Musculoskeletal transcriptomic signature-based model for sports injury assessment

Abstract

The application discloses a method for constructing a sports injury evaluation model based on a musculoskeletal transcriptome feature, divides experimental mice into a resting control group, a 28-day sports training group and a 28-day sports training + 14-day training stop group, obtains muscle fiber morphology, gait parameters, sports ability, bone microstructure and differential gene data through standardization treadmill intervention, combination of HE staining, immunohistochemistry, small animal gait analysis, Micro-CT bone microstructure detection and single cell transcriptome sequencing, verifies a key signal path by using an inhibitor intervention, establishes a gait parameter-sports ability-muscle / bone state correlation model and forms a 'phenotype-molecule-pathway-function' closed loop verification system. The application can quantize the dynamic influence of sports and training stop on muscles and bones, excavates core regulation genes of sports adaptation and de-adaptation, realizes precise evaluation of sports injury risks, and provides a standardized method and molecular basis for sports prescription optimization, muscle and bone health protection and sports injury mechanism research.

Description

Technical Field

[0001] This invention relates to the field of sports injury assessment technology, and more specifically, to a method for constructing a sports injury assessment model based on musculoskeletal transcriptome features. Background Technology

[0002] Physical exercise is widely recognized as a powerful non-pharmacological intervention for promoting human health and longevity. Extensive epidemiological and clinical evidence confirms the multiple health benefits of exercise, including a significant reduction in the risk of major non-communicable diseases such as cardiovascular disease, type 2 diabetes, and some cancers, while also delaying age-related functional decline. Despite this strong evidence, a sedentary lifestyle remains a widespread global health crisis, causing millions of premature deaths each year. This stark paradox—the clear therapeutic value of exercise versus its underutilization in the long term—highlights a core need: to go beyond simply recording the benefits of exercise and to elucidate the fundamental molecular mechanisms regulating the establishment, maintenance, and decline of these adaptive changes after cessation of training. A deeper understanding of this adaptive plasticity is crucial for optimizing exercise prescriptions and developing health protection strategies for the unavoidable sedentary period.

[0003] Exercise can influence the muscle immune microenvironment by regulating immune cells, participate in tissue repair and inflammation regulation, and induce long-term adaptive changes, including muscle fiber structural remodeling and continuous changes in systemic immune status. These changes are crucial for maintaining muscle function. However, the specific regulatory mechanisms, especially the patterns of muscle fiber morphological changes under different exercise modes, are still unclear, and no correlation model has been established between initial changes and exercise capacity, muscle and skeletal status.

[0004] Therefore, this invention provides a method for constructing a sports injury assessment model based on musculoskeletal transcriptome features. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a sports injury assessment model based on musculoskeletal transcriptome characteristics, clarify its specific regulatory mechanisms, especially the laws governing changes in muscle fiber morphology under different exercise modes, and establish a correlation model between initial changes and exercise capacity, muscle and skeletal status.

[0006] The above-mentioned technical objective of this invention is achieved through the following technical solution: a method for constructing a sports injury assessment model based on musculoskeletal transcriptome characteristics, comprising the following steps and methods:

[0007] S1: All animals underwent acclimatization rearing. The ambient temperature was controlled at 22-25℃, the relative humidity at 40%-60%, and the light cycle was alternating between 12 hours of light and 12 hours of darkness. Mice had free access to food and water. After one week of acclimatization rearing, the animals were randomly divided into a resting control group (SC) and an exercise training group. The exercise training group was further divided into a 28-day exercise training group (ET) and a 28-day exercise training + 14-day off-training group (DT).

[0008] S2: The resting control group was kept in the cage and fed normally without exercise. The exercise training group was given exercise intervention according to the preset exercise intensity, duration and frequency. During the exercise intervention, the exercise status and health status were closely observed.

[0009] S3: Muscle tissue was collected from each group of animals. After fixation with 4% paraformaldehyde, gradient dehydration, and paraffin embedding, paraffin sections were prepared, stained with hematoxylin and eosin (HE), and the morphology of muscle fibers was observed under an optical microscope. The cross-sectional area of ​​the muscle fibers was also measured.

[0010] S4: After dewaxing, hydration, and antigen retrieval, paraffin sections were incubated with primary antibody at 4°C overnight, then with secondary antibody at room temperature for 30 min. DAB staining was performed, followed by hematoxylin counterstaining, dehydration and clearing, and mounting with neutral resin. The distribution of myofibril types was observed under an optical microscope.

