CN115245507A - Pharmaceutical composition and application thereof, and application of disulfiram and/or disulfiram salt - Google Patents
Pharmaceutical composition and application thereof, and application of disulfiram and/or disulfiram salt Download PDFInfo
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- CN115245507A CN115245507A CN202210471368.0A CN202210471368A CN115245507A CN 115245507 A CN115245507 A CN 115245507A CN 202210471368 A CN202210471368 A CN 202210471368A CN 115245507 A CN115245507 A CN 115245507A
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- osteoarthritis
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- disulfiram
- pharmaceutical composition
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Abstract
The invention relates to the technical field of prevention and treatment of osteoarthritis, in particular to a pharmaceutical composition and application thereof, and application of disulfiram and/or a salt of disulfiram. The disulfiram and/or the salt of disulfiram in the pharmaceutical composition provided by the invention can reduce the risk of joint replacement, has a delay effect on the occurrence and the progression of osteoarthritis, and can be used for preventing and treating osteoarthritis.
Description
Technical Field
The invention relates to the technical field of prevention and treatment of osteoarthritis, in particular to a pharmaceutical composition and application thereof in preparation of medicines, and application of disulfiram and/or disulfiram salt in preparation of medicines.
Background
Osteoarthritis is a degenerative joint disease characterized mainly by degeneration of articular cartilage, subchondral bone sclerosis and osteophyte formation, and has main clinical manifestations of joint pain, limited movement and joint deformation after movement, which often involve weight-bearing joints.
Epidemiological investigation studies in China show that the prevalence of osteoarthritis in people over 65 years old exceeds 50%, about 80% of osteoarthritis patients have certain restricted mobility, and 25% of osteoarthritis patients have significant effects on daily life, and the annual cost for treating osteoarthritis is as high as 1500 billion yuan. The prevalence of osteoarthritis will increase further as the global aging population and obese population increase.
At present, no clear, effective and safe medicine capable of delaying the progress of the osteoarthritis exists at home and abroad. The latest international authoritative guidelines for osteoarthritis clearly state that many drugs which have been widely used for osteoarthritis, such as hyaluronic acid, glucosamine, chondroitin sulfate and diacerein, have been widely disputed worldwide due to the emergence of the latest high-quality evidence of evidence-based medicine, and even are considered to be uncertain in efficacy or not recommended for use, the treatment of osteoarthritis in early and middle stages often only has the effect of relieving pain and improving functions.
In addition, since osteoarthritis patients are mostly middle-aged and elderly people, and other systemic diseases such as digestive system diseases and cardiovascular system diseases are often accompanied, and the use of first-line therapeutic drugs (symptom relief) for osteoarthritis such as Non-steroidal anti-inflammatory drugs (NSAIDs) is liable to cause gastrointestinal side effects and increase the risk of cardiovascular events, a safe and effective therapeutic drug for early-and-middle-term osteoarthritis patients is urgently needed to be explored.
Disulfiram is a Drug approved by the Food and Drug Administration (FDA), has been used for over 60 years in clinical application, and has good safety and tolerance, definite pharmacokinetics, and strong pharmacological action. Disulfiram acts on acetaldehyde dehydrogenase (ALDH) in cytoplasm and mitochondria to prevent acetaldehyde from being oxidized, so that the concentration of acetaldehyde in blood of a drinker is increased by 5-10 times, and strong discomfort is generated to achieve the aim of abstinence.
In addition to the treatment of alcohol addiction, a large number of observational studies in recent years show that disulfiram has anti-tumor activity and is effective on various malignant tumors, such as prostate cancer, breast cancer, colon cancer and the like, and clinical tests prove that disulfiram can be used for treating highly malignant breast cancer. That is, disulfiram may have a use beyond specification, but there is no literature reporting on whether disulfiram can prevent and treat osteoarthritis.
Disclosure of Invention
The invention aims to solve the problem that no clear, effective and safe medicament capable of delaying the progress of osteoarthritis exists in the prior osteoarthritis prevention and/or treatment technology.
In order to achieve the above object, the present invention provides, in a first aspect, the use of disulfiram and/or a salt of disulfiram for the preparation of a medicament having at least one function selected from the group consisting of a function of protecting chondrocytes, a function of protecting cartilage, a function of alleviating cartilage degeneration caused by osteoarthritis, a function of alleviating pain symptoms caused by osteoarthritis, a function of reducing the risk of joint replacement, a function of preventing osteoarthritis and a function of treating osteoarthritis.
The second aspect of the invention provides a pharmaceutical composition, which contains the following components stored in a mixed manner or independently:
the composition comprises a component A and a component B, wherein the component A is disulfiram and/or a salt of disulfiram, and the component B is hyaluronic acid and/or a non-steroidal anti-inflammatory drug; in the pharmaceutical composition, the content weight ratio of the component A to the component B is 1:0.2-1.
The third aspect of the present invention provides the use of the pharmaceutical composition according to the second aspect described above for the preparation of a medicament having at least one function selected from the group consisting of a function of protecting chondrocytes, a function of protecting cartilage, a function of relieving cartilage degeneration caused by osteoarthritis, a function of alleviating pain symptoms caused by osteoarthritis, a function of reducing the risk of joint replacement, a function of preventing osteoarthritis, and a function of treating osteoarthritis.
