Application of LB-100 in preparation of medicine for treating osteoarthritis

Abstract

The invention relates to the technical field of biological medicines, and discloses application of LB-100 in preparation of a medicine for treating osteoarthritis, the LB-100 can inhibit PP2A so as to realize continuous phosphorylation of an AMPK signal channel, inhibit expression of matrix metalloproteinase in cartilage cells and reduce damage of an articular cartilage structure in the osteoarthritis, and the LB-100 can be used for treating the osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of LB-100 in the preparation of a medicament for treating osteoarthritis. Background Art

[0002] Osteoarthritis is a chronic degenerative disease caused by the progressive destruction of articular cartilage and subchondral bone tissue, characterized by pathological features such as articular cartilage damage, limited mobility, osteophyte formation, and chronic pain. Currently, there is no drug that can effectively cure osteoarthritis, and artificial joint replacement has become the ultimate helpless choice for osteoarthritis.

[0003] Previous studies have identified multiple molecular signaling pathways involved in the regulation of the pathological process of osteoarthritis, such as the AMPK, Wnt / β-catenin, and TGF-β signaling pathways. These signaling pathways mainly participate in the regulation of the pathological process of osteoarthritis by directly or indirectly affecting anabolism and catabolism in cartilage. For example, matrix metalloproteinases MMP3, MMP13, ADAMTS4, ADAMTS5, etc. secreted by chondrocytes can promote the degradation of the extracellular matrix of chondrocytes and accelerate the structural damage of articular cartilage in the course of osteoarthritis. In mouse and human osteoarthritis models, the upregulated expression of matrix metalloproteinases (MMPs) and aggrecanases in chondrocytes and macrophages promotes articular cartilage damage. However, more and more evidence shows that energy metabolism disorders, especially mitochondrial dysfunction, play an important role in the pathological process of OA. Although chondrocytes mainly rely on anaerobic respiration (glycolysis) to obtain energy, mitochondria still play an important role in energy generation pathways such as fatty acid oxidation in chondrocytes. In addition, mitochondria also generate an appropriate amount of reactive oxygen species (ROS) to participate in the maintenance of the energy balance homeostasis of chondrocytes, which is important for the homeostasis of articular cartilage. Reports show that compared with normal articular cartilage, the expression of energy metabolism-related factors such as AMPK (AMP-activated protein kinase) and SIRT1 (Sirtuin 1) in chondrocytes under the pathological state of OA is significantly reduced. The AMPK signaling pathway is the main energy metabolism regulatory signaling pathway in cells. When the intracellular ATP level decreases and the levels of AMP and ADP increase, as an energy sensor, the AMPK signaling pathway is activated, and then by phosphorylating multiple substrates (such as PGC-1α, ACC, mTOR, etc.), it promotes glycolysis and fatty acid oxidation, while inhibiting the synthesis of fat and protein, and improving the energy metabolism level of cells. Some studies have shown that AMPK promotes the expression of genes related to mitochondrial biogenesis by activating PGC-1α, and increases the number and function of mitochondria in chondrocytes. Inhibiting the AMPK signaling pathway breaks the balance between the decomposition and anabolism of the extracellular matrix of chondrocytes, promotes matrix degradation, and exacerbates cartilage tissue damage. The above studies indicate that the AMPK signaling pathway is an important potential molecular target in the treatment of OA. Summary of the Invention

[0004] In view of this, the main object of the present invention is to provide an application of LB-100 in the preparation of a drug for treating osteoarthritis, which can solve the problem in the prior art that there is currently no effective drug for curing osteoarthritis, and the treatment method for osteoarthritis is artificial joint replacement.

[0005] To achieve the above object, the technical solution of the present invention is realized as follows:

[0006] Use of LB-100 in the preparation of a medicament for treating osteoarthritis.

[0007] The said LB-100 can inhibit the expression of PP2A, thereby enabling the continuous phosphorylation of the AMPK signaling pathway, and further alleviating the course of osteoarthritis.

[0008] The said LB-100 can effectively activate the mRNA level expression of the downstream target genes Pparα, Hif1, and Glut4 of the AMPK signaling pathway, and further alleviate the course of osteoarthritis.

[0009] The said LB-100 reduces the destruction of cartilage structure by inhibiting the mRNA expression of matrix metalloproteinases Mmp3, Mmp13, Adamts4, and Adamts5 in chondrocytes.

