Application of catalpa glycoside in preparation of medicine for treating and relieving diseases related to mitochondrial dysfunction

Abstract

The invention provides application of catalpa glycoside in preparation of drugs for treating and relieving diseases related to mitochondrial dysfunction, and belongs to the technical field of medicine preparation. The catalpa glycoside can improve the mitochondrial membrane potential, reduce the active oxygen level in cartilage cells, improve the ATP content in the cartilage cells and up-regulate the expression level of fusion and division related proteins in the cartilage cells, so that the mitochondrial dysfunction is improved, cartilage repair is promoted, and the catalpa glycoside can be applied to preparation of drugs for treating and relieving arthritis and has a good application prospect. The disease progress of arthritis can be effectively relieved or delayed.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and relates to the related technology of using catalpa glycosides for drug preparation, and specifically to the application of catalpa glycosides in preparing drugs for treating and alleviating diseases related to mitochondrial dysfunction. Background Art

[0002] Arthritis is a common joint disease that has a significant impact on the daily quality of life of patients. Arthritis generally refers to inflammatory diseases that occur in human joints and surrounding tissues, which can be caused by inflammation, infection, degeneration, trauma or other factors. Knee osteoarthritis (KOA) is the most common type of arthritis and is recognized as one of the main causes of disability in the elderly. It specifically refers to osteoarthritis that occurs in the knee joint. Knee osteoarthritis is a disease based on degenerative lesions of the knee cartilage, which is mainly manifested as pain, swelling, stiffness and dysfunction of the knee joint. With aging, overuse of joints, obesity, joint injuries and genetic factors, the knee cartilage gradually wears away, which in turn causes inflammatory reactions and pain. As a traditional topical preparation, anti-swelling and analgesic patches have been widely used in the treatment of arthritis. It can effectively relieve symptoms such as joint swelling, pain and stiffness through local administration.

[0003] The recently released "Guidelines for the Diagnosis and Treatment of Osteoarthritis in China (2024 Edition)" (Osteoarthritis Committee of the Chinese Geriatric Association, etc., 2024) points out that in the step-by-step treatment of osteoarthritis, repair therapy is mainly used in the early or mild stages of the disease. This includes the use of cartilage repair drugs, joint injection therapy, and cartilage repair surgery (such as platelet-rich plasma therapy and stem cell therapy). However, these treatments are usually expensive and may be accompanied by side effects. In clinical practice, it is particularly important to explore drugs that can effectively relieve joint pain and swelling and promote cartilage repair. In modern research on swelling and pain relief patches, it was found that ziziphi zanthoxylum bungeanum is one of the ingredients in the preparation that can penetrate the skin and be absorbed, but whether ziziphi zanthoxylum bungeanum can be used as an active ingredient in swelling and pain relief patches to relieve arthritis symptoms (such as pain and swelling) requires further study. Summary of the invention

[0004] In view of the above background technology, whether ziziphus glycosides can be used as an active ingredient in anti-edema and analgesic patches to relieve arthritis symptoms (such as pain and swelling) still requires further research. In response to this technical problem, the present invention proposes the use of ziziphus glycosides in the preparation of drugs for treating and alleviating diseases related to mitochondrial dysfunction, with the aim of providing a drug that can effectively treat and relieve arthritis and promote cartilage repair.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The invention discloses an application of catalpa glycosides in the preparation of drugs for treating and alleviating diseases related to mitochondrial dysfunction, wherein the diseases related to mitochondrial dysfunction include arthritis caused by mitochondrial dysfunction.

[0007] It is further defined that the catalpa glycoside treats and alleviates diseases related to mitochondrial dysfunction by improving mitochondrial membrane potential.

[0008] It is further defined that the catalpa glycosides treat and alleviate diseases related to mitochondrial dysfunction by reducing the level of reactive oxygen species in chondrocytes.

[0009] It is further defined that the catalpa glycoside improves the treatment and alleviates diseases related to mitochondrial dysfunction by increasing the ATP content in chondrocytes.

[0010] It is further defined that the catalpa glycosides can treat and alleviate diseases related to mitochondrial dysfunction by upregulating the expression levels of fusion and fission related proteins in chondrocytes.

[0011] It is further defined that the related proteins include one or more of NDUFS 6, AMPK and MYL 3.

[0012] It is further defined that the catalpa glycoside upregulates the expression level of AMPK protein in chondrocytes by regulating the relative expression level of p-AMPK / AMPK protein.

[0013] In a further embodiment, the arthritis includes osteoarthritis or osteochondritis caused by osteochondritis.

