relative expression of MMP-3; Figure 8G is a statistical diagram of relative expression of MMP-2; Figure 8H is a relative expression of
  • Anti-p-JAK2 CST, Cat.no.3771S
  • anti-JAK2 CST, Cat.no.3230S
  • anti-p-STAT3 CST, Cat.no.9145S
  • anti-STAT3 CST, Cat.no.9139S
  • anti-p-p65 CST, Cat.no.3033S
  • anti-p65 CST, Cat.no.4764S
  • anti-p-AKT CST, Cat.no.9275S
  • Anti-AKT Abcam, Cat.no.ab32505)
  • antibody detects the expression of JAK2, STAT3, AKT, p65 total protein and phosphorylated protein in cells after TNF- ⁇ stimulation for 0.5h.
  • Figure 9A is the western example of PFD intervention TNF- ⁇ stimulated MH7A cells to express JAK2, STAT3, AKT, p65 total protein and phosphorylated protein provided in Experimental Example 9.
  • the CCK-8 method was used to detect the proliferation activity of PFD intervention EA.hy926 cells
  • EA.hy926 cells with good growth status configure 3000 cells/90 ⁇ l cell suspension in a 96-well plate, place in an incubator for pre-culture (37°C, 5% CO 2 ), set up five experimental groups and a blank control Group, each group has 3 multiple wells. After 24 hours, PFD with drug concentration of 10 ⁇ mol/L, 25 ⁇ mol/L, 50 ⁇ mol/L, 100 ⁇ mol/L and 200 ⁇ mol/L was added to the five experimental groups respectively. The blank control group did not add PFD. . After 72h, add 10 ⁇ l of CCK-8 reagent to each well. Use a microplate reader to detect the absorbance of each well at 450nm within 30min-2h. Repeat twice, subject to the agreement of the experimental results.
  • Fig. 10 is the percentage graph of cell survival rate of EA.hy926 cells 72h after PFD intervention by the CCK-8 method provided in Experimental Example 10.
  • the abscissa represents the PFD concentration of different groups, the ordinate represents the relative cell survival rate of the blank group; ** represents P ⁇ 0.01)
  • PFD with different concentration gradients is used to interfere with the proliferation activity of EA.hy926 cells.
  • the experimental results show: In the concentration range of ⁇ 100 ⁇ mol/L, PFD has no significant inhibition on the proliferation activity of EA.hy926 cells, while PFD at a concentration of 200 ⁇ mol/L significantly inhibits the proliferation activity of EA.hy926 cells. Therefore, ⁇ 100 ⁇ mol/L is selected as PFD vs. EA.
  • the safe concentration range of hy926 cells is selected as PFD vs. EA.
  • the safe concentration range of hy926 cells is selected as PFD vs. EA.
  • Figure 11A is an experimental graph of the effect of PFD on the formation of EA.hy926 cell matrigel tubes provided in Experimental Example 11 (the abscissa represents the PFD concentration);
  • Figure 11B is the experiment Example 11 provides a quantitative statistical diagram of the network area of EA.hy926 cell matrigel (where the abscissa represents the PFD concentration, and the ordinate represents the ratio of the network area of each group to the blank group; * means P ⁇ 0.05, ** means P ⁇ 0.01);
  • Figure 11C is a quantitative statistical diagram of PFD on the branch length of EA.hy926 cell matrigel tube provided by Experimental Example 11 (where the abscissa represents the PFD concentration, and the ordinate represents the ratio of the branch length of each component tube to the blank group; * represents P ⁇ 0.05, ** means P ⁇ 0.01).
  • the scratch test was used to detect the effect of PFD on the migration of EA.hy926 cells
  • EA.hy926 cells Take an equal amount of well-growing EA.hy926 cells into a 6-well plate and culture them in an incubator (37°C, 5% CO 2 ). After the cells are 90% confluent, use a 200 ⁇ l pipette tip to make a vertical scratch. The cell debris was cleaned and cultured in serum-free medium, and then PFD at concentrations of 0, 50 ⁇ mol/L, and 100 ⁇ mol/L were added for intervention. After 24 hours, the effect of PFD on scratch recovery was observed under the microscope.
  • Figure 12A is an intuitive diagram provided by Experimental Example 12 using the scratch test to detect the effect of different concentrations of PFD on the recovery of the scratch width of EA.hy926 cells (the abscissa represents the PFD concentration);
  • Figure 12B It is the relative migration distance statistics chart of different concentrations of PFD intervention EA.hy926 cells provided by Experimental Example 12 (the abscissa represents the PFD concentration, and the ordinate represents the ratio of the recovery width of each group of cells to the blank group; * means P ⁇ 0.05, ** Indicates P ⁇ 0.01). It can be seen from the figure that PFD at a concentration of 100 ⁇ mol/L significantly inhibits scratch recovery, indicating that PFD at a concentration of 100 ⁇ mol/L can significantly inhibit the migration of EA.hy926 cells.
  • EA.hy926 cells Take an equal amount of well-growing EA.hy926 cells and inoculate them into the upper chamber of Transwell, and add PFD at a concentration of 50 ⁇ mol/L and 100 ⁇ mol/L to the lower chamber.
  • the upper chamber uses serum-free medium and the lower chamber contains 2%.
  • the culture medium of fetal calf serum after incubating for 4 hours, take out the chamber and wipe the cells on the membrane with a cotton swab, fix with 4% paraformaldehyde and stain with crystal violet, observe under the microscope and statistically analyze the number of cells passing through the membrane.
  • Figure 13A is an experimental graph of the transwell experiment to detect the effect of different concentrations of PFD on EA.hy926 cell penetration provided by Experimental Example 13 (the abscissa represents the PFD concentration);
  • Figure 13B is the experiment Example 13 provides different concentrations of PFD intervention EA.hy926 cell relative penetrating number statistics PFD (the abscissa represents the PFD concentration, the ordinate represents the ratio of the number of cells penetrating the membrane in each group relative to the blank group; * means P ⁇ 0.05, ** means P ⁇ 0.01).
  • Anti-MMP-2 CST, Cat.no.40994S
  • anti-MMP-9 Proteintech, Cat.no.10375-2-AP
  • anti-VEGF Abcam, Cat.no.ab46154
  • Figure 14A is the western blot of PFD intervention EA.hy926 cells expressing matrix metalloproteinases (MMP-2, MMP-9) and VEGF provided in Experimental Example 14.
  • MMP-2 matrix metalloproteinases
  • MMP-9 matrix metalloproteinases
  • PFD significantly reduces the expression of VEGF, MMP-2 and MMP-9 proteins, which is consistent with the observed formation and migration of inhibitory tubes. It is suggested that PFD can inhibit the matrigel formation and migration of EA.hy926 cells by down-regulating the expression of angiogenic factors.
  • the Western blot method was used to detect the total protein and phosphorylated protein levels of JAK2, STAT3 and AKT in PFD intervention EA.hy926 cells:
  • Anti-p-JAK2 CST, Cat.no.3771S
  • anti-JAK2 CST, Cat.no.3230S
  • anti-p-STAT3 CST, Cat.no.9145S
  • anti-STAT3 CST, Cat.no.9139S
  • anti-p-AKT CST, Cat.no.9275S
  • Anti-AKT Abcam, Cat.no.ab32505
  • Figure 15A is the western blot of PFD intervention EA.hy926 cells expressing JAK2, STAT3, AKT total protein and phosphorylated protein provided by Experimental Example 15.
