Application of carvacrol in reducing cartilage cell apoptosis and treating osteoarthritis

Abstract

The invention discloses application of carvacrol in reduction of cartilage cell apoptosis and treatment of osteoarthritis, and belongs to the field of medicine.The application method comprises the following specific steps that S1, animal models are established and grouped, and inflammatory factors, cartilage changes, matrix metabolism and hepatorenal toxicity of the animal models in each group are detected after the animal models are cultured for one month; s2, detecting the influence of CAR with different concentrations on ADTC5 cells and the influence of CAR in an inflammatory environment on cell proliferation; s3, constructing a cell inflammation model, carrying out CAR treatment, extracting mRNA, carrying out RT-qPCR detection, and evaluating the influence of CAR on inflammatory factors; s4, detecting the influence of the CAR on cell apoptosis in an inflammatory environment by using a flow cytometry, a TUNEL staining method and a Western blot method; compared with a traditional treatment method, the carvacrol can reduce cartilage matrix degradation and inhibit inflammatory factor expression, has little toxic and side effects on liver and kidney tissues, and is low in drug price.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis. Background Art

[0002] Osteoarthritis is a chronic degenerative disease characterized by cartilage degeneration, osteophyte formation, and synovitis. The most common sites of osteoarthritis are the hip and knee joints, which bear heavy loads, followed by the interphalangeal joints and spine. As the disease worsens, the lesions will affect the subchondral bone, joint capsule, synovium, and muscles around the joints. Joint pain, swelling, stiffness, limited mobility, loss of function, and deformity are the main symptoms of osteoarthritis patients. Patients in the middle and late stages may experience joint deformities, thoracic vertebrae curvature, cervical vertebrae deformities, scoliosis, and even heart and lung dysfunction. Knee arthritis is the most common osteoarthritis, which has a great impact on the patients' lives. The joint stiffness and progressive loss of knee joint function caused by it seriously affect the patients' quality of life.

[0003] The main treatment options for osteoarthritis include physical therapy, drug therapy and surgical treatment. Currently, clinical drug treatments mainly include oral nonsteroidal anti-inflammatory drugs such as acetaminophen, diclofenac and celecoxib; local treatments mainly use capsaicin cream, intra-articular injections of glucocorticoids, steroids, hyaluronic acid and its derivatives, and opioids such as codeine; the main surgical methods for the treatment of osteoarthritis in clinical practice include arthroscopic joint cleaning, high fibula osteotomy, high tibial osteotomy, partial or total knee replacement, etc. However, the current clinical treatment options have failed to effectively and thoroughly treat knee osteoarthritis. The main purpose is to relieve pain and delay disease progression, and commonly used drugs have certain adverse effects on patients. Therefore, exploring treatment options for osteoarthritis is an urgent problem that needs to be solved worldwide. Summary of the invention

[0004] The purpose of the present invention is to solve the defects in the prior art and to propose the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis.

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

[0006] The application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis, the specific steps of the application method are as follows:

[0007] S1: Establish animal models and divide them into groups. After one month of culture, detect inflammatory factors, cartilage changes, matrix metabolism and liver and kidney toxicity in each group of animal models;

[0008] S2: Detect the effects of different concentrations of CAR on ADTC5 cells, as well as the effects of CAR on cell proliferation in an inflammatory environment;

[0009] S3: Construct a cell inflammation model and perform CAR treatment, then extract mRNA for RT-qPCR detection to evaluate the effect of CAR on inflammatory factors;

[0010] S4: Flow cytometry, TUNEL staining and Western blot were used to detect the effect of CAR on cell apoptosis in an inflammatory environment.

[0011] As a further embodiment of the present invention, the specific steps of detecting inflammatory factors, cartilage changes, matrix metabolism and hepato-renal toxicity in each group of animal models described in S1 are as follows:

[0012] Q1.1: Anterior cruciate ligament rupture surgery was performed on the left knee joints of 40 12-week-old C57BL / 6J male mice. The treated mice were divided into a control group and a treatment group. In the control group, the right knee joint of the mice was used as a blank control group, and the left knee joint was used as a model group. Carvacrol, i.e., CAR, was intraperitoneally injected into the treatment group mice at a standard of 50 mg / kg / d, and the left knee joint of the treatment group mice was used as a treatment observation group;

[0013] Q1.2: One month after the intervention, serum of mice was collected for enzyme-linked immunosorbent assay to evaluate the changes in the levels of inflammatory factors IL-6, IL-10, TNF-a and VEGF in the serum. The integrity of the knee cartilage of living mice was detected by magnetic resonance imaging. Hematoxylin-eosin staining and safranin O-fast green staining were used to evaluate the effect of CAR on the knee cartilage and subchondral bone of mice.

[0014] Q1.3: Immunofluorescence staining was used to evaluate the effect of CAR on metabolic markers of knee cartilage matrix in OA mice, and various indicators of liver and kidney function in mouse serum were detected. HE staining was used to observe changes in liver and kidney function tissue structure, and various experimental data were recorded.

[0015] As a further embodiment of the present invention, the specific steps of detecting the effects of different concentrations of CAR on ADTC5 cells and the effects of CAR on cell proliferation in an inflammatory environment described in S2 are as follows:

[0016] Q2.1: Use EDTA-trypsin to digest the second generation chondrocytes in the logarithmic growth phase, resuspend them in complete culture medium, aspirate 20 μL and place it on the counting plate, then use a cell counter to count the cells;

[0017] Q2.2: According to the calculation results, a specified amount of cell suspension was pipetted into a 15 mL centrifuge tube, and complete culture medium was added to mix thoroughly. The cells were seeded in a 96-well plate at a requirement of 5000 cells / 100 μL per well. Each group was designed with 6 replicates, and 2 columns were designed as controls, 1 column as a complete blank control, and 1 column as a simple CCK8 control. Three 96-well plates were seeded, and then the 96-well plates were placed in a 37°C incubator for 24 hours, 48 ​​hours, and 72 hours, respectively.