[0011] S5: Using an animal gait analysis system, animals are placed on the system's transparent walkway and allowed to walk freely. The system automatically collects gait images and data, and analyzes stride length, stride frequency, and time spent in the air using the system's built-in software; endurance tests and maximum speed tests are conducted on the movement capabilities of each group.

[0012] S6: After fixing the femur of the animal, a CT scan was performed to obtain three-dimensional images. The bone microstructure parameters were analyzed using CTAn software, and single-cell transcriptomics analysis was also performed.

[0013] S7: Gene validation was conducted. For the immune cell repair-related signaling pathways screened from the transcriptome, specific inhibitors of these pathways were selected and injected intraperitoneally into the exercise group. "Exercise + Inhibitor Group", "Exercise + Solvent Control Group" and "Blank Control Group" were set up. Bone density was detected by CT and the phosphorylation level of Smad1 / 5 / 8 was detected to verify the key role of this pathway in the regulation of bone by exercise. For muscle-related pathways, inhibitor intervention was used. The changes in the cross-sectional area of ​​muscle fibers were observed by HE staining to clarify the pathway regulatory effect and form a complete mechanism chain of "gene-pathway-phenotype". A correlation model of "gait parameters-exercise ability-muscle / bone status" was established.

[0014] The present invention is further configured as follows: In step S2, the ET group and the DT group undergo moderate-intensity treadmill training; before formal training, a 3-day adaptation period is conducted with training at 10 m / min and 15 min / day; the formal 28-day training plan is 15 m / min and 60 min / day, 7 days a week, with an incline of 10%; after the DT group completes 28 days of training, they completely stop treadmill exercise and resume normal cage activities for 14 days.

[0015] The present invention is further configured such that: in step S4, the primary antibody and the secondary antibody are MyHC-IIb, a fast-twitch muscle fiber-specific antibody, or MyHC-I, a slow-twitch muscle fiber-specific antibody.

[0016] The present invention is further configured as follows: In step S6, single-cell transcriptomics analysis, RNA is extracted from muscle and bone tissue using the Trizol method, RNA purity is detected by Nanodrop2000, and RNA integrity is detected by agarose gel electrophoresis; qualified RNA samples are subjected to Illumina NovaSeq high-throughput sequencing to obtain raw sequencing data; the raw data are quality controlled to obtain Clean Reads, which are compared with a reference genome to calculate gene expression levels, screen differentially expressed genes, and perform GO and KEGG enrichment analysis; the cell subpopulation gene expression data of single-cell transcriptomics, the differential gene data of bulk transcriptomics, and the differential metabolite data of metabolomics are subjected to canonical correlation analysis using the R language "omicade4" package to discover cross-omics co-regulatory modules.

[0017] In summary, this invention has the following beneficial effects: It achieves systematic quantification and comprehensive comparison of multi-dimensional exercise parameters: It breaks through the limitations of existing research that focuses on a single exercise parameter, and cross-combines the three core parameters of exercise intensity, duration, and frequency to form multiple differentiated intervention programs, and sets dynamic tracking nodes such as 2 weeks, 4 weeks, and 8 weeks to accurately locate the optimal exercise parameters for promoting muscle function and bone health; At the same time, it constructs a comprehensive comparison system of "blank control + intra-group parameter comparison + subdivided index comparison" to avoid the bias of single control and enhance the persuasiveness of mechanism research.

[0018] Breaking away from the simplistic descriptive model of traditional gait research, this study combines the CatWalkXT gait analysis system with the treadmill motor ability test to establish a correlation model of "gait parameters-motor ability-muscle / bone status," quantifying the impact of exercise on limb coordination and motor efficiency. Simultaneously, it abandons isolated transcriptome analysis, correlating differentially expressed gene results with phenotypic data such as muscle fibers and bone microstructure, and then validating them through qRT-PCR, forming a closed loop of "phenotypic observation-molecular screening-functional verification," thus improving the reliability of molecular mechanism discovery.