  • the abscissa represents the PFD concentration of different groups; the ordinate represents the target protein corresponding to each color band);
  • Figure 15B, Figure 15C, and Figure 15D are the western blot detection of PFD intervention EA.hy926 cells provided by Experimental Example 15.
  • FIG. 15 A semi-quantitative statistical graph showing the relative total protein levels of phosphorylated proteins expressing JAK2, STAT3, and AKT (where the abscissa represents the PFD concentration of different groups; the ordinate represents the target protein expression ratio of each group to the blank group; * represents P ⁇ 0.05 , ** means P ⁇ 0.01), where Figure 15B is a statistical graph of JAK2 phosphorylated protein relative to total protein expression; Figure 15C is a statistical graph of STAT3 phosphorylated protein relative to total protein expression; Figure 15D is a relative expression of AKT phosphorylated protein Statistical graph of total protein level expression.
  • PFD can significantly alleviate the pathological changes in the joints of CIA rats.
  • PFD can significantly inhibit the secretion of inflammatory factors, matrix metalloproteinases and VEGF in MH7A cells in a dose-dependent manner.
  • it can also significantly inhibit EA.hy926 Cell formation and migration.
  • an intragastric dose of PFD 500 mg/kg/day can significantly alleviate pathological changes such as joint swelling, synovial hyperplasia, inflammatory cell infiltration, and joint destruction in CIA rats.
  • PFD can effectively reduce the expression of VEGF, IL-6, IL-8, IL-1 ⁇ , MMP-1, MMP-3 secreted by TNF- ⁇ stimulated MH7A cells within the safe concentration range of 100 ⁇ mol/L-1mmol/L , And is concentration-dependent.
  • PFD has significant anti-EA.hy926 cell matrix gel-forming function at 50 ⁇ mol/L and 100 ⁇ mol/L concentration, which may be because PFD inhibits the expression of VEGF, MMP-2 and MMP-9 in EA.hy926 cells.
  • PFD PFD at a concentration of 100 ⁇ mol/L significantly inhibits scratch recovery; PFD at 50 ⁇ mol/L and 100 ⁇ mol/L can significantly reduce the number of EA.hy926 cells passing through the membrane, and the higher the concentration, the less the number of passing through the membrane. This may be because PFD inhibited the expression of MMP-2 and MMP-9 in EA.hy926 cells.

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Abstract

一种PFD在制备防治RA的药物中的应用，所述PFD的化学结构式如下式1所示，或者为下式1所示结构的盐。所述PFD通过抑制炎症反应和抑制血管增生等关键病理环节可有效抑制RA进程，抑制EA.hy926细胞的成血管作用，并显著降低TNF-α诱导MH7A细胞分泌的炎症因子、软骨破坏因子和成血管相关因子表达量升高，从而对RA具有明显的抑制作用。

Description

吡非尼酮在制备防治类风湿性关节炎的药物中的应用 技术领域
本申请涉及医药技术领域，具体涉及一种吡非尼酮在制备防治类风湿性关节炎的药物中的应用，以及一种用于防治类风湿关节炎的药物。
背景技术
类风湿性关节炎(Rheumatoid Arthritis，RA)是一种病因未明的慢性、以滑膜增生、炎性细胞浸润、血管翳形成、软骨和骨性损伤为主的系统性疾病。其特征在于手、足小关节的多关节、对称性、侵袭性关节炎症，经常伴有关节外器官受累及血清类风湿因子阳性，可以导致关节畸形及功能丧失，属于自身免疫炎性疾病。患病早期有关节红肿热痛和功能障碍表现，到达晚期时，关节可出现不同程度的僵硬畸形，并伴有骨和骨骼肌的萎缩，极易致残。从病理改变的角度来看，RA是一种主要累及关节滑膜(以后可波及到关节软骨、骨组织、关节韧带和肌键)，其次为浆膜、心、肺及眼等结缔组织的广泛性炎症性疾病。RA的全身性表现除关节病变外，还有发热、疲乏无力、心包炎、皮下结节、胸膜炎、动脉炎、周围神经病变等。
RA的治疗方法主要是采用激素和抗风湿缓解病情药物进行抗炎和免疫治疗，但是现有治疗方法的不良反应较大且难以根治。此外，目前RA治疗药物功能单一，并不能有效阻止RA进展，且副作用较大。因此，寻找具有多效功能且副作用小的药物，对于RA的治疗具有重要意义。由于滑膜细胞参与RA几乎所有的病理过程，包括炎症和骨破坏，而肌腱膜与关节的渐进性破坏密切相关，因此近年来的研究主要集中在针对滑膜炎症和血管生成的治疗策略上。与开发新药相比，老药新用可降低开发风险，对难治性疾病的治疗具有明显优势。例如基于蛋白质相互作用，已经对几种治疗COVID-19的临床药物进行了评估验证，从而避免了费时和昂贵的新药物开发。
