Use of sema3e protein in preparation of drugs for treating rheumatoid arthritis and promoting cartilage regeneration

Abstract

The application provides application of SEMA3E protein in preparation of a drug for treating rheumatoid arthritis and promoting cartilage regeneration, and belongs to the technical field of biological medicine. The amino acid sequence of the SEMA3E protein is shown as SEQ ID No. 1. Research results show that one month after injection of the SEMA3E protein solution, the joint swelling degree of the double hind limbs of an animal model is reduced, the joint space and integrity are restored to normal, the cartilage thickness is increased, and the phenomenon of synovial cell infiltration and invasion into the cartilage is eliminated. That is, the SEMA3E protein can well reduce joint cavity inflammation and improve damaged joints.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of SEMA3E protein in the preparation of drugs for treating rheumatoid arthritis and promoting cartilage regeneration. Background Technology

[0002] Osteoarthritis is a general term for diseases characterized by joint structural destruction and functional impairment. Typical symptoms include joint pain, swelling, stiffness, and limited mobility; severe cases can lead to deformity and disability. Based on etiology, it can be classified into degenerative (e.g., osteoarthritis), inflammatory (e.g., rheumatoid arthritis), metabolic (e.g., gouty arthritis), and traumatic types. Among these, rheumatoid arthritis (RA), a systemic autoimmune disease that has become increasingly prevalent in recent years, involves multiple factors in its pathogenesis, including genetic susceptibility, pathogen infection, environmental exposure, and abnormal immune regulation. This disease not only causes irreversible functional damage but also carries a significantly higher risk of death than the general population. Global epidemiological data shows that the overall prevalence of RA has reached 1% and is showing a continuous upward trend, accompanied by an abnormally high mortality rate.

[0003] Currently, while conventional clinical medications can alleviate some symptoms, they have significant limitations in improving long-term prognosis. For example, they are difficult to effectively halt disease progression, cannot completely reverse the pathological state, and are prone to complications such as gastrointestinal damage, cardiovascular toxicity, drug dependence, metabolic disorders, and abnormal weight. This often results in a mismatch between high treatment costs and expected efficacy. For end-stage patients, while total joint replacement surgery is an important treatment, this invasive procedure itself carries risks such as deep vein thrombosis and postoperative infection. Furthermore, the limited lifespan of the prosthesis (usually 15-20 years), unsatisfactory recovery of motor function, and the potential need for revision surgery place multiple challenges on patients, both physically and financially.

[0004] Currently, research on the repair and regeneration of cartilage damage secondary to rheumatoid arthritis (RA) still faces core challenges. While tissue engineering therapies such as microfracture techniques based on animal models and autologous chondrocyte transplantation can recruit bone marrow mesenchymal stem cells (MSCs) to achieve directed differentiation of cartilage and generate repair tissue, their efficacy is critically limited by the fact that the abundance of MSCs in the bone marrow microenvironment is less than 0.001%. Clinical data show a significant decline in knee joint function scores 24 months post-surgery. Histopathological analysis further reveals that the newly formed tissue mainly exhibits a fibrocartilage phenotype, lacking the stable expression of type II collagen characteristic of natural hyaline cartilage. This phenotypic deviation leads to dual defects in the mechanical properties and biological function of the repaired tissue.

[0005] In the field of cartilage regeneration research, existing strategies mainly focus on two major directions: growth factor regulation and biomaterial development, but significant limitations remain. Growth factor intervention studies have confirmed that molecules such as FGF-18 can exert a proliferative effect by activating the PI3K / AKT signaling pathway in chondrocytes, while simultaneously inhibiting MMP-mediated matrix degradation. However, the cartilage-like tissues induced by these molecules exhibit phenotypic heterogeneity, and long-term exposure may lead to potential risks such as abnormal vascular infiltration. While three-dimensional biomimetic scaffold technology enhances the load-bearing capacity of newly formed cartilage through mechanically adaptive design, the acidic metabolites released during scaffold degradation can induce local microenvironmental homeostasis imbalance, thus accelerating the degeneration of residual cartilage. These limitations indicate that research on cartilage regeneration secondary to rheumatoid arthritis (RA) still faces key bottlenecks, and novel systemic treatment strategies are still needed in the field of joint injury repair.