Compared with the existing osteoarthritis prevention and/or treatment technology, the pharmaceutical composition containing disulfiram and/or the salt of disulfiram provided by the invention has at least the following advantages:
the disulfiram and/or the salt of disulfiram in the pharmaceutical composition provided by the invention can improve metabolic dysfunction, protect chondrocytes and cartilage, relieve cartilage degeneration caused by osteoarthritis, relieve pain symptoms caused by osteoarthritis and reduce joint replacement risk, and can be used for preventing and treating osteoarthritis.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
FIG. 1 is a graph showing the results of apoptosis of mouse chondrocytes after different intervention treatments. Wherein, fig. 1A is a protein imprinting diagram of an endothelin-N terminal (GSDMD-N) protein in mouse chondrocytes after different intervention treatments, fig. 1B is a result diagram of the content of GSDMD-N protein in mouse chondrocytes after different intervention treatments, fig. 1C is a result diagram of LDH secretion in mouse chondrocytes after different intervention treatments, fig. 1D is a result diagram of IL-1 β secretion in mouse chondrocytes after different intervention treatments, and fig. 1E is a result diagram of IL-18 secretion in mouse chondrocytes after different intervention treatments;
FIG. 2 is a graph showing the results of qRT-PCR detection after reverse transcription of mouse chondrocyte RNA after different intervention treatments. Wherein, fig. 2A is a result diagram of Acan mRNA expression amount after reverse transcription of mouse chondrocyte RNA after different intervention treatment, fig. 2B is a result diagram of Timp-3 mRNA expression amount after reverse transcription of mouse chondrocyte RNA after different intervention treatment, fig. 2C is a result diagram of Timp-1 mRNA expression amount after reverse transcription of mouse chondrocyte RNA after different intervention treatment, fig. 2D is a result diagram of Adamts-4 mRNA expression amount after reverse transcription of mouse chondrocyte RNA after different intervention treatment, and fig. 2E is a result diagram of Adamts-5 mRNA expression amount after reverse transcription of mouse chondrocyte RNA after different intervention treatment; FIG. 2F is a graph showing the result of Mmp-3 mRNA expression level after reverse transcription of mouse chondrocyte RNA after different intervention treatments; FIG. 2G is a graph showing the result of the expression level of Mmp-13 mRNA after reverse transcription of mouse chondrocyte RNA after different intervention treatments;
FIG. 3 is a graph showing the results of different protein contents in mouse chondrocytes after different intervention treatments. Wherein, fig. 3A is a western blot diagram of different proteins in mouse chondrocytes after different intervention treatments, fig. 3B is a result diagram of iNOS protein content in mouse chondrocytes after different intervention treatments, fig. 1C is a result diagram of TIMP-3 protein content in mouse chondrocytes after different intervention treatments, and fig. 1D is a result diagram of MMP-3 protein content in mouse chondrocytes after different intervention treatments;
FIG. 4 is a graph showing the results of different protein contents in mouse chondrocytes after different intervention treatments. Wherein, fig. 4A is a western blot diagram of different proteins in mouse chondrocytes after different intervention treatments, fig. 4B is a result diagram of Col-2 protein content in mouse chondrocytes after different intervention treatments, fig. 4C is a result diagram of Aggrecan protein content in mouse chondrocytes after different intervention treatments, and fig. 4D is a result diagram of MMP-13 protein content in mouse chondrocytes after different intervention treatments;
FIG. 5 is a graph showing the results of different protein secretion amounts in mouse chondrocytes after different intervention treatments. Wherein, FIG. 5A is a result chart of ADAMTS-4 protein secretion in mouse chondrocytes after different intervention treatments, and FIG. 5B is a result chart of MMP-13 protein secretion in mouse chondrocytes after different intervention treatments;
FIG. 6 is a graph of the cartilage degeneration of knee joint of mice detected by safranin fast green staining after different intervention treatments;
FIG. 7 is a graph of OARSI scores of femur (femur) and tibia (tibia) in knee cartilage of mice following different intervention treatments;
FIG. 8 is a graph showing the expression of different proteins in the cartilage of the knee joint of mice after different intervention treatments;
FIG. 9 is a graph of the quantification of different protein expression in mouse knee cartilage following different intervention treatments.
Wherein, fig. 9A is a quantitative result diagram of the GSDMD protein expression in the mouse knee joint cartilage after different intervention treatments, fig. 9B is a quantitative result diagram of the MMP-3 protein expression in the mouse knee joint cartilage after different intervention treatments, fig. 9C is a quantitative result diagram of the ADAMTS-5 protein expression in the mouse knee joint cartilage after different intervention treatments, and fig. 9D is a quantitative result diagram of the MMP-13 protein expression in the mouse knee joint cartilage after different intervention treatments;
FIG. 10 is a graph of HE staining to detect synovial hyperplasia of mouse knee joint after different intervention treatments;
FIG. 11 is a graph showing the results of Synovitis scores (Synovitis score) of knee joints of mice after different intervention treatments;
FIG. 12 is a graph of synovial focal death and inflammatory cell infiltration in the knee joints of mice after different intervention treatments;
figure 13 is a graph of synovial fibrosis in the knee joints of mice following different intervention treatments.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.
As described above, the first aspect of the present invention provides the use of disulfiram and/or a salt of disulfiram for the preparation of a medicament having at least one function selected from the group consisting of a function of protecting chondrocytes, a function of protecting cartilage, a function of alleviating cartilage degeneration caused by osteoarthritis, a function of alleviating pain symptoms caused by osteoarthritis, a function of reducing the risk of joint replacement, a function of preventing osteoarthritis and a function of treating osteoarthritis.
Preferably, the osteoarthritis is selected from at least one of knee joint osteoarthritis, hip joint osteoarthritis, ankle joint osteoarthritis, shoulder joint osteoarthritis, elbow joint osteoarthritis, hand joint osteoarthritis.
As described above, the second aspect of the present invention provides a pharmaceutical composition comprising two or more of the following components stored in admixture or independently:
the composition comprises a component A and a component B, wherein the component A is disulfiram and/or a salt of disulfiram, and the component B is hyaluronic acid and/or a non-steroidal anti-inflammatory drug; in the pharmaceutical composition, the content weight ratio of the component A to the component B is 1:0.2-1.
Preferably, the salt of disulfiram is disulfiram hydrochloride.
Preferably, the pharmaceutical composition also contains an excipient, and the content weight ratio of the component A to the excipient is 1:10-100.
Preferably, the excipient is selected from at least one of anhydrous lactose, magnesium stearate, microcrystalline cellulose, sodium starch glycolate and stearic acid.
