Application of SEMA3E protein in preparation of medicine for treating rheumatoid arthritis and promoting cartilage regeneration

Abstract

The invention provides application of SEMA3E protein in preparation of medicines for treating rheumatoid arthritis and promoting cartilage regeneration, and belongs to the technical field of biological medicines. The amino acid sequence of the SEMA3E protein is as shown in SEQ ID No. 1. A research result shows that after the SEMA3E protein solution is injected for one month, the swelling degree of joints of double hind limbs of an animal model is reduced, the joint space and integrity are recovered to be normal, the cartilage thickness is increased, and the phenomena that synovial cells infiltrate and invade cartilage are eliminated. That is, the SEMA3E protein can well relieve articular cavity inflammation and improve damaged joints.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of SEMA3E protein in preparing drugs for treating rheumatoid arthritis and promoting cartilage regeneration. Background Art

[0002] Osteoarticular diseases are a general term for conditions characterized by structural destruction and dysfunction of joints. Typical symptoms include joint pain, swelling, stiffness, and limited mobility, which can lead to deformity and disability in severe cases. Based on etiology, they can be categorized as degenerative (e.g., osteoarthritis), inflammatory (e.g., rheumatoid arthritis), metabolic (e.g., gouty arthritis), and traumatic. Rheumatoid arthritis (RA), a systemic autoimmune disease that has become increasingly prevalent in recent years, has a pathogenesis involving multiple factors, including genetic susceptibility, pathogen infection, environmental exposure, and abnormal immune regulation. This condition not only causes irreversible functional impairment but also carries a significantly higher risk of mortality than the general population. Global epidemiological data show that the overall prevalence of RA has reached 1% and continues to rise, accompanied by an abnormally elevated mortality rate.

[0003] While conventional clinical medications can alleviate some symptoms, they have significant limitations in improving long-term outcomes. These include difficulty effectively halting disease progression, inability to completely reverse the pathological state, and the potential for complications such as gastrointestinal damage, cardiovascular toxicity, drug dependence, metabolic disorders, and weight loss. This often creates a dilemma where high treatment costs are mismatched with the expected efficacy. While total joint replacement surgery is an important treatment option for terminally ill patients, this invasive procedure carries inherent risks such as deep vein thrombosis and postoperative infection. This, combined with the limited lifespan of the prosthesis (typically 15-20 years), suboptimal motor function recovery, and the potential need for revision surgery, presents patients with multiple challenges, both physical and financial, and financially.

[0004] Currently, research on the repair and regeneration of cartilage damage secondary to RA still faces core challenges. While tissue engineering therapies such as microfracture technology and autologous chondrocyte transplantation based on animal models can achieve directed cartilage differentiation and generate repair tissue by recruiting bone marrow mesenchymal stem cells (MSCs), their effectiveness is critically limited by the fact that the abundance of MSCs in the bone marrow microenvironment is less than 0.001%. Clinical data show that knee joint function scores significantly decline 24 months after surgery. Histopathological analysis further revealed that the newly formed tissue primarily exhibits a fibrocartilage phenotype and lacks the stable expression of type II collagen, which is unique to native hyaline cartilage. This phenotypic shift leads to defects in both the mechanical properties and biological function of the repaired tissue.

[0005] In the field of cartilage regeneration research, existing strategies are mainly focused on two major directions: growth factor regulation and biomaterial development, but there are still significant limitations. Growth factor intervention studies have confirmed that molecules such as FGF-18 can exert a proliferative effect by activating the PI3K / AKT signaling pathway of chondrocytes, while inhibiting MMPs-mediated matrix degradation. However, the induced cartilage-like tissue has phenotypic heterogeneity, and long-term exposure may cause potential risks such as abnormal vascular infiltration. Although three-dimensional biomimetic scaffold technology improves the load capacity of new cartilage through mechanical adaptability design, the acidic metabolites released during the degradation of the scaffold can induce an imbalance in the local microenvironment homeostasis, which in turn accelerates the degeneration of the residual cartilage. These limitations show that there are still key bottlenecks in the research on secondary cartilage regeneration in RA, and the field of joint injury repair still needs new systematic treatment strategies.

