Application of calcium ion chelating agent in preparation of medicine for treating individual osteoarthritis

Abstract

The invention discloses application of a calcium ion chelating agent in preparation of a medicine for treating individual osteoarthritis, and belongs to the technical field of biological medicine. Research finds that the calcium ion chelating agent can effectively improve osteoarthritis, and further provides application of the calcium ion chelating agent to preparation of the medicine for treating osteoarthritis, and a foundation is laid for development of the novel medicine capable of effectively treating osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a calcium ion chelator in the preparation of a drug for treating osteoarthritis in an individual. Background Art

[0002] Osteoarthritis is the most common joint degenerative disease, and more than 303 million people worldwide suffer from osteoarthritis. With the development of population aging, the prevalence of osteoarthritis is also increasing, bringing heavier medical burdens and decision-making burdens to society and patients. Currently, the treatment of osteoarthritis mainly includes disease education, rehabilitation guidance, drug treatment, and surgical treatment, etc. Although current drug treatments can relieve or control the pain and inflammation of osteoarthritis, they cannot reverse the progression of the disease, and long-term medication may also cause side effects. Joint replacement surgery has always been a conventional treatment method for end-stage osteoarthritis, but the timing of the surgery is crucial because the service life of the replaced artificial joint is also limited.

[0003] There are many known risk factors for osteoarthritis, such as age, gender, obesity, genetics, injury, and abnormal load. Currently, the treatment of osteoarthritis mainly focuses on pain control and late-stage artificial joint replacement, and there is still a lack of therapeutic drugs that can relieve or reverse the progression of osteoarthritis. Therefore, in order to better treat this world problem, it is crucial to deeply explore the pathogenesis of osteoarthritis, find new therapeutic targets, and develop innovative drugs that can effectively treat osteoarthritis.

[0004] Calcium ion chelators can prevent cell damage by reducing intracellular calcium overload, but there is currently no relevant literature report on the role of calcium ion chelators in the treatment of osteoarthritis. The research group of this project found in experiments that calcium ion chelators can effectively improve osteoarthritis. In view of this, calcium ion chelators have the potential to become new therapeutic targets for osteoarthritis and are expected to be applied to the clinical treatment of osteoarthritis. Summary of the Invention

[0006] In order to overcome the deficiencies of the above-mentioned existing background art, the purpose of the present invention is to provide the application of a calcium ion chelator in the preparation of a drug for treating osteoarthritis in an individual. The present invention discovers through research that calcium ion chelators can effectively improve osteoarthritis, and thus provides the use of a calcium ion chelator for the preparation of a drug for treating osteoarthritis, laying a foundation for the development of new drugs that can effectively treat osteoarthritis.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The application of a calcium ion chelator in the preparation of a drug for treating osteoarthritis in an individual.

[0009] Use of a calcium ion chelator in the preparation of a medicament for treating osteoarthritis in an individual caused by calcium ion overload in chondrocytes.

[0010] Preferably, the calcium ion chelator is used to slow down the osteoarthritis process by chelating the overload calcium ions in chondrocytes and maintaining the calcium ion homeostasis in chondrocytes.

[0011] Preferably, the calcium ion chelator is selected from one or more mixtures of ethylenediaminetetraacetic acid and its derivatives, ethylene glycol bis(2-aminoethyl ether) tetraacetic acid and its derivatives, 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid and its derivatives.

[0012] Preferably, the calcium ion chelator is a small molecule drug that can chelate calcium ions in chondrocytes and reduce the intracellular calcium ion level in chondrocytes.

[0013] Preferably, the calcium ion chelator is BAPTA-AM.

[0014] Preferably, the individual osteoarthritis includes age-related primary osteoarthritis and secondary osteoarthritis caused by injury, inflammation, genetics, metabolism, and endocrine diseases.

[0015] Preferably, the individual is a mammal.

[0016] Preferably, the individual is a human or a mouse.

[0017] The beneficial effects achieved by the present invention are as follows:

[0018] For the osteoarthritis disease that currently lacks effective treatment methods, the present invention provides a new treatment strategy: The inventors have found that the calcium ion chelator BAPTA-AM can effectively alleviate chondrocyte apoptosis and cartilage matrix degradation caused by osteoarthritis. It is found in in vitro experiments that BAPTA-AM can protect chondrocytes by inhibiting calcium ion influx under inflammatory conditions, and it is also found in in vitro experiments that BAPTA-AM can significantly improve traumatic osteoarthritis when administered into the joint cavity. In view of this, chelating excessive calcium ions in chondrocytes and maintaining the calcium ion homeostasis in chondrocytes have the potential to become new targets for the treatment of osteoarthritis, and BAPTA-AM as a calcium ion chelator is also expected to be applied to the clinical treatment of osteoarthritis. Description of the Drawings

[0019] Figure 1 It is a safranin-fast green staining result diagram of the knee joint tissues of OA mice in the BAPTA-AM treatment group and OA mice in the normal saline control group (scale = 100 μm).