Preferably, the dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form.
Preferably, the dosage form of the pharmaceutical composition is selected from at least one of injection, tablets, capsules, granules and granules.
As described above, the third aspect of the present invention provides the use of the pharmaceutical composition according to the aforementioned second aspect for the preparation of a medicament having at least one function selected from the group consisting of a function of protecting chondrocytes, a function of protecting cartilage, a function of alleviating cartilage degeneration caused by osteoarthritis, a function of alleviating pain symptoms caused by osteoarthritis, a function of reducing the risk of joint replacement, a function of preventing osteoarthritis, and a function of treating osteoarthritis.
Preferably, the osteoarthritis is selected from at least one of knee joint osteoarthritis, hip joint osteoarthritis, ankle joint osteoarthritis, shoulder joint osteoarthritis, elbow joint osteoarthritis, hand joint osteoarthritis.
The present invention will be described in detail below by way of examples.
All animal experimental schemes in the invention have been approved by the ethical committee of animal experiments in Hunan ya Hospital, southern university, and all experiments are carried out according to animal ethical criteria and approved animal experimental system design.
Male 12-week-old C57BL/6J mice required for the experiments in the invention were purchased from Splakeda, inc. in south China, and were housed in the department of laboratory animals, university of Central and south China, SPF class, at room temperature 22 + -3 deg.C, with 12-hour light/dark cycles.
Disulfiram (disulfiram) used in the experiments of the invention, dimethyl sulfoxide (DMSO) and olive oil (olive oil) as solvents were purchased from sigma.
The study methods in the following examples are as follows:
(1) Study object
25,505 study subjects over 40 years old were included in the study, excluding patients who had previously suffered from cancer and had undergone hip or knee replacement surgery.
(2) Determination of dosing of disulfiram
Patients taking disulfiram or other alcohol withdrawal drugs (acamprosate, naltrexone, baclofen and topiramate) for the first time are identified by a drug code.
The disulfiram group is a treatment group, other abstinent drug groups are control groups, and the definition of 'first taking drug' is the first prescription of the abstinent drug after entering a queue, namely, a study object with the history of the abstinent drug prescription before entering a study queue is excluded.
(3) Determination of outcome
The outcome of this study was a new hip or knee replacement surgery during follow-up due to osteoarthritis, identified and defined by the disease code.
(4) Statistical analysis
The study was based on a prospective cohort study comparing the risk of new hip or knee replacement surgery for osteoarthritis in the disulfiram group with other drug withdrawal groups.
The characteristics of the two groups of baselines are compared, the quantitative data are statistically described by means of mean and standard deviation, and the qualitative data are statistically described by means of percentage. To simulate clinical trials, we used Inverse Probability Weighting (IPW) to balance potential confounding factors between the two groups, in inverse probability weighting analysis we used the predicted probabilities of the propensity scoring model to calculate stable inverse probability weighting, and asymmetric truncation excluded those subjects who almost always received disulfiram or other withdrawal medications (propensity scoring close to 0 or 1).
The included confounding factors in the trend scoring model were: general demographic data (age, sex, and townsend deprivation index), lifestyle information (smoking and drinking), body mass index, complications and drug use prior to the date of treatment, and health care availability within the year prior to the date of treatment.
The study used a Cox proportional Risk regression model to assess the risk of developing osteoarthritis-induced hip or knee replacement surgery in the disulfiram group versus the other drug withdrawal groups, and calculated the Risk ratio (HR) and its 95% confidence interval (95% confidence interval,95% CI).
All statistical analyses were performed using SAS9.4 software, differences were considered statistically significant when P <0.05, and the tests were all two-sided.
Example 1: results recording
A total of 25,505 subjects (1,724 disulfiram group, 23,781 other drug withdrawal groups) were included in the study, including 11,197 males and 14,308 females, with an average age of 53.3 ± 9.6 years. In general, the two groups of people who have passed IPW have better balance of basic characteristics, and the standard mean difference of all mixed factors is less than 0.1, which is detailed in Table 1.
Cox proportional hazards regression analysis results showed that the incidence of hip or knee replacement surgery due to osteoarthritis was significantly reduced in the disulfiram group compared to the other abstinent drug groups throughout the follow-up procedure, with results of 0.53 (95% ci.
Example 2: chondrocyte extraction and culture, establishment of scorching model and pharmaceutical intervention
(1) Instruments and containers used in the experiment are sterilized, and the experiment operation is carried out under the aseptic condition.