[0019] To address the gap in existing research on the correlation between immune cells and muscle changes, this study combines data on muscle fiber type and inflammation level to explore the mechanism by which exercise affects muscle repair through immune balance, enriching the research dimensions of muscle regulation. At the same time, it breaks through the limitations of macroscopic research on the effects of exercise on bones and uses KEGG pathway analysis to uncover key regulatory pathways, providing new molecular targets for bone health. Attached Figure Description

[0020] Figure 1 In this embodiment of the invention, (A) experimental design: C57BL / 6J male mice were divided into 3 groups: resting control group (SC), 28-day exercise training group (28ET), and 28-day exercise + 14-day rest group (28ET+14DT). Skeletal muscle and bone were collected for histological, micro-CT, and transcriptome sequencing analysis. (B) Representative cross-sectional images of adhesion protein staining in the gastrocnemius muscle layer of mice in the SC, 28ET, and 28ET+14DT groups; scale bar: 100 μm. (C) Statistical quantification of muscle fiber cross-sectional area (CSA). (D) Representative micro-CT imaging images of distal femoral trabeculae. (E–I) Quantitative analysis of trabeculae-related indicators: bone mineral density (BMD), trabecular bone number (Tb.N), bone volume fraction (BV / TV), bone surface area / bone volume (BS / BV), and trabecular bone separation (Tb.Sp)).

[0021] Figure 2 The figures in this embodiment are: (A) Average expression change curves of genes in cluster 1 in the three groups (SC, 28ET, and 28ET+14DT), indicating that even after training stops following exercise intervention, these genes continue to be upregulated. (B) Expression heatmaps of genes in cluster 1 in the three groups. (C) GO biological process enrichment analysis of genes in cluster 1, with enriched entries concentrated on the regulation of catabolism. (D) Protein-protein interaction (PPI) network of genes in cluster 1 constructed using STRING and visualized in Cytoscape; core genes selected by maximum cluster centrality (MCC) are marked in orange. Note: A_TPM = SC group, B_TPM = 28ET group, C_TPM = 28ET+14DT group.

[0022] Figure 3In this embodiment of the invention, (A) the average expression trend of genes in cluster 2 in the three groups (SC, 28ET, and 28ET+14DT) shows that the genes are significantly downregulated after exercise, and the inhibitory effect persists during the training stoppage phase. (B) Heatmap of gene expression in cluster 2 in the three groups. (C) GO enrichment of genes in cluster 2 in biological processes: enrichment of immune regulation, leukocyte activation, lymphocyte differentiation, cell adhesion, extracellular matrix assembly, and various inflammation-related biological processes. (D) PPI network of genes in cluster 2 constructed by STRING and visualized by Cytoscape; core genes screened by MCC are marked in orange. Note: A_TPM=SC group, B_TPM=28ET group, C_TPM=28ET+14DT group.

[0023] Figure 4 The following are the average expression curves of genes in cluster 4 in the three groups (SC, 28ET, and 28ET+14DT) of this invention: (A) The genes are upregulated in the exercise-induced manner, and their expression drops after training is stopped. (B) The expression heatmap of genes in cluster 4 in the three groups. (C) GO biological process enrichment of genes in cluster 4: enrichment of muscle system processes, muscle contraction, skeletal muscle development, myofibril assembly, sarcomere construction, and myocyte differentiation. (D) PPI network of genes in cluster 4 constructed by STRING and visualized by Cytoscape; core genes screened by MCC are marked in orange. Note: A_TPM=SC group, B_TPM=28ET group, C_TPM=28ET+14DT group.

[0024] Figure 5 In this embodiment of the invention, (A) a circular heatmap displays the relative expression levels of three candidate genes: a resting control (A), 28 days of exercise (B), and 28 days of exercise followed by 14 days of rest (C). Expression values ​​are standardized using Z-scores, with red representing high expression and blue representing low expression. Genes are hierarchically clustered based on temporal expression patterns. The selected genes include immune regulatory factors, stress response genes, and inflammation-related molecules, visually demonstrating exercise-induced co-transcriptional remodeling, and that this remodeling characteristic is partially maintained after rest.

[0025] Figure 6 The line graph in this embodiment of the invention shows the normalized average expression trajectory of gene clusters 1-6 in three groups: SC, 28ET, and 28ET+14DT. The magenta curve represents the average gene expression within the cluster, the gray shading represents the range of expression fluctuation within the cluster, and the dashed line represents the normalized baseline expression. The temporal expression patterns of genes in each group are significantly different, and can be divided into continuous, transient, delayed, and biphasic responses, reflecting the diverse transcriptional responses of genes to exercise and training stoppage.