技术问题
本申请实施例的目的之一在于：提供一种吡非尼酮(PFD)在制备防治RA的药物中的应用，旨在解决目前RA治疗药物功能单一，并不能有效阻止RA进展，且副作用较大的问题。
本申请实施例的目的之二在于：提供一种用于防治类风湿关节炎的药物。
技术解决方案
为解决上述技术问题，本申请实施例采用的技术方案是：
第一方面，提供了一种PFD在制备防治RA的药物中的应用，所述PFD的化学结构式如下式1所示，或者为下式1所示结构的盐
Figure PCTCN2020115952-appb-000001
在一些实施例中，所述PFD通过抑制EA.hy926细胞成血管作用发挥防治RA的作用。
在一些实施例中，所述PFD通过抑制EA.hy926细胞JAK2/STAT3通路和AKT通路发挥防治RA的作用。
在一些实施例中，所述PFD通过抑制EA.hy926细胞分泌基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)和血管内皮生长因子(vascular endothelial growth factor，VEGF)，发挥防治RA的作用。
在一些实施例中，所述PFD通过抑制EA.hy926细胞成血管作用发挥防治RA的作用，且所述PFD的有效浓度为10μmol/L-100μmol/L。
在一些实施例中，所述PFD通过抑制滑膜细胞的磷酸化发挥防治RA的作用。
在一些实施例中，所述PFD通过抑制人RA成纤维样滑膜细胞(MH7A细胞)的STAT3磷酸化和AKT磷酸化发挥防治RA的作用。
在一些实施例中，所述PFD通过降低肿瘤坏死因子α(TNF-α)诱导的MH7A分泌的炎症因子、软骨破坏因子和成血管相关因子的升高，发挥防治RA的作用。
在一些实施例中，所述炎症因包括白介素6(IL-6)、白介素8(IL-8)、白介素1β(IL-1β)。在一些实施例中，所述软骨破坏因子包括基质金属蛋白酶1(MMP-1)和基质金属蛋白酶3(MMP-3)。在一些实施例中，所述成血管因子包括VEGF、基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)。
在一些实施例中，所述PFD通过降低TNF-α诱导的MH7A分泌的炎症因子IL-6、IL-8、IL-1β、MMP-1、MMP-3、MMP-2、MMP-9和VEGF的升高发挥防治RA的作用。
在一些实施例中，所述PFD通过降低TNF-α诱导的MH7A细胞分泌的炎症因子IL-6、IL-8、IL-1β、MMP-1、MMP-3、MMP-2、MMP-9和VEGF的升高发挥防治RA的作用，且所述PFD的有效浓度为10μmol/L-1mmol/L。
在一些实施例中，所述药物为以所述PFD作为活性成分，与药学上可接受的辅助剂制成的药物组合物。
在一些实施例中，所述药物组合物作为片剂、丸剂、胶囊剂、颗粒剂、粉 剂、液剂、乳剂、悬浮剂、软膏剂、注射剂、皮肤贴剂的口服或非口服用药物制备。
第二方面，提供了一种用于防治类风湿关节炎的药物，包括PFD，且所述PFD的化学结构式如下式1所示，或者为下式1所示结构的盐
Figure PCTCN2020115952-appb-000002
在一些实施例中，还包括药学上可接受的辅助剂。
在一些实施例中，所述药物的剂型为片剂、丸剂、胶囊剂、颗粒剂、粉剂、液剂、乳剂、悬浮剂、软膏剂、注射剂、皮肤贴剂中的一种。
在一些实施例中，所述辅助剂包括填充剂、崩解剂、粘合剂、乳化剂、润滑剂、助流剂、矫味剂、矫嗅剂、着色剂中的一种或几种。
本申请实施例提供的PFD在制备防治RA的药物中的应用的有益效果在于：所述PFD通过抑制炎症反应和抑制血管增生形成等关键病理环节可有效抑制RA进程，抑制EA.hy926细胞的成血管作用，并显著降低TNF-α诱导MH7A细胞分泌的炎症因子、软骨破坏因子和成血管相关因子表达量升高，从而对RA具有明显的抑制作用。
本申请实施例提供的一种用于防治类风湿关节炎的药物的有益效果在于：以所述PFD作为活性成分，所述PFD通过抑制炎症反应和抑制血管翳形成等关键病理环节可有效抑制RA进程，抑制EA.hy926细胞的成血管作用，并显著降低TNF-α诱导MH7A细胞分泌的炎症因子、软骨破坏因子和成血管相关因子表达量升高，从而对RA具有明显的抑制作用。
附图说明
图1A是实验例1提供的胶原诱导关节炎(CIA)大鼠连续给药21天后足部肿胀的直观图；
图1B是实验例1提供的造模成功后的大鼠，每隔7天测量的大鼠足部厚度(毫米)的折线图；
图2A是实验例2提供的CIA大鼠膝关节切片苏木精、伊红染色直观图；
图2B是实验例2提供的CIA大鼠膝关节滑膜炎症量化图；
图3A是实验例3提供的CIA大鼠膝关节切片甲苯胺蓝染色直观图；
图3B是实验例3提供的CIA大鼠膝关节软骨破坏量化图；
图4A是实验例4提供的CIA大鼠膝关节三维重建直观图；
图4B是实验例4提供的CIA大鼠膝关节软骨下骨影像学评分；
图4C是实验例4提供的CIA大鼠膝关节软骨下骨至远端1.5mm范围的的骨体积分数柱状图；
图5是实验例5提供的采用CCK-8法检测PFD干预MH7A细胞72h后的细胞存活率百分比图；
图6A是实验例6提供的采用酶联免疫吸附(ELISA)法检测PFD药物干预TNF-α刺激MH7A细胞所分泌的IL-1β表达量统计图；
图6B是实验例6提供的采用ELISA法检测PFD药物干预TNF-α刺激MH7A细胞所分泌的IL-6表达量统计图；
图6C是实验例6提供的采用ELISA法检测PFD药物干预TNF-α刺激MH7A细胞所分泌的IL-8表达量统计图；
图6D是实验例6提供的采用ELISA法检测PFD药物干预TNF-α刺激MH7A细胞所分泌的VEGF表达量统计图；
图7A是实验例7提供的PFD干预TNF-α刺激MH7A细胞表达基质金属蛋白酶和血管内皮生长因子的western blot显色图；
图7B是实验例7提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达MMP-1的半定量统计图；
图7C是实验例7提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达MMP-3的半定量统计图；
图7D是实验例7提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达MMP-2的半定量统计图；
图7E是实验例7提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达MMP-9的半定量统计图；
图7F是实验例7提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达VEGF的半定量统计图；
图8A是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α刺激的MH7A细胞在RNA水平上IL-1β相对表达量的实验结果统计图；
图8B是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α刺激的MH7A细胞在RNA水平上IL-6相对表达量的实验结果统计图；
图8C是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α刺激的MH7A细胞在RNA水平上IL-8相对表达量的实验结果统计图；
图8D是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α刺激的MH7A细胞在RNA水平上VEGF相对表达量的实验结果统计图；
