A method for resolving olfactory neural circuit and joint connection

Abstract

This invention discloses a method for analyzing the connection between olfactory neural circuits and joints. By activating multi-level synaptic connections between the olfactory bulb, piriform cortex, and joints, it achieves efficient cartilage regeneration and osteoarthritis treatment. Specifically, the method includes: using multi-level retrograde pseudorabies virus (PRV) and anterograde herpes simplex virus (HSV) tracing technology to analyze the neural circuit connections between the olfactory neural circuits and joints; further, by chemogenetic activation of glutamatergic neurons in the olfactory bulb's mitral cell layer or the piriform cortex, with continuous intervention for 8 weeks, significantly promoting cartilage damage repair and slowing the progression of osteoarthritis. This invention reveals for the first time the neural functional connections between the olfactory system and joints, providing an innovative strategy for non-invasive treatment of osteoarthritis, applicable to drug development and clinical translation.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of neuroscience, regenerative medicine and osteoarthritis treatment, and relates to a method for analyzing the connection between olfactory nerve circuits and joints. Specifically, it relates to an olfactory nerve circuit regulation method based on chemogenetics and its application in regulating osteoarthritis and promoting cartilage regeneration. Background Technology

[0002] Osteoarthritis (OA) is one of the most common age-related diseases, characterized by pain, stiffness, and loss of joint function. The prevalence of knee osteoarthritis in my country is 8.1%, affecting approximately 140 million people nationwide. Normally, the end of the knee joint is covered by a layer of smooth, translucent articular cartilage of a certain thickness, which plays an important role in shock absorption and reducing friction. Due to aging, obesity, and overuse of the joints, this articular cartilage gradually degenerates and decreases, leading to knee osteoarthritis. Osteoarthritis is most common in the elderly (approximately 70% of cases are over 55 years of age), and with the increasing aging of the population, the prevalence of OA is expected to continue to rise. In recent years, my country has successively formulated and published the "Clinical Practice Guidelines for Pain Management of Osteoarthritis in China (2020 Edition)," the "Guidelines for the Diagnosis and Treatment of Osteoarthritis in China (2021 Edition)," and the "Expert Consensus on Clinical Drug Treatment of Osteoarthritis" to promote the standardized diagnosis and treatment of OA. However, whether it's basic treatments such as exercise therapy, physical therapy, and mobility-assisted therapy, drug treatments such as analgesics and traditional Chinese medicine, or reconstructive treatments such as joint debridement and total joint replacement, all these approaches only address the symptoms and not the root cause, failing to truly achieve hyaline cartilage regeneration. Despite groundbreaking progress in basic research related to osteoarthritis, problems such as long treatment cycles, poor repair outcomes, and difficulties in clinical translation remain unavoidable. Therefore, finding a treatment method that can achieve hyaline cartilage regeneration and is easily translated into clinical practice is a crucial issue that urgently needs to be addressed in the field of osteoarthritis.

[0003] As the most advanced organ in humans, the brain dominates all bodily activities and participates in the regulation of processes including metabolism, immune responses, inflammatory pathways, and social behavior, ensuring the balance between the body and its environment and maintaining essential conditions for survival. Circuit tracing and regulation based on the nervous system is currently one of the most cutting-edge research hotspots in the life sciences, with related research results possessing extremely high translational value and excellent clinical application potential. Numerous studies have found that external environmental stimuli can induce activity in related sensory nervous systems, thereby regulating bodily homeostasis. For example, light regulates glucose metabolism through the retina-hypothalamus-brown adipose tissue axis; low-intensity sound-to-noise ratio inhibits the projection of glutamatergic neurons in the auditory cortex to the thalamus, alleviating pain in mice; and moderate electroacupuncture stimulation of the "Zusanli" acupoint in mice can induce a neural network-mediated systemic anti-inflammatory response. Compared to other senses, smell is the only sense that does not require traversing the thalamus to directly reach the cerebral cortex, meaning that interventions related to smell may trigger a faster response in the nervous system, thereby achieving rapid regulation of the body.

[0004] Although some regulatory mechanisms related to bone metabolism in the nervous system have been reported and progress has been made, such as the neural mechanism by which the subfornical organ (SFO) in specific brain regions intervenes in bone metabolism by regulating parathyroid hormone, there are currently no reports of promoting cartilage regeneration or treating osteoarthritis based on the nervous system. This is mainly because elucidating the relevant circuits between cartilage and nerves and using the nervous system for intervention remains an unresolved problem. Thanks to the iteration of photosensitive genes and artificially designed receptors, optogenetics and chemogenetics have made great strides, making it possible to artificially regulate and intervene in neural activity, and have become indispensable tools in the field of neuroscience. Therefore, using advanced neural-related technologies to map brain-joint neural circuits and intervene in specific brain regions may be of great significance for developing effective drugs and products for the treatment of osteoarthritis. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for regulating olfactory neural circuits based on chemogenetics and its applications.

[0006] This invention provides a method for analyzing the connection between olfactory nerve circuits and joints, connecting the brain with the nerves of joints and cartilage. The method includes the following steps:

[0007] Injecting pseudorabies virus (PRV) or rabies virus (RV) across multiple levels into the joint cavity marks the neural connections from the joint to the brain.

[0008] By injecting multilevel anterograde herpes simplex virus (HSV) or adeno-associated virus (AAV) into the olfactory epithelium or olfactory bulb mitral cell layer, the neural connections from the olfactory nerve circuit to the joints are marked.

[0009] The marked brain tissue and joints were fixed and made into frozen sections to verify the neural synaptic connections between specific brain regions and joints, and to analyze the neural connections between specific brain regions and joints.

[0010] Furthermore, the specific brain regions in the brain include one or more of the following: olfactory bulb, agranular insular cortex, piriform cortex, hippocampus, entorhinal cortex, central aqueduct, locus coeruleus, zona indeterminate, hypothalamus;

[0011] Furthermore, significant neuronal activation was observed in the olfactory-related brain regions (including the olfactory bulb, piriform cortex, and locus coeruleus, etc.);

[0012] Preferably, the neurons that retrograde from the joint to the piriform cortex are glutamatergic neurons;

[0013] Furthermore, the pseudorabies virus includes PRV carrying red or green fluorescence, etc.

