Use of secreted frizzled related protein in preparation of arthritis treating drugs and arthritis treating drugs

Abstract

The application provides application of a secreted frizzled-related protein in preparation of an arthritis treatment drug and the arthritis treatment drug, and belongs to the technical field of biotechnology. The application research finds that the secreted frizzled-related protein has a good arthritis treatment effect, can significantly up-regulate expression levels of a type II collagen marker COL2A1 and a SOX family transcription factor SOX5 in damaged chondrocytes, and realizes strengthening of chondrocyte proliferation ability by promoting expression of a cell cycle related protein Ki67. Meanwhile, the SFRP can target and regulate a human synovial macrophage polarization phenotype, significantly inhibit expression of an iNOS marker of a proinflammatory M1 macrophage, and then reduce release of inflammatory factors in an inflammatory microenvironment of synovial tissue, target and regulate synovial inflammation, and significantly relieve joint pain.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the application of secretory coil-related proteins in the preparation of arthritis treatment drugs and arthritis treatment drugs. Background Technology

[0002] Arthritis is the most common painful and debilitating disease among the elderly, characterized by loss of articular cartilage, subchondral bone dysfunction, and synovitis. Articular cartilage allows for painless bone movement, but severe cartilage damage caused by age-related degeneration or overuse rarely heals. Current treatment interventions primarily focus on short-term symptom relief, and almost all patients eventually require joint replacement surgery. With the increasing aging of my country's population, the incidence of arthritis is rising annually, placing a heavy burden on socioeconomic and medical resources. Therefore, developing drugs that effectively relieve joint pain and range of motion, ultimately achieving cartilage regeneration, is crucial for improving the quality of life for elderly patients and reducing the socioeconomic and medical burden.

[0003] While traditional arthritis treatments can alleviate pain and inflammation to some extent, they often have limitations. For example, medication may cause side effects and is not very effective in stopping disease progression; physical therapy usually only provides temporary relief and cannot fundamentally repair damaged joint tissue. Therefore, research into treatments for osteoarthritis has emerged.

[0004] The WNT signaling pathway plays a crucial role in joint development, formation, and maintenance. During the embryonic period, mesenchymal cells derived from the lateral mesoderm migrate and aggregate under the highly coordinated interaction of several signaling molecules, including fibroblast growth factors (FGFs, such as FGF8), Indian hedgehog protein (IHH), bone morphogenetic protein (BMP), and the Wnt pathway. They differentiate into chondrocytes expressing genes such as SOX9 and COL2, forming cartilage plates that persist throughout the fetal period. As the cartilage plates continue to grow, WNT and IHH downregulate the expression of type II collagen in chondrocytes and initiate the expression of type X collagen and alkaline phosphatase, resulting in the emergence of mast chondrocytes that secrete VEGF, IHH, and RANK. The extracellular matrix gradually mineralizes and degrades, followed by the infiltration of blood vessels, osteoclasts, and osteoblasts, leading to endochondral ossification and the formation of the skeletal outline. After undergoing joint cavitation, a joint is formed. Throughout joint development and maintenance, the Wnt signaling pathway is precisely spatiotemporally regulated, maintaining a balanced state that governs chondrocyte hypertrophy and osteoblast proliferation, playing a crucial role in cartilage formation and the initiation and maintenance of endochondral ossification. During arthritis, Wnt signaling in chondrocytes is overactivated, promoting inflammation, aging, hypertrophy, osteoblast differentiation, and osteophyte formation, thus contributing to osteoarthritis (OA). Therefore, maintaining low Wnt levels is essential for chondrocyte identity and preventing hypertrophy. Targeting the overactive Wnt signaling pathway during OA has become a powerful and promising strategy for the prevention and treatment of OA.

[0005] Therefore, developing new, efficient, and safe treatments for arthritis has become an urgent need in the medical field. Summary of the Invention

[0006] The purpose of this invention is to provide an application of secretory spherical coil-related protein in the preparation of arthritis treatment drugs and the arthritis treatment drugs, wherein SFRP can significantly reduce synovial inflammation, significantly slow down the degree of cartilage degeneration, promote chondrocyte proliferation, restore cartilage structure, and pathological examination has revealed obvious cartilage regeneration, providing a new treatment strategy for intervening in the pathological process of arthritis.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of a secretory coil-related protein in the preparation of an arthritis treatment drug.

