Method for evaluating food for alleviating bone and joint damage

Abstract

The application discloses a kind of food relieving bone joint damage evaluation method, it is related to food efficacy evaluation technical field, for solving the problem of single dimension of existing evaluation method, unstable model leads to poor reliability of conclusion.The application builds composite modeling and multi-modal analysis mechanism, first, a stable animal model is established by using chemical injury combined with forced exercise training, a two-stage increasing dosing regimen is set, and behavior dynamic trend data and histology, imaging multi-modal results are collected synchronously.Then, based on the weight assigned to osteoarthritis stage, a weighted composite score is generated, and a perturbation sensitivity index is calculated, a multi-index collaborative influence map is constructed, a key evaluation node is located, a graded evaluation conclusion is generated based on the food action characteristics and efficacy determination threshold, and false positives caused by single index fluctuations are avoided.Finally, a full-cycle closed-loop evaluation process is formed, which improves the scientificity, reproducibility and self-adaptive ability of efficacy determination in complex biological systems.

Description

Technical Field

[0001] This invention relates to the field of food efficacy evaluation technology, and more specifically, to an evaluation method for food's ability to alleviate bone and joint injuries. Background Technology

[0002] Currently, in the field of food efficacy evaluation, methods for evaluating the relief effect of bone and joint injuries based on single indicators or static observations have been applied. These methods typically establish animal models through single modeling methods such as chemical induction or mechanical injury, and then measure indicators such as cartilage histological scores or inflammatory factors at fixed time points to determine whether the test substance has a relieving effect. First, existing technical solutions focus on optimizing the accuracy of the end indicators, determining efficacy through standardized operating procedures and statistical hypothesis testing. Second, their technical implementation involves the synergistic application of animal behavior, histopathology, and biostatistics, but often neglect the temporal correlation between indicators and fail to dynamically couple macroscopic functional recovery with microscopic structural repair.

[0003] However, existing technologies have significant limitations in terms of evaluation dimensions. On the one hand, the contradiction between a single end-point indicator and the spontaneous repair of damage in animal models makes evaluation conclusions susceptible to false positives or false negatives, as the model may reconstruct itself during the intervention period, obscuring the intervention effect. On the other hand, the pathological heterogeneity caused by different modeling methods makes it difficult for the model to maintain a stable degree of damage throughout the entire intervention period, and there is a lack of synchronous dynamic monitoring of functional recovery and microstructural repair. More importantly, this single evaluation dimension and model instability prevent the system from forming a comprehensive efficacy assessment across multiple scales and the entire cycle, thereby weakening the scientific rigor and reproducibility of food efficacy evaluation and making it difficult to accurately reflect the true alleviating effect of food on bone and joint damage. Summary of the Invention

[0004] In order to overcome the above-mentioned defects of the prior art, embodiments of the present invention provide an evaluation method for food to alleviate bone and joint injuries to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for evaluating the relief of bone and joint injuries by food, characterized by comprising the following steps: S1. When the test substance information of the food to be evaluated and the evaluation index of bone and joint injury are received, according to the preset food action characteristics, a composite modeling method of sequential chemical damage induction and forced exercise training is adopted to construct a bone and joint injury animal model that can maintain the preset target histological score range and the cartilage full-thickness defect volume fraction measured by micro-CT is less than the preset threshold within the preset intervention period. S2. The animal model of bone and joint injury was divided into a model control group and a drug administration group. The drug administration group was set up with a drug administration regimen that included two administration phases. At the end of each administration phase, each preset evaluation index was measured and recorded. The differences of each preset evaluation index were compared using preset statistical methods. At the same time, the effect quantity evaluation criteria were preset. S3. Before the start of the drug administration phase and throughout the entire intervention process of the first and second drug administration phases, the mechanical pain threshold, cold pain threshold, and gait parameters of the animal model of bone and joint injury are measured according to the preset monitoring cycle to form dynamic trend data. S4. After completing the first and second drug administration phases, cartilage, subchondral bone, synovium, and dorsal root ganglion tissues were simultaneously collected from the animal model of bone and joint injury. Histochemical staining, micro-CT scanning, immunohistochemistry, and immunofluorescence staining were performed to obtain multimodal analysis results covering cartilage histological scores, subchondral bone microstructural parameters, synovial inflammation scores, and dorsal root ganglion neuropeptide immunopositive signal intensity. S5. Based on the results of multimodal analysis, a weighted comprehensive scoring system is established. The single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are uniformly scored according to preset standards. Preset weights are assigned to each indicator according to the preset osteoarthritis stage. The comprehensive score is calculated, and the effective threshold is set when the comprehensive score reaches the preset efficacy judgment threshold and the scores of the single measurement results and dynamic trend data both reach the preset single-item effective threshold. Statistical verification is performed by comparing the differences with the model control group and evaluating the effect quantity to determine whether the effective threshold is met. When the effective threshold is met, the efficacy evaluation conclusion of the food to be evaluated in relieving osteoarthritis is output.

[0006] In a preferred embodiment, upon receiving the test substance information and bone and joint injury evaluation indicators of the food to be evaluated, a composite modeling method involving sequential chemical injury induction and forced exercise training is adopted based on the preset food action characteristics. Specifically, this includes: The system receives test substance information of the food to be evaluated and bone and joint injury evaluation indicators. Based on the preset food action characteristics, it looks up the corresponding chemical damage induction and forced exercise training from the preset mapping relationship table. The mapping relationship table records the correspondence between different food action characteristics and chemical damage induction schemes and forced exercise training. According to the selected chemical damage induction method, the corresponding chemical damage agent is administered to the experimental animals to complete the chemical damage induction operation; After the chemical damage induction procedure is completed, the experimental animals are subjected to forced exercise training using pre-set exercise equipment according to the selected forced exercise training. Forced exercise training and chemical damage induction are carried out in sequence to form a composite modeling method.

[0007] In a preferred embodiment, to construct an animal model of bone and joint injury that maintains a preset target histological score range throughout the entire intervention period and whose full-thickness cartilage defect volume fraction measured by micro-CT is less than a preset threshold, specifically includes: Throughout the pre-set intervention period, histological scoring and micro-CT measurements were performed on the experimental animals to obtain the target histological score and the volume fraction of full-thickness cartilage defects. When the target histological score exceeds the preset target histological score range, or when the volume fraction of the full-thickness cartilage defect reaches the preset threshold, based on the direction and degree of the target histological score exceeding the target histological score range, and the difference between the volume fraction of the full-thickness cartilage defect and the preset threshold, the corresponding level of chemical damage induction or forced exercise training is selected from the preset damage regulation mapping table to construct an animal model of bone and joint injury in which the target histological score is maintained within the preset target histological score range and the volume fraction of the full-thickness cartilage defect is less than the preset threshold within the preset intervention period.

[0008] In a preferred embodiment, the animal model of bone and joint injury is divided into a model control group and a drug administration group. The drug administration group is given a dosing regimen consisting of two administration phases. At the end of each administration phase, pre-defined evaluation indicators are measured and recorded. Pre-defined statistical methods are used to compare the differences among the pre-defined evaluation indicators. Simultaneously, pre-defined effect evaluation criteria are established, specifically including: Animal models of bone and joint injury were divided into a model control group and a drug administration group. The drug administration group was given a dosing regimen that included a first dosing phase and a second dosing phase. In the first administration phase, a preset maintenance dose is administered via gavage for the preset number of days of the first administration phase. In the second administration phase, the drug is administered via gavage, with a preset increment added at preset intervals, for a preset number of days in the second administration phase. Each pre-defined evaluation index was measured and recorded at the end of the two dosing phases. The differences between the preset evaluation indicators are compared using preset statistical methods, and the effect quantity evaluation standards are preset.

