Application of 1, 5-dicaffeoylquinic acid in preparation of medicine for improving and treating diseases related to mitochondrial dysfunction

Abstract

The invention provides application of 1, 5-dicaffeoylquinic acid in preparation of drugs for improving and treating diseases related to mitochondrial dysfunction, and belongs to the technical field of medicine preparation. By reducing the active oxygen level in cartilage cells, improving the mitochondrial membrane potential, increasing the ATP content in the cartilage cells, promoting the mitochondrial structure recovery, up-regulating the expression level of NDUFA6 protein, up-regulating the expression level of MYL 3 protein and regulating the relative expression level of p-AMPK / AMPK protein, the invention improves and treats the mitochondrial dysfunction, and can be used for treating the chondrial dysfunction. The compound can be applied to preparation of drugs for improving and treating mitochondrial dysfunction.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparation, and relates to a technology for using 1,5-dicaffeoylquinic acid for pharmaceutical preparation, and specifically to an application of 1,5-dicaffeoylquinic acid in preparing pharmaceuticals for improving and treating diseases related to mitochondrial dysfunction. Background Art

[0002] Chondrocytes rely on nucleotide triphosphates (ATP) produced by mitochondria to maintain their normal physiological functions, including matrix synthesis and decomposition, cell morphology maintenance, etc. When mitochondrial function is impaired, ATP production decreases, resulting in insufficient energy supply to chondrocytes, which may in turn cause chondrocyte dysfunction and apoptosis. In addition, mitochondrial dysfunction increases the generation of reactive oxygen species (ROS), triggers oxidative stress, damages biomacromolecules in chondrocytes, and accelerates chondrocyte aging and death. At the same time, mitochondrial dysfunction may also affect the apoptosis pathway, leading to excessive apoptosis of chondrocytes, which is an important pathological feature of cartilage diseases such as osteoarthritis.

[0003] At present, there is no specific therapeutic drug for chondrocyte diseases caused by mitochondrial dysfunction, and the research and development of related drugs is still in the exploratory stage. Therefore, for those skilled in the art, it is urgent to further study the molecular mechanism of mitochondrial dysfunction, further study the drugs related to the treatment of chondrocyte diseases caused by mitochondrial dysfunction, and develop drugs that can effectively alleviate or repair mitochondrial dysfunction, so as to provide new strategies and directions for the treatment of cartilage diseases such as osteoarthritis. Summary of the invention

[0004] In view of the above background technology, there is no specific therapeutic drug for chondrocyte diseases caused by mitochondrial dysfunction in the prior art, and the research and development of related drugs is still in the exploratory stage. There is a technical problem that further research is needed on drugs related to the treatment of chondrocyte diseases caused by mitochondrial dysfunction. In view of this technical problem, the present invention proposes the use of 1,5-dicaffeoylquinic acid in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction.

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

[0006] The invention discloses an application of 1,5-dicaffeoylquinic acid in the preparation of a drug for improving and treating a disease associated with mitochondrial dysfunction, wherein the disease associated with mitochondrial dysfunction refers to a cartilage disease caused by mitochondrial dysfunction.

[0007] It is further defined that the 1,5-dicaffeoylquinic acid is used in the preparation of a drug for improving and treating mitochondrial dysfunction by reducing the level of reactive oxygen species in chondrocytes.

[0008] It is further defined that the 1,5-dicaffeoylquinic acid is used in the preparation of drugs for improving and treating mitochondrial dysfunction by increasing mitochondrial membrane potential.

[0009] It is further defined that the 1,5-dicaffeoylquinic acid is used in the preparation of a drug for improving and treating mitochondrial dysfunction by increasing the ATP content in chondrocytes.

[0010] It is further defined that the 1,5-dicaffeoylquinic acid is used in the preparation of drugs for improving and treating mitochondrial dysfunction by promoting the recovery of mitochondrial structure.

[0011] It is further defined that the 1,5-dicaffeoylquinic acid is used in the preparation of drugs for improving and treating mitochondrial dysfunction by upregulating the expression level of NDUFA6 protein.