[0026] Note: A_TPM = group SC, B_TPM = group 28ET, C_TPM = group 28ET+14DT.

[0027] Figure 7 The bubble chart in this embodiment of the invention shows the cellular component enrichment results of gene cluster 1 (GO). The enriched entries include the mitotic spindle, mitochondrial inner membrane intermembrane space, organelle envelope lumen, spindle mid-region, and U2 snRNP, indicating that this gene cluster is involved in the construction of intracellular structures, the mitotic complex, and the RNA processing complex. The bubble size represents the number of enriched genes, the color corresponds to the corrected P-value, and the horizontal axis represents the gene enrichment ratio.

[0028] Figure 8 In this embodiment of the invention, the bubble chart sequentially displays the cellular components of GO genes in cluster 2, GO molecular function, and KEGG pathway enrichment results. The enriched items are mainly concentrated in cell membrane and extracellular matrix components, transporter and receptor activity, and immune and stress-related signaling pathways. The bubble size represents the number of enriched genes, the color corresponds to the corrected P-value, and the horizontal axis represents the gene enrichment ratio.

[0029] Figure 9 This embodiment of the invention uses a bubble chart to display the GO biological process enrichment entries for genes in cluster 3, mainly enriching processes such as kidney development, epigenetic regulation of gene expression, negative regulation of kinase activity, heterochromatin formation, and pyrimidine nucleotide synthesis and metabolism. The size of the bubble represents the number of enriched genes, the color corresponds to the corrected P-value, and the horizontal axis represents the gene enrichment ratio.

[0030] Figure 10 In this embodiment of the invention, the bubble diagram sequentially displays the GO cellular components, GO molecular functions, and KEGG pathway enrichment results for genes in cluster 4. The enriched items primarily focus on sarcomere / myofibril structure and actin-filament binding function, while also enriching focal adhesion, actin cytoskeleton regulation, and muscle contraction-related pathways. Bubble size represents the number of enriched genes, color corresponds to the corrected P-value, and the horizontal axis represents the gene enrichment ratio.

[0031] Figure 11 In this embodiment of the invention, the bubble chart includes four enrichment results for genes in cluster 5: GO biological processes, GO cellular components, GO molecular functions, and the KEGG pathway. This gene cluster is enriched for oxidative phosphorylation, aerobic respiration, ATP synthesis and metabolism, mitochondrial respiratory complex, actin / calmodulin binding, and muscle structure-related functions. The KEGG pathway is enriched for oxidative phosphorylation and thermogenic pathways. The bubble size represents the number of enriched genes, the color corresponds to the corrected P-value, and the horizontal axis represents the gene enrichment ratio.

[0032] Figure 12The bubble chart in this embodiment of the invention illustrates the enrichment of GO genes in cluster 6 through biological processes and cellular components: biological processes are enriched in leukocyte-mediated immunity, lymphocyte-mediated immunity, adaptive immune responses, and histone H3 acetylation; cellular components are enriched in the SAGA complex and the histone / protein acetyltransferase complex, suggesting that this cluster of genes synergistically participates in immune regulation and epigenetic modification. The bubble size represents the number of enriched genes, the color corresponds to the corrected P-value, and the horizontal axis represents the gene enrichment ratio. Detailed Implementation

[0033] The following is in conjunction with the appendix Figure 1-12 The present invention will be described in further detail below.

[0034] Example: A method for constructing a sports injury assessment model based on musculoskeletal transcriptome features, such as... Figures 1-12 As shown, the procedure includes the following steps: S1: All animals were acclimatized and kept in an environment with a temperature of 22-25℃, a relative humidity of 40%-60%, and a light cycle of 12 hours of light and 12 hours of darkness. Mice had free access to food and water. After acclimatizing for one week, the animals were randomly divided into a resting control group and an exercise training group. The exercise training group was further divided into a 28-day exercise training group and a 28-day exercise training + 14-day no-training group.

[0035] S2: The resting control group was kept in the cage and fed normally without exercise. The exercise training group was given exercise intervention according to the preset exercise intensity, duration and frequency. During the exercise intervention, the exercise status and health status were closely observed.