[0018] Q2.3: After the cells adhere to the wall, remove the old culture medium and add the culture medium with CAR to 96-well plates. Group them according to the drug concentrations of 0, 25μM, 50μM and 100μM, and add a group of pure culture medium.

[0019] Q2.4: After 24 hours of culture, take out the first 96-well plate and add 10 μL of CCK8 reagent to each well according to the instructions. Gently shake and cover with tin foil to avoid light. Continue to culture for 2 hours. After 2 hours, take out the 96-well plate and detect the absorbance of each well on an ELISA reader with a wavelength set to 450 nm. Similarly, add CCK8 to the second plate after 48 hours of CAR treatment and detect the absorbance value. Add CCK8 reagent to the third plate at 72 hours and use an ELISA reader to detect the absorbance value.

[0020] Q2.5: Calculate the mean OD value of the full culture medium plus CCK8 group, and subtract the mean of the CCK8 group from the OD value of each well in the experimental group. The difference is used for statistics.

[0021] As a further embodiment of the present invention, the specific steps of extracting mRNA for RT-qPCR detection in S3 are as follows:

[0022] Q3.1: The effective concentrations of CAR treatment obtained from the CCK8 experiment were 25μM, 50μM and 100μM. After that, the blank group was set to be directly added to the whole culture, the model group was set to be the whole culture plus IL-1β, and the treatment group was added with CAR 12.5μM+IL-1β and 25μM+IL-1β respectively;

[0023] Q3.2: 300,000 cells were added to a six-well plate. After 12 hours of culture, intervention was started after the cells attached to the wall. CAR was added to the treatment group. After 2 hours, 10 ng / μL of IL-1β was added to the treatment group and the model group, respectively, with a final concentration of 10 ng / mL. The cells were then placed in an incubator at 37°C for 24 hours. After cell stimulation was completed, total RNA was extracted according to the EZB kit method.

[0024] Q3.3: Take a 0.2 mL PCR tube without enzyme, prepare the reaction solution according to 4 μL of 4×gDNA wiper Mix, 500 ng of RNA sample and 16 μL of DEPC water, add 4 μL of 5×HiScriptⅡqRT SuperMixⅡ to the reaction solution, mix the prepared reaction system by pipetting and centrifuging briefly;

[0025] Q3.4: Place the reaction system in a PCR instrument for reverse transcription. After reverse transcription at 50°C for 15 minutes, reverse transcribe the RNA template into cDNA. Then, maintain it at 85°C for 5 seconds to inactivate the reverse transcriptase and terminate the reaction. Then, adjust the temperature to 4°C. After the reverse transcription reaction is completed, transfer the obtained cDNA to a -20°C refrigerator for long-term storage.

[0026] Q3.5: Take a 0.5mL PCR tube, and prepare Mix+primers required for IL-6, COX2, iNOS and GAPDH 4 gene detection according to the standard configuration of 5μL 2×Taq Pro Universal SYBR Qpcr Master Mix, 0.2μL+0.2μL Forward+Reverse, 0.6μL cDNA and 4μL ddH2O, and mix well for use. Prepare the amount of cDNA+ddH2O for the control group, model group, 12.5μM treatment group and 25μM treatment group according to each system, and mix well for use;

[0027] Q3.6: Use a 200μL PCR 96-well plate, set up the plate, and add 5.4μL of Mix and primer mixture to each well. Each gene has 12 wells, and each well is added with 4.6μL of cDNA+ddH2O. Each group has 12 wells. When adding different genes in different groups, the tip needs to be changed. After loading, seal the plate with a sealing film, centrifuge at 3000g for 5 minutes at 4℃, and detect on a fluorescent quantitative PCR instrument;

[0028] Q3.7: Using QuantStudio TM 3. Perform RT-qPCR experiment, detect the CT value of each well, and calculate the relative expression of each indicator mRNA using the 2-ΔΔCt formula, where ΔCt = Ct target gene - Ct internal reference gene.

[0029] As a further embodiment of the present invention, the specific steps of detecting the effect of CAR on apoptosis in an inflammatory environment by flow cytometry in S4 are as follows:

[0030] Q4.1: Chondrocytes were seeded in 12-well plates, with 250,000 cells per well. They were divided into a blank group, a model group with IL-1β, and a treatment group with 25 μM IL-1β+CAR. After the cells adhered to the plate, they were digested with EDTA-free trypsin.

[0031] Q4.2: After the cell intervention is completed, the cells are collected and the cell suspension is centrifuged. After centrifugation, PBS buffer is added to resuspend the cells. Repeat twice, then 500 μl of Binding buffer is added to the cells, and then 5 μl of Annexin V-FITC is added and gently mixed;

[0032] Q4.3: Add 5 μL of Propidium lodide before loading, mix gently, incubate at room temperature in the dark for 5 to 15 minutes, centrifuge and resuspend in 100 μL of PBS buffer in the flow tube;

[0033] Q4.4: Within 1 hour, use flow cytometry for quantitative detection, with the excitation wavelength of Ex=488nm, the emission wavelength of Em=530nm, the Annexin V-FITC green fluorescence through the FITC channel, and the PI red fluorescence through the FL3 channel, and record the experimental results.

[0034] As a further embodiment of the present invention, the specific steps of TUNEL staining described in S4 to detect the effect of CAR on cell apoptosis in an inflammatory environment are as follows:

[0035] Q5.1: First, add 10 μL of culture medium to each well of a 24-well plate, then place the slide on the droplet and press it tightly, inoculate the resuspended chondrocytes on the slide of the 24-well plate, add 5,000 cells to each well, and group them into a blank group, an IL-1β model group, and an IL-1+CAR carvacrol treatment group, and culture them in an incubator;

[0036] Q5.2: After the cells adhered, the old culture medium was removed, and the treatment group was added with a full culture medium containing 25 μM CAR, and the other groups were replaced with a new full culture medium. After 2 hours, 10 ng / mL IL-1β was added to the model group and the treatment group, respectively. The plates were returned to the incubator and cultured for 48 hours before being harvested.

