CN111991393A - Application of pirfenidone in preparation of medicine for preventing and treating rheumatoid arthritis - Google Patents

Abstract

The application relates to the technical field of medicines, in particular to application of pirfenidone in preparation of a medicine for preventing and treating rheumatoid arthritis and a medicine for preventing and treating rheumatoid arthritis. The application of the pirfenidone in preparing the medicine for preventing and treating rheumatoid arthritis is that the chemical structural formula of the pirfenidone is shown as the following formula 1, or the chemical structural formula of the pirfenidone is shown as the salt shown as the following formula 1

The pirfenidone can effectively inhibit RA process by inhibiting inflammatory reaction, inhibiting angiogenesis and other key pathological links, and remarkably reduce tumor necrosis factor alpha to induce human RA fibroblastsThe inflammatory factors secreted by the cells, such as interleukin 6, interleukin 8, interleukin 1 beta and the like, are increased, the abnormal increase of the expression level of the vascular endothelial growth factor secreted by the human RA fibroblasts induced by the tumor necrosis factor alpha is obviously reduced, and the anti-tumor agent has an obvious inhibiting effect on rheumatoid arthritis.

Description

Application of pirfenidone in preparation of medicine for preventing and treating rheumatoid arthritis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of pirfenidone in preparation of a medicine for preventing and treating rheumatoid arthritis, and a medicine for preventing and treating rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is a chronic, systemic disease of unknown etiology, dominated by synovial hyperplasia, inflammatory cell infiltration, pannus formation, cartilage and bone damage. The medicine is characterized in that the arthritis is multiarticular, symmetrical and invasive in hand and foot small joints, and is often accompanied with positive serum rheumatoid factor of external organs of joints, which can cause joint deformity and function loss, and belongs to autoimmune inflammatory diseases. The early stage of the disease is associated with the red swelling and pain of the joints and the manifestation of dysfunction, when the later stage is reached, the joints can have different degrees of rigid deformity and are accompanied with the atrophy of bones and skeletal muscles, which is very easy to cause disability. From the pathological point of view, RA is a widespread inflammatory disease that mainly involves the synovial membrane of joints (which may later be spread to articular cartilage, bone tissue, articular ligaments and musculature), and secondly the connective tissues of serosa, heart, lung and eye. The systemic manifestations of RA include, in addition to arthropathy, fever, fatigue, weakness, pericarditis, subcutaneous nodules, pleuritis, arteritis, peripheral neuropathy, etc.

The treatment method of RA mainly adopts hormone and antirheumatic disease-relieving medicines to carry out anti-inflammatory and immunotherapy, but the existing treatment method has large adverse reaction and is difficult to radically cure. In addition, the current RA treatment drugs have single function, cannot effectively prevent the progress of RA, and have large side effects. Therefore, the search for drugs with pleiotropic functions and little side effects is of great significance for the treatment of RA. Since synovial cells are involved in almost all pathological processes of RA, including inflammation and bone destruction, while the myotendinous membrane is closely associated with progressive destruction of the joint, research in recent years has focused mainly on therapeutic strategies for synovial inflammation and angiogenesis. Compared with the development of new medicines, the new application of old medicines can reduce the development risk and has obvious advantages for treating intractable diseases. Several clinical drugs for treating COVID-19 have been validated for evaluation, e.g., based on protein interactions, thereby avoiding the time-consuming and expensive development of new drugs.

Disclosure of Invention

The application aims to provide application of pirfenidone in preparation of a medicine for preventing and treating rheumatoid arthritis, and aims to solve the problems that the existing medicine for treating rheumatoid arthritis is single in function, cannot effectively prevent RA from progressing and has large side effects.

It is another object of the present application to provide a medicament for the prevention and treatment of rheumatoid arthritis.

In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:

in a first aspect, an application of PFD in preparing a medicament for preventing and treating RA is provided, wherein the chemical structural formula of the PFD is shown as the following formula 1, or a salt with the structure shown as the following formula 1

In some embodiments, the effective concentration of pirfenidone on cells is

10umol/L-1mmol/L。

In some embodiments, the PFD acts to prevent RA by inhibiting the angiogenic effect of ea.hy926 cells.

In some embodiments, the PFD acts to prevent RA by inhibiting ea.hy926 cells JAK2/STAT3 pathway and AKT pathway.

In some embodiments, the PFD acts to prevent RA by inhibiting ea.hy926 cells from secreting matrix metalloproteinase 2(MMP-2), matrix metalloproteinase 9(MMP-9), and Vascular Endothelial Growth Factor (VEGF).

In some embodiments, the PFD acts to prevent RA by inhibiting the angiogenic effect of ea.hy926 cells, and the effective concentration of the PFD is between 10 μmol/L and 100 μmol/L.

In some embodiments, the PFD acts to control RA by inhibiting phosphorylation of synovial cells.

In some embodiments, the PFD plays a role in controlling RA by inhibiting STAT3 phosphorylation and AKT phosphorylation of human RA fibroblast-like synoviocytes (MH7A cells).

In some embodiments, the PFD acts to prevent RA by reducing tumor necrosis factor alpha (TNF- α) -induced elevation of inflammatory, cartilage destruction and angiogenesis-related factors secreted by MH 7A.

In some embodiments, the inflammatory agent comprises interleukin 6(IL-6), interleukin 8(IL-8), interleukin 1 beta (IL-1 beta). In some embodiments, the cartilage destruction factor comprises matrix metalloproteinase 1(MMP-1) and matrix metalloproteinase 3 (MMP-3). In some embodiments, the angiogenic factor comprises VEGF, matrix metalloproteinase 2(MMP-2), and matrix metalloproteinase 9 (MMP-9).

In some embodiments, the PFD plays a role in preventing RA by decreasing TNF- α -induced increases in the inflammatory factors IL-6, IL-8, IL-1 β, MMP-1, MMP-3, MMP-2, MMP-9, and VEGF secreted by MH 7A.

In some embodiments, the PFD acts to prevent RA by decreasing TNF-alpha induced elevations of the inflammatory factors IL-6, IL-8, IL-1 β, MMP-1, MMP-3, MMP-2, MMP-9, and VEGF secreted by MH7A cells, and the effective concentration of the PFD is 10 μmol/L to 1 mmol/L.

In some embodiments, the medicament is a pharmaceutical composition made with the PFD as an active ingredient, and pharmaceutically acceptable adjuvants.

In some embodiments, the pharmaceutical composition is prepared as an oral or non-oral medicament in the form of a tablet, pill, capsule, granule, powder, liquid, emulsion, suspension, ointment, injection, skin patch.

In a second aspect, a medicament for preventing and treating rheumatoid arthritis is provided, which comprises PFD, wherein the chemical structural formula of the PFD is shown as the following formula 1, or a salt with the structure shown as the following formula 1

In some embodiments, a pharmaceutically acceptable adjuvant is also included.

In some embodiments, the pharmaceutical formulation is one of a tablet, a pill, a capsule, a granule, a powder, a liquid, an emulsion, a suspension, an ointment, an injection, and a skin patch.

In some embodiments, the adjuvants include one or more of fillers, disintegrants, binders, emulsifiers, lubricants, glidants, flavoring agents, odorants, colorants.

