Application of oridonin in treatment of osteoarthritis

Abstract

The present application relates to the technical field of biological medicine, and discloses an application of oridonin in treating osteoarthritis. The oridonin can target and inhibit M1 type polarization of synovial macrophages, down-regulate multiple pro-inflammatory factors such as TNF-alpha and IL-1beta, simultaneously relieve joint swelling and pain, reduce synovial inflammatory infiltration, repair damaged cartilage and reverse subchondral bone sclerosis. The local administration in the joint cavity is high in safety and free of liver toxicity. The oridonin is further combined with low-intensity pulsed ultrasound (LIPUS), the drug joint penetration capacity is improved by the acoustic window effect of ultrasound, and the two produce significant synergistic therapeutic effect, which is better than that of single drug or single physiotherapy in analgesic anti-inflammatory effect and cartilage repair effect. Further, the present application provides a new targeted therapeutic drug and rehabilitation combined scheme for osteoarthritis, which has good clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of oridonin in the treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease characterized by degeneration of articular cartilage, synovitis, and subchondral bone remodeling. Clinical manifestations include joint pain, swelling, and limited mobility; in severe cases, it can lead to joint deformities. With the increasing aging of the population, the incidence of OA is rising annually, becoming a significant global public health issue. Statistics show that there were approximately 595 million OA patients worldwide in 2020, and the number is expected to continue to grow.

[0003] Currently, OA treatment primarily focuses on symptom relief, with common regimens including nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of hyaluronic acid, or corticosteroids. However, these methods cannot effectively halt disease progression, and long-term use may lead to side effects. Advanced OA often requires joint replacement, which carries perioperative risks and high costs.

[0004] Recent studies have shown that synovitis is a key factor in the pathogenesis of osteoarthritis (OA). Macrophages, as the most abundant immune cells in the synovium, exhibit an imbalance between their M1 (pro-inflammatory) and M2 (anti-inflammatory) polarization, which is the core mechanism leading to the persistent chronic inflammation progression of OA. M1 macrophages secrete large amounts of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6), inducing chondrocyte inflammatory responses and promoting the overexpression of matrix-degrading enzymes, resulting in the degradation of the extracellular matrix of chondrocytes.

[0005] Oridonin A, a diterpenoid active ingredient extracted from the traditional Chinese medicine Rabdosia rubescens, possesses strong anti-inflammatory activity and has been proven to be a covalent NLRP3 inhibitor. Existing studies have shown that oridonin A exerts its anti-inflammatory effects by inhibiting inflammatory signaling pathways such as NF-κB and MAPK, and can regulate macrophage polarization. However, the low solubility and bioavailability of oridonin A limit its clinical application, and there is currently a lack of systematic research on the therapeutic effect of oridonin A on osteoarthritis (OA) through the regulation of macrophage polarization. Summary of the Invention

[0006] In view of this, the present invention proposes an application of oridonin in the treatment of osteoarthritis, aiming to solve at least one of the problems in the current background art.

[0007] This invention proposes the use of oridonin in the preparation of drugs for the prevention and / or treatment of osteoarthritis.

[0008] Preferably, the osteoarthritis is traumatic knee osteoarthritis induced by anterior cruciate ligament transection.

[0009] Preferably, the drug treats osteoarthritis by inhibiting M1 polarization of synovial macrophages.

[0010] Preferably, the inhibition of macrophage M1 polarization includes: downregulating the expression of M1 markers iNOS and CD86, and downregulating the gene expression of pro-inflammatory factors iNOS, IL-1β, TNF-α, IL-6, and IL-12β.

[0011] Preferably, the administration method of oridonin A is intra-articular injection.

[0012] Preferably, the dosage in the drug administration method is as follows: the effective dose for animals is 10 mg / kg, administered once a week for 4 consecutive weeks; the safe working concentration for in vitro cells is 2 μmol / L.

[0013] Preferably, it also includes the synergistic use of oridonin in combination with low-intensity pulsed ultrasound; The parameters of the low-intensity pulsed ultrasound are: frequency 1.5MHz, sound intensity 30mW / cm². 2 Duty cycle 20%, single intervention 20 minutes, once a day.

[0014] The present invention also provides the use of oridonin in the preparation of drugs for reducing the expression levels of pro-inflammatory factors iNOS, IL-1β, TNF-α, IL-12β and IL-6.