[0037] Q5.3: Remove the culture medium from the stimulated cells and add 4% polyformaldehyde to fix for 25 minutes. After fixation, remove PFA and add 1% permeabilization solution to permeabilize the membrane for 10 minutes at room temperature. Discard the permeabilization solution and wash with PBS buffer for 3 times, 5 minutes each time. Then, add 100 μL of TdT Equilibration Buffer to each slide and treat at 37°C for 30 minutes.

[0038] Q5.4: Preparation of labeling working solution. Calculate the sample volume and prepare it centrally. Prepare the amount of each sample according to 35μL TdTEquilibration Buffer, 10μL Labeling Solution and 5μL TdT Enzyme. Mix thoroughly. Use absorbent paper to dry the water around the sample. Then add 50μL TdT enzyme reaction solution and place it in a wet box. React at 37℃ in the dark for 60 minutes.

[0039] Q5.5: Take 4 μL of 25 μg / mL DAPI Reagent and add it to 96 μL PBS. Mix well in the dark. Immerse the reacted sample in 1× PBS and rinse three times for 5 min each time in the dark. Then use absorbent paper to absorb the moisture around the sample. Then add 50 μL of DAPI working solution and incubate at room temperature for 5 min. After the reaction is completed, rinse with PBS buffer for 3 times for 5 min each time. Then seal the slice with anti-fade sealing agent and air-dry at room temperature in the dark for 10 min. Set the excitation wavelength of ImageD2 fluorescence microscope to 450-500 nm and the emission wavelength to 515-565 nm, and take pictures.

[0040] As a further embodiment of the present invention, the specific steps of the Western blot method described in S4 for detecting the effect of CAR on cell apoptosis in an inflammatory environment are as follows:

[0041] Q6.1: Prepare a cell lysis solution mixture with Ripa cell lysis buffer, Pmsf cell lysis buffer, phosphatase inhibitors and protease inhibitors at a ratio of 100:1:1:1, then inoculate the cells into a 6-well plate. After the cell intervention, place the 6-well plate on ice and wash twice with PBS buffer;

[0042] Q6.2: Add 100 μl of cell lysis solution mixture to each well of the 6-well plate, and use a scraper to scrape the cells. Collect the liquid into an EP tube. After scraping each group of cells, wash the scraper with PBS buffer and wipe it dry with gauze, and then scrape the next group of cells;

[0043] Q6.3: Perform cell lysis under the preset parameter settings for a total of 3 cycles. Place the ultrasonically lysed specimen in water to cool it down. Centrifuge the cooled specimen at 12,000 rpm at 4°C for 15 minutes. After centrifugation, transfer the supernatant to a new 1.5EP tube and discard the precipitate.

[0044] Q6.4: Add 25 μL of loading buffer to each new EP tube and cook it in a 100℃ heater for 10 min. Then store the obtained protein molecules in a -80℃ refrigerator for later use. Assemble the gel rack and glass plate and perform a leak test. After the leak test is passed, prepare 1.5 mm, 15-well SDS-PAGE separation gel and concentration gel of corresponding concentration according to the molecular weight of the target protein.

[0045] Q6.5: Add freshly prepared TBE to the inner tank of the electrophoresis tank and used TBE to the outer tank. Then pull out the comb between the glass plates and use a pipette to add the marker protein and each group of samples to each well. Set the voltage to 80 volts for electrophoresis. When the protein sample drops to the junction of the separation gel and the concentrated gel, adjust the voltage to 120 volts. When the bromophenol blue band is about to electrophoresed to the bottom of the separation gel, turn off the power and stop the electrophoresis.

[0046] Q6.6: Soak the transfer clip and support pad in TB, take out the glass plate, pry it open along the gap of the glass plate with a prying plate, remove the long glass plate, take out the gel and put it in the tray containing TB, cut out the gel where each target protein is located according to the position of the marker band, and transfer the cut gel to the support pad;

[0047] Q6.7: According to the size of the gel, cut out the PVDF membrane of the required size, and soak the PVDF membrane in anhydrous methanol for 15 to 30 seconds to fully activate the PVDF membrane. Place the transfer clip, sponge, filter paper, gel strip, PVDF membrane, filter paper, sponge and transfer clip in order, place the PVDF membrane close to the gel, remove bubbles, cover with the transfer clip, and place it in the transfer tank;

[0048] Q6.8: Place the electrophoresis apparatus in a container filled with ice, pour the prepared TB into the electrophoresis tank until the TB completely covers the strips, set the current to 200mA constant current, and the electrophoresis time is the molecular weight of the target protein KDa + 20min. Prepare a blocking solution containing 5% fresh skimmed milk powder. After the transfer is completed, place the membrane in the blocking solution, shake it slowly at room temperature, block it for 1h, then transfer the strips to the diluted primary antibody working solution, mark it, shake it slowly in a 4℃ cold storage, and incubate overnight;

[0049] Q6.9: Take out the PVDF membrane and wash it three times with TBST solution, each time for 15 minutes; dilute the secondary antibody and diluent at a ratio of 1:5000, place the PVDF membrane in the secondary antibody and incubate for 1 hour, take out the PVDF membrane, wash it again with TBST solution for 3 times, each time for 15 minutes, add the luminescent liquid AB liquid at a ratio of 1:1 into the light-proof box, place the strip in ECL luminescent liquid, and obtain the luminescent density image on the chemiluminometer.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis, anterior cruciate ligament rupture (ACLT) surgery was performed on the left knee joints of 40 12-week-old C57BL / 6J male mice. In the control group, the right knee joint of the mice was the blank control group (Sham), and the left knee joint was the model group (OA). The mice in the treatment group were given intraperitoneal injection of carvacrol (CAR) (50 mg / kg / d), and the left knee joint was used as the treatment observation group (OA+CAR). After one month of intervention, the inflammatory factors, changes in knee cartilage, effects on cartilage matrix metabolism and liver and kidney toxicity in the mice were detected. Then, the effects of different concentrations of CAR on ADTC5 cells and the effects of CAR on cell proliferation in an inflammatory environment were detected, and CAR on in vitro inflammatory factors were detected. Flow cytometry, TUNEL staining and Western blot methods were used to detect the effects of CAR on cell apoptosis in an inflammatory environment. Compared with traditional treatment methods, carvacrol can reduce cartilage matrix degradation, inhibit the expression of inflammatory factors, have fewer toxic and side effects on liver and kidney tissues, and have low drug prices. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0053] Figure 1 This is a flowchart of the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis proposed by the present invention;