[0014] Furthermore, the herpes simplex virus includes HSV carrying green fluorescence, etc.

[0015] Furthermore, the virus is administered via injection.

[0016] Furthermore, the PRV injection method is local injection into the joint cavity.

[0017] Furthermore, the PRV injection volume is 1-10 microliters; preferably, it is 3 microliters.

[0018] Furthermore, the PRV is injected into the joint cavity and sampled several days later; preferably, it is 6 days later.

[0019] Furthermore, the HSV injection method is stereotactic brain injection.

[0020] Furthermore, the HSV injection volume is 100-500 nanoliters; preferably, it is 100 nanoliters.

[0021] Furthermore, the HSV is injected into the joint cavity and a sample is taken several days later; preferably, it is taken after 6 days.

[0022] Further, the fixation includes: fixation in a preservative or protein fixative; preferably, fixation in 4% PFA.

[0023] Furthermore, the preparation of the frozen sections includes: preparing frozen sections after treatment with sucrose solution and OCT embedding.

[0024] Furthermore, the verification method includes one or more methods such as fluorescent staining; preferably, it is an immunofluorescence co-localization method.

[0025] This invention also provides a method for chemically and genetically regulating olfactory neural circuits, comprising the following steps:

[0026] Injecting adeno-associated viruses carrying the chemogenetic gene hM3Dq or hM4Di receptors into the olfactory bulb mitral cell layer and / or piriform cortex to express artificially designed protein receptors specifically activated by the designed drug;

[0027] The designed drug activates the artificially designed protein receptor, thereby activating or inhibiting neuronal activity and regulating the olfactory-joint neural circuit.

[0028] Furthermore, the designed drug includes one or more of artificially designed G protein-coupled receptor-specific ligands and chemically-based genetic activators; preferably, it is clozapine nitric oxide.

[0029] Furthermore, the chemical genetic material includes one or more of the following: a gene encoding an activating artificial protein receptor, a gene encoding an inhibitory artificial protein receptor; preferably, it is hM3Dq or hM4Di.

[0030] Furthermore, the protein receptor includes protein receptors that can activate neuronal activity under the activation of the designed drug; preferably, the artificially designed protein receptor is hM3Dq or hM4Di.

[0031] Furthermore, the activation (continuous activation of the target neuron) time of the designed drug is 8-20 weeks to simulate the activation or inhibition effect of neural circuits; preferably, it is 8 weeks.

[0032] The present invention also provides a method for promoting cartilage regeneration by chemically genetically regulating the olfactory nerve circuit, the method comprising the following steps: injecting adeno-associated virus carrying a viral hM3Dq receptor or hM4Di receptor carrying a chemical genetic gene into the olfactory bulb mitral cell layer and piriform cortex and surrounding area of ​​a joint with injury, expressing an artificially designed protein receptor specifically activated by a designed drug;

[0033] The designed drug activates the artificially designed protein receptor, thereby activating or inhibiting neuronal activity, regulating the olfactory-joint neural circuit, promoting cartilage regeneration, or slowing the progression of osteoarthritis.

[0034] Furthermore, the joint injury includes one or more of the following: articular cartilage defects, osteoarthritis, post-traumatic joint injury, and degenerative joint injury; preferably, it is articular cartilage defects induced by microfracture surgery or osteoarthritis induced by anteromedial meniscus detachment.

[0035] Furthermore, the artificially designed protein receptor is a protein receptor that can activate or inhibit neuronal activity under the activation effect of the designed drug; preferably, the artificially designed protein receptor is hM3Dq or hM4Di.

[0036] Furthermore, the designed drug includes one or more of artificially designed G protein-coupled receptor-specific ligands and chemically-based genetic activators; preferably, it is clozapine nitric oxide.

[0037] Furthermore, the drug is administered via one or more of the following methods: drinking water administration, intraperitoneal injection, subcutaneous injection, intravenous injection, and gavage administration; preferably, it is administered via drinking water.

[0038] Furthermore, the dosage of the designed drug is 3-8 mg / kg body weight; preferably, it is 5 mg / kg body weight. Considering that the weight of different experimental subjects (such as mice, rats, rabbits, etc.) varies greatly and the water intake of individuals varies, the volume of administration is not strictly limited. The concentration of the administration solution can be flexibly adjusted according to the weight and daily water intake of the experimental subjects to ensure that the daily intake dose reaches the above range; preferably, it is 5 mg / kg administered through drinking water.

[0039] Furthermore, the drug is administered for 8-20 weeks to mimic the activation or inhibition effects of neural circuits, promoting cartilage regeneration or slowing the progression of osteoarthritis; preferably, for 8 weeks.

[0040] In this invention, activation of neurons in the olfactory bulb mitral cell layer and glutamatergic neurons in the piriform cortex leads to cartilage regeneration exhibiting at least one of the following effects:

[0041] Increased thickness of articular cartilage;

[0042] The degree of cartilage damage has been alleviated;

[0043] Cartilage catabolism is reduced (e.g., decreased expression of catabolism markers COL1 and MMP13);

[0044] Increased cartilage anabolic metabolism (e.g., elevated expression of anabolic markers ACAN and COL2);

[0045] The regenerated cartilage has a hardness close to that of natural hyaline cartilage.

[0046] The present invention also proposes a drug / drug composition comprising a virus, which needs to be used in combination with the designed drug to achieve the beneficial technical effects of the present invention—that is, by means of virus-mediated expression of chemical genetic material in target neurons, and then by means of designed drug to activate artificial protein receptors, the olfactory neural circuit is regulated, thereby promoting cartilage regeneration and slowing the progression of osteoarthritis.

[0047] Furthermore, the virus includes one or more of adeno-associated virus (AAV) or lentivirus; preferably, the virus is an AAV.