(2) A 4-day-old newborn mouse (purchased from slykh scenda, hunan) was sacrificed by cervical dislocation and then sterilized in 75% ethanol for 2 hours; placing the sterilized mouse on sterile gauze, taking out the two hind limbs of the mouse from the hip joint part by using forceps, removing the skin on the surface, then placing the mouse in a culture dish containing Phosphate buffer solution (Phosphate Buffered Saline-PBS, purchased from Pronosaxi corporation), scraping the soft tissue around the articular cartilage by using a No. 10 surgical blade, and placing the transparent cartilage tissue in a new culture dish containing the PBS buffer solution;
(3) PBS buffer was aspirated, 4mL of type II collagenase (purchased from bionorxx, solubilized with DMEM (Dulbecco's modified Eagle's medium)/F-12 medium, purchased from Gibco, inc.) was added, and the cartilage tissue mass was minced with a scalpel. Placing the culture dish in 5% CO at 37 deg.C 2 Digesting in an incubator overnight;
(4) Taking out the culture dish the next day, adding 4mL of complete culture medium (prepared from DMEM/F-12 culture medium and Fetal Bovine Serum (FBS), wherein the proportion of the fetal bovine serum is 10%, and purchased from Thermo company) to terminate digestion, then sucking the culture dish to a 0.45 mu m disposable filter by using a Pasteur tube, and filtering to obtain filtrate containing mouse chondrocytes;
(5) Centrifuging at 1000 revolutions per minute (rpm) for 5 minutes, removing supernatant, washing mouse chondrocytes by using complete culture medium, centrifuging at 1000rpm for 5 minutes, and adding the cells suspended by using the complete culture medium to obtain mouse chondrocyte suspension;
(6) Mouse chondrocytes at 1X 10 5 PermL in a cell culture dish, and then placing the dish at 37 ℃ in 5% CO 2 Carrying out cell culture in a constant-temperature incubator;
(7) Changing new complete culture medium after 1 day, changing the culture solution 1 time every 2 days, and observing the shape and growth condition of mouse chondrocytes under a microscope;
(8) When the adherence of the culture dish cells reaches about 80%, passage is carried out, the culture solution in the culture dish is completely sucked, the cells are washed for 2 times by PBS buffer solution, and then the PBS buffer solution is sucked off; then 4mL of type II collagenase was added and the plates were placed at 37 ℃ in 5% CO 2 The incubators were digested for 30 minutes at constant temperature. The culture dish is placed upside down under a microscope for observation, 2mL of complete culture medium is added when the circular cells float and flow, and the culture dish is blown repeatedly until the thin film formed by the cells is completely separated from the bottom of the culture dish. Then sucking the cells into a 15mL centrifuge tube, centrifuging the cells at 1000rpm for 5 minutes, removing the supernatant, washing the cells with complete culture medium, centrifuging the cells at 1000rpm for 5 minutes, and adding the cells suspended by the complete culture medium to obtain the mouse chondrocyte suspension. Finally 1 × 10 5 PermL on a 6-well cell culture plate, and then put in 5% CO at 37 ℃ 2 Culturing in a constant-temperature incubator;
(9) Changing a new complete culture medium after 1 day, changing the culture medium for 1 time every 2 days, and observing the morphology and growth condition of mouse chondrocytes under a microscope;
(10) After the cells grow to a proper condition, starving the mouse chondrocytes for 24 hours to ensure that the cells synchronously appear in a non-proliferation period and a non-activity period;
(11) The 1 st generation mouse chondrocytes were cultured in complete medium and then modelled by randomly giving different interventions, specifically as follows:
adding 1 μ L1 μ g/mL Lipopolysaccharide (LPS) or equal volume of sterile water for 24 hr, and then 60 μ L5 mM Adenosine Triphosphate (Adenosine Triphosphate-ATP) or equal volume of sterile water for 0.5 hr; and 0.8 μ L of 20 μ M necrotic sulfonamide (Necrosulfonamide-NSA) or an equal volume of DMSO or different concentrations of disulfiram intervention was administered to the former molding groups.
Example 3: mouse chondrocyte supernatant lactate dehydrogenase (lactate dehydrogenase-LDH) assay
(1) Inoculating a proper amount of cells into a 96-hole cell culture plate according to the size and the growth speed of mouse chondrocytes, so that the cell density is not more than 80% when the cells are detected;
(2) The culture solution is aspirated, the fresh culture medium is replaced after the washing is carried out once by using PBS (phosphate buffer solution), and each culture well is divided into the following groups:
cell-free medium wells (background blank control wells), wells for subsequent lysis (maximum enzyme activity control wells), and wells for different intervention treatments (drug treatment sample wells) given different interventions according to the experimental procedure in example 2 were included and labeled.
1.25 hours prior to the predetermined assay time point, the cell culture plate was removed from the cell incubator, 10 μ L of 10X lysis buffer provided by the kit (purchased from Thermo) was added to the "maximum enzyme activity control well", 10 μ L of sterile water was added to the "drug treated sample well", followed by gentle tapping and mixing, and then continued incubation in the cell incubator for 45 minutes;
(3) And transferring 50 mu L of cell culture medium in the cell culture plate to an enzyme label plate after reaching a preset detection time point, adding 50 mu L of reaction mixture provided by the kit into each hole to be detected, lightly beating and mixing, incubating for 30 minutes at room temperature in a dark place, adding 50 mu L of stop solution provided by the kit into each hole to be detected, lightly beating and mixing uniformly, and placing in an enzyme label instrument (model Epoch, manufacturer Biotek) for detection.
Example 4: enzyme-linked immunosorbent assay (ELISA) detection of mouse chondrocyte supernatant
(1) The number of microplates required for the assay was determined and the microplates were washed 2 times with wash buffer (purchased from Biosharp);
(2) Standard dilutions were performed on microplates: adding 100 mu L of sample diluent provided by a kit (purchased from R & D Systems company) into all micropores to be detected, adding 100 mu L of prepared standard solution (provided by a stock solution kit) into a first transverse micropore of a micropore plate, sequentially sucking 100 mu L of liquid in the former micropore into the latter micropore to create standard diluent, and discarding 100 mu L of liquid from the last micropore;
(3) Add 100 μ Ι _ of sample dilution to blank wells in duplicate, then 50 μ Ι _ of sample dilution and 50 μ Ι _ of sample are added to sample wells in duplicate; adding 50 μ L of biotin conjugate provided by the kit into the blank and sample wells, covering with a microplate lid, and incubating at room temperature for 2 hours;
(4) Absorbing away the biotin conjugate in the blank hole and the sample hole, washing the microplate for 4 times by using a washing buffer solution, adding streptavidin-HRP (horse radish peroxidase) provided by a 100 mu L kit into the blank hole and the sample hole, covering a microplate cover, and incubating for 1 hour at room temperature;
(5) Sucking away streptavidin-HRP in the blank wells and the sample wells, and washing for 4 times with a washing buffer; adding 100 mu L of TMB substrate solution provided by the kit into the blank holes and the sample holes, and incubating the microplate at room temperature in a dark place for 10 minutes;
(6) After adding 100. Mu.L of the stop solution provided by the kit to the blank well and the sample well, the absorbance values of the blank well and the sample well were measured at 450nm using a microplate reader.