[0009] In a preferred embodiment, before the start of the drug administration phase and throughout the intervention process of the first and second drug administration phases, mechanical pain threshold, cold pain threshold, and gait parameters of the bone and joint injury animal model are measured according to a preset monitoring cycle to generate dynamic trend data, specifically including: Before the start of the drug administration phase, the initial mechanical pain threshold of the animal model of bone and joint injury was determined by an electronic pain meter, the initial cold pain threshold of the animal model of bone and joint injury was determined by the cold plate method, and the initial gait parameters of the animal model of bone and joint injury were determined by a gait analysis device. The results of each measurement were recorded in a preset data recording table. During the first administration phase, the mechanical pain threshold, cold pain threshold, and gait parameters of the animal model of bone and joint injury were repeatedly measured using an electronic pain meter, cold plate method, and gait analysis equipment according to the preset monitoring cycle, and the results of each measurement were recorded in the preset data recording table. During the second administration phase, the same monitoring cycle was followed, and the mechanical pain threshold, cold pain threshold and gait parameters of the animal model of bone and joint injury were measured using an electronic pain meter, cold plate method and gait analysis equipment. The results of each measurement were recorded in a preset data recording table. Each measurement result is compared with the initial mechanical pain threshold, initial cold pain threshold, initial gait parameters, and the measurement results of the previous monitoring cycle to form dynamic trend data.

[0010] In a preferred embodiment, after completing the first and second drug administration phases, cartilage, subchondral bone, synovium, and dorsal root ganglion tissues are simultaneously collected from the bone and joint injury animal model. Histochemical staining, micro-CT scanning, immunohistochemistry, and immunofluorescence staining are performed on these tissues to obtain multimodal analysis results covering cartilage histological scores, subchondral bone microstructural parameters, synovial inflammation scores, and dorsal root ganglion neuropeptide immunopositive signal intensity. Specifically, these results include: After completing the first and second administration phases, the animal model of bone and joint injury was euthanized, and the cartilage, subchondral bone, synovium, and lumbar dorsal root ganglion tissue of the knee joint were isolated and collected. The collected cartilage tissue was subjected to histochemical staining, and the morphological changes of the cartilage tissue were observed and recorded under a microscope. The degree of cartilage damage was scored according to the preset histological scoring criteria to obtain the cartilage histological score. The collected subchondral bone tissue was subjected to micro-computed tomography to obtain three-dimensional microstructure images of the subchondral bone. The number of trabeculae, trabeculae thickness, trabeculae separation degree and bone volume fraction parameters were measured and recorded to obtain the microstructure parameters of the subchondral bone. The collected synovial tissue was subjected to immunohistochemical staining. The proliferation status of synovial cells, the degree of inflammatory cell infiltration, and the formation of pannus were observed under a microscope. The degree of synovial inflammation was scored according to the preset synovial inflammation scoring criteria to obtain the synovial inflammation score. The collected dorsal root ganglion tissue was subjected to immunofluorescence staining. The number of neurons with positive neuropeptide immunosuppression and the fluorescence intensity were observed and counted under a fluorescence microscope. The positive signal intensity was evaluated according to the preset positive signal intensity grading standard to obtain the dorsal root ganglion neuropeptide immunosuppression signal intensity. The cartilage histological score, subchondral bone microstructural parameters, synovial inflammation score, and dorsal root ganglion neuropeptide immunopositive signal intensity were combined to construct a multimodal analysis result.

[0011] In a preferred embodiment, a weighted comprehensive scoring system is established based on the results of multimodal analysis. Single measurements, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are uniformly scored according to preset standards. Preset weights are assigned to each indicator based on a preset osteoarthritis stage, and a comprehensive score is calculated. Specifically, this includes: Based on the results of multimodal analysis, a weighted comprehensive scoring system is established, which includes preset weights and comprehensive score calculation rules. Based on the preset osteoarthritis stage, the measured mechanical pain threshold, cold pain threshold, gait parameters, dynamic trend data, and multimodal analysis results are assigned corresponding preset weights. The single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are scored according to preset standards to obtain scores for the single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters. The scores are then combined with preset weights and calculated according to the comprehensive scoring rules to obtain the comprehensive score.

[0012] In a preferred embodiment, an effective threshold is set as follows: the overall score reaches a preset efficacy judgment threshold, and the scores of both the single measurement result and the dynamic trend data reach preset single-item effective thresholds. Statistical verification is performed by comparing the differences with the model control group and evaluating the effect quantity to determine whether the effective threshold is met. When the effective threshold is met, the efficacy evaluation conclusion for the food to be evaluated in alleviating bone and joint damage is output, specifically including: Set effective thresholds, which include the comprehensive score reaching the preset efficacy judgment threshold, and the scores of the single measurement results of mechanical pain threshold, cold pain threshold and gait parameters and the scores of dynamic trend data all reaching the preset single effective threshold. The differences between the drug-treated group and the model control group were compared and the effect size was assessed. The results of the difference comparison and the effect size assessment were statistically validated to determine whether the effective threshold was met simultaneously. If the effective threshold is met, the evaluation conclusion on the efficacy of the food to be evaluated in alleviating bone and joint damage is output.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. By jointly constructing a multimodal weighted comprehensive score and dynamic trend data, this method achieves comprehensive and precise quantification and cross-stage collaborative assessment of the repair effect of osteoarthritis. It is a stable pathological model constructed based on a chemical injury-forced movement composite model, effectively avoiding the problems of high model heterogeneity and interference from spontaneous repair during the intervention period caused by single modeling methods. Combined with the temporal dynamic monitoring of mechanical pain threshold, cold pain threshold, and gait parameters, and through the weighted fusion of dynamic trend data and multimodal histological and imaging parameters, it establishes a multi-scale physical state correlation between macroscopic functional recovery and microscopic pathological changes. Compared with existing technologies, it can identify asynchronous functional and structural recovery characteristics in real time at different stages of osteoarthritis. Through adaptive allocation of preset weights and joint constraints of effective thresholds, it significantly improves the objectivity and reproducibility of evaluation conclusions, suppressing false positives or false negatives caused by fluctuations in a single indicator.

[0014] 2. By employing a synergistic design combining a two-stage incremental dosing regimen with efficacy dose-statistical validation, this approach addresses the shortcomings of existing evaluation methods, such as simplistic dosing regimens, unclear dose-response relationships, and strong subjectivity in efficacy conclusions. It is based on the temporal adaptation of the maintenance dose in the first dosing stage and the progressively incremental dosing in the second dosing stage, allowing for real-time matching of the metabolic kinetic characteristics of the evaluated food at different stages of action. Through dual locking of pre-set statistical methods and efficacy dose assessment criteria, combined with synergistic constraints of inter-group difference comparisons and large effect dose threshold determination, it ensures that efficacy conclusions consistently match the actual efficacy intensity of the test substance and the stability of experimental data, avoiding misjudgments of effectiveness due to insufficient sample size or data fluctuations. While maintaining the scientific rigor of the evaluation method, it significantly reduces the waste of subsequent research resources caused by false positive conclusions, ensuring that the efficacy determination conclusions of the evaluated food in complex biological systems have reliable statistical support and biological significance. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a flowchart of an evaluation method for alleviating bone and joint damage using food according to the present invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Figure 1 A flowchart of an evaluation method for food-induced osteoarthritis relief according to the present invention is provided, which includes the following steps: S1. When the test substance information of the food to be evaluated and the evaluation index of bone and joint injury are received, according to the preset food action characteristics, a composite modeling method of sequential chemical damage induction and forced exercise training is adopted to construct a bone and joint injury animal model that can maintain the preset target histological score range and the cartilage full-thickness defect volume fraction measured by micro-CT is less than the preset threshold within the preset intervention period. S2. The animal model of bone and joint injury was divided into a model control group and a drug administration group. The drug administration group was set up with a drug administration regimen that included two administration phases. At the end of each administration phase, each preset evaluation index was measured and recorded. The differences of each preset evaluation index were compared using preset statistical methods. At the same time, the effect quantity evaluation criteria were preset. S3. Before the start of the drug administration phase and throughout the entire intervention process of the first and second drug administration phases, the mechanical pain threshold, cold pain threshold, and gait parameters of the animal model of bone and joint injury are measured according to the preset monitoring cycle to form dynamic trend data. S4. After completing the first and second drug administration phases, cartilage, subchondral bone, synovium, and dorsal root ganglion tissues were simultaneously collected from the animal model of bone and joint injury. Histochemical staining, micro-CT scanning, immunohistochemistry, and immunofluorescence staining were performed to obtain multimodal analysis results covering cartilage histological scores, subchondral bone microstructural parameters, synovial inflammation scores, and dorsal root ganglion neuropeptide immunopositive signal intensity. S5. Based on the results of multimodal analysis, a weighted comprehensive scoring system is established. The single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are uniformly scored according to preset standards. Preset weights are assigned to each indicator according to the preset osteoarthritis stage. The comprehensive score is calculated, and the effective threshold is set when the comprehensive score reaches the preset efficacy judgment threshold and the scores of the single measurement results and dynamic trend data both reach the preset single-item effective threshold. Statistical verification is performed by comparing the differences with the model control group and evaluating the effect quantity to determine whether the effective threshold is met. When the effective threshold is met, the efficacy evaluation conclusion of the food to be evaluated in relieving osteoarthritis is output.

