Method for analyzing curative effect, safety and action mechanism of nanoneedle on knee osteoarthritis and application of nanoneedle

Abstract

The invention discloses a method for analyzing the curative effect, safety and action mechanism of a nanoneedle on knee osteoarthritis and application of the nanoneedle. The invention develops a research scheme for the curative effect, safety, action mechanism and the like of the nanoneedle in the knee osteoarthritis model, and specifically comprises (1) selection of an animal model and a control group, design of a treatment scheme and the like, and (2) subjective and objective curative effect verification indexes combined with qualitative and quantitative, such as behavioristics, pathological sections, inflammatory factors and the like. And (3) a mechanism research method based on scientific hypothesis and unbiased multi-omics analysis are combined, an existing onset mechanism is verified, a new path is found, the curative effect, the safety and the action mechanism of the nanoneedle on knee osteoarthritis can be scientifically and systematically analyzed, and popularization and application of the nanoneedle are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an analysis method for the efficacy, safety and action mechanism of a nano needle for knee osteoarthritis. Background Art

[0002] Knee osteoarthritis is a common degenerative disease in clinical practice. In addition to Western medical treatments (such as intra-articular injection of glucocorticoids, etc.), acupuncture therapy is a clinically proven alternative treatment option. At the same time, traditional medical clinical operations also include acupoint injection therapy for this pathological process.

[0003] Traditional acupuncture or electroacupuncture, warm acupuncture, fire needle and other treatments: Acupuncture is the use of needles (usually filiform needles) to pierce the patient's body at a certain angle, and stimulate the body's local area through needle manipulations such as lifting, inserting, and twisting, thereby playing the role of dredging the meridians, qi and blood, and regulating the body's yin and yang. At the same time, based on conventional acupuncture, electroacupuncture, warm acupuncture, fire needle and other methods have been derived to meet the needs of treating different symptoms. However, the mechanism of action and efficacy of traditional acupuncture are still controversial. In some situations, the therapeutic effect of drug therapy in the acute phase will be more significant. Acupoint injection: It is a modern Chinese medicine external treatment method evolved from traditional acupuncture. It is a treatment method that uses a syringe to inject drugs into the selected acupoints or parts. Compared with traditional acupuncture, acupoint injection enhances the intensity of stimulation, prolongs the duration of action, and enhances the therapeutic effect of meridians. However, if there are problems in the existing acupoint injection method in the injection amount of drugs, infection control, syndrome differentiation and acupoint selection, it will directly affect the efficacy and (or) safety of the operation. External application of Chinese medicine patch: External application of Chinese medicine patch is to apply the prepared ointment to specific acupoints or lesions, which continuously exerts its effects through meridians and drugs, improves microcirculation, reduces local tissue inflammatory factors, and improves proprioception. However, this treatment method is a transdermal drug delivery technology, and it is difficult for drugs to enter deep into the acupoints, so it is often used as an auxiliary treatment. Traditional acupuncture plus Western medicine treatment plan: Currently, Western medicine mainly uses surgical operations or oral and topical non-steroidal anti-inflammatory drugs, hyaluronic acid injections, etc. as the main treatment methods. In order to enhance the treatment effect, traditional acupuncture plus oral and topical non-steroidal anti-inflammatory drugs, hyaluronic acid injections and other treatment plans are often used in clinical practice. This treatment method is relatively cumbersome and causes prolonged treatment time, and may cause toxic side effects due to systemic administration.