Example 5: reverse transcription of mouse chondrocyte RNA and real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) detection
(1) Taking 2 mu L of RNA sample, and quantitatively detecting the concentration of RNA by adopting an enzyme-labeling instrument;
(2) Removing genomic DNA from RNA samples: mu.g of the RNA sample, 2. Mu.L of 5 XgDNA Eraser Buffer, 1. Mu.L of gDNA Eraser were added to a centrifuge tube and RNA was usedse Free dH 2 O is added to 10 mu L of constant volume, and the mixture reacts for 2 minutes at 42 ℃ after being fully and evenly mixed;
(3) Preparing a reverse transcription reaction mixture: mu.L of PrimeScript RT Enzyme Mix I, 1. Mu.L of RT Primer Mix *4 4. Mu.L of 5 XPrimeScript Buffer 2 (for Real Time) and 4. Mu.L of RNase Free dH 2 Fully and uniformly mixing O;
(4) Adding the reverse transcription reaction mixture in the step (3) into the centrifugal tube in the step (2), reacting at 37 ℃ for 15 minutes, and reacting at 85 ℃ for 5 seconds;
(5) Diluting the mixture (cDNA) obtained in the step (4) according to the need, and fully and uniformly mixing;
(6) qRT-PCR reaction System: well mixing 1. Mu.L of cDNA, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer, 0.8. Mu.L of ROX, 2.4. Mu.L of MIX and 1.8. Mu.L of RNase Free dH 2O;
(7) Setting a qRT-PCR reaction program, and amplifying cDNA by using a Thermo fisher QuantStudio 3 fluorescent quantitative PCR instrument, wherein an internal reference is beta-actin.
Example 6: mouse chondrocyte total protein extraction and Western Blot
(1) Taking the mouse chondrocytes in the step (11) in the example 2, sucking out the culture medium, adding PBS buffer solution for washing for 2 times, and then adding 120 mu L of cell lysate for lysis for 15 minutes;
(2) Sucking the liquid containing the cell lysate into a 1.5mL centrifuge tube, heating at 95 ℃ for 10 minutes, and then carrying out ultrasonic treatment by using an ultrasonic cell disruption instrument, wherein each tube is used for 3 times, and each time is 5 seconds; centrifuging at 25 deg.C at 10000rpm for 10 min, collecting supernatant, placing in a new 1.5mL centrifuge tube, and freezing at-80 deg.C;
(3) Protein concentration was determined by protein quantitation (Bicinchoninic Acid Assay-BCA) method:
(4) SDS-PAGE gel electrophoresis: after protein quantification, taking protein liquid with proper volume according to concentration, adding a loading buffer into a new centrifuge tube, and heating at 95 ℃ for 5 minutes to denature the protein;
designing a sample application sequence, recording, applying a sample, changing into 130V constant voltage electrophoresis after 80V constant voltage electrophoresis for 40 minutes until the loading buffer migrates to the position 1.0cm at the bottom of the separation gel, and turning off a power supply;
(5) Film transferring: cutting an adhesive tape to a proper size, putting the adhesive tape into a film transfer liquid for balancing, cutting a polyvinylidene fluoride film slightly larger than the adhesive tape in advance, soaking the polyvinylidene fluoride film in methanol for 10 seconds for activation before film transfer, then putting the film transfer liquid for balancing, putting a film transfer device in the order of anode carbon electrode-sponge-filter paper-gel-filter paper-sponge-cathode carbon electrode for clamping, paying attention to each step to avoid the formation of bubbles, and after switching on a power supply, carrying out 290mA constant current 80 minutes film transfer;
(6) And (3) sealing: after the membrane transfer is completed, the membrane is washed 3 times by using Tris-HCl buffer salt solution (Tris Buffered Saline + Tween-20-TBST) buffer solution containing Tween 20, the side of the polyvinylidene fluoride membrane with protein faces upwards, and then the membrane is blocked for 1 hour by using 5% skimmed milk (dissolved by TBST buffer solution);
(7) Antibody incubation: adding primary antibodies diluted according to a certain proportion, wherein the primary antibodies are respectively as follows:
clean Gasdermin D (Asp 276) antibody (No. 10137, available from Cell Signaling Technology, at a concentration of 1; then adding the diluted secondary antibodies according to the proportion, wherein the secondary antibodies are respectively as follows:
anti-mouse IgG, HRP-linked antibody (trade name 7076, available from Cell signalling Technology at a concentration of 1;
(8) And (3) developing: according to the following steps: 1, the surface of the polyvinylidene fluoride membrane with protein faces upwards, and the developing solution enters a dark room for development after full reaction.
Example 7:
1. establishment of osteoarthritis model
The medial meniscal tibial ligament of the right hind limb of the 57BL/6J mouse was cut using the medial meniscal Destabilization (DMM) method as an experimental group, and the joint capsule of only the right hind limb of the 57BL/6J mouse was cut as a sham group.
2. Experiment grouping
2.1, randomly dividing 35 mice 57BL/6J into 5 groups, 7 mice in each group, wherein the specific grouping conditions are as follows:
1) Sham group: only performing false operation treatment;
2) Solvent intragastric administration group: mice 3 days after constructing the knee osteoarthritis model by DMM surgery were treated with gastric lavage with a solvent (DMSO: olive oil (v/v) = 0.05;
3) Disulfiram (50 mg/kg) gavage group: carrying out intragastric administration treatment on the mice 3 days after constructing the knee osteoarthritis model by DMM (digital Mobile mechanical Module) operation, wherein the administration treatment is carried out 1 time per day;
4) Disulfiram (100 mg/kg) gavage group: carrying out intragastric administration treatment on mice 3 days after constructing a knee osteoarthritis model by DMM (digital multiplex surgery) for 1 time each day;
5) Disulfiram (200 mg/kg) gavage group: mice 3 days after constructing the knee osteoarthritis model by DMM surgery were treated with disulfiram (200 mg/kg) gavage 1 time per day.