[0010] The said LB-100 can effectively inhibit the formation of new osteophytes during the course of arthritis.

[0011] The effective concentration of the said LB-100 for treating chondrocytes is 1-5 μm, and the time for treating cells with LB-100 is 12 hours.

[0012] The effective concentration of the said LB-100 for treating chondrocytes is 2 μm.

[0013] The use of LB-100 of the present invention in the preparation of a medicament for treating osteoarthritis has the following beneficial effects:

[0014] Research has confirmed that using LB-100 in the preparation of a medicament for treating osteoarthritis can effectively reduce osteophyte hyperplasia in osteoarthritis mice, alleviate cartilage structure damage, effectively promote the activation of the AMPK signaling pathway, inhibit the overexpression of matrix metalloproteinases in chondrocytes, and reduce the destruction of the extracellular matrix of chondrocytes, providing a potential drug small molecule for osteoarthritis cartilage damage. It is expected to solve the problem that there is currently no effective drug for curing osteoarthritis, and the treatment method for osteoarthritis is artificial joint replacement. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0016] Figure 1 Flow chart for modeling and treating osteoarthritis mice;

[0017] Figure 2After treating osteoarthritis mice with LB-100, micro-CT showed the changes in osteophyte bone mass of the knee joint;

[0018] Figure 3 After treating osteoarthritis mice with LB-100, safranin O-fast green staining map of the knee joint and statistical graph of histological index scores;

[0019] Figure 4 Immunohistochemical result map of AMPK+ cells in the mouse knee joint, statistical graph, and qPCR detection result map of downstream genes Pparα, Hif1, and Glut4 of the AMPK signaling pathway in chondrocytes.

[0020] Figure 5 qPCR detection result map of matrix metalloproteinase genes Mmp3, Mmp13, Adamts4, and Adamts5 in chondrocytes. Detailed implementation manners

[0021] The method of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments of the present invention.

[0022] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0023] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless otherwise clearly specified in the context, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "include" and / or "comprise" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects and do not necessarily describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the implementation manners of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units does not necessarily have to be limited to those clearly listed steps or units, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices.

[0025] For ease of description, spatial relative terms such as "above", "over", "on the upper surface", "upper" etc. can be used here to describe the spatial positional relationship of a device or feature shown in the figure with other devices or features. It should be understood that spatial relative terms are intended to encompass different orientations in use or operation in addition to the orientation depicted in the figure for the device. For example, if the device in the attached drawing is inverted, the device described as "above" or "over" other devices or structures will then be positioned "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both the orientations of "above" and "below". The device can also be positioned in other different ways (rotated 90 degrees or in other orientations), and corresponding interpretations are made for the spatial relative descriptions used here.

[0026] Referring to Figures 1-5 , the present invention provides a technical solution:

[0027] The application of LB-100 in the preparation of drugs for treating osteoarthritis. LB-100 is a water-soluble inhibitor of PP2A (protein phosphatase 2A).

[0028] LB-100 can inhibit the expression of PP2A, thereby continuously phosphorylating the AMPK signaling pathway, and further alleviating the course of osteoarthritis.

[0029] LB-100 can effectively activate the mRNA level expression of the downstream target genes of the AMPK signaling pathway: Pparα, Hif1 and Glut4, and further alleviate the course of osteoarthritis.

[0030] Since matrix metalloproteinases can exacerbate the pathological phenotype of osteoarthritis by degrading the extracellular matrix of chondrocytes, LB-100 reduces the destruction of cartilage structure by inhibiting the mRNA expression of matrix metalloproteinases Mmp3, Mmp13, Adamts4 and Adamts5 in chondrocytes.

[0031] LB-100 can effectively inhibit the formation of new osteophytes during the course of arthritis and alleviate cartilage structure damage. Its mechanism is related to LB-100 inhibiting PP2A, thereby continuously phosphorylating the AMPK signaling pathway.

[0032] Inhibition of the AMPK signaling pathway in chondrocytes is induced by the potent pro-inflammatory cytokine IL-1β (Interleukin-1beta). Overexpression of matrix metalloproteinases in chondrocytes is induced by IL-1β. The concentration of IL-1β (pro-inflammatory cytokine) used to treat cells is 20 ng / ml, and the treatment time is 12 hours. The effective concentration of LB-100 for treating chondrocytes is 1 - 5 μm, and the treatment time is 12 hours.