[0004] The drug-device combination design of nanoneedles refers to the modification of drug molecules on the surface of acupuncture needles through physical and chemical means to achieve the dual effects of acupuncture and drug delivery. It is considered to be a promising emerging treatment method. However, the research on its efficacy, safety, and mechanism of action is a new scientific field. In addition, the in vivo and in vitro experimental procedures and scientific and objective data analysis methods of nanoneedles have not yet been reported. Therefore, it is of great significance to design analysis methods for the efficacy, safety, and mechanism of action of drug-loaded nanoneedles for knee osteoarthritis. Summary of the invention

[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides an analysis method for the efficacy, safety, and mechanism of action of a nanoneedle for knee osteoarthritis and its application, which is of great significance for the application of drug-loaded nanoneedles in the treatment of knee osteoarthritis.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for analyzing the efficacy, safety, and mechanism of action of a nanoneedle for knee osteoarthritis, the method comprising:

[0008] Acupuncture was performed on an animal model of knee osteoarthritis using drug-loaded nanoneedles as the experimental group, and a control group was set up, which included a normal animal group, a non-intervention group of the animal model of knee osteoarthritis, an acupuncture group using ordinary acupuncture needles, and a drug acupoint injection group; the efficacy verification indicators of the animal models in the experimental group and the control group were tested; based on the mechanism research method based on scientific hypothesis and the unbiased multi-omics analysis method, the mechanism of action of the nanoneedles was analyzed.

[0009] In the present invention, a research scheme for the efficacy, safety, mechanism of action, etc. of nanoneedles in a knee osteoarthritis model was developed, specifically including (1) the selection of animal models, control groups, and treatment scheme design, etc., (2) subjective and objective, qualitative and quantitative efficacy verification indicators: behavior, pathological sections, inflammatory factors, etc., (3) combining a mechanism research method based on scientific hypotheses with unbiased multi-omics analysis to verify existing mechanisms of action and discover new pathways, which can scientifically and systematically analyze the efficacy, safety, and mechanism of action of nanoneedles for knee osteoarthritis, and facilitate the promotion and application of nanoneedles.

[0010] It can be understood that the type of drug loaded by the nanoneedle can be adjusted, including the introduction of active drug molecules or combinations with central or peripheral analgesia, single or multiple active products of traditional Chinese medicine, traditional Chinese medicine extracts, combinations of traditional Chinese and Western medicine treatment plans, etc.; the introduction of hormone active molecules such as triamcinolone acetonide, etc.; the introduction of nerve nutrition active molecules such as vitamin B12, hyaluronic acid, etc.

[0011] Preferably, the drug comprises lidocaine.

[0012] In addition, in addition to lidocaine, other similar analgesic drugs include: central analgesics: including two major categories, non-peptide and peptide. Non-peptide includes opioid alkaloids and synthetic analgesics, such as morphine, codeine, fentanyl, tramadol, buprenorphine, ion channel modulators, etc.; peripheral analgesics: salicylates such as acetyl salicylic acid, sodium salicylate, etc.; acetanilides such as phenacetin, paracetamol, and pyraclostrobin; pyrazolones such as clofenamic acid, mefenamic acid, etc.; indoles such as indomethacin, anthracene; aromatic propionic acids such as ibuprofen, etc.; natural products: such as extract monomers and combinations thereof from Corydalis yanhusuo, Hemerocallis quinquefolia, Bupleurum chinense, Pueraria lobata, and Lophatherum gracile.

[0013] In the present invention, lidocaine is delivered by nanoneedles and acupuncture is performed to treat knee osteoarthritis. The specific efficacy and related mechanisms of the lidocaine nanoneedles are observed in a model of knee osteoarthritis in mice induced by sodium iodoacetate, and the results are effective. The efficacy and safety of the nanoneedles in preclinical models are verified, providing an important basis for the selection of clinical indications for this technology.

[0014] Preferably, the control group also includes a group using hormone injection into the knee joint cavity.

[0015] In the present invention, mice, rats and other rodents or large animals are all suitable for constructing models of the present invention.

[0016] Preferably, the animal model of knee osteoarthritis comprises a mouse knee osteoarthritis model.

[0017] Preferably, the efficacy verification indicators include paw retraction time in response to heat, inflammatory factors, cartilage degeneration level, synovial inflammation level and knee joint bone structure.

[0018] Preferably, the hypothesized pathway in the mechanism research method based on the scientific hypothesis is the HMGB1 / TLR4 pathway.