[0009] Preferably, the secretory curl-related protein includes one of secretory curl-related protein 1, secretory curl-related protein 2, secretory curl-related protein 3, and secretory curl-related protein 4.

[0010] The present invention also provides an arthritis treatment drug comprising secreted coil-related protein.

[0011] Preferably, the arthritis treatment drug includes drugs that reduce articular cartilage damage, promote cartilage regeneration, or promote collagen regeneration.

[0012] Preferably, the arthritis treatment drug may be added with a pharmaceutically acceptable carrier.

[0013] Preferably, the dosage form of the arthritis treatment drug is one of the following: powder, solution, capsule, granule, tablet, emulsion, or suspension.

[0014] The beneficial effects of this invention compared to the prior art are as follows:

[0015] (1) This invention found that secreted coil-related protein has a good therapeutic effect on arthritis. It can significantly upregulate the expression levels of type II collagen marker COL2A1 and SOX family transcription factor SOX5 in damaged chondrocytes, and enhance the proliferation capacity of chondrocytes by promoting the expression of cell cycle-related protein Ki67. At the same time, SFRP can target and regulate the polarization phenotype of human synovial macrophages, significantly inhibit the expression of pro-inflammatory M1 macrophage marker iNOS, and thus reduce the release of inflammatory factors in the inflammatory microenvironment of synovial tissue.

[0016] (2) The SFRP of the present invention can significantly upregulate signaling pathways such as cartilage formation and collagen formation, and downregulate signaling pathways such as Wnt and osteogenic differentiation. It can also significantly reduce synovial inflammation and slow down the degree of cartilage degeneration by inhibiting abnormal proliferation of synovial fibroblasts and infiltration of inflammatory cells, promote chondrocyte proliferation, restore cartilage structure, and pathological examination has revealed obvious cartilage regeneration. The treatment method is simple, low-cost, and effective, providing a new treatment strategy for the intervention of the pathological process of arthritis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Calcein / PI staining was used to verify the function of SFRP2 in primary rat chondrocytes after injury.

[0019] Figure 2 For the detection of pain level in arthritis rats after SFRP2 treatment;

[0020] Figure 3 Macroscopic images of joints in rats with osteoarthritis after SFRP2 treatment;

[0021] Figure 4 H&E and safranin staining of joints in rats with arthritis after SFRP2 treatment;

[0022] Figure 5 The signaling pathways in cartilage are upregulated after SFRP2 treatment;

[0023] Figure 6 The signaling pathways in cartilage were downregulated after SFRP2 treatment;

[0024] Figure 7 For the in vitro human chondrocyte injury after SFRP1 function verification in Example 1 of this invention, COL2A1 and SOX5 staining were used for identification.

[0025] Figure 8 Ki67 staining was used to verify the function of SFRP1 in human chondrocytes after injury in vitro in Example 1 of this invention.

[0026] Figure 9 This is an iNOS staining image used to verify the function of SFRP1 in human synovial macrophages after in vitro treatment in Example 1 of this invention.

[0027] Figure 10 This is a Safranin O staining image of the joints of rats in the SFRP1 and saline groups in Experiment Example 2 of this invention;

[0028] Figure 11 This is a toluidine blue staining image of the joints of rats in the SFRP1 and saline groups in Experiment Example 2 of this invention;

[0029] Figure 12 The figures show the results of joint swelling and range of motion measurements before, after, and after treatment in Experiment Example 2 of this invention.

[0030] Figure 13 These are ultrasound images of the cynomolgus monkey joints before, after, and after treatment in Embodiment 2 of the present invention.

[0031] Figure 14 These are MRI scans and PD-FS images of the joints before, after, and after treatment in Embodiment 2 of the present invention.

[0032] Figure 15 These are PD-FS images of the longitudinal MRI scans of the joint before, after, and after treatment in Embodiment 2 of the present invention.

[0033] Figure 16 These are T1 images of the joints before, after, and after treatment in Embodiment 2 of the present invention.

[0034] Figure 17 These are T1 images of longitudinal MRI scans of the joint before modeling, after modeling, and after treatment in Embodiment 2 of the present invention. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example 1

[0041] Example 1 of this invention tested the therapeutic effect of secretory curl-related protein 2, and the specific steps are as follows:

[0042] (1) Ten male SD rats aged 7-8 weeks (Kunming University of Science and Technology, Institute of Primate Translational Medicine) were raised in an SPF-grade environment;

[0043] (2) Main reagents

[0044] Sodium iodoacetate: purchased from ACROS ORGANICS, a subsidiary of Thermo Fisher Scientific; 0.9% saline solution: purchased from Guangxi Yuyuan Pharmaceutical Co., Ltd.