[0014] It is further defined that the catalpa glycoside is combined with a pharmaceutically acceptable carrier or excipient to form a preparation for use in treating and alleviating diseases related to mitochondrial dysfunction.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The invention discloses an application of catalpa glycosides in the preparation of drugs for treating and alleviating diseases related to mitochondrial dysfunction. By analyzing and studying catalpa glycosides, it is found that catalpa glycosides can increase mitochondrial membrane potential, reduce the level of reactive oxygen in chondrocytes, increase the ATP content in chondrocytes, and upregulate the expression level of fusion and fission-related proteins in chondrocytes, thereby improving mitochondrial dysfunction and promoting cartilage repair. The invention can be applied to the preparation of drugs for treating and alleviating arthritis, and can effectively alleviate or delay the progression of arthritis. The invention develops a new idea and approach for drugs for treating arthritis, and promotes the further development of drugs for treating arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The viability of chondrocytes stimulated with different concentrations of IL-1β for 24 h;

[0018] Figure 2 To detect the level of TNF-α inflammatory factor after chondrocytes were stimulated with different concentrations of IL-1β using ELISA technology;

[0019] Figure 3 The images are the development of COL2A1 protein expression after chondrocytes were stimulated with different concentrations of IL-1β;

[0020] Figure 4 Schematic diagram of the relative expression levels of MMP-13 / GAPDH proteins detected by WB technology after chondrocytes were stimulated with different concentrations of IL-1β;

[0021] Figure 5 The changes in chondrocyte proliferation after 24h treatment with 6 compounds;

[0022] Figure 6 Fluorescence images of each group detected by DCFH-DA fluorescent probe;

[0023] Figure 7 This is the fluorescence intensity diagram of each group detected by DCFH-DA fluorescent probe;

[0024] Figure 8 The JC-1 probe staining method was used to detect the changes in mitochondrial membrane potential in each group;

[0025] Fig. 9 This is a statistical graph of mitochondrial membrane potential in each group detected by JC-1 probe staining;

[0026] Fig.10 Comparison of ATP content test results in each group;

[0027] Fig.11 Flow cytometry was used to detect cell apoptosis in each group;

[0028] Fig.12 Flow cytometry was used to detect the cell apoptosis rate in each group;

[0029] Fig.13 The images are the development images of protein expression in each group detected by immunoblotting;

[0030] Fig.14 Schematic diagram of MYL3 protein expression levels in each group;

[0031] Fig.15 Schematic diagram of the NDUFA6 protein expression level in each group;

[0032] Fig.16 Schematic diagram of the relative expression levels of p-AMPK / AMPK proteins in each group. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the implementation modes described below.

[0034] Catalposide, with a chemical name of Catalposide and a CAS number of 6736-85-2, is an iridoid glycoside compound extracted from plants of the Bignoniaceae family. Its molecular formula is C 22 H 26 O 12 , with a molecular weight of 482.43. Catalpa glycosides have a wide range of applications in medicine and scientific research. As an effective (HO)-1 inducer, catalpa glycosides exhibit a variety of biological activities, including antioxidant, anti-apoptotic, anti-microbial, anti-tumor and anti-inflammatory properties. Studies have shown that catalpa glycosides can inhibit the production of TNF-α, IL-1β and IL-6 in lipopolysaccharide-activated RAW 264.7 macrophages, as well as the activation of NF-κB (p65), thereby playing an important role in inflammation and immune regulation. In addition, catalpa glycosides have also been found to have anti-tumor activity and have significant inhibitory effects on a variety of tumor cell lines.

[0035] Mitochondrial dysfunction accompanied by elevated levels of reactive oxygen species (ROS) is closely associated with knee osteoarthritis (KOA). Mitochondria are ubiquitous organelles in cells that not only regulate cellular energy production but also participate in cellular metabolism and function, playing a vital role in cell survival and death. The energy generated by the synthesis of adenosine triphosphate (ATP) in mitochondria comes from a series of redox reactions involving the electron transport chain (ETC), a process coordinated by four key electron carrier protein complexes (ETC complex I, ETC complex II, ETC complex III, and ETC complex IV) to complete electron transfer. In particular, ETC complex I and ETC complex III are the main sites of reactive oxygen species (ROS) generation in mitochondria. In knee osteoarthritis (KOA), the electron transport chain (ETC) complex is affected, resulting in impaired electron transfer and energy metabolism, reduced adenosine triphosphate (ATP) production, and elevated levels of reactive oxygen species (ROS), indicating dysfunction and abnormality of mitochondrial function. This abnormality induces the production of other proinflammatory stimuli, including cytokines IL-1β, IL-6, and PGE2. ETC complex I mediates ROS generation, and its deficiency leads to increased reactive oxygen species (ROS) production and weakened antioxidant defense capacity, which further impairs mitochondrial function.

[0036] Studies have shown that catalpa glycosides can increase mitochondrial membrane potential, reduce the level of reactive oxygen species in chondrocytes, increase the ATP content in chondrocytes, and upregulate the expression levels of fusion- and fission-related proteins in chondrocytes, thereby improving mitochondrial dysfunction and promoting cartilage repair. It can be used to prepare drugs for the treatment and relief of arthritis.

[0037] Arthritis includes osteoarthritis or cartilage arthritis caused by osteochondritis.

[0038] Example 1

[0039] This example experimentally verifies the use of catalpa glycosides in the preparation of drugs for treating and alleviating diseases related to mitochondrial dysfunction, wherein the diseases related to mitochondrial dysfunction include arthritis caused by mitochondrial dysfunction.

