Use of ufm1 protein or its encoding gene in preparation of medicine for treating osteoarthritis

Abstract

The application discloses application of UFM1 protein or a coding gene thereof in preparation of a medicine for treating osteoarthritis, and belongs to the technical field of biotechnology.The application proves that knockout of UFM1 accelerates aging of human chondrocytes, overexpression of specific UFM1 protein or a coding gene thereof can relieve aging of human chondrocytes, and specific knockout of UFM1 in a mouse experimental model can accelerate osteoarthritis of the mouse, and injection of UFM1 recombinant protein into a joint cavity of the mouse can relieve osteoarthritis of the mouse.Therefore, the UFM1 protein or the coding gene thereof can be used as a drug target, and has a good prospect in screening and preparation of the medicine for treating osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of UFM1 protein or its encoding gene in the preparation of drugs for treating osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a common chronic disease, also known as degenerative joint disease. It is a condition caused by the degeneration and damage of articular cartilage, leading to joint pain, stiffness, and dysfunction. It primarily affects the elderly, but can also impact younger people. The most common sites of osteoarthritis are the knee, hip, spine, fingers, and the base of the thumb. Its causes are diverse, including overuse of joints, age, genetic factors, joint injury, and inflammatory diseases. Osteoarthritis is a chronic joint disease with a significantly increased incidence with age. It is characterized by cartilage destruction, synovitis, and subchondral bone remodeling, primarily manifesting as progressive joint destruction, osteophyte formation, leading to joint pain, deformity, and disuse atrophy. Symptoms progressively worsen, and in severe cases, can result in joint deformities and even loss of function. The main affected joints include the hip, knee, and shoulder. Osteoarthritis is a significant cause of chronic, non-traumatic disability in the middle-aged and elderly population.

[0003] Currently, the main treatments for osteoarthritis include medication and surgery, but both have limitations. Medication is the primary treatment for osteoarthritis, mainly including nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and analgesics. These medications can effectively relieve pain and inflammation, improving patients' quality of life. However, the main problem with medication is that it cannot change the course of the disease, only temporarily relieving symptoms. Furthermore, long-term use of these medications may produce side effects. Surgical treatment, such as joint replacement surgery, is a treatment option for patients with severe osteoarthritis. Surgery can effectively improve joint function, relieve pain, and improve patients' quality of life. However, surgical treatment also has some problems. First, the surgery carries significant risks, including surgical complications and anesthesia risks. Second, the recovery period after surgery is long, requiring long-term rehabilitation training. Furthermore, the lifespan of a joint replacement is limited, generally around 15-20 years; therefore, younger patients may need a second or even multiple surgeries. Finally, the cost of surgical treatment is high, which may be unaffordable for some patients with limited financial resources. Therefore, there is an urgent need for new treatment methods for osteoarthritis.

[0004] UFMylation, a protein modification similar to ubiquitination and SUMOylation, is a relatively recent discovery. It involves the C-terminal peptide chain of a ubiquitin-like protein called Small ubiquitin-like modifier 1 (SUMO1) linking to a target protein. Unlike other ubiquitin-related modifications, UFMylation primarily participates in protein synthesis and quality control within the endoplasmic reticulum and Golgi apparatus. UFMylation is a highly regulated process involving multiple steps, including activation of UFM1ylation enzymes (Uba5 and Ufc1), binding of the carrier protein UFM1 to the target protein, and enzymatic regulation of the modification process. Through UFMylation, UFM1 can covalently link to the target protein, forming a UFM1-target protein structure, and participate in protein modification and regulation. Although the specific functions and mechanisms of UFMylation are not fully understood, studies have shown that it plays an important role in many cellular processes. Research has found that UFMylation is involved in various biological processes, including apoptosis, cell cycle regulation, DNA damage repair, and protein quality control. Furthermore, UFMylation modification is also associated with antiviral immune responses, mitochondrial function, tumorigenesis, and neurodegenerative diseases. Future research will further elucidate the importance of UFMylation modification in cell biology and is expected to provide new strategies and targets for the treatment of related diseases. In the field of osteoarthritis treatment, research on UFMylation modification is still in its early stages. Future research is expected to reveal the specific role of UFMylation modification in the occurrence and development of osteoarthritis, and whether it can become a target for osteoarthritis treatment. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides the use of recombinant UFM1 protein as a drug target in the treatment of osteoarthritis, and the application of this UFM1 protein or its encoding gene in the preparation of drugs for treating osteoarthritis, thereby solving the technical problem that existing drugs are not effective in treating osteoarthritis.