[0012] It is further defined that the 1,5-dicaffeoylquinic acid is used in the preparation of a drug for improving and treating mitochondrial dysfunction by upregulating the expression level of MYL 3 protein.

[0013] It is further defined that the 1,5-dicaffeoylquinic acid is used to overregulate the relative expression level of p-AMPK / AMPK protein in the preparation of drugs for improving and treating mitochondrial dysfunction.

[0014] In further embodiments, the cartilage disease caused by mitochondrial dysfunction includes osteoarthritis, cartilage tissue damage, chondromalacia or osteochondritis.

[0015] It is further defined that the 1,5-dicaffeoylquinic acid is combined with a pharmaceutically acceptable carrier or excipient to form a preparation for use in preparing a drug for improving and treating mitochondrial dysfunction.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The invention discloses an application of 1,5-dicaffeoylquinic acid in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction. By analyzing and studying 1,5-dicaffeoylquinic acid, it is found that 1,5-dicaffeoylquinic acid can reduce the level of reactive oxygen in chondrocytes, increase mitochondrial membrane potential, increase the ATP content in chondrocytes, promote mitochondrial structural recovery, up-regulate the expression level of NDUFA6 protein, up-regulate the expression level of MYL 3 protein, and regulate the relative expression level of p-AMPK / AMPK protein, thereby improving and treating mitochondrial dysfunction. The invention can be applied in the preparation of drugs for improving and treating mitochondrial dysfunction in chondrocytes, and provide a new strategy and direction for the treatment of cartilage diseases such as osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The viability of chondrocytes stimulated with different concentrations of IL-1β for 24 h;

[0019] Figure 2 To detect the level of TNF-α inflammatory factor after chondrocytes were stimulated with different concentrations of IL-1β using ELISA technology;

[0020] Figure 3 The images are the development of COL2A1 protein expression after chondrocytes were stimulated with different concentrations of IL-1β;

[0021] 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;

[0022] Figure 5 The changes in chondrocyte proliferation after 24h treatment with 6 compounds;

[0023] Figure 6 Fluorescence images of each group detected by DCFH-DA fluorescent probe;

[0024] Figure 7 This is the fluorescence intensity diagram (ROS content) of each group detected by DCFH-DA fluorescent probe;

[0025] Figure 8 The JC-1 probe staining method was used to detect the changes in mitochondrial membrane potential in each group;

[0026] Fig. 9 This is a statistical graph of mitochondrial membrane potential in each group detected by JC-1 probe staining;

[0027] Fig.10 Comparison of ATP content test results in each group;

[0028] Fig.11 Ultrastructural analysis of mitochondria in each group (M represents mitochondria, N represents nucleus; red arrows represent single mitochondria);

[0029] Fig.12 Flow cytometry was used to detect cell apoptosis in each group;

[0030] Fig.13 Flow cytometry was used to detect the cell apoptosis rate in each group;

[0031] Fig.14 The images are developed by immunoblotting to detect the protein expression in each group;

[0032] Fig.15 Schematic diagram of MYL3 protein expression levels in each group;

[0033] Fig.16Schematic diagram of the NDUFA6 protein expression level in each group;

[0034] Fig.17 Schematic diagram of the relative expression levels of p-AMPK / AMPK proteins in each group. DETAILED DESCRIPTION

[0035] 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.

[0036] 1,5-Dicaffeoylquinic acid is a compound with multiple biological activities. Its CAS number is 30964-13-7 and its molecular formula is C 25 H 24 O 12 , with a molecular weight of 516.46. 1,5-dicaffeoylquinic acid has significant pharmacological and pharmacodynamic properties, including antioxidant, antiviral, antifibrotic, smooth muscle contraction inhibition, and lipid-lowering effects. In particular, it performs well in antiviral aspects, and has significant inhibitory effects on hepatitis B virus, HIV, and coronavirus, and is not absorbed by human cells, showing good safety. In addition, the compound also has liver-protecting effects, can enhance human bile secretion, promote amino acid metabolism, and has an auxiliary effect on the treatment of chronic hepatitis and lowering cholesterol. Based on these pharmacological properties, 1,5-dicaffeoylquinic acid shows broad prospects in clinical applications.