[0054] Figure 2 An in vivo experimental technical roadmap for the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis proposed by the present invention;

[0055] Figure 3 The in vitro experimental technical roadmap for the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis proposed by the present invention;

[0056] Figure 4 A comparison diagram of carvacrol inhibiting cartilage degeneration in the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis proposed by the present invention;

[0057] Figure 5 A comparison diagram of carvacrol inhibiting cartilage matrix degradation in the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis proposed by the present invention;

[0058] Figure 6 A comparison diagram of carvacrol's application in reducing chondrocyte apoptosis and treating osteoarthritis in the present invention showing that carvacrol inhibits overexpression of cartilage inflammatory factors;

[0059] Figure 7 A comparison chart of liver and kidney toxicity detection of carvacrol proposed in the present invention in reducing chondrocyte apoptosis and treating osteoarthritis;

[0060] Figure 8 A comparison diagram of the effects of carvacrol on cartilage matrix metabolism in reducing chondrocyte apoptosis and treating osteoarthritis in the present invention;

[0061] Fig. 9 A comparison diagram of carvacrol's application in reducing chondrocyte apoptosis and treating osteoarthritis in the present invention, showing that carvacrol inhibits the damage of chondrocytes caused by inflammation;

[0062] Fig.10 A comparative diagram of the relative expression of mRNA of inflammatory factors inhibited by carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis according to the present invention;

[0063] Fig.11 This is a comparison diagram of carvacrol's application in reducing chondrocyte apoptosis and treating osteoarthritis in the present invention, showing that carvacrol reduces the apoptosis rate of chondrocytes in an inflammatory environment;

[0064] Fig.12 This is a TUNEL detection diagram of carvacrol reducing chondrocyte apoptosis, which is the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis proposed by the present invention;

[0065] Fig.13 This is a Western detection diagram of carvacrol inhibiting chondrocyte apoptosis protein in the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis proposed by the present invention. DETAILED DESCRIPTION

[0066] Reference Figure 1-Figure 13 , the application of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis, the specific steps of the application method are as follows:

[0067] Animal models were established and divided into groups. After one month of culture, the inflammatory factors, cartilage changes, matrix metabolism and hepato-renal toxicity of each group of animal models were detected.

[0068] Specifically, anterior cruciate ligament rupture surgery was performed on the left knee joints of 40 12-week-old C57BL / 6J male mice, and the treated mice were divided into a control group and a treatment group. In the control group, the right knee joint of the mouse was used as a blank control group, and the left knee joint was used as a model group. Carvacrol, i.e., CAR, was injected intraperitoneally into the treatment group mice at a standard of 50 mg / kg / d, and the left knee joints of the treatment group mice were used as a treatment observation group. After one month of intervention, the mouse serum was collected for enzyme-linked immunosorbent assay to evaluate the changes in the levels of inflammatory factors IL-6, IL-10, TNF-a and VEGF in the serum, and then the integrity of the knee cartilage of living mice was detected by magnetic resonance imaging. Hematoxylin-eosin staining and safranin O-fast green staining were used to evaluate the effects of CAR on the knee cartilage and subchondral bone of mice. Immunofluorescence staining was used to evaluate the effects of CAR on metabolic markers of knee cartilage matrix in OA mice. The various indicators of liver and kidney function in the serum of mice were detected, and HE staining was used to observe the changes in the tissue structure of liver and kidney function, and various experimental data were recorded.

[0069] The effects of different concentrations of CAR on ADTC5 cells and the effect of CAR on cell proliferation in an inflammatory environment were detected.

[0070] Specifically, the second generation chondrocytes in the logarithmic growth phase were digested with EDTA-trypsin, and then resuspended with complete culture medium. 20 μL was aspirated and placed on a counting plate, and then the cells were counted using a cell counter. According to the calculation results, a specified amount of cell suspension was aspirated into a 15 mL centrifuge tube, and complete culture medium was added to mix thoroughly. According to the requirement of 5000 cells / 100 μL per well, the cells were seeded in a 96-well plate. Each group was designed with 6 repeated wells, and 2 columns were designed as controls, 1 column was a complete blank control, and 1 column was a simple CCK8 control. Three 96-well plates were seeded, and then the 96-well plates were placed in a 37°C incubator and cultured for 24 hours, 48 ​​hours, and 72 hours, respectively. After the cells adhered to the wall, the old culture medium was aspirated, and the culture medium with CAR was added to the wells. The 96-well plate was grouped according to the drug concentrations of 0, 25μM, 50μM and 100μM, and a group of simple culture medium was added. After 24 hours of culture, the first 96-well plate was taken out and 10μL of CCK8 reagent was added to each well according to the instructions. The plate was gently shaken and wrapped with tin foil to avoid light. The culture was continued for 2 hours. After 2 hours, the 96-well plate was taken out and the absorbance of each well was detected on an ELISA reader with the wavelength set to 450nm. Similarly, CCK8 was added to the second plate after 48 hours of CAR treatment, and the absorbance value was detected. CCK8 reagent was added to the third plate at 72 hours, and the absorbance values ​​were detected using an ELISA reader. The mean OD value of the full culture plus CCK8 group was calculated, and the OD value of each well in the experimental group was subtracted from the mean of the CCK8 group. The difference was used for statistics.