[0040] 1. Experimental Materials

[0041] 1.1 Experimental cells and drugs

[0042] Primary chondrocytes were obtained from the cartilage tissue of the lower limbs of healthy male SD rats aged 4 weeks. SD rats were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., weighing approximately (135±5) g, with a license number of SCXK(Beijing)2021-0006. Experimental operations and corresponding treatment of experimental animals were strictly carried out in accordance with the Guiding Opinions on the Ethical Treatment of Laboratory Animals. All standard product information is shown in Table 1.

[0043] Table 1 Standard product information

[0044]

[0045] 1.2 Experimental Reagents

[0046] (1) Preparation of main reagents:

[0047] 0.2% collagenase type II solution: Add 50 mg collagenase powder into 25 mL DMEM / F12 medium, vortex to fully dissolve and mix, and store in a refrigerator at 4°C for later use.

[0048] DMEM / F12 (50 mL) complete medium containing 15% fetal bovine serum: add 7.5 mL of fetal bovine serum to 42.5 mL of DMEM / F12 medium, then add 500 μL of double antibody, mix well and store in a refrigerator at 4°C for later use.

[0049] (2) Detailed information of reagents

[0050] Detailed information on reagents is provided in Table 2.

[0051] Table 2 Detailed information of reagents

[0052]

[0053]

[0054] (3) Experimental instruments and equipment

[0055] Detailed information on the experimental instruments and equipment is shown in Table 3.

[0056] Table 2 Detailed information of experimental instruments and equipment

[0057]

[0058]

[0059] 2. Experimental methods

[0060] 2.1 Isolation, culture, cryopreservation and recovery of primary chondrocytes from SD rats

[0061] (1) Isolation of primary chondrocytes from SD rats

[0062] SD rats were killed by cervical dislocation, and the hair on the lower limbs and abdomen was shaved with a shaver. The limbs were removed and immediately soaked in 75% ethanol for 5 minutes to remove the fur and muscles on the surface of the legs. The knee joint segment was cut and quickly moved into the cell room. Under the aseptic conditions of the clean bench, sterile scissors and tweezers were used to peel off the muscles and ligaments around the joints, expose the joint cavity, remove the surface synovium, cut the articular cartilage, place it in a culture dish with 6mL double-antibody PBS, wash it 4-5 times, and cut it into 1mm with ophthalmic scissors. 3 For small pieces of about 1000 pieces, add 0.25% trypsin to digest for 40 minutes, discard the supernatant, add 0.2% type II collagenase (dissolved and diluted in DMEM / F12 medium) to the remaining tissue, place it in a constant temperature shaker at 37°C, digest for 4-5 hours, until the cartilage tissue pieces disappear, and obtain a cell suspension. After repeated blowing, pass through a 210-mesh filter in turn. Collect the filtrate, transfer it to a centrifuge tube, centrifuge it at 1200r / min for 8 minutes, discard the supernatant, resuspend it with DMEM / F12 double-antibody complete medium to obtain a single cell suspension, plant the cells in a T25 cell culture flask, and place it in a 37°C, 5% CO2 culture flask according to the counting density. 2 Culture in a cell culture incubator, and change the medium after the extracted cells adhere to the wall for 4-5 days. In the later stage, the medium can be changed once every other day.

[0063] (2) Subculture of primary chondrocytes from SD rats

[0064] When the cell density reaches more than 80% fusion, the cells are subcultured. When subcultured, the supernatant is first aspirated, 2 mL of PBS solution is added to gently wash the cells 3 times, trypsin digestion solution is added for 2-3 minutes, the cell morphology is observed to be wrinkled or round under an optical microscope, and double volume of complete culture medium is added to stop digestion. The cells are gently blown off with a pipette to form a suspension, collected in a 15 mL centrifuge tube and centrifuged at 1000 r / min for 3 minutes, the supernatant is poured out, fresh complete culture medium is added again, and the cells are gently blown to resuspend, and the cells are evenly inoculated in a cell culture dish at a ratio of 1:2 for culture. After the cells are subcultured 3 times, the experiment is carried out.

[0065] (3) Cryopreservation of primary chondrocytes from SD rats

[0066] Chondrocytes with good growth status and in the logarithmic growth phase (2nd-3rd generation) were selected for cryopreservation. The early operation was the same as cell passaging, that is, the chondrocytes were digested with trypsin and collected in a centrifuge tube for centrifugation. After the centrifugation, the supernatant was removed as much as possible, and the cell freezing solution was added and gently pipetted to resuspend, and the cell density was controlled to 1x10 6 / ml. Pack into cell cryopreservation tubes and label them, seal them with sealing film, transfer them into cell cryopreservation boxes, place them in a -80℃ refrigerator overnight, and transfer them to liquid nitrogen for storage after 24 hours. At the same time, cells are cryopreserved at the 2nd-3rd generation.

[0067] (4) Cell recovery of primary chondrocytes from SD rats

[0068] Before the operation, take out the culture medium from the 4℃ refrigerator and place it at room temperature. Adjust the water bath temperature to 37℃, take out the cryopreserved tube, quickly place it in a 37℃ water bath, gently shake (within 2 minutes) to thaw the cells, place the frozen cells in a 15ml centrifuge tube in the clean bench, add 6mL of complete culture medium, gently blow to mix, centrifuge at 1000r / min for 3 minutes, discard the supernatant, add 6mL of complete culture medium, gently blow to resuspend, and evenly inoculate in cell culture dishes for culture.

