Application of verbascoside in preparation of medicine for up-regulating low-expression protein component related diseases in cartilage cells

Abstract

The invention provides application of verbascoside in preparation of a medicine for up-regulating protein component related diseases with low expression in chondrocytes, and belongs to the technical field of medicine preparation, analysis and research on the verbascoside find that the verbascoside can up-regulate expression of NDUFA6 protein, AMPK protein and MYL3 protein in the chondrocytes, so that the verbascoside can be used for treating chondrocytes related diseases, and the application of the verbascoside to the preparation of the medicine for up-regulating the low-expression protein component related diseases in the chondrocytes. Therefore, the oxidative stress level in the cartilage cells is reduced, the mitochondrial dysfunction in the cartilage cells is relieved, the ATP content in the cartilage cells is increased, the compound can be used for preparing the medicine for treating or relieving the cartilage cell degeneration, and the progress of the cartilage cell degeneration is effectively relieved or delayed.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and relates to the technology of verbascoside for pharmaceutical preparation, specifically the application of verbascoside in the preparation of drugs for treating diseases related to the protein components with low expression in chondrocytes. Background Art

[0002] Chondrocytes are the only cell type in articular cartilage, responsible for synthesizing and secreting extracellular matrix (ECM) to maintain the structure and function of cartilage. However, with aging or under the influence of external factors, chondrocytes may undergo a series of changes, leading to chondrocyte degeneration, resulting in discomfort such as pain, stiffness, swelling or inflammation at the joint. Among them, the low expression of proteins such as NDUFA6, AMPK and MYL3 is an important factor contributing to chondrocyte degeneration. NDUFA6 is one of the subunits of mitochondrial respiratory chain complex I and is crucial for maintaining the normal function and energy metabolism of mitochondria. In chondrocytes, the low expression of NDUFA6 may lead to impaired mitochondrial function, thereby affecting the energy metabolism and physiological functions of chondrocytes, resulting in the inability of chondrocytes to effectively synthesize and secrete ECM, thus accelerating the degeneration and wear of cartilage. AMPK is an energy sensor that can regulate the metabolic activities of cells according to the change of the ratio of AMP / ATP in cells. In chondrocytes, AMPK maintains the energy balance and homeostasis of cells by regulating multiple metabolic pathways. The low expression of AMPK may lead to a decrease in the ability of chondrocytes to regulate energy metabolism, thereby affecting the proliferation, differentiation and metabolic activities of chondrocytes. The role of MYL3 in chondrocytes is mainly reflected in its participation in physiological processes such as the assembly of the cytoskeleton and cell movement. In addition, MYL3 also protects chondrocytes from degeneration by inhibiting clathrin-mediated endocytosis (CME). In chondrocytes, the low expression of MYL3 may lead to enhanced CME, thereby inducing the activation of the Notch signaling pathway and promoting the senescence and dysfunction of chondrocytes.

[0003] The abnormal protein expression of chondrocytes is one of the important reasons for the occurrence and development of cartilage diseases. Although there have been attempts to treat such diseases by drugs in the prior art, these drug treatment methods often can only temporarily relieve pain symptoms and cannot effectively improve or delay the progression of the disease fundamentally. Therefore, for those skilled in the art, it is particularly urgent to deeply explore new drugs for treating abnormal protein expression of chondrocytes. Summary of the Invention

[0004] As described in the above background art, in the prior art, treating abnormal protein expression in chondrocytes with drugs can only relieve pain symptoms temporarily, but cannot effectively improve or delay the progression of the disease fundamentally. There is still a technical problem that new drugs for treating abnormal protein expression in chondrocytes need to be explored deeply. In view of this technical problem, the present invention provides the use of acteoside in the preparation of drugs for diseases related to upregulating the low-expressed protein components in chondrocytes.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The use of acteoside in the preparation of drugs for diseases related to upregulating the low-expressed protein components in chondrocytes according to the present invention is characterized in that the diseases related to the low-expressed protein components in chondrocytes include chondral diseases caused by the low expression of protein components in chondrocytes.