[0045] 30% H2O2: Purchased from Thermo Fisher Scientific.

[0046] Hot and cold plate pain meter: purchased from Beijing Zhongshi Technology, model: ZS-CTE

[0047] (3) In vitro evaluation of the effect of SFRP1 on rat primary chondrocytes

[0048] Rat chondrocytes were used at a concentration of 1×10⁻⁶. 5 Primary rat chondrocytes were seeded at a density of 1,000 μmol / L in 6-well plates. When the cell confluence reached 70%-80%, H2O2 (500 μmol / L) was added to the culture medium for 24 hours. After 24 hours, the H2O2 was removed, and SFRP2 (56 ng / mL) was added to the culture medium. After 48 hours, the primary rat chondrocytes were stained with Calcein / PI to assess cell viability. The results are as follows: Figure 1 As shown.

[0049] Figure 1 The results showed that H2O2 injury caused massive chondrocyte death in rats (PI positive), but 72 hours after SFRP2 treatment, chondrocyte proliferation significantly increased (Calcein positive). Figure 1 Therefore, SFRP2 significantly promotes the survival and proliferation of chondrocytes after injury.

[0050] (2) Establishment and evaluation of rat arthritis model

[0051] Ten 7-week-old male SD rats were acclimatized for two weeks, then anesthetized with 2% isoflurane. After anesthesia, the rats' knee joints were disinfected with povidone-iodine. Sodium iodoacetate (18 mg / kg, dissolved in 0.9% physiological saline to a concentration of 0.25 μL) was injected into the joint cavity to induce knee arthritis in the rats.

[0052] Two weeks after sodium monoiodide injection, 10 arthritic rats were randomly divided into two groups. One group received intra-articular injection of SFRP2 (25 μg / kg, 100 μL); the other group received an equal volume of physiological saline intra-articular injection. Pain intensity was assessed using hot and cold plate tests. The results are as follows: Figure 2 As shown.

[0053] Figure 2The results showed that H2O2 injury significantly increased joint pain in rats, manifested as prolonged response time to hot and cold plate stimuli. After SFRP2 treatment, arthritic rats experienced significant pain relief, manifested as rapid response to hot and cold plate stimuli.

[0054] Eight weeks later, rats were euthanized using carbon dioxide, and knee joints were collected to assess the histological characteristics of osteoarthritis (OA) and synovitis. Paraffin sections of the left and right joints were fixed with 4% PFA and 10% formalin, respectively. Samples were embedded in paraffin and then cut into 4–6 mm thick sections. Morphology of regenerated cartilage was assessed using safranin O and toluidine blue staining according to standard procedures. Results are shown below. Figure 3 , 4 As shown.

[0055] Figure 3 , 4 The results showed that an osteoarthritis model in rats was established using iodoacetate. The saline injection group exhibited severe joint wear, while intra-articular injection of SFRP2 significantly reduced articular cartilage damage. Figure 3 Pathological analysis, including H&E and Safranin O staining, confirmed the disappearance of articular cartilage and severe synovial hyperplasia in the saline group, while cartilage regeneration was observed in the SFRP2 treatment group. Figure 4 ).

[0056] Transcriptomic analysis was performed on cartilage samples from the normal control group, the saline treatment group, and the SFRP2 treatment group to elucidate the molecular mechanism of SFRP2 treatment for osteoarthritis in rats. The results are as follows: Figure 5 , 6 As shown.

[0057] Figure 5 , 6 Transcriptomic analysis showed that SFRP2 treatment significantly upregulated signaling pathways such as chondrogenesis and collagen formation, and significantly downregulated signaling pathways such as Wnt and osteogenic differentiation. Therefore, SFRP2 can effectively treat arthritis in rats.

[0058] Example 2

[0059] Example 2 of this invention tested the effect of secretory curl-related protein 1 (SFRP1), and the specific steps are as follows:

[0060] (1) Ten male SD rats aged 7-8 weeks were housed in an SPF-grade environment; three cynomolgus monkeys aged 10-12 years were free from B herpesvirus, simian retrovirus, simian leukemia virus and immunodeficiency virus infection. Both rats and cynomolgus monkeys were obtained from the Institute of Primate Translational Medicine, Kunming University of Science and Technology.