图8E是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α刺激的MH7A细胞在RNA水平上MMP-1相对表达量的实验结果统计图；
图8F是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α 刺激的MH7A细胞在RNA水平上MMP-3相对表达量的实验结果统计图；
图8G是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α刺激的MH7A细胞在RNA水平上MMP-2相对表达量的实验结果统计图；
图8H是实验例8提供的采用QRT-PCR法检测不同浓度PFD干预TNF-α刺激的MH7A细胞在RNA水平上MMP-9相对表达量的实验结果统计图；
图9A是实验例9提供的PFD干预TNF-α刺激MH7A细胞表达JAK2、STAT3、AKT、p65总蛋白和磷酸化蛋白的western blot显色图；
图9B是实验例9提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达JAK2磷酸化蛋白相对总蛋白水平的半定量统计图；
图9C是实验例9提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达STAT3磷酸化蛋白相对总蛋白水平的半定量统计图；
图9D是实验例9提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达AKT磷酸化蛋白相对总蛋白水平的半定量统计图；
图9E是实验例9提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达p65磷酸化蛋白相对总蛋白水平的半定量统计图；
图10是实验例10提供的采用CCK-8法检测PFD干预人脐静脉细胞融合细胞(EA.hy926)72h后的细胞存活率百分比图；
图11A是实验例11提供的PFD对EA.hy926细胞基质胶成管影响的实验直观图；
图11B是实验例11提供EA.hy926细胞基质胶成管网状面积的定量统计图；
图11C是实验例11提供的PFD对EA.hy926细胞基质胶成管分支长度的定量统计图；
图12A是实验例12提供的采用划痕实验检测不同浓度PFD影响EA.hy926细胞划痕宽度恢复的直观图；
图12B是实验例12提供的不同浓度PFD干预EA.hy926细胞的相对迁移距离统计图；
图13A是实验例13提供的采用transwell实验检测不同浓度PFD对EA.hy926细胞穿膜影响的实验结果图；
图13B是实验例13提供的不同浓度PFD干预EA.hy926细胞相对穿膜数量统计图；
图14A是实验例14提供的PFD干预EA.hy926细胞表达基质金属蛋白酶和VEGF的western blot显色图；
图14B是实验例14提供的western blot检测PFD干预EA.hy926细胞表达VEGF的半定量统计图；
图14C是实验例14提供的western blot检测PFD干预EA.hy926细胞表达 MMP-2的半定量统计图；
图14D是实验例14提供的western blot检测PFD干预EA.hy926细胞表达MMP-9的半定量统计图；
图15A是实验例15提供的PFD干预EA.hy926细胞表达JAK2、STAT3、AKT总蛋白和磷酸化蛋白的western blot显色图；
图15B是实验例15提供的western blot检测PFD干预EA.hy926细胞表达JAK2磷酸化蛋白相对总蛋白水平的半定量统计图；
图15C是实验例15提供的western blot检测PFD干预EA.hy926细胞表达STAT3磷酸化蛋白相对总蛋白水平的半定量统计图；
图15D是实验例15提供的western blot检测PFD干预EA.hy926细胞表达AKT磷酸化蛋白相对总蛋白水平的半定量统计图。
附图中，M为浓度单位“mol/L”的缩写，如：μM表示“μmol/L”。
本发明的实施方式
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。
PFD最初用于治疗炎症性疾病，后来发现对特发性肺纤维化和肾纤维化等纤维化性疾病有很好的疗效，且副作用较小。由于其广泛的抗炎、抗纤维化和抗氧化作用，新的适应症不断被开发。PFD在体内中可有效抑制成纤维样滑膜细胞增殖，减轻局部炎症细胞浸润，减少胶原沉积。此外，它还可以调节伤口愈合，抑制内皮细胞血管生成。有鉴于此，
本申请实施例第一方面提供了一种PFD在制备防治RA的药物中的应用，PFD的化学结构式如下式1所示，或者为下式1所示结构的盐
Figure PCTCN2020115952-appb-000003
本申请实施例中，PFD通过抑制炎症反应和抑制血管增生等关键病理环节，有效抑制RA进程。
在一种可能的实施方式中，PFD通过抑制EA.hy926细胞成血管作用发挥防治RA的作用。通过直接抑制靶细胞-EA.hy926细胞的成血管作用，可以达到预防和治疗RA的作用。
在一些实施例中，PFD通过抑制EA.hy926细胞JAK2/STAT3通路和AKT 通路发挥防治RA的作用。示例性的，PFD通过抑制EA.hy926细胞分泌MMP-2、MMP-9和VEGF，发挥防治RA的作用。
在一些实施例中，PFD通过抑制EA.hy926细胞成血管作用发挥防治RA的作用，且PFD的有效浓度为10μmol/L-100μmol/L。示例性的，PFD的有效浓度为10μmol/L、20μmol/L、30μmol/L、40μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、80μmol/L、100μmol/L等具体浓度，但不限于此。
在一种可能的实施方式中，PFD通过抑制滑膜细胞的磷酸化发挥防治RA的作用。
在一些实施例中，PFD通过抑制MH7A的STAT3磷酸化和AKT磷酸化，显著减低炎症因子升高和成血管作用，发挥防治RA的作用。
在一些实施例中，在PFD通过降低TNF-α诱导的MH7A分泌的炎症因子、软骨破坏因子和成血管相关因子的升高，发挥防治RA的作用。在这种情况下，PFD可以通过抑制炎症因子减缓RA关节肿胀，通过抑制软骨破坏因子减轻关节软骨破坏，预防和治疗RA，通过降低成血管相关因子抑制关节滑膜血管翳形成，发挥防治RA的作用。最终，通过三重作用，发挥有效的防止RA的作用。
在一些实施例中，炎症因包括IL-6、IL-8和IL-1β。在这种情况下，PFD通过抑制TNF-α诱导的MH7A细胞分泌炎症因子IL-6、IL-8、IL-1β，降低关节肿胀，发挥防治RA的作用。
在一些实施例中，软骨破坏因子包括MMP-1和MMP-3。在这种情况下，PFD通过抑制TNF-α诱导的MH7A细胞分泌MMP-1、MMP-3，减轻关节软骨破坏，发挥防治RA的作用。
在一些实施例中，成血管相关因子包括VEGF、MMP-2和MMP-9。在这种情况下，PFD通过抑制TNF-α诱导的MH7A细胞分泌VEGF、MMP-2和MMP-9，抑制关节滑膜血管翳形成，发挥防治RA的作用。
在一些实施例中，PFD通过降低TNF-α诱导的MH7A细胞分泌的IL-6、IL-8、IL-1β、MMP-1、MMP-3、MMP-2、MMP-9和VEGF的升高发挥防治RA的作用。
在一些实施例中，PFD通过降低TNF-α诱导的MH7A细胞分泌的IL-6、IL-8、IL-1β、MMP-1、MMP-3、MMP-2、MMP-9和VEGF的升高发挥防治RA的作用，且PFD防治RA的效果呈浓度依赖性。具体的，PFD的有效浓度为10μmol/L-1mmol/L。在10μmol/L-1mmol/L安全浓度范围内，浓度越高，PFD抑制TNF-α诱导的MH7A细胞分泌IL-6、IL-8、IL-1β、MMP-1、MMP-3、MMP-2、MMP-9和VEGF的作用越强。示例性的，PFD的有效浓度为10μmol/L、20μmol/L、50μmol/L、80μmol/L、100μmol/L、200μmol/L、250μmol/L、500μmol/L、800μmol/L、1mmol/L等具体浓度，但不限于此。