[0071] A cellular inflammation model was constructed and CAR treatment was performed. Then mRNA was extracted for RT-qPCR detection to evaluate the effect of CAR on inflammatory factors.

[0072] Specifically, the effective concentrations of CAR treatment obtained from the CCK8 experiment were 25μM, 50μM and 100μM. Then the blank group was set to be directly added to the whole culture, the model group was set to be the whole culture plus IL-1β, and the treatment groups were added with CAR 12.5μM+IL-1β and 25μM+IL-1β, respectively. 300,000 cells were added to the six-well plate. After 12 hours of culture, the intervention began after the cells attached to the wall, and CAR was added to the treatment group. After 2 hours, 10ng / μL of IL-1β was added to the treatment group and the model group, respectively, with a final concentration of 10ng / mL. Then, the cells were placed in an incubator at 37°C for 24 hours. After the cell stimulation was completed, total RNA was extracted according to the EZB kit method. A 0.2mL PCR tube without enzyme was taken, and the reaction solution was prepared according to 4μL of 4×gDNA wiper MiX, 500ng of RNA sample and 16μL of DEPCwater. 4μL of 5×HiScriptⅡqRT SuperMixⅡ, then blow and mix the prepared reaction system and centrifuge briefly, place the reaction system in a PCR instrument for reverse transcription reaction, reverse transcribe at 50℃ for 15 minutes, reverse transcribe the RNA template into cDNA, and then maintain it at 85℃ for 5 seconds to inactivate the reverse transcriptase and terminate the reaction, then adjust the temperature to 4℃. After the reverse transcription reaction, transfer the obtained cDNA to a -20℃ refrigerator for long-term storage, take a 0.5mL PCR tube, and follow 5μL of 2×Taq Pro Universal SYBR QPCR Master Mix, 0.2μL+0.2μL of Forward+Reverse, 0.6μL of cDNA and 4μL of ddH2O were used to prepare the Mix+primers required for the detection of IL-6, COX2, iNOS and GAPDH four genes, and mixed well for use. The amount of cDNA+ddH2O in the control group, model group, 12.5μM treatment group and 25μM treatment group was configured according to each system, and mixed well for use. A 200μL PCR96-well plate was used to arrange the plate. Three replicate wells were prepared for each sample and each gene. 5.4μL of the mixture of Mix and primers was first added to each well. Each gene had 12 wells, and 4.6μL of cDNA+ddH2O was added to each well. There were 12 wells in each group. At the same time, the tip of the gun needed to be changed when adding different genes in different groups. The sample was sealed with a sealing film. Centrifuged at 3000g for 5min at 4°C, detected on a fluorescent quantitative PCR instrument, and analyzed with QuantStudio TM3. Perform RT-qPCR experiment, detect the CT value of each well, and calculate the relative expression of each indicator mRNA using the 2-ΔΔCt formula, where ΔCt = Ct target gene - Ct internal reference gene.

[0073] Flow cytometry, TUNEL staining and Western blot were used to detect the effect of CAR on cell apoptosis in an inflammatory environment.

[0074] Specifically, chondrocytes were seeded in 12-well plates with 250,000 cells in each well and grouped into a blank group, a model group with IL-1β, and a treatment group with 25 μM IL-1β+CAR. After the cells adhered to the wall, the cells were digested with trypsin without EDTA. After the cell intervention was completed, the cells were collected and the cell suspension was centrifuged in a centrifuge. After centrifugation, PBS buffer was added and resuspended, and the process was repeated twice. Then, 500 μl of Binding buffer was added to the cells, and 5 μl of Annexin V-FITC was added and gently mixed. Before being put on the machine, 5 μl of Propidium lodide was added and gently mixed. The cells were incubated at room temperature in the dark for 5 to 15 min. After centrifugation, the cells were resuspended in a flow tube with 100 μl of PBS buffer. Within 1 hour, the cells were quantitatively detected using a flow cytometer. The excitation wavelength was Ex=488 nm, the emission wavelength was Em=530 nm, the green fluorescence of Annexin V-FITC passed through the FITC channel, the red fluorescence of PI passed through the FL3 channel, and the experimental results were recorded.

[0075] Specifically, 10 μL of culture medium was first added to each well of a 24-well plate, and then the slide was placed on the droplet and pressed tightly. The resuspended chondrocytes were inoculated on the slide of the 24-well plate, and 5,000 cells were added to each well. The cells were divided into a blank group, an IL-1β model group, and an IL-1+CAR carvacrol treatment group, and placed in an incubator for culture. After the cells adhered to the wall, the old culture medium was removed, and a full culture medium containing 25 μM CAR was added to the treatment group, and new full culture medium was used for other groups. After 2 hours, 10 ng / mL IL-1β was added to the model group and the treatment group, respectively. The cells were returned to the incubator for culture for 48 hours, and the plates were collected. The culture medium was removed from the stimulated cells, and 4% polyformaldehyde was added for fixation for 25 minutes. After fixation, PFA was removed, and 1% permeabilization solution was added. The membrane was broken for 10 minutes at room temperature, and the membrane breaking solution was discarded. The cells were washed 3 times with PBS buffer, each for 5 minutes, and then 100 μL of TdTEquilibration was added to each slide. Buffer, treated at 37℃ for 30min, preparation of labeling working solution, calculated sample volume and centralized configuration, and prepared 35μL TdT Equilibration Buffer, 10μL Labeling Solution and 5μL TdT Enzyme configures the amount of each sample and mixes it thoroughly. Use absorbent paper to absorb the moisture around the sample, then add 50 μL of TdT enzyme reaction solution, put it in a wet box, and react for 60 min in the dark at 37°C. Take 4 μL of 25 μg / mL DAPI Reagent and add it to 96 μL PBS. Mix it in the dark. Under the light-proof condition, immerse the reacted sample slices in 1× PBS and rinse three times, 5 min each time, then use absorbent paper to absorb the moisture around the sample, then add 50 μL of DAPI working solution, incubate at room temperature for 5 min, rinse with PBS buffer for 3 times, 5 min each time after the reaction, and then seal the slice with anti-fade sealing agent, air-dry at room temperature in the dark for 10 min, set the excitation wavelength of ImageD2 fluorescence microscope to 450-500 nm, the emission wavelength to 515-565 nm, and take pictures.