[0006] As a classic family of axonal guidance molecules, semaphorins (SEMAs) have expanded their biological functions from their initial role in neural guidance to diverse areas such as immune regulation, tumor microenvironment remodeling, and metabolic homeostasis maintenance. Of particular interest is that, despite considerable research into the SEMA family's applications in immune regulation and tumor intervention, its direct mechanism of action in cartilage repair remains to be elucidated. Summary of the Invention

[0007] The purpose of this invention is to provide the application of SEMA3E protein in the preparation of drugs for treating rheumatoid arthritis and promoting cartilage regeneration. The SEMA3E protein can be used in rodents to rapidly improve the occurrence of inflammation and joint damage in their bodies.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides the use of SEMA3E protein in the preparation of a medicament for treating rheumatoid arthritis, wherein the amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0010] Preferably, the effective concentration of the SEMA3E protein is 0.1~1.0 mg / ml.

[0011] The present invention also provides the use of SEMA3E protein in the preparation of drugs that promote cartilage regeneration, wherein the amino acid sequence of SEMA3E protein is shown in SEQ ID No. 1.

[0012] Preferably, the effective concentration of the SEMA3E protein is 0.1~1.0 mg / ml.

[0013] This invention also provides a method for preparing a medicament for treating rheumatoid arthritis, comprising the following steps:

[0014] The drug was obtained by mixing SEMA3E protein with physiological saline.

[0015] The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0016] Preferably, the dosage form of the drug includes a solution.

[0017] This invention also provides a method for preparing a drug that promotes cartilage regeneration, comprising the following steps:

[0018] The drug was obtained by mixing SEMA3E protein with physiological saline.

[0019] The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0020] Preferably, the dosage form of the drug includes a solution.

[0021] The beneficial effects of this invention compared to the prior art are as follows:

[0022] This invention provides the application of SEMA3E protein in the preparation of drugs for treating rheumatoid arthritis and promoting cartilage regeneration. Experimental results show that the SEMA3E protein can rapidly and effectively restore damaged joints to normal and intact state. Furthermore, by observing the swelling of the hind limbs and the degree of damage to the joint surface in rats with rheumatoid arthritis, and performing joint pathological staining and joint histochemical staining analysis, it was found that the SEMA3E protein can be used in rodents to rapidly improve the occurrence of inflammation and joint damage in their bodies. Attached Figure Description

[0023] 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.

[0024] Figure 1 A flowchart illustrating the treatment of rheumatoid arthritis rats with SEMA3E protein;

[0025] Figure 2 The swelling of the hind limb paws in rats with rheumatoid arthritis;

[0026] Figure 3 The extent of cartilage damage in the femoral limbs of rats with rheumatoid arthritis;

[0027] Figure 4 The synovial inflammation of the knee joint in rats with rheumatoid arthritis;

[0028] Figure 5 Safranin and Fast Green staining of the knee joints of rats with rheumatoid arthritis;

[0029] Figure 6 Toluidine blue staining of the knee joint cartilage in rats with rheumatoid arthritis;

[0030] Figure 7 The results are shown in the preliminary experiment.

[0031] Figure 8 The swelling of the hind limb paws in rats with rheumatoid arthritis after injection of SEMA3E protein solution;

[0032] Figure 9 The extent of cartilage damage in the femoral limbs of rats with rheumatoid arthritis after injection of SEMA3E protein solution;

[0033] Figure 10 The synovial inflammation of the knee joint in rats with rheumatoid arthritis after injection of SEMA3E protein solution;

[0034] Figure 11 Safranin and Fast Green staining of the knee joints of rheumatoid arthritis rats after injection of SEMA3E protein solution;

[0035] Figure 12 Toluidine blue staining of the knee joint cartilage of rheumatoid arthritis rats after injection of SEMA3E protein solution. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] This invention provides the use of SEMA3E protein in the preparation of a medicament for treating rheumatoid arthritis, wherein the amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0042] In this invention, the amino acid sequence of the SEMA3E protein is ALDRTEERLAYGIESNSTLLECTPRSLQAKVIWFVQKGRDVRKEEVKTDDRVVKMDLGLLFLRVRKSDAGTYFCQTVEHNFVHTVRKIT (SEQ ID No. 1), and the effective concentration of the SEMA3E protein is preferably 0.1~1.0 mg / ml, more preferably 0.2~0.8 mg / ml, even more preferably 0.4~0.6 mg / ml, and still more preferably 0.5 mg / ml.