[0019] In step S1, when the test substance information and bone and joint injury evaluation indicators of the food to be evaluated are received, a composite modeling method of sequentially performing chemical injury induction and forced exercise training is adopted according to the preset food action characteristics. The specific implementation is as follows: First, the test substance information of the food to be evaluated is received, such as the batch number of a freeze-dried plant extract (XX-2023), and the evaluation indicators for bone and joint injury, including OARSI histological score, full-thickness cartilage defect volume fraction, mechanical pain threshold, cold pain threshold, and gait parameters. Based on the pre-defined characteristics of the food's action, including mild onset of action, need for long-term intervention, and chronic regulation, mild onset of action means that the food has a slow onset of action in the body, and usually requires continuous administration for at least 2 weeks to observe preliminary efficacy. Need for long-term intervention means that the food needs continuous administration for at least 4 weeks to achieve stable efficacy. Chronic regulation means that the food mainly acts on the chronic pathological process of bone and joint injury, such as inhibiting cartilage matrix degradation, regulating the balance of inflammatory factors, and promoting cartilage repair, rather than acute anti-inflammatory and analgesic effects. The corresponding chemical injury induction and forced exercise training are then found from the pre-established mapping table.

[0020] This mapping table, established through preliminary experiments, records the correspondence between the effects of different foods and chemical damage induction, including the type, concentration, injection volume, and injection site of the chemical damage agent, as well as the forced exercise training, including the type, speed, incline, daily duration, and number of days of exercise equipment. For example, for mild effects, the chemical damage induction corresponding to the mapping table is a single injection of sodium iodoacetate solution (2 mg / mL, 50 μL) into the right knee joint cavity of SPF-grade male SD rats weighing 180-220g. The corresponding forced exercise training is to begin on the 7th day after chemical damage induction, using an electric treadmill at a speed of 15 m / min and an incline of 0°, for 30 minutes daily for 7 consecutive days.

[0021] For example, for chronic regulation, the chemical injury induction corresponding to the mapping table is to inject a single dose of sodium iodoacetate solution (1.5 mg / mL, 50 μL) into the right knee joint cavity of SPF-grade male SD rats weighing 180 to 220 g. The corresponding forced exercise training is to start on the 7th day after the completion of chemical injury induction, using an electric treadmill to train the experimental animals at a speed of 10 m / min and an incline of 0° for 20 minutes daily for 10 consecutive days. According to the selected chemical injury induction, 50 μL of sodium iodoacetate solution is drawn with a 1 mL syringe and injected into the right knee joint cavity of the rat through the lateral space of the infrapatellar ligament to complete the chemical injury induction operation.

[0022] After the chemical injury induction procedure was completed and a 7-day recovery period was completed, the experimental animals were placed on a pre-set exercise device, such as an electric treadmill, for treadmill training according to the selected forced exercise training protocol. The daily training time was fixed from 9:00 to 9:30 am, the treadmill speed was maintained at 15 m / min, and the incline was maintained at 0°. The training was carried out continuously for 7 days. Forced exercise training and chemical injury induction were carried out in sequence to form a composite modeling method.

[0023] In step S1, the construction of an animal model of bone and joint injury that maintains a preset target histological score range throughout the entire intervention period and whose full-thickness cartilage defect volume fraction measured by micro-CT is less than a preset threshold is specifically implemented as follows: The animals entered a preset intervention period of 28 days. During this period, histological scoring and micro-CT measurements were performed on the experimental animals to obtain the target histological score, which was based on the OARSI histological scoring standard. At the same time, the full-thickness cartilage defect volume fraction was obtained. The full-thickness cartilage defect volume fraction was calculated as the percentage of the defect volume to the full-thickness cartilage volume by using three-dimensional reconstruction software after scanning the knee joint sample with micro-CT. The preset target histological score range was set to 2 to 5 points, and the preset threshold for the full-thickness cartilage defect volume fraction was set to 10%.

[0024] When the detection finds that the target histological score exceeds the preset target histological score range, such as a target histological score below 2 points or above 5 points, or when the volume fraction of the full-thickness cartilage defect reaches or exceeds the preset threshold, the system automatically selects the corresponding level of chemical damage induction or forced exercise training from the preset damage control mapping table for adjustment based on the direction and degree of the target histological score exceeding the target histological score range, i.e., the absolute difference between the target histological score and the target histological score range. For example, an absolute difference of less than 0.5 points, 0.5 points to 1.5 points, and more than 1.5 points correspond to a degree of score deviation of mild, moderate, and severe, respectively. At the same time, based on the difference between the volume fraction of the full-thickness cartilage defect and the preset threshold, for example, a difference of less than 2% indicates a mild defect deviation, 2% to 5% indicates a moderate defect deviation, and more than 5% indicates a severe defect deviation.

[0025] The damage regulation mapping table was established in the preliminary experiments. Corresponding levels of chemical damage induction or forced exercise training were pre-set for different degrees of score deviation and defect deviation. Specifically, when the target histological score was below 2 points and the deviation was mild, the concentration of sodium iodoacetate in chemical damage induction was increased from 1.5 mg / mL to 2.0 mg / mL, or the forced exercise training speed was increased from 10 m / min to 12 m / min. When the target histological score was below 2 points and the deviation was moderate, the concentration of sodium iodoacetate in chemical damage induction was increased to 2.5 mg / mL, or the forced exercise training speed was increased to 14 m / min. When the target histological score was below 2 points and the deviation was severe, the concentration of sodium iodoacetate in chemical damage induction was increased to 3.0 mg / mL, or the forced exercise training speed was increased to 16 m / min, while the daily duration was extended to 40 minutes. When the target histological score is higher than 5 and the deviation is mild, the concentration of sodium iodoacetate in chemical injury induction is reduced from 2.0 mg / mL to 1.5 mg / mL, or the daily duration of forced exercise training is shortened from 30 minutes to 25 minutes. When the target histological score is higher than 5 and the deviation is moderate, the concentration of sodium iodoacetate in chemical injury induction is reduced to 1.0 mg / mL, or the daily duration of forced exercise training is shortened to 20 minutes. When the target histological score is higher than 5 and the deviation is severe, the concentration of sodium iodoacetate in chemical injury induction is reduced from 2.0 mg / mL to 0.5 mg / mL, or the daily duration of forced exercise training is shortened from 30 minutes to 15 minutes.

[0026] For deviations where the volume fraction of full-thickness cartilage defects reaches or exceeds a preset threshold of 10%, the corresponding adjustment scheme is selected from the mapping table. Specifically, when the defect deviation is mild, the concentration of sodium iodoacetate in chemical injury induction is reduced by 0.5 mg / mL. If the current sodium iodoacetate concentration is already below 0.5 mg / mL, chemical induction is suspended, or the forced exercise training speed is reduced by 2 m / min. When the defect deviation is moderate, the concentration of sodium iodoacetate in chemical injury induction is reduced by 1.0 mg / mL. If the current sodium iodoacetate concentration is already below 0.5 mg / mL, chemical induction is suspended, or the forced exercise training speed is reduced by 4 m / min, and the daily duration is shortened by 5 minutes. When the defect deviation is severe, chemical injury induction is suspended, and the forced exercise training speed is reduced to 50% of the original speed, and the daily duration is shortened to less than 15 minutes.