[0036] S3: Muscle tissue was collected from each group of animals. After fixation with 4% paraformaldehyde, gradient dehydration, and paraffin embedding, paraffin sections were prepared, stained with hematoxylin and eosin (HE), and the morphology of muscle fibers was observed under an optical microscope. The cross-sectional area of ​​the muscle fibers was also measured.

[0037] S4: After dewaxing, hydration, and antigen retrieval, paraffin sections were incubated with primary antibody at 4°C overnight, then with secondary antibody at room temperature for 30 min. DAB staining was performed, followed by hematoxylin counterstaining, dehydration and clearing, and mounting with neutral resin. The distribution of myofibril types was observed under an optical microscope.

[0038] S5: Using an animal gait analysis system, animals are placed on the system's transparent walkway and allowed to walk freely. The system automatically collects gait images and data, and analyzes stride length, stride frequency, and time spent in the air using the system's built-in software; endurance tests and maximum speed tests are conducted on the movement capabilities of each group.

[0039] S6: After fixing the femur of the animal, a CT scan was performed to obtain three-dimensional images. The bone microstructure parameters were analyzed using CTAn software, and single-cell transcriptomics analysis was also performed.

[0040] S7: Gene validation was conducted. For the immune cell repair-related signaling pathways screened from the transcriptome, specific inhibitors of these pathways were selected and injected intraperitoneally into the exercise group. "Exercise + Inhibitor Group", "Exercise + Solvent Control Group" and "Blank Control Group" were set up. Bone density was detected by CT and the phosphorylation level of Smad1 / 5 / 8 was detected to verify the key role of this pathway in the regulation of bone by exercise. For muscle-related pathways, inhibitor intervention was used. The changes in the cross-sectional area of ​​muscle fibers were observed by HE staining to clarify the pathway regulatory effect and form a complete mechanism chain of "gene-pathway-phenotype". A correlation model of "gait parameters-exercise ability-muscle / bone status" was established.

[0041] In step S2, the 28-day exercise training group and the 28-day exercise training + 14-day training stop group underwent moderate-intensity treadmill training; before formal training, a 3-day adaptation period was conducted with training at 10 m / min and 15 min / day; the formal 28-day training plan was 15 m / min and 60 min / day, 7 days a week, with an incline of 10%; after completing the 28-day training, the DT group completely stopped treadmill exercise and resumed normal cage activities for 14 days.

[0042] In step S4, the primary and secondary antibodies added are either fast-twitch muscle fiber-specific antibody MyHC-IIb or slow-twitch muscle fiber-specific antibody MyHC-I.

[0043] Step S6, single-cell transcriptomics analysis, involved extracting RNA from muscle and bone tissue using the Trizol method, detecting RNA purity with Nanodrop2000, and checking RNA integrity using agarose gel electrophoresis. Qualified RNA samples were subjected to Illumina NovaSeq high-throughput sequencing to obtain raw sequencing data. The raw data underwent quality control to obtain Clean Reads, which were compared with a reference genome to calculate gene expression levels, screen for differentially expressed genes, and perform GO and KEGG enrichment analyses. Canonical correlation analysis was performed using the R language "omicade4" package on the single-cell transcriptome gene expression data, bulk transcriptome differential gene data, and metabolome differential metabolite data to identify cross-omics co-regulatory modules.

[0044] Specifically, 8-week-old male C57BL / 6J mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. After one week of acclimatization, the SPF-grade C57BL / 6 mice were randomly divided into three groups of 10 mice each, based on factors such as weight and sex. The groups were: a resting control group (SC, n=5), a 28-day exercise training group (ET, n=5), and a 28-day exercise training + 14-day off-training group (DT, n=5).

[0045] The resting group was kept in cages throughout the entire process. The ambient temperature was controlled at 22-25℃, relative humidity at 40%-60%, and the light cycle was 12 hours of light / 12 hours of darkness. Mice had free access to food and water. Mice in the exercise treatment group underwent exercise intervention using a treadmill, following a pre-set exercise intensity, duration, and frequency. Mice in the control group were fed normally and did not receive any exercise. During the exercise intervention, the mice's movement and health status were closely monitored, and any abnormalities were addressed promptly.