[0006] The technical solution of the present invention is as follows:

[0007] Application of UFM1 protein or its encoding gene in the preparation of drugs for treating osteoarthritis.

[0008] The amino acid sequence of the UFM1 protein is shown in SEQ ID NO.1. The gene sequence encoding the UFM1 protein is shown in SEQ ID NO.2.

[0009] In applications, when preparing drugs for treating osteoarthritis using UFM1 protein, the drugs are administered via in-situ injection at the joint or via intravenous infusion.

[0010] When preparing drugs for treating osteoarthritis, the gene encoding the UFM1 protein is overexpressed in chondrocytes for drug delivery.

[0011] The present invention also provides a medicament for treating osteoarthritis, wherein the active substance is UFM1 protein or its encoding gene.

[0012] The amino acid sequence of the UFM1 protein is shown in SEQ ID NO.1. The gene sequence encoding the UFM1 protein is shown in SEQ ID NO.2.

[0013] Specifically, when the active substance is UFM1 protein, it is administered via in-situ injection at the joint or intravenous infusion.

[0014] When the active substance is the gene encoding the UFM1 protein, it is administered by overexpressing the gene encoding the UFM1 protein in chondrocytes.

[0015] Preferably, when the active substance is the gene encoding the UFM1 protein, the gene encoding the UFM1 protein is introduced into chondrocytes via a retroviral vector, a lentiviral vector, or an adenovirus vector.

[0016] The beneficial effects of this invention are:

[0017] This invention demonstrates that knocking out UFM1 accelerates chondrocyte senescence, while overexpression of specific UFM1 protein or its encoded gene can alleviate chondrocyte senescence. Furthermore, specific knockout of UFM1 in a mouse model accelerates osteoarthritis, while injection of recombinant UFM1 protein into the joint cavity of mice alleviates osteoarthritis. Therefore, UFM1 protein or its encoded gene can serve as a drug target and shows great promise in screening and developing drugs for treating osteoarthritis. Attached Figure Description

[0018] Figure 1 The table shows the expression levels of UFM1 in the knee joints of wild-type and accelerated-aging mice; A represents the expression levels of UFM1 in the knee joints of normal mice at 2, 12, and 18 months of age. Scale bar: 100 μm; B represents the expression levels of UFM1 in the knee joints of 6-month-old wild-type and accelerated-aging mice (TERC). - / - UFM1 expression level in the knee joint, scale bar: 100μm.

[0019] Figure 2The changes in various indicators after UFM1 knockout in human chondrocytes are shown. A shows the expression of UFM1, Sox9, Col2a1, Mmp13, p21, and p53 in human chondrocytes after UFM1 knockout, detected by quantitative real-time PCR; scale bar: 50 μm. B shows the expression of F-actin and p53 in human chondrocytes after UFM1 knockout, detected by immunofluorescence. C shows the senescence level of cells in human chondrocytes after UFM1 knockout, detected by β-galactosidase staining. * indicates p < 0.05, ** indicates p < 0.01.