[0019] Preferably, the unbiased multi-omics analysis method comprises performing multi-omics analysis of transcriptomics, proteomics and metabolomics.

[0020] In the present invention, strategies commonly used in the art for introducing drugs into acupuncture needles are applicable to the present invention, such as laser engraving, hydrogel, trocar or nanogold.

[0021] Preferably, the method for preparing the drug-loaded nanoneedle comprises:

[0022] Nano adhesive polysaccharide is deposited on acupuncture needles to obtain nano needles, and the nano needles are contacted with drug solution to obtain the drug-loaded nano needles.

[0023] As a preferred technical solution, the method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis includes the following steps:

[0024] (1) depositing nano-adhesive polysaccharides on acupuncture needles to obtain nano-needles, and contacting the nano-needles with a drug solution to obtain the drug-loaded nano-needles;

[0025] (2) Acupuncture was performed on a mouse model of knee osteoarthritis using drug-loaded nanoneedles as the experimental group, and a control group was set up, including a normal animal group, a knee osteoarthritis animal model non-intervention group, an acupuncture group using ordinary acupuncture needles, and a drug acupoint injection group;

[0026] (3) Detect the paw retraction time, inflammatory factors, cartilage degradation level, synovial inflammation level and knee joint bone structure in the experimental and control groups of mice with knee osteoarthritis;

[0027] (4) Based on the mechanism research method based on scientific hypothesis, the HMGB1 / TLR4 pathway was analyzed, and multi-omics analysis of transcriptomics, proteomics and metabolomics was performed to analyze the mechanism of action of the nanoneedle.

[0028] In a second aspect, the present invention provides the use of the method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis described in the first aspect in analyzing markers and drug targets for knee osteoarthritis.

[0029] In a third aspect, the present invention provides a marker associated with knee osteoarthritis, wherein the marker comprises HMGB1, TLR-4, CDC42, rac1, Akt1, Akt2 and mmp9.

[0030] In the present invention, it was found that the expression of HMGB1 and TLR-4 genes was upregulated in the mouse knee osteoarthritis model, and the expression was restored after treatment, indicating that they can be used as markers for knee osteoarthritis. In addition, through proteomic analysis, it was found that the expression of CDC42, rac1, Akt2 and mmp9 proteins was upregulated in the mouse knee osteoarthritis model, and the expression of Akt1 protein was downregulated in the mouse knee osteoarthritis model.

[0031] In a fourth aspect, the present invention provides use of the markers associated with knee osteoarthritis and / or detection reagents thereof described in the third aspect in the preparation of a knee osteoarthritis detection product.

[0032] In a fifth aspect, the present invention provides the use of the markers associated with knee osteoarthritis described in the third aspect as targets in screening drugs for preventing or treating knee osteoarthritis.

[0033] Preferably, the screening comprises determining whether the candidate drug can be used to prevent or treat knee osteoarthritis based on the effects of the candidate drug on the marker before and after use.

[0034] In the present invention, through multi-omics mechanism analysis, it was found that the mechanism of action of lidocaine nanoneedles in treating knee osteoarthritis may be related to reactive oxygen species, mitochondrial function, SmallGTPnase protein family and HMGB / TLR4 pathways.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention has developed a research scheme for the efficacy, safety, mechanism of action, etc. of nanoneedles in a knee osteoarthritis model, which specifically includes (1) the selection of animal models, control groups, and treatment scheme design, etc., (2) subjective and objective, qualitative and quantitative efficacy verification indicators: behavior, pathological sections, inflammatory factors, etc., (3) combining a mechanism research method based on scientific hypotheses with unbiased multi-omics analysis to verify existing mechanisms of action and discover new pathways, which can scientifically and systematically analyze the efficacy, safety, and mechanism of action of nanoneedles for knee osteoarthritis, and is conducive to the promotion and application of nanoneedles. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The results of in vitro release experiments of lidocaine-loaded nanoneedles and common acupuncture needles are shown;

[0038] Figure 2 This is a graph showing the time of retraction of the affected limb of the mouse in Example 3 in response to heat;