The application of the PFD in preparing the medicine for preventing and treating RA provided by the embodiment of the application has the beneficial effects that: the PFD can effectively inhibit RA progress, inhibit angiogenesis of EA.hy926 cells by inhibiting inflammatory reaction, inhibiting angiogenesis formation and other key pathological links, and remarkably reduce the increase of the expression level of inflammatory factors, cartilage destruction factors and angiogenesis-related factors secreted by MH7A cells induced by TNF-alpha, thereby having obvious inhibition effect on RA.

The medicine for preventing and treating rheumatoid arthritis provided by the embodiment of the application has the beneficial effects that: the PFD is used as an active ingredient, can effectively inhibit RA process, inhibit angiogenesis of EA.hy926 cells and remarkably reduce the increase of the expression level of inflammatory factors, cartilage destruction factors and angiogenesis-related factors secreted by MH7A cells induced by TNF-alpha through inhibiting inflammatory reaction, inhibiting pannus formation and other key pathological links, thereby having obvious inhibition effect on RA.

Drawings

FIG. 1A is a visual image of swelling of the feet of collagen-induced arthritis (CIA) rats after 21 days of continuous administration provided in Experimental example 1;

FIG. 1B is a line graph showing the thickness (mm) of the feet of rats measured every 7 days after the molding provided in Experimental example 1 was successfully completed;

FIG. 2A is a visual chart of hematoxylin and eosin staining of CIA rat knee joint sections provided in Experimental example 2;

FIG. 2B is a diagram of quantification of synovial inflammation of CIA rat knee joint provided in Experimental example 2;

FIG. 3A is a visual chart of toluidine blue staining of CIA rat knee joint sections provided in Experimental example 3;

FIG. 3B is a graph showing the quantification of cartilage destruction in knee joints of CIA rats provided in Experimental example 3;

FIG. 4A is a three-dimensional reconstruction visual diagram of the CIA rat knee joint provided in Experimental example 4;

FIG. 4B is CIA rat knee joint subchondral bone imaging score provided in Experimental example 4;

FIG. 4C is a bar graph of bone body integral numbers for CIA rat knee joint subchondral bone to distal 1.5mm range provided in Experimental example 4;

FIG. 5 is a graph showing the percentage of cell survival after 72h of PFD intervention in MH7A cells using the CCK-8 method provided in Experimental example 5;

FIG. 6A is a statistical chart of IL-1. beta. expression levels secreted from MH7A cells stimulated by TNF-. alpha.with PFD drug intervention detected by enzyme-linked immunosorbent assay (ELISA) as provided in Experimental example 6;

FIG. 6B is a statistical chart of the expression level of IL-6 secreted by MH7A cells stimulated by PFD drug intervention TNF-alpha by ELISA method provided in Experimental example 6;

FIG. 6C is a statistical chart of the expression level of IL-8 secreted by MH7A cells stimulated by PFD drug intervention TNF-alpha by ELISA method provided in Experimental example 6;

FIG. 6D is a statistical chart of the expression level of VEGF secreted by MH7A cells stimulated by PFD drug intervention TNF-alpha by ELISA method provided in Experimental example 6;

FIG. 7A is a western blot color of the PFD intervention TNF- α provided in Experimental example 7 stimulating MH7A cells to express matrix metalloproteinase and vascular endothelial growth factor;

FIG. 7B is a semi-quantitative statistical chart of the western blot detection PFD intervention TNF-alpha stimulation of MH7A cells to express MMP-1 provided in Experimental example 7;

FIG. 7C is a semi-quantitative statistical chart of the western blot detection PFD provided in Experimental example 7 interfering with TNF- α stimulation of MH7A cells to express MMP-3;

FIG. 7D is a semi-quantitative statistical chart of the western blot detection PFD provided in Experimental example 7 interfering with TNF-. alpha.stimulation of MH7A cells to express MMP-2;

FIG. 7E is a semi-quantitative statistical chart of the western blot detection PFD intervention TNF- α provided in Experimental example 7 to stimulate the expression of MMP-9 by MH7A cells;

FIG. 7F is a semi-quantitative statistical chart of the western blot detection PFD intervention TNF- α stimulation of MH7A cells to express VEGF provided in Experimental example 7;

FIG. 8A is a statistical chart of the results of experiments using QRT-PCR method to detect IL-1 β relative expression at RNA level of MH7A cells with different concentrations of PFD intervening TNF- α stimulation provided in Experimental example 8;

FIG. 8B is a statistical chart of the results of experiments using QRT-PCR method to detect the relative expression of IL-6 at RNA level of MH7A cells with different concentrations of PFD intervening TNF-alpha stimulation provided in Experimental example 8;

FIG. 8C is a statistical chart of the results of experiments using QRT-PCR method to detect the relative expression of IL-8 at RNA level of MH7A cells with different concentrations of PFD intervening TNF-alpha stimulation provided in Experimental example 8;

FIG. 8D is a statistical chart of the results of experiments using QRT-PCR method to detect the relative expression of VEGF on RNA level of MH7A cells with different concentrations of PFD intervening TNF-alpha stimulation provided in Experimental example 8;

FIG. 8E is a statistical chart of the results of experiments using QRT-PCR method to detect the relative MMP-1 expression of MH7A cells at RNA level at different concentrations of PFD-interfering TNF-alpha stimulation provided in Experimental example 8;

FIG. 8F is a statistical chart of the results of experiments using QRT-PCR method to detect the relative MMP-3 expression of MH7A cells at RNA level at different concentrations of PFD interfering TNF-alpha stimulation provided in Experimental example 8;

FIG. 8G is a graph showing the statistics of the results of experiments using QRT-PCR method to detect the relative MMP-2 expression of MH7A cells at RNA level at different concentrations of MH7A cells interfering with TNF-alpha stimulation by PFD provided in Experimental example 8;

FIG. 8H is a graph showing the statistics of the results of experiments performed in experiment 8 to determine the relative MMP-9 expression level of MH7A cells at RNA level, which were stimulated by TNF- α and intervened by PFD at different concentrations, by QRT-PCR;

FIG. 9A is a western blot color development of total protein and phosphorylated protein from PFD intervention TNF- α as provided in Experimental example 9 to stimulate the expression of JAK2, STAT3, AKT, p65 in MH7A cells;

FIG. 9B is a semi-quantitative statistical plot of relative total protein levels of the western blot assay PFD-mediated stimulation of MH7A cells expressing JAK2 phosphorylated protein by TNF- α provided in Experimental example 9;

FIG. 9C is a semi-quantitative statistical plot of the relative total protein levels of STAT3 phosphorylated protein expressed by MH7A cells stimulated by the western blot detection PFD provided in Experimental example 9 to TNF- α;

FIG. 9D is a semi-quantitative statistical plot of the relative total protein levels of the western blot assay PFD provided in Experimental example 9 interfering with TNF-. alpha.stimulation of MH7A cells to express AKT phosphorylated protein;

FIG. 9E is a semi-quantitative statistical plot of the relative total protein levels of p65 phosphorylated protein expressed by MH7A cells stimulated by PFD intervention in the western blot assay provided in Experimental example 9 by TNF- α;

