Application of freshwater mussel glucan HBP-A in treatment of knee osteoarthritis

Abstract

The invention provides an application of freshwater mussel glucan HBP-A in treatment of knee osteoarthritis. Furthermore, the invention also provides a pharmaceutical composition for preventing or treating knee osteoarthritis, and the pharmaceutical composition consists of freshwater mussel glucan HBP-A and pharmaceutically acceptable carrier auxiliary materials. The HBP-A in the preparation can down-regulate a p38-MAPK signal channel key target spot, reduce overexpression of meniscus hypertrophy and calcification related protein and gene markers, and delay or inhibit meniscus pathological degeneration. The injection prepared from the HBP-A is used for knee joint cavity injection, and can improve the metabolism of meniscus, delay or inhibit the degeneration of meniscus and change the pathological process of knee osteoarthritis while relieving the symptoms of knee osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of mussel glucan HBP-A in the therapeutic drugs for treating knee osteoarthritis, as well as a pharmaceutical preparation containing mussel glucan HBP-A or its salt. Background Art

[0002] Due to its complex anatomical structure, the knee joint has become a high-incidence site of osteoarthritis. Knee osteoarthritis (KOA) is a clinically typical chronic osteoarticular disease, which has imposed a huge socio-economic burden on individuals and countries. The pathogenesis and prevention of KOA have become the focus of research in this field and a hot issue of concern to medical practitioners. Trauma, sports injuries, obesity, and advanced age are considered important pathogenic factors of KOA, and these factors will all lead to a foreseeable result - the mechanical imbalance of the knee joint. The mechanical imbalance will change the stability, muscle morphology, and mobility of the knee joint, increase the abnormal mechanical load on the intra-articular tissues, and thus further aggravate the development of KOA. However, current research on the occurrence and progression of KOA caused by abnormal mechanical injury mainly focuses on articular cartilage, while the importance of other structures is often overlooked.

[0003] As one of the important structures of the knee joint, the meniscus plays a key role in the occurrence and development of KOA. The meniscus can mechanically transmit, absorb shock, and maintain joint stability. After the meniscus is injured, biomechanical changes usually occur in the joint. The ability of the injured meniscus to transmit load within the joint is weakened, and the abnormal mechanical load caused by joint instability will in turn exacerbate the damage to other tissues and structures within the joint, leading to knee joint degeneration and even developing into post-traumatic osteoarthritis (PTOA). Therefore, the meniscus may play a "bridge" role in the occurrence and progression of abnormal mechanical load and osteoarthritis. Research shows that abnormal mechanical injury can lead to cell death, proteoglycan loss, and the production of matrix degrading enzymes in meniscus tissue, indicating that abnormal mechanical injury can accelerate the degeneration of the meniscus. At the same time, the degeneration of the meniscus will further damage articular cartilage, which is an important factor in triggering or exacerbating KOA. Relevant research reports that there is calcification in the meniscus of KOA patients, which will lead to narrowing of the joint space and friction with cartilage, and is a key pathological factor in the occurrence and development of KOA. Previously, our research in a guinea pig knee joint mechanical injury model has confirmed that abnormal mechanical load will cause meniscus hypertrophy and calcification, thus promoting the occurrence of KOA. This indicates that meniscus hypertrophy and calcification caused by abnormal mechanical injury play an important role in the occurrence and development of KOA. Inhibiting the pathological degeneration of the meniscus has become a potential new treatment target for KOA. Summary of the Invention

[0004] The present invention is made to solve the above problems, and aims to provide a new medical use of mussel glucan HBP-A and its carrier, as well as a pharmaceutical composition containing mussel glucan HBP-A or its salt.

[0005] In the first aspect of the present invention, there is provided the use of mussel glucan HBP-A or its salt in the preparation of a therapeutic drug for knee osteoarthritis.

[0006] Preferably, the therapeutic drug for knee osteoarthritis is a drug for slowing down or inhibiting meniscus degeneration.

[0007] More preferably, the drug for slowing down or inhibiting meniscus degeneration is a drug for treating meniscus overhypertrophy or pathological calcification, or a drug for treating medial meniscus or articular cartilage injury and degeneration.

[0008] Even more preferably, the drug for treating meniscus overhypertrophy or pathological calcification is a drug for inhibiting the overexpression of the p38-MAPK signaling pathway.

