Application of adamantane derivative LW6 in construction of osteoarthritis model

Abstract

The invention provides an application of an adamantane derivative LW6 in construction of an osteoarthritis model. Experimental results show that in a low-oxygen environment, HIF-1alpha participates in adaptive reaction of cartilage cells to insufficient oxygen, and the cartilage cells are protected by adjusting extracellular matrix components, inhibiting matrix degradation and other processes. It is found through experiments that when the treatment concentration of the LW6 medicine reaches 20 [mu] m / ml, expression of HIF1a in cartilage cells under hypoxia can be completely inhibited, expression of cartilage extracellular matrix protein COL2a1 is reduced, expression of MMP13 protein is increased, and degradation of cartilage extracellular matrix is accelerated. Therefore, the LW6 can be used for constructing an osteoarthritis model.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of an adamantane derivative LW6 or a pharmaceutically acceptable salt thereof in constructing an osteoarthritis model. Background Art

[0002] Osteoarthritis (OA) is a degenerative joint disease that is common in the elderly and is characterized by progressive cartilage degeneration, synovitis, osteophyte formation, and subchondral bone sclerosis. At the same time, OA is also the primary cause of joint pain and chronic disability in the elderly. Its prevalence increases with age, and has even reached 50% in people over 65 years old.

[0003] Osteoarthritis not only affects the quality of life of individual patients, but also brings heavy social and economic burdens. Therefore, the prevention and treatment of osteoarthritis faces major challenges.

[0004] Animal models with similar manifestations to human osteoarthritis are of great significance for the study of osteoarthritis. The current animal models mainly include surgical models of traumatic arthritis (medial meniscus resection surgery); chemical models of cartilage destruction (papain intraarticular injection); degenerative arthritis models, etc. Although these modeling methods can simulate the disease manifestations of osteoarthritis, they only exist in the local joint cavity and cannot explore the relationship between osteoarthritis and systemic metabolism.

[0005] In the development of osteoarthritis (OA), degradation of the extracellular matrix of cartilage cells is the main cause of impaired joint structure and function. HIF1A (hypoxia-inducible factor 1α) is an important regulatory factor for cells in hypoxic environments, especially in cartilage, a naturally hypoxic tissue, where HIF1A plays a key role. HIF1A is stabilized under hypoxic conditions and helps chondrocytes resist external stress and reduce matrix degradation by activating a series of genes related to cell survival, matrix synthesis and metabolism. It can be seen that HIF1A plays a protective role in maintaining the stability of the extracellular matrix of cartilage cells and preventing the progression of osteoarthritis. Summary of the invention

[0006] In order to solve the problems in the prior art, the present invention provides the use of adamantane derivative LW6 or a pharmaceutically acceptable salt thereof in constructing an osteoarthritis model. Experiments have found that in a hypoxic environment, HIF-1α participates in the adaptive response of chondrocytes to oxygen deficiency, and protects chondrocytes by regulating extracellular matrix components, inhibiting matrix degradation, and other processes. However, surprisingly, a specific concentration of LW6 inhibits HIF-1α, destroying this protective regulation, resulting in a decrease in the extracellular matrix of chondrocytes. Therefore, the use of a specific concentration of LW6 can be used to construct an osteoarthritis model.

[0007] The present invention adopts the following technical solution:

[0008] The present invention provides use of an adamantane derivative LW6 or a pharmaceutically acceptable salt thereof in constructing an osteoarthritis model.

[0009] The adamantane derivative LW6 of the present invention has the following structural formula:

[0010]

[0011] According to one embodiment of the present invention, LW6 of the present invention inhibits the expression of HIF1a in chondrocytes under hypoxia, resulting in decreased expression of cartilage extracellular matrix protein COL2a1 and increased expression of MMP13 protein, thereby accelerating the degradation of cartilage extracellular matrix.

[0012] According to one embodiment of the present invention, the pharmaceutically acceptable salt of the present invention is a salt formed by LW6 and an acid. The acid is selected from sulfuric acid, phosphoric acid, nitric acid, hydrochloric acid, methanesulfonic acid, hydrobromic acid, hydroiodic acid, benzenesulfonic acid, pyrosulfuric acid, ethanesulfonic acid, p-toluenesulfonic acid, acetic acid, pyruvic acid, maleic acid, aspartic acid, trifluoroacetic acid, propionic acid, caproic acid, benzoic acid, salicylic acid, tartaric acid, stearic acid, cinnamic acid, cyclopentanepropionic acid, lactic acid, oxalic acid, dodecyl sulfuric acid, fumaric acid, D-gluconic acid, 2-naphthalenesulfonic acid, citric acid, malonic acid, succinic acid, and malic acid.