[0069] 2.2 Detection of IL-1β toxicity to chondrocytes using CCK-8 assay

[0070] The CCK-8 method was used to detect the viability of chondrocytes after stimulation with different concentrations of IL-1β. 4 The density of cells / well was inoculated in a 96-well plate overnight. After 24 hours, the cells were fully attached to the wall. After 24 hours of stimulation with different concentrations of IL-1β (0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL), the previous culture medium was discarded, and 10% 100 μL of CCK-8 solution (diluted with culture medium) was added to the cell wells in sequence. The cells were placed in an incubator for incubation for 1.5 hours, and the OD value was measured at a wavelength of 450 nm using an enzyme reader after being taken out.

[0071] Experimental results: Before induction, chondrocytes were normal. After 10ng / mL IL-1β induced chondrocytes with a 24-hour decrease in cell number, and the decrease was significant after 20ng / mL. CCK8 method was used to detect the effects of IL-1β concentration groups (0ng / m1, 5ng / m1, 10ng / m1, 20ng / m1, 40ng / m1, 80ng / m1, 160ng / m1, 320ng / m1 and 640ng / m1) on the activity of rat chondrocytes at 24 hours. Figure 1 After 24 hours of IL-1β intervention, compared with the empty control group (0ng / m1), IL-1β at concentrations of 5ng / m1, 10ng / m1, 20ng / m1, 40ng / m1, 80ng / m1, 160ng / m1, 320ng / m1 and 640ng / m1 did not produce toxic effects on rat chondrocytes, the cells did not show growth inhibition, and the percentage of live cells was not statistically different from that of the empty control group.

[0072] 2.3 CCK-8 method was used to detect the toxicity of six compounds (6-feruloyl catalpol, chlorogenic acid, verbascoside, catalpol, picroside II and 1,5-dicaffeoquinic acid) on chondrocytes

[0073] The CCK-8 method was used to detect the viability of chondrocytes stimulated by 6 compounds at different concentrations (0 μM, 25 μM, 50 μM and 100 μM). The specific operation method was the same as in Section 2.2.

[0074] Experimental results: Figure 5 As shown, it can be seen that the administration concentration of 50 μM for the six compounds is within the safe range, so 50 μM is used as the subsequent administration concentration.

[0075] 2.4 Drug administration

[0076] IL-1β-induced chondrocyte degeneration model was constructed with 6 transdermal component intervention groups. Cell suspension was prepared and plated. 20x10 4 Cells were administered as follows: chondrocytes were divided into 8 groups, 6 drugs were co-cultured with IL-1β, one model group, one control group, and 6 drug groups and the model group were co-cultured with serum-free medium containing 10ng / ml IL-1β for 24h. The grouping is as follows:

[0077] (1) Control group (normal culture medium);

[0078] (2) model group (medium containing 10 ng / L IL-1β);

[0079] (3) chlorogenic acid group (medium containing 10 ng / L IL-1β + 50 μmol / L chlorogenic acid);

[0080] (4) 6-feruloyl catalpol group (medium containing 10 ng / L IL-1β + 50 μmol / L 6-feruloyl catalpol)

[0081] (5) catalpa glycoside group (medium containing 10 ng / L IL-1β + 50 μmol / L catalpa glycoside);

[0082] (6) Picroside II group (medium containing 10 ng / L IL-1β + 50 μmol / L picroside II);

[0083] (7) 1,5-dicaffeoylquinic acid group (medium containing 10 ng / L IL-1β + 50 μmol / L 1,5-dicaffeoylquinic acid);

[0084] (8) Verbascoside group (medium containing 10 ng / L IL-1β + 50 μmol / L Verbascoside).

[0085] 2.5 Establishment of IL-1β-induced chondrocyte injury model in SD rat knee joint

[0086] Grouping: Chondrocytes were plated in 6-well plates, with 200,000 cells in each well. After the control group (only culture medium) and the generation of cells adhered to the wall, the conditions of 24 hours of IL-1β induction of chondrocytes were investigated. The ELISA technique was used to detect the levels of TNF-α inflammatory factors after 24 hours of stimulation with IL-1β at different concentrations, and the WB technique was used to detect the levels of COL2A1 and MMP13 proteins after 24 hours of stimulation with IL-1β at different concentrations to determine the conditions for IL-1β-induced cartilage degeneration.

[0087] (1) ELISA was used to detect the level of TNF-α inflammatory factor after 24 h of stimulation with different concentrations of IL-1β

[0088] The TNF-α content in chondrocytes of each group was detected by enzyme-linked immunosorbent assay (ELISA). The detection method was performed according to the instructions in the kit, as follows:

[0089] ① Take out the reagent kit and equilibrate it at room temperature for 30 minutes. At the same time, take out the sample and thaw it naturally at room temperature. The sample is first centrifuged in a centrifuge at 3000g / min for 10 minutes, and the supernatant is taken for detection.

[0090] ② Take out the ELISA plate, set up the standard sample well, blank well and sample well on the ELISA plate. Perform gradient dilution of the standard sample, take out 50μL of each concentration of standard sample and add it to the corresponding standard sample well, a total of 6 concentration gradients, and make 1 duplicate well for each concentration.