[0020] Figure 3 This study investigated the changes in various indicators after overexpressing wild-type, D2, and D3 UFM1 in human chondrocytes, respectively. A represents the expression of UFM1, Sox9, Col2a1, Mmp13, p21, and p53 in human chondrocytes after overexpressing wild-type, D2, and D3 UFM1, respectively, using quantitative real-time PCR. B represents the expression of F-actin and p53 in human chondrocytes after overexpressing wild-type, D2, and D3 UFM1, respectively, using immunofluorescence. C represents the senescence level of human chondrocytes after overexpressing wild-type, D2, and D3 UFM1, respectively, using β-galactosidase staining. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0021] Figure 4 The study investigated how specific knockout of UFM1 in the articular cartilage of mice accelerates osteoarthritis in mice; where A represents a conditional knockout mouse with UFM1 in cartilage. f1 / f1 CCol2a1 CreERT2 The construction process of ); B is UFM1 in the sham group and DMM group. f1 / f1 and (UFM1) f1 / f1 CCol2a1 CreERT2 C represents the results of safranin-fast green staining and micro-ct scanning in mice; C represents the detection of UFM1 in the sham group and DMM group, respectively. f1 / f1 and (UFM1) f1 / f1 CCol2al CreERT2 Expression of CCOl2a1, Mmp3, UFM1 and p16 in the articular cartilage of mice.

[0022] Figure 5The study investigated the effects of intra-articular injection of recombinant UFM1 protein on the relief of osteoarthritis in mice. Image A shows micro-CT scans of mice in the Sham, DMM+PBS, and DMM+UFM1 rp groups; image B shows safranin-fast green staining of mice in the Sham, DMM+PBS, and DMM+UFM1 rp groups; and image C shows tissue fluorescence staining of UFM1 and Col2a1 in mice in the Sham, DMM+PBS, and DMM+UFM1 rp groups. Detailed Implementation

[0023] Example 1: UFM1 protein expression decreased in the cartilage of aging mice.

[0024] In this embodiment, the decreased expression of UFM1 in the cartilage of aging mice was determined by safranin-fast green staining and immunofluorescence.

[0025] The ethics review approval number for this study is: ZJU20230414.

[0026] Wild-type C57 mice were euthanized at 2, 12, and 18 months of age. The knee joints were removed, and the soft tissues, including muscles, were carefully dissected using ophthalmic scissors and forceps. The joints were fixed in a fixative solution for one day. Starting the next day, the joint specimens were placed in a decalcification solution, which was changed every two days. After two weeks of decalcification, the joint specimens were paraffin-embedded and sectioned. Finally, the sections were dewaxed with xylene and graded alcohols, and then stained with safranin and fast green to assess the wear and tear of the knee joints. Immunofluorescence assays using UFM1 antibody were performed to detect UFM1 expression in the knee joints. The results showed that UFM1 expression in the articular cartilage decreased with increasing age in mice. Figure 1 A).

[0027] Purchased TERC knockout mouse heterozygotes (TERC) from Jicui Pharmaceutical Co., Ltd. + / - ), and by naturally breeding heterozygous mice, TERC mice homozygous (TERC mice) were produced. - / - From the next generation naturally bred from heterozygous TERC knockout mice, wild-type mice (WT) and homozygous TERC mice (TERC) were selected. - / -Mice were euthanized at 6 months of age, and their knee joints were removed. The soft tissues, including muscles, of the leg were carefully dissected using ophthalmic scissors and forceps. The joints were fixed in a fixative solution for one day. Starting the next day, the joint specimens were placed in a decalcification solution, which was changed every two days. After two weeks of decalcification, the joint specimens were paraffin-embedded and sectioned. Finally, the sections were dewaxed with xylene and graded alcohols, and then stained with safranin and fast green to assess the wear and tear of the knee joints. Immunofluorescence assays using UFM1 antibody were performed to detect UFM1 expression in the knee joints. The results showed that UFM1 expression in the articular cartilage of accelerated-aging mice was decreased compared to wild-type mice. Figure 1 B).

[0028] Example 2: UFM1 knockout accelerates human chondrocyte senescence

[0029] In this embodiment, the role of UFM1 knockout in human chondrocyte senescence was determined in vitro by qRT-PCR, immunofluorescence, and β-galactosidase staining.