[0046] Mice in both the ET and DT groups underwent moderate-intensity treadmill training (LE8710MTS treadmill, Panlab). A 3-day acclimatization period (10 m / min, 15 min / day) was given before formal training. The formal 28-day training regimen was: 15 m / min, 60 min / day, 7 days a week, with a 10% incline. After completing 28 days of training, the DT group completely stopped treadmill exercise and resumed normal cage activity for 14 days.

[0047] Mouse tissues were collected at designated time points (day 28 in the ET group, day 42 in the DT group, and corresponding time points in the SC group) to rapidly separate skeletal muscle (gastrocnemius) from the distal femur. Muscle tissue was fixed in 4% paraformaldehyde, dehydrated in a gradient manner, embedded in paraffin, and then sectioned in paraffin for HE staining. Muscle fiber morphology was observed under an optical microscope, and the cross-sectional area of ​​muscle fibers was measured using Image-Pro Plus software. Muscle tissue used for histological analysis was embedded in OCT and flash-frozen in isopentane cooled to liquid nitrogen; tissue used for RNA extraction was immediately flash-frozen in liquid nitrogen and stored at -80°C. After soft tissue removal, the femur was fixed in 4% paraformaldehyde (PFA) for micro-CT scanning.

[0048] Paraffin sections were dewaxed, hydrated, and antigen-retrieved. Primary antibodies (such as MyHC-IIb for fast-twitch muscle fibers and MyHC-I for slow-twitch muscle fibers) were added, and the sections were incubated overnight at 4°C. Secondary antibodies were added, and the sections were incubated at room temperature for 30 minutes. DAB staining was performed, followed by hematoxylin counterstaining, dehydration and clearing, and mounting with neutral resin. The distribution of muscle fiber types was observed under an optical microscope. Frozen gastrocnemius muscles were sectioned transversely at a thickness of 10 μm (CM1950 cryostat, LEICA). Anti-laminus adhesion protein antibody (abcam) staining was used to visualize the muscle membrane and quantify muscle fiber size. Images were acquired using a fluorescence microscope (Nikon A1 HD25). The cross-sectional area (CSA) of a single muscle fiber was calculated using ImageJ software. At least 500 muscle fibers were analyzed per mouse to calculate the average CSA.

[0049] The CatWalk-XT small animal gait analysis system was used. Mice were placed on the system's transparent walkway and allowed to walk freely. The system automatically collected gait images and data, and the built-in software analyzed parameters such as stride length, cadence, and time spent in the air. The treadmill speed started at 5 m / min, increasing by 1 m / min every 5 minutes. The time and distance from when the mouse began running until it could no longer run were recorded. For maximum speed testing, the treadmill speed started at 5 m / min, increasing by 2 m / min every 2 minutes. The maximum speed at which the mouse could no longer run was recorded.

[0050] After fixation, mouse femurs underwent Micro-CT scanning to acquire three-dimensional images. Bone microstructural parameters (BV / TV, Tb.Th, Tb.N, Tb.Sp, etc.) were analyzed using CTAn software. The distal femur microstructure was assessed using a high-resolution micro-CT system (SkyScan 1176, Bruker). Scanning parameters: voltage 50 kV, current 500 μA, isotropic voxel size 10 μm. After reconstruction using NRecon software, a standardized region of interest (ROI) was selected in the trabecular bone region of the distal femoral metaphysis: starting 0.5 mm proximal to the growth plate and extending 1.5 mm proximally. Three-dimensional morphometric analysis was performed using Best CT analysis software to calculate bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular spacing (Tb.Sp), and bone surface area / volume ratio (BS / BV).

[0051] RNA was extracted from muscle and bone tissues using the Trizol method. RNA purity was assessed using Nanodrop2000, and RNA integrity was checked by agarose gel electrophoresis. Qualified RNA samples were sent to a sequencing company for Illumina NovaSeq high-throughput sequencing to obtain raw sequencing data. Quality control was performed on the raw data to obtain Clean Reads, which were aligned with a reference genome to calculate gene expression levels, screen for differentially expressed genes, and perform GO and KEGG enrichment analyses. Canonical correlation analysis (CCA) was performed on cell subset gene expression data from single-cell transcriptomics, differentially expressed gene data from bulk transcriptomics, and differentially expressed metabolite data from metabolomics using the R language "omicade4" package to identify cross-omics co-regulatory modules.