[0048] In one specific embodiment, the drug / drug composition includes one or more of AAV-hsyn-hM3Dq-EGFP or AAV-hsyn-hM4Di-EGFP; all of the above viruses can exert a pharmaceutical effect, achieving the beneficial technical effects of the present invention: wherein AAV-hsyn-EGFP serves as a control virus to exclude the influence of the virus itself on neuronal activity and cartilage regeneration; AAV-hsyn-hM3Dq-EGFP is used to mediate the expression of the activating receptor hM3Dq, thereby activating the target neuron; AAV-hsyn-hM4Di-EGFP is used to mediate the expression of the inhibitory receptor hM4Di, thereby inhibiting the target neuron; while the above viral vectors are not the first of their kind proposed in this invention, their use in mediating the regulation of the olfactory neural circuit, and subsequently in cartilage regeneration and osteoarthritis treatment, is an innovative application proposed for the first time in this invention.

[0049] Preferably, in the above-mentioned drug / drug composition, by designing the drug to activate hM3Dq or hM4Di, and activating or inhibiting the neuronal activity of the olfactory bulb mitral cell layer, joint injury can be treated and / or prevented and / or alleviated and / or improved.

[0050] Preferably, in the above-mentioned drug / drug composition, by designing the drug to activate hM3Dq or hM4Di, the activity of piriform cortex glutamatergic neurons can be activated or inhibited, thereby treating and / or preventing and / or alleviating and / or improving joint damage.

[0051] Furthermore, the drug / drug composition further comprises a designed drug, which includes one or more of artificially designed G protein-coupled receptor-specific ligands, chemically-genetically-specific activators, etc.; preferably, it is clozapine nitric oxide.

[0052] Furthermore, the administration method of the drug / drug composition includes one or more of the following: injection, water administration, intraperitoneal injection, subcutaneous injection, intravenous injection, and gavage administration; preferably, the virus is administered via stereotactic injection into the brain or intra-articular injection, and the drug is administered via water administration.

[0053] Furthermore, the dosage of the drug in the drug / drug composition is designed to be 3-8 mg / kg body weight; preferably, it is 5 mg / kg body weight / day administered via drinking water.

[0054] Furthermore, the administration period of the drug / drug composition is 8-20 weeks; preferably, it is 8 weeks.

[0055] Furthermore, the drug / drug composition also includes a pharmaceutically acceptable carrier, etc.

[0056] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0057] Specifically, the drug / drug composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0058] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0059] Specifically, the drug / drug composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0060] Specifically, the drug / drug composition can be formulated into injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field.

[0061] Specifically, the drug / drug composition can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, via injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it can be introduced into the body after being mixed with or encapsulated by other substances. Preferably, it is administered by injection. The drug / drug composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0062] The dosage level of the drug / drug composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments thereof. The selected dosage level and regimen are rationally adjusted based on various factors including the activity and stability (i.e., half-life) of the cellular drug / drug composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0063] The therapeutically effective dose of the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0064] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0065] Furthermore, the drug / drug composition can be used alone and / or in combination with other drugs.

[0066] The other drugs include at least one of the following: organic diphosphates, chemotherapeutic agents, radiopharmaceuticals, TNF-antagonists, nonsteroidal anti-inflammatory drugs, steroids, antioxidants, angiogenesis inhibitors, matrix metalloproteinase inhibitors, vitamins, selective estrogen receptor modulators, estrogen-progesterone, androgens, calcitonin, antibiotics, cathepsin K inhibitors, inhibin, integrin receptor antagonists, osteoblast anabolic agents, selective serotonin reuptake inhibitors, glucosamine, and hyaluronic acid.

[0067] The present invention also provides the application of the methods and drugs / drug compositions described above in the preparation of drugs for treating and / or preventing and / or alleviating and / or improving joint damage, cartilage regeneration or alleviating osteoarthritis, etc.; in addition, it can also be applied to the preparation of scientific research reagents and experimental tools related to the regulation of olfactory nerve circuits, as well as the development of related products for the study of cartilage regeneration mechanisms.

[0068] The present invention also provides the application of the methods and drugs / drug compositions described above in the treatment and / or prevention and / or relief and / or improvement of joint damage, cartilage regeneration or slowing down osteoarthritis, etc.; in addition, it can also be applied to scientific research on the interaction mechanism between olfactory nerve circuits and joints.

[0069] This invention also provides a method for treating and / or preventing and / or alleviating and / or improving joint damage, promoting cartilage regeneration, or slowing the progression of osteoarthritis. This method employs the chemogenetic modulation of the olfactory nerve circuit as described above, or administers the drug / drug composition as described above. The joint damage includes articular cartilage defects, osteoarthritis, post-traumatic joint injury, degenerative joint injury, etc., and is particularly suitable for articular cartilage defects induced by microfracture surgery and osteoarthritis induced by anteromedial meniscus detachment.

[0070] This invention also proposes a cartilage regeneration and osteoarthritis treatment system based on olfactory nerve circuit regulation. This system includes the methods described above for analyzing the connection between the olfactory nerve circuit and the joint, the methods for chemically and genetically regulating the olfactory nerve circuit, and the drugs / drug compositions described above. It can achieve full-chain coverage from circuit analysis and mechanism verification to therapeutic application, providing a complete technical solution for the treatment of osteoarthritis.

[0071] In this invention, the cartilage regeneration exhibits at least one of the following effects:

[0072] Increased thickness of articular cartilage;

[0073] The degree of cartilage damage has been alleviated;

[0074] Cartilage catabolism decreases;

[0075] Increased cartilage synthesis metabolism;

[0076] The regenerated cartilage has a hardness close to that of natural hyaline cartilage.

[0077] The contents described in this invention fully demonstrate the unique improvements of the method. Its unique innovative concept, the technical difficulties overcome, and the breakthroughs achieved are as follows: 1. It overcomes the technical difficulties in existing technologies, such as the inability to analyze the neural connections between the brain and joints and the difficulty in using the nervous system to regulate cartilage regeneration. It reveals for the first time the bidirectional neuroanatomical connections and functional regulation relationship between the olfactory system and joints; 2. It innovatively combines chemogenetics with neural circuit tracing technology to construct a technical system of "circuit analysis-precise regulation-therapeutic application," achieving specific regulation of the olfactory neural circuit, thereby promoting cartilage regeneration and slowing the progression of osteoarthritis; 3. It provides a non-invasive osteoarthritis treatment strategy that is easily clinically translated, offering advantages such as ease of operation, fewer side effects, better repair effects, and long-term intervention compared to existing treatment methods; 4. It clarifies the key roles of neurons in the olfactory bulb mitral cell layer and glutamatergic neurons in the piriform cortex in cartilage regeneration, providing new specific targets for osteoarthritis treatment.