[0030] Joint specimens from patients undergoing joint replacement surgery at the Sir Run Run Shaw Hospital affiliated with Zhejiang University School of Medicine were randomly collected. Cartilage tissue was separated and fragmented using a scalpel, and the fragments were digested with 0.2% type II collagenase to culture normal human chondrocytes in vitro. To investigate the role of UFM1 knockout in human chondrocyte senescence, two UFM1 shRNAs at different sites were constructed and mixed with packaging plasmids. These were co-transfected into HEK-293T cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). The culture medium was changed 6 hours after transfection. Forty-eight hours after transfection, the supernatant was centrifuged at 3000 rpm for 10 minutes, supplemented with 10 μg / mL polybrene (SolarBio, Beijing, China), and then added to human chondrocytes for infection. Finally, cells were selected with 2 μg / mL puromycin for 24 hours to construct a stable UFM1 knockout human chondrocyte cell line. RNA was extracted from the chondrocytes and analyzed using qRT-PCR technology. The expression of UFM1, Sox9, Col2a1, p21, p53, and Mmp13 in cells was detected using the Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology). Immunofluorescence assays were used to detect the expression of F-actin and p53 in cells. β-galactosidase staining was used to determine the proportion of senescent cells. Results showed that knocking out UFM1 in human chondrocytes decreased the expression of matrix synthases Sox9 and Col2a1, while increasing the expression of matrix-degrading enzyme Mmp13 and aging-related factors p21 and p53. Figure 2 A and Figure 2 B), the proportion of senescent chondrocytes increases ( Figure 2 C). The primer sequences and UFM1shRNA sequences used in the qRT-PCR experiment are shown in Table 1.

[0031] Table 1

[0032]

[0033] Example 3: Overexpression of UFM1 alleviates human chondrocyte senescence

[0034] In this embodiment, the role of UFM1 overexpression in human chondrocyte senescence was determined by qRT-PCR, immunofluorescence, and β-galactosidase staining.

[0035] The UFM1 protein forms its active form by cleaving the C-terminal amino acids to expose glycine residues. Therefore, we constructed a wild-type UFM1 plasmid (amino acid sequence shown in SEQ ID NO.1, gene sequence shown in SEQ ID NO.2), cleaved the last two amino acids of the UFM1 plasmid (UFM1-D2), representing the active state of the UFM1 protein, and cleaved the last three amino acids of the UFM1 plasmid (UFM1-D3), representing the silent state of the UFM1 protein. These plasmids were then transfected into human chondrocytes (transfection steps were the same as for UFM1 shRNA). After extracting cellular RNA, qRT-PCR was performed... The expression of UFM1, Sox9, Col2a1, p21, p53, and Mmp13 in cells was detected using the Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology); the expression of F-actin and p53 in cells was detected by immunofluorescence; and the proportion of senescent cells was detected by β-galactosidase staining (the experimental procedure was the same as in Example 2). The results showed that after overexpression of wild-type and activated UFM1 proteins in human chondrocytes, respectively, the expression of matrix synthases Sox9 and Col2a1 increased, while the expression of matrix degradative enzyme Mmp13 and aging-related factors p21 and p53 decreased. Figure 3 A and Figure 3 B) The proportion of senescent chondrocytes increases, but this effect disappears when UFM1 silent protein is overexpressed in human chondrocytes. Figure 3 C).

[0036] Example 4: Specific knockout of UFM1 in mouse articular cartilage accelerates the progression of osteoarthritis in mice.