[0091] ③ Add 50 μL of centrifuged cell supernatant to the sample well, cover with sealing film, and incubate in a 37°C constant temperature incubator for 1 hour.

[0092] ④After incubation, remove the ELISA plate and discard the liquid in the wells, pat dry on filter paper, add 300 μL of plate washing solution to each well, shake gently, soak for 30 seconds, discard the plate washing solution, and wash the plate again for a total of 6 times.

[0093] ⑤ After washing, add biotinylase-labeled secondary antibody to all wells except the blank well, 50 μL / well, shake gently, cover with sealing film, and incubate in a 37°C constant temperature incubator for 0.5 hour.

[0094] ⑥After incubation, remove the ELISA plate, discard the liquid in the wells, pat dry on filter paper, and wash the plate according to the above-mentioned washing method for a total of 6 times. Add 50μL / well of color development solution A to each well, then add 50μL / well of color development solution B, shake and mix, and incubate in a 37℃ constant temperature incubator for 20 minutes.

[0095] ⑦ After the incubation is completed, take out the ELISA plate, peel off the sealing film, add the stop solution directly to each well, 50μL / well, shake and mix, and measure the absorbance value directly on the ELISA instrument. The measurement must be completed within 20 minutes.

[0096] ⑧ Fit the standard curve according to the standard sample concentration and the OD value of the standard sample well, then fit the standard equation according to the standard curve, substitute the OD value of each measurement well into the equation, calculate the concentration value of the analyte in each measurement well, and perform statistical analysis.

[0097] Test results: See Figure 2 , compared with the blank group, the TNF-α level in the 10 ng / mL IL-1β group was significantly increased (P<0.001).

[0098] (2) Western blotting was used to detect the protein levels of COL2A1 and MMP-13 after 24 h of stimulation with different concentrations of IL-1β

[0099] Preparation: 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 6 The cells were lysed with 100 μl of buffer and the protein concentration obtained was approximately 2-4 mg / ml.

[0100] The specific steps are as follows:

[0101] ① Extraction of cell protein: After washing and centrifugation, add PIRA lysis buffer, PMSF (phenylmethylsulfonyl fluoride): phosphatase inhibitor: protease inhibitor = 100:1:1 protein lysis buffer, lyse the cells on ice for 30 minutes, scrape them with a cell scraper, and ultrasonicate for 1 minute. Centrifuge at 10000r / min for 10 minutes to take the protein supernatant. Mix the loading buffer and the collected protein supernatant at a volume ratio of 1:4, heat them in a constant temperature oscillator at 100℃ for 8 minutes, cool them at room temperature, and freeze them in a -20℃ refrigerator.

[0102] ② Clean the glass plate, check for leaks in the gel maker, and prepare SDS-PAG electrophoresis gel.

[0103] ③ Loading: Add the protein marker and extracted protein sample into the gel wells respectively and fill them with electrophoresis fluid.

[0104] ④Electrophoresis: Turn on the electrophoresis instrument and run at 80V constant voltage for about 45 minutes. A blue marker will appear. Change the voltage to 120V and continue electrophoresis to the bottom of the separation gel, which will take about 50 minutes.

[0105] ⑤ Transfer: Cut the PVDF membrane to a suitable size and activate it in anhydrous methanol. Cut the gel according to the protein marker and transfer the membrane at 220mA.

[0106] ⑥ Blocking: After transfer, place the membrane in 5% milk blocking solution (1g skim milk powder plus 20ml TPBS) and block at 37℃ for 2h.

[0107] ⑦ Incubate with primary antibody: discard the milk blocking solution, pour in TBST, place on a shaker and wash at room temperature for 3 times, each time for 10 minutes. Add diluted COL2A1 and MMP-13 primary antibodies (the dilution ratio is 1:1500) to the antibody incubation box, place the membrane in the incubation box to incubate the antibody at 4°C overnight; wash the membrane: place the membrane in a box filled with TBST and wash it 3 times, each time for 10 minutes.

[0108] ⑧ Incubate with secondary antibody: add the secondary antibody to 5% milk blocking solution (milk blocking solution: secondary antibody = 1:10000), place the membrane in the prepared secondary antibody, and incubate on a shaker at 37°C for 2 hours; wash the membrane: wash 3 times with TBST and once with PBS, each time for 10 minutes.

[0109] ⑨ Development: Use a chemiluminescence instrument to expose the membrane coated with developer (1:1) to visualize protein expression and save the image.

[0110] Experimental results: see Figure 3 and Figure 4The expression of cartilage matrix decomposition factor MMP13 protein was detected by WB technology. Compared with the blank group, the MMP13 protein was significantly increased (P<0.001). Therefore, 10 ng / mL IL-1β induction for 24 h was selected as the optimal time and dose for modeling in the later experiments.