[0078] The innovative reagent applications and experimental methods involved in this invention are supplemented as follows: 1. An innovative combination of PRV, HSV, and AAV viruses is used for tracing the olfactory neural circuit and joint connections, clarifying key parameters such as the specific injection site, injection volume, and sampling time, thus improving the accuracy and reliability of tracing; 2. A standardized experimental method for chemically and genetically regulating the olfactory neural circuit has been established, including optimization of parameters such as virus injection coordinates, administration method, dosage, and administration time, which can stably achieve the activation or inhibition of target neurons; 3. Two standardized animal models (microfracture surgery-induced articular cartilage defect model and anteromedial meniscus detachment-induced osteoarthritis model) have been constructed, and corresponding histological staining and scoring methods have been established, providing a reliable experimental basis for the verification of the technical solutions; 4. Evaluation indicators for cartilage regeneration have been clarified, including macroscopic morphology, histological scoring, expression of synthetic / catabolistic biomarkers, and cartilage stiffness, achieving multi-dimensional and precise evaluation of cartilage regeneration effects.

[0079] Compared with the prior art, the beneficial effects of the present invention include:

[0080] A comprehensive analysis of the structural connections between the olfactory neural circuit and joints: Multiple neural tracing techniques revealed neural connections between several brain regions and joints, particularly key centers of the olfactory neural circuit such as the olfactory bulb (OB), piriform cortex (PIR), and locus coeruleus (LC), which exhibit multi-level synaptic connections with joints. These results indicate a bidirectional neuroanatomical connection between key brain regions of the olfactory system (including the olfactory bulb, piriform cortex, and locus coeruleus) and the knee joint, suggesting that the olfactory system may participate in the physiological or pathological regulation of joints through specific neural pathways. Clarification of the functional regulation of cartilage regeneration by the olfactory neural circuit: Based on the above results, further chemogenetic methods were used to activate neurons. Activation of neurons in the olfactory bulb effectively promoted articular cartilage regeneration, and specific activation of mitral cells in the olfactory bulb also effectively promoted cartilage damage repair. The piriform cortex plays a crucial role in the olfactory neural circuit by synaptarily connecting multiple olfactory-related brain regions. Viral tracing results showed that piriform cortex glutamatergic neurons are connected to joints, and chemogenetic activation of piriform cortex glutamatergic neurons can effectively promote articular cartilage regeneration and slow the progression of surgery-induced osteoarthritis. This is consistent with the aforementioned structural connections between the olfactory neural circuit and the joint, jointly verifying the function of olfactory neural circuit activation in regulating cartilage regeneration. Attached Figure Description

[0081] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0082] Figure 1 This diagram illustrates intra-articular PRV injection and PRV retrograde flow. The structural diagram shows the distribution of PRV-EGFP-labeled neurons in different regions, with locations referenced to the anterior fontanelle. The left side shows a standard brain atlas structure, with green areas representing the locations of PRV-infected brain regions. The right side shows the positive signal data of brain slices corresponding to the brain regions shown in the diagram; green signals represent neurons infected with PRV-EGFP. Scale bar = 1 mm.

[0083] Figure 2 This image shows brain scans of HSV-EGFP-positive and PRV-mRFP-positive regions. Green fluorescence represents HSV-positive neurons, red fluorescence represents PRV-positive neurons, and white arrows indicate double-positive neurons. The scans also include the olfactory bulb (OB), zona indeterminate (ZI), paraventricular nucleus of the hypothalamus (PVN), lateral hypothalamus (LHA), piriform cortex (PIR), central aqueduct (PAG), locus coeruleus (LC), and inferior olive complex (IOC).

[0084] Figure 3Results of chemogenetic activation of the olfactory bulb mitral cell layer to promote articular cartilage regeneration: A: Schematic diagram of the chemogenetic activation experiment of the olfactory bulb mitral cell layer. B: Representative image of the femur of MF mice, with red circles representing the injury sites. C: Verification of AAV-hsyn-hM3Dq-EGFP injection sites, with green signals representing post-infection neurons, scale bar = 200 μm. D: Representative image of Safranin-Fix-Green staining of the mouse knee joint after chemogenetic activation of the mitral cell layer, scale bar = 100 μm. E: Statistical analysis of Safranin-Fix-Green scores of the mouse knee joint, data represent mean ± SD. p<0.001, Student's t-test was used. FG: Representative image of immunofluorescence staining results of mouse knee joint after chemogenetic activation of the mitral cell layer, blue: DAPI, red: ACAN or COL2, scale bar = 100μm.

[0085] Figure 4 Results of chemogenetic activation of glutamatergic neurons in the piriform cortex to promote articular cartilage regeneration: A: Representative images of femur and Safranin-Fix-Green staining in MF mice, scale bar = 100 μm. B: Safranin-Fix-Green score statistics for mouse knee joints, data represent mean ± SD, ns indicates no difference. p<0.001, one-way ANOVA test was used. C: Representative image of immunofluorescence staining results of mouse knee joint after chemically activated piriform cortex glutamatergic neurons, blue: DAPI, red: ACAN or COL2, scale bar = 100 μm.

[0086] Figure 5 Results of chemogenetic activation of glutamatergic neurons in the piriform cortex to slow the progression of osteoarthritis: A: Representative images of Safranin-Fix-Green staining of bone and joint in DMM mice, scale bar = 100 μm. B: Statistical analysis of Safranin-Fix-Green scores in mouse knee joints, data represent mean ± SD, ns indicates no difference. p<0.001, one-way ANOVA test was used. C: Representative images of immunofluorescence and immunohistochemical staining results of mouse knee joint after chemogenetic activation of piriform cortex glutamatergic neurons. Blue: DAPI, red: ACAN or COL2, yellow: COL1 or MMP13, scale bar = 100 μm. Detailed Implementation

[0087] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0088] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0089] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0090] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents. Unless otherwise specified, the experimental methods in the following examples are all conventional methods.