[0015] The present invention also provides a medicament for treating osteoarthritis, comprising oridonin and pharmaceutically acceptable excipients.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Oridonin not only relieves clinical symptoms such as joint swelling and mechanical / thermal pain, but also improves the core pathological changes of synovitis, cartilage defects and subchondral bone sclerosis, achieving an integrated approach of pain relief, anti-inflammation and cartilage repair, breaking through the limitation of traditional drugs that only relieve symptoms.

[0017] (2) This invention is the first to systematically demonstrate that oridonin targets the M1 polarization of synovial macrophages as the core target and blocks the progression of OA from the source of inflammation: inhibiting the release of pro-inflammatory factors and cutting off the vicious cycle of macrophage inflammation-cartilage degradation, which is different from traditional drugs that simply inhibit cartilage inflammation.

[0018] (3) The present invention also provides a method of administration by local intra-articular administration, which avoids the liver and gastrointestinal toxicity of oral anti-inflammatory drugs. Animal experiments have confirmed that after intervention with a dose of 10mg / kg for 4 consecutive weeks, the liver organ coefficient and liver tissue HE staining of mice were not different from those of the normal group, and there was no hepatocellular damage or inflammatory infiltration, which is suitable for long-term chronic intervention of OA.

[0019] (4) In addition, the LIPUS acoustic effect enhances the diffusion and absorption of oridonin in joint tissues, making up for its poor water solubility and low bioavailability; the combined regimen has significantly better analgesic, anti-inflammatory and cartilage repair effects than single drug / single physical therapy, and takes effect faster; and cell experiments have confirmed that there is no macrophage toxicity at a concentration of 2 μmol / L and it does not inhibit immune cell activity, which is different from the defect of high-dose anti-inflammatory drugs that damage normal immune homeostasis. Attached Figure Description

[0020] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 Statistical graph of knee joint diameter in mice at different intervention time points; Figure 2 The results of mechanical pain threshold and thermal pain threshold detection in mice are shown in the figure. Figure 3 Safranin O-Fix Green staining and OARSI score map of knee joint; Figure 4 HE staining of synovial tissue and synovitis scoring chart; Figure 5 Micro-CT reconstruction and quantitative indicators of tibial subchondral bone; Figure 6 HE staining of mouse liver and statistical graph of liver organ coefficients; Figure 7 Flowchart for the assay and experiment of RAW264.7 macrophage CCK8 cell viability; Figure 8 Figures showing macrophage morphology, iNOS immunofluorescence, and CD86 flow cytometry results; Figure 9 The graph shows the relative expression levels of M1 pro-inflammatory factor mRNA in macrophages detected by qRT-PCR. Figure 10 Image of iNOS immunofluorescence assay in knee joint synovium. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0022] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] This invention provides the use of oridonin A in the preparation of drugs for the prevention and / or treatment of osteoarthritis.

[0027] In this invention, the osteoarthritis is traumatic knee osteoarthritis induced by anterior cruciate ligament transection.

[0028] In this invention, the drug treats osteoarthritis by inhibiting M1 polarization of synovial macrophages.

[0029] In this invention, the inhibition of macrophage M1 polarization includes: downregulating the expression of M1 markers iNOS and CD86, and downregulating the gene expression of pro-inflammatory factors iNOS, IL-1β, TNF-α, IL-6, and IL-12β.

[0030] In this invention, the administration method of oridonin A is intra-articular injection.

[0031] In this invention, the dosage of the drug in the method of administration is as follows: the effective dose for animals is 10 mg / kg, administered once a week for 4 consecutive weeks; the safe working concentration for in vitro cells is 2 μmol / L.

[0032] This invention also includes the synergistic use of oridonin in combination with low-intensity pulsed ultrasound; The parameters of the low-intensity pulsed ultrasound are: frequency 1.5MHz, sound intensity 30mW / cm². 2 Duty cycle 20%, single intervention 20 minutes, once a day.

[0033] The present invention also provides the use of oridonin in the preparation of drugs for reducing the expression levels of pro-inflammatory factors iNOS, IL-1β, TNF-α, IL-12β and IL-6.