[0111] 2.6 Detection of reactive oxygen species activity in each group using the fluorescent probe DCFH-DA of the ROS detection kit

[0112] The ROS detection kit uses the fluorescent probe DCFH-DA to detect the reactive oxygen species in the treated cells. Detection principle: DCFH-DA itself has no fluorescence and can pass through the cell membrane freely. After entering the cell, it can be hydrolyzed by the esterase in the cell to generate DCFH. DCFH cannot penetrate the cell membrane, which makes it easy for the probe to be loaded into the cell. The reactive oxygen species in the cell can oxidize the non-fluorescent DCFH to generate fluorescent DCF. Specific operation: Since the cells are adherent cultured, the probe is loaded in situ, and DCFH-DA is diluted with serum-free culture medium at 1:1000 to a final concentration of 10μmol / L. Remove the cell culture medium from the cells after administration, wash them twice with serum-free culture medium for 3 minutes each time, and add 1.5mL of diluted DCFH-DA. Incubate in a cell culture incubator at 37℃ for 25 minutes. Wash the cells with PBA three times for 3 minutes each time to fully remove the DCFH-DA that has not entered the cells, and cover the cells with PBS. ROS detection: The labeled cells in the well plate are directly placed under fluorescence microscopy for observation. Using an excitation wavelength of 488 nm and an emission wavelength of 525 nm, the fluorescence spectrum of DCF is very similar to that of FITC, and DCF can be detected using the parameter settings of FITC.

[0113] Experimental results: see Figure 6 and Figure 7 The relative fluorescence intensity of DCFH-DA in chondrocytes treated with IL-1β was significantly increased (P<0.001), while that in cells treated with the six drug groups (chlorogenic acid, picroside II, 6-feruloyl catalpol, catalpol, verbascoside and 1,5-dicaffeoylquinic acid) was significantly decreased (P<0.001), indicating that catalpol can reduce the level of reactive oxygen species (ROS) in chondrocytes.

[0114] 2.7 Fluorescence microscopy observation of the six transdermal ingredients inhibiting IL-1β-induced mitochondrial membrane potential fluorescence

[0115] JC-1 is a fluorescent probe widely used to detect mitochondrial membrane potential ΔΨm. It can detect cell mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is a monomer and can produce green fluorescence. The relative ratio of red and green fluorescence is used to measure the proportion of mitochondrial depolarization. The decrease in mitochondrial membrane potential is a landmark event in the early stage of cell apoptosis.

[0116] The experimental steps are as follows:

[0117] (1) Preparation of JC-1 staining working solution: The amount of JC-1 staining working solution required for each well of a six-well plate is 1 ml, and the amount of JC-1 staining working solution required for other culture vessels is similar; for cell suspension, 0.5 ml of JC-1 staining working solution is required for every 500,000 to 1 million cells. Take an appropriate amount of JC-1 (200X) and dilute JC-1 at a ratio of 8 ml of ultrapure water per 50 μl of JC-1 (200X). Vortex vigorously to fully dissolve and mix JC-1. Then add 2 ml of JC-1 staining buffer (5X) and mix to obtain JC-1 staining working solution.

[0118] (2) For one well of the six-well plate, remove the culture medium, wash the cells once with PBS solution, add 1 ml of cell culture medium, which may contain serum and phenol red, add 1 ml of JC-1 staining working solution, and mix thoroughly. Incubate at 37°C in a cell culture incubator for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) according to the ratio of adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5X), and place it in an ice bath. After the incubation at 37°C, remove the supernatant and wash twice with JC-1 staining buffer (1X). Add 2 ml of cell culture medium, which may contain serum and phenol red, and observe under a fluorescence microscope.

[0119] Experimental results: see Figure 8 and Fig. 9 Compared with the control group, the red fluorescence intensity of JC-1 polymer in chondrocytes in the IL-1β group was significantly weakened, and the green fluorescence intensity of C-1 monomer was significantly enhanced, and the ratio was statistically significant (P<0.001), indicating that IL-1β has an adverse effect on mitochondrial function, resulting in a decrease in mitochondrial membrane potential of chondrocytes. After intervention with 6 transdermal components, compared with the model group, the red fluorescence intensity of JC-1 polymer in chondrocytes in the 6 compound intervention groups was significantly enhanced, and the green fluorescence intensity of JC-1 monomer was significantly weakened, indicating that catalpa glycoside treatment restored the decrease in mitochondrial membrane potential caused by IL-1β and has mitochondrial protective function.

[0120] 2.8 ATP content detection in each group

[0121] As the most important energy molecule, ATP plays an important role in various physiological and pathological processes of cells. Changes in ATP levels will affect cell function. Usually, when cells are in apoptosis, necrosis, or some toxic state, ATP levels will decrease. Usually, a decrease in ATP levels indicates that mitochondrial function is damaged or decreased. During cell apoptosis, a decrease in ATP levels usually occurs simultaneously with a decrease in mitochondrial membrane potential. The experimental steps are as follows:

[0122] (1) Preparation of cell samples: Aspirate the culture medium and add 200 μL of lysis solution to each well of a 6-well plate to lyse the cells. Usually, cells will lyse immediately after contact with the lysis solution. After lysis, centrifuge at 4°C and 12,000 r / min for 5 minutes and take the supernatant for subsequent determination.

[0123] (2) Preparation for standard curve determination: melt the reagents to be used in an ice bath, and dilute the ATP standard solution into an appropriate concentration gradient with concentrations of 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM and 320 μM. In subsequent experiments, the concentration range of the standard can be appropriately adjusted according to the ATP concentration in the sample.