[0091] Example 1: Virus Tracing Strategy Analysis of Neural Circuit Structures in the Olfactory System and Joints

[0092] 1. Stereoscopic injection of viral strain into the brain

[0093] (1) First, the experimental mice were anesthetized by intraperitoneal injection of aphthylamine. After the mice were anesthetized, the scalp was cut along the midline to expose the skull, which was then disinfected with alcohol.

[0094] (2) Subsequently, the anesthetized mouse was placed steadily on the special adapter of the stereotaxic instrument and fixed with the ear rod to ensure that its head remained fixed.

[0095] (3) To ensure precise positioning, locate the anterior and posterior fontanelles on the mouse skull, adjust the skull to a horizontal position, and determine the zero point. After determining the zero point, determine the injection location based on the coordinates of the target brain region. The coordinates of the olfactory bulb mitral cell layer are AP 4.25mm, ML -0.25mm, and DV 1.75mm.

[0096] (4) At the marked location, use a craniotomy drill to make a small hole to expose the dura mater. If bleeding occurs, stop the bleeding immediately.

[0097] (5) Take the required HSV virus out of the refrigerator, pick it up with a glass electrode, and fix it on the stereotaxic instrument. By adjusting the position of the cantilever of the stereotaxic instrument, slowly move the glass electrode containing the HSV virus to the target brain region, slowly lower it until it just touches the surface of the skull, and then return the Z-axis of the stereotaxic instrument to zero.

[0098] (6) By re-determining the zero point and coordinates, the electrode is moved above the target brain region and slowly inserted into the brain tissue to the predetermined depth. 100 nl of HSV virus is injected at an injection rate of 50 nl / min.

[0099] (7) After injection, let the mouse stand for 10 minutes, then remove the electrode, suture the mouse scalp and place it on a heating plate to wake it up.

[0100] 2. Intra-articular injection

[0101] (1) The mice were anesthetized using a gas anesthesia device. After anesthesia, the skin on the surface of the joints was cut open.

[0102] (2) After exposing the joint muscles, use a microsyringe to puncture the joint cavity below the synovium of the mouse.

[0103] (3) Take 3 μl of PRV virus and inject it slowly, leaving it in place for 2 min.

[0104] (4) After the virus injection is completed, slowly withdraw the micro-syringe and suture the skin.

[0105] 3. Frozen slices

[0106] (1) After the experimental period ended, the mice were euthanized by dislocation of the neck, and their brain tissue and joints were collected.

[0107] (2) After cutting open the scalp, slowly peel back the mouse skull along the midline. After exposing the brain tissue, carefully separate the brain tissue, ensuring its integrity. The procedure for collecting joint tissue samples is the same as above.

[0108] (3) Mouse brain tissue and joints were fixed in 4% PFA.

[0109] (4) After 6 hours, the mouse brain tissue was immersed in a 30% sucrose solution. After 48 hours, when the brain tissue had completely sunk to the bottom of the sucrose solution, it was embedded using OCT and then stored in a -80°C freezer.

[0110] (5) After the joint tissue is decalcified, it is dehydrated using sucrose solution and then embedded in OCT.

[0111] (6) The brain tissue was cut into 40 μm sections and the joint tissue was cut into 10 μm sections, and stored in a -20℃ refrigerator.

[0112] Pseudorabies virus (PRV), as a retrograde transsynaptic virus, has been widely used in peripheral-to-central neural circuit tracing studies. After infecting neurons, the virus replicates and is expressed intracellularly. Upon transport to the synapse, it retrogradely crosses the synapse to enter the next higher-level neuron and begins a new replication and transsynaptic process, enabling tracing across multiple neuronal levels. To investigate neural connections between joints and the brain, PRV-EGFP was injected into the joint cavity of MF-induced model mice. Five days after viral injection, brain tissue was collected after cardiac perfusion for whole-brain sectioning.

[0113] Experimental results showed that multiple brain regions exhibited significant green PRV positive signals. The positive signals were located using a standard mouse brain atlas (slice coordinates referenced the anterior fontanelle). These regions included the olfactory bulb (OB), agranular insular cortex (IP), piroid cortex (PIR), anterior part of the basolateral amygdaloid nucleus (BLA), central amygdala (CeA), paraventricular nucleus of hypothalamus (PVN), posterior part of the basolateral amygdaloid nucleus (BLP), hippocampus (Hippo), entorhinal cortex (ENT), central aqueductal gray (PAG), and locus coeruleus (LC). These results indicate the existence of neural connections between the central nervous system and joints, with a large number of PRV-positive neurons present in olfactory-related brain regions (OB, PIR, ENT, Hippo, and LC). Figure 1 ).

[0114] Herpes simplex virus (HSV) possesses specific anterograde transsynaptic properties, enabling efficient and stable anterograde transsynaptic labeling of neural networks. To further elucidate the connection between the olfactory neural network and the joint, PRV-mRFP virus was injected into the joint cavity, followed by HSV-EGFP virus injected into the olfactory bulb MCL (monk's lamina layer, the first-order structure of the olfactory neural circuit). Seven days later, brain and joint tissues were collected from mice after cardiac perfusion and frozen sectioned. HSV-EGFP infection of brain regions downstream of olfaction enables labeling of the olfactory neural circuit, while PRV-mRFP infection of brain regions with neural connections to the joint. If HSV-EGFP and PRV-mRFP co-localize, the brain region is both part of the olfactory neural network and innervates the joint. Fluorescence co-labeling experiments showed that HSV-EGFP and PRV-mRFP expressed positive signals in multiple identical brain regions with significant signal overlap, involving the olfactory bulb (OB), zona incerta (ZI), paraventricular nucleus of hypothalamus (PVN), lateral hypothalamus (LHA), piriform cortex (PIR), central aqueduct (PAG), locus coeruleus (LC), and inferior olivary complex (IOC). These results are consistent with previous experimental findings, further indicating the existence of direct or indirect multi-level neural connections between the central nervous system and joints. Core brain regions of the olfactory neural circuit, such as OB, PIR, and LC, may regulate joint function through connections between these brain regions and joints (see appendix). Figure 2 Furthermore, this finding provides direct neural circuit evidence for inhaled aromatherapy's regulation of cartilage regeneration through the olfactory neural circuit, further supporting the potential of aromatherapy for cartilage regeneration.