[0037] As one of the core transdermal absorption ingredients of the swelling and pain relief patch, the pharmacological properties of 1,5-dicaffeoylquinic acid are highly consistent with its topical efficacy. In the swelling and pain relief patch, this ingredient reaches the lesion site directly through transdermal delivery technology: it can reduce the level of reactive oxygen in chondrocytes, increase mitochondrial membrane potential, increase ATP content in chondrocytes, promote mitochondrial structural recovery, upregulate the expression level of NDUFA6 protein, upregulate the expression level of MYL 3 protein, and regulate the relative expression level of p-AMPK / AMPK protein, thereby improving the treatment of cartilage mitochondrial dysfunction. Based on this, the swelling and pain relief patch can not only target the improvement of muscle joint inflammation and pain, but also provide innovative solutions for the treatment of diseases such as osteoarthritis and soft tissue injuries through local antiviral and metabolic regulation effects, and further highlight the unique value of 1,5-dicaffeoylquinic acid in the development of transdermal improvement and treatment of mitochondrial dysfunction preparations.

[0038] Example 1

[0039] Experimental verification was conducted on the application of 1,5-dicaffeoylquinic acid in the preparation of drugs to improve and treat mitochondrial dysfunction by reducing the level of reactive oxygen in chondrocytes, the application of 1,5-dicaffeoylquinic acid in the preparation of drugs to improve and treat mitochondrial dysfunction by reducing the level of reactive oxygen in chondrocytes, the application of 1,5-dicaffeoylquinic acid in the preparation of drugs to improve and treat mitochondrial dysfunction by increasing the ATP content in chondrocytes, and the application of 1,5-dicaffeoylquinic acid in the preparation of drugs to improve and treat mitochondrial dysfunction by promoting the recovery of mitochondrial structure.

[0040] 1. Experimental Materials

[0041] 1.1 Experimental cells and drugs

[0042] Primary chondrocytes were derived from the lower limb cartilage tissue of 4-week-old healthy male SD rats. SD rats were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. A total of 30 SPF-grade SD rats were purchased, weighing approximately (135±5) g, with a license number of SCXK (Beijing) 2021-0006. The experimental operation and corresponding treatment of experimental animals were strictly referred to the Guiding Opinions on the Kindness of Experimental Animals. All standard product information is shown in Table 1.

[0043] Table 1 Standard product information

[0044]

[0045] 1.2 Experimental Reagents

[0046] (1) Configuration of main reagents

[0047] Preparation of 0.2% type II collagenase solution: Add 50 mg type II 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] Preparation of DMEM / F12 (50 mL) complete culture medium containing 15% fetal bovine serum: add 7.5 mL of fetal bovine serum to 42.5 mL of DMEM / F12 culture 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 the reagents used in the experiments is shown in Table 2.

[0051] Table 2 Detailed information of reagents

[0052]

[0053]

[0054] (3) Detailed information on instruments and equipment

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

[0056] Table 3 Detailed information of instruments and equipment

[0057]

[0058]

[0059] 2. Experimental methods

[0060] 2.1 Isolation 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 sterile 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 100 μg of cartilage tissue, add 0.25% trypsin to digest for 40 minutes, discard the supernatant, add 0.2% type II collagenase (dissolved and diluted in DMEM / F12 culture medium) to the remaining tissue, place it in a constant temperature shaker at 37°C, digest for 4h-5h, until the cartilage tissue pieces disappear, and obtain a cell suspension. After repeated blowing and beating, pass through a 210-mesh filter in turn. Collect the filtrate, transfer it to a centrifuge tube, centrifuge it at 1200r / min for 8min, discard the supernatant, resuspend it with DMEM / F12 double-antibody complete culture medium to obtain a single cell suspension, plant the cells in a T25 cell culture bottle, and culture it in a 37°C, 5% CO2 cell culture incubator by counting the density. After the cells are extracted and attached to the wall for 4-5 days, the medium is changed, and the medium can be changed every other day in the later stage.