[0007] Further defined, the low-expressed protein components in chondrocytes are NDUFA6 protein, AMPK protein and MYL3 protein.

[0008] Further defined, the chondral diseases include chondral degeneration diseases caused by the low expression of NDUFA6 protein, AMPK protein and MYL3 protein in chondrocytes.

[0009] Further defined, the chondral degeneration diseases include osteoarthritis, cartilage injury, chondromalacia, chondritis or systemic joint diseases.

[0010] Further defined, acteoside upregulates the expression of AMPK protein in chondrocytes by regulating the relative expression level of p-AMPK / AMPK protein.

[0011] Further defined, the molecular formula of acteoside is C 22 H 26 O 13 , with a molecular weight of 498.43 and a CAS number of 50932-20-2.

[0012] Further defined, the use of a preparation formed by acteoside and a pharmaceutically acceptable carrier or excipient in the preparation of drugs for treating or preventing chondral diseases.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The application of verbascoside in the preparation of drugs for treating diseases related to protein components with low expression in chondrocytes. Through the analysis and research of verbascoside, it is found that verbascoside can up-regulate the expression of NDUFA6 protein, AMPK protein and MYL3 protein in chondrocytes, thereby reducing the level of oxidative stress in chondrocytes, alleviating mitochondrial dysfunction in chondrocytes and increasing the ATP content in chondrocytes. It can be used to prepare drugs for treating and alleviating chondrocyte degeneration, and effectively alleviate or delay the progression of osteoarthritis or joint bony injury caused by chondrocyte degeneration. It has developed a new idea and approach for the development of therapeutic drugs for chondrocyte degeneration, and promoted the further development of drugs for osteoarthritis or joint bony injury. Brief Description of the Drawings

[0015] Figure 1 To detect the apoptosis of cells in each group by flow cytometry;

[0016] Figure 2 To detect the apoptosis rate of cells in each group by flow cytometry;

[0017] Figure 3 To detect the developed image of the protein expression in each group by Western blot;

[0018] Figure 4 To show the schematic diagram of the expression level of MYL3 protein in each group;

[0019] Figure 5 To show the schematic diagram of the expression level of NDUFA6 protein in each group;

[0020] Figure 6 To show the schematic diagram of the relative expression level of p-AMPK / AMPK protein in each group. Detailed Embodiments

[0021] The technical solutions of the present invention will be further explained below in conjunction with the drawings and embodiments, but the present invention is not limited to the embodiments described below.

[0022] Verbascoside is a natural glycoside substance. It exists in certain plants and is mainly composed of glucose and verbascoside alcohol. Its molecular formula is C 22 H 26 O 13 , with a molecular weight of 498.43 and a CAS number of 50932-20-2. The structural formula of verbascoside is

[0023] The verbascoside of the present application and another compound with the same name (molecular formula: C 29 H 36 O 15, Molecular weight: 624.59, CAS No.: 61276-17-3) Although they have the same Chinese name, there are significant differences in molecular formula, molecular weight, CAS number, as well as uses and efficacy.

[0024] Hereinafter, the acteoside in this application is represented by Verproside, and another acteoside with the same name is represented by Verbascoside.

[0025] There is currently no clear report on the specific uses and efficacy of Verproside.

[0026] Uses of Verbascoside: It is widely present in various medicinal plants, such as Cistanche deserticola, Rehmannia glutinosa, Ligustrum lucidum, Verbena officinalis, etc., and is one of the main active ingredients of these plants. Therefore, it is often used in the fields of medicine, health care, etc. as a raw material for drugs or health products. Efficacy of Verbascoside: ① It has the ability to inhibit the proliferation, invasion and migration of tumor cells, and can promote the apoptosis of tumor cells. By targeting mesenchymal epidermal transition factor (c-Met), it effectively inhibits epithelial-mesenchymal transition (EMT), thereby inhibiting the proliferation, migration and invasion of malignant tumors such as glioblastoma. ② Anti-inflammatory, Verbascoside can significantly inhibit the release of inflammatory mediators, reduce the inflammatory response, and effectively slow down the inflammatory process by intervening in the NF-κB pathway and inhibiting the activation of AP-1 in macrophages. ③ Neuroprotection, Verbascoside shows neuroprotective effects in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, by reducing the deposition of β-amyloid (Aβ), improving the normal synaptic activity between neurons, and protecting nerve cells from oxidative damage and apoptosis. ④

[0027] Verbascoside also has various pharmacological effects such as antioxidant, anti-skin damage, improving immune function, enhancing memory, reducing blood lipid and laxative effects.