[0039] Figure 3 This is a diagram of the inflammation analysis results in Example 3;

[0040] Figure 4A The result of Safranin O-Fast Green staining of the knee joint tissue of the affected limb of the mouse in Example 3;

[0041] Figure 4B This is a semi-quantitative result diagram of the degree of cartilage degradation in mice in Example 3;

[0042] Figure 5A The result of HE staining of the knee joint tissue of the affected limb of the mouse in Example 3;

[0043] Figure 5B This is a semi-quantitative result diagram of evaluating synovial inflammation in Example 3;

[0044] Figure 6 This is a high-resolution X-ray tomography image of the knee joint of the affected limb of the mouse in Example 3;

[0045] Fig. 7A This is a result diagram of the HMGB1 gene mRNA level in the synovial fluid of the affected knee joint cavity of mice in Example 4;

[0046] Figure 7BThis is a result diagram of the mRNA level of TLR-4 gene in the synovial fluid of the affected knee joint cavity of mice in Example 4;

[0047] Figure 8 This is a bubble chart of GO enrichment analysis based on proteomics differential protein analysis in Example 4. DETAILED DESCRIPTION

[0048] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0049] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0050] Example 1

[0051] This embodiment prepares lidocaine nano needles, and loads lidocaine on acupuncture needles, specifically including: modifying the surface of ordinary acupuncture needles (millimacules) with cyclic molecules (cyclodextrin) that can form inclusion complex structures to form cavities; then connecting the cyclic molecules to the surface of acupuncture needles through chemical bonds (methyl salicylate), so that the acupuncture needles have the ability to load and release small molecules, becoming nano needles. Soak them in a saturated lidocaine solution for 24 hours, take them out and dry them naturally in a sterile environment to obtain "lidocaine needles".

[0052] Similarly, the nanoneedles are immersed in other drug solutions such as central analgesics: including two major categories, non-peptide and peptide, non-peptide including opioid alkaloids and synthetic analgesics, such as morphine, codeine, fentanyl, tramadol, buprenorphine, ion channel regulators, etc.; peripheral analgesics: salicylates such as acetyl salicylic acid, sodium salicylate, etc.; acetanilide such as phenacetin, paracetamol, pyrrolidone, etc.; pyrazolones such as clofenamic acid, mefenamic acid, etc.; indoles such as indomethacin, anthracene; aromatic propionic acids such as ibuprofen, etc.; natural products: such as extract monomers from Corydalis yanhusuo, Hemerocallis quinquefolia, Bupleurum chinense, Pueraria lobata, and Lophatherum gracile and their combinations, to prepare needles loaded with different drugs.

[0053] Example 2

[0054] This example is about the in vitro release of lidocaine nanoneedles.

[0055] In order to observe the effect of "lidocaine needle" in releasing lidocaine in vitro and the specific quantitative analysis, we divided "lidocaine needle" and ordinary acupuncture needle into 2 groups, with 5 needles in each group. The two groups of acupuncture needles were placed in 1×PBS solution, and the lidocaine release of the two groups of acupuncture needles was observed at 30s, 1min, 2min, 3min, 4min, 5min, 10min, 15min, 20min, 25min, and 30min, a total of 11 time points. The lidocaine content at each time point was accurately measured by HPLC, and a curve was drawn to specifically quantify the loading and release of lidocaine drugs by nanoneedles. The results are shown in the figure. Figure 1 As shown, the lidocaine content released from the lidocaine needle into the PBS solution over time in the in vitro release experiment of the lidocaine needle is shown. The horizontal axis is time, and the vertical axis is the amount of lidocaine measured by high performance liquid chromatography. There are 5 acupuncture needles in the experiment, so the lidocaine loading amount of each acupuncture needle is 50μg, that is, the 30-minute release amount of the lidocaine nanoneedle is: 50μg / acupuncture needle.

[0056] Example 3

[0057] This example observes the efficacy of lidocaine nanoneedles in treating a mouse model of knee osteoarthritis.