FIG. 10 is a graph showing the percentage of cell survival after PFD intervention in human umbilical vein cell fusion cells (EA.hy926) for 72h measured by the CCK-8 method provided in Experimental example 10;

fig. 11A is an experimental visual presentation of the effect of PFD on ea.hy926 cell matrigel tubulation provided in experimental example 11;

fig. 11B is a quantitative statistical plot of the reticular area of ea.hy926 cell matrigel provided by experimental example 11;

fig. 11C is a quantitative statistical plot of PFD versus ea.hy926 cell matrigel tubulation branch length as provided in experimental example 11;

fig. 12A is a visual chart of the test of the recovery of the scratch width of ea.hy926 cells affected by PFD with different concentrations using the scratch test provided in experimental example 12;

fig. 12B is a statistical plot of the relative migration distances of different concentrations of PFD-interfering ea.hy926 cells provided in experimental example 12;

fig. 13A is a graph of the results of experiments performed using the transwell assay to determine the effect of PFD at different concentrations on ea.hy926 cell membrane penetration provided in experimental example 13;

fig. 13B is a statistical plot of relative transmembrane numbers of different concentrations of PFD intervention ea.hy926 cells provided in experimental example 13;

fig. 14A is a western blot chromogenic map of PFD intervention ea.hy926 cells expressing matrix metalloproteinase and VEGF as provided in experimental example 14;

fig. 14B is a semi-quantitative statistical plot of the western blot detection PFD intervention ea.hy926 cells expressing VEGF provided in experimental example 14;

FIG. 14C is a semi-quantitative statistical chart of the western blot detection PFD intervention EA.hy926 cells expressing MMP-2 provided in Experimental example 14;

FIG. 14D is a semi-quantitative statistical chart of the western blot detection PFD intervention EA.hy926 cells expressing MMP-9 provided in Experimental example 14;

fig. 15A is a western blot chromogenic map of PFD-intervened ea.hy926 cells expressing JAK2, STAT3, AKT total protein and phosphorylated protein as provided in experimental example 15;

figure 15B is a semi-quantitative statistical plot of the relative total protein levels of the western blot detection PFD intervention ea.hy926 cells expressing JAK2 phosphorylated protein provided in experimental example 15;

fig. 15C is a semi-quantitative statistical plot of the expression of STAT3 phosphorylated protein by western blot detection PFD intervention ea.hy926 cells versus total protein level provided in experimental example 15;

fig. 15D is a semi-quantitative statistical plot of the relative total protein levels of the western blot assay PFD intervention ea.hy926 cells provided in experimental example 15 expressing AKT phosphorylated protein.

In the drawings, M is an abbreviation for the concentration unit "mol/L", such as: μ M means "μmol/L".

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

PFD is used for treating inflammatory diseases at first, and is found to have good curative effect on fibrosis diseases such as idiopathic pulmonary fibrosis and renal fibrosis and the like later, and has small side effect. New indications are constantly being developed due to their broad anti-inflammatory, anti-fibrotic and anti-oxidant effects. PFD can effectively inhibit the proliferation of fibroblast synovial cells in vivo, relieve local inflammatory cell infiltration and reduce collagen deposition. In addition, it can regulate wound healing and inhibit endothelial cell angiogenesis. In view of this, it is preferable that,

the first aspect of the embodiments of the present application provides an application of PFD in the preparation of a medicament for preventing and treating RA, wherein the chemical structural formula of PFD is shown in formula 1 below, or a salt having a structure shown in formula 1 below

In the embodiment of the application, the PFD effectively inhibits the RA process by inhibiting inflammatory reaction, inhibiting vascular proliferation and other key pathological links.

In one possible embodiment, PFD acts to prevent RA by inhibiting the angiogenic effect of ea.hy926 cells. The effect of preventing and treating RA can be achieved by directly inhibiting the angiogenesis of target cells, namely EA.hy926 cells.

In some embodiments, PFD plays a role in preventing RA by inhibiting ea.hy926 cells JAK2/STAT3 pathway and AKT pathway. Illustratively, PFD plays a role in preventing RA by inhibiting EA.hy926 cells from secreting MMP-2, MMP-9 and VEGF.

In some embodiments, the PFD acts to prevent RA by inhibiting the angiogenic effect of ea.hy926 cells, and the effective concentration of the PFD is between 10 μmol/L and 100 μmol/L. Illustratively, the effective concentration of PFD is 10. mu. mol/L, 20. mu. mol/L, 30. mu. mol/L, 40. mu. mol/L, 50. mu. mol/L, 60. mu. mol/L, 70. mu. mol/L, 80. mu. mol/L, 90. mu. mol/L, 100. mu. mol/L, etc., but is not limited thereto.

In one possible embodiment, PFD acts to control RA by inhibiting phosphorylation of synovial cells.

In some embodiments, PFD exerts an effect of preventing RA by significantly reducing inflammatory factor elevation and angiogenesis by inhibiting STAT3 phosphorylation and AKT phosphorylation of MH 7A.

In some embodiments, PFD acts to prevent RA by reducing TNF- α -induced elevation of inflammatory, cartilage destruction and angiogenesis-related factors secreted by MH 7A. In this case, PFD can relieve RA joint swelling by inhibiting inflammatory factors, relieve joint cartilage destruction by inhibiting cartilage destruction factors, prevent and treat RA, inhibit the formation of pannus in joint synovium by reducing angiogenesis-related factors, and prevent and treat RA. Finally, the three-fold effect is exerted to effectively prevent RA.

In some embodiments, the inflammatory agent comprises IL-6, IL-8, and IL-1 β. Under the condition, PFD can reduce joint swelling and play a role in preventing and treating RA by inhibiting MH7A cells induced by TNF-alpha from secreting inflammatory factors IL-6, IL-8 and IL-1 beta.

In some embodiments, the cartilage destruction factor comprises MMP-1 and MMP-3. Under the condition, PFD can reduce the damage of articular cartilage and play a role in preventing and treating RA by inhibiting MH7A cells induced by TNF-alpha from secreting MMP-1 and MMP-3.

In some embodiments, the angiogenesis-related factors include VEGF, MMP-2, and MMP-9. Under the condition, PFD can inhibit the formation of pannus of joint synovium by inhibiting TNF-alpha induced MH7A cells from secreting VEGF, MMP-2 and MMP-9, thereby playing a role in preventing and treating RA.

In some embodiments, PFD plays a role in preventing RA by decreasing TNF- α -induced increases in IL-6, IL-8, IL-1 β, MMP-1, MMP-3, MMP-2, MMP-9, and VEGF secreted by MH7A cells.

In some embodiments, PFD acts to prevent RA by decreasing TNF- α -induced increases in IL-6, IL-8, IL-1 β, MMP-1, MMP-3, MMP-2, MMP-9, and VEGF secreted by MH7A cells, and the effect of PFD in preventing RA is concentration dependent. Specifically, the effective concentration of PFD is 10. mu. mol/L-1 mmol/L. Within the safe concentration range of 10. mu. mol/L-1mmol/L, the higher the concentration, the stronger the PFD effect of inhibiting TNF-alpha induced secretion of IL-6, IL-8, IL-1. beta., MMP-1, MMP-3, MMP-2, MMP-9 and VEGF by MH7A cells. Illustratively, the effective concentration of PFD is 10. mu. mol/L, 20. mu. mol/L, 50. mu. mol/L, 80. mu. mol/L, 100. mu. mol/L, 200. mu. mol/L, 250. mu. mol/L, 500. mu. mol/L, 800. mu. mol/L, 1mmol/L, and the like, but is not limited thereto.