[0115] Example 2 uses chemogenetic methods to elucidate the neural circuit function of the olfactory system in regulating joint regeneration.

[0116] 1. Chemical genetic virus injection

[0117] (1) After anesthetizing the mice, adjust the skull to a horizontal position and determine the zero point. Bilateral chemogenetic virus injection was performed according to the following coordinates: olfactory bulb mitral cell layer (AP 4.25mm, ML ±0.25mm, DV 1.75mm), piriform cortex (AP -1.07mm, ML ±3.40mm, DV 5.10mm).

[0118] (2) Inject 200 nl of chemical genetic virus (AAV-hsyn-EGFP, AAV-hsyn-hM3Dq-EGFP or AAV-hsyn-hM4Di-EGFP) at each site at a rate of 50 nl / min.

[0119] (3) Ten minutes after the injection, slowly remove the glass electrode and suture the mouse scalp.

[0120] 2. CNO administration

[0121] (1) Two weeks after the virus was stably expressed, the body weight of the mice was taken and CNO solution was prepared with physiological saline at a concentration of 5 mg / kg.

[0122] (2) To ensure CNO activity, frozen CNO solution was added to the mice’s drinking water daily for eight weeks.

[0123] 3. Prepare laboratory animals

[0124] Animal model of cartilage injury (Microfracture, MF)

[0125] (1) Eight-week-old C57BL / 6J mice were selected and anesthetized by intraperitoneal injection of 300 μl of aphthylamine.

[0126] (2) After ensuring that the mouse is under general anesthesia, use sterile scissors to cut open the skin of the knee joint, and then use a sterile scalpel blade to cut open the muscle and remove the muscle and patella to expose the femur.

[0127] (3) A hole was drilled in the center of the femoral cartilage using a polished 26G puncture needle to create a cartilage defect with a depth of 1 mm and a diameter of 0.5 mm.

[0128] (4) Postoperatively, the patella and muscles were repositioned and sutured. The mouse was placed on a heated plate at a constant 37°C to promote recovery. After recovery, the mouse was returned to its original cage.

[0129] (5) The sham surgery group only cut the skin after anesthesia, without performing cartilage damage surgery, and then sutured it.

[0130] (6) Eight weeks after the operation, the knee joints of the mice were collected for photography and section staining.

[0131] Example 3: Preparation of an animal model of osteoarthritis (Destabilization of the Medial Meniscus, DMM)

[0132] (1) Eight-week-old C57BL / 6J mice were selected and anesthetized by intraperitoneal injection of 300 μl of aphthylamine.

[0133] (2) After ensuring that the mouse is under general anesthesia, use sterile scissors to cut open the skin of the knee joint, and then use a sterile scalpel blade to cut open the muscle and remove the muscle and patella to expose the joint cavity.

[0134] (3) Use a scalpel blade to cut the medial collateral ligament of the meniscus and peel off the surrounding tissue, so that the meniscus is in a free state and returns to its original position.

[0135] (4) Postoperatively, the patella and muscles were repositioned and sutured. The mouse was placed on a heated plate at a constant 37°C to promote recovery. After recovery, the mouse was returned to its original cage.

[0136] (5) In the sham surgery group, the skin was only cut after anesthesia, without medial meniscus dissection, and then sutured.

[0137] (6) Eight weeks after the operation, the knee joints of the mice were collected for section staining.

[0138] Example 4 Histological staining scoring

[0139] 1. Safranin-Fixed Green Staining

[0140] (1) Select slices containing damaged areas and place them on a baking machine at 65°C for 1 hour.

[0141] (2) After baking, perform gradient dewaxing. Soak the slices in xylene I for 5 minutes, xylene II for 5 minutes, xylene: anhydrous ethanol (1:1) for 5 minutes, then dewax them using gradient ethanol (soaking in 100%, 95%, 85%, and 75% ethanol for 5 minutes each), and finally rinse with ddH2O for 5 minutes to completely remove wax and other residues.

[0142] (3) Preheat 0.1% safranin solution and 0.05% fast green solution to 37°C. Then place the dewaxed sections in the safranin staining vat for 15 minutes and rinse with tap water 2-3 times. Next, place the sections in the fast green staining vat for 15 minutes and rinse with tap water 2-3 times to ensure uniform and clear staining.

[0143] (4) Use 1% glacial acetic acid to separate the colors for 8 seconds, then rinse with tap water to ensure even color separation.

[0144] (5) After staining, the sections were subjected to gradient dehydration. The sections were soaked in 95% ethanol, anhydrous ethanol, and xylene-anhydrous ethanol (1:1) for 2 minutes, followed by xylene II and xylene I for 5 minutes.

[0145] (6) After dehydration, use neutral resin to seal the film, air dry the residual xylene in a fume hood and take a picture.

[0146] 2. Immunofluorescence staining

[0147] (1) The baking and dewaxing steps are the same as above.

[0148] (2) Use an immunohistochemical pen to draw circles around the dewaxed tissue section to ensure that no liquid is lost.

[0149] (3) Add 30 μl of proteinase K repair solution to each tissue, incubate at 37°C for 30 minutes, and wash three times with PBS.

[0150] (4) Block with 5% goat serum for 1 hour.

[0151] (5) After sealing, use a suction pump to remove the sealing solution and add the corresponding primary antibody, and incubate overnight at 4°C.

[0152] (6) After the primary antibody is recovered, the slides are washed three times with PBS, and then incubated with the corresponding secondary antibody and DAPI for 1 hour.

[0153] (7) After incubation with the secondary antibody, wash three times with PBS and mount with an anti-fluorescence quencher.

[0154] 3. Immunohistochemical staining

[0155] (1) The baking and dewaxing steps are the same as above.

[0156] (2) Use an immunohistochemical pen to draw circles around the dewaxed tissue section to ensure that no liquid is lost.

[0157] (3) Add 30 μl of proteinase K repair solution to each tissue, incubate at 37°C for 30 minutes, and wash three times with PBS.

[0158] (4) Use 3% hydrogen peroxide to block endogenous peroxidase, treat in the dark for 10 minutes, and wash three times with PBS.

[0159] (5) After blocking with 5% goat serum, add the corresponding primary antibody and incubate overnight at 4°C.