[0013] According to one embodiment of the present invention, the concentration of LW6 described in the present invention is ≥20 um / ml; preferably, the concentration of LW6 is 20-80 um / ml.

[0014] According to one embodiment of the present invention, LW6 is administered by injection or oral gavage. Preferably, the injection is intraperitoneal injection or intravenous injection.

[0015] Beneficial effects:

[0016] The present invention provides an application of an adamantane derivative LW6 in constructing an osteoarthritis model. Experimental results show that in a hypoxic environment, HIF-1α participates in the adaptive response of chondrocytes to oxygen deficiency, and protects chondrocytes by regulating extracellular matrix components, inhibiting matrix degradation, and the like. However, LW6 inhibits HIF-1α, destroying this protective regulation, causing the chondrocyte extracellular matrix to be more easily reduced. This experiment found that LW6 drug treatment concentration of 20um / ml will completely inhibit the expression of HIF1a in chondrocytes under hypoxia, resulting in decreased expression of chondrocyte extracellular matrix protein COL2a1, increased expression of MMP13 protein, and accelerated degradation of chondrocyte extracellular matrix. Therefore, the use of a specific concentration of LW6 can be used to construct an osteoarthritis model. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is an exploration of the effective concentration of LW6 under hypoxia.

[0018] Figure 2 This is a statistical analysis chart of the effects of HIF1a protein.

[0019] Figure 3 The effect of 20um / ml concentration of LW6 inhibitor on the expression of HIF1a, MMP13, and CoL2a proteins in osteoarthritis chondrocytes under hypoxia.

[0020] Figure 4 Statistical analysis of the effect of LW6 inhibitor on HIF1a protein in osteoarthritis chondrocytes under hypoxia.

[0021] Figure 5 Statistical analysis of the effect of LW6 inhibitor on MMP13 protein in osteoarthritis chondrocytes under hypoxia.

[0022] Figure 6 Statistical analysis of the effect of LW6 inhibitor on CoL2a1 protein in osteoarthritic chondrocytes under hypoxia. DETAILED DESCRIPTION

[0023] In order to further illustrate the present invention and its advantages, the technical scheme of the present invention is further illustrated by specific embodiments below, and it should be understood that these embodiments only help to understand the present invention and should not be regarded as specific limitations of the present invention. The raw materials and reagents used in the present invention are all commercially available products. Unless otherwise specified, the parts described in the present invention are all weight parts, and the percentages are all mass percentages.

[0024] Example 1

[0025] 1. Primary extraction of mouse knee chondrocytes

[0026] Several C57BL / 6J suckling mice were killed by rapid cervical dislocation and then immersed in 75% ethanol for 10 minutes for comprehensive disinfection. In the clean bench, sterile micro-scissors were first used to cut the skin of the mouse legs, and then forceps were used to quickly separate the muscles on the surface of the mouse femur and tibia, and the knee joints on both sides were separated. The extracted joint tissue was soaked in PBS containing 5% double antibody for 5 minutes, and then the PBS was discarded, and 0.25% trypsin was added and incubated at 37°C and 5% CO 2 Digest in an incubator for 10 minutes. After discarding the pancreatic enzyme, add enough PBS to soak the cartilage tissue to prevent it from drying out. Use micro-tweezers to further thoroughly remove the muscle and ligament tissue around the cartilage to ensure that clean articular cartilage is obtained. Soak and rinse the articular cartilage with PBS 3 times and cut the cartilage into 1mm 3 Add enough 0.1% collagenase type II to the culture dish and place it in a 37°C, 5% CO 2Incubate overnight in a cell incubator until all cartilage blocks are fully digested. The next day, complete culture medium was added at a ratio of 1:2 to terminate cartilage digestion. The mixture was fully blown with a pipette to fully disperse the cartilage tissue. After filtering through a 70 μm mesh, the mixture containing cells was transferred to a centrifuge tube and centrifuged at 1500 rpm for 5 minutes in a low-speed centrifuge at room temperature. The supernatant was discarded and the cells were re-inoculated with the complete culture medium. The mixture was placed at 37°C and 5% CO. 2 The cells were routinely cultured in an incubator, and the culture medium was replaced every 2-3 days. When the cell density reached about 80%-90%, the cells were passaged for subsequent experiments.