[0034] The present invention also provides a medicament for treating osteoarthritis, comprising oridonin and pharmaceutically acceptable excipients.

[0035] Example 1 I. Preparation Stage (1) Animal experiment grouping: After 1 week of adaptive feeding, 30 mice were randomly divided into 5 groups: ① artificial hand The treatment groups were: ① SHAM group; ② Osteoarthritis group (OA group); ③ Low-intensity pulsed ultrasound therapy group (OA+LIPUS group); ④ Oridonin therapy group (OA+ORI group); ⑤ Low-intensity pulsed ultrasound combined with oridonin therapy group (OA+LIPUS+ORI group).

[0036] (2) Animal modeling: The osteoarthritis (OA) model was constructed by transecting the anterior cruciate ligament (ACLT) of the right knee joint of mice to simulate the pathological process of human OA.

[0037] The specific operating steps are as follows: ① All mice must be fasted and deprived of water for at least 8 hours. ② Mice are anesthetized using isoflurane gas, the anesthetized mice are fixed in place, the hair in the surgical area is removed, and the skin is prepared. ③ The right knee joint of the mouse is opened layer by layer using surgical instruments. The patellar ligament is gently separated using ophthalmic forceps to fully expose the knee joint cavity. The anterior cruciate ligament is cut with ophthalmic shears. ④ After surgery, the patellar ligament is repositioned, and the joint capsule, each layer of tissue, and skin are sutured sequentially with absorbable sutures. The wound skin is disinfected three times with iodine disinfectant. For mice in the SHAM group, only the right knee joint is opened and the incision is sutured after the above steps; the anterior cruciate ligament is not treated. The other four groups are modeled using the standard ACLT method. After the operation, all mice are placed in an incubator to await awakening. After they have fully recovered, they are returned to their cages in the animal house for normal rearing.

[0038] II. Intervention Phase (1) Experimental intervention method: ① Starting 2 weeks after surgery, ORI (10 mg / kg) was injected intra-articularly into the right knee joint of mice in the ORI group and LIPUS+ORI group once a week for 4 weeks. SHAM group, Both the OA group and the LIPUS group were injected with the same volume of physiological saline, with the same injection frequency and cycle as the two groups mentioned above. ② For the LIPUS group and the LIPUS+ORI group, the ultrasound coupling agent on the surface of the low-intensity pulsed ultrasound (LIPUS) instrument probe was evenly applied and placed in front of the knee joint during modeling. The frequency was set to 1.5 kHz, the duty cycle to 20%, and the intensity to 30 mW / cm². 2 ③ The mice were treated for 20 minutes daily for 28 consecutive days. ③ Simultaneously, a non-output LIPUS probe was placed on the knee joint of mice in the SHAM, OA, and ORI groups as a control.

[0039] (2) Measurement of mouse indicators: The diameter of the mouse knee joint can reflect the degree of joint swelling. The body weight and knee joint diameter of the mice were measured and recorded 1 day before the start of treatment and at the end of 1, 2, 3 and 4 weeks after treatment. The test results are as follows: Figure 1 As shown.

[0040] based on Figure 1 The results showed that, one day before treatment, the diameter of the right knee joint in the sham-operated group (SHAM group) was significantly smaller than that in the other modeling groups (OA group, OA+LIPUS group, OA+ORI group, and OA+LIPUS+ORI group), indicating that OA modeling successfully induced knee joint swelling in mice. Furthermore, there was no significant difference in the diameter of the right knee joint in the OA group, LIPUS group, OA+ORI group, and OA+LIPUS+ORI group before treatment, suggesting that the baseline values ​​were consistent across groups. As treatment progressed, the degree of knee joint swelling in the LIPUS group, ORI group, and LIPUS+ORI group was significantly reduced compared to the OA group, indicating that the treatment measures in each group had a positive effect on alleviating knee joint swelling. The combined treatment group showed the most significant effect.

[0041] (3) Mechanical pain threshold measurement: The mechanical pain threshold of each group of mice was measured 1 day before the start of treatment and at the end of 1, 2, 3 and 4 weeks after treatment.