[0076] Specifically, Ripa cell lysis buffer, Pmsf cell lysis buffer, phosphatase inhibitors and protease inhibitors were prepared into a cell lysis buffer mixture at a ratio of 100:1:1:1, and then the cells were inoculated in a 6-well plate. After the cell intervention was completed, the 6-well plate was placed on ice and washed twice with PBS buffer. 100 μl of the cell lysis buffer mixture was added to each well of the 6-well plate, and the cells were scraped with a scraper to collect the liquid into an EP tube. After scraping each group of cells, the scraper was washed with PBS buffer and wiped dry with gauze, and then another group of cells was scraped. Under the preset parameter settings, cell lysis was performed for a total of 3 cycles. The ultrasonically lysed specimen was placed in water to cool down, and then the cooled specimen was centrifuged at 12000 rpm at 4°C for 15 minutes. After the centrifugation, the supernatant was transferred to a new 1.5EP tube, and the precipitate was discarded. In each new EP tube, 25 μL of loading buffer, and boil it in a 100℃ heater for 10 minutes, then store the obtained protein molecules in a -80℃ refrigerator for later use, assemble the gel rack and glass plate and perform a leak test. After the leak test is passed, prepare the corresponding concentration of 1.5mm, 15-well SDS-PAGE separation gel and stacking gel, add freshly prepared TBE to the inner tank of the electrophoresis tank, add used TBE to the outer tank, then pull out the comb between the glass plates, use a pipette to add the marker protein and each group of samples to each well, and set the voltage to 80 volts for electrophoresis. When the protein sample drops to the junction of the separation gel and the stacking gel, adjust the voltage to 120 volts. When the bromophenol blue band is about to electrophoresed to the bottom of the separation gel, turn off the power and stop the electrophoresis, and remove the transfer clip and support pad. Soak in TB, take out the glass plate, pry it open along the gap of the glass plate with a prying plate, take out the long glass plate, take out the gel and put it in the tray containing TB, cut out the gel where each target protein is located according to the position of the Marker band, transfer the cut gel to the support pad, cut out the PVDF membrane of the required size according to the size of the gel, and soak the PVDF membrane in anhydrous methanol for 15 to 30 seconds to fully activate the PVDF membrane, and place the transfer clip, sponge, filter paper, gel band, PVDF membrane, filter paper, sponge and Place the transfer clip in sequence, place the PVDF membrane close to the gel, remove bubbles, cover with the transfer clip, put it in the transfer tank, put the electrophoresis instrument in a container filled with ice, pour the prepared TB into the electrophoresis tank until TB completely covers the strips, set the current to 200mA constant current, and the electrophoresis time is the molecular weight of the target protein KDa + 20min. Prepare a blocking solution containing 5% fresh skim milk powder. After the transfer is completed, put the membrane in the blocking solution, shake it slowly at room temperature, block it for 1h, and then transfer the strips to the diluted primary antibody working medium. Liquid, mark well, shake slowly in a 4℃ cold storage, incubate overnight, remove the PVDF membrane, wash with TBST solution 3 times, 15 minutes each time; dilute the secondary antibody and diluent at a ratio of 1:5000, place the PVDF membrane in the secondary antibody and incubate for 1 hour, remove the PVDF membrane, wash with TBST solution 3 times again, 15 minutes each time, add the luminescent liquid AB liquid in a 1:1 ratio into the light-proof box, place the strip in the ECL luminescent liquid, and obtain the luminescent density image on the chemiluminometer. .

Claims

1. Use of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis, characterized in that: The specific steps of this application method are as follows: S1: Establish animal models and divide them into groups. After one month of culture, detect inflammatory factors, cartilage changes, matrix metabolism and liver and kidney toxicity in each group of animal models; S2: Detect the effects of different concentrations of CAR on ADTC5 cells, as well as the effects of CAR on cell proliferation in an inflammatory environment; S3: Construct a cell inflammation model and perform CAR treatment, then extract mRNA for RT-qPCR detection to evaluate the effect of CAR on inflammatory factors; S4: Flow cytometry, TUNEL staining and Western blot were used to detect the effect of CAR on cell apoptosis in an inflammatory environment.

2. The use of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis according to claim 1, characterized in that: The specific steps for detecting inflammatory factors, cartilage changes, matrix metabolism, and hepato-renal toxicity in each group of animal models described in S1 are as follows: Q1.1: Anterior cruciate ligament rupture surgery was performed on the left knee joints of 40 12-week-old C57BL / 6J male mice. The treated mice were divided into a control group and a treatment group. In the control group, the right knee joint of the mice was used as a blank control group, and the left knee joint was used as a model group. Carvacrol, i.e., CAR, was intraperitoneally injected into the treatment group mice at a standard of 50 mg / kg / d, and the left knee joint of the treatment group mice was used as a treatment observation group; Q1.2: One month after the intervention, serum of mice was collected for enzyme-linked immunosorbent assay to evaluate the changes in the levels of inflammatory factors IL-6, IL-10, TNF-a and VEGF in the serum. The integrity of the knee cartilage of living mice was detected by magnetic resonance imaging. Hematoxylin-eosin staining and safranin O-fast green staining were used to evaluate the effect of CAR on the knee cartilage and subchondral bone of mice. Q1.3: Immunofluorescence staining was used to evaluate the effect of CAR on metabolic markers of knee cartilage matrix in OA mice, and various indicators of liver and kidney function in mouse serum were detected. HE staining was used to observe changes in liver and kidney function tissue structure, and various experimental data were recorded.