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

[0064] When the cell density reaches more than 80% fusion, the cells are passaged. When passaged, the culture medium is first aspirated, and 2 mL of PBS solution is added to gently wash the cells 3 times. Trypsin digestion solution is added for 2-3 minutes, and the cell morphology is observed to be wrinkled or round under an optical microscope. Double volume of complete culture medium is added to stop digestion. The cells are gently blown off with a pipette to form a suspension. The cells are collected in a 15 mL centrifuge tube and centrifuged at 1000 r / min for 3 minutes. The supernatant is poured out, and fresh complete culture medium is added again. 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 passaged 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. 4The cells / well were seeded in a 96-well plate overnight. After 24 hours, the cells were fully attached to the wall and stimulated with different concentrations (0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL, 640 ng / mL) of IL-1β for 24 hours. 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 turn. The cells were placed in an incubator for incubation for 1.5 hours. After being taken out, the OD value was measured at a wavelength of 450 nm using a microplate reader.

[0071] Experimental results: Chondrocytes were normal before induction, the number of chondrocytes decreased after 24 hours induced by 10ng / mL IL-1β, and the number of chondrocytes decreased significantly after 24 hours induced by 20ng / mL IL-1β. The CCK-8 method was used to detect the effects of various IL-1β concentration groups (0ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 40ng / mL, 80ng / mL, 160ng / mL, 320ng / mL, 640ng / mL) on the activity of rat chondrocytes within 24 hours. Figure 1 As shown, after 24 hours of IL-1β intervention, compared with the blank group (0 ng / m1), IL-1β at concentrations of 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL, and 640 ng / mL 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 blank 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, therefore, 50 μM was used as the subsequent administration concentration.

[0075] 2.4 Establishment of IL-1β-injured chondrocyte model in SD rat knee joint

[0076] Grouping: Chondrocytes were plated in 6-well plates, with 200,000 cells in each well. The blank group (only culture medium) and the IL-1β groups with different concentrations (5ng / ml, 10ng / ml, 20ng / ml, 40ng / ml) were used to induce chondrocytes for 24 hours after the cells adhered to the wall. The TNF-α inflammatory factor level after 24 hours of stimulation with IL-1β at different concentrations was detected by ELISA technology, and the COL2A1 and MMP13 protein levels after 24 hours of stimulation with IL-1β at different concentrations were detected by WB technology to determine the conditions for IL-1β to induce cartilage degeneration.

[0077] (1) ELISA was used to detect the level of TNF-α inflammatory factor after 24h stimulation with different concentrations of IL-1β

[0078] 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:

[0079] ①Take 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. Centrifuge the sample first at 3000g / min for 10 minutes, and take the supernatant for detection.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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. Then add 50μL / well of color development solution A to each well, and then add 50μL / well of color development solution B, shake and mix, and incubate in a 37℃ constant temperature incubator for 20 minutes.

[0085] 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.

[0086] ⑧ Fit the standard curve according to the concentration of the standard and the OD value of the standard 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.

[0087] Experimental 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).

[0088] (2) Western blotting was used to detect the levels of COL2A1 and MMP-13 proteins after 24 h of stimulation with different concentrations of IL-1β.

[0089] After 24 hours of stimulation with IL-1β at different concentrations (0 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 40 ng / ml), chondrocytes were collected and total cell protein was extracted.

[0090] 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 are lysed with 100 μl of buffer and the protein concentration obtained is approximately 2-4 mg / ml. The steps are as follows:

[0091] 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.

[0092] ② Clean the glass plate, check for leaks in the gel maker, and prepare SDS-PAG electrophoresis gel.

[0093] ③ Loading: Add the protein marker and extracted protein sample into the gel wells respectively and fill them with electrophoresis fluid.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] ⑧ Incubate with secondary antibody: add the secondary antibody to 5% milk blocking solution (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.