在一些实施例中，PFD在制备防治RA的药物中的应用时，药物为以PFD作为活性成分，与药学上可接受的辅助剂制成的药物组合物。即可以理解为防治RA的药物，包括活性成分和要学上可接受的辅助剂，其中，活性成分为PFD。
在一些实施例中，PFD在制备防治RA的药物中的应用时，所述药物组合物的剂型可以根据给药方式灵活设置，可以作为片剂、丸剂、胶囊剂、颗粒剂、粉剂、液剂、乳剂、悬浮剂、软膏剂、注射剂、皮肤贴剂的口服或非口服用药物制备。
本申请实施例提供的PFD在制备防治RA的药物中的应用的有益效果在于：PFD通过抑制炎症反应和抑制血管翳形成等关键病理环节可有效抑制RA进程，抑制EA.hy926细胞的成血管作用，并显著降低TNF-α诱导MH7A细胞分泌的炎症因子、软骨破坏因子和成血管相关因子表达量升高，从而对RA具有明显的抑制作用。
第二方面，本申请实施例提供了一种用于防治类风湿关节炎的药物，包括PFD，且PFD的化学结构式如下式1所示，或者为下式1所示结构的盐
Figure PCTCN2020115952-appb-000004
用于防治类风湿关节炎的药物中，上式1所示的PFD，或者为上式1所示的PFD的盐，作为防治类风湿关节炎的活性成分发挥作用。
此时，所述PFD通过抑制STAT3和AKT的磷酸化，显著减低下游炎症信号通路和成血管通路，共同发挥防治RA的作用RA。具体的，一方面，PFD通过抑制EA.hy926细胞分泌MMP-2、MMP-9和VEGF，抑制成血管作用，发挥防治RA的作用；另一方面，PFD通过抑制TNF-α诱导的MH7A细胞分泌炎症因子IL-6、IL-8、IL-1β，软骨破坏因子MMP-1、MMP-3，以及成血管相关因子VEGF、MMP-2、MMP-9，减缓RA关节肿胀、减轻关节软骨破坏，抑制关节滑膜血管翳形成，共同发挥防治RA的作用。
在一些实施例中，所述用于防治类风湿关节炎的药物，除了包括所述PFD或所述PFD的盐以外，还包括药学上可接受的辅助剂，以方便其制备成满足各种给药途径的剂型。
在一些实施例中，所述药物的剂型为片剂、丸剂、胶囊剂、颗粒剂、粉剂、液剂、乳剂、悬浮剂、软膏剂、注射剂、皮肤贴剂中的一种。
在一些实施例中，所述辅助剂包括填充剂、崩解剂、粘合剂、乳化剂、润滑剂、助流剂、矫味剂、矫嗅剂、着色剂中的一种或几种，但不限于此。
本申请实施例提供的一种用于防治类风湿关节炎的药物的有益效果在于：以所述PFD作为活性成分，所述PFD通过抑制滑膜炎症反应和抑制血管翳形成等关键病理环节可有效抑制RA进程，显抑制EA.hy926细胞的成血管通路，并显著降低TNF-α诱导MH7A细胞分泌的炎症因子、软骨破坏因子和成血管相关因子表达量升高，从而对RA具有明显的抑制作用。
为了说明本申请所述的技术方案，以下结合具体附图及实施例进行详细说明。
实验例1
测量分析PFD对CIA大鼠的足部肿胀的改善作用
雄性Lewis大鼠(8周龄)24只，在无病原体环境中饲养。将24只大鼠随机分为4组(6只/组)：正常组(Normal，第一组)和3个CIA模型组(第二组至第四组)。首先建立CIA大鼠模型：用玻璃注射器将等量牛II型胶原和不完全佐剂混匀，在大鼠尾巴距离根部2cm处皮下注射200μl混悬液，7天后，再按上述方法，每只老鼠尾根部注射100μl混悬液，强化免疫反应，再经14天后确定造模成功。CIA模型建立成功后，第二组老鼠灌胃羧甲基纤维素钠混悬液，10毫升/千克/天(CIA)，第三组老鼠给予甲氨蝶呤作为阳性对照药，0.1毫克/千克/3天腹腔注射(CIA-MTX)，第四组大鼠灌胃PFD，500毫克/千克/天(CIA-PFD)，连续给药21天后对大鼠安乐死并进行下肢取材。确定造模成功后每隔7天测量大鼠后爪厚度。
结果如图1A、图1B所示，其中，图1A为CIA大鼠连续给药21天后足部肿胀的直观照片(横坐标表示组别)；图1B为是实验例1提供的：大鼠造模成功后，每隔7天测量大鼠足部厚度的折线图(其中，横坐标表示开始造模时间，纵坐标表示足部厚度(毫米))。由图可见：与正常组相比，CIA大鼠足部明显肿胀，经甲氨蝶呤(MTX)和PFD治疗后足肿胀减轻(P<0.05)。
实验例2
采用苏木精、伊红染色检测PFD对CIA大鼠的关节滑膜炎症的作用
实施例1的大鼠安乐死后，用4％多聚甲醛固定右侧膝关节，10％EDTA脱钙40天，依次经过常规脱水、石蜡包埋、5-μm切片。然后对切片进行苏木精、伊红(H&E)染色：膝关节石蜡切片使用二甲苯和梯度酒精经常规脱蜡、复水后，使用苏木素染色液染色7分钟，冲洗多余染液后，再使用尹红染色1分钟，然后使用梯度酒精脱水、二甲苯透明、再进行中性树胶封片，最后对切片进行拍照分析。关节滑膜炎症评分标准为：0＝无炎症，1＝滑膜衬里层略增厚或有炎性细胞浸润，2＝滑膜衬里层略增厚伴有炎性细胞浸润，3＝滑膜衬里层增厚，滑膜间隙炎性细胞浸润，4＝滑膜炎性细胞严重浸润。
结果如图2A、图2B所示，其中，图2A是实验例2提供的CIA大鼠膝关节切片苏木精、伊红染色直观图(横坐标表示组别；*表示P<0.05)；图2B是实验例2提供的CIA大鼠膝关节滑膜炎症量化图(其中，横坐标表示组别，纵坐标表示膝关节滑膜炎症评分)。由图可见：与正常组相比，CIA大鼠滑膜增生和炎性细胞浸润明显，而PFD可显著减轻膝关节滑膜增生和炎性细胞浸润(P<0.05)。
实验例3
采用甲苯胺蓝染色分析PFD对CIA大鼠的关节软骨破坏的影响
甲苯胺蓝(TB)染色：实施例1的大鼠安乐死后，将膝关节石蜡切片使用二甲苯和梯度酒精经常规脱蜡、复水后，使用甲苯胺蓝液染色10min，洗去多余染液，梯度酒精脱水，二甲苯透明，中性树胶封片，显微镜下观察拍照并分析关节软骨侵蚀程度，其中关节软骨损伤评价标准为：0＝无破坏，1＝点状破坏，2＝局部轻度至中度破坏，3＝较大范围破坏，4＝广泛破坏。
结果如图3A、图3B所示，其中，图3A是实验例3提供的CIA大鼠膝关节切片甲苯胺蓝染色直观图(横坐标表示组别；*表示P<0.05)；图3B是实验例3提供的CIA大鼠膝关节软骨破坏量化图(其中，横坐标表示组别，纵坐标表示软骨破坏评分)。由图可见：与正常组相比，CIA大鼠关节软骨破坏、间隙变窄，而PFD可显著减轻膝关节软骨破坏(P<0.05)。
实验例4
采用微计算机断层扫描技术分析PFD对CIA大鼠关节骨破坏的影响
微计算机断层扫描：对大鼠安乐死后，将膝关节取材，经4％多聚甲醛固定右膝关节1天，使用Micro-CT(SkyScan1176)扫描膝关节部位骨组织。扫描参数为：x射线管电压为65kV，管电流为385μA，像素尺寸为12.63μm。将扫描后的图像进行三维重建并评估分析，其中关节损伤评价标准为:0＝无损伤，1＝轻度，2＝中度，3＝重度。再选取从胫骨软骨下骨至远端1.5mm的切片，分析BV/TV比值，评估关节软骨下骨组织结构状态。
结果如图4A、图4B、图4C所示，其中图4A是实验例4提供的CIA大鼠膝关节三维重建直观图(横坐标表示组别；*表示P<0.05)；图4B是实验例4提供的CIA大鼠膝关节软骨下骨影像学评分(其中，横坐标表示组别，纵坐标表示骨破坏影像学评分)；图4C是实验例4提供的CIA大鼠膝关节软骨下骨至远端1.5mm范围的的骨体积分数柱状图(其中，横坐标表示组别，纵坐标表示骨体积分数)。由图可见：与正常组相比，CIA大鼠关节软骨下骨侵蚀，关节破坏明显，BV/TV值降低，骨量减少，而PFD可显著减轻膝关节软骨下骨损伤，提高BV/TV比值，增加骨量(P<0.05)。