[0058] (1) A mouse model of knee osteoarthritis was established by injecting 0.5 mg of sodium iodoacetate (MIA) into the hind limb joint cavity of mice (8-12 week old C57BL / 6 female mice, purchased from Spefoc (Beijing) Biotechnology Co., Ltd.).

[0059] (2) A control group was set up, including normal mice, a knee osteoarthritis model non-intervention group, a conventional acupuncture treatment group, a lidocaine acupoint injection group, and a standard treatment group (hormone injection into the knee joint cavity), and the control groups were compared with the lidocaine nanoneedle treatment group.

[0060] (3) Treatment plan: Both the conventional acupuncture treatment group and the lidocaine nanoneedle treatment group were treated with electroacupuncture. The mice were punctured at the Zusanli and Yanglingquan points on the affected limbs and connected to an electroacupuncture instrument. The parameters were adjusted as follows: sparse-dense wave, frequency 2 Hz, output 2 mA, 30 minutes / time / day. The treatment lasted for 28 days starting from the third day after modeling. The lidocaine acupoint injection group was injected with lidocaine (Zusanli and Yanglingquan points) every day starting from the third day after modeling, 50 μg / acupoint, for 28 days. The standard treatment group (hormone injection into the knee joint cavity) was injected with triamcinolone acetonide (25 μg) solution into the affected knee joint cavity of the mice starting from the third day after modeling. The treatment was performed once a week for a total of 4 times. No intervention measures were taken for normal mice and the knee osteoarthritis model group. After 28 days of treatment, all mice were killed and samples were collected.

[0061] (4) Efficacy observation indicators:

[0062] ① Behavioral indicators: Use a hot and cold plate analgesia instrument (SA705) to observe whether the thermal pain sensation of the affected limb of the mouse is sensitized, that is, measure the time it takes for the affected limb of the mouse to shrink its foot in response to heat. Test once before modeling, and test once every 5 days after modeling until the end of the experiment, and draw a curve. The results are shown in the figure. Figure 2 As shown, the horizontal axis is the time (days) after the injection of MIA to model knee osteoarthritis in mice, and the vertical axis is the time (seconds) of the mice's paw retraction in response to heat measured by a hot and cold plate analgesia meter. It can be seen that 28 days after lidocaine nanoneedle electroacupuncture treatment of mice with knee osteoarthritis, the thermal pain sensation of the affected limbs of the mice was significantly restored (Normal = normal mice, NT = untreated mice after knee osteoarthritis modeling, nd-Acu = mice treated with lidocaine nanoneedle electroacupuncture), indicating that lidocaine nanoneedle treatment of mice with knee osteoarthritis improved their thermal pain sensation and prolonged the mice's thermal paw retraction reaction time.

[0063] ② Inflammatory factors: After the experiment, the mice were killed and the joint cavity of the affected side of each group of mice was sampled and homogenized, and the supernatant was extracted. The ELISA method was used to detect the levels of IL-6, IL-1β, and TNF-α in the fluid of the knee joint cavity of the affected limb of the mice. Figure 3 The results show that after treating mice with knee osteoarthritis with lidocaine nanoneedle, the excessive release of inflammatory factors IL-6, IL-1β, and TNF-α in the synovial fluid of the knee joint cavity of mice with knee osteoarthritis was corrected. The horizontal axis represents 6 groups, including: normal group (Normol), knee osteoarthritis model group (NT), lidocaine nanoneedle electroacupuncture group (nd-Acu), ordinary acupuncture needle electroacupuncture group (Acu), lidocaine acupoint injection group (Lido) and standard treatment (hormone joint cavity injection) group (TAA). The vertical axis represents the content of IL-6, IL-1β, and TNF-α in the synovial fluid of the knee joint cavity of mice. It can be seen that after 28 days of lidocaine nanoneedle electroacupuncture treatment of mice with knee osteoarthritis, the release of inflammatory factors in the knee joint cavity of mice was controlled. The lidocaine nanoacupuncture electroacupuncture group had the most obvious improvement, followed by the ordinary electroacupuncture group (*, p<0.05; **, p<0.01; ***, p<0.001; ns=no significant difference).