[0061] (2) Main reagents

[0062] Sodium iodoacetate: purchased from ACROS ORGANICS, a subsidiary of Thermo Fisher Scientific; 0.9% saline solution: purchased from Guangxi Yuyuan Pharmaceutical Co., Ltd.

[0063] Human chondrocyte cell line: purchased from Pronosei Life Sciences Co., Ltd.

[0064] Human synovial macrophage cell line: purchased from Shanghai Cell Bank.

[0065] Lipopolysaccharide (LPS): Purchased from Thermo Fisher Scientific.

[0066] 30% H2O2: Purchased from Thermo Fisher Scientific.

[0067] (3) In vitro evaluation of the effect of SFRP1 on human chondrocytes

[0068] Human chondrocytes (Pricela, China) were used at a concentration of 1×10⁻⁶. 5 Human chondrocytes were seeded at a density of 1,000 μmol / L in 6-well plates. When the cell confluence reached 70%-80%, the culture medium was treated with H2O2 (500 μmol / L) for 24 hours. After 24 hours, the H2O2 was removed, and SFRP1 (20 g / L) was added to the culture medium. After 48 hours, the human chondrocytes were subjected to COL2A1, SOX5, and Ki67 immunofluorescence staining to assess the number and proliferative capacity of the proliferating chondrocytes. The results are as follows: Figure 7 , 8 As shown.

[0069] Figure 7 , 8 The results showed that after H2O2 injury, the number of positive cells for type II collagen (COL2A1) and the key transcription factor SOX5 in cartilage development was significantly reduced, but 48 hours after SFRP1 treatment, the number of positive cells for COL2A1 and SOX5 was significantly increased. Figure 7 Therefore, SFRP1 significantly increased the expression of COL2A1 and SOX5 in chondrocytes after injury. Simultaneously, Ki67 staining, a typical marker for chondrocyte proliferation, also revealed a significant decrease in the number of Ki67-positive cells after H2O2 injury, while SFRP1 treatment significantly increased Ki67 expression. Figure 8 Therefore, SFRP1 can promote the proliferation of damaged chondrocytes.

[0070] Human synovial macrophages (BLUEFBIO, China) were used at a rate of 1×10⁻⁶. 5Human chondrocytes were seeded at a density of 1,000 cells per well in 6-well plates. When the cell confluence reached 70%-80%, LPS (20 μg / mL) was added to the culture medium and the cells were treated for 24 hours. After 24 hours, LPS was removed and SFRP1 (20 μg / mL) was added to the culture medium. After 48 hours, iNOS immunofluorescence staining was performed on nitric oxide synthase, a key marker of human chondrocyte M1 macrophages, to evaluate the anti-inflammatory effect of SFRP1 on synovial macrophages.

[0071] Specifically, cells were fixed with 4% PFA for 10 minutes, permeabilized with 0.25% PBST for 30 minutes, blocked with 2% BSA for 1 hour, washed with PBS, and incubated overnight with primary antibody. The next day, the primary antibody was washed off, secondary antibody was added, and the cells were incubated at room temperature for 1 hour. Finally, confocal microscopy was used for imaging and examination. The results are as follows: Figure 9 As shown.

[0072] Figure 9 The results showed that the expression level of iNOS in normal human synovial macrophages was very low. After LPS treatment, synovial macrophages transformed into pro-inflammatory M1 macrophages, exhibiting high expression of iNOS. However, after SFRP1 treatment, the expression level of iNOS was significantly reduced. Figure 9 Therefore, SFRP1 can significantly inhibit the differentiation of synovial macrophages into M1 cells, thereby suppressing synovial inflammation.

[0073] (4) Establishment and evaluation of rat arthritis model

[0074] Ten 7-week-old male SD rats were acclimatized for two weeks, then anesthetized with 2% isoflurane. After anesthesia, the rats' knee joints were disinfected with povidone-iodine. Sodium iodoacetate (18 mg / kg, dissolved in 0.9% physiological saline to a concentration of 0.25 μL) was injected into the joint cavity to induce knee arthritis in the rats.