[0052] For immune cell repair-related signaling pathways (related to bone formation) identified through transcriptome screening, specific inhibitors of these pathways (such as Dorsomorphin) were selected and administered via intraperitoneal injection to mice in the exercise group. "Exercise + Inhibitor Group", "Exercise + Solvent Control Group", and "Blank Control Group" were established. Bone mineral density was measured using Micro-CT, and Smad1 / 5 / 8 phosphorylation levels were detected using Western blot to verify the key role of this pathway in exercise-regulated bone. Similarly, for muscle-related pathways, inhibitor intervention was used, and changes in muscle fiber cross-sectional area were observed using HE staining to clarify the pathway regulatory effects, forming a complete "gene-pathway-phenotype" mechanism chain.

[0053] RT-PCR: Using extracted total RNA as a template, cDNA was synthesized by reverse transcription. qRT-PCR was performed using specific primers, with GAPDH as an internal reference gene, and the relative expression level of the target gene was calculated.

[0054] 28 days of treadmill training (ET), followed by 14 days of voluntary timeout (DT) to capture adaptation and de-adaptation responses. Figure 1 A). First, assess the effect of exercise on skeletal muscle (the primary tissue responding to mechanical load).

[0055] Histological analysis of the gastrocnemius muscle revealed a significant hypertrophic response induced by training. Compared to the resting control group (SC), the ET group showed significantly thicker and denser muscle fibers, indicating substantial protein deposition and tissue growth (Figure 1B). Quantitative analysis of muscle fiber cross-sectional area (CSA) confirmed this conclusion, showing a significant increase in CSA in the ET group (Figure 1C). After 14 days of training cessation, the mean muscle fiber CSA significantly decreased, demonstrating the principle of reversibility; however, muscle mass remained significantly higher than the resting baseline, suggesting that some structural benefits were preserved. High-resolution micro-CT of the distal femoral trabeculae provided a three-dimensional view of the adaptive response (Figure 1D). The three-dimensional reconstruction clearly showed that the ET group had a denser and more complex trabecular network, while the SC and DT groups were relatively sparse.

[0056] Exercise training significantly improved bone mineral density (BMD) and bone volume fraction (BV / TV) (core indicators of bone mass and structural integrity) (Figures 1E and 1F). These improvements were mediated by a significant increase in trabecular bone number (Tb.N) and a decrease in trabecular bone spacing (Tb.Sp), reflecting the formation of new trabecular bone and the densification of existing trabecular structures (Figures 1G and 1H), suggesting structural optimization to improve load-bearing capacity. Highly similar to the trend of muscle changes, these key skeletal parameters significantly declined back to baseline after training cessation, but remained significantly better than the resting control group. Notably, the bone surface area / volume ratio (BS / BV) in the ET group was significantly higher than that in the SC group; although the mean value in the DT group showed a slight decreasing trend, there was no significant difference between the DT and ET groups (Figure 1I). This indicates that although overall bone mass decreases after exercise cessation, the complexity of exercise-induced trabecular bone structure exhibits significant structural continuity.

[0057] Unsupervised clustering analysis of dynamic transcriptomes successfully divided differentially expressed genes into co-expressed gene modules, each with unique temporal expression characteristics. Figure 6 ).

[0058] Among them, Module 1 (Cluster 1) exhibited a highly valuable expression pattern: the gene was significantly upregulated after 28 days of training, and not only maintained high expression after 14 days of training cessation, some even increased further (Figures 2A and 2B). Unlike transient stimulus-response genes, it was not affected by phenotypic reversibility, suggesting that it constitutes a core component of transcriptional "memory" traces, responsible for the long-term retention of adaptive benefits.

[0059] Gene Ontology (GO) annotation revealed a significant enrichment of "positive regulation of catabolic processes" and "positive regulation of cellular catabolic processes" in the Biological Processes (BP) pathway (Figure 2C). In the context of post-exercise adaptation, the sustained upregulation of catabolic pathways does not indicate tissue depletion, but rather reflects an enhanced state of cellular quality control and macromolecular turnover. This enhanced catabolic capacity is crucial for the sustained clearance of damaged proteins and organelles, thereby maintaining the functional and metabolic remodeling required for the training phenotype.