[0009] The HBP-A salt of the present invention refers to a pharmaceutically acceptable salt of HBP-A, such as hydrochloride, sulfate, citrate, etc.

[0010] The research results of the present invention show that compared with the model group, the width of the meniscus, the area and intensity of meniscus calcification in the HBP-A group of guinea pigs were significantly reduced, and the scores of meniscus and articular cartilage injury were also significantly reduced. The protein and gene expression levels of matrix metalloproteinase 13 (MMP13), Runt-related transcription factor 2 (Runx2), Indian hedgehog protein (Ihh), alkaline phosphatase (ALP) and ankylosis homolog (ANKH) in the HBP-A group were significantly lower than those in the model group. In vitro studies have shown that after HBP-A intervention, apoptosis, hypertrophy and calcification of rat meniscus fibrocartilage cells (PMFs) with abnormal mechanical injury can be significantly improved. The results of Western blot and RT-qPCR show that compared with the model group, the expression of meniscus fibrocartilage cell hypertrophy, calcification and p38-MAPK signaling pathway-related markers in the HBP-A group was significantly inhibited. Therefore, HBP-A can slow down meniscus hypertrophy and calcification caused by abnormal mechanical load, and its mechanism of action may be transduced through the p38-MAPK signaling pathway.

[0011] The use of mussel glucan HBP-A or its salt in the preparation of a therapeutic drug for knee osteoarthritis described in the present invention also has the following technical features: the therapeutic drug for knee osteoarthritis uses HBP-A or its salt as the sole active ingredient or is a pharmaceutical composition containing mussel glucan HBP-A or its salt. The content of HBP-A or its salt is 0.1-99 wt%.

[0012] In addition, the drug or pharmaceutical composition of the present invention can be formulated into any dosage form with pharmaceutically common excipients, such as joint cavity injection preparations, granules, capsules, or suspensions, etc., and preferably joint cavity injection preparations.

[0013] The second aspect of the present invention lies in providing a pharmaceutical preparation for preventing or treating KOA, which consists of HBP-A or its salt and pharmaceutically acceptable excipients. The excipient is preferably a drug carrier that enhances the sustained-release system. Preferably, the drug is prepared in the form of a joint cavity injection preparation.

[0014] Functions and effects of the present invention

[0015] The main research results of the present invention are as follows: (1) Abnormal mechanical injury activates the p38-MAPK signaling pathway and causes the degradation of HDAC4 by upregulating the expression of its downstream substrate caspase-3, which is an important molecular biological mechanism for the upregulation of markers related to meniscus hypertrophy and calcification; (2) The active ingredient HBP-A in mussel meat plays a role in slowing down or inhibiting meniscus overhypertrophy and calcification and protecting cartilage by regulating the upstream and downstream targets of the p38-MAPK signaling pathway.

[0016] The present invention verifies the specific signal transduction mechanism by which abnormal mechanical stimulation activates the p38-MAPK signaling pathway to cause meniscus hypertrophy and calcification, and explores the molecular mechanism of the protective effect of HBP-A on meniscus pathological degeneration by regulating the p38-MAPK signaling pathway. Meniscus injury is an early event in the pathological development of KOA. This study reveals the potential therapeutic effect of HBP-A in the treatment of KOA based on meniscus hypertrophy and calcification.

[0017] In addition, as a small molecule compound from natural sources, HBP-A has low toxicity and side effects and a relatively mature extraction process, which is beneficial to reducing the preparation cost of KOA treatment drugs, thereby reducing the medical expenses of patients to a certain extent. Description of the drawings

[0018] Figure 1 Shows the changes in the meniscus after intra-articular injection of HBP-A in KOA guinea pigs: HBP-A alleviates meniscus overhypertrophy and pathological calcification caused by abnormal mechanical injury. Among them, (A and B) Measurement and quantitative analysis of the width of medial and lateral meniscus hypertrophy after intervention with different concentrations of HBP-A, and quantitative comparison of the left and right knee joints in the medial or lateral meniscus; (C and D) Alizarin red staining and quantitative analysis show the calcification and its area of the medial meniscus of the right knee joint after intervention with different concentrations of HBP-A.