[0028] Therefore, although Verproside and Verbascoside have the same Chinese name, there are significant differences in molecular formula, molecular weight, CAS number, as well as uses and efficacy. When conducting research and application, the two should be clearly distinguished to avoid confusion. This application mainly focuses on the research of Verproside, which has been less studied currently.

[0029] Mitochondrial dysfunction leading to elevated levels of reactive oxygen species (ROS) is closely associated with knee osteoarthritis (KOA). Mitochondria are ubiquitous intracellular organelles that not only regulate cellular energy production but also participate in cellular metabolism and functions, which are crucial for cell survival and death. Mitochondrial energy generated through adenosine triphosphate (ATP) synthesis consists of a series of redox reactions that involve the coordinated transfer of electrons through the electron transport chain (ETC) via four key electron carrier protein complexes (including ETC complex I, ETC complex II, ETC complex III, and ETC complex IV). In particular, ETC complex I and ETC complex III are the major sites of ROS formation in mitochondria. ETC complex I mediates ROS production, and defects in ETC complex I lead to increased ROS production and reduced antioxidant defense, thereby impairing mitochondrial function. Studies have demonstrated that the NDUFA6 gene has been identified as a key gene involved in the assembly and function of mitochondrial complex I. Research has shown that the stabilization of the TMH1-2ND3 loop by the auxiliary subunit LYRM6 (NDUFA6) is crucial for the energy conversion of mitochondrial complex I. In addition, mitochondrial dysfunction leads to increased release of ROS, resulting in reduced AMPK activity. AMPK is generally considered an essential regulator of cellular energy homeostasis. Studies have shown that AMPK activation can inhibit oxidative stress and various inflammatory responses in chondrocytes.

[0030] It has been found through research that verbascoside has a good effect on enhancing the expression of NDUFA6 protein, AMPK protein, and MYL3 protein in chondrocytes, thereby reducing the level of oxidative stress in chondrocytes, alleviating mitochondrial dysfunction in chondrocytes, and increasing the content of high-energy phosphate compound ATP in chondrocytes, and can be used to prepare drugs for treating and alleviating chondrocyte degeneration.

[0031] Example 1

[0032] This example experimentally verifies the use of verbascoside in the preparation of drugs for treating or preventing cartilage diseases by improving chondrocyte apoptosis.

[0033] 1. Experimental materials

[0034] The Annexin V-FITC apoptosis detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The chemiluminescence instrument was the Shanghai Tanon 4800 full-automatic chemiluminescence image analysis system. The BD FACSymphony A1 flow cytometer was from BD Medical Devices (Shanghai) Co., Ltd. The NDUFA6 antibody and MYL3 antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd. The AMPK antibody and p-AMPK antibody were purchased from immunoway Co., Ltd.

[0035] 2. Experimental methods

[0036] 2.1 Administration method

[0037] For the construction of the IL-1β-induced chondrocyte degeneration model, three administration groups were prepared. Cell suspensions were prepared and plated. 20x10 4 cells were added to each well of a 6-well plate. The administration method was as follows: The chondrocytes were divided into 5 groups, including 3 groups co-cultured with drugs and IL-1β, one model group, and one control group. Except for the control group, the 3 administration groups and the model group were respectively co-cultured with serum-free medium containing 10 ng / ml of IL-1β for 24 h.

[0038] The grouping is as follows:

[0039] (1) Control group (ordinary medium);

[0040] (2) Model group (medium containing 10 ng / L IL-1β);

[0041] (3) Catalpol group (medium containing 10 ng / L IL-1β + 50 μmol / L catalpol);

[0042] (4) 1,5-Dicaffeoylquinic acid group (medium containing 10 ng / L IL-1β + 50 μmol / L 1,5-dicaffeoylquinic acid);

[0043] (5) Acteoside group (medium containing 10 ng / L IL-1β + 50 mol / L acteoside).