[0027] When both the target histological score deviation and the full-thickness cartilage defect volume fraction deviation occur simultaneously, adjustments are made first according to the deviation level of the full-thickness cartilage defect volume fraction. Only when the full-thickness cartilage defect volume fraction does not exceed the preset threshold are adjustments made according to the deviation degree of the target histological score. Through the above dynamic adjustments, a bone and joint injury animal model is finally constructed in which the target histological score is always maintained within the preset target histological score range of 2 to 5 points and the full-thickness cartilage defect volume fraction is always less than the preset threshold of 10% within the preset intervention period.

[0028] In step S2, the animal model of bone and joint injury is divided into a model control group and a drug administration group. The drug administration group is given a dosing regimen consisting of two administration phases. At the end of each administration phase, pre-set evaluation indicators are measured and recorded. Pre-set statistical methods are used to compare the differences among the pre-set evaluation indicators. Simultaneously, pre-set effect evaluation criteria are implemented. Specifically, the implementation is as follows: The adjusted animal models of bone and joint injury were randomly divided into a model control group and a drug administration group. The drug administration group was set up with a dosing regimen that included a first dosing phase and a second dosing phase. The first dosing phase lasted for 14 days, and the second dosing phase lasted for 14 days. The total number of days for the first and second dosing phases was 28 days, which was consistent with the intervention period.

[0029] During the first dosing phase, from day 1 to day 14, the drug was administered via gavage at a predetermined maintenance dose, which was determined based on the pre-experimental safe dose of the test substance. Specifically, the maintenance dose was 200 mg / kg body weight, prepared with purified water as the gavage solution, with a gavage volume of 10 mL / kg body weight, once daily, for the predetermined number of days of the first dosing phase.

[0030] During the second dosing phase, from day 15 to day 28, the administration method continued by gavage. At preset intervals, which were set to 2 days, a preset increment of 50 mg / kg body weight was added. Specifically, the dose was 250 mg / kg body weight on day 15, 300 mg / kg body weight on day 17, 350 mg / kg body weight on day 19, 400 mg / kg body weight on day 21, 450 mg / kg body weight on day 23, 500 mg / kg body weight on day 25, 550 mg / kg body weight on day 27, and 550 mg / kg body weight on day 28, continuing until the end of the second dosing phase.

[0031] At the end of the first and second dosing phases, pre-defined evaluation indicators were measured and recorded. These indicators included OARSI histological score, full-thickness cartilage defect volume fraction, mechanical pain threshold, cold pain threshold, and gait parameters. The mechanical pain threshold was measured using an electronic Von Frey instrument to determine the right hind foot withdrawal threshold of the bone and joint injury animal model. The cold pain threshold was measured using the acetone spray method to determine the right hind foot withdrawal latency of the bone and joint injury animal model. Gait parameters were determined using animal footprint analysis. Specifically, the bone and joint injury animal model was placed in a transparent walking tunnel and allowed to walk freely. A high-speed camera recorded the footprint trajectory from below the tunnel. The average walking speed, support time, and stride length were calculated by analyzing the footprints and contact area, and the time series. At least three valid walking processes were collected for each sample, and the average value was used for statistical analysis.

[0032] Pre-defined statistical methods were used to compare the differences between the pre-defined evaluation indicators. Specifically, the normality test and homogeneity of variance test were first performed on the data of each pre-defined evaluation indicator of the model control group and the drug administration group at each time point. If the data of each pre-defined evaluation indicator satisfies normal distribution and homogeneity of variance, the independent samples t test was used to compare the differences between the model control group and the drug administration group.

[0033] If the data for each pre-set evaluation index do not meet the normal distribution or have unequal variances, the Mann-Whitney U test will be used for comparison. Simultaneously, for all pre-set evaluation indices, a pre-set effect size assessment standard will be used, which employs Cohen's d effect size. This effect size is calculated by dividing the difference between the mean of the model control group and the mean of the treatment group by the pooled standard deviation. The pooled standard deviation is calculated by taking the square root of the sum of the squares of the standard deviations of the model control group and the treatment group, divided by the sum of the sample sizes of the model control group and the treatment group minus 2. In the pre-set effect size assessment standard, an absolute value of Cohen's d effect size less than 0.2 is defined as a small effect size, an absolute value between 0.2 and 0.5 is defined as a moderate effect size, and an absolute value greater than 0.5 is defined as a large effect size.

[0034] When the p-value of the independent samples t-test or Mann-Whitney U test is less than 0.05, the p-value refers to the probability of obtaining the current sample statistic and more extreme results when the null hypothesis is true. It determines that the difference is statistically significant and reports the corresponding Cohen's d effect size and its effect size rank. The effect size ranks include small effect size, moderate effect size, and large effect size. Through the above statistical methods, we can draw conclusions on whether each preset evaluation index has a statistically significant difference between the model control group and the treatment group and the effect size rank, and determine the strength of the difference according to the effect size criteria.

[0035] In step S3, before the start of the drug administration phase and throughout the entire intervention process of the first and second drug administration phases, the mechanical pain threshold, cold pain threshold, and gait parameters of the bone and joint injury animal model are measured according to a preset monitoring cycle to generate dynamic trend data. Specifically, the implementation is as follows: Before the start of the drug administration phase, specifically before gavage administration on day 1 of the first drug administration phase, the initial mechanical pain threshold of the bone and joint injury animal model was determined using an electronic analgesic device, such as the IITC Model 2390 from the United States. The method involved placing the probe of the electronic analgesic device perpendicularly to the center of the right hind foot of the bone and joint injury animal model and applying pressure at a constant rate until the animal model exhibited a foot withdrawal response. The pressure value at this point was recorded as the initial mechanical pain threshold. The initial cold pain threshold was determined using the cold plate method, where the bone and joint injury animal model was placed in a container pre-cooled to 4°C. On the cold plate, timing begins from the moment the plate is placed until the animal model of bone and joint injury exhibits its first foot withdrawal or licking response. This time is recorded as the initial cold pain threshold. Gait analysis equipment is used to measure the initial gait parameters of the animal model of bone and joint injury, specifically including average walking speed, support time, and stride length. At least three valid walking processes are collected for each sample, and the average of the three is taken as the initial gait parameters. The results of each measurement are recorded in a pre-set data recording table, which is saved in spreadsheet format. The recorded content includes the measurement date, drug administration stage identifier, animal number, and values ​​of each measured index.

[0036] During the first administration phase, according to a pre-set monitoring cycle (every 3 days), mechanical pain threshold, cold pain threshold, and gait parameter measurements were repeatedly performed on animal models of bone and joint injuries using an electronic analgesic device, the cold plate method, and gait analysis equipment. For example, the IITC Model 2390 electronic analgesic device was used to measure the mechanical pain threshold in the animal models of bone and joint injuries. The measurement method involved perpendicularly pointing the probe of the electronic analgesic device to the center of the right hind foot of the animal model and applying pressure at a constant speed until the animal model exhibited a foot withdrawal response. The pressure value at this point was recorded as the mechanical pain threshold. The cold plate method was used to determine the cold pain threshold in animal models of bone and joint injuries. The method involved placing the animal model of bone and joint injury on a cold plate pre-cooled to 4°C. Timing was started from the moment the animal model was placed on the cold plate until it exhibited its first foot withdrawal or licking response. This time was recorded as the cold pain threshold. Gait parameters of the animal models of bone and joint injuries were measured using a gait analysis device, including average walking speed, support time, and stride length. At least three valid walking processes were collected for each sample, and the average of the three was taken as the gait parameters. The mechanical pain threshold, cold pain threshold, and gait parameters obtained from each measurement were recorded in a pre-set data recording table.