[0064] ③Pathological level: After the mice were killed at the end of the experiment, the knee joints of the affected side of each group of mice were sampled, fixed and decalcified for half a month, and then the knee joint tissues were embedded in paraffin. The changes in the knee cartilage and synovial bursa of the mice were observed by safranin O-fast green staining and HE staining of paraffin sections of the knee joints, and semi-quantitative analysis was performed. Figure 4A and Figure 4B It was demonstrated that lidocaine nanoneedles treated mice with knee osteoarthritis and corrected the level of femoral and tibial plateau cartilage degeneration in mice with knee osteoarthritis. Figure 4AThese are the results of Safranin O-Fast Green staining of the knee joint tissues of the affected limbs of 6 groups of mice. The femoral and tibial plateau cartilage of normal mice were stained uniformly (red in the figure), indicating that the cartilage was normal. The cartilage of mice with knee osteoarthritis was degenerated, and the staining became lighter or even absent. After treatment, the cartilage degeneration improved to varying degrees, and lidocaine nanoneedles had the best therapeutic effect. Figure 4B This is a semiquantitative table for evaluating the degree of cartilage degeneration (OARSI table). The horizontal axis represents 6 groups, including: normal group, knee osteoarthritis model group, lidocaine nanoneedle electroacupuncture group, common acupuncture needle electroacupuncture group, lidocaine acupoint injection group and standard treatment (hormone joint cavity injection) group. The vertical axis represents the degree of cartilage degeneration in the affected side of the knee joint of mice. The higher the score, the more severe the degeneration. It can be seen that after 28 days of lidocaine nanoneedle electroacupuncture treatment of mice with knee osteoarthritis, the degree of cartilage degeneration in the femoral and tibial plateau of the knee joint of mice was significantly improved. The lidocaine nanoneedle electroacupuncture group had the most obvious improvement, followed by the common electroacupuncture group (*, p<0.05; **, p<0.01; ***, p<0.001).

[0065] Figure 5A and Figure 5B It was demonstrated that lidocaine nanoneedles treated mice with knee osteoarthritis and corrected the level of synovial inflammation in the knee joints of mice with knee osteoarthritis. Figure 5A The results of HE staining of the knee joint tissues of the affected limbs of 6 groups of mice are shown in the figure. As shown by the black arrows, the synovial surface of the knee joints of normal mice is smooth, usually with a single layer of lining cells and a normal number of interstitial cells, while the number of cell layers and cell density of the synovial bursa of mice with knee osteoarthritis increased significantly, indicating the formation of synovial inflammation. After treatment, the synovial inflammation improved to varying degrees, and the lidocaine nanoneedle had the best therapeutic effect. Figure 5B This is a semi-quantitative table for evaluating synovial inflammation (synovitis score table). The horizontal axis is 6 groups, including: normal group, knee osteoarthritis model group, lidocaine nanoneedle electroacupuncture group, common acupuncture needle electroacupuncture group, lidocaine acupoint injection group and standard treatment (hormone joint cavity injection) group. The vertical axis is the score of the degree of synovitis in the affected knee joint of mice. The higher the score, the more severe the inflammation. As can be seen from the figure, after 28 days of lidocaine nanoneedle electroacupuncture treatment of knee osteoarthritis mice, the degree of synovial inflammation in the knee joint of mice was significantly improved. The lidocaine nanoneedle electroacupuncture group had the most obvious improvement, followed by the lidocaine group and the common electroacupuncture group (among which *, p<0.05; **, p<0.01; ***, p<0.001).