In some embodiments, when the PFD is used for preparing a medicament for preventing and treating RA, the medicament is a pharmaceutical composition prepared from the PFD as an active ingredient and pharmaceutically acceptable adjuvants. I.e. a medicament which can be understood as preventing RA, comprising an active ingredient and a pharmaceutically acceptable adjuvant, wherein the active ingredient is PFD.

In some embodiments, when the PFD is used for preparing a medicament for preventing and treating RA, the dosage form of the pharmaceutical composition can be flexibly set according to the administration mode, and the pharmaceutical composition can be prepared as an oral or non-oral medicament of tablets, pills, capsules, granules, powders, liquids, emulsions, suspensions, ointments, injections and skin patches.

The application of the PFD in preparing the medicine for preventing and treating RA provided by the embodiment of the application has the beneficial effects that: PFD can effectively inhibit RA process, inhibit angiogenesis of EA.hy926 cell by inhibiting inflammatory reaction, inhibiting pannus formation and other key pathological links, and significantly reduce the increase of inflammatory factor, cartilage destruction factor and angiogenesis related factor expression level secreted by MH7A cell induced by TNF-alpha, thereby having obvious inhibition effect on RA.

In a second aspect, the embodiments of the present application provide a medicament for preventing and treating rheumatoid arthritis, which comprises PFD, wherein the chemical structural formula of PFD is shown in formula 1 below, or a salt with the structure shown in formula 1 below

In the drug for preventing and treating rheumatoid arthritis, the PFD represented by the above formula 1 or a salt of the PFD represented by the above formula 1 acts as an active ingredient for preventing and treating rheumatoid arthritis.

At this time, the PFD significantly reduces the downstream inflammatory signaling pathway and the angiogenetic pathway by inhibiting phosphorylation of STAT3 and AKT, and exerts an effect RA of preventing RA in combination. Specifically, on one hand, the PFD inhibits the angiogenesis function by inhibiting EA.hy926 cells from secreting MMP-2, MMP-9 and VEGF, thereby playing a role in preventing and treating RA; on the other hand, PFD inhibits MH7A cells induced by TNF-alpha to secrete inflammatory factors IL-6, IL-8, IL-1 beta, cartilage destruction factors MMP-1 and MMP-3, and angiogenesis-related factors VEGF, MMP-2 and MMP-9, slows RA joint swelling, relieves joint cartilage destruction, inhibits the formation of a synovium of joints, and plays a role in preventing and treating RA together.

In some embodiments, the drug for the prevention and treatment of rheumatoid arthritis includes pharmaceutically acceptable adjuvants in addition to the PFD or the salt of the PFD, so as to facilitate the preparation thereof into dosage forms satisfying various administration routes.

In some embodiments, the pharmaceutical formulation is one of a tablet, a pill, a capsule, a granule, a powder, a liquid, an emulsion, a suspension, an ointment, an injection, and a skin patch.

In some embodiments, the adjuvant includes one or more of a filler, a disintegrant, a binder, an emulsifier, a lubricant, a glidant, a flavoring agent, a coloring agent, but is not limited thereto.

The medicine for preventing and treating rheumatoid arthritis provided by the embodiment of the application has the beneficial effects that: the PFD is used as an active ingredient, can effectively inhibit RA process by inhibiting key pathological links such as synovium inflammatory reaction, pannus formation and the like, obviously inhibits the angiogenetic pathway of EA.hy926 cells, and obviously reduces the increase of the expression level of inflammatory factors, cartilage destruction factors and angiogenetic related factors secreted by MH7A cells induced by TNF-alpha, thereby having obvious inhibition effect on RA.

The technical solutions described in the present application are described in detail below with reference to specific drawings and embodiments.

Experimental example 1

Measurement and analysis of the improving Effect of PFD on swelling of feet in CIA rats

Male Lewis rats (8 weeks old) 24 were housed in a pathogen-free environment. 24 rats were randomly divided into 4 groups (6/group): normal group (Normal, first group) and 3 CIA model groups (second to fourth group). Firstly, establishing a CIA rat model: mixing equal amount of bovine type II collagen and incomplete adjuvant, injecting 200 μ l suspension subcutaneously at a position 2cm away from the root of rat tail for 7 days, injecting 100 μ l suspension at the root of each old rat tail according to the above method, enhancing immunoreaction, and determining molding success after 14 days. After the CIA model was successfully established, the second group of rats was gavaged with 10 ml/kg/day (CIA) of sodium carboxymethyl cellulose suspension, the third group of rats was given methotrexate as a positive control, 0.1 mg/kg/3 days of intraperitoneal injection (CIA-MTX), the fourth group of rats was gavaged with PFD, 500 mg/kg/day (CIA-PFD), and the rats were euthanized and lower limb-drawn after 21 consecutive days of dosing. And determining that the thickness of the hind paw of the rat is measured every 7 days after the model is successfully made.

The results are shown in FIG. 1A and FIG. 1B, in which FIG. 1A is a visual photograph of swelling of feet (abscissa indicates group) of CIA rats after 21 days of continuous administration; FIG. 1B is provided for Experimental example 1: after the successful modeling of the rat, a line graph of the thickness of the foot of the rat was measured every 7 days (wherein the abscissa represents the time for starting the modeling and the ordinate represents the thickness (mm) of the foot). As can be seen from the figure: in comparison to the normal group, CIA rats had significant swelling in the feet, and the swelling was reduced after treatment with Methotrexate (MTX) and PFD (P < 0.05).

Experimental example 2

Method for detecting effect of PFD (pulse frequency detector) on joint synovitis of CIA (common animal and animal) rats by adopting hematoxylin and eosin staining

After euthanizing the rats of example 1, the right knee joints were fixed with 4% paraformaldehyde, decalcified with 10% EDTA for 40 days, and then subjected to conventional dehydration, paraffin embedding, and 5- μm sectioning in this order. Sections were then stained with hematoxylin, eosin (H & E): the paraffin sections of the knee joints are dewaxed and rehydrated by xylene and gradient alcohol in a conventional way, stained by hematoxylin staining solution for 7 minutes, stained by yihong for 1 minute after washing excessive staining solution, dehydrated by gradient alcohol, transparent by xylene, sealed by neutral gum, and finally photographed and analyzed. The joint synovial inflammation scoring standard is as follows: 0-no inflammation, 1-a slight thickening of the synovial lining layer or inflammatory cell infiltration, 2-a slight thickening of the synovial lining layer with inflammatory cell infiltration, 3-a thickening of the synovial lining layer, inflammatory cell infiltration in the synovial space, and 4-a severe infiltration of synovial inflammatory cells.