[0043] The present invention also provides the use of SEMA3E protein in the preparation of drugs that promote cartilage regeneration, wherein the amino acid sequence of SEMA3E protein is shown in SEQ ID No. 1.

[0044] In this invention, the effective concentration of the SEMA3E protein is preferably 0.1~1.0 mg / ml, more preferably 0.2~0.8 mg / ml, even more preferably 0.4~0.6 mg / ml, and even more preferably 0.5 mg / ml.

[0045] This invention also provides a method for preparing a medicament for treating rheumatoid arthritis, comprising the following steps:

[0046] The SEMA3E protein was mixed with physiological saline to obtain the drug;

[0047] The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0048] In this invention, the dosage form of the drug preferably includes a solution.

[0049] This invention also provides a method for preparing a drug that promotes cartilage regeneration, comprising the following steps:

[0050] The drug was obtained by mixing SEMA3E protein with physiological saline.

[0051] The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0052] In this invention, the dosage form of the drug includes a solution.

[0053] Example 1

[0054] 1.1 Establishment of a rat model of rheumatoid arthritis

[0055] Collagen-induced arthritis (CIA) models, with their high degree of biomimicry in pathology and immunology, have irreplaceable advantages in revealing disease mechanisms, screening therapeutic targets, and evaluating drug efficacy, and have become a classic modeling method in the field of RA research.

[0056] We established a rat RA model by referring to the classic CIA modeling method, as follows:

[0057] This study used SD rats aged 6 to 8 weeks, weighing between 260 and 280 grams. Six rats were used in each group. Male SD rats (purchased from the Experimental Animal Center of Kunming University of Science and Technology) were anesthetized by abdominal injection of Afodin (purchased from Nanjing Aibei Biotechnology Co., Ltd.). Animals were kept warm during anesthesia. Subsequently, a mixed inducing agent (a 1:1 volume mixture of type II collagen (purchased from Chondrex) and complete Freund's adjuvant (purchased from Sigma)) was injected subcutaneously at multiple sites on the rat's back once a week for two weeks. The inflammatory process was monitored weekly, and the severity of arthritis was assessed based on a paw pad edema and arthritis score: 0 = normal; 1 = mild ankle edema and redness; 2 = mild edema and redness from the ankle to the metacarpal or metacarpophalangeal joints; 3 = moderate edema and redness from the ankle to the metacarpal or metacarpophalangeal joints; 4 = severe ankle edema and stiffness, with difficulty in movement. Twenty-eight days after modeling, the knee joints and paws of rats were characterized and photographed. SD model rats with significant swelling, a disease score ≥4, and joint flexion <150° were included in the experiment after 28 days. Four weeks later, the hind limb paws of the model rats were observed, and the rats were sacrificed to observe their hind limb knee joints. HE staining (staining reagents purchased from Wuhan Sewell Biotechnology Co., Ltd.), safranin-fast green staining (staining reagents purchased from Wuhan Sewell Biotechnology Co., Ltd.), and toluidine blue staining (staining reagents purchased from Wuhan Sewell Biotechnology Co., Ltd.) were then performed for analysis. Results are as follows: Figures 2 to 6 As shown.

[0058] The results showed that, compared with the untreated normal control group (Control group), the paw swelling of the model rats was significantly increased. Upon euthanizing the rats and opening the hind limb knee joints, significant synovial hyperplasia was observed in the model rats compared to the Control group. HE staining revealed extensive immune cell infiltration in the synovium of the hind limb joints, with substantial synovial invasion of cartilage tissue and significant cartilage defects. Safranin-Fix-Green staining showed joint destruction, reduced joint space, rough cartilage surface, and damage caused by synovial invasion. Toluidine blue staining showed a significant reduction in cartilage expression in the model rats.

[0059] 1.2 Preparation of SEMA3E protein solution

[0060] Weigh 45 μg (25 μg / kg / joint) and 90 μg (50 μg / kg / joint) of SEMA3E lyophilized powder (purchased from Abbexa, the amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1) using an analytical balance based on animal body weight. Dissolve the SEMA3E lyophilized powder in 480 μl of 0.9% physiological saline, and gently shake to mix thoroughly to obtain a SEMA3E protein solution. The prepared SEMA3E protein solution should be injected within 30 minutes.