[0160] (6) After the primary antibody is recovered, the slides are washed three times with PBS and incubated with the corresponding immunohistochemical secondary antibody for 1 hour.

[0161] (7) After incubation with the secondary antibody, wash three times with PBS, incubate with SABC for 30 minutes, and wash three times with PBS.

[0162] (8) Prepare the DAB colorimetric solution according to the steps of the kit. Prepare it fresh for use and perform DAB colorimetric reaction.

[0163] (9) After the tissue shows a yellow signal, use PBS to block the color development reaction and record the time to ensure that the color development time of each tissue with the same antibody is consistent.

[0164] (10) After the staining is completed, hematoxylin is used to stain the cell nuclei and differentiate them in reverse blue.

[0165] (11) After the inversion is completed, the slides are dehydrated, sealed and photographed, following the same steps as above.

[0166] Histological scoring

[0167] The joint scoring of mice after MF (Multiple Molecular Joint) was based on the scoring principles of the International Society for Cartilage Repair (ICRS).

[0168]

[0169] The DMM post-articular scoring in mice was based on the scoring principles of the Osteoarthritis Research Institute International (OARSI).

[0170]

[0171] In all animal experiments, mice were randomly assigned to eliminate error. All data were statistically processed and plotted using GraphPad Prism 10 software, and are presented as mean ± standard deviation (Mean ± SD). Student's t-test was used to compare statistical significance between two independent groups. One-way ANOVA was used for comparative analyses of multiple samples. Two-way ANOVA was used for comparative analyses of multiple groups. The significance level was set at 1 / 3 for all data analyses. p<0.05, p<0.01, p<0.001.

[0172] The olfactory bulb is the first-level center for transmitting olfactory information to the brain, and mitral cells are the most important neurons in the olfactory bulb that output neural signals. To further explore the role of the olfactory neural circuit in regulating cartilage regeneration, this invention used chemogenetics to simulate the activation of olfactory bulb neurons induced by essential oil stimulation in MF mice. Specifically, AAV-hsyn-hM3Dq-EGFP was injected into the mitral cell layer of the olfactory bulb, followed by administration of CNO to achieve neuronal activation for eight weeks. The control group was injected with AAV-hsyn-EGFP. From the macroscopic results, compared with the control virus, mice injected with the chemically activated virus showed significant filling at the cartilage defect site, level with the surrounding normal cartilage, indicating a better cartilage damage repair effect. In addition, the slicing verification of the injection site showed that the AAV chemogenetic virus effectively infected neurons in the olfactory bulb. Safranin-Fix-Green staining and immunofluorescence staining results showed that, compared with the MF group, activation of neurons in the mitral cell layer of the olfactory bulb effectively promoted cartilage damage repair in mice, with a significant increase in scores and a large number of ACAN and COL2 positive signals at the damaged site. These results indicate that the olfactory bulb capillary cell layer plays an important role in regulating cartilage regeneration (see appendix). Figure 3 ).

[0173] To further understand the effect of piriform cortex glutamatergic neurons on cartilage regeneration, this invention conducted a histological evaluation of cartilage repair in MF-induced model mice. Regarding the filling of the damaged area, chemogenetic activation of piriform cortex glutamatergic neurons significantly promoted cartilage repair, almost resembling natural cartilage, while the control and inhibition groups showed obvious cavities. Similarly, Safranin-Fix-Green staining results indicated that activation of piriform cortex glutamatergic neurons resulted in more cartilage matrix at the damaged area, and a better osteochondral structure was formed compared to the control and inhibition groups. In terms of scoring, activation of piriform cortex glutamatergic neurons significantly increased the cartilage repair score in mice, while there was no difference between the control and inhibition groups. Furthermore, the activated group showed more ACAN and COL2 positive signals at the damaged area, indicating that the repaired cartilage tissue was close to hyaline cartilage, further demonstrating that activation of piriform cortex glutamatergic neurons is highly effective in promoting articular cartilage regeneration (see appendix). Figure 4 ).

[0174] Safranin-Fix-Green staining results in osteoarthritis-inducing mice showed that, compared to the control and inhibition groups, the progression of osteoarthritis was significantly slowed after chemogenetic activation of piriform cortex glutamatergic neurons, with cartilage tissue remaining at the joints. OARSI scores indicated that mice injected with the control or inhibitory virus after DMM had higher osteoarthritis scores, while scores significantly decreased eight weeks after chemogenetic activation of glutamatergic neurons, suggesting that activation of piriform cortex glutamatergic neurons can significantly slow the progression of osteoarthritis in mice. Furthermore, cartilage anabolic and catabolistic marker staining results showed abundant positive signals for COL2 and ACAN in the cartilage surface of the activation group, effectively inhibiting the expression of catabolism markers (COL1 and MMP13). These results indicate that activation of piriform cortex glutamatergic neurons can maintain the homeostasis of anabolic and catabolistic metabolism in the joints, slowing the progression of osteoarthritis in mice (see appendix). Figure 5 ).

[0175] In summary, this invention, through chemogenetic methods, reveals the regulatory role of olfactory bulb mitral cells and piriform cortex glutamatergic neurons in cartilage regeneration within the olfactory neural circuit. The primary olfactory center (olfactory bulb mitral cells) and the higher olfactory center (piriform cortex) form a functional circuit, and activation of this circuit may promote cartilage regeneration by modulating the local joint microenvironment. This section provides new evidence for the interaction between the nervous and joint systems, suggesting the potential application value of olfactory stimulation and neural modulation strategies in the treatment of osteoarthritis.

[0176] In summary, this invention, through multi-faceted exploration, systematically elucidates the structural and functional connections between the olfactory neural network and joints, providing important evidence for understanding central regulatory mechanisms and developing new therapeutic targets. These findings are significant for understanding the role of the nervous system in the pathogenesis of cartilage-related diseases and lay the foundation for developing non-pharmacological intervention strategies based on neural circuit regulation.