[0020] Figure 2Schematic diagram of osteoarthritis scores of OA mice in the BAPTA-AM treatment group and OA mice in the normal saline control group (n = 10, * indicates P < 0.05 or ** indicates P < 0.01).

[0021] Figure 3 Schematic diagram of the results of using Flou-4-AM to detect the calcium ion level chelated by BAPTA-AM in chondrocytes. (Scale bar = 50 μm).

[0022] Figure 4 Schematic diagram of the results of toluidine blue and safranin staining of chondrocytes after intervention with IL-1β, calcium chloride, and BAPTA-AM (Scale bar = 200 μm).

[0023] Figure 5 Schematic diagram of the results of detecting the expression of anabolic, catabolic, and apoptosis indexes of chondrocytes by Western blot after intervention of chondrocytes with IL-1β and BAPTA-AM (n = 3, * indicates P < 0.05 or ** indicates P < 0.01).

[0024] Figure 6 Schematic diagram of the results of detecting the expression of anabolic, catabolic, and apoptosis indexes of chondrocytes by Western blot after intervention of chondrocytes with calcium chloride and BAPTA-AM (n = 10, * indicates P < 0.05 or ** indicates P < 0.01).

[0025] Figure 7 Schematic diagram of the results of detecting the expression of inflammatory indexes by Western blot after intervention of chondrocytes with IL-1β, calcium chloride, and BAPTA-AM (n = 10, * indicates P < 0.05 or ** indicates P < 0.01). Detailed implementation manners

[0026] The present invention will be further described in detail below in conjunction with the accompanying drawings, examples, and experimental examples. Of course, the protection scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiments. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them in as much detail as possible here. The following examples further illustrate the present invention rather than limit the present invention. Any formal but not substantial equivalent transformation made based on the concept of the present invention should be regarded as falling within the scope of the technical solution of the present invention.

[0027] In the following examples, the test methods or testing methods described, unless otherwise specified, are all conventional methods; the reagents and materials described, unless otherwise specified, are all obtained from conventional commercial channels or prepared by conventional methods.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] Currently, the treatment of osteoarthritis mainly focuses on pain control and late-stage artificial joint replacement, and there is still a lack of therapeutic drugs that can relieve or reverse the process of osteoarthritis. Therefore, in order to better treat this world problem, it is crucial to deeply explore the pathogenesis of osteoarthritis, find new therapeutic targets, and develop innovative drugs that can effectively treat osteoarthritis.

[0030] Definition and use of terms

[0031] Calcium ion chelator: In the present invention, the calcium ion chelator refers to one or a mixture of ethylene diamine tetraacetic acid and its derivatives, ethylene glycol bis(2-aminoethyl ether) tetraacetic acid and its derivatives, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and its derivatives. The Chinese name of BAPTA-AM is: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl ester).

[0032] Individual: In the present invention, the term "individual" refers to mammals, including but not limited to rats, mice, non-human primates, humans, dogs, cats, horses, cows, sheep, pigs, goats. Preferably humans or mice.

[0033] Treatment:

[0034] As used in the present invention, "treatment" means reducing the degree of cartilage degeneration in osteoarthritis, or curing osteoarthritis to make it normal, or slowing down the process of osteoarthritis.

[0035] The present invention has demonstrated through the following examples that by maintaining intracellular calcium ion homeostasis by reducing the intracellular calcium ion level, the cartilage degeneration in osteoarthritis can be significantly alleviated.

[0036] To better explain the present invention, the following is a detailed description in conjunction with specific examples.

[0037] Example 1 Effect of intra-articular injection of BAPTA-AM into the knee joint cavity on the disease process of osteoarthritis in mice:

[0038] Experimental animals and materials:

[0039] 1. Experimental animals:

[0040] Germline, strain: Wild-type mice (WT, C57BL / 6) bred in the animal house of Tongji Hospital;

[0041] Reproductive age: 8 - 10 weeks old.

[0042] 2. Experimental materials:

[0043] Sodium pentobarbital: Cyagen Biosciences Inc.; BAPTA-AM: SelleckChemicals.

[0044] 3. Experimental methods:

[0045] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg / kg). Subsequently, a murine osteoarthritis model was established by destabilization of the medial meniscus (DMM) surgery. One week after modeling, BAPTA-AM at 1.5 μg / kg or 15 μg / kg was injected into the knee joint cavity once a week for 8 weeks. Nine weeks after modeling, the mice were sacrificed, and the knee joints were removed and fixed in tissue fixative. The fixed knee joint specimens were decalcified and sectioned. Safranin-O fast green staining experiments were performed on the tissue sections to detect cartilage damage. Two pathologists independently evaluated the severity of osteoarthritis in each safranin-O fast green stained section in a blinded manner using the Osteoarthritis Research Society International (OARSI) scoring system.