[0019] Figure 2It shows the pathological staining of the meniscus and cartilage after injecting HBP-A into the knee joint cavity of KOA guinea pigs: HBP-A alleviated the injury and degeneration of the medial meniscus and tibial articular cartilage caused by abnormal mechanical injury. Among them, (A and C) Safranin O / Fast Green staining and OA meniscus injury grading showed the injury of the medial meniscus of the right knee joint and the effects of different concentrations of HBP-A intervention; (B and D) The overall morphology of the tibial articular cartilage, Safranin O / Fast Green staining, and OA cartilage injury score described the injury of the tibial articular cartilage of the right knee joint and the effects of different concentrations of HBP-A intervention.

[0020] Figure 3 It shows the expression of markers related to meniscus hypertrophy and calcification after injecting HBP-A into the knee joint cavity of KOA guinea pigs: HBP-A reduced the overexpression of proteins and mRNAs related to pathological hypertrophy and calcification caused by abnormal mechanical injury. Among them, (A and B) Immunohistochemical staining and quantitative analysis showed the expression of hypertrophy-related protein markers MMP13, Ihh and calcification-related markers Runx2, ALP, ANKH in the meniscus. (C) RT-qPCR showed the mRNA expression levels of meniscus hypertrophy markers IL-1β, MMP13, Ihh and calcification marker Runx2.

[0021] Figure 4 It shows the cell morphology, pathological staining, etc. after HBP-A intervention on PMFs cells: 0.3 mg / ml HBP-A could improve the apoptosis and morphological changes of PMFS cells induced by 10% tensile force (model group): (A and B) Flow cytometry and analysis showed that apoptosis of PMFs in the model group increased significantly, and 0.3 mg / ml HBP-A and SB could reduce apoptosis of PMFs in the model group to varying degrees; (C and E) Phalloidin staining showed the morphology of PMF in different groups; (D and F-G) Alizarin red and toluidine blue staining showed the calcification and hypertrophy of PMF in different groups.

[0022] Figure 5 It shows the protein expression of markers related to meniscus hypertrophy and calcification and key targets of the p38-MAPK signaling pathway after HBP-A intervention on PMFs cells: HPB-A changed the expression levels of proteins related to PMFs calcification, hypertrophy and p38-MAPK signaling pathway: (A-H) Western blotting results of Caspase3, p38, HDAC4, MMP13, Ihh, ALP, ANKH: HBP-A may reduce the hypertrophy and calcification of PMFs by inhibiting the p38-MAPK signaling pathway.

[0023] Figure 6Shows the expression of key targets of the p38-MAPK signaling pathway and genes related to hypertrophy and calcification in the meniscus after HBP-A intervention on PMFs cells: HBP-A effectively changes the expression of genes related to hypertrophy and calcification in PMFs by inhibiting the overexpression of the p38-MAPK signaling pathway: (A-I) The RT-qPCR results of p38, Caspase3, HDAC4, Runx2, IL-1β, MMP13, Ihh, ANKH, and ALP are consistent with the expression trends of their related proteins. HBP-A can inhibit the p38-MAPK signaling pathway and reduce the hypertrophy and calcification of PMFs. Detailed implementation mode

[0024] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such methods being well known to those of ordinary skill in the art and being described in many publications.

[0025] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described in the detailed implementation mode are for illustrative purposes only.

[0026] 1. Preparation of HBP-A

[0027] HBP-A is prepared by processes such as boiling, centrifugation, extraction, lyophilization, and chromatography of mussel meat. The specific method can be seen in Patent ZL200610028598.0. After the mussel meat is crushed, it is extracted with hot water for 3 hours, and the temperature is controlled between 80-100 °C; the obtained extract is filtered or centrifuged to retain the supernatant, and 1-3 volumes of industrial alcohol are added with stirring, and then centrifuged again to retain the precipitate; the precipitate is dissolved in water and further separated by DESE-Sepharose FF or DEAE-Cellulose ion exchange gel, and water → 2M sodium chloride solution is used as the eluent; the sugar-containing part eluate obtained in the water elution part is dried to obtain mussel glucan.

[0028] The mussel glucan is used as an active ingredient to prepare an intra-articular injection preparation.