[0037] During the second administration phase, following the same monitoring cycle as the first administration phase (every 3 days), mechanical pain threshold, cold pain threshold, and gait parameter measurements were performed on the animal model of bone and joint injury using an electronic analgesic device, the cold plate method, and gait analysis equipment. These measurements were repeated. For example, the IITC Model 2390 electronic analgesic device was used to measure the mechanical pain threshold in the animal model of bone and joint injury. The measurement method involved perpendicularly pointing the probe of the electronic analgesic device to the center of the right hind foot of the animal model of bone and joint injury and applying pressure at a uniform rate until the bone and joint were damaged. Animal models of bone and joint injuries exhibited a foot withdrawal response, and the pressure value at this time was recorded as the mechanical pain threshold. The cold pain threshold of the animal models of bone and joint injuries was determined using the cold plate method. The method involved placing the animal model of bone and joint injuries on a cold plate pre-cooled to 4°C, and timing was started from the moment the animal model of bone and joint injuries exhibited its first foot withdrawal or foot-licking response. This time was recorded as the cold pain threshold. Gait parameters of the animal models of bone and joint injuries were measured using gait analysis equipment, specifically including average walking speed, support time, and stride length. At least three valid walking processes were collected for each sample, and the average of the three was taken as the gait parameters. The mechanical pain threshold, cold pain threshold, and gait parameters obtained from each measurement were recorded in a pre-set data recording table.

[0038] The data recording table compares each measurement result with the initial mechanical pain threshold, initial cold pain threshold, initial gait parameters, and the measurement results of the previous monitoring period to form dynamic trend data. Specifically, for the mechanical pain threshold, the difference between the mechanical pain threshold and the initial mechanical pain threshold is first calculated, and then the difference between this difference and the corresponding difference in the previous monitoring period is calculated to obtain the rate of change of the mechanical pain threshold over time. Similarly, for the cold pain threshold, the difference between the cold pain threshold and the initial cold pain threshold is calculated, and then the difference between this difference and the difference in the previous monitoring period is calculated to obtain the rate of change of the cold pain threshold. For the gait parameters, including average walking speed, support time, and stride length, the percentage change of average walking speed relative to the average walking speed of the initial gait parameter, the percentage change of support time relative to the support time of the initial gait parameter, and the percentage change of stride length relative to the stride length of the initial gait parameter are calculated, and then the difference between each percentage change and the corresponding percentage change in the previous monitoring period is calculated.

[0039] All calculated differences, differences between differences, percentage changes, and differences of percentage changes are arranged in chronological order into tables or line graphs to form dynamic trend data. This dynamic trend data is used to evaluate the dynamic evolution characteristics of mechanical pain threshold, cold pain threshold, and gait function of animal models with bone and joint injury over the entire dosing period.

[0040] In step S4, after completing the first and second drug administration phases, cartilage, subchondral bone, synovium, and dorsal root ganglion tissues are simultaneously collected from the animal model of bone and joint injury. Histochemical staining, micro-CT scanning, immunohistochemistry, and immunofluorescence staining are performed on these tissues to obtain multimodal analysis results covering cartilage histological scores, subchondral bone microstructural parameters, synovial inflammation scores, and the intensity of positive neuropeptide immunogenic signals in the dorsal root ganglion. Specifically, the implementation is as follows: After completing the first and second drug administration phases, the animal models of bone and joint injury were euthanized, and cartilage, subchondral bone, synovial membrane, and lumbar dorsal root ganglion tissue were isolated and collected from the knee joint. The cartilage was taken from the full-thickness cartilage of the femoral condyle and tibial plateau articular surfaces; the subchondral bone was taken from the cancellous bone region below the cartilage layer to the growth plate; the synovial membrane was taken from the synovial layer of the medial wall of the knee joint capsule; and the lumbar dorsal root ganglion tissue was taken from the bilateral dorsal root ganglia of the L4 to L6 segments.

[0041] The collected cartilage tissue was subjected to histochemical staining, specifically hematoxylin-eosin staining and safranin O-fast green staining. Morphological changes in the cartilage tissue were observed and recorded under a microscope, including the number of chondrocytes, layered structure, integrity of the tide line, and matrix staining intensity. The degree of cartilage damage was scored according to the preset histological scoring standard, namely the OARSI histological scoring standard, to obtain a cartilage histological score, which ranged from 0 to 6 points.

[0042] The collected subchondral bone tissue was subjected to micro-computed tomography (CT) scan with the following parameters set: voltage 50 kV, current 200 μA, and resolution 10 μm. Three-dimensional microstructure images of the subchondral bone were obtained. The number of trabeculae, trabecular thickness, trabecular separation, and bone volume fraction were measured and recorded using three-dimensional reconstruction software to obtain the microstructure parameters of the subchondral bone.

[0043] Immunohistochemical staining was performed on the collected synovial tissue. Macrophages were labeled with anti-CD68 antibody to assess the degree of inflammatory cell infiltration, and proliferating cells were labeled with anti-Ki-67 antibody to assess the synovial cell proliferation status. The proliferation status of synovial cells, the degree of inflammatory cell infiltration, and the formation of pannus were observed under a microscope. The synovial inflammation was scored according to a pre-set synovial inflammation scoring standard. The scoring standard is divided into three levels: 0 indicates normal, 1 indicates mild inflammation (a small amount of inflammatory cell infiltration and no pannus), 2 indicates moderate inflammation (a moderate amount of inflammatory cell infiltration and a small amount of pannus), and 3 indicates severe inflammation (a large amount of inflammatory cell infiltration and extensive pannus formation). The synovial inflammation score was obtained.

[0044] The collected dorsal root ganglion tissue was subjected to immunofluorescence staining. Neuropeptides were labeled with anti-calcitonin gene-related peptide antibodies. The number of neurons and fluorescence intensity of calcitonin gene-related peptide immunopositive were observed and counted under a fluorescence microscope. The fluorescence intensity was measured by image analysis software to obtain the average gray value of each positive neuron. At the same time, three cell-free areas (i.e., areas without tissue cells or blank areas with background fluorescence) on the same slice were selected and their average gray values ​​were measured as background values. The mean of the three cell-free areas was taken as the final background value. The positive signal intensity was evaluated according to a preset grading standard. This grading standard divides the fluorescence intensity into weak positive (average gray value is 1.5 to 2 times the background value), moderate positive (average gray value is 2 to 3 times the background value), and strong positive (average gray value is more than 3 times the background value). The dorsal root ganglion neuropeptide immunopositive signal intensity was obtained.

[0045] The obtained cartilage histological scores, subchondral bone microstructural parameters, synovial inflammation scores, and dorsal root ganglion neuropeptide immunopositive signal intensity were summarized to construct a multimodal analysis result.

[0046] In step S5, a weighted comprehensive scoring system is established based on the multimodal analysis results. The single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are uniformly scored according to preset standards. Preset weights are assigned to each indicator based on the preset osteoarthritis stage, and the comprehensive score is calculated. The specific implementation is as follows: Based on the results of multimodal analysis, a weighted comprehensive scoring system was established. This system includes preset weights and comprehensive score calculation rules. The preset weights are determined according to the preset osteoarthritis stages, which include early stage, middle stage and late stage. Each stage corresponds to a set of preset weight values, and the sum of the preset weight values ​​of each stage is 1.

[0047] The comprehensive score is calculated by multiplying the score of each preset evaluation indicator by its corresponding preset weight, and then summing all the products to obtain the weighted comprehensive score. Specifically, the preset evaluation indicators include the single measurement results and dynamic trend data of mechanical pain threshold, cold pain threshold, and gait parameters, as well as the cartilage histology score, subchondral bone microstructure parameters, synovial inflammation score, and dorsal root ganglion neuropeptide immunopositive signal intensity from multimodal analysis results.

[0048] Based on the preset osteoarthritis stage, each preset evaluation index is assigned a corresponding preset weight. Taking the early stage as an example, the early stage is defined as the period from day 1 to day 14 after the completion of the composite modeling mode. The specific preset weights for this early stage are set as follows: the weight of a single measurement result of mechanical pain threshold is 0.15, the weight of a single measurement result of cold pain threshold is 0.15, and the weights of the three single measurement results of gait parameters, namely average walking speed, support time and stride length, are all 0.05, for a total of 0.15.