The results are shown in fig. 2A and fig. 2B, wherein fig. 2A is a visual representation of hematoxylin and eosin staining of CIA rat knee joint sections provided in experimental example 2 (abscissa indicates group;. indicates P < 0.05); fig. 2B is a quantitative graph of the synovial inflammation of the knee joints of CIA rats provided in experimental example 2 (wherein the abscissa represents the group and the ordinate represents the synovial inflammation score of the knee joints). As can be seen from the figure: compared with the normal group, the synovial hyperplasia and inflammatory cell infiltration of the CIA rats are obvious, and the PFD can obviously reduce the synovial hyperplasia and inflammatory cell infiltration of the knee joints (P < 0.05).

Experimental example 3

Analysis of the effect of PFD on articular cartilage destruction in CIA rats by toluidine blue staining

Toluidine Blue (TB) staining: after euthanasia of rats of example 1, paraffin sections of knee joints were dewaxed and rehydrated conventionally using xylene and gradient alcohol, stained for 10min using toluidine blue, excess stain was washed off, dehydrated by gradient alcohol, transparent xylene, and sealed with neutral gum, photographed under microscope observation and analyzed for the extent of erosion of articular cartilage, wherein the evaluation criteria for articular cartilage damage were: 0 is no destruction, 1 is punctate destruction, 2 is local mild to moderate destruction, 3 is wide destruction, and 4 is wide destruction.

The results are shown in fig. 3A and 3B, wherein fig. 3A is a visual depiction of toluidine blue staining of CIA rat knee joint sections provided in experimental example 3 (abscissa indicates group;. indicates P < 0.05); fig. 3B is a graph showing the quantification of cartilage destruction in knee joints of CIA rats provided in experimental example 3 (wherein the abscissa represents the group and the ordinate represents the cartilage destruction score). As can be seen from the figure: in CIA rats articular cartilage destruction, gap narrowing, and PFD significantly reduced knee cartilage destruction compared to normal group (P < 0.05).

Experimental example 4

Analyzing the influence of PFD on the joint bone destruction of CIA rat by adopting microcomputer tomography

Microcomputer tomography: after euthanasia of rats, the knee joints were harvested, the right knee joints were fixed with 4% paraformaldehyde for 1 day, and the bone tissue at the knee joint sites was scanned using Micro-CT (SkyScan 1176). The scanning parameters are: the x-ray tube voltage was 65kV, the tube current was 385 μ A, and the pixel size was 12.63 μm. The scanned images were reconstructed three-dimensionally and evaluated for analysis, wherein the evaluation criteria of joint damage were 0 ═ no damage, 1 ═ mild, 2 ═ moderate, and 3 ═ severe. And selecting a section of 1.5mm from the subchondral bone of the tibia to the far end, analyzing the BV/TV ratio, and evaluating the structural state of the subchondral bone of the joint.

The results are shown in fig. 4A, 4B and 4C, wherein fig. 4A is a three-dimensional reconstruction visual map of the CIA rat knee joint provided in experimental example 4 (the abscissa represents the group;. indicates P < 0.05); FIG. 4B is the CIA rat knee joint subchondral bone imaging score provided in Experimental example 4 (wherein the abscissa represents the group and the ordinate represents the bone destruction imaging score); FIG. 4C is a bar graph of bone volume fraction for CIA rats ranging from subchondral bone to the distal end of the knee joint 1.5mm provided in Experimental example 4 (wherein the abscissa represents the group and the ordinate represents the bone volume fraction). As can be seen from the figure: compared with the normal group, the CIA rat has obvious articular subchondral bone erosion, obvious joint destruction, reduced BV/TV value and reduced bone mass, and the PFD can obviously relieve the articular subchondral bone damage of the knee joint, improve the BV/TV ratio and increase the bone mass (P is less than 0.05).

Experimental example 5

Experimental test for detecting activity of PFD on MH7A by CCK-8 method

MH7A cells with good growth status were selected, counted and planted at 3000/90. mu.l/well in 96-well plates at 37 ℃ with 5% CO₂Culturing in an incubator with concentration, setting five experimental groups and a blank control group, wherein each group is provided with 3 multiple holes, adding PFD with drug concentration of 100 mu mol/L, 250 mu mol/L, 500 mu mol/L, 1mmol/L and 2mmol/L into the five experimental groups respectively after 24h, and adding no blank control group. And adding 10 mul of CCK-8 solution into each hole after 72 hours, detecting the absorbance of each hole at 450nm within 30min-2 hours by using an enzyme-labeling instrument, and repeating twice until the experimental results are matched.

The results are shown in FIG. 5, which is a graph of the percentage of cell survival after PFD intervention in MH7A cells for 72h by using CCK-8 method provided in Experimental example 5. (the abscissa indicates the concentration of PFD in different groups and the ordinate indicates the cell viability of each group relative to the blank;. indicates P < 0.05). As can be seen from the figure: in the concentration range of 100 mu mol/L-1mmol/L, the proliferation activity of MH7A cells is not obviously inhibited by PFD, while the proliferation activity of MH7A cells is obviously inhibited by PFD with the concentration of 2mmol/L, so that PFD with the concentration of 100 mu mol/L-1mmol/L is selected for subsequent experiments.

Experimental example 6

ELISA method is adopted to detect inflammatory and angiogenetic factors secreted by TNF-alpha stimulation MH7A cells interfered by PFD drugs

PFD with the concentration of 0, 100 mu mol/L, 250 mu mol/L, 500 mu mol/L and 1mmol/L is selected for intervention of MH7A cells for 2h in the experiment respectively, and then TNF-alpha is used for stimulation for 24h, cell culture fluid is sucked into a centrifuge for 3000R/min, cell culture supernatant is centrifugally extracted under the conditions of 4 ℃ and 10min, and then inflammatory factors and VEGF expression in the cell culture supernatant are detected by using kits such as IL-6(R & D Systems, cat.D6050), IL-8(R & D Systems, cat.D8000C), IL-1 beta (R & D Systems, cat.D DLB50) and VEGF (R & D Systems, cat.DVE00) respectively.

Results As shown in FIGS. 6A, 6B, 6C and 6D, the statistical graphs of the expression levels of inflammatory and angiogenic factors secreted by MH7A cells stimulated by PFD drug-mediated TNF-. alpha.by ELISA method provided in Experimental example 6 (wherein, the first row on the abscissa indicates the addition of TNF-. alpha. (-without addition, +: addition) at a concentration of 20ng/ml for each group; the second row on the abscissa indicates the concentration of PFD for each group; the ordinate indicates the expression concentration of cytokine in the culture solution; indicates P < 0.05; indicates P < 0.01): wherein FIG. 6A is the IL-1. beta. expression level; FIG. 6B shows the expression level of IL-6; FIG. 6C shows the expression level of IL-8; FIG. 6D shows the expression level of VEGF. As can be seen from the figure, the experimental results show that: the PFD can obviously reduce the increase of inflammatory factors such as IL-6, IL-8, IL-1 beta and the like secreted by MH7A cells induced by TNF-alpha, and obviously reduce the abnormal increase of VEGF expression level secreted by MH7A cells induced by TNF-alpha, and the concentration of the VEGF expression level is dependent, namely the higher the concentration is in the safe concentration range of 100 mu mol/L-1mmol/L, the stronger the inhibition effect is.