[0029] 2. Effects of HBP-A on the meniscus and PMFs of KOA guinea pigs

[0030] The anterior cruciate ligament of the right knee joint of 3-month-old guinea pigs was transected (ACLT). Three days after successful establishment of the model, 200 μl of different concentrations (divided into three different concentration groups: 7.5 mg / ml, 15 mg / ml, and 30 mg / ml) were injected into the right knee joint cavity twice a week for 10 weeks. After the treatment ended, the guinea pigs were euthanized with excessive carbon dioxide, and the meniscus and tibia were taken to observe the gross appearance, and alizarin red and safranin O / fast green staining were used to evaluate the damage of the meniscus and tibial articular cartilage.

[0031] Meniscal fibrochondrocytes (PMFs) were isolated from the knee joints of 1-month-old rats and the phenotypes of PMFs were identified after culturing for 48 hours. After passing the identification, PMFs were obtained by the same method. The PMFs were subjected to 24-hour cyclic mechanical (uniaxial sine wave with a 10% elongation rate and a frequency of 0.5 Hz) stretching using a Flexcell FX-5000 tension system, and then intervened with 0.3 mg / ml HBP-A and the p38-MAPK signaling pathway inhibitor SB203580. Flow cytometry and TRITC Phalloidin staining were used to evaluate the apoptosis and morphological changes of PMFs, and alizarin red and toluidine blue staining were used to explore the calcification and hypertrophy of PMFs.

[0032] The results showed that after injecting HBP-A into the knee joint cavity of KOA guinea pigs, the meniscal hypertrophy and calcification were significantly reduced, and the meniscal and cartilage damage was significantly improved ( Figure 1 , Figure 2 ). After HBP-A intervened in PMFs with abnormal mechanical stimuli, their morphology gradually tended to be normal, the apoptosis was significantly improved, and the hypertrophy and calcification were significantly reduced ( Figure 4 ).

[0033] 3. Effects of HBP-A on molecular markers related to meniscal hypertrophy and calcification

[0034] The protein and gene expression levels of markers related to meniscal hypertrophy and calcification were detected by immunohistochemical staining, Western blot, RT-qPCR and their quantitative analysis. The results showed that after intervention with HBP-A in vivo and in vitro, the overexpression of the protein and gene levels of markers related to meniscal hypertrophy and calcification could be significantly reduced ( Figure 3 , Figure 5 , Figure 6 ).

[0035] The protein and gene levels of the targets related to the p38-MAPK signaling pathway were detected by Western blot, RT-qPCR and their quantitative analysis after the intervention of HBP-A. The results showed that abnormal mechanical stimulation caused overactivation of the p38-MAPK signaling pathway, upregulation of the expression of related target proteins and genes, and HBP-A could downregulate the overexpression of the p38-MAPK signaling pathway ( Figure 5 , Figure 6 ), thereby reducing the pathological degradation of PMFs.

[0036] The parts not described in the present invention are the same as the prior art or are implemented by using the prior art. The applicant declares that the present invention illustrates the detailed method of the present invention through the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Use of mussel glucan HBP-A or its salt in the preparation of a therapeutic drug for knee osteoarthritis.

2. The application according to claim 1, wherein The therapeutic drug for knee osteoarthritis is a drug for slowing down or inhibiting meniscus degeneration.

3. The application according to claim 2, characterized in that, The drug for slowing down or inhibiting meniscus degeneration is a drug for treating meniscus hypertrophy or pathological calcification, or a drug for treating medial meniscus or articular cartilage injury and degeneration.

4. The application according to claim 3, wherein The drug for treating meniscus hypertrophy or pathological calcification is a drug for inhibiting the overexpression of the p38-MAPK signaling pathway.

5. The application according to any one of claims 1 to 3, characterized in that, The therapeutic drug for knee osteoarthritis is a drug composition taking mussel glucan HBP-A or its salt as the sole active ingredient or containing mussel glucan HBP-A or its salt.

6. The application according to claim 4, characterized in that The content of mussel glucan HBP-A or its salt in the therapeutic drug for knee osteoarthritis is 0.1-99 wt%.

7. The application according to claim 1, characterized in that The preparation form of the therapeutic drug for knee osteoarthritis is selected from injection preparations.

8. The application according to claim 1, wherein The injection preparation is selected from intra-articular injection preparations.

9. A pharmaceutical composition for preventing or treating knee osteoarthritis, characterized in that, It is composed of mussel glucan HBP-A or its salt and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, wherein The excipients include but are not limited to drug carriers for increasing the sustained and controlled release system.

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