[0099] ⑨ Development: Use a chemiluminescence instrument to expose the membrane coated with developer (1:1) to visualize protein expression and save the image.

[0100] Experimental results: see Figure 3 and Figure 4 The expression of cartilage matrix decomposition factor MMP13 protein was determined by WB. Compared with the blank group, the MMP13 protein was significantly increased (P<0.001).

[0101] Therefore, 10 ng / mL IL-1β was selected to induce 24 h as the optimal time and dose for modeling in later experiments.

[0102] 2.5 Drug administration

[0103] 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, and except for the control group, 6 drug groups and the model group were co-cultured with serum-free medium containing 10ng / ml IL-1β for 24h. The specific groups are as follows:

[0104] (1) Control group (normal culture medium);

[0105] (2) model group (medium containing 10 ng / L IL-1β);

[0106] (3) chlorogenic acid group (medium containing 10 ng / L IL-1β + 50 μmol / L chlorogenic acid);

[0107] (4) 6-feruloyl catalpol group (medium containing 10 ng / L IL-1β + 50 μmol / L 6-feruloyl catalpol);

[0108] (5) catalpa glycoside group (medium containing 10 ng / L IL-1β + 50 μmol / L catalpa glycoside);

[0109] (6) Picroside II group (medium containing 10 ng / L IL-1β + 50 μmol / L picroside II);

[0110] (7) 1,5-dicaffeoylquinic acid group (medium containing 10 ng / L IL-1β + 50 μmol / L 1,5-dicaffeoylquinic acid);

[0111] (8) Verbascoside group (medium containing 10 ng / L IL-1β + 50 μmol / L Verbascoside).

[0112] 2.6 Detection of reactive oxygen species activity in each group using the fluorescent probe DCFH-DA of the ROS detection kit

[0113] The ROS detection kit uses the fluorescent probe DCFH-DA to detect the reactive oxygen species in the treated cells. 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. After the cells are administered, the cell culture medium is removed, and the cells are washed twice with serum-free culture medium for 3 minutes each time, and 1.5mL of diluted DCFH-DA is added. Incubate in a cell culture incubator at 37℃ for 25 minutes. Wash the cells three times with PBA 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.

[0114] Experimental results: Changes in mitochondrial membrane potential directly affect the level of reactive oxygen species (ROS) in chondrocytes, which was detected using a reactive oxygen species detection kit. Figure 6 and Figure 7It can be seen that the relative fluorescence intensity of DCFH-DA in chondrocytes treated with IL-1β increased significantly (P<0.001), while the relative fluorescence intensity of DCFH-DA in cells treated with 1,5-dicaffeoylquinic acid decreased significantly (P<0.001). Therefore, 1,5-dicaffeoylquinic acid can reduce the level of reactive oxygen species (ROS) in chondrocytes.

[0115] 2.7 Fluorescence microscopy observation of the six transdermal ingredients inhibiting IL-1β-induced mitochondrial membrane potential fluorescence

[0116] JC-1 is an ideal 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 (J-aggregates), 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. 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. 9The normality of mitochondrial function is closely related to the changes in mitochondrial membrane potential. In this study, the JC-1 probe staining method was used to detect the effects of six transdermal components on mitochondrial membrane potential in the IL-1β-induced cartilage degeneration model of rat chondrocytes. The results showed that compared with the control group, the red fluorescence intensity of JC-1 polymers in chondrocytes in the IL-1β group was significantly weakened, and the green fluorescence intensity of JC-1 monomers was significantly enhanced, and the ratio was statistically significant (P<0.001). Compared with the model group, in the chondrocytes in the 1,5-dicaffeoylquinic acid group, the red fluorescence intensity of JC-1 polymers was significantly enhanced, and the green fluorescence intensity of JC-1 monomers was significantly weakened. Therefore, 1,5-dicaffeoylquinic acid can restore the decrease in mitochondrial membrane potential caused by IL-1β, promote the increase of mitochondrial membrane potential, 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 with ATP detection lysis buffer to an appropriate concentration gradient. The specific concentration depends on the concentration of ATP in the sample. The detection concentrations are 2.5μM, 5μM, 10μM, 20μM, 40μM, 80μM, 160μM and 320μM. In subsequent experiments, the concentration range of the standard can be appropriately adjusted according to the concentration of ATP 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 well or detection tube. Leave at room temperature for 3-5 minutes to consume all the background ATP, thereby reducing the background. 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 well, quickly mix with a micropipette, and after an interval of 2 seconds, use the luminometer function to measure the RLU value.