[0046] 4. Experimental results:

[0047] Specifically, the safranin-O fast green staining results of the knee joint tissues of OA mice in the BAPTA-AM treatment group and OA mice in the normal saline control group were as Figure 1 shown. Combining Figure 1 it can be seen that after DMM surgery, compared with the normal saline control group, the degree of medial knee joint cartilage degeneration in OA mice in the BAPTA-AM treatment group was significantly reduced.

[0048] The osteoarthritis scoring results were as Figure 2 shown. From Figure 2 it can be seen that the osteoarthritis score of OA mice in the BAPTA-AM treatment group was lower than that of OA mice in the normal saline control group, indicating that the degree of cartilage degeneration was greatly reduced.

[0049] Example 2 Effects of BAPTA-AM intervention on intracellular calcium levels, anabolism, catabolism, apoptosis, and inflammation in chondrocytes:

[0050] This example explores the therapeutic effect of BAPTA-AM on osteoarthritis in in vitro experiments. In this invention, chondrocytes were stimulated with IL-1β for 24 h to induce an inflammatory OA phenotype, calcium ion overload was induced by calcium chloride, and different concentrations of BAPTA-AM were added to the culture medium. Western blot was used to detect the expression of molecular markers related to anabolism, catabolism, apoptosis, and inflammation in chondrocytes after BAPTA-AM intervention.

[0051] Specifically, 5 μM and 10 μM of BAPTA-AM were added to the culture medium of primary mouse chondrocytes, and OA was induced by stimulating with IL-1β or calcium chloride at the same time. Chondrocytes were collected, and total protein was extracted for Western blot detection.

[0052] Combined with Figure 3 it can be seen that BAPTA-AM can significantly reduce the calcium ion level in chondrocytes of the calcium chloride group.

[0053] Combined with Figure 4 it can be seen that BAPTA-AM can alleviate the morphological changes and the reduction in the number of chondrocytes caused by IL-1β and calcium chloride.

[0054] To further evaluate the role of inhibiting BAPTA-AM in the progression of osteoarthritis, in this invention, Western blot was used to detect the expression of molecular markers related to anabolism, catabolism, apoptosis, and inflammation in chondrocytes after 24 h of stimulation with IL-1β or calcium chloride by BAPTA-AM.

[0055] Combined with Figure 5 , Figure 6 and Figure 7 it can be seen that, compared with the IL-1β or calcium chloride group, BAPTA-AM intervention can rescue the anabolic index COL2A1, reduce the catabolic marker MMP13 and the expression of the apoptosis key proteins Cleaved-Caspase-3 and P53, and reduce the expression of the inflammatory indexes INOS and COX2.

[0056] In summary, the above experimental data show that BAPTA-AM can slow down the process of osteoarthritis by inhibiting chondrocyte degeneration.

[0057] Therefore, BAPTA-AM studied in this invention provides a new treatment strategy for osteoarthritis, which currently lacks effective treatment methods, and lays a foundation for the development of new drugs that can effectively treat osteoarthritis.

[0058] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the protection scope of the present invention should be defined by the scope of the claims. For those of ordinary skill in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as within the protection scope of the present invention.

Claims

1. Use of a calcium ion chelator in the preparation of a medicament for treating osteoarthritis in an individual.

2. The application according to claim 1, characterized in that, The calcium ion chelator is used to chelate the overloaded calcium ions in chondrocytes and maintain the calcium ion homeostasis in chondrocytes, thereby slowing down the process of osteoarthritis.

3. The application according to claim 1, wherein: The calcium ion chelator is a small molecule drug capable of chelating calcium ions in chondrocytes and reducing the calcium ion level in chondrocytes.

4. The application according to claim 3, wherein: The calcium ion chelator is selected from one or more mixtures of ethylenediaminetetraacetic acid and its derivatives, ethylene glycol bis(2-aminoethyl ether) tetraacetic acid and its derivatives, 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid and its derivatives.

5. The application according to claim 4, characterized in that: The calcium ion chelator is selected as BAPTA-AM.

6. The application according to claim 1, wherein: The individual osteoarthritis includes age-related primary osteoarthritis and secondary osteoarthritis caused by injury, inflammation, genetics, metabolism, and endocrine diseases.

7. The application according to claim 1, characterized in that: The individual is a mammal.

8. The application according to claim 8, characterized in that: The individual is a human or a mouse.

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