[0042] The specific procedure is as follows: Mice are placed individually in transparent plastic cages with a metal mesh bottom, allowing them to acclimatize for 20-30 minutes. Then, using von Frey fibers, pressure is gradually applied vertically to the center of the mouse's right paw through the mesh openings at the bottom of the device until the mouse exhibits a paw retraction action. The instrument automatically records the applied pressure intensity (g). Measurements are repeated three times with at least 5 minutes intervals between each mouse, and the results are recorded. The average pressure intensity (g) from the three measurements is used as the mouse's mechanical pain threshold for subsequent statistical analysis.

[0043] Thermal pain threshold measurement: Thermal pain threshold was measured in mice in each group on day 1 before treatment and at the end of weeks 1, 2, 3, and 4 after treatment. Note that the interval between thermal pain threshold measurement and mechanical pain threshold measurement should be at least 1 hour to avoid tissue damage and interference with the results.

[0044] The specific operation is as follows: Set the hot plate temperature to 55℃, and wait for the temperature to stabilize at (55±0.2)℃. A single mouse was placed in a transparent plastic box on a hot plate, and timing was started. The mouse's behavior was closely observed, and the time when the mouse's right hind leg first exhibited shaking, paw raising, licking, or jumping was recorded, with a cutoff time of 30 seconds. Measurements were repeated three times for each mouse, with at least 5 minutes between each measurement, and the results were recorded. The average of the three thermal pain response times (in seconds) was used as the mouse's thermal pain threshold for subsequent statistical analysis.

[0045] The above test results are as follows Figure 2 As shown in the figure, A represents the thermal pain threshold of each group of mice; B represents the mechanical pain threshold of each group of mice. Note: ns P>0.05, *P<0.05, **P<0.01, ***P<0.001.

[0046] In the experiment, the thermal pain threshold was assessed by the time (s) of the mouse's paw withdrawal response to thermal stimulation, and the mechanical pain threshold was assessed by the force (g) of the mouse's paw withdrawal response to mechanical stimulation. The lower the thermal pain threshold and the lower the mechanical pain threshold, the higher the mouse's pain sensitivity, and the more pronounced the pain symptoms may be.

[0047] based on Figure 2 The results showed that, one day before treatment, the mechanical pain threshold of mice in the sham-operated group (SHAM group) was significantly higher than that of other modeling groups (OA group, OA+LIPUS group, OA+ORI group, OA+LIPUS+ORI group), indicating that OA modeling successfully induced pain response in mice. Furthermore, there were no significant differences in mechanical and thermal pain thresholds among the OA, OA+LIPUS, OA+ORI, and LIPUS+ORI groups before treatment, suggesting that the baseline values ​​of each group were consistent. After 1 and 2 weeks of treatment, the mechanical and thermal pain thresholds in the LIPUS and ORI treatment groups were not significantly relieved compared to the OA group, while the mechanical and thermal pain thresholds in the LIPUS+ORI group were significantly lower than those in the OA group, indicating that LIPUS combined with ORI treatment could alleviate pain symptoms in mice in the early stages compared to LIPUS or ORI treatment alone. As the intervention continued, statistically significant improvements in mechanical and thermal pain thresholds began to appear in the LIPUS and ORI groups at the end of week 3, with the combined treatment group showing the most significant improvement. The above results suggest that combined therapy has a synergistic effect in improving OA-related pain.

[0048] (4) Testing of knee cartilage damage in mice: Safranin O-Fix-Green cartilage staining + OARSI scoring was used. The specific procedure was as follows: knee joint decalcification, paraffin sectioning, SOFG staining, microscopic image acquisition, and OARSI score calculation. The stained sections and score bar chart are shown below. Figure 3 As shown, Figure 3 A. Safranin-Fix-Green staining of joint pathological sections from each group of mice (scale bar: top image 250μm, bottom image 100μm); B. Statistical analysis of OARIS Score for each group of mice. Note: ***P<0.001.

[0049] based on Figure 3 It can be seen that: in the OA group, there was a large area of ​​cartilage damage and a large amount of red-stained matrix loss; in the OR single-drug group, the area of ​​cartilage defect was significantly reduced and the OARSI score was lower than that in the OA group, but the cartilage integrity was not as good as that in the LIPUS combined with ORI group, and the single drug could only slightly delay cartilage degeneration.

[0050] (5) Synovial HE staining for synovitis scoring: The specific procedure is as follows: HE staining of synovial tissue sections, scoring based on three indicators: inflammatory infiltration, synovial hyperplasia, and pannus. The images and scores are as follows. Figure 4 .