[0006] Semaphorins (SEMAs), a family of classic axon guidance molecules, have expanded their biological functions from their initial role in neural guidance to diverse areas such as immune regulation, tumor microenvironment remodeling, and maintenance of metabolic homeostasis. Notably, while the SEMA family has been extensively explored in the fields of 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 the present invention is to provide the use 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 quickly improve the occurrence of inflammation and joint damage in their bodies.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides use of a SEMA3E protein in preparing a drug for treating rheumatoid arthritis. The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0009] Preferably, the effective concentration of the SEMA3E protein is 0.1-1.0 mg / ml.

[0010] The present invention also provides the use of SEMA3E protein in preparing a drug for promoting cartilage regeneration. The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0011] Preferably, the effective concentration of the SEMA3E protein is 0.1-1.0 mg / ml.

[0012] The present invention also provides a method for preparing a drug for treating rheumatoid arthritis, comprising the following steps: Mixing the SEMA3E protein with physiological saline to obtain the drug; The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0013] Preferably, the dosage form of the drug includes a solution.

[0014] The present invention also provides a method for preparing a drug for promoting cartilage regeneration, comprising the following steps: Mixing the SEMA3E protein with physiological saline to obtain the drug; The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0015] Preferably, the dosage form of the drug includes a solution.

[0016] The beneficial effects of the present invention compared with the prior art are: The present invention provides the use of SEMA3E protein in the preparation of a drug for treating rheumatoid arthritis and promoting cartilage regeneration. Experimental results show that the SEMA3E protein can quickly and effectively restore damaged joints to normal and complete state. Furthermore, by observing the swelling of the soles of the hind limbs of rats with rheumatoid arthritis and the degree of damage to the joint surfaces, and performing joint pathological staining and joint histochemical staining analysis, it was found that the SEMA3E protein can be used in rodents to quickly improve the occurrence of inflammation and joint damage in their bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 Flow chart for the treatment of rheumatoid arthritis rats with SEMA3E protein; Figure 2 The swelling of the hind paws of rats with rheumatoid arthritis; Figure 3 This is the damage of femoral cartilage in rats with rheumatoid arthritis; Figure 4 This is the synovial inflammation of the limb and knee joints of rats with rheumatoid arthritis; Figure 5 The figure shows the safranin fast green staining of the knee joints of rats with rheumatoid arthritis. Figure 6 Toluidine blue staining of the knee joint cartilage of rats with rheumatoid arthritis; Figure 7This is the result diagram of the preliminary experiment; Figure 8 The swelling of the hind limbs of rats with rheumatoid arthritis after injection of SEMA3E protein solution; Figure 9 This is the damage of femoral cartilage in the limbs of rats with rheumatoid arthritis after injection of SEMA3E protein solution; Figure 10 The synovial inflammation of the limb and knee joints of rheumatoid arthritis rats after injection of SEMA3E protein solution; Figure 11 The figure shows the safranin fast green staining of the knee joints of rheumatoid arthritis rats after injection of SEMA3E protein solution; Figure 12 Toluidine blue staining of the knee joint cartilage of rheumatoid arthritis rats after injection of SEMA3E protein solution. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] The present invention provides use of a SEMA3E protein in preparing a drug for treating rheumatoid arthritis. The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0025] In the present 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, further preferably 0.4-0.6 mg / ml, and even more preferably 0.5 mg / ml.

[0026] The present invention also provides the use of SEMA3E protein in preparing a drug for promoting cartilage regeneration. The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0027] In the present 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.

[0028] The present invention also provides a method for preparing a drug for treating rheumatoid arthritis, comprising the following steps: Mixing the SEMA3E protein with physiological saline to obtain the drug; The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0029] In the present invention, the dosage form of the drug preferably includes a solution.