[0033] The optimal effective concentration of LB-100 for treating chondrocytes is 2 μm.

[0034] The effective concentration of LB-100 for intra-articular injection treatment of osteoarthritis mice is 40 mg / 20 g body weight. The injection frequency is once a week for a total of 4 injections.

[0035] Sources of biomaterials in the present invention. The chondrocyte cell line ATDC-5 was purchased from the American type culture collection company. DMEM medium, fetal bovine serum, penicillin, streptomycin, and trypsin were purchased from Gibico. LB-100 and IL-1β were purchased from Selleckchem. The AMPK antibody was purchased from Abcam. The primers used for qPCR were purchased from Sangon Biotech (Shanghai) Co., Ltd. The LABORAS platform was purchased from Metris. 10-week-old specific pathogen-free (SPF) male C57BL / 6 mice: weighing 25 - 30 g, were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0036] Example 1

[0037] Verify the prevention or treatment of cartilage structure damage in an osteoarthritis mouse model by LB-100.

[0038] 1. Establishment of animal model. All animal care and experimental procedures were carried out in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) of the Experimental Animal Science Center of the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Ten-week-old specific pathogen-free (SPF) male C57BL / 6 mice were selected for the study. The animal house environment was set at a temperature of (22 - 25°C), with a 12-hour / 12-hour light / dark cycle. The animals had free access to water and food in the cage. The mice were allowed to acclimatize to the new environment for one week, and then underwent medial meniscus destabilization (DMM) surgery on the medial side of the knee joint or only the medial patellar ligament was incised. Four weeks later, an osteoarthritis mouse model was successfully established, and then intra-articular injection of PBS or LB-100 was given for treatment for 4 weeks. After the administration treatment (week 6), Von Frey experiments were performed every two weeks, and after the behavioral experiment at week 16, all mice were euthanized. The right knee joint was isolated for histological evaluation.

[0039] 2. Construction and grouping of osteoarthritis mouse models. The mice were randomly divided into two groups: Group 1, Sham group (n≥3), only the medial patellar ligament was incised, and after exposing the joint cavity, the tissue and skin were sutured. The first group was randomly divided into two subgroups, with n≥3 mice in each subgroup, namely (1) Sham+PBS group;

[0040] (2) Sham+LB-100 group. Group 2 (n≥3) received medial meniscus tibial ligament resection to construct an osteoarthritis (DMM) mouse model. After the mice were anesthetized, fixed, and disinfected, the medial patellar ligament was incised with a microblade to expose the joint cavity. The fat pad between the femoral condyles was bluntly dissected to expose the intercondylar area, and the medial meniscus tibial ligament was transected with a microblade, and then the tissue and skin were sutured. The surgical limb was not fixed after the operation, and the mice were allowed to move freely in the cage, and penicillin was injected intraperitoneally to prevent infection. Group 2 was randomly divided into two subgroups, with n≥3 mice in each subgroup, namely (1) DMM+PBS group; (2) DMM+LB-100 group.

[0041] 3. Injection of osteoarthritis mice. Starting from 4 weeks after the operation, LB-100 was injected into the joint cavities of the mice in Sham+LB-100 and DMM+LB-100 once a week for a total of 4 times, and the injection volume was... The mice in Sham+PBS group and DMM+PBS group were treated with an equal dose of phosphate buffered saline (PBS) using the same administration method. According to the body weight of the mice, the concentration of each injection volume was 40mg / 20g. For example, a 20g mouse was injected with 20μl of LB-100 with a concentration of 2.0mg / ul.