Experimental example 7

Western blot method is adopted to detect the level of matrix metalloproteinase and angiogenetic factor expressed by TNF-alpha stimulated MH7A cells of PFD drug intervention

Equal amount of well-grown cells were plated on 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator with concentration, after 80% of cells are fused, changing liquid, respectively interfering MH7A cells for 2h by PFD with gradient of 0, 100 mu mol/L, 250 mu mol/L, 500 mu mol/L and 1mmol/L, then stimulating MH7A cells for 24h by TNF-alpha with concentration of 20ng/ml, extracting total protein in cells, sequentially carrying out electrophoresis, membrane transfer, immune closure, primary antibody incubation, secondary antibody incubation and other series of operations to obtain a membrane with target protein, carrying out color reaction,

matrix metalloproteinase and VEGF expression levels in cells 24h after TNF-alpha stimulation were detected using 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) antibodies.

As shown in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and FIG. 7F, wherein FIG. 7A is a western blot color map of the expression of matrix metalloproteinase and VEGF by the MH7A cells stimulated by PFD intervention TNF-alpha provided in Experimental example 7 (wherein, the first row of abscissa indicates the addition of TNF-alpha at a concentration of 20ng/ml (no addition, addition); the second row of abscissa indicates the concentration of PFD in different groups; the ordinate indicates the target protein corresponding to each color development band); FIGS. 7B, 7C, 7D, 7E and 7F are semi-quantitative statistical graphs of the western blot assay PFD provided in Experimental example 7 for interfering with TNF- α stimulation MH7A cells expressing matrix metalloprotease and angiogenin (wherein, the first row on the abscissa indicates TNF- α addition at a concentration of 20ng/ml for different groups (-: no addition, +: addition); the second row on the abscissa indicates the concentration of PFD for different groups; the ordinate indicates the ratio of expression of the target protein for each group to the blank; P < 0.05; P <0.01), wherein FIG. 7B is a statistical graph of the relative expression of MMP-1; FIG. 7C is a statistical graph showing the relative expression of MMP-3; FIG. 7D is a statistical graph showing the relative expression of MMP-2; FIG. 7E is a statistical graph of the relative expression of MMP-9; FIG. 7F is a statistical graph of the relative expression of VEGF. As can be seen, TNF- α stimulation of MH7A cells increased the expression of IL1, IL6, IL8, VEGF, MMP-1, MMP-2, MMP-3, and MMP-9, while PFD significantly and dose-dependently reduced the expression levels of these cytokines.

Experimental example 8

QRT-PCR method is adopted to detect the level of cell factor expressed by MH7A cells stimulated by TNF-alpha intervention of PFD drugs

Equal amount of well-grown cells were plated on 6-well plates at 37 ℃ with 5% CO₂Culturing under the condition of concentration, after 80% fusion of cells, changing liquid, then respectively interfering MH7A cells for 2h by PFD with gradient of 0, 100 mu mol/L, 250 mu mol/L, 500 mu mol/L and 1mmol/L, then stimulating for 6h by TNF-alpha with concentration of 20ng/ml, extracting RNA on ice, measuring concentration, removing qRNA according to the instruction of Takara kit (Code No. RR047A), obtaining cDNA by reverse transcription reaction, finally amplifying by qPCR (real-time fluorescence quantitative PCR) to obtain cycle number (Cq), obtaining delta Cq value according to target gene and internal reference GAPDH and subsequent 2^-ΔΔAnd Cq calculating the relative expression quantity, thereby obtaining the difference of the target gene expression quantity between the experimental group and the control group.

FIG. 8 is a statistical graph of the results of experiments using QRT-PCR to detect the cytokine expression level of MH7A cells stimulated by PFD-interfering TNF-alpha at RNA level as provided in Experimental example 8 (wherein, the first row on the abscissa indicates the addition of TNF-alpha at a concentration of 20ng/ml (— no addition, +: addition); the second row on the abscissa indicates the concentration of PFD in different groups; the ordinate indicates the expression level of cytokine in the culture fluid relative to the blank group;. indicates P <0.05,. indicates P < 0.01): wherein FIG. 8A is a statistical graph of the relative expression levels of IL-1 β; FIG. 8B is a statistical graph showing the relative expression levels of IL-6; FIG. 8C is a statistical graph of the relative expression levels of IL-8; FIG. 8D is a statistical graph of the relative expression of VEGF; FIG. 8E is a statistical graph showing the relative expression of MMP-1; FIG. 8F is a statistical graph showing the relative expression of MMP-3; FIG. 8G is a statistical graph showing the relative expression of MMP-2; FIG. 8H is a graph showing the statistics of relative MMP-9 expression. As can be seen, TNF-alpha stimulation of MH7A cells increased the production of IL1, IL6, IL8, VEGF, MMP-1, MMP-2, MMP-3 and MMP-9, while PFD significantly and dose-dependently reduced the expression level of the above cytokines, and the mRNA and protein expression levels were in consistent trend, indicating that PFD regulates the expression of cytokines at the transcriptional level.

Experimental example 9

Western blot method is adopted to detect the protein levels of total protein and phosphorylated protein of MH7A cells expressed by TNF-alpha intervention of PFD drugs and JAK2, STAT3, AKT and p65

Equal amount of well-grown cells were plated on 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator with concentration, after 80% of cells are fused, changing liquid, respectively interfering MH7A cells for 2 hours by PFD with gradient of 0, 100 mu mol/L, 250 mu mol/L, 500 mu mol/L and 1mmol/L, then stimulating MH7A cells for 0.5 hours by TNF-alpha with concentration of 20ng/ml, extracting total protein in the cells, sequentially carrying out electrophoresis, membrane transfer, immune closure, primary antibody incubation, secondary antibody incubation and other series of operations to obtain a membrane with target protein, and carrying out color reaction.

The expression levels of JAK2, STAT3, AKT, total p65 and phosphorylated protein in cells after 0.5h of TNF- α stimulation were detected using primary antibody (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)).