Mice were sacrificed 12 weeks post-surgery and knee joint specimens of mice were excised for subsequent testing.
Example 8: taking materials from knee joint and embedding
(1) Killing a mouse by adopting a cervical dislocation method, taking down a knee joint, removing redundant soft tissues by using an ophthalmological scissors, simultaneously paying attention to avoid damaging a knee joint cavity, fixing the knee joint at 135 degrees by using an iron wire, putting the knee joint into a 15mL centrifuge tube filled with 4% paraformaldehyde aqueous solution, and fixing the centrifuge tube on a shaking table at 4 ℃ for overnight;
(2) Placing the fixed tissue into an embedding box, washing the tissue with tap water for 1 hour, and then carrying out decalcification treatment on the tissue with 0.5 mol/L15% ethylenediaminetetraacetic acid (decalcification solution) for 7 days;
(3) And (3) carrying out gradient ethanol dehydration on the tissue after decalcification treatment, wherein the specific procedures are as follows in sequence: 50% ethanol water solution for 2 hours; 70% ethanol aqueous solution for 2 hours; 80% ethanol aqueous solution for 2 hours; 95% ethanol aqueous solution for 2 hours; 100% ethanol I, overnight; 100% ethanol II,2 hours;
(4) And (3) carrying out transparency on the tissue dehydrated by the ethanol, namely replacing the ethanol by adopting dimethylbenzene, wherein the specific procedures are as follows in sequence: xylene I,20 minutes; xylene II,20 minutes;
(5) And (3) waxing the transparent tissue, namely replacing dimethylbenzene by paraffin, wherein the specific procedures are as follows in sequence: treating paraffin I at 65 ℃ for 1.5 hours; treating paraffin II at 65 ℃ for 2 hours; treating the paraffin III for 2 hours at 65 ℃;
(6) The tissues after being soaked in wax are embedded by a paraffin embedding machine in a sagittal position, and the integrity of wax blocks is ensured to be free from cracks and bubbles during embedding; the embedded tissue was then sectioned with a paraffin microtome to a slice thickness of 3 μm.
Example 9: pathology detection
Staining scoring was performed by selecting one section at 40 μm intervals from the beginning of appearance to disappearance of the cartilage surface, and selecting 5 consecutive sections per knee joint for the following procedure:
(1) Placing paraffin sections of knee joint tissues in a thermostat at 65 ℃ for baking for 2 hours, and then placing the paraffin sections in dewaxing liquid (trade name YA0031, purchased from solarbibo company) for dewaxing treatment for 2 times, wherein each time of soaking is 20 minutes;
(2) And (3) carrying out gradient ethanol water combination and cleaning on the tissue section subjected to dewaxing treatment, wherein the specific procedures are as follows in sequence: 100% ethanol aqueous solution for 3 minutes; 90% ethanol aqueous solution for 3 minutes; 80% ethanol aqueous solution for 3 minutes; 70% ethanol aqueous solution for 3 minutes; 50% ethanol aqueous solution for 3 minutes; then washing for 3 minutes by adopting a PBS aqueous solution;
(3) The washed tissue sections were stained with hematoxylin-eosin (HE, available from Sigma Aldrich), safranin fast green (available from Sigma Aldrich), sirius scarlet (model ab150681, available from Abcam), specifically:
i) HE staining to assess synovitis: placing the washed tissue slices in hematoxylin dye to be soaked for 3 minutes, and then washing the tissue slices for 15 minutes by using tap water; then placing the mixture in a differentiation solution for differentiation for 30 seconds, and washing the mixture for 15 minutes by using tap water; then placing the mixture in eosin dye for soaking for 30 seconds, and then washing the mixture for 15 minutes by using tap water; soaking in 95% ethanol, 100% ethanol and xylene for 1 min, wiping off excessive xylene at the edge of the slice, quickly dripping 2 drops of neutral gum, and sealing with cover glass;
II) Safranin fast green (Safranin O-fast green) staining to assess cartilage degeneration: placing the washed tissue slices in a fast green dye for soaking for 3 minutes, and then quickly rinsing with 1% glacial acetic acid; soaking in safranine dye for 1 min, washing with tap water for 15 min, sequentially soaking in 95% ethanol, 100% ethanol and xylene for 1 min, wiping off excessive xylene at the edge of the slice, dripping 2 drops of neutral gum rapidly, and sealing with cover glass;
III) Sirius red (Sirius) staining to assess synovial fibrosis: placing the washed tissue slices in sirius red dye to be soaked for 5 minutes and then washing the tissue slices with tap water for 15 minutes; then sequentially soaking in 95% ethanol, 100% ethanol and xylene for 1 min, finally wiping off the excessive xylene at the edge of the slice, quickly dripping 2 drops of neutral gum, and sealing the slice with a cover glass;
(4) Cartilage destruction scoring was performed on stained tissue sections, specifically: cartilage (femur and tibia) destruction scoring was performed on safranin fast green stained sections by two scorers under blind conditions using the international association for osteoarthritis research scoring system (OARSI, scale 0-6), and if any divergence was present, a third scorer was added and the divergence was resolved by a few obedients to the majority principle after discussion.
Synovitis was scored on HE stained sections using the synovial lining layer and a cell density scoring system (grade 0-6) in the same manner.