[0066] ④ Bone and cartilage morphology: After the mice were killed at the end of the experiment, the knee joints of the affected side of each group of mice were sampled and fixed. Before decalcification, a high-resolution X-ray tomography imaging system was used to perform high-resolution X-ray tomography on the affected knee joints of the mice to observe the changes in the femur, tibial platform and joint cavity of the knee joint. Figure 6The lidocaine nanoneedle device was used to treat mice with knee osteoarthritis, and the structural changes of the knee joint bones of the mice with knee osteoarthritis were corrected. In the sagittal plane of the knee joint tissue, osteophytes were formed at the femoral end of the mice with knee osteoarthritis compared with normal mice; in the coronal plane, it can be seen that the knee joint cavity of the mice with knee osteoarthritis has been significantly reduced. After the lidocaine nanoneedle device treatment, the above conditions have improved, indicating that the lidocaine nanoneedle device treatment also has a certain effect on improving bone structure changes.

[0067] Through the data at all levels mentioned above, it can be comprehensively evaluated that the lidocaine nanoneedle treatment of the knee osteoarthritis mouse model after 28 days showed significant efficacy in behavior, pathology, inhibition of inflammatory factor release and bone structure changes.

[0068] Example 4

[0069] This example studies the mechanism of action of lidocaine nanoneedles.

[0070] ① Mechanism research based on scientific hypothesis: Based on scientific hypothesis, the HMGB1 / TLR4 pathway is a key pathway for inflammation, and the mechanism of action of lidocaine nanoneedles is verified by this pathway. After 28 days of treatment and experiment, the mice were killed, and the joint cavity of the affected side of each group of mice was sampled and homogenized. The mRNA content of HMGB1 and TLR4 was tested by qRT-PCR to verify the mechanism of action of lidocaine nanoneedles in the TLR4-NF-κB-HMGB1 pathway.

[0071] Fig. 7A and Figure 7B The study of the anti-inflammatory mechanism of lidocaine nanoneedle in treating knee osteoarthritis mice based on scientific hypothesis in the TLR4-NF-κB-HMGB1 pathway. The horizontal axis is 6 groups, including: normal group, knee osteoarthritis model group, lidocaine nanoneedle electroacupuncture group, common acupuncture needle electroacupuncture group, lidocaine acupoint injection group and standard treatment (hormone joint cavity injection) group; the vertical axis is the HMGB1 ( Fig. 7A ) and TLR-4( Figure 7B ) mRNA levels; compared with normal mice, the levels of both in mice with knee osteoarthritis were significantly increased, indicating that the inflammatory mechanism of the TLR4-NF-κB-HMGB1 pathway was activated, and after treatment, the mRNA levels of HMGB1 and TLR-4 in each group of mice had different degrees of callback, especially in the lidocaine nanoneedle group, indicating that the mechanism of action of lidocaine nanoneedle may be in the TLR4-NF-κB-HMGB1 pathway (*, p<0.05; **, p<0.01; ***, p<0.001).

[0072] ② Unbiased multi-omics analysis: After the experimental treatment, the affected knee cartilage of the mice was taken for multi-omics analysis of transcriptomics, proteomics and metabolomics. Figure 8 The bubble chart of GO (Gene Ontology) enrichment analysis based on proteomics differential protein analysis was displayed, and the top 10 related factors of biological process (BP), cellular component (CC) and molecular function (MF) were intercepted (the principle of selecting differential proteins among the three groups of normal mice group, knee osteoarthritis model group and lidocaine nanoneedle treatment group was: p<0.05; fold change>1.20or<0.83). It can be seen that the mechanism of action of lidocaine nanoneedle is highly correlated with mitochondria, redox, etc. (as shown by the red arrow in the figure), indicating that the mechanism of action of lidocaine nanoneedle in the treatment of knee osteoarthritis may be related to reactive oxygen species, mitochondrial function, SmallGTPnase protein family, HMGB / TLR4 and other pathways; through proteomics analysis, it was found that CDC42, rac1, Akt2 and mmp9 proteins were upregulated in the mouse knee osteoarthritis model, and Akt1 protein was downregulated in the mouse knee osteoarthritis model; it can suggest new methods and medication directions for the treatment of knee osteoarthritis.