[0124] (3) Preparation of ATP detection working solution: Prepare an appropriate amount of ATP detection working solution according to the ratio of 100 μL ATP detection working solution for each sample or standard. Melt the reagent to be used in an ice bath. Take an appropriate amount of ATP detection reagent and dilute the ATP detection reagent in a ratio of 1:9. The diluted ATP detection reagent is the ATP detection working solution used in subsequent experiments and is temporarily stored in an ice bath.

[0125] (4) Determination of ATP concentration: Add 100 μL of ATP detection working solution to the detection wells or detection tubes. Leave at room temperature for 3-5 minutes to consume all the background ATP. Add 100 μL of ATP detection working solution to 16 detection wells or detection tubes (8 for standards and 8 for samples) at one time to save time. Add 20 μL of sample or standard to the detection wells, quickly mix with a micropipette, and after an interval of 2 seconds, use the luminometer function to determine the RLU value.

[0126] Experimental results: Fig.10As shown in the results, compared with the control group, the ATP content in chondrocytes induced by IL-1β was significantly reduced, and the difference was statistically significant (P<0.001); compared with the IL-1β group, each treatment group had different degrees of increase, among which picroside II, catalpa glycoside, 1,5-dicaffeoylquinic acid and verbascoside were statistically significant (P<0.05, P<0.01, P<0.001 and P<0.001). This proves that catalpa glycoside can increase the ATP content in chondrocytes.

[0127] Example 2

[0128] This example experimentally verifies that catalpa glycoside improves mitochondrial dysfunction by upregulating the expression levels of fusion- and fission-related proteins in chondrocytes.

[0129] 1. Experimental Materials

[0130] Annexin V-FITC apoptosis detection kit was purchased from Shanghai Bio-Tech Co., Ltd., the chemiluminescence instrument was Shanghai Tanon 4800 fully automatic chemiluminescence image analysis system, the BD FACSymphony A1 flow cytometer was from Becton Dickinson Medical (Shanghai) Co., Ltd., NDUFA6 antibody and MYL3 antibody were purchased from Wuhan Mitaka Biotechnology Co., Ltd., and AMPK antibody and p-AMPK antibody were purchased from Immunoway Co., Ltd.

[0131] 2. Experimental methods

[0132] 2.1 Administration

[0133] IL-1β-induced chondrocyte degeneration model was established to construct three drug-dosing groups, prepare cell suspension, plate, and add 20x10 4 cells, and the drug administration method was as follows: the chondrocytes were divided into 5 groups, 3 drugs and IL-1β were co-cultured, a model group and a control group; except for the control group, the 3 drug groups and the model group were added with 10 ng / ml IL-1β serum-free culture medium and co-cultured for 24 hours.

[0134] The groups are as follows:

[0135] (1) Control group (normal culture medium);

[0136] (2) model group (medium containing 10 ng / L IL-1β);

[0137] (3) catalpa glycoside group (medium containing 10 ng / L IL-1β + 50 μmol / L catalpa glycoside);

[0138] (4) 1,5-dicaffeoylquinic acid group (medium containing 10 ng / L IL-1β + 50 μmol / L 1,5-dicaffeoylquinic acid);

[0139] (5) Verbascoside group (medium containing 10 ng / L IL-1β + 50 mol / L Verbascoside).

[0140] 2.2 Flow cytometry

[0141] Digest the cells to be tested with trypsin, collect the cells into a centrifuge tube, wash the collected cells with PBS and centrifuge, remove the supernatant, add 585μl of Annexin V-FITC binding solution to resuspend the cells, then add 15μl of Annexin V-FITC and 30μl of propidium iodide (PI) staining solution, mix well, incubate at room temperature in the dark for 10 minutes, and detect using a flow cytometer.

[0142] Experimental results: see Fig.11 and Fig.12 Compared with the control group, the apoptosis rate of chondrocytes in the IL-1β group was significantly increased (P<0.001), and compared with the model group, the apoptosis rate of chondrocytes in catalpa glycosides, verbascoside and 1,5-dicaffeoylquinic acid was significantly decreased (P<0.01, P<0.001, P<0.001). The IL-1β-induced chondrocyte degeneration model can lead to cartilage damage, mitochondrial dysfunction, increased levels of tissue protein peroxidation, and ultimately produce cytotoxic effects, and may mediate chondrocyte apoptosis. Flow cytometry showed that catalpa glycosides can improve chondrocyte apoptosis.

[0143] 2.3 Immunoblotting

[0144] Collect cells co-cultured with catalpa glycosides, verbascoside, 1,5-dicaffeoylquinic acid and IL-1β for 24 hours, and extract total cell protein. Preparation: 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 6 The cells were lysed with 100 μl of buffer and the protein concentration was approximately 2-4 mg / ml. The specific steps are as follows:

[0145] ① Extraction of cell protein: After washing and centrifugation, add PIRA lysis buffer, PMSF (phenylmethylsulfonyl fluoride): phosphatase inhibitor: protease inhibitor = 100:1:1 protein lysis buffer, lyse the cells on ice for 30 minutes, scrape them with a cell scraper, and ultrasonicate for 1 minute. Centrifuge at 10000r / min for 10 minutes to take the protein supernatant. Mix the loading buffer and the collected protein supernatant at a volume ratio of 1:4, heat them in a constant temperature oscillator at 100℃ for 8 minutes, cool them at room temperature, and freeze them in a -20℃ refrigerator.