Example 10: immunohistochemical analysis
(1) Placing the paraffin section of the knee joint tissue in a thermostat at 65 ℃ for baking for 2 hours, and then placing the paraffin section in dewaxing liquid for dewaxing treatment for 2 times, wherein each time of soaking is 20 minutes;
(2) And (3) carrying out gradient ethanol water combination and cleaning on the tissue slices subjected to dewaxing treatment, wherein the specific procedures are as follows in sequence: 100% ethanol aqueous solution for 3 minutes; 90% ethanol aqueous solution for 3 minutes; 80% ethanol aqueous solution for 3 minutes; 70% ethanol aqueous solution for 3 minutes; 50% ethanol aqueous solution for 3 minutes; then washing for 3 minutes by adopting a PBS aqueous solution;
(3) Drawing a circle around the tissue in the washed tissue section by using an immunohistochemical pen, then adding 150 mu L of pepsin digestive juice, repairing for 15 minutes at room temperature, and washing for 2 times by using PBS (phosphate buffer solution) for 3 minutes each time; then adding 150 mu L of endogenous peroxidase blocking agent, incubating for 10 minutes at room temperature, and washing for 2 times with PBS buffer solution, 3 minutes each time; adding 150 μ L of sealing agent, incubating with normal goat serum working solution at room temperature for 15 min for sealing, and discarding the serum;
(4) The blocked tissue sections were incubated overnight at 4 ℃ with the following antibodies (primary antibodies), respectively, and then washed 2 times for 3 minutes in PBS buffer:
a GSDMD antibody (designation ab219800, available from Abcam at a concentration of 1;
(5) Adding 150 mu L of biotin-labeled goat anti-rabbit IgG polymer (secondary antibody) into the tissue section after primary antibody incubation, incubating for 15 minutes at room temperature, washing for 2 times with PBS buffer solution, and each time for 3 minutes;
(6) Adding 150 mu L of horseradish enzyme labeled streptavidin working solution into the tissue section incubated by the secondary antibody, incubating for 15 minutes at room temperature, and washing for 3 minutes each time for 2 times by using PBS buffer solution;
(7) Adding a ready-prepared color development liquid (DAB concentrated solution: DAB release solution (v/v) =1 20) into the marked tissue section, and washing with PBS under a microscope to control the color development degree;
(8) Placing the developed tissue slices in hematoxylin dye to be soaked for 30 seconds; differentiating by 1% hydrochloric acid ethanol solution for 30 seconds, and washing by tap water until the blue color is returned; then sequentially soaking in 95% ethanol, 100% ethanol and xylene for 1 min, wiping off excessive xylene at the edge of the slice, quickly dripping 2 drops of neutral gum, and sealing with cover glass.
Example 11: immunofluorescence assay
(1) Placing the paraffin section of the knee joint tissue in a thermostat at 65 ℃ for baking for 2 hours, and then placing the paraffin section in dewaxing liquid for dewaxing treatment for 2 times, wherein each time of soaking is 20 minutes;
(2) And (3) carrying out gradient ethanol water combination and cleaning on the tissue section subjected to dewaxing treatment, wherein the specific procedures are as follows in sequence: 100% ethanol aqueous solution for 3 minutes; 90% ethanol aqueous solution for 3 minutes; 80% ethanol aqueous solution for 3 minutes; 70% ethanol aqueous solution for 3 minutes; 50% ethanol aqueous solution for 3 minutes; then washing for 3 minutes by adopting a PBS aqueous solution;
(3) Drawing a circle around the tissue in the washed tissue section by using an immunohistochemical pen, then adding 150 mu L of pepsin digestive juice, repairing for 15 minutes at room temperature, and washing for 2 times by using PBS (phosphate buffer solution) for 3 minutes each time; then 150. Mu.L of 4% Bovine Serum Albumin (BSA) was added and incubated at room temperature for 1 hour for blocking, and the serum was discarded;
(4) The blocked tissue sections were incubated with the following antibodies (primary antibodies) at 4 ℃ overnight and then washed 5 times with PBS buffer for 3 minutes each:
GSDMD antibody (designation ab219800, from Abcam at a concentration of 1;
(5) After the primary antibody incubation, 150 μ L of Alexa 488 (No. ab150077, purchased from Abcam, concentration 1);
(6) After the secondary antibody incubation, 100. Mu.L of the anti-fluorescence quencher was added to the tissue sections, covered with a cover slip, and mounted with nail polish.
Results of the experiment
All data in this invention are expressed as mean ± standard deviation. Data analysis was analyzed using software SAS9.4 using statistical methods of one-way variance analysis or Tukey's post-hoc analysis, where time and intercross effects between groups were assessed using two-way repeated variance analysis, all P values were two-sided P values, and the thresholds for statistical differences were P <0.05, # P <0.01, # P <0.001, # P <0.0001.
Table 1: basic information at baseline (n =25,505)
n: the number of samples; IPW: inverse probability weighting method
FIG. 1 is a graph of the results of apoptosis of mouse chondrocytes following different intervention treatments. As can be seen from fig. 1, disulfiram significantly inhibited LPS and 5mM ATP-induced chondrocyte GSDMD-N protein expression (fig. 1B); while disulfiram reduced the release of LDH in the chondrocyte media induced by LPS and 5mM ATP in a dose-dependent manner (fig. 1C). Furthermore, disulfiram also inhibited the LPS and 5mM ATP-induced production of IL-1 β and IL-18 in chondrocyte culture media in a dose-dependent manner (fig. 1D and 1E), and these results indicate that disulfiram was able to inhibit the occurrence of chondrocyte apoptosis and reduce the release of inflammatory factors by reducing the expression of GSDMD-N and inhibiting membrane perforation.
FIG. 2 is a graph of qRT-PCR detection results after reverse transcription of mouse chondrocyte RNA after different intervention treatments. As can be seen in FIG. 2, disulfiram was able to significantly reverse LPS and 5mM ATP-induced decreases in chondrocyte Acan and Timp-3 (FIGS. 2A and 2B), as well as increases in the sum of Timp-1, adamts-4, adamts-5, mmp-3 and Mmp-13 (FIGS. 2C-2G).