[0042] 4. Evaluation by micro-CT examination. As Figure 2 shown, the imaging evaluation was used to assess the effect of LB-100 on the osteophyte bone mass of the mouse knee joint. After the mouse knee joint was fixed in 4% paraformaldehyde for 48h, the position was fixed with transparent plastic wrap, moistened with 70% ethanol, and then placed in a Micro-CT scanning tube for scanning. Quantitative analysis was performed using analysis software and a three-dimensional reconstruction platform. The region of interest was the calcified meniscus and synovial tissue, which was the osteophyte. The detected parameter was the volume (BV) of the calcified meniscus and synovial tissue. Figure 2 A shows the 3D reconstruction images of the knee joints of each group by Micro-CT, Figure 2Figure B shows the statistical chart of osteophyte volume in the knee joint. The abscissas are successively the Sham+PBS group, the Sham+LB-100 group, the DMM+PBS model group, and the DMM+LB-100 group. The results show that compared with the Sham group, the osteophyte formation in the knee joint of the DMM+PBS group mice increased (p<0.001), indicating that the medial meniscus modeling of the mouse knee joint led to knee joint instability, changes in the micro-structure of the knee joint, and abnormal bone remodeling. Compared with DMM+PBS, the osteophyte formation in the DMM+LB-100 group was less, and LB-100 could effectively reduce the formation of osteophytes (p<0.01), indicating that LB-100 could effectively relieve bone destruction and absorption, reduce abnormal bone remodeling, and thus play a role in protecting the bone structure of the knee joint.

[0043] 5. Histological examination. After the mice were euthanized, the right knee joint tissues were isolated and fixed in 4% paraformaldehyde solution for 3 days, decalcified in ETDA solution for 4 weeks, with the solution changed every 5 days during this period, then dehydrated with gradient ethanol and embedded in paraffin, and cut into 5-μm thick sagittal sections. The cartilage degeneration of the mouse knee joint was evaluated by safranin O-fast green staining and the expression of AMPK + cells was observed by IHC staining. The staining method was carried out strictly according to the kit instructions and the morphological changes of the tissues were observed under an ordinary light microscope. The safranin O-fast green staining of the right knee joint of the mouse is as Figure 3 shown in Figure A. It can be observed that the surface of the tibial cartilage in the Sham+PBS group (normal group) was bright and intact, while in the DMM+PBS group, the cartilage surface almost disappeared and the subchondral bone tissue was revealed. In the DMM+LB-100 treatment group, there were fewer defect lesions on the cartilage surface of the joint and most of the cartilage tissue was retained. In the DMM-induced osteoarthritis model group, the joint destruction and deformity were very serious, while in the LB-100 treatment group, they were significantly reduced.

[0044] The sections were scored for degenerative changes (cartilage degeneration, cartilage area) by different researchers. The cartilage degeneration scoring criteria refer to the Osteoarthritis Research Society International (OARSI) scoring. Figure 3 Figure B is the OARSI score; Figure 3 Figure C is the tibial cartilage area scale; the results show that compared with the Sham+PBS group, a large amount of matrix loss, a decrease in the number of chondrocytes, and a serious reduction in the cartilage area were observed in the DMM+PBS group mice, and the OARSI, (both P<0.001). After treatment with intra-articular injection of LB-100, the cartilage surface of the tibial plateau in the DMM+LB-100 group mice was continuous, the loss of matrix was inhibited, the staining of the cartilage layer was improved, the hypertrophy and apoptosis of chondrocytes were significantly reduced, the area of articular cartilage was significantly increased, and the OARSI, score was significantly decreased.

[0045] The immunohistochemical staining of the right knee joint of the mouse is asFigure 4 As shown in A, it can be observed that AMPK in chondrocytes in the Sham+LB-100 group was significantly increased compared with that in the Sham+PBS group (normal group), + and AMPK in chondrocytes in the DMM+LB-100 treatment group was also significantly increased compared with that in the DMM+PBS group. + cells were also significantly increased.

[0046] Example 2

[0047] Verify the effect of LB-100 on the AMPK signaling pathway in IL-1β-induced ATDC-5 cells.