[0075] Two weeks after sodium monoiodide injection, 10 arthritic rats were randomly divided into two groups. One group received intra-articular injection of SFRP1 (30 g / L, 100 μL); the other group received an equal volume of physiological saline. Eight weeks later, the rats were euthanized using carbon dioxide, and knee joints were collected to assess the histological characteristics of osteoarthritis (OA) and synovitis. Paraffin sections of the left and right joints were fixed with 4% PFA and 10% formalin, respectively. The samples were embedded in paraffin and then cut into 4-6 mm thick sections. Morphology of regenerated cartilage was assessed using safranin O and toluidine blue staining according to standard procedures. The results are as follows: Figure 10 , 11 As shown.

[0076] Figure 10 , 11The results showed that SFRP1 injection significantly reduced synovitis and cartilage damage in an OA rat model. After SFRP1 treatment, synovial hyperplasia and inflammatory infiltration were significantly reduced, while the saline injection group showed severe synovitis and hyperplasia (black arrows) and cartilage defects (blue arrows). Toluidine blue and Safranin O staining confirmed that after SFRP1 injection, cartilage structure was restored and the tidal line was clearly visible (red arrows). Therefore, SFRP1 is an effective treatment for arthritis in rats.

[0077] (5) Establishment and evaluation of cynomolgus monkey arthritis model

[0078] Weigh sodium iodoacetate according to the animal's weight using an analytical balance, at a dose of 18 mg / kg / dose / joint. Dissolve the sodium iodoacetate in 0.9% physiological saline to a concentration of 0.25 μL. Gently shake to mix thoroughly. Anesthetize the animal with ketamine at a dose of 15 mg / kg. Disinfect the knee joint with povidone-iodine after anesthesia. Inject the prepared sodium iodoacetate into the knee joint cavity using a 450 nm diameter syringe under ultrasound guidance. After injection, press the injection site with an alcohol swab. Observe the animals for one month after sodium iodoacetate injection, monitoring the progression and spontaneous healing of osteoarthritis in the cynomolgus monkeys. One month later, assess the range of motion and swelling of the joints using ultrasound and MRI to evaluate model establishment.

[0079] One month after sodium iodoacetate injection, successful modeling was confirmed by ultrasound and MRI. Three cynomolgus monkeys were then injected with SFRP1 (30 g / L, 1 mL) into their left joints, and an equal volume of saline into their right joints. They were observed for two months. Two months later, the range of motion, swelling, and ultrasound and MRI scans of the cynomolgus monkeys' joints were performed to evaluate the treatment effect. The results are as follows: Figures 12-17 As shown.

[0080] Figures 12-17 The results showed that one month after iodoacetate injection, joint swelling in cynomolgus monkeys increased significantly, and joint mobility decreased significantly. Two months after SFRP1 treatment, joint swelling decreased significantly, and joint mobility increased. Figure 12 Ultrasound examination revealed that normal hyaline cartilage is a uniform anechoic layer lining the bone cortex, with thin and sharp edges, exhibiting continuous and regular hyperechoic appearance. One month after injection of monoiodoacetate, focal and asymmetrical thinning of the knee joints was observed in all three monkeys. However, two months after SFRP1 injection, significant femoral smoothing and cartilage regeneration were observed. Figure 13 MRI scans revealed severe tibial edema and synovial hyperplasia in animals after intra-articular injection of monoiodoacetate (red arrow, pd-fs). Figure 14 , Figure 15 ) and thinning or degeneration of articular cartilage (T1) Figure 16 , Figure 17 Two months after SFRP1 injection (PD-FS), synovial hyperplasia effectively subsided, bone edema significantly improved, and T1 signal showed obvious cartilage signal (yellow arrow). However, in the saline group, synovial hyperplasia remained severe, joint space narrowed, and no cartilage signal was observed (red arrow). Figure 14 , Figure 15 , Figure 16 , Figure 17 ).

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a secreted coil-related protein in the preparation of an arthritis treatment drug, characterized in that, The secretory curl-related protein is either secretory curl-related protein 1 or secretory curl-related protein 2, and the arthritis treatment drug is a drug that reduces articular cartilage damage and promotes cartilage regeneration.

2. The application according to claim 1, characterized in that, The arthritis treatment drug is supplemented with a pharmaceutically acceptable carrier.

3. The application according to claim 1, characterized in that, The dosage form of the arthritis treatment drug is one of the following: powder, solution, capsule, granule, tablet, emulsion, or suspension.

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