[0037] In this example, UFM1 was used in both control and cartilage UFM1 conditional knockout mice (UFM1f1 / f1 Col2a1 CreERT2 The DMM model was constructed to simulate post-traumatic osteoarthritis in mice. The specific steps were as follows: Twelve-week-old male mice were anesthetized and the knee joint was exposed via a medial capsular incision. In the DMM group, the medial meniscus ligament was transversely incised, rinsed with PBS, and the joint cavity was sutured. In the Sham group, the knee joint was exposed, rinsed with PBS, and the joint cavity was sutured. Figure 4 A) Eight weeks after modeling, mice were euthanized, and the knee joints were removed. The soft tissues, including muscles, of the mouse leg were carefully dissected using ophthalmic scissors and forceps. After fixation in fixative for one day, the number of osteophytes in the mouse knee joint was detected by micro-CT scanning. After the micro-CT scan, the mouse joint tissue was placed in decalcification solution for two weeks. The mouse joint specimens were then paraffin-embedded and sectioned. Finally, the mouse sections were dewaxed with xylene and graded alcohols, and safranin-fast green staining was performed to detect the degree of wear in the mouse knee joint. Immunofluorescence experiments were conducted using antibodies against UFM1, Col2a1, Mmp3, and p16 to detect the expression of UFM1, Col2a1, Mmp3, and p16 in the mouse knee cartilage. The results showed that specific knockout of UFM1 in mouse articular cartilage accelerates the progression of osteoarthritis in mice, specifically manifested as UFM1... f1 / f1 Col2a1 CreERT2 Mice showed more severe joint wear and more osteophyte formation on the articular cartilage surface after modeling. Figure 4 B) Chondrocyte extracellular matrix Col2a1 expression was lower, while matrix-degrading enzyme Mmp3 and aging-related factor p16 expression were higher. Figure 4 C).

[0038] Example 5: Progress of UFM1 wild-type recombinant protein injection in mice to alleviate osteoarthritis

[0039] In this example, the presence of wild-type recombinant UFM1 protein (synthesized by Hangzhou Huaan Biotechnology Co., Ltd., amino acid sequence shown in SEQ ID NO.1) injected into the joint cavity of arthritic mice was investigated to determine whether UFM1 has a delaying effect on the progression of osteoarthritis in mice.

[0040] Twenty-four wild-type C57 mice were used. Sixteen mice were randomly selected for DMM surgery to establish an osteoarthritis model, while the remaining eight mice underwent sham surgery, which involved opening the joint cavity, rinsing it with PBS, and then suturing the cavity closed. One week later, the 16 mice in the DMM surgery group were randomly divided into two groups of eight each. One group received intra-articular injections of PBS, while the other group received intra-articular injections of UFM1 wild-type recombinant protein (UFM1 rp). Injections were repeated every two weeks. After eight weeks, the mice were euthanized, and the knee joints were removed. The soft tissues, including the leg muscles, were carefully dissected using ophthalmic scissors and forceps. After fixation with fixative for one day, the number of osteophytes in the knee joint was detected by micro-CT scanning. After the micro-CT scan, the mouse joint tissue was placed in decalcification solution for two weeks. The mouse joint specimens were then paraffin-embedded and sectioned. Finally, mouse tissue sections were dewaxed with xylene and graded alcohols, and then stained with safranin and fast green to detect the degree of wear in the mouse knee joints. Immunofluorescence assays using UFM1 and Col2a1 antibodies were performed to detect the expression of UFM1 and Col2a1 in the knee cartilage. The results showed that local injection of wild-type recombinant UFM1 protein into the joint cavity of mice delayed the progression of osteoarthritis. Specifically, compared with mice injected with PBS, mice injected with recombinant UFM1 protein showed less joint wear after modeling. Figure 5 B), less osteophyte formation occurs on the surface of articular cartilage ( Figure 5 A) More Col2a1 is expressed in the extracellular matrix of chondrocytes. Figure 5 C).

Claims

1. The application of UFM1 protein or its encoding gene in the preparation of drugs for treating osteoarthritis. When preparing drugs for treating osteoarthritis using the UFM1 protein encoding gene, the UFM1 protein encoding gene is overexpressed and administered. The amino acid sequence of the UFM1 protein is shown in SEQ ID NO.

1. The gene sequence encoding the UFM1 protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, When preparing drugs for treating osteoarthritis using UFM1 protein, the drugs are administered via in-situ injection at the joint or via intravenous infusion. When preparing drugs for treating osteoarthritis, the gene encoding the UFM1 protein is overexpressed in chondrocytes for drug delivery.

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