[0126] Experimental results: Changes in mitochondrial membrane potential affect the generation of ATP. The effects of six transdermal ingredients on ATP in the rat chondrocyte degeneration model were detected by microplate reader. Fig.10 As shown in the results, compared with the control group, the ATP content in IL-1β-induced chondrocytes 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). Therefore, 1,5-dicaffeoylquinic acid can promote the increase of ATP content.

[0127] 2.9 Transmission electron microscopy observation of mitochondrial morphology

[0128] (1) Sample collection and fixation: Collect cells by centrifugation. The pellet should be at least the size of a mung bean. Remove the culture medium and add electron microscopy fixative. Resuspend and mix at 4°C for 2-4 hours. Store and transport at 4°C.

[0129] (2) Agar pre-embedding: Centrifuge the cells or bacteria, discard the supernatant and add 0.1M phosphate buffer PBS (PH7.4), mix and rinse for 3 minutes, then centrifuge again, repeat the washing 3 times. Prepare 1% agarose solution by heating and dissolving in advance, add it to the EP tube after cooling slightly, and pick up the precipitate with tweezers before the agarose solidifies, suspend it and encapsulate it in the agarose.

[0130] (3) Post-fixation: Fix with 1% osmium hydroxide prepared in 0.1 M phosphate buffer PBS (pH 7.4) at room temperature in the dark for 2 h. Rinse with 0.1 M phosphate buffer PBS (pH 7.4) 3 times, 15 min each time.

[0131] (4) Dehydration at room temperature: Dehydrate the tissue in ascending order by immersing it in 30%-50%-70%-80%-95%-100%-100% alcohol for 20 min each time, and 100% acetone twice for 15 min each time.

[0132] (5) Infiltration embedding: acetone: 812 embedding agent = 1:1 at 37℃ for 2-4h, acetone: 812 embedding agent = 1:2 at 37℃ overnight, pure 812 embedding agent at 37℃ for 5-8h. Pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate and place in an oven at 37℃ overnight.

[0133] (6) Polymerization: Place the embedded plate in a 60°C oven for polymerization for 48 h, and remove the resin block for later use.

[0134] (7) Ultrathin sectioning: The resin block was sliced ​​into 60-80 nm ultrathin sections using an ultrathin sectioning machine and the sections were scooped out using a 150-mesh copper mesh.

[0135] (8) Staining: Stain the copper mesh in 2% uranyl acetate saturated alcohol solution in the dark for 8 min; wash 3 times with 70% alcohol; wash 3 times with ultrapure water; stain 8 min with 2.6% lead citrate solution in the dark for 8 min; wash 3 times with ultrapure water, and dry with filter paper. Place the copper mesh slices in a copper mesh box and dry overnight at room temperature.

[0136] (9) Take out the copper mesh and observe it under a transmission electron microscope to collect images.

[0137] Experimental results: see Fig.11 The results of electron microscopic observation of mitochondrial ultrastructure showed that the empty group had a large number of mitochondria, short rod-shaped, complete mitochondrial cristae structure, neat arrangement, intact membrane and uniform matrix; the model group had severe mitochondrial damage, sparse matrix, dissolution and fading, mitochondrial swelling, mitochondrial cristae collapse, and fuzzy mitochondrial membrane structure. Compared with the IL-1β group, each drug-treated group had different degrees of recovery, reduced mitochondrial damage, uniform matrix recovery, parallel cristae arrangement, reduced mitochondrial swelling, and complete membrane recovery. Among them, 6-feruloyl catalpol, catalpol, verbascoside and 1,5-dicaffeoylquinic acid recovered the best. Therefore, 1,5-dicaffeoylquinic acid can promote the recovery of mitochondrial structure.