[0030] The present invention also provides a method for preparing a drug for promoting cartilage regeneration, comprising the following steps: Mixing the SEMA3E protein with physiological saline to obtain the drug; The amino acid sequence of the SEMA3E protein is shown in SEQ ID No. 1.

[0031] In the present invention, the dosage form of the drug includes a solution.

[0032] Example 1 1.1 Determination of the rheumatoid arthritis rat model The collagen-induced arthritis (CIA) model, with its high degree of biomimetic pathology and immunology, has irreplaceable advantages in revealing disease mechanisms, screening therapeutic targets, and evaluating drug efficacy, and has become a classic modeling method in the field of RA research.

[0033] We established a rat RA model based on the classic CIA modeling method, as follows: This study used 6- to 8-week-old Sprague-Dawley (SD) rats weighing 260 to 280 grams, with six rats per group. Male Sprague-Dawley (purchased from the Experimental Animal Center of Kunming University of Science and Technology) were anesthetized with avertin (purchased from Nanjing Aibei Biotechnology Co., Ltd.) injected intraperitoneally. Animals were kept warm at all times during anesthesia. Subsequently, a mixed inducer (type II collagen (purchased from Chondrex) and complete Freund's adjuvant (purchased from Sigma) mixed at a volume ratio of 1:1 was injected subcutaneously at multiple sites on the back of the rats once weekly for two consecutive weeks. The inflammatory process was monitored weekly, and arthritis severity was assessed using footpad edema and arthritis scores: 0 = normal; 1 = mild edema and redness of the ankle joint; 2 = mild edema and redness from the ankle joint to the metacarpal or metacarpophalangeal joint; 3 = moderate edema and redness from the ankle joint to the metacarpal or metacarpophalangeal joint; and 4 = severe edema and stiffness of the ankle joint, making movement difficult. 28 days after modeling, the knee joints and soles of the rats were observed and photographed. After 28 days, SD model mice with obvious swelling, disease score ≥ 4 points, and joint flexion < 150° were included in the experiment. Four weeks later, the soles of the hind limbs of the model mice were observed, and the rats were killed to observe their hind limb knee joints, and then HE staining (staining reagent purchased from Wuhan Seville Biotechnology Co., Ltd.), safranin fast green staining (staining reagent purchased from Wuhan Seville Biotechnology Co., Ltd.), and toluidine blue staining (staining reagent purchased from Wuhan Seville Biotechnology Co., Ltd.) were performed for analysis. The results are as follows Figures 2 to 6 shown.

[0034] The results showed that compared with the untreated normal control group (Control group), the swelling of the model rats' paws increased significantly. When the rats were sacrificed and the knee joints were opened, the model rats' joints showed significant synovial hyperplasia compared with the Control group. HE staining revealed that the synovium in the hind limb joints of the model rats was infiltrated with a large number of immune cells, and a large amount of synovium invaded the cartilage tissue in the joints, resulting in obvious cartilage defects. Safranin fast green staining showed that the model rats' joints were damaged, the joint space was reduced, the cartilage surface was rough, and the synovium invaded the cartilage, causing damage. Toluidine blue staining showed that the expression of cartilage in the model rats was significantly reduced.

[0035] 1.2 Preparation of SEMA3E protein solution 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 SEMA3E protein is shown in SEQ ID No. 1) based on the animal's body weight using an analytical balance. Dissolve the SEMA3E lyophilized powder in 480 μl of 0.9% saline and gently shake to mix thoroughly to prepare the SEMA3E protein solution. Inject the prepared SEMA3E protein solution within 30 minutes.