[0044] 2.2 Flow cytometry

[0045] The cells to be detected were digested with trypsin, and the cells were collected into a centrifuge tube. The collected cells were washed with PBS and centrifuged to remove the supernatant. The cells were resuspended with 585 μl of Annexin V-FITC binding solution. Subsequently, 15 μl of Annexin V-FITC and 30 μl of propidium iodide (PI) staining solution were added, mixed well, and incubated in the dark at room temperature for 10 min. Detection was performed using a flow cytometer.

[0046] 2.3 Experimental results

[0047] See Figure 1 and Figure 2 , compared with the control group, the apoptosis rate of chondrocytes in the IL-1β group was significantly increased (P < 0.001). Compared with the model group, the apoptosis rate of chondrocytes in the acteoside group was significantly decreased (P < 0.001). In Figure 1 and Figure 2In this model, the IL-1β-induced chondrocyte degeneration model can lead to cartilage damage, resulting in mitochondrial dysfunction, increasing the level of protein peroxidation in body tissues, ultimately producing cytotoxic effects, and may lead to chondrocyte apoptosis. It was found by flow cytometry that acteoside can improve chondrocyte apoptosis.

[0048] Example 2

[0049] In this example, the use of acteoside in the preparation of drugs for the treatment or prevention of cartilage diseases by upregulating the low-expressed protein components in chondrocytes was experimentally verified.

[0050] 1. Experimental materials

[0051] Same as the first part of Example 1.

[0052] 2. Experimental methods

[0053] 2.1 Administration method

[0054] Same as the first part of Example 1.

[0055] 2.2 Western blotting

[0056] Collect the cells after co-culturing catalpol, acteoside, 1,5-dicaffeoylquinic acid and IL-1β for 24 h, and extract the total cellular proteins. Preparation work: Turn on the low-temperature high-speed centrifuge in advance and pre-cool it at 4°C; prepare RIPA lysis buffer containing 2% PMSF, and according to every 10 6 cells use 100 μl of lysis buffer, and the obtained protein concentration is about 2 - 4 mg / ml. The specific steps are as follows:

[0057] ① Extraction of cellular proteins: After the cells are washed and centrifuged, add PIRA lysis buffer, a protein lysis buffer prepared by mixing PMSF (phenylmethylsulfonyl fluoride): phosphatase inhibitor: protease inhibitor = 100:1:1. The cells are lysed on ice for 30 min, then scraped off with a cell scraper and sonicated for 1 min. Centrifuge at 10000 r / min for 10 min to separate, and take the protein supernatant. Mix the loading buffer and the collected protein supernatant at a volume ratio of 1:4, place it in a thermostatic oscillator and heat at 100°C for 8 min, cool at room temperature and then store at -20°C in a refrigerator.

[0058] ② Clean the glass plates and check the leak of the gel maker, and prepare the SDS-PAG electrophoresis gel.

[0059] ③ Loading: Add the protein Marker and the extracted protein samples into the gel wells respectively, and fill up the electrophoresis buffer.

[0060] ④Electrophoresis: Turn on the electrophoresis apparatus. After constant voltage electrophoresis at 80 V for about 45 min, the blue Marker appears. Then change the voltage to 120 V and continue electrophoresis until the bottom of the separating gel, which takes about 50 min.

[0061] ⑤Transfer membrane: Cut the PVDF membrane to an appropriate size and activate it in anhydrous methanol. Cut the gel according to the protein Marker and transfer the membrane at a constant current of 220 mA.

[0062] ⑥Blocking: After the membrane transfer is completed, put the membrane into a 5% milk blocking solution (1 g of skim milk powder plus 20 ml of TPBS) and block it at 37 °C for 2 h.