3. The use of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis according to claim 1, characterized in that: The specific steps described in S2 for detecting the effects of different concentrations of CAR on ADTC5 cells and the effects of CAR on cell proliferation in an inflammatory environment are as follows: Q2.1: Use EDTA-trypsin to digest the second generation chondrocytes in the logarithmic growth phase, resuspend them in complete culture medium, aspirate 20 μL and place it on the counting plate, then use a cell counter to count the cells; Q2.2: According to the calculation results, a specified amount of cell suspension was pipetted into a 15 mL centrifuge tube, and complete culture medium was added to mix thoroughly. The cells were seeded in a 96-well plate at a requirement of 5000 cells / 100 μL per well. Each group was designed with 6 replicates, and 2 columns were designed as controls, 1 column as a complete blank control, and 1 column as a simple CCK8 control. Three 96-well plates were seeded, and then the 96-well plates were placed in a 37°C incubator for 24 hours, 48 ​​hours, and 72 hours, respectively. Q2.3: After the cells adhere to the wall, remove the old culture medium and add the culture medium with CAR to 96-well plates. Group them according to the drug concentrations of 0, 25μM, 50μM and 100μM, and add a group of pure culture medium. Q2.4: After 24 hours of culture, take out the first 96-well plate and add 10 μL of CCK8 reagent to each well according to the instructions. Gently shake and cover with tin foil to avoid light. Continue to culture for 2 hours. After 2 hours, take out the 96-well plate and detect the absorbance of each well on an ELISA reader with a wavelength set to 450 nm. Similarly, add CCK8 to the second plate after 48 hours of CAR treatment and detect the absorbance value. Add CCK8 reagent to the third plate at 72 hours and use an ELISA reader to detect the absorbance value. Q2.5: Calculate the mean OD value of the full culture medium plus CCK8 group, and subtract the mean of the CCK8 group from the OD value of each well in the experimental group. The difference is used for statistics.

4. The use of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis according to claim 3, characterized in that: The specific steps for extracting mRNA for RT-qPCR detection described in S3 are as follows: Q3.1: The effective concentrations of CAR treatment obtained from the CCK8 experiment were 25μM, 50μM and 100μM. After that, the blank group was set to be directly added to the whole culture, the model group was set to be the whole culture plus IL-1β, and the treatment group was added with CAR 12.5μM+IL-1β and 25μM+IL-1β respectively; Q3.2: 300,000 cells were added to a six-well plate. After 12 hours of culture, intervention was started after the cells attached to the wall. CAR was added to the treatment group. After 2 hours, 10 ng / μL of IL-1β was added to the treatment group and the model group, respectively, with a final concentration of 10 ng / mL. The cells were then placed in an incubator at 37°C for 24 hours. After cell stimulation was completed, total RNA was extracted according to the EZB kit method. Q3.3: Take a 0.2 mL PCR tube without enzyme, prepare the reaction solution according to 4 μL of 4×gDNA wiper Mix, 500 ng of RNA sample and 16 μL of DEPC water, add 4 μL of 5×HiScriptⅡqRT SuperMixⅡ to the reaction solution, mix the prepared reaction system by pipetting and centrifuging briefly; Q3.4: Place the reaction system in a PCR instrument for reverse transcription. After reverse transcription at 50°C for 15 minutes, reverse transcribe the RNA template into cDNA. Then, maintain it at 85°C for 5 seconds to inactivate the reverse transcriptase and terminate the reaction. Then, adjust the temperature to 4°C. After the reverse transcription reaction is completed, transfer the obtained cDNA to a -20°C refrigerator for long-term storage. Q3.5: Take a 0.5mL PCR tube, and prepare Mix+primers required for IL-6, COX2, iNOS and GAPDH 4 gene detection according to the standard configuration of 5μL 2×Taq Pro Universal SYBR Qpcr Master Mix, 0.2μL+0.2μL Forward+Reverse, 0.6μL cDNA and 4μL ddH2O, and mix well for use. Prepare the amount of cDNA+ddH2O for the control group, model group, 12.5μM treatment group and 25μM treatment group according to each system, and mix well for use; Q3.6: Use a 200μL PCR 96-well plate, set up the plate, and add 5.4μL of Mix and primer mixture to each well. Each gene has 12 wells, and each well is added with 4.6μL of cDNA+ddH2O. Each group has 12 wells. When adding different genes in different groups, the tip needs to be changed. After loading, seal the plate with a sealing film, centrifuge at 3000g for 5 minutes at 4℃, and detect on a fluorescent quantitative PCR instrument; Q3.7: Using QuantStudio TM 3. Perform RT-qPCR experiment, detect the CT value of each well, and calculate the relative expression of each indicator mRNA using the 2-ΔΔCt formula, where ΔCt = Ct target gene - Ct internal reference gene.

5. The use of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis according to claim 1, characterized in that: The specific steps of flow cytometry detection of the effect of CAR on cell apoptosis in an inflammatory environment described in S4 are as follows: Q4.1: Chondrocytes were seeded in 12-well plates, with 250,000 cells per well. They were divided into a blank group, a model group with IL-1β, and a treatment group with 25 μM IL-1β+CAR. After the cells adhered to the plate, they were digested with EDTA-free trypsin. Q4.2: After the cell intervention is completed, collect the cells and centrifuge the cell suspension. After centrifugation, add PBS buffer to resuspend the cells. Repeat twice. Then add 500μl of Binding buffer to the cells, add 5μL of Annexin V-FITC, and mix gently. Q4.3: Add 5 μL of Propidium lodide before loading, mix gently, incubate at room temperature in the dark for 5 to 15 minutes, centrifuge and resuspend in 100 μL of PBS buffer in the flow tube; Q4.4: Within 1 hour, use flow cytometry for quantitative detection, with the excitation wavelength of Ex=488nm, the emission wavelength of Em=530nm, the Annexin V-FITC green fluorescence through the FITC channel, and the PI red fluorescence through the FL3 channel, and record the experimental results.