[0036] 1.3 Preliminary Experiment 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) without modeling were selected for drug dosage verification. The specific operation is as follows: Each rat was subcutaneously injected with type II collagen (purchased from Chondrex) to establish a rheumatoid arthritis rat model. 28 days after modeling, it was determined that each joint of each rat had obvious swelling, a disease score ≥ 4 points, and a joint flexion < 150°. Subsequently, 25 μg / kg / joint of 1.2-prepared SEMA3E protein solution was injected into the left leg joint cavity, and 50 μg / kg / joint of 1.2-prepared SEMA3E protein solution was injected into the right leg joint cavity, once a week for a total of 2 weeks. Four weeks after the injection was completed, the knee flexion of the left and right joints of the treated rats was determined and compared with those before treatment to determine whether it was > 150°, the disease score was < 4 points, and the swelling of the hind limb soles was reduced. The results are as follows Figure 7 shown.

[0037] The results showed that the function of the right knee joint of the rats recovered well, with a stretching angle of >150°; the swelling of the hind limb soles was reduced more significantly; therefore, the final dosage of SEMA3E protein was 50 μg / kg / joint to ensure better improvement of the rheumatoid arthritis rat model.

[0038] 1.4 Injection Anesthetize the rats 28 days after modeling in 1.1 with 280 mg / kg of avertin. After anesthesia, disinfect the knee joints with iodine. Inject the SEMA3E protein solution prepared in 1.2 into the knee joint cavity at a dose of 50 μg / kg / joint using a 1 ml syringe with a 0.4 mm diameter. Inject once weekly for two weeks. After injection, press the needle opening with an alcohol swab.

[0039] At the same time, a sham group injected with normal saline was set up, with 6 rats in each group.

[0040] 1.5 Therapeutic evaluation in a rat model of rheumatoid arthritis After the injection of SEMA3E protein solution, the animals were kept normally for one month to observe the treatment and improvement of the rheumatoid arthritis rat model. The swelling of the hind limbs was observed and photographed in the fourth week after the drug injection. Figures 8 to 12 shown.

[0041] Depend on Figure 8 It can be seen that the swollen paws of rats injected with SEMA3E protein solution returned to normal significantly compared to the Sham group injected with saline. Figure 9 It can be seen that after the treatment, the rats were killed and the knee joints of the hind limbs were opened. Observation of the cartilage surface showed that compared with the Sham group, there was no invading synovium on the cartilage surface in the SEMA3E-treated group, the cartilage damage was reduced, and the cartilage surface was intact and smooth. Figure 10 It can be seen that HE staining found that the infiltration of a large number of immune cells in the synovial membrane of the hind limb joints decreased, the synovial membrane was loose and the cells were evenly distributed. Figure 11 and Figure 12 Safranin fast green staining revealed intact joint surfaces, increased joint space, and smooth cartilage. Toluidine blue staining showed that cartilage expression had returned to normal levels. These results indicate that one month after SEMA3E protein injection, joint swelling in both hind limbs of the animal model decreased, joint space and integrity returned to normal, cartilage thickness increased, and synovial cell infiltration and invasion of the cartilage subsided. SEMA3E protein effectively reduces joint inflammation and improves damaged joints.

[0042] As can be seen from the above examples, the present invention provides the use 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 quickly improve the occurrence of inflammation and joint damage in the body.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of SEMA3E protein in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The amino acid sequence of the SEMA3E protein is shown in SEQ ID No.

1.

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

3. Use of SEMA3E protein in the preparation of a drug for promoting cartilage regeneration, characterized in that: The amino acid sequence of the SEMA3E protein is shown in SEQ ID No.

1.

4. The use according to claim 3, characterized in that The effective concentration of the SEMA3E protein is 0.1-1.0 mg / ml.

5. A method for preparing a drug for treating rheumatoid arthritis, characterized in that: The steps include: Mixing the SEMA3E protein with physiological saline to obtain the drug; The amino acid sequence of the SEMA3E protein is shown in SEQ ID No.

1.

6. The preparation method according to claim 5, characterized in that The dosage form of the drug includes a solution.

7. A method for preparing a drug for promoting cartilage regeneration, characterized in that: The steps include: Mixing the SEMA3E protein with physiological saline to obtain the drug; The amino acid sequence of the SEMA3E protein is shown in SEQ ID No.

1.

8. The preparation method according to claim 7, characterized in that The dosage form of the drug includes a solution.

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