[0063] ⑦Incubate with primary antibody: Discard the milk blocking solution, pour in TBST, place it on a shaker and wash it 3 times at room temperature, 10 min each time. Add the diluted primary antibodies of COL2A1 and MMP-13 (the dilution ratio is 1:1500) to the antibody incubation box, put the membrane into the incubation box to incubate the antibody, overnight at 4 °C; Wash the membrane: Put the membrane into the box filled with TBST and wash the membrane 3 times, 10 min each time.

[0064] ⑧Incubate with secondary antibody: Add the secondary antibody to the 5% milk blocking solution (milk blocking solution: secondary antibody = 1:10000), put the membrane into the prepared secondary antibody, and incubate it on a shaker at 37 °C for 2 h; Wash the membrane: Wash it 3 times with TBST and 1 time with PBS, 10 min each time.

[0065] ⑨Developing: Expose the membrane coated with the developing solution (1:1) using a chemiluminescence instrument, the protein expression can be seen, and save the picture.

[0066] 2.3 Experimental results

[0067] See Figure 3 、 Figure 4 、 Figure 5 and Figure 6 Compared with the control group, the expressions of NDUFA6 protein, AMPK protein and MYL3 protein in chondrocytes of the IL-1β group were all decreased (P<0.001). Compared with the model group, acteoside (P<0.01) significantly corrected the abnormal protein expression in mitochondrial respiratory chain complex I (NDUFA6), and at the same time increased the phosphorylation level of AMPK protein and the expression level of MYL3 protein. It is shown that acteoside can up-regulate the expressions of NDUFA6 protein, AMPK protein and MYL3 protein in chondrocytes, thereby reducing oxidative stress in chondrocytes, increasing the mitochondrial membrane potential level, increasing ATP content, alleviating mitochondrial dysfunction in chondrocytes, reducing apoptosis of chondrocytes, alleviating the progression of osteoarthritis, providing an innovative strategy for the treatment of osteoarthritis, developing a new idea and approach for the development of therapeutic drugs for chondrocyte degeneration, and promoting the further development of drugs for chondrocyte degeneration.

[0068] In the present invention, preferably, the use of verbascoside in the preparation of a medicament for treating or preventing cartilage diseases in the form of a preparation with a pharmaceutically acceptable carrier or excipient.

[0069] In the present invention, the diseases related to the protein components with low expression in chondrocytes include cartilage diseases caused by the low expression of protein components in chondrocytes. The cartilage diseases include cartilage degeneration diseases caused by the low expression of NDUFA6 protein, AMPK protein and MYL3 protein in chondrocytes. Among them, the cartilage degeneration diseases include osteoarthritis, cartilage injury, chondromalacia, osteochondritis or systemic joint diseases.

[0070] It should be noted that the verbascoside in the specification drawings refers to verbascoside in the content of the present application.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of verbascoside in the preparation of a drug for upregulating a protein component underexpressed in chondrocytes and related diseases, characterized in that: The diseases associated with the protein component low expression in chondrocytes include cartilage diseases caused by low expression of the protein component in chondrocytes.

2. The use according to claim 1, characterized in that The protein components with low expression in the chondrocytes are NDUFA6 protein, AMPK protein and MYL3 protein.

3. The use according to claim 1, characterized in that The cartilage diseases include cartilage degeneration diseases caused by low expression of NDUFA6 protein, AMPK protein and MYL3 protein in chondrocytes.

4. The use according to claim 3, characterized in that The cartilage degenerative diseases include osteoarthritis, cartilage damage, chondromalacia, osteochondritis or systemic joint diseases.

5. The use according to claim 3, characterized in that: The verbascoside upregulates the expression of AMPK protein in chondrocytes by regulating the relative expression level of p-AMPK / AMPK protein.

6. The use according to any one of claims 1 to 5, characterized in that: The molecular formula of the verbascoside is C 22 H 26 O 13 , molecular weight is 498.43 and CAS number is 50932-20-2.

7. The use according to any one of claims 1 to 5, characterized in that The verbascoside is combined with a pharmaceutically acceptable carrier or auxiliary material to form a preparation for preparing a drug for treating or preventing cartilage diseases.

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