[0049] In the dynamic trend data, the weight of the rate of change of mechanical pain threshold was 0.10, the weight of the rate of change of cold pain threshold was 0.10, the weight of the difference in percentage changes of gait parameters was 0.05, the weight of cartilage histology score in the multimodal analysis results was 0.05, the weight of each of the subchondral bone microstructure parameters (trabecular number, trabecular thickness, trabecular separation, and bone volume fraction) was 0.025, totaling 0.10, the weight of synovial inflammation score was 0.05, and the weight of the intensity of positive neuropeptide immunogenic signal in the dorsal root ganglion was 0.10. The intermediate stage was defined as days 15 to 28 after the completion of the composite modeling model. During this intermediate stage, the weights of cartilage and subchondral bone structural indicators were increased. Specifically, the preset weights were adjusted as follows: the weight of a single measurement of mechanical pain threshold was 0.10; the weight of a single measurement of cold pain threshold was 0.10; the total weight of a single measurement of gait parameters was 0.10; in the dynamic trend data, the weights of the rate of change of mechanical pain threshold and the rate of change of cold pain threshold were 0.05; the total weight of the difference in percentage changes of gait parameters was 0.05; in the multimodal analysis results, the weight of cartilage histology score was 0.15; the total weight of each subchondral bone microstructure parameter was 0.20; the weight of synovial inflammation score was 0.10; and the weight of the intensity of the dorsal root ganglion neuropeptide immunopositive signal was 0.10.

[0050] The late stage was defined as days 29 to 42 after the completion of the composite modeling. The preset weights for this stage were as follows: a single measurement result of mechanical pain threshold was weighted at 0.05; a single measurement result of cold pain threshold was weighted at 0.05; a total weight of a single measurement result of gait parameters was weighted at 0.05; in the dynamic trend data, the rate of change of mechanical pain threshold was weighted at 0.05; the rate of change of cold pain threshold was weighted at 0.05; the total weight of the difference in percentage changes of gait parameters was weighted at 0.05; in the multimodal analysis results, the weight of cartilage histology score was 0.20; the total weight of each index of subchondral bone microstructure parameters was 0.25; the weight of synovial inflammation score was 0.10; and the weight of the intensity of positive neuropeptide immunogenic signal in the dorsal root ganglion was 0.15.

[0051] After assigning preset weights, the single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are uniformly scored according to preset standards. The preset standards adopt a percentage scoring method, that is, the original measured value of each preset evaluation indicator is converted into a score between 0 and 100. The conversion rules are preset according to the normal range and pathological range of each preset evaluation indicator. For example, for mechanical pain threshold, the average right hind foot withdrawal threshold of 60g in the animal model of bone and joint injury is used as 100 points, and the lowest average value of 10g in the model control group is used as 0 points. The measured value is mapped to the score using a linear interpolation method. Specifically, the score is equal to the measured value minus 10g divided by 60g minus 10g and then multiplied by 100. If the measured value is lower than 10g, the score is 0 points, and if it is higher than 60g, the score is 100 points.

[0052] For the cold pain threshold, the average foot withdrawal latency of 30 seconds in the animal model of bone and joint injury was set as 100 points, and the shortest average latency of 5 seconds in the model control group was set as 0 points. The same linear interpolation method was used to calculate the score. For the average walking speed in the gait parameters, the average speed of 40 cm / s in the animal model of bone and joint injury was set as 100 points, and the lowest speed of 10 cm / s in the severely injured model was set as 0 points. The score was calculated by linear interpolation.

[0053] For the support time, the average support time of 0.25 seconds in the animal model of bone and joint injury was set as 100 points, and the longest average support time of 0.50 seconds in the model control group was set as 0 points. The score is equal to the difference between 0.50 seconds and the measured value, divided by the difference between 0.50 seconds and 0.25 seconds, and then multiplied by 100. If the measured value is higher than 0.50 seconds, the score is 0 points, and if it is lower than 0.25 seconds, the score is 100 points.

[0054] For stride length, the average stride length of 15cm in the animal model of bone and joint injury is set as 100 points, and the shortest average stride length of 5cm in the model control group is set as 0 points. The score is equal to the difference between the measured value and 5cm, divided by the difference between 15cm and 5cm, and then multiplied by 100. If the measured value is lower than 5cm, the score is 0 points, and if it is higher than 15cm, the score is 100 points. For the rate of change of mechanical pain threshold in dynamic trend data, the direction of improvement is taken as positive. The score corresponding to the maximum rate of improvement is set as 100 points, and the score corresponding to no change or deterioration is set as 0 points. Specifically, a linear mapping is performed through a pre-calibrated numerical range.

[0055] The maximum rate of improvement was defined as the average rate of change of the mechanical pain threshold in the animal model of bone and joint injury in the preliminary experiment, set at +0.5 g / day, which is 100 points. The score corresponding to no change or deterioration was set at the average rate of change of the mechanical pain threshold in the model control group, set at -1.0 g / day, which is 0 points. If the measured rate of change was higher than +0.5 g / day, the score was 100 points; if it was lower than -1.0 g / day, the score was 0 points. When it was between the two, the score was equal to the measured rate of change minus negative 1.0 g / day, divided by 0.5 g / day minus negative 1.0 g / day, and then multiplied by 100. That is, the score was equal to the measured rate of change plus 1.0, divided by 1.5, and then multiplied by 100.

[0056] For the cartilage histology score in the multimodal analysis results, 0 points (normal) was taken as 100 points, and 6 points (most severe) was taken as 0 points. The score was equal to 6 minus the measured value, divided by 6, and then multiplied by 100. For the bone volume fraction in the subchondral bone microstructure parameters, 35% of the average value of the animal model of bone and joint injury was taken as 100 points, and the lowest value of 10% of the severe osteoporosis model was taken as 0 points. Linear interpolation was used for calculation. Trabecular bone number, trabecular bone thickness, and trabecular bone separation were linearly mapped using corresponding normal and pathological thresholds. Specifically, for trabecular bone number, the average trabecular bone number in the subchondral bone region of the right knee in the animal model of bone and joint injury was set as 100 points, and the lowest average number in the model control group was set as 1.5 1 / mm as 0 points. A linear interpolation method was used to calculate the score, i.e., the score equals the measured value minus 1.5 divided by 4.0 minus 1.5 and then multiplied by 100. If the measured value is lower than 1.5, the score is 0 points, and if it is higher than 4.0, the score is 100 points. For trabecular bone thickness, the normal average value was set as 80 μm as 100 points, and the lowest average value in the model control group was set as 30 μm as 0 points. A linear interpolation method was used to calculate the score, i.e., the measured value minus 30 divided by 80 minus 30 and then multiplied by 100. If the measured value is lower than 30, the score is 0 points, and if it is higher than 80, the score is 100 points.

[0057] For trabecular separation, the normal average value, such as 200 μm, is taken as 100 points, and the highest average value of the model control group, such as 500 μm, is taken as 0 points. Linear interpolation is used for calculation. Since a larger separation indicates a worse structure, the score decreases as the separation increases. The specific formula is: score equals 500 minus the measured value divided by 500 minus 200 and then multiplied by 100. If the measured value is higher than 500, the score is 0 points, and if it is lower than 200, the score is 100 points. For synovitis, grade 0 (normal) is taken as 100 points, and grade 3 (most severe) is taken as 0 points. The score is equal to 3 minus the measured grade divided by 3 and then multiplied by 100.

[0058] The intensity of the dorsal root ganglion neuropeptide immunopositive signal was graded from a strong positive score of 100 to a negative score of 0, with weak positive corresponding to 33 points, moderate positive to 67 points, strong positive to 100 points, and negative to 0 points. Using the above preset criteria, scores were obtained for single measurements of mechanical pain threshold, cold pain threshold, gait parameters, dynamic trend data, and multimodal analysis results. Each score was then multiplied by a preset weight assigned according to the stage of osteoarthritis, and all products were summed to calculate a weighted composite score.