[0048] ATDC5 cells in good growth state were seeded into 6-well plates. When their density grew to 70%-80%, they were divided into a blank treatment group, an LB-100 treatment group, an IL-1β treatment group, and an IL-1β+LB-100 treatment group, with 3 replicates in each group. According to the grouping requirements, the blank treatment group was treated with complete medium for 12 h, the LB-100 treatment group was replaced with complete medium containing 2 μM LB-100 and treated for 12 h, the IL-1β treatment group was replaced with complete medium containing 20 ng / ml IL-1β, and the IL-1β+LB-100 treatment group was replaced with complete medium containing 20 ng / ml IL-1β and 2 μM LB-100 and treated for 12 h. Then total RNA was extracted. The qRT-PCR experiment was carried out strictly according to the kit instructions. qPCR was used to detect the mRNA expression levels of Pparα (as shown in Figure 4 A), Hif1 (as shown in Figure 4 B), and Glut4 (as shown in Figure 4 C). The ordinate is the upregulation fold of Pparα, Hif1, or Glut4 with β-Actin as the reference gene, and the abscissa is successively the blank treatment group, the LB-100 treatment group, the IL-1β treatment group, and the IL-1β+LB-100 treatment group. The expression levels of Pparα, Hif1, and Glut4 in the blank treatment group were set to 1. AMPK has been proven to be related to the osteoarthritic process and cartilage disorder. As shown in Figure 4 shown, the levels of Pparα, Hif1, and Glut4 were significantly decreased in the IL-1β group (p < 0.001), but when 2 μM LB-100 was applied, Pparα, Hif1, and Glut4 in the drug administration group were all significantly increased (p < 0.001). The above results show that LB-100 can activate the mRNA level expression of the target genes Pparα, Hif1, and Glut4 of the AMPK signaling pathway in IL-1β-induced chondrocyte line ATDC-5, thereby alleviating the course of osteoarthritis.

[0049] Example 3

[0050] Effect of LB-100 on the extracellular matrix of IL-1β-induced ATDC-5 cells

[0051] The test conditions of Example 3 were the same as those of Example 2. The results are as Figure 5 shown in the quantitative PCR detection results of ATDC-5 cell extracellular matrix-related molecules. Figure 5 A is the mRNA transcription level of MMP3; Figure 5 B is the mRNA transcription level of MMP13; Figure 5 C is the mRNA transcription level of ADAMTS4; Figure 5 D is the mRNA transcription level of ADAMTS5. The ordinate is the upregulation fold with β-Actin as the reference gene, and the abscissa is successively the blank treatment group, LB-100 treatment group, IL-1β treatment group, and IL-1β + LB-100 treatment group. Among them, the blank treatment group was injected with PBS, the LB-100 treatment group was injected with LB-100, the IL-1β treatment group was injected with IL-1β, and the IL-1β + LB-100 treatment group was injected with IL-1β and LB-100, and β-Actin was used as the internal reference. As Figure 5 shown, Mmp3, Mmp13, Adamts4, and Adamts5 increased significantly in the IL-1β group. When 2 μm LB-100 was applied, the expressions of Mmp3, Mmp13, Adamts4, and Adamts5 all decreased significantly (p < 0.001). The above results suggest that LB-100 can reduce the pathological phenotype of osteoarthritis by degrading the chondrocyte extracellular matrix.

[0052] In summary, the present invention discovers that LB-100 is used to prepare a drug for treating osteoarthritis, which can continuously phosphorylate the AMPK signaling pathway by inhibiting PP2A, inhibit the expression of matrix metalloproteinases in chondrocytes, and reduce the destruction of articular cartilage structure in osteoarthritis, providing an important reference for the guiding treatment of osteoarthritis.

[0053] The above is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention.

Claims

1. Application of LB-100 in the preparation of drugs for the treatment of osteoarthritis.

2. The use of LB-100 according to claim 1 in the preparation of a drug for treating osteoarthritis, characterized in that: The LB-100 can inhibit the expression of PP2A, thereby causing the AMPK signaling pathway to continue to be phosphorylated.

3. The use of LB-100 according to claim 1 in the preparation of a drug for treating osteoarthritis, characterized in that: The LB-100 can effectively activate the mRNA level expression of downstream target genes of the AMPK signaling pathway in the chondrocyte cell line ATDC-5: Pparα, H i f1 and Glut4.

4. The use of LB-100 according to claim 1 in preparing a drug for treating osteoarthritis, characterized in that: The LB-100 inhibits the mRNA expression of matrix metalloproteinases Mmp3, Mmp13, Adamts4 and Adamts5 in chondrocytes.

5. The use of LB-100 according to claim 1 in the preparation of a drug for treating osteoarthritis, characterized in that: The LB-100 can effectively inhibit the formation of new osteophytes during the course of arthritis.

6. Use of LB-100 according to any one of claims 1 to 5 in the preparation of a drug for treating osteoarthritis, characterized in that: The effective concentration of LB-100 for treating chondrocytes is 1-5 μM, and the LB-100 treatment time for cells is 12 hours.

7. The use of LB-100 according to claim 6 in preparing a drug for treating osteoarthritis, characterized in that: The effective concentration of LB-100 for treating chondrocytes is 2 μM.

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