The results are shown in FIGS. 9A, 9B, 9C and 9D, wherein FIG. 9A is a western blot chromatogram of the PFD-mediated TNF- α provided in Experimental example 9 stimulating MH7A cells to express JAK2, STAT3, AKT, p65 total protein and phosphorylated protein (wherein the first row on the abscissa indicates the addition of TNF- α at a concentration of 20ng/ml in different groups (— no addition, +: addition), the second row on the abscissa indicates the concentration of PFD in different groups, the third row on the abscissa indicates the addition of transforming growth factor- β at a concentration of 10ng/ml in different groups (— no addition, +: addition), and the ordinate indicates the target protein corresponding to each chromogenic band); FIGS. 9B, 9C, 9D, 9E are semi-quantitative statistical graphs of the relative amounts of JAK2 phosphorylated protein to total protein levels, as determined by the western blot assay provided in Experimental example 9, wherein PFD intervention in TNF- α stimulated the expression of JAK2, STAT3, AKT, P65 total protein and phosphorylated protein levels in MH7A cells (where the first row on the abscissa indicates the addition of TNF- α at a concentration of 20ng/ml for different groups (— no addition, +: addition), the second row on the abscissa indicates the concentration of PFD for different groups, and the third row on the abscissa indicates the addition of transforming growth factor- β at a concentration of 10ng/ml for different groups (— no addition, +: addition), and the ordinate indicates the ratio of target protein expression for each group to a blank group; P <0.05, P <0.01), and FIG. 9B is a statistical graph of the relative amounts of JAK2 phosphorylated protein to total protein expression; figure 9C is a statistical graph of STAT3 phosphorylated protein relative to total protein level expression; FIG. 9D is a statistical graph of the amount of AKT phosphorylated protein expressed relative to the total protein level; FIG. 9E is a statistical graph of the amount of p65 phosphorylated protein expressed relative to the total protein level. As can be seen, the phosphorylation levels of AKT and p65 in MH7A cells were significantly increased following TNF-alpha stimulation. PFD treatment can down-regulate the expression of phosphorylated STAT3, AKT, and p 65. The expression levels of STAT3, AKT, and p65 phosphorylation were effectively restored after TGF- β intervention (FIGS. 9C-9E). These results show that: PFD may inhibit downstream STAT3, AKT-NF-kB mediated inflammatory signaling pathways, possibly through inhibition of TGF-beta targets.

Experimental example 10

Detecting the proliferation activity of EA.hy926 cell interfered by PFD by CCK-8 method

Selecting EA.hy926 cells with good growth state, preparing 3000/90 μ l cell suspension in 96-well plate, placing in incubator for pre-culture (37 deg.C, 5% CO)₂) Five experimental groups and a blank control group are set, each group is provided with 3 multiple holes, PFD with the drug concentration of 10 mu mol/L, 25 mu mol/L, 50 mu mol/L, 100 mu mol/L and 200 mu mol/L is respectively added in the five experimental groups after 24 hours, and the blank control group is not added. And adding 10 mul of CCK-8 reagent into each hole after 72 hours, detecting the absorbance of each hole at 450nm within 30min-2 hours by using an enzyme-labeling instrument, and repeating twice until the experimental results are matched.

The results are shown in fig. 10, which is a graph of the percentage of cell survival after PFD intervention ea.hy926 cells were detected for 72h by CCK-8 provided in experimental example 10. (the abscissa indicates the concentration of PFD in different groups and the ordinate indicates the cell viability in the relative blank group;. indicates P <0.01) as can be seen from the graph: PFD with different concentration gradients is used for interfering the proliferation activity of EA.hy926 cells, and the experimental result shows that: in the concentration range of less than or equal to 100 mu mol/L, PFD has no obvious inhibition on the proliferation activity of EA.hy926 cells, while PFD with the concentration of 200 mu mol/L obviously inhibits the proliferation activity of EA.hy926 cells, so that the concentration range of less than or equal to 100 mu mol/L is selected as the safe concentration range of PFD on the EA.hy926 cells.

Experimental example 11

Detecting the effect of PFD on EA.hy926 cell tubulation by matrigel tubulation experiment

And paving matrigel in a 24-well plate, inoculating an equivalent cell suspension after 30 minutes, interfering EA.hy926 cells by PFD with the concentrations of 0, 50 mu mol/L and 100 mu mol/L respectively in the experiment, and observing the interference effect on tube formation under a 6-8 hour rear mirror.

The results are shown in fig. 11A, fig. 11B, and fig. 11C, in which fig. 11A is an experimental visual chart of the effect of PFD on ea.hy926 cell matrigel tubulation provided in experimental example 11 (the abscissa indicates PFD concentration); FIG. 11B is a quantitative statistical plot of the tubular network area of the matrigel of EA.hy926 cells provided in Experimental example 11 (where the abscissa represents PFD concentration and the ordinate represents the ratio of each network area to the blank group;. represents P <0.05 and. represents P < 0.01); fig. 11C is a quantitative statistical plot of PFD versus ea.hy926 cell matrigel tubulation branch length provided in experimental example 11 (where the abscissa represents PFD concentration and the ordinate represents the relative blank fraction of each component tube branch length;. represents P <0.05 and. represents P < 0.01). As can be seen from the figure: the PFD at the concentration of 50 mu mol/L and 100 mu mol/L obviously inhibits the tube forming effect of EA.hy926 cells, and the higher the concentration is, the more obvious the tube forming effect of the EA.hy926 cells is inhibited by the PFD.

Experimental example 12

Scratch test was used to test the effect of PFD on EA.hy926 cell migration

Equal amount of EA.hy926 cells with good growth state are spread on a 6-well plate and cultured in an incubator (37 ℃ and 5% CO)₂) After the cells are 90% fused, vertically scratching the cells by using a 200 mu L pipette tip, cleaning cell fragments, culturing the cells by using a serum-free culture medium, adding PFD with the concentrations of 0, 50 mu mol/L and 100 mu mol/L for intervention, and observing the influence of the PFD on scratch recovery under a mirror after 24 hours.

The results are shown in fig. 12A and 12B, wherein fig. 12A is a visual chart of test results of experimental example 12, which are obtained by testing the recovery of ea.hy926 cell scratch width affected by PFD at different concentrations through a scratch test (the abscissa represents PFD concentration); fig. 12B is a statistical plot of the relative migration distances of the different concentrations of PFD-interfered ea.hy926 cells provided in experimental example 12 (PFD concentration is shown on the abscissa, scratch recovery width versus blank fraction value for each group of cells; P < 0.05; P < 0.01). It can be seen from the figure that the marked recovery inhibition of the PFD at the concentration of 100. mu. mol/L indicates that the migration of EA.hy926 cells can be significantly inhibited by the PFD at the concentration of 100. mu. mol/L.

Experimental example 13

Transwell experiment was used to examine the effect of PFD on EA.hy926 cell migration

Equal amounts of well-grown ea.hy926 cells were inoculated into the upper chamber of a Transwell, PFD was added at a concentration of 50 μmol/L and 100 μmol/L to the lower chamber, serum-free medium was used for the upper chamber, medium containing 2% fetal bovine serum was used for the lower chamber, after 4 hours of incubation, the cells on the membrane were wiped clean with a cotton swab, fixed with 4% paraformaldehyde and stained with crystal violet, and the number of cells passing through the membrane was observed and statistically analyzed under a microscope.

As shown in fig. 13A and fig. 13B, fig. 13A is an experimental visual chart provided in experimental example 13, which uses a transwell experiment to detect the effect of PFD at different concentrations on the cell membrane penetration of ea.hy926 (the abscissa represents PFD concentration); fig. 13B is a statistical plot PFD of relative transmembrane number of different concentrations of PFD-interfered ea.hy926 cells provided in experimental example 13 (PFD concentration on abscissa, transmembrane number per group relative to blank group ratio on ordinate;. P <0.05, P < 0.01).

As can be seen from the figure: PFD at concentrations of 50 μmol/L and 100 μmol/L significantly reduced the number of transmembrane of ea.hy926 cells, and the higher the concentration, the lower the number of transmembrane, indicating that PFD significantly inhibited the migration of ea.hy926 cells.