[0059] In step S5, the effective threshold is set as follows: the comprehensive score reaches the preset efficacy judgment threshold, and the scores of both the single measurement result and the dynamic trend data reach the preset single-item effective threshold. Statistical verification is performed by comparing the differences with the model control group and evaluating the effect quantity to determine whether the effective threshold is met. When the effective threshold is met, the efficacy evaluation conclusion of the food to be evaluated in alleviating bone and joint damage is output. The specific implementation is as follows: An effective threshold is set, which includes the overall score reaching the preset therapeutic effect judgment threshold, and the scores of the single measurement results of mechanical pain threshold, cold pain threshold and gait parameters and the scores of dynamic trend data all reaching the preset single effective threshold.

[0060] The preset efficacy judgment threshold is set at 60 points. This threshold is determined based on the average weighted composite score of animal models with bone and joint injuries in the preliminary experiment under known effective interventions. For example, the known effective intervention is the positive control drug glucosamine sulfate, administered by gavage at 200 mg / kg body weight daily for 28 consecutive days. The average composite score was 62 points, which was rounded down to 60 points. The preset single-item effectiveness threshold is set at 50 points. This threshold is determined based on the fact that, at the end of the treatment period, the average scores of the single measurement results and dynamic trend data of mechanical pain threshold, cold pain threshold, and gait parameters in the model control group (i.e., the animal model with bone and joint injuries without any intervention) were all between 30 and 40 points. 50 points is set as a threshold exceeding the average of the model control group and having biological significance. That is, only when each single-item score is not lower than 50 points is the food considered to have produced effective improvement in the corresponding dimension. Each single-item score must reach the preset single-item effectiveness threshold of 50 points.

[0061] After setting the effective threshold, the differences between the pre-set evaluation indicators of the drug-treated group and the model control group were compared and the effect size was assessed. The results of the difference comparison and effect size assessment were statistically validated to determine whether the effective threshold was met simultaneously. The difference comparison and effect size assessment used the pre-set statistical methods, namely, normality tests and homogeneity of variance tests were performed on the last measurement data of each pre-set evaluation indicator. If the data met the requirements of normal distribution and homogeneity of variance, an independent samples t-test was used; otherwise, a Mann-Whitney U test was used.

[0062] The effect size was assessed using Cohen's d effect size, which is calculated by dividing the difference between the mean of each pre-set evaluation index in the model control group and the mean of each pre-set evaluation index in the drug treatment group by the pooled standard deviation. The pooled standard deviation is equal to the sum of the squares of the standard deviations of the model control group and the drug treatment group, divided by the square root of the sum of the sample sizes of the model control group and the drug treatment group minus 2.

[0063] In the pre-defined effect size assessment criteria, an absolute value of Cohen's d effect size less than 0.2 is defined as a small effect size, 0.2 to 0.5 as a moderate effect size, and greater than 0.5 as a large effect size. The specific rules for statistical validation are as follows: for the composite score, the p-value of the independent samples t-test or Mann-Whitney U test between the treatment group and the model control group must be less than 0.05, and the absolute value of the Cohen's d effect size of the composite score must be greater than 0.5 (i.e., a large effect size). For the scores of single measurements of mechanical pain threshold, cold pain threshold, and gait parameters, as well as the scores of each dynamic trend data item, the p-value of the inter-group comparison of the single measurements of mechanical pain threshold, cold pain threshold, and gait parameters, as well as the scores of each dynamic trend data item, must all be less than 0.05, and the absolute value of the Cohen's d effect size of the single measurements of mechanical pain threshold, cold pain threshold, and gait parameters, as well as the scores of each dynamic trend data item, must all be greater than 0.5 (i.e., a large effect size). When all the above conditions are met simultaneously, statistical validation is considered successful, and the treatment group is deemed to have simultaneously met the effective threshold.

[0064] When the effective thresholds are met simultaneously, the efficacy evaluation conclusion for the food to be evaluated in alleviating bone and joint injury is output. Specifically, if the overall score reaches the preset efficacy judgment threshold of 60 points, and all individual scores reach their respective preset individual effective thresholds, and the p-value for inter-group differences is less than 0.05 and the effect size reaches the maximum effect size, then the efficacy evaluation conclusion for the food to be evaluated in alleviating bone and joint injury is output as having the efficacy in alleviating bone and joint injury, and the efficacy level is effective.

[0065] If the overall score reaches the preset efficacy judgment threshold of 60 points, but not all individual scores reach the preset individual effective threshold, or the p-value of the inter-group difference comparison is less than 0.05 but the effect size does not reach the maximum effect size, then the output conclusion of the efficacy evaluation of the food to be evaluated in relieving bone and joint damage is that it has a trend of relieving bone and joint damage, but the efficacy intensity needs to be further verified.

[0066] If the overall score does not reach the preset efficacy judgment threshold of 60 points, or the p-value of the intergroup difference is greater than or equal to 0.05, the output conclusion of the efficacy evaluation of the food to be evaluated in relieving bone and joint damage is that it has no effect on relieving bone and joint damage.

[0067] The above embodiments can be implemented, in whole or in part, by software, hardware, firmware, or any other combination thereof. When implemented using software, the above embodiments can be implemented, in whole or in part, in the form of a computer program product.

[0068] Those skilled in the art will recognize that the modules and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, computer software, and a combination of electronic hardware and software. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0069] In addition, the functional modules in the various embodiments of this application can be integrated into one processing module, or each module can exist physically separately, or two or more modules can be integrated into one module.

[0070] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0071] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for evaluating the ability of food to alleviate bone and joint injuries, characterized in that, Includes the following steps: S1. When the test substance information of the food to be evaluated and the evaluation index of bone and joint injury are received, according to the preset food action characteristics, a composite modeling method of sequential chemical damage induction and forced exercise training is adopted to construct a bone and joint injury animal model that can maintain the preset target histological score range and the cartilage full-thickness defect volume fraction measured by micro-CT is less than the preset threshold within the preset intervention period. S2. The animal model of bone and joint injury was divided into a model control group and a drug administration group. The drug administration group was set up with a drug administration regimen that included two administration phases. At the end of each administration phase, each preset evaluation index was measured and recorded. The differences of each preset evaluation index were compared using preset statistical methods. At the same time, the effect quantity evaluation criteria were preset. S3. Before the start of the drug administration phase and throughout the entire intervention process of the first and second drug administration phases, the mechanical pain threshold, cold pain threshold, and gait parameters of the animal model of bone and joint injury are measured according to the preset monitoring cycle to form dynamic trend data. S4. After completing the first and second drug administration phases, cartilage, subchondral bone, synovium, and dorsal root ganglion tissues were simultaneously collected from the animal model of bone and joint injury. Histochemical staining, micro-CT scanning, immunohistochemistry, and immunofluorescence staining were performed to obtain multimodal analysis results covering cartilage histological scores, subchondral bone microstructural parameters, synovial inflammation scores, and dorsal root ganglion neuropeptide immunopositive signal intensity. S5. Based on the results of multimodal analysis, a weighted comprehensive scoring system is established. The single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are uniformly scored according to preset standards. Preset weights are assigned to each indicator according to the preset osteoarthritis stage. The comprehensive score is calculated, and the effective threshold is set when the comprehensive score reaches the preset efficacy judgment threshold and the scores of the single measurement results and dynamic trend data both reach the preset single-item effective threshold. Statistical verification is performed by comparing the differences with the model control group and evaluating the effect quantity to determine whether the effective threshold is met. When the effective threshold is met, the efficacy evaluation conclusion of the food to be evaluated in relieving osteoarthritis is output.

2. The evaluation method for the effect of food on alleviating bone and joint damage according to claim 1, characterized in that: In step S1, when the test substance information and bone and joint injury evaluation indicators of the food to be evaluated are received, a composite modeling method of sequentially inducing chemical damage and forced exercise training is adopted according to the preset food action characteristics. Specifically, this includes: The system receives test substance information of the food to be evaluated and bone and joint injury evaluation indicators. Based on the preset food action characteristics, it looks up the corresponding chemical damage induction and forced exercise training from the preset mapping relationship table. The mapping relationship table records the correspondence between different food action characteristics and chemical damage induction schemes and forced exercise training. According to the selected chemical damage induction protocol, the corresponding chemical damage agent is administered to the experimental animals to complete the chemical damage induction operation; After the chemical damage induction procedure is completed, the experimental animals are subjected to forced exercise training using pre-set exercise equipment according to the selected forced exercise training. Forced exercise training and chemical damage induction are carried out in sequence to form a composite modeling method.