实验例5
采用CCK-8法检测PFD对MH7A的活性实验测试
选生长状态良好的MH7A细胞，计数后在96孔板中每孔种植3000个/90μl，于37℃、5％CO 2浓度的培养箱中培养，设置五个实验组和一个空白对照组，每组设3个复孔，24h后在五个实验组中分别添加药物浓度为100μmol/L、250μmol/L、500μmol/L、1mmol/L和2mmol/L的PFD，空白对照组不添加。72h后每孔加10μl CCK-8液，30min-2h内使用酶标仪于450nm处检测各孔吸光度，重复两次，以实验结果吻合为准。
结果如图5是实验例5提供的采用CCK-8法检测PFD干预MH7A细胞72h后的细胞存活率百分比图。(横坐标表示不同组别PFD浓度，纵坐标表示各组相对空白组细胞存活率；*表示P<0.05)。由图可见：在100μmol/L-1mmol/L浓度范围内，PFD对MH7A细胞增殖活性无明显抑制，而2mmol/L浓度的PFD明显抑制MH7A细胞的增殖活性，因此，选定100μmol/L-1mmol/L浓度的PFD进行后续实验。
实验例6
采用ELISA法检测PFD药物干预TNF-α刺激MH7A细胞所分泌的炎症和成血管因子
实验分别选用0、100μmol/L、250μmol/L、500μmol/L、1mmol/L浓度的PFD干预MH7A细胞2h，再用TNF-α刺激24h后，吸取细胞培养液于离心机中3000转/分钟，4℃、10分钟的条件下离心提取细胞培养上清液，再分别用IL-6(R&D Systems，cat.D6050)、IL-8(R&D Systems，cat.D8000C)、IL-1β(R&D Systems，cat.DLB50)和VEGF(R&D Systems，cat.DVE00)等试剂盒检测细胞培养上清液中炎症因子和VEGF表达。
结果如图6A、图6B、图6C、图6D是实验例6提供的采用ELISA法检测PFD药物干预TNF-α刺激MH7A细胞所分泌的炎症和成血管因子表达量统计图(其中，横坐标第一行表示不同组别添加20ng/ml浓度的TNF-α(-：无添加，+：添加)；横坐标第二行表示不同组别PFD浓度；纵坐标表示培养液中细胞因子表达浓度；*表示P<0.05，**表示P<0.01)：其中图6A是IL-1β表达量；图6B是IL-6表达量；图6C是IL-8表达量；图6D是VEGF表达量。由图可见，实验结果表明：PFD可显著降低TNF-α诱导MH7A细胞分泌的IL-6、IL-8、IL-1β等炎症因子升高，显著降低TNF-α诱导MH7A细胞分泌的VEGF表达量异常升高，且呈浓度依赖性，即在100μmol/L-1mmol/L安全浓度范围内，浓度越高，抑制作用越强。
实验例7
采用Western blot法检测PFD药物干预TNF-α刺激MH7A细胞表达基质金属蛋白酶和成血管因子水平
取等量生长状态良好的细胞铺种6孔板，于37℃、5％CO 2浓度的培养箱中培养，待细胞80％融合后，换液，然后分别选用0、100μmol/L、250μmol/L、 500μmol/L、1mmol/L梯度的PFD干预MH7A细胞2h，再用20ng/ml浓度的TNF-α刺激24h，然后提取细胞内总蛋白，依次通过电泳，转膜，免疫封闭，一抗、二抗孵育等一系列操作得到具有目的蛋白的膜，进行显色反应，
使用抗MMP-1(CST,Cat.no.54376S)、抗MMP-2(CST,Cat.no.40994S)、抗MMP-3(Abcam,Cat.no.ab53015)、抗MMP-9(Proteintech,Cat.no.10375-2-AP)、抗VEGF(Abcam,Cat.no.ab46154)抗体检测TNF-α刺激24h后细胞中基质金属蛋白酶因子和VEGF表达水平。
如图7A、图7B、图7C、图7D、图7E、图7F所示，其中，图7A是实验例7提供的PFD干预TNF-α刺激MH7A细胞表达基质金属蛋白酶和VEGF的western blot显色图(其中，横坐标第一行表示不同组别添加20ng/ml浓度的TNF-α(-：无添加，+：添加)；横坐标第二行表示不同组别PFD浓度；纵坐标表示各显色条带所对应的目标蛋白)；图7B、图7C、图7D、图7E、图7F是实验例7提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达基质金属蛋白酶和成血管因子的半定量统计图(其中，横坐标第一行表示不同组别添加20ng/ml浓度的TNF-α(-：无添加，+：添加)；横坐标第二行表示不同组别PFD浓度；纵坐标表示各组别相对空白组的目标蛋白表达比值；*表示P<0.05，**表示P<0.01)，其中图7B是MMP-1相对表达量统计图；图7C是MMP-3相对表达量统计图；图7D是MMP-2相对表达量统计图；图7E是MMP-9相对表达量统计图；图7F是VEGF相对表达量统计图。由图可见，TNF-α刺激MH7A细胞增加了IL1、IL6、IL8、VEGF、MMP-1、MMP-2、MMP-3和MMP-9的表达，而PFD显著且呈剂量依赖性地降低了上述细胞因子的表达水平。
实验例8
采用QRT-PCR法检测PFD药物干预TNF-α刺激MH7A细胞表达细胞因子水平
取等量生长状态良好的细胞铺种6孔板，于37℃、5％CO 2浓度的条件下培养，待细胞80％融合后，换液，然后分别选用0、100μmol/L、250μmol/L、500μmol/L、1mmol/L梯度的PFD干预MH7A细胞2h，再用20ng/ml浓度的TNF-α刺激6h，冰上提取RNA，测量浓度，按Takara试剂盒(Code No.RR047A)说明书去除qRNA，再通过逆转录反应得到cDNA，最后通过qPCR(实时荧光定量PCR)进行扩增，得到循环数(Cq)，根据目的基因与内参GAPDH的ΔCq值，以及后续的2 -ΔΔCq计算相对表达量，从而得到实验组与对照组之间目的基因表达量的差异。
如图8是实验例8提供的采用QRT-PCR法检测PFD干预TNF-α刺激MH7A细胞在RNA水平的细胞因子表达量的实验结果统计图(其中，横坐标第一行表示不同组别添加20ng/ml浓度的TNF-α(-：无添加，+：添加)；横 坐标第二行表示不同组别PFD浓度；纵坐标表示培养液中细胞因子相对空白组表达量；*表示P<0.05，**表示P<0.01)：其中图8A是IL-1β相对表达量统计图；图8B是IL-6相对表达量统计图；图8C是IL-8相对表达量统计图；图8D是VEGF相对表达量统计图；图8E是MMP-1相对表达量统计图；图8F是MMP-3相对表达量统计图；图8G是MMP-2相对表达量统计图；图8H是MMP-9相对表达量统计图。由图可见，TNF-α刺激MH7A细胞增加了IL1、IL6、IL8、VEGF、MMP-1、MMP-2、MMP-3和MMP-9的产生，而PFD显著且呈剂量依赖性地降低了上述细胞因子的表达水平，且mRNA和蛋白表达水平变化趋势一致，说明PFD在转录水平上调控细胞因子的表达。
实验例9
采用Western blot法检测PFD药物干预TNF-α刺激MH7A细胞表达JAK2、STAT3、AKT、p65总蛋白和磷酸化蛋白水平
取等量生长状态良好的细胞铺种6孔板，于37℃、5％CO 2浓度的培养箱中培养，待细胞80％融合后，换液，然后分别选用0、100μmol/L、250μmol/L、500μmol/L、1mmol/L梯度的PFD干预MH7A细胞2h，再用20ng/ml浓度的TNF-α刺激0.5h，然后提取细胞内总蛋白，依次通过电泳，转膜，免疫封闭，一抗、二抗孵育等一系列操作得到具有目的蛋白的膜，进行显色反应。