6. The use of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis according to claim 1, characterized in that: The specific steps of TUNEL staining to detect the effect of CAR on cell apoptosis in an inflammatory environment as described in S4 are as follows: Q5.1: First, add 10 μL of culture medium to each well of a 24-well plate, then place the slide on the droplet and press it tightly, inoculate the resuspended chondrocytes on the slide of the 24-well plate, add 5,000 cells to each well, and group them into a blank group, an IL-1β model group, and an IL-1+CAR carvacrol treatment group, and culture them in an incubator; Q5.2: After the cells adhered, the old culture medium was removed, and the treatment group was added with a full culture medium containing 25 μM CAR, and the other groups were replaced with a new full culture medium. After 2 hours, 10 ng / mL IL-1β was added to the model group and the treatment group, respectively. The plates were returned to the incubator and cultured for 48 hours before being harvested. Q5.3: Remove the culture medium from the stimulated cells and add 4% polyformaldehyde to fix for 25 minutes. After fixation, remove PFA and add 1% permeabilization solution to permeabilize the membrane for 10 minutes at room temperature. Discard the permeabilization solution and wash with PBS buffer for 3 times, 5 minutes each time. Then, add 100 μL of TdT Equilibration Buffer to each slide and treat at 37°C for 30 minutes. Q5.4: Preparation of labeling working solution. Calculate the sample volume and prepare it centrally. Prepare the amount of each sample according to 35μL TdT Equilibration Buffer, 10μL Labeling Solution and 5μL TdT Enzyme. Mix thoroughly. Use absorbent paper to dry the water around the sample. Then add 50μL TdT enzyme reaction solution and place it in a wet box. React at 37℃ in the dark for 60min. Q5.5: Take 4 μL of 25 μg / mL DAPI Reagent and add it to 96 μL PBS. Mix well in the dark. Immerse the reacted sample in 1×PBS and rinse three times for 5 min each time in the dark. Then use absorbent paper to absorb the moisture around the sample. Then add 50 μL of DAPI working solution and incubate at room temperature for 5 min. After the reaction is completed, rinse with PBS buffer for 3 times for 5 min each time. Then seal the slice with anti-fade sealing agent and air-dry at room temperature in the dark for 10 min. Set the excitation wavelength of ImageD2 fluorescence microscope to 450-500 nm and the emission wavelength to 515-565 nm, and take pictures.

7. The use of carvacrol in reducing chondrocyte apoptosis and treating osteoarthritis according to claim 1, characterized in that: The specific steps of the Western blot method described in S4 to detect the effect of CAR on cell apoptosis in an inflammatory environment are as follows: Q6.1: Prepare a cell lysis solution mixture with Ripa cell lysis buffer, Pmsf cell lysis buffer, phosphatase inhibitors and protease inhibitors at a ratio of 100:1:1:1, then inoculate the cells into a 6-well plate. After the cell intervention, place the 6-well plate on ice and wash twice with PBS buffer; Q6.2: Add 100 μl of cell lysis solution mixture to each well of the 6-well plate, and use a scraper to scrape the cells. Collect the liquid into an EP tube. After scraping each group of cells, wash the scraper with PBS buffer and wipe it dry with gauze, and then scrape the next group of cells; Q6.3: Perform cell lysis under the preset parameter settings for a total of 3 cycles. Place the ultrasonically lysed specimen in water to cool it down. Centrifuge the cooled specimen at 12,000 rpm at 4°C for 15 minutes. After centrifugation, transfer the supernatant to a new 1.5EP tube and discard the precipitate. Q6.4: Add 25 μL of loading buffer to each new EP tube and cook it in a 100℃ heater for 10 min. Then store the obtained protein molecules in a -80℃ refrigerator for later use. Assemble the gel rack and glass plate and perform a leak test. After the leak test is passed, prepare 1.5 mm, 15-well SDS-PAGE separation gel and concentration gel of corresponding concentration according to the molecular weight of the target protein. Q6.5: Add freshly prepared TBE to the inner tank of the electrophoresis tank and used TBE to the outer tank. Then pull out the comb between the glass plates and use a pipette to add the marker protein and each group of samples to each well. Set the voltage to 80 volts for electrophoresis. When the protein sample drops to the junction of the separation gel and the concentrated gel, adjust the voltage to 120 volts. When the bromophenol blue band is about to electrophoresed to the bottom of the separation gel, turn off the power and stop the electrophoresis. Q6.6: Soak the transfer clip and support pad in TB, take out the glass plate, pry it open along the gap of the glass plate with a prying plate, remove the long glass plate, take out the gel and put it in the tray containing TB, cut out the gel where each target protein is located according to the position of the marker band, and transfer the cut gel to the support pad; Q6.7: According to the size of the gel, cut out the PVDF membrane of the required size, and soak the PVDF membrane in anhydrous methanol for 15 to 30 seconds to fully activate the PVDF membrane. Place the transfer clip, sponge, filter paper, gel strip, PVDF membrane, filter paper, sponge and transfer clip in order, place the PVDF membrane close to the gel, remove bubbles, cover with the transfer clip, and place it in the transfer tank; Q6.8: Place the electrophoresis apparatus in a container filled with ice, pour the prepared TB into the electrophoresis tank until the TB completely covers the strips, set the current to 200mA constant current, and the electrophoresis time is the molecular weight of the target protein KDa + 20min. Prepare a blocking solution containing 5% fresh skimmed milk powder. After the transfer is completed, place the membrane in the blocking solution, shake it slowly at room temperature, block it for 1h, then transfer the strips to the diluted primary antibody working solution, mark it, shake it slowly in a 4℃ cold storage, and incubate overnight; Q6.9: Take out the PVDF membrane and wash it three times with TBST solution, each time for 15 minutes; dilute the secondary antibody and diluent at a ratio of 1:5000, place the PVDF membrane in the secondary antibody and incubate for 1 hour, take out the PVDF membrane, wash it again with TBST solution for 3 times, each time for 15 minutes, add the luminescent liquid AB liquid at a ratio of 1:1 into the light-proof box, place the strip in ECL luminescent liquid, and obtain the luminescent density image on the chemiluminometer.

Images