[0138] From the above analysis, it can be seen that 1,5-dicaffeoylquinic acid can reduce the level of ROS in chondrocytes. The normal mitochondrial function is closely related to the change of mitochondrial membrane potential. 1,5-dicaffeoylquinic acid can restore the decrease of mitochondrial membrane potential caused by IL-1β, indicating that 1,5-dicaffeoylquinic acid has mitochondrial protective function. The change of mitochondrial membrane potential affects the generation of ATP. Compared with the IL-1β group, the ATP content of each treatment group increased to varying degrees. Among them, picroside II, catalpa glycoside, 1,5-dicaffeoylquinic acid and verbascoside were statistically significant. Therefore, 1,5-dicaffeoylquinic acid can promote the increase of ATP.

[0139] Example 2

[0140] Experimental verification was conducted on the application of 1,5-dicaffeoylquinic acid in the preparation of drugs to improve and treat mitochondrial dysfunction by up-regulating the expression level of NDUFA6 protein, the application of 1,5-dicaffeoylquinic acid in the preparation of drugs to improve and treat mitochondrial dysfunction by up-regulating the expression level of MYL 3 protein, and the application of 1,5-dicaffeoylquinic acid in the preparation of drugs to improve and treat mitochondrial dysfunction by overregulating the relative expression level of p-AMPK / AMPK protein.

[0141] 1. Experimental Materials

[0142] 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 BD Medical (Shanghai) Co., Ltd., NDUFA6 and MYL3 antibodies were purchased from Wuhan Mitaka Biotechnology Co., Ltd., and AMPK and p-AMPK antibodies were purchased from Immunoway Co., Ltd.

[0143] 2. Experimental methods

[0144] 2.1 Administration

[0145] IL-1β-induced chondrocyte degeneration model was constructed with three transdermal component intervention groups. Cell suspension was prepared and plated. 20x10 4 Cells were administered as follows: chondrocytes were divided into 5 groups, 3 drugs and IL-1β were co-cultured, and except for the control group, 3 drug groups and the model group were added with serum-free medium containing 10ng / ml IL-1β and co-cultured for 24h. The specific groups are as follows:

[0146] 1. Control group (normal culture medium);

[0147] 2. Model group (medium containing 10 ng / L IL-1β);

[0148] 3. Catalpa glycoside group (medium containing 10 ng / L IL-1β + 50 μmol / L catalpa glycoside);

[0149] 4. 1,5-dicaffeoylquinic acid group (medium containing 10 ng / L IL-1β+50 μmol / L1,5-dicaffeoylquinic acid);

[0150] 5. Verbascoside group (medium containing 10ng / L IL-1β+50mol / L Verbascoside).

[0151] 2.2 Flow cytometry

[0152] 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.

[0153] Experimental results: see Fig.12 and Fig.13 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 catalpa glycoside, 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, and increased levels of tissue protein peroxidation, ultimately producing cytotoxic effects and possibly mediating chondrocyte apoptosis. Flow cytometry showed that catalpa glycoside, verbascoside and 1,5-dicaffeoylquinic acid can improve chondrocyte apoptosis. Therefore, 1,5-dicaffeoylquinic acid can improve chondrocyte apoptosis.

[0154] 2.3 Immunoblotting

[0155] Collect the cells after 24 hours of co-culture of the three transdermal components (catalpa glycosides, verbascosides and 1,5-dicaffeoylquinic acid) and IL-1β, and extract the 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:

[0156] 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 obtain 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.

[0157] ② Clean the glass plate, check for leaks in the gel maker, and prepare SDS-PAG electrophoresis gel.