[0051] based on Figure 4 It can be seen that the OA group had severe synovial hyperplasia and a large number of inflammatory cell infiltrations; the ORI monotherapy group had a synovial inflammation grade reduced to mild, but the degree of improvement was limited and could not completely inhibit excessive synovial hyperplasia.

[0052] (6) Micro-CT subchondral bone detection, the specific operation is as follows: the specimen is scanned by SCANCO-μ80, the three-dimensional bone structure is reconstructed and BV / TV, Tb.Th, Tb.N are statistically analyzed, and the images and quantitative indicators are as follows. Figure 5 In the figure, A. Three-dimensional reconstructed image of the subchondral bone of the medial tibia in mice scanned by Micro-CT; B. Evaluation indicators of subchondral bone mass: bone volume fraction (BV / TV), trabecular thickness (Tb.Th) increase, and trabecular number (Tb.N). Note: **P<0.01, ***P<0.001.

[0053] based on Figure 5 It can be seen that: in the OA group, the trabeculae thickened and the bone mass increased significantly; oridonin alone only slightly improved subchondral bone sclerosis, and the decrease in the three bone indicators was much lower than that in the combined intervention group.

[0054] (6) Liver HE staining and organ coefficient: The specific procedure is as follows: the liver is separated, weighed, and the organ coefficient is calculated. Liver sections are stained with HE. The results are as follows. Figure 6As shown in the figure: A. HE staining shows the liver lesions in each group of mice (scale bar: 100μm); B. Liver organ coefficient = organ mass (g) / mouse body mass (g) × 100%. Note: ns P>0.05.

[0055] based on Figure 6 It can be seen that the liver cells in each group are arranged in a regular manner and there is no significant difference in organ coefficient (ns), which proves that the local administration of 10 mg / kg oridonin into the joint cavity has no hepatotoxicity and the drug has good safety.

[0056] Example 2 I. Complete Step-by-Step Preparation and Operation in the Preliminary Stage The first step involves the simultaneous preparation of experimental materials, instruments, cells, and animals. (1) Cell-related preparation Frozen RAW264.7 mouse macrophages were sterilized under ultraviolet light in a clean bench for 30 min, rapidly thawed in a 37℃ water bath, centrifuged at 1000 rpm for 3 min, and resuspended in DMEM complete medium containing 10% fetal bovine serum + 1% double antibiotics and high glucose for passage. Cells with normal viability after 3 passages or more were selected for use. Modeling induction solution was prepared: 100 ng / mL LPS + 20 ng / mL IFN-γ. A 2 μmol / L oridonin cell working solution was prepared, and the concentration was verified to be non-cytotoxic by CCK8 pre-experiment.

[0057] (2) Animal-related preparations Thirty 12-week-old SPF-grade male C57BL / 6J mice, weighing 20-25g, were purchased and acclimatized in a constant temperature and humidity animal room for 7 days. An ACLT anterior cruciate ligament transection was performed to construct a traumatic OA model. The mice were fed for 14 days post-surgery to allow for extensive infiltration of M1 macrophages in the synovium. Oridonin A animal injection solution was prepared at a dose of 10mg / kg with sterile physiological saline and sterilized using a 0.22μm filter membrane.

[0058] (3) Instrument debugging The low-intensity pulse ultrasound device has the following preset parameters: frequency 1.5MHz, pulse repetition frequency 1kHz, duty cycle 20%, and sound intensity 30mW / cm². 2 Turn on the machine and preheat for 10 minutes; prepare the following equipment: flow cytometer, fluorescence inverted microscope, fluorescence quantitative PCR instrument, CCK8 microplate reader, and a complete set of immunofluorescence slide reagents.

[0059] The second step is to set up the groups (5 groups in vivo and 5 groups in vitro, one-to-one correspondence). Animal groups in vivo (n=6 / group): SHAM sham operation group, OA model group, OA + LIPUS alone group, OA + ORI alone group, OA + LIPUS + ORI combined group; In vitro cell groups: M0 blank control group, M1 induced model group, M1 + LIPUS alone group, M1 + ORI alone (2μM) group, M1 + LIPUS + ORI combined group.