[0146] ② Clean the glass plate, check for leaks in the gel maker, and prepare SDS-PAG electrophoresis gel.

[0147] ③ Loading: Add the protein marker and the extracted protein sample into the gel wells respectively and fill them with electrophoresis fluid.

[0148] ④Electrophoresis: Turn on the electrophoresis instrument and run at 80V constant voltage for about 45 minutes. A blue marker will appear. Change the voltage to 120V and continue electrophoresis to the bottom of the separation gel, which will take about 50 minutes.

[0149] ⑤ Transfer: Cut the PVDF membrane to a suitable size and activate it in anhydrous methanol. Cut the gel according to the protein marker and transfer the membrane at 220mA.

[0150] ⑥ Blocking: After transfer, place the membrane in 5% milk blocking solution (1g skim milk powder plus 20ml TPBS) and block at 37℃ for 2h.

[0151] ⑦ Incubate with primary antibody: discard the milk blocking solution, pour in TBST, place on a shaker and wash at room temperature for 3 times, each time for 10 minutes. Add diluted COL2A1 and MMP-13 primary antibodies (the dilution ratio is 1:1500) to the antibody incubation box, place the membrane in the incubation box to incubate the antibody at 4°C overnight; wash the membrane: place the membrane in a box filled with TBST and wash it 3 times, each time for 10 minutes.

[0152] ⑧ Incubate with secondary antibody: add the secondary antibody to 5% milk blocking solution (milk blocking solution: secondary antibody = 1:10000), place the membrane in the prepared secondary antibody, and incubate on a shaker at 37°C for 2 hours; wash the membrane: wash 3 times with TBST and once with PBS, each time for 10 minutes.

[0153] ⑨ Development: Use a chemiluminescence instrument to expose the membrane coated with developer (1:1) to visualize protein expression and save the image.

[0154] Experimental results: see Fig.13 , Fig.14 , Fig.15 and Fig.16Compared with the control group, the protein expressions of NDUFA6, AMPK, and MYL3 in chondrocytes in the IL-1β group were decreased (P<0.001). Compared with the model group, catalpa glycosides (P<0.01) significantly corrected the abnormal protein expression in mitochondrial respiratory chain complex I (NDUFA6), and upregulated the phosphorylation level of AMPK and the expression level of MYL3 protein. These results suggest that catalpa glycosides may improve mitochondrial function of chondrocytes by increasing the expression of mitochondrial complex 1, thereby delaying the development of KOA. In addition, catalpa glycosides treatment significantly reduced the levels of inflammatory cytokines and reactive oxygen species (ROS), while increasing the phosphorylation level of AMPK, alleviating oxidative stress in chondrocytes and inhibiting the degradation of cartilage matrix.

[0155] In the present invention, preferably, catalpa glycosides and pharmaceutically acceptable carriers or excipients are used to form a preparation for the preparation of drugs for treating and alleviating chondrocyte degeneration.

[0156] In the present invention, diseases associated with mitochondrial dysfunction include arthritis caused by mitochondrial dysfunction, wherein the arthritis includes osteoarthritis or osteochondritis caused by osteochondritis.

[0157] It should be noted that the verbascoside in the drawings of the specification refers to the verbascoside in the content of the present application.

[0158] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of catalpa glycosides in the preparation of drugs for treating and alleviating diseases related to mitochondrial dysfunction, characterized in that: The mitochondrial dysfunction-related diseases include arthritis caused by mitochondrial dysfunction.

2. The use according to claim 1, characterized in that: The catalpa glycosides can treat and alleviate diseases related to mitochondrial dysfunction by increasing mitochondrial membrane potential.

3. The use according to claim 1, characterized in that: The catalpa glycosides can treat and alleviate diseases related to mitochondrial dysfunction by reducing the level of reactive oxygen species in chondrocytes.

4. The use according to claim 1, characterized in that: The catalpa glycoside improves the treatment and alleviates diseases related to mitochondrial dysfunction by increasing the ATP content in chondrocytes.

5. The use according to claim 1, characterized in that: The catalpa glycosides can treat and alleviate diseases related to mitochondrial dysfunction by upregulating the expression levels of fusion and fission related proteins in chondrocytes.

6. The use according to claim 5, characterized in that: The related proteins include one or more of NDUFS 6, AMPK and MYL 3.

7. The use according to claim 6, characterized in that: The catalpa glycosides upregulate the expression level of AMPK protein in chondrocytes by regulating the relative expression level of p-AMPK / AMPK protein.

8. The use according to claim 1, characterized in that: The arthritis includes osteoarthritis or osteochondritis caused by osteochondritis.

9. The use according to any one of claims 1 to 8, characterized in that: The catalpa glycoside is combined with a pharmaceutically acceptable carrier or excipient to form a preparation for use in treating and alleviating mitochondrial dysfunction-related diseases.

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