[0061] 1.3 Preliminary Experiment

[0062] To verify the dosage of SEMA3E protein, three 6-8 week old male SD rats (purchased from the Experimental Animal Center of Kunming University of Science and Technology) that had not undergone modeling were selected for drug dosage verification. The specific procedures are as follows:

[0063] A rat model of rheumatoid arthritis was established by subcutaneous injection of type II collagen (purchased from Chondrex). After 28 days of modeling, each rat exhibited significant swelling in every joint, a disease score ≥4, and joint flexion <150°. Subsequently, the left leg joint was injected intra-articularly with 25 μg / kg / joint of a 1.2-prepared SEMA3E protein solution, and the right leg joint was injected intra-articularly with 50 μg / kg / joint of a 1.2-prepared SEMA3E protein solution, once weekly for two weeks. Four weeks after the injections, the rats were compared with pre-treatment levels to determine whether the knee flexion of both joints was >150°, the disease score <4, and the reduction in hind limb paw swelling. Results are as follows: Figure 7 As shown.

[0064] The results showed that the rats' right knee joints recovered better, with a stretching angle >150°; the swelling of the hind limb paws was reduced more significantly; therefore, the final dosage of SEMA3E protein was selected as 50 μg / kg / joint to ensure better improvement in the rat model of rheumatoid arthritis.

[0065] 1.4 Injection

[0066] Rats were anesthetized with 280 mg / kg afodin 28 days after the model was established in step 1.1. The knee joints were then disinfected with povidone-iodine after anesthesia. Using a 1 ml syringe with a diameter of 0.4 mm, the prepared SEMA3E protein solution (step 1.2) was injected into the knee joint cavity at a dose of 50 μg / kg / joint. Injections were given once a week for two weeks. After injection, the injection site was pressed with an alcohol swab.

[0067] Meanwhile, a Sham group was set up, with 6 animals in each group receiving saline injections.

[0068] 1.5 Treatment evaluation of a rat model of rheumatoid arthritis

[0069] Animals were fed normally for one month after injection of SEMA3E protein solution to observe the treatment and improvement of a rat model of rheumatoid arthritis. Swelling of the hind limbs was observed and photographed at the fourth week after drug injection. Results are as follows: Figures 8 to 12 As shown.

[0070] Depend on Figure 8It was found that, compared with the Sham group injected with saline, the swollen paws of rats injected with SEMA3E protein solution significantly recovered to normal. Figure 9 It was found that after euthanizing the treated rats and opening the knee joint of the hind limbs, observation revealed reduced synovial hyperplasia; cartilage surface observation showed that, compared with the Sham group, the SEMA3E treatment group had no intrusive synovial membrane on the cartilage surface, reduced cartilage damage, and an intact and smooth cartilage surface. Figure 10 It can be seen that HE staining revealed a decrease in the large-scale infiltration of immune cells in the synovium of the hind limb joints, and the synovium was loose with uniformly distributed cells. Figure 11 and Figure 12 Safranin-Fix Green staining showed that the joint surface was intact, the joint space increased, and the cartilage was smooth. Toluidine Blue staining showed that cartilage expression returned to normal levels. These results indicate that one month after injection of SEMA3E protein solution, the swelling of the hind limb joints in the animal model decreased, the joint space and integrity returned to normal, cartilage thickness increased, and synovial cell infiltration and invasion of the cartilage subsided. SEMA3E protein can effectively reduce intra-articular inflammation and improve damaged joints.

[0071] As can be seen from the above embodiments, the present invention provides the application of SEMA3E protein in the preparation of drugs for treating rheumatoid arthritis and promoting cartilage regeneration. The SEMA3E protein can be used in rodents and can rapidly improve the occurrence of inflammation and joint damage in vivo.

[0072] 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 SEMA3E protein in the preparation of drugs for treating rheumatoid arthritis, characterized in that, The amino acid sequence of the SEMA3E protein is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The effective concentration of the SEMA3E protein is 0.1~1.0 mg / ml.

3. A method for preparing a medicament for treating rheumatoid arthritis, characterized in that, Includes the following steps: The drug was obtained by mixing SEMA3E protein with physiological saline. The amino acid sequence of the SEMA3E protein is shown in SEQ ID No.

1.

4. The preparation method according to claim 3, characterized in that, The dosage form of the drug includes a solution.

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