3. The evaluation method for the effect of food on alleviating bone and joint damage according to claim 2, characterized in that: Step S1 involves constructing an animal model of bone and joint injury that maintains a preset target histological score range throughout the entire intervention period, and whose full-thickness cartilage defect volume fraction measured by micro-CT is less than a preset threshold. Specifically, this includes: Throughout the pre-set intervention period, histological scoring and micro-CT measurements were performed on the experimental animals to obtain the target histological score and the volume fraction of full-thickness cartilage defects. When the target histological score exceeds the preset target histological score range, or when the volume fraction of the full-thickness cartilage defect reaches the preset threshold, based on the direction and degree of the target histological score exceeding the target histological score range, and the difference between the volume fraction of the full-thickness cartilage defect and the preset threshold, the corresponding level of chemical damage induction or forced exercise training is selected from the preset damage regulation mapping table to construct an animal model of bone and joint injury in which the target histological score is maintained within the preset target histological score range and the volume fraction of the full-thickness cartilage defect is less than the preset threshold within the preset intervention period.

4. The evaluation method for the relief of bone and joint injuries by food according to claim 1, characterized in that: In step S2, the animal model of bone and joint injury is divided into a model control group and a drug administration group. The drug administration group is given a dosing regimen consisting of two administration phases. At the end of each administration phase, pre-set evaluation indicators are measured and recorded. Pre-set statistical methods are used to compare the differences among the pre-set evaluation indicators. Simultaneously, pre-set effect evaluation criteria are established, specifically including: Animal models of bone and joint injury were divided into a model control group and a drug administration group. The drug administration group was given a dosing regimen that included a first dosing phase and a second dosing phase. In the first administration phase, a preset maintenance dose is administered via gavage for the preset number of days of the first administration phase. In the second administration phase, the drug is administered via gavage, with a preset increment added at preset intervals, for a preset number of days in the second administration phase. Each pre-defined evaluation index was measured and recorded at the end of the two dosing phases. The differences between the preset evaluation indicators are compared using preset statistical methods, and the effect quantity evaluation standards are preset.

5. The evaluation method for the relief of bone and joint injuries by food according to claim 1, characterized in that: In step S3, before the start of the drug administration phase and throughout the intervention process of the first and second drug administration phases, mechanical pain threshold, cold pain threshold, and gait parameters of the bone and joint injury animal model are measured according to a preset monitoring cycle to generate dynamic trend data, specifically including: Before the start of the drug administration phase, the initial mechanical pain threshold of the animal model of bone and joint injury was determined by an electronic pain meter, the initial cold pain threshold of the animal model of bone and joint injury was determined by the cold plate method, and the initial gait parameters of the animal model of bone and joint injury were determined by a gait analysis device. The results of each measurement were recorded in a preset data recording table. During the first administration phase, the mechanical pain threshold, cold pain threshold, and gait parameters of the animal model of bone and joint injury were repeatedly measured using an electronic pain meter, cold plate method, and gait analysis equipment according to the preset monitoring cycle, and the results of each measurement were recorded in the preset data recording table. During the second administration phase, the same monitoring cycle was followed, and the mechanical pain threshold, cold pain threshold and gait parameters of the animal model of bone and joint injury were measured using an electronic pain meter, cold plate method and gait analysis equipment. The results of each measurement were recorded in a preset data recording table. Each measurement result is compared with the initial mechanical pain threshold, initial cold pain threshold, initial gait parameters, and the measurement results of the previous monitoring cycle to form dynamic trend data.

6. The evaluation method for the relief of bone and joint injuries by food according to claim 1, characterized in that: In step S4, after completing the first and second drug administration phases, cartilage, subchondral bone, synovium, and dorsal root ganglion tissues are simultaneously collected from the bone and joint injury animal model. Histochemical staining, micro-CT scanning, immunohistochemistry, and immunofluorescence staining are performed on these tissues to obtain multimodal analysis results covering cartilage histological scores, subchondral bone microstructural parameters, synovial inflammation scores, and the intensity of positive neuropeptide immunogenic signals in the dorsal root ganglion. Specifically, these include: After completing the first and second administration phases, the animal model of bone and joint injury was euthanized, and the cartilage, subchondral bone, synovium, and lumbar dorsal root ganglion tissue of the knee joint were isolated and collected. The collected cartilage tissue was subjected to histochemical staining, and the morphological changes of the cartilage tissue were observed and recorded under a microscope. The degree of cartilage damage was scored according to the preset histological scoring criteria to obtain the cartilage histological score. The collected subchondral bone tissue was subjected to micro-computed tomography to obtain three-dimensional microstructure images of the subchondral bone. The number of trabeculae, trabeculae thickness, trabeculae separation degree and bone volume fraction parameters were measured and recorded to obtain the microstructure parameters of the subchondral bone. The collected synovial tissue was subjected to immunohistochemical staining. The proliferation status of synovial cells, the degree of inflammatory cell infiltration, and the formation of pannus were observed under a microscope. The degree of synovial inflammation was scored according to the preset synovial inflammation scoring criteria to obtain the synovial inflammation score. The collected dorsal root ganglion tissue was subjected to immunofluorescence staining. The number of neurons with positive neuropeptide immunosuppression and the fluorescence intensity were observed and counted under a fluorescence microscope. The positive signal intensity was evaluated according to the preset positive signal intensity grading standard to obtain the dorsal root ganglion neuropeptide immunosuppression signal intensity. The cartilage histological score, subchondral bone microstructural parameters, synovial inflammation score, and dorsal root ganglion neuropeptide immunopositive signal intensity were combined to construct a multimodal analysis result.

7. The evaluation method for the relief of bone and joint injuries by food according to claim 1, characterized in that: In step S5, a weighted comprehensive scoring system is established based on the multimodal analysis results. Single measurements, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are uniformly scored according to preset standards. Preset weights are assigned to each indicator based on the preset osteoarthritis stage, and a comprehensive score is calculated. Specifically, this includes: Based on the results of multimodal analysis, a weighted comprehensive scoring system is established, which includes preset weights and comprehensive score calculation rules. Based on the preset osteoarthritis stage, the measured mechanical pain threshold, cold pain threshold, gait parameters, dynamic trend data, and multimodal analysis results are assigned corresponding preset weights. The single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters are scored according to preset standards to obtain scores for the single measurement results, dynamic trend data, and multimodal analysis results of mechanical pain threshold, cold pain threshold, and gait parameters. The scores are then combined with preset weights and calculated according to the comprehensive scoring rules to obtain the comprehensive score.

8. The evaluation method for the relief of bone and joint injuries by food according to claim 7, characterized in that: In step S5, the effective threshold is set as follows: the comprehensive score reaches the preset efficacy judgment threshold, and the scores of both the single measurement result and the dynamic trend data reach the preset single-item effective threshold. Statistical verification is performed by comparing the differences with the model control group and evaluating the effect quantity to determine whether the effective threshold is met. When the effective threshold is met, the efficacy evaluation conclusion of the food to be evaluated in alleviating bone and joint damage is output, specifically including: Set effective thresholds, which include the comprehensive score reaching the preset efficacy judgment threshold, and the scores of the single measurement results of mechanical pain threshold, cold pain threshold and gait parameters and the scores of dynamic trend data all reaching the preset single effective threshold. The drug administration group and the model control group were compared for differences and the effect size was evaluated. The results of the difference comparison and the effect size evaluation were statistically validated to determine whether the effective threshold was met simultaneously. If the effective threshold is met, the evaluation conclusion on the efficacy of the food to be evaluated in alleviating bone and joint damage is output.

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