Experimental example 14

Western blot method is adopted to detect the levels of matrix metalloproteinase and angiogenetic factor expressed by PFD drug intervention EA.hy926 cells

Equal amount of well-grown cells were plated on 6-well plates at 37 ℃ with 5% CO₂Culturing in culture box with concentration, changing solution after 80% cell fusion, then interfering EA.hy926 cell with PFD with concentration of 0, 50 μmol/L, 100 μmol/L for 24h, extracting total protein in cell, sequentially performing electrophoresis, membrane transfer, immune closure, primary antibody and secondary antibody incubation, and the like to obtain the final productThe membrane containing the target protein is subjected to a color reaction.

Matrix metalloproteinase factor and VEGF expression levels after 24h intervention of the EA.hy926 cells by PFD were detected using anti-MMP-2 (CST, Cat.no.40994S), anti-MMP-9 (Proteintetech, Cat.no.10375-2-AP), anti-VEGF (Abcam, Cat.no. ab46154) antibodies.

The results are shown in fig. 14A, 14B, 14C, and 14D, where fig. 14A is a western blot chromogenic graph of PFD-interfered ea.hy926 cells expressing matrix metalloproteinases (MMP-2 and MMP-9) and VEGF provided in experimental example 14 (where the abscissa indicates the concentration of PFD in different groups; and the ordinate indicates the target protein corresponding to each chromogenic band); FIGS. 14B, 14C, and 14D are semi-quantitative statistical graphs of the western blot detection PFD intervention EA.hy926 cells expressing matrix metalloproteinase and VEGF provided in Experimental example 14 (wherein, the horizontal axis represents PFD concentration of different groups; the vertical axis represents the target protein expression ratio of each group to blank group; P < 0.05; P <0.01), wherein FIG. 14B is a statistical graph of VEGF relative expression; FIG. 14C is a statistical graph showing the relative expression of MMP-2; FIG. 14D is a graph showing the statistics of relative MMP-9 expression. As can be seen, PFD significantly reduced the expression of VEGF, MMP-2, and MMP-9 proteins, consistent with the observed formation and migration of inhibitory tubes. It is suggested that PFD may inhibit matrigel tubulation and migration of ea.hy926 cells by down-regulating the expression of angiogenic factors.

Experimental example 15

Detecting the expression of JAK2, STAT3 and AKT total protein and phosphorylated protein levels of PFD intervention EA.hy926 cells by using a Western blot method:

equal amount of well-grown cells were plated on 6-well plates and cultured in an incubator (37 ℃ C., 5% CO)₂) And after 80% of cells are fused, changing the solution, respectively interfering the EA.hy926 cells for 0.5h by PFD with the concentrations of 0, 50 mu mol/L and 100 mu mol/L, then extracting total protein in the cells, sequentially carrying out electrophoresis, membrane transfer, immune blocking, primary antibody incubation, secondary antibody incubation and other series of operations to obtain the membrane with the target protein, and carrying out color reaction.

The expression amount of JAK2, STAT3, AKT total protein and phosphorylated protein after 0.5h of PFD intervention EA.926 cells was detected using primary antibodies (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)).

The results are shown in fig. 15A, fig. 15B, fig. 15C and fig. 15D, wherein fig. 15A is a western blot chromogenic map of PFD-interfering ea.hy926 cells expressing JAK2, STAT3, AKT total protein and phosphorylated protein provided in experimental example 15 (wherein the abscissa represents PFD concentrations of different groups; and the ordinate represents target proteins corresponding to respective chromogenic bands); FIGS. 15B, 15C, and 15D are semi-quantitative statistical graphs of relative total protein levels of the western blot detection PFD intervention EA.hy926 cells expressing JAK2, STAT3, and AKT phosphorylated proteins provided in Experimental example 15 (wherein, the abscissa represents PFD concentrations of different groups; the ordinate represents target protein expression ratios of each group to a blank group; P < 0.05; P <0.01), wherein FIG. 15B is a statistical graph of relative total protein level expression of JAK2 phosphorylated proteins; figure 15C is a statistical graph of STAT3 phosphorylated protein relative to total protein level expression; FIG. 15D is a statistical graph of the amount of AKT phosphorylated protein expressed relative to the total protein level.

As can be seen from the figure: PFD significantly inhibited the expression of phosphorylated JAK2, STAT3, and AKT in a dose-dependent manner, suggesting that PFD may inhibit the expression of vascular factors by inhibiting the JAK2/STAT3 and AKT pathways.

In conclusion, PFD can obviously relieve pathological changes of joints of CIA rats, has obvious effects of inhibiting MH7A cells from secreting inflammatory factors, matrix metalloproteinase and VEGF in vitro experiments, is dose-dependent, and can also obviously inhibit EA.hy926 cells from forming tubes and migrating. Specifically, the lavage dose of PFD500 mg/kg/day can obviously relieve pathological changes of joint swelling, synovial membrane hyperplasia, inflammatory cell infiltration, joint destruction and the like of a CIA rat. In vitro experiments; PFD can effectively reduce the expression of VEGF, IL-6, IL-8, IL-1 beta, MMP-1 and MMP-3 secreted by MH7A cells stimulated by TNF-alpha in a safe concentration range of 100 mu mol/L-1mmol/L, and is in concentration dependence.

PFD had significant anti-ea.hy926 cell matrigel tubulation at 50 μmol/L and 100 μmol/L concentrations, probably because PFD inhibited ea.hy926 cells from expressing VEGF, MMP-2 and MMP-9. PFD at a concentration of 100. mu. mol/L significantly inhibited scratch recovery; PFD at concentrations of 50 μmol/L and 100 μmol/L significantly reduced the number of transmembrane membranes of ea.hy926 cells, and the higher the concentration, the lower the number of transmembrane membranes, probably because PFD inhibited ea.hy926 cells from expressing MMP-2 and MMP-9.

The above are merely alternative embodiments of the present application and are not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement or the like made within the spirit and principle of the present application shall be included in the scope of the claims of the present application.

Claims (7)

1. An application of pirfenidone in preparing a medicament for preventing and treating rheumatoid arthritis, wherein the chemical structural formula of pirfenidone is shown as formula 1 below, or a salt shown as formula 1 below

2. The use according to claim 1, wherein said effective concentration of pirfenidone on cells is from 10umol/L to 1 mmol/L.

3. The use of claim 1 or 2, wherein the medicament is a pharmaceutical composition comprising pirfenidone as an active ingredient and pharmaceutically acceptable adjuvants.

4. Use according to claim 3, wherein the pharmaceutical composition is prepared as a medicament for oral or non-oral administration in the form of tablets, pills, capsules, granules, powders, liquids, emulsions, suspensions, ointments, injections, skin patches.

5. A medicine for preventing and treating rheumatoid arthritis is characterized by comprising pirfenidone, wherein the chemical structural formula of the pirfenidone is shown as formula 1 below, or a salt shown as formula 1 below

6. The medicament of claim 5, further comprising a pharmaceutically acceptable adjuvant.

7. The medicament of claim 5 or 6, wherein the medicament is in the form of one of tablets, pills, capsules, granules, powders, liquids, emulsions, suspensions, ointments, injections and skin patches.

Images