[0027] 2. Drug concentration exploration

[0028] Use DMSO to fully dissolve LW6 drug and prepare it into a concentration of 10mM / ML. Use six-well plates to randomly divide the chondrocytes into control, DMSO, 10um, 20um, 40um, and 80um groups. Use Lw6 to treat chondrocytes in normoxia for 12 hours and then put them into a hypoxic incubator (1% pO 2 ) was cultured for 24 h to induce HIF1a stabilization. Protein was then extracted for Western Blot experiment to detect HIF1a expression. Figure 1 Statistical analysis of HIF1a protein effects is shown in Figure 2 The Western blot results and statistical analysis of HIF1a protein showed that HIF1a was significantly inhibited when the concentration of LW6 was 20um / ml under hypoxia.

[0029] 3. Explore the effect of LW6 on osteoarticular chondrocytes under hypoxia

[0030] IL-1β (10ng / ml) was used to treat primary chondrocytes of the mouse knee joint to simulate osteoarticular chondrocyte injury in vitro and construct an OA model. The cells were randomly divided into the Control group, IL-1β group, IL-1β+HX group, IL-1β+HX+LW6 group and IL-1β+HX+DMSO group. After 12h of normoxic pretreatment with LW6, IL-1β was added for intervention and the IL-1β+HX group, IL-1β+HX+LW6 group and IL-1β+HX+DMSO group were placed in a hypoxic incubator. The Control group and IL-1β group were placed in a normoxic incubator and cultured for 24h respectively. After that, proteins were extracted and Western Blot experiments were performed to detect the expression of HIF1a, MMP13 and CoL2a1 proteins in different groups. The effect of 20um / ml concentration of LW6 inhibitor on the expression of HIF1a, MMP13 and CoL2a proteins in osteoarthritis chondrocytes under hypoxia is shown in Figure 2. Figure 3Statistical analysis of the effect of LW6 inhibitor on HIF1a protein in osteoarthritis chondrocytes under hypoxia is shown in Figure 4 Statistical analysis of the effect of LW6 inhibitor on MMP13 protein in osteoarthritis chondrocytes under hypoxia is shown in Figure 5 Statistical analysis of the effect of LW6 inhibitor on CoL2a1 protein in osteoarthritis chondrocytes under hypoxia is shown in Figure 6 Western blot results of CoL2α1, HIF1a, and MMP13 proteins: The results showed that the expression of HIF1a was decreased in the LW6-treated group under hypoxia, the expression of cartilage extracellular matrix protein CoL2a1 was decreased, and the expression of matrix metalloproteinase MMP13 was increased, indicating that LW6 inhibited HIF1a and increased the degradation of extracellular matrix.

Claims

1. Use of an adamantane derivative LW6 or a pharmaceutically acceptable salt thereof in constructing an osteoarthritis model; the adamantane derivative LW6 has the following structural formula:

2. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by LW6 and an acid; the acid is selected from sulfuric acid, phosphoric acid, nitric acid, hydrochloric acid, methanesulfonic acid, hydrobromic acid, hydroiodic acid, benzenesulfonic acid, pyrosulfuric acid, ethanesulfonic acid, p-toluenesulfonic acid, acetic acid, pyruvic acid, maleic acid, aspartic acid, trifluoroacetic acid, propionic acid, hexanoic acid, benzoic acid, salicylic acid, tartaric acid, stearic acid, cinnamic acid, cyclopentanepropionic acid, lactic acid, oxalic acid, dodecyl sulfuric acid, fumaric acid, D-gluconic acid, 2-naphthalenesulfonic acid, citric acid, malonic acid, succinic acid or malic acid.

3. The use according to claim 1 or 2, characterized in that: LW6 inhibits the expression of HIF1a in chondrocytes under hypoxia, resulting in decreased expression of cartilage extracellular matrix protein COL2a1 and increased expression of MMP13 protein, thereby accelerating the degradation of cartilage extracellular matrix.

4. The use according to claim 3, characterized in that: The concentration of LW6 is ≥20um / ml.

5. The use according to claim 4, characterized in that: The concentration of LW6 is 20-80um / ml.

6. The use according to claim 4 or 5, characterized in that: LW6 was administered by injection or oral gavage.

7. The use according to claim 4 or 5, characterized in that: The injection is intraperitoneal injection or intravenous injection.

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