[0158] ③ Loading: Add the protein marker and extracted protein sample into the gel wells respectively and fill them with electrophoresis fluid.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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;

[0164] ⑨ Development: Use a chemiluminescence instrument to expose the membrane coated with developer (1:1) to visualize protein expression and save the image.

[0165] Experimental results: see Fig.14 , Fig.15 , Fig.16 and Fig.17 Compared with the control group, the expressions of NDUFA6, AMPK, and MYL3 proteins in chondrocytes in the IL-1β group were decreased (P<0.001). Compared with the model group, 1,5-dicaffeoylquinic acid (P<0.01) significantly corrected the abnormal protein expression in mitochondrial respiratory chain complex I (NDUFA6), and increased the phosphorylation level of AMPK and the expression level of MYL3 protein, as shown in Figure 2. Figure 2 These results suggest that 1,5-dicaffeoylquinic acid may improve mitochondrial function in chondrocytes by increasing the expression of mitochondrial complex 1, thereby delaying the development of KOA. In addition, 1,5-dicaffeoylquinic acid treatment significantly reduced inflammatory cytokine levels and ROS content, while increasing the phosphorylation level of AMPK, alleviating oxidative stress in chondrocytes and inhibiting the degradation of cartilage matrix.

[0166] In the present invention, preferably, 1,5-dicaffeoylquinic acid is combined with a pharmaceutically acceptable carrier or excipient to form a preparation for use in preparing a drug for improving and treating mitochondrial dysfunction.

[0167] In the present invention, diseases related to mitochondrial dysfunction refer to cartilage diseases caused by mitochondrial dysfunction, wherein the cartilage diseases caused by mitochondrial dysfunction include osteoarthritis, cartilage tissue damage, chondromalacia or osteochondritis.

[0168] It should be noted that the verbascoside in the drawings of the specification refers to the verbascoside in the present application.

[0169] 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 1,5-dicaffeoylquinic acid in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The mitochondrial dysfunction-related disease refers to a cartilage disease caused by mitochondrial dysfunction.

2. The use of 1,5-dicaffeoylquinic acid according to claim 1 in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is used in preparing drugs for improving and treating mitochondrial dysfunction by reducing the level of reactive oxygen in chondrocytes.

3. The use of 1,5-dicaffeoylquinic acid according to claim 1 in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is used in preparing drugs for improving and treating mitochondrial dysfunction by increasing mitochondrial membrane potential.

4. The use of 1,5-dicaffeoylquinic acid according to claim 1 in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is used in preparing drugs for improving and treating mitochondrial dysfunction by increasing the ATP content in chondrocytes.

5. The use of 1,5-dicaffeoylquinic acid according to claim 1 in preparing drugs for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is used in preparing drugs for improving and treating mitochondrial dysfunction by promoting the recovery of mitochondrial structure.

6. The use of 1,5-dicaffeoylquinic acid according to claim 1 in preparing a drug for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is used in preparing drugs for improving and treating mitochondrial dysfunction by upregulating the expression level of NDUFA6 protein.

7. The use of 1,5-dicaffeoylquinic acid according to claim 1 in preparing a drug for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is used in preparing a drug for improving and treating mitochondrial dysfunction by upregulating the expression level of MYL 3 protein.

8. The use of 1,5-dicaffeoylquinic acid according to claim 1 in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is used to overregulate the relative expression level of p-AMPK / AMPK protein in the preparation of drugs for improving and treating mitochondrial dysfunction.

9. The use of 1,5-dicaffeoylquinic acid according to claim 1 in preparing a drug for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The cartilage diseases caused by mitochondrial dysfunction include osteoarthritis, cartilage tissue damage, chondromalacia or osteochondritis.

10. Use of 1,5-dicaffeoylquinic acid according to any one of claims 1 to 9 in the preparation of drugs for improving and treating diseases related to mitochondrial dysfunction, characterized in that: The 1,5-dicaffeoylquinic acid is combined with a pharmaceutically acceptable carrier or auxiliary material to form a preparation for preparing a drug for improving and treating mitochondrial dysfunction.

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