[0073] In summary, the present invention has developed a research scheme for the efficacy, safety, mechanism of action, etc. of nanoneedles in a knee osteoarthritis model, which specifically includes (1) the selection of animal models, control groups, and treatment scheme design, etc., (2) subjective and objective, qualitative and quantitative efficacy verification indicators: behavior, pathological sections, inflammatory factors, etc., (3) combining a mechanism research method based on scientific hypotheses with unbiased multi-omics analysis to verify existing mechanisms of action and discover new pathways, which can scientifically and systematically analyze the efficacy, safety, and mechanism of action of nanoneedles for knee osteoarthritis, and is conducive to the promotion and application of nanoneedles.

[0074] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for analyzing the efficacy, safety, and mechanism of action of a nanoneedle for knee osteoarthritis, characterized in that: The method comprises: Acupuncture was performed on an animal model of knee osteoarthritis using drug-loaded nanoneedles as an experimental group, and a control group was set up, the control group including a normal animal group, a knee osteoarthritis animal model non-intervention group, an acupuncture group using ordinary acupuncture needles, and a drug acupoint injection group; Detect efficacy validation indicators of animal models in experimental and control groups; The mechanism of action of nanoneedles is analyzed based on a mechanism research method based on scientific hypotheses and an unbiased multi-omics analysis method.

2. The method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis according to claim 1, characterized in that: The drugs include lidocaine; Preferably, the control group also includes a group using hormone injection into the knee joint cavity.

3. The method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis according to claim 1 or 2, characterized in that: The knee osteoarthritis animal model includes a mouse knee osteoarthritis model; Preferably, the efficacy verification indicators include paw retraction time in response to heat, inflammatory factors, cartilage degeneration level, synovial inflammation level and knee joint bone structure.

4. The method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis according to any one of claims 1 to 3, characterized in that: The hypothesized pathway in the mechanism research method based on the scientific hypothesis is the HMGB1 / TLR4 pathway; Preferably, the unbiased multi-omics analysis method comprises performing multi-omics analysis of transcriptomics, proteomics and metabolomics.

5. The method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis according to any one of claims 1 to 4, characterized in that: The method for preparing the drug-loaded nanoneedle comprises: Nano adhesive polysaccharide is deposited on acupuncture needles to obtain nano needles, and the nano needles are contacted with drug solution to obtain the drug-loaded nano needles.

6. The method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) depositing nano-adhesive polysaccharides on acupuncture needles to obtain nano-needles, and contacting the nano-needles with a drug solution to obtain the drug-loaded nano-needles; (2) Acupuncture was performed on a mouse model of knee osteoarthritis using drug-loaded nanoneedles as the experimental group, and a control group was set up, including a normal animal group, a knee osteoarthritis animal model non-intervention group, an acupuncture group using ordinary acupuncture needles, and a drug acupoint injection group; (3) Detect the paw retraction time, inflammatory factors, cartilage degradation level, synovial inflammation level and knee joint bone structure in the experimental and control groups of mice with knee osteoarthritis; (4) Based on the mechanism research method based on scientific hypothesis, the HMGB1 / TLR4 pathway was analyzed, and multi-omics analysis of transcriptomics, proteomics and metabolomics was performed to analyze the mechanism of action of the nanoneedle.

7. Use of the method for analyzing the efficacy, safety, and mechanism of action of the nanoneedle for knee osteoarthritis according to any one of claims 1 to 6 in analyzing markers and drug targets for knee osteoarthritis.

8. A marker associated with knee osteoarthritis, characterized in that: The markers include HMGB1, TLR-4, CDC42, rac1, Akt1, Akt2 and mmp9.

9. Use of the marker associated with knee osteoarthritis and / or its detection reagent according to claim 8 in the preparation of a knee osteoarthritis detection product.

10. Use of the marker associated with knee osteoarthritis according to claim 8 as a target in screening drugs for preventing or treating knee osteoarthritis; Preferably, the screening comprises determining whether the candidate drug can be used to prevent or treat knee osteoarthritis based on the effects of the candidate drug on the marker before and after use.

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