[0060] II. Step-by-step procedure for in vitro macrophage intervention (conducted simultaneously with animal intervention). Step 1: Cell plating and modeling: RAW264.7 cells were plated at a density of 6 × 10⁻⁶ cells / cm². 5 Inoculate 6-well plates with 1×10⁶ cells / well. 5 Cells were seeded per well in 24-well plates using a 1.4 × 10⁶ cell culture medium. 4 Inoculate 1 cell / well into a 96-well CCK8 assay plate and incubate at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere to the plate. Except for the M0 group which was given sterile PBS, the other groups were inoculated with an induction medium containing 100 ng / mL LPS and 20 ng / mL IFN-γ to construct the M1 polarization model.

[0061] Step 2: Grouped drug administration and ultrasound intervention: M0 group: Only an equal volume of sterile PBS was added, without ultrasound stimulation; Group M1: Induction solution + PBS, place a non-energy ultrasound probe for 20 min; M1+LIPUS group: induction solution + PBS, probe coated with coupling agent and attached to the bottom of culture plate, sonic stimulation with standard parameters for 20 min; M1+ORI group: induction solution + 2μmol / L oridonin A, placed without energy probe for 20min; M1+LIPUS+ORI group: induction solution + 2μmol / L oridonin A, start standard parameter ultrasound within 10min after drug addition, and continue for 20min.

[0062] After all interventions were completed, the cells were returned to the incubator for another 12 hours of incubation.

[0063] III. Testing (1) CCK8 cell viability assay: 10 μL of CCK8 solution was added to each of 96 wells and incubated for 1 h. The absorbance was measured at 450 nm using a microplate reader. Cell viability data were plotted, and the results are shown below. Figure 7 As shown. Based on Figure 7 It can be seen that 2 μmol / L OR, standard LIPUS, and the combination of the two do not inhibit the activity of RAW264.7 cells, and the intervention regimen is safe and non-toxic at the cellular level.

[0064] (2) Light microscopy cell morphology imaging: The morphology of each group of macrophages was photographed at 100x and 400x magnification using an inverted microscope. iNOS cell immunofluorescence (ICF): Cells were fixed in 4% paraformaldehyde, permeabilized, blocked, incubated with iNOS primary antibody overnight at 4°C, incubated with fluorescent secondary antibody in the dark, and stained with DAPI on the nuclei.

[0065] Flow cytometry (FCM) detection of CD86: Cells from each group were collected and resuspended, and CD86 fluorescent antibody was incubated at 4°C in the dark for 30 min. The average fluorescence intensity of CD86-positive cells was then detected by flow cytometry.

[0066] The above test results are as follows Figure 8 As shown, the results include: A. Morphological images of macrophages in each group under a light microscope (scale bar: top image 200μm, bottom image 50μm); B. iNOS immunofluorescence images of macrophages in each group (scale bar: 50μm); C. CD86 expression of macrophages in each group detected by flow cytometry; D. Statistical analysis of the average fluorescence intensity of each experimental group in the flow cytometry experiment. Note: ***P<0.001 based on Figure 8 It was found that the average fluorescence intensity of CD86 in the combined group decreased significantly, proving that the combined treatment could significantly reduce the expression of surface markers on M1 macrophages. The iNOS green fluorescence in the M1 group was extremely strong, and the fluorescence intensity in the combined group was significantly lower than that in the single drug and single ultrasound groups, indicating that the expression of M1 markers was significantly inhibited. M1 group cells were hypertrophic and had a large number of pseudopodia processes; single intervention only partially restored cell morphology, while the cells in the combined group shrank and became rounded, with a morphology close to that of resting M0 macrophages.

[0067] (3) qRT-PCR detection of pro-inflammatory factors: Total RNA was extracted from cells in each group using Trizol, and cDNA was prepared by reverse transcription. iNOS, IL-1β, TNF-α, IL-6, and IL-12β were amplified, and their relative mRNA expression levels were detected. The results are shown below. Figure 9 As shown: Based on Figure 9 It can be seen that LIPUS combined with ORI can synergistically and significantly downregulate all M1-related pro-inflammatory genes, with a better inhibitory effect than single intervention methods.