使用一抗(Anti-p-JAK2(CST,Cat.no.3771S)、anti-JAK2(CST,Cat.no.3230S)、anti-p-STAT3(CST,Cat.no.9145S)、anti-STAT3(CST,Cat.no.9139S)、anti-p-p65(CST,Cat.no.3033S)anti-p65(CST,Cat.no.4764S)、anti-p-AKT(CST,Cat.no.9275S)and Anti-AKT(Abcam,Cat.no.ab32505))抗体检测TNF-α刺激0.5h后细胞中JAK2、STAT3、AKT、p65总蛋白和磷酸化蛋白的表达量。
结果如图9A、图9B、图9C、图9D所示，其中，图9A是实验例9提供的PFD干预TNF-α刺激MH7A细胞表达JAK2、STAT3、AKT、p65总蛋白和磷酸化蛋白的western blot显色图(其中，横坐标第一行表示不同组别添加20ng/ml浓度的TNF-α(-：无添加，+：添加)；横坐标第二行表示不同组别PFD浓度；横坐标第三行表示不同组别添加10ng/ml浓度的转化生长因子-β(-：无添加，+：添加)；纵坐标表示各显色条带所对应的目标蛋白)；图9B、图9C、图9D、图9E是实验例9提供的western blot检测PFD干预TNF-α刺激MH7A细胞表达JAK2、STAT3、AKT、p65总蛋白和磷酸化蛋白水平的半定量统计图(其中，横坐标第一行表示不同组别添加20ng/ml浓度的TNF-α(-：无添加，+：添加)；横坐标第二行表示不同组别PFD浓度；横坐标第三行表示不同组别添加10ng/ml浓度的转化生长因子-β(-：无添加，+：添加)；纵坐标表示各组别相对空白组的目标蛋白表达比值；*表示P<0.05，**表示P<0.01)，其中图9B是JAK2磷酸化蛋白相对总蛋白水平表达量统计图；图9C是STAT3磷酸化蛋白相对总蛋白水平表达量统计图；图9D是AKT磷酸化蛋 白相对总蛋白水平表达量统计图；图9E是p65磷酸化蛋白相对总蛋白水平表达量统计图。由图可见，经TNF-α刺激后，MH7A细胞中AKT和p65的磷酸化水平显著升高。PFD处理可下调磷酸化STAT3、AKT和p65的表达。而经TGF-β干预后，STAT3、AKT、p65磷酸化表达水平有效恢复(图9C-9E)。这些结果表明：PFD可能通过抑制TGF-β靶点，抑制下游STAT3,AKT-NF-κB介导的炎症信号通路。
实验例10
采用CCK-8法检测PFD干预EA.hy926细胞的增殖活性
选生长状态良好的EA.hy926细胞，在96孔板中配置3000个/90μl的细胞悬液，放置培养箱中预培养(37℃，5％CO 2)，设置五个实验组和一个空白对照组，每组设3个复孔，24h后在五个实验组中分别添加药物浓度为10μmol/L、25μmol/L、50μmol/L、100μmol/L和200μmol/L的PFD，空白对照组不添加。72h后每孔加10μl CCK-8试剂，30min-2h内使用酶标仪于450nm处检测各孔吸光度，重复两次，以实验结果吻合为准。
结果如图10是实验例10提供的采用CCK-8法检测PFD干预EA.hy926细胞72h后的细胞存活率百分比图。(横坐标表示不同组别PFD浓度，纵坐标表示相对空白组细胞存活率；**表示P<0.01)由图可见：用不同浓度梯度的PFD干预EA.hy926细胞的增殖活性，实验结果表明：在≤100μmol/L浓度范围内，PFD对EA.hy926细胞增殖活性无明显抑制，而200μmol/L浓度的PFD明显抑制EA.hy926细胞的增殖活性，因此选定≤100μmol/L为PFD对EA.hy926细胞的安全浓度范围。
实验例11
采用基质胶成管实验检测PFD对EA.hy926细胞成管的影响
在24孔板中铺基质胶，30分钟后接种等量细胞悬液，实验分别选用0、50μmol/L、100μmol/L浓度的PFD干预EA.hy926细胞，6-8小时后镜下观察其对成管的干预作用。
结果如图11A、图11B、图11C所示，其中，图11A是实验例11提供的PFD对EA.hy926细胞基质胶成管影响的实验直观图(横坐标表示PFD浓度)；图11B是实验例11提供EA.hy926细胞基质胶成管网状面积的定量统计图(其中横坐标表示PFD浓度，纵坐标表示各组网状面积相对空白组比值；*表示P<0.05，**表示P<0.01)；图11C是实验例11提供的PFD对EA.hy926细胞基质胶成管分支长度的定量统计图(其中横坐标表示PFD浓度，纵坐标表示各组成管分支长度相对空白组比值；*表示P<0.05，**表示P<0.01)。由图可见：在50μmol/L和100μmol/L浓度的PFD显著抑制EA.hy926细胞的成管作用，且浓度越高，PFD抑制EA.hy926细胞的成管作用越明显。
实验例12
采用划痕实验检测PFD对EA.hy926细胞迁移的影响
取等量生长状态良好的EA.hy926细胞铺种6孔板，于培养箱中培养(37℃、5％CO 2)，待细胞90％融合后，使用200μl移液枪头进行垂直划痕，清洗干净细胞碎片并使用无血清培养基进行培养，然后分别添加0、50μmol/L、100μmol/L浓度的PFD进行干预，24小时后镜下观察PFD对划痕恢复的影响。
结果如图12A、图12B所示，其中，图12A是实验例12提供的采用划痕实验检测不同浓度PFD影响EA.hy926细胞划痕宽度恢复的直观图(横坐标表示PFD浓度)；图12B是实验例12提供的不同浓度PFD干预EA.hy926细胞的相对迁移距离统计图(横坐标表示PFD浓度，纵坐标表示各组细胞划痕恢复宽度相对空白组比值；*表示P<0.05，**表示P<0.01)。由图可见所示100μmol/L浓度的PFD显著抑制划痕恢复表明100μmol/L浓度的PFD显可著抑制EA.hy926细胞的迁移作用。
实验例13
采用transwell实验检测PFD对EA.hy926细胞迁移的影响
取等量生长状态良好的EA.hy926细胞接种到Transwell的上室中，取50μmol/L和100μmol/L浓度的PFD添加到下室，其中上室使用无血清培养基，下室使用含2％胎牛血清的培养基，孵育4小时后，取出小室用棉签拭净膜上面细胞后，使用4％多聚甲醛固定并使用结晶紫染色，显微镜下观察并统计分析细胞过膜数量。
如图13A、图13B所示，其中，图13A是实验例13提供的采用transwell实验检测不同浓度PFD对EA.hy926细胞穿膜影响的实验直观图(横坐标表示PFD浓度)；图13B是实验例13提供的不同浓度PFD干预EA.hy926细胞相对穿膜数量统计图PFD(横坐标表示PFD浓度，纵坐标表示各组细胞穿膜数量相对空白组比值；*表示P<0.05，**表示P<0.01)。
由图可见：50μmol/L和100μmol/L浓度的PFD可显著减少EA.hy926细胞过膜数量，且浓度越高，过膜数量越少，表明PFD可显著抑制EA.hy926细胞的迁移作用。
实验例14
采用Western blot法检测PFD药物干预EA.hy926细胞表达基质金属蛋白酶和成血管因子水平
取等量生长状态良好的细胞铺种6孔板，于37℃、5％CO 2浓度的培养箱中培养，待细胞80％融合后，换液，然后分别选用0、50μmol/L、100μmol/L浓度的PFD干预EA.hy926细胞24h，然后提取细胞内总蛋白，依次通过电泳，转膜，免疫封闭，一抗、二抗孵育等一系列操作得到具有目的蛋白的膜，进行显色反应。
使用抗MMP-2(CST,Cat.no.40994S)、抗 MMP-9(Proteintech,Cat.no.10375-2-AP)、抗VEGF(Abcam,Cat.no.ab46154)抗体检测PFD干预EA.hy926细胞24h后基质金属蛋白酶因子和VEGF表达水平。