Fig. 3, 4 and 5 are graphs showing the results of different protein contents in mouse chondrocytes after different intervention treatments. As can be seen from the figures, disulfiram was able to significantly inhibit the increased expression of MMP-3, MMP-13, and iNOS proteins in LPS and 5mM ATP-induced chondrocytes (FIG. 3A-FIG. 3D), and promote the decreased expression of Aggrecan, col-2, and TIMP-3 proteins in LPS and 5mM ATP-induced chondrocytes (FIG. 4A-FIG. 4D). In addition, release of ADAMTS-4 and MMP-13 was significantly increased in LPS and 5mM ATP-induced chondrocyte media, while disulfiram inhibited the production of ADAMTS-4 and MMP-13 in LPS and 5mM ATP-induced chondrocyte media in a dose-dependent manner (FIGS. 5A and 5B). These results indicate that disulfiram can reduce chondrocyte apoptosis-induced cellular catabolism and inflammation, and enhance anabolism.
FIG. 6 is a graph of the cartilage degeneration of knee joint of mice detected by safranin fast green staining after different intervention treatments. As can be seen from the figure, mice in the DMM + vehicle group showed significant cartilage degeneration at 12 weeks post-surgery, with an OARSI score higher than that of the sham + vehicle group, while disulfiram reduced knee cartilage degeneration caused by DMM surgery in a dose-dependent manner.
Fig. 7 is a graph of OARSI scores of femur (femur) and tibia (tibia) in knee cartilage of mice after different intervention treatments. As can be seen from the figure, disulfiram reduced the OARSI score in a dose-dependent manner.
FIG. 8 is a graph showing the expression of different proteins in mouse knee cartilage after different intervention treatments, and FIG. 9 is a graph showing the quantitative results of different protein expression in mouse knee cartilage after different intervention treatments. The expression of GSDMD, MMP-3, ADAMTS-5, and MMP-13 was significantly increased in knee joint chondrocytes 12 weeks after surgery in the DMM + vehicle group, while disulfiram was able to significantly reduce the expression of GSDMD, MMP-3, ADAMTS-5, and MMP-13 (FIGS. 9A-9D). These results indicate that disulfiram is able to inhibit chondrocyte apoptosis and delay progression of cartilage degeneration in a DMM-induced mouse OA model.
FIG. 10 is a graph of HE staining for detecting mouse knee joint synovial hyperplasia after different intervention treatments. As can be seen from the graph, the collagen deposition was significantly increased in the DMM + vehicle group compared to the sham + vehicle group, while the increased collagen deposition was alleviated in the disulfiram-dried group.
FIG. 11 is a graph of mouse knee Synovitis scores (Synovitis score) results after different intervention treatments. As can be seen from the figure, disulfiram reduced synovial hyperplasia and inflammatory cell infiltration in the knee joint after DMM surgery in a dose-dependent manner, reducing the synovitis score (fig. 11).
Figure 12 is a graph of synovial scorch and inflammatory cell infiltration in mice knee joints following different intervention treatments. As can be seen from the figure, disulfiram reduced the expression of GSDMD and F4/80 (macrophage marker) in synovial tissue.
Figure 13 is a graph of synovial fibrosis in the knee joints of mice following different intervention treatments. As can be seen from the figure, disulfiram reduced the expression of GSDMD and Vimeintin (fibroblast markers) in synovial tissue.
The results show that disulfiram can inhibit synovial membrane cell apoptosis in a mouse OA model induced by DMM, relieve synovitis and synovial membrane fibrosis, and delay the generation and development of OA.
As can be seen from the results in table 1, the risk of hip or knee replacement surgery due to osteoarthritis is lower in the people taking disulfiram than in the people taking other drugs for abstinence. The disulfiram and/or the salt of disulfiram can reduce the risk of joint replacement, has a delay effect on the occurrence and the progression of osteoarthritis, and can be used for preventing and treating osteoarthritis.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. Use of disulfiram and/or a salt of disulfiram for the manufacture of a medicament, characterized in that the medicament is a medicament having at least one function selected from the group consisting of a function for protecting chondrocytes, a function for protecting cartilage, a function for relieving cartilage degeneration caused by osteoarthritis, a function for alleviating pain symptoms caused by osteoarthritis, a function for reducing the risk of joint replacement, a function for preventing osteoarthritis and a function for treating osteoarthritis.
2. The use according to claim 1, wherein the osteoarthritis is selected from at least one of knee joint osteoarthritis, hip joint osteoarthritis, ankle joint osteoarthritis, shoulder joint osteoarthritis, elbow joint osteoarthritis, hand joint osteoarthritis.
3. The pharmaceutical composition is characterized by comprising the following components which are stored in a mixing way or independently:
the composition comprises a component A and a component B, wherein the component A is disulfiram and/or a salt of disulfiram, and the component B is hyaluronic acid and/or a non-steroidal anti-inflammatory drug; in the pharmaceutical composition, the content weight ratio of the component A to the component B is 1:0.2-1.
4. A pharmaceutical composition according to claim 3, wherein the salt of disulfiram is disulfiram hydrochloride.
5. The pharmaceutical composition according to claim 3 or 4, further comprising an excipient, wherein the content ratio of the component A to the excipient is 1:10-100.
6. The pharmaceutical composition of claim 5, wherein the excipient is selected from at least one of anhydrous lactose, magnesium stearate, microcrystalline cellulose, sodium starch glycolate, stearic acid.
7. The pharmaceutical composition according to any one of claims 3 to 6, wherein the pharmaceutical composition is in the form of an oral dosage form or an injectable dosage form.
8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is in a dosage form selected from at least one of an injection, a tablet, a capsule, a granule and a granule.
9. Use of the pharmaceutical composition according to any one of claims 3 to 8 for the preparation of a medicament which is a medicament having at least one function selected from the group consisting of a function of protecting chondrocytes, a function of protecting cartilage, a function of relieving cartilage degeneration caused by osteoarthritis, a function of alleviating pain symptoms caused by osteoarthritis, a function of reducing the risk of joint replacement, a function of preventing osteoarthritis and a function of treating osteoarthritis.
10. The use of claim 9, wherein the osteoarthritis is selected from at least one of knee joint osteoarthritis, hip joint osteoarthritis, ankle joint osteoarthritis, shoulder joint osteoarthritis, elbow joint osteoarthritis, hand joint osteoarthritis.
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