[0177] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0178] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0179] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0180] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. A method for analyzing the connection between olfactory nerve circuits and joints, characterized in that, The method includes the following steps: Injecting pseudorabies virus (PRV) or rabies virus (RV) across multiple levels into the joint cavity to mark the neural connections from the joint to the brain. Injecting multilevel anterograde herpes simplex virus (HSV) or adeno-associated virus (AAV) across the olfactory epithelium or mitral cell layer of the olfactory bulb marks the neural connections from the olfactory nerve circuit to the joints. The marked brain tissue and joints were fixed and made into frozen sections to verify the neural synaptic connections between specific brain regions and the joints, and to analyze the neural connections between specific brain regions and the joints.

2. The method according to claim 1, characterized in that, The PRV carries a red or green fluorescent label; and / or, the HSV carries a green fluorescent label; and / or, the injection volumes of the PRV and HSV are 1-10 μL and 100-500 nanoL, respectively; and / or, the specific brain regions include the olfactory bulb, agranular insular cortex, piriform cortex, hippocampus, entorhinal cortex, central aqueduct, locus coeruleus, zona indeterminate, and hypothalamus; and / or, the virus is administered by injection; the PRV is injected intra-articularly. And / or, the fixation includes: fixing in a preservative or protein fixative; and / or, the preparation of the frozen sections includes: preparing frozen sections after being treated with sucrose solution and OCT embedding; and / or, the verification method includes a fluorescent staining method.

3. The method according to claim 2, characterized in that, The neurons in the olfactory bulb mitral cell layer and the glutamatergic neurons in the piriform cortex form direct or indirect neural connections with the joints through viral tracing.

4. A method for chemically genetically regulating olfactory neural circuits, characterized in that, Includes the following steps: Injecting adeno-associated virus carrying the viral receptor hM3Dq or hM4Di, which carries a chemical genetic gene, into the olfactory bulb mitral cell layer and / or piriform cortex to express an artificially designed protein receptor specifically activated by a designed drug; the artificially designed protein receptor is hM3Dq or hM4Di. The designed drug activates the artificially designed protein receptor, thereby activating or inhibiting neuronal activity and regulating the olfactory-joint neural circuit. CNO continuously activates or inhibits the target neurons for 8-20 weeks to simulate the activation or inhibition effect of the neural circuit.

5. The method as described in claim 4, characterized in that, The designed drug includes one or more of the following: artificially designed G protein-coupled receptor-specific ligands, chemically-genetically-specific activators, etc.

6. A drug / drug composition, characterized in that, It contains viruses, AAV-hsyn-EGFP, AAV-hsyn-hM3Dq-EGFP, or AAV-hsyn-hM4Di-EGFP.

7. The pharmaceutical / pharmaceutical composition according to claim 6, characterized in that, The virus is an adeno-associated virus or a lentivirus; and / or, the drug / drug composition is designed to activate hM3Dq or hM4Di, thereby activating or inhibiting neuronal activity in the olfactory bulb mitral cell layer, to treat and / or prevent and / or alleviate and / or improve joint injury; or is designed to activate hM3Dq or hM4Di, thereby activating or inhibiting glutamatergic neuronal activity in the piriform cortex, to treat and / or prevent and / or alleviate and / or improve joint injury.

8. The pharmaceutical / pharmaceutical composition according to claim 7, characterized in that, The designed drug includes one or more of artificially designed G protein-coupled receptor-specific ligands and chemically-based genetic activators; and / or, the administration method of the designed drug includes one or more of injection, water administration, intraperitoneal injection, subcutaneous injection, intravenous injection, and gavage administration; and / or, the administration period of the designed drug is 8-20 weeks; and / or, the dosage of the designed drug is 3-8 mg / kg body weight.

9. The use of the drug / drug composition according to any one of claims 6-8 in the preparation of a drug for treating and / or preventing and / or alleviating and / or improving joint damage, or promoting cartilage regeneration or alleviating osteoarthritis; or in the preparation of research reagents and experimental tools related to the regulation of olfactory nerve circuits, and in the development of related products for the study of cartilage regeneration mechanisms.

10. The application as described in claim 9, characterized in that, The joint injuries include articular cartilage defects, osteoarthritis, post-traumatic joint injuries, and degenerative joint injuries.

11. A method for promoting cartilage regeneration by chemically and genetically regulating the olfactory nerve circuit, characterized in that, The method includes the following steps: Injecting adeno-associated viruses carrying chemogenetic genes into the olfactory bulb mitral cell layer and / or piriform cortex of damaged joints with hM3Dq or hM4Di receptors to express artificially designed protein receptors specifically activated by the designed drugs. The designed drug activates the artificially designed protein receptor, thereby activating or inhibiting neuronal activity, regulating the olfactory-joint neural circuit, promoting cartilage regeneration, or slowing the progression of osteoarthritis.

12. The method as described in claim 11, characterized in that, The joint injury includes one or more of the following: articular cartilage defects, osteoarthritis, post-traumatic joint injury, and degenerative joint injury; and / or, the artificially designed protein receptor is a protein receptor that can activate or inhibit neuronal activity under the activation of the designed drug; and / or, the virus is one of adeno-associated virus or lentivirus; and / or, the designed drug includes one or more of the following: artificially designed G protein-coupled receptor specific ligands and chemical genetic activators; and / or, the administration period is 8-20 weeks; and / or, the administration method of the designed drug is one or more of the following: oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection, and gavage; and / or, the dosage of the designed drug is 3-8 mg / kg body weight; and / or, after activation of neurons in the olfactory bulb mitral cell layer and glutamatergic neurons in the piriform cortex, cartilage regeneration exhibits at least one of the following effects: Increased thickness of articular cartilage; The degree of cartilage damage has been alleviated; Cartilage catabolism decreases; Increased cartilage synthesis metabolism; The regenerated cartilage has a hardness close to that of natural hyaline cartilage.

13. A method for treating and / or preventing and / or alleviating and / or improving joint damage, promoting cartilage regeneration, or slowing the progression of osteoarthritis, characterized in that, The method employs the chemical genetic regulation of the olfactory neural circuit as described in claim 4, or administers the drug / drug composition as described in any one of claims 6-8.

14. A cartilage regeneration and osteoarthritis treatment system based on olfactory nerve circuit regulation, characterized in that, The system includes the method for analyzing olfactory neural circuits and joint connections as described in claim 1, the method for chemically and genetically regulating olfactory neural circuits as described in claim 4, and the drug / drug composition as described in any one of claims 6-8.

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