结果如图14A、图14B、图14C、图14D所示，其中，图14A是实验例14提供的PFD干预EA.hy926细胞表达基质金属蛋白酶(MMP-2、MMP-9)和VEGF的western blot显色图(其中，横坐标表示不同组别PFD浓度；纵坐标表示各显色条带所对应的目标蛋白)；图14B、图14C、图14D是实验例14提供的western blot检测PFD干预EA.hy926细胞表达基质金属蛋白酶和VEGF的半定量统计图(其中，横坐表示不同组别PFD浓度；纵坐标表示各组别相对空白组的目标蛋白表达比值；*表示P<0.05，**表示P<0.01)，其中图14B是VEGF相对表达量统计图；图14C是MMP-2相对表达量统计图；图14D是MMP-9相对表达量统计图。由图可见，PFD在显著降低了VEGF、MMP-2和MMP-9蛋白的表达，与观察到的抑制管的形成和迁移相一致。提示PFD可通过下调血管生成因子的表达抑制EA.hy926细胞的基质胶成管和迁移。
实验例15
采用Western blot法检测PFD干预EA.hy926细胞表达JAK2、STAT3和AKT总蛋白和磷酸化蛋白水平：
取等量生长状态良好的细胞铺种6孔板，于培养箱中培养(37℃、5％CO 2)至细胞80％融合后，换液，然后分别选用0、50μmol/L、100μmol/L浓度的PFD干预EA.hy926细胞0.5h，然后提取细胞内总蛋白，依次通过电泳，转膜，免疫封闭，一抗、二抗孵育等一系列操作得到具有目的蛋白的膜，进行显色反应。
使用一抗(Anti-p-JAK2(CST,Cat.no.3771S)、anti-JAK2(CST,Cat.no.3230S)、anti-p-STAT3(CST,Cat.no.9145S)、anti-STAT3(CST,Cat.no.9139S)、anti-p-AKT(CST,Cat.no.9275S)and Anti-AKT(Abcam,Cat.no.ab32505))抗体检测PFD干预EA.hy926细胞0.5h后JAK2、STAT3、AKT总蛋白和磷酸化蛋白的表达量。
结果如图15A、图15B、图15C、图15D所示，其中，图15A是实验例15提供的PFD干预EA.hy926细胞表达JAK2、STAT3、AKT总蛋白和磷酸化蛋白的western blot显色图(其中，横坐标表示不同组别PFD浓度；纵坐标表示各显色条带所对应的目标蛋白)；图15B、图15C、图15D是实验例15提供的western blot检测PFD干预EA.hy926细胞表达JAK2、STAT3、AKT磷酸化蛋白相对总蛋白水平的半定量统计图(其中，横坐标表示不同组别PFD浓度；纵坐标表示各组别相对空白组的目标蛋白表达比值；*表示P<0.05，**表示P<0.01)，其中图15B是JAK2磷酸化蛋白相对总蛋白水平表达量统计图；图15C是STAT3磷酸化蛋白相对总蛋白水平表达量统计图；图15D是AKT磷酸化蛋白相对总蛋白水平表达量统计图。
由图可见：PFD以剂量依赖性的方式显著抑制磷酸化JAK2、STAT3和AKT的表达，表明PFD可能通过抑制JAK2/STAT3和AKT通路抑制成血管因子的表达。
综上可见，PFD可明显缓解CIA大鼠关节病理改变，体外实验中PFD具有明显的抑制MH7A细胞分泌炎症因子、基质金属蛋白酶和VEGF作用，且呈剂量依赖性，此外还可明显抑制EA.hy926细胞成管和迁移作用。具体的，PFD 500mg/kg/天的灌胃剂量可明显缓解CIA大鼠关节肿胀、滑膜增生、炎性细胞浸润、关节破坏等病理改变。体外实验中；PFD在100μmol/L-1mmol/L安全浓度范围内能有效降低TNF-α刺激MH7A细胞分泌的VEGF、IL-6、IL-8、IL-1β、MMP-1、MMP-3表达，且呈浓度依赖性。
PFD在50μmol/L和100μmol/L浓度具有显著的抗EA.hy926细胞基质胶成管功能，这可能是因为PFD抑制EA.hy926细胞表达VEGF、MMP-2和MMP-9。PFD在100μmol/L浓度的PFD显著抑制划痕恢复；50μmol/L和100μmol/L浓度的PFD可显著减少EA.hy926细胞过膜数量，且浓度越高，过膜数量越少，这可能是因为PFD抑制EA.hy926细胞表达MMP-2和MMP-9。
以上仅为本申请的可选实施例而已，并不用于限制本申请。对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的权利要求范围之内。

Claims (17)

  1. PFD在制备防治RA的药物中的应用，所述PFD的化学结构式如下式1所示，或者为下式1所示结构的盐
    Figure PCTCN2020115952-appb-100001
  2. 根据权利要求1所述的应用，其特征在于，所述PFD通过抑制血管内皮细胞成血管作用发挥防治RA的作用。
  3. 根据权利要求2所述的应用，其特征在于，所述PFD通过抑制EA.hy926细胞JAK2/STAT3通路和AKT通路发挥防治RA的作用。
  4. 根据权利要求3所述的应用，其特征在于，所述PFD通过抑制EA.hy926细胞分泌MMP-2、MMP-9和VEGF发挥防治RA的作用。
  5. 根据权利要求2至4任一项所述的应用，其特征在于，所述PFD通过抑制EA.hy926细胞成血管作用发挥防治RA的作用，且所述PFD的有效浓度为10μmol/L-100μmol/L。
  6. 根据权利要求1所述的应用，其特征在于，所述PFD通过抑制滑膜细胞磷酸化化发挥防治RA的作用。
  7. 根据权利要求1所述的应用，其特征在于，所述PFD通过抑制MH7A细胞的STAT3磷酸化和AKT磷酸化发挥防治RA的作用。
  8. 根据权利要求7所述的应用，其特征在于，所述PFD通过降低TNF-α诱导的MH7A分泌的炎症因子、软骨破坏因子和成血管相关因子的升高，发挥防治RA的作用。
  9. 根据权利要求8所述的应用，其特征在于，所述炎症因包括IL-6、IL-8、IL-1β；
    所述软骨破坏因子包括MMP-1和MMP-3；
    所述成血管相关因子包括VEGF、MMP-2和MMP-9。
  10. 根据权利要求8所述的应用，其特征在于，所述PFD通过降低TNF-α诱导的MH7A分泌IL-6、IL-8、IL-1β、MMP-1、MMP-3、MMP-2、MMP-9和VEGF的升高发挥防治RA的作用。
  11. 根据权利要求9所述的应用，其特征在于，所述PFD的有效浓度为10μmol/L-1mmol/L。
  12. 根据权利要求1至4、6至11任一项所述的应用，其特征在于，所述药物为以所述PFD作为活性成分，与药学上可接受的辅助剂制成的药物组合物。
  13. 根据权利要求12所述的应用，其特征在于，所述药物组合物作为片剂、丸剂、胶囊剂、颗粒剂、粉剂、液剂、乳剂、悬浮剂、软膏剂、注射剂、皮肤贴剂的口服或非口服用药物制备。
  14. 一种用于防治类风湿关节炎的药物，其特征在于，包括PFD，且所述PFD的化学结构式如下式1所示，或者为下式1所示结构的盐
    Figure PCTCN2020115952-appb-100002
  15. 根据权利要求11或12所述的药物，其特征在于，还包括药学上可接受的辅助剂。
  16. 根据权利要求15所述的药物，其特征在于，所述药物的剂型为片剂、丸剂、胶囊剂、颗粒剂、粉剂、液剂、乳剂、悬浮剂、软膏剂、注射剂、皮肤贴剂中的一种。
  17. 根据权利要求16所述的药物，其特征在于，所述辅助剂包括填充剂、崩解剂、粘合剂、乳化剂、润滑剂、助流剂、矫味剂、矫嗅剂、着色剂中的一种或几种。
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