[0068] III. Step-by-step in vivo animal intervention (synchronized with cell experiments over a 4-week cycle) Step 1: Weekly Joint Injection Procedure Mice were anesthetized every morning, and joint cavity was punctured via the lateral patellar approach: the SHAM, OA, and LIPUS groups were injected with an equal volume of physiological saline; the ORI single-drug and combination groups were injected with 10 mg / kg oridonin A injection, injected for 1 min, and the needle was removed after 30 s in place.

[0069] The second step is daily ultrasound intervention (to be completed within 10 minutes after injection, with an interval of ≤15 minutes). The probe was coated with 0.3mm coupling agent and fitted to the affected knee of a mouse. The operating frequencies were 1.5MHz, 1kHz, 20% duty cycle, and 30mW / cm².2 Parameters were continuously monitored with ultrasound for 20 minutes; the SHAM, OA, and ORI single-drug groups underwent sham procedures with only a non-energy probe. The complete intervention lasted for 4 weeks, with injections once a week and ultrasound daily.

[0070] IV. Step-by-step procedure for in vivo iNOS immunofluorescence detection of synovial membrane After 4 weeks of intervention, mice were euthanized, and the synovial tissue of the knee joint was completely isolated, fixed, dewaxed, and repaired with sodium citrate antigen. The tissue was permeabilized with 0.1% Triton, blocked with goat serum, incubated with iNOS primary antibody at 4°C overnight, incubated with fluorescent secondary antibody in the dark, stained with DAPI, and iNOS immunofluorescence images of the synovium were acquired by fluorescence microscopy.

[0071] The results are as follows Figure 10 As shown, based on Figure 10 It can be seen that: the high expression of iNOS fluorescence in the synovium of OA model mice indicates a large number of M1 macrophages infiltrating; the iNOS fluorescence in the synovium of the LIPUS combined with ORI treatment group was significantly reduced, and the M1 polarization of synovial macrophages in vivo was synergistically inhibited.

[0072] In summary: Oridonin combined with standard parameter LIPUS can simultaneously reverse the morphology of M1 macrophages, downregulate iNOS and CD86 specific markers, and inhibit multiple pro-inflammatory genes such as IL-1β and TNF-α, and the intervention concentration has no cytotoxic effect on macrophages; at the in vivo animal synovial tissue level: this combined intervention can significantly reduce the expression of iNOS, a marker of M1 macrophages, in the synovial membrane of traumatic OA mice, and reduce the infiltration of pro-inflammatory immune cells in the synovial membrane; ORI or LIPUS alone can only mildly inhibit macrophage M1 polarization. The combination of the two has a synergistic inhibitory effect of 1+1>2. This combination can improve chronic inflammation of synovial membrane in osteoarthritis by regulating macrophage polarization, which is verified at both the cellular and tissue levels.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. The use of oridonin in the preparation of drugs for the prevention and / or treatment of osteoarthritis.

2. The application according to claim 1, characterized in that, The osteoarthritis mentioned is traumatic knee osteoarthritis induced by anterior cruciate ligament transection.

3. The application according to claim 1, characterized in that, The drug treats osteoarthritis by inhibiting M1 polarization of synovial macrophages.

4. The application according to claim 3, characterized in that, The inhibition of macrophage M1 polarization includes: downregulating the expression of M1 markers iNOS and CD86, and downregulating the gene expression of pro-inflammatory factors iNOS, IL-1β, TNF-α, IL-6, and IL-12β.

5. The application according to any one of claims 1-4, characterized in that, The administration method of oridonin A is intra-articular injection.

6. The application according to claim 5, characterized in that, The dosage for administration of the drug is as follows: the effective dose for animals is 10 mg / kg, administered once a week for 4 consecutive weeks; the safe working concentration for in vitro cells is 2 μmol / L.

7. The application according to claim 1, characterized in that, This also includes the synergistic use of oridonin in combination with low-intensity pulsed ultrasound; The parameters of the low-intensity pulsed ultrasound are: frequency 1.5MHz, sound intensity 30mW / cm². 2 Duty cycle 20%, single intervention 20 minutes, once a day.

8. The use of oridonin in the preparation of a drug for reducing the expression levels of pro-inflammatory factors iNOS, IL-1β, TNF-α, IL-12β and IL-6.

9. A drug for treating osteoarthritis, characterized in that, It contains oridonin A and pharmaceutically acceptable excipients.

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