[0060] Cellular component (CC) enrichment analysis showed that the encoded protein was significantly localized in the "mitochondrial intermembrane space" and "spindle" ( Figure 7 The enrichment of mitochondrial components is highly consistent with metabolic function, suggesting a sustained enhancement of oxidative capacity. In terminally differentiated tissues such as mature skeletal muscle, spindle-related proteins are enriched, suggesting their role in maintaining cytoskeleton integrity, intracellular transport, and repositioning in sarcomere structures. These processes are crucial for preserving movement-induced structural benefits.

[0061] To identify potential core factors regulating this module, a protein-protein interaction (PPI) network was constructed. Topological analysis identified a highly connected set of core genes, including the antioxidant enzyme Sod1, the tyrosine kinase Syk, the metabolic core regulator Hif1a, and the proto-oncogene Abl1 (Figure 2D). These genes play a central role in oxidative stress management (Sod1), cellular signal integration (Syk), and coordination of hypoxia and exercise-metabolic adaptation (Hif1a), highlighting their function as key nodes coordinating the sustained activity of the module.

[0062] In summary, the persistent gene regulatory module functionally drives metabolic remodeling and homeostasis, spatially located in mitochondria and the cytoskeleton, and is regulated by a core network of stress and metabolic response proteins.

[0063] Further, a co-expression module (module 2, Cluster 2) was identified that was strongly and persistently suppressed by exercise. This module was basally highly expressed in resting animals, significantly downregulated after 28 days of training, and remained deeply suppressed even after training was stopped. Figure 3 (A, B). This potent and persistent transcriptional silencing suggests that chronic exercise exerts a long-term regulatory repression on the biological functions encoded by this module.

[0064] Multilevel enrichment analysis revealed an unexpected dual functional profile. GO enrichment analysis showed that this module is mainly involved in biological processes related to leukocyte activation (such as "positive regulation of leukocyte activation", "B cell activation", "lymphocyte proliferation", and "monocyte proliferation"), as well as extracellular matrix (ECM) tissue-related processes (such as "extracellular matrix tissue", "extracellular structural tissue", and "cell-substrate adhesion regulation") (Figure 3C).

[0065] KEGG pathway enrichment analysis also validated this conclusion, suggesting that the module genes are involved in pathways such as the "B cell receptor signaling pathway" and "cell adhesion molecules". Figure 8 A). Furthermore, the enrichment of molecular functions (MF) includes "immunoreceptor activity," "cytokine receptor activity," "carbohydrate binding," "extracellular matrix structural components," and "metallopeptidase activity," further deepening the above understanding. Figure 8 B).

[0066] To resolve functional dichotomy and identify core driving factors of the modules, this application constructed a PPI network. The results clearly show that the core topology of this network is entirely dominated by B cell-related genes (Figure 3d, orange nodes). Predicted core genes include:

[0067] Pax5 and Irf4: Key transcription factors for B cell lineage orientation and plasma cell differentiation;

[0068] Cd19, Cd79a, Cd79b, and Bln: These constitute the B cell receptor (BCR) complex and core signaling components;

[0069] Ms4a1, Ptprc, Tnfrsf13c, and Pou2af1: B cell markers that may play an important role in B cell differentiation and activation.

[0070] The analysis results suggest that the gene clusters within this module are mainly regulated by B cell-related genes, while the synchronous regulation of extracellular matrix (ECM) genes may be a downstream effect of B cell activity in the tissue microenvironment.

[0071] From a physiological perspective, the sustained suppression of this key immune module provides a compelling molecular explanation for the anti-inflammatory benefits of exercise. Long-term exercise systematically reduces this B-cell-centric immune surveillance tension, establishing a lower baseline level of inflammation, thereby enhancing the body's anti-inflammatory reserves and overall anti-inflammatory capacity.

[0072] In addition to the persistent and transient modules, a unique gene program (Cluster 3) was identified, characterized by a distinctive “delayed downregulation” trajectory. While gene expression in Cluster 3 remained largely stable or slightly elevated during active training, it underwent a dramatic and significant decline after exercise cessation (Figure 6). Functional enrichment analysis revealed that this module was primarily associated with “epigenetic regulation of gene expression,” “heterochromatin formation,” and “negative regulation of kinase activity” (Figure 9). The specific collapse of Cluster 3 during cessation of training suggests that the body initiates an active molecular silencing process—potentially mediated by chromatin remodeling—when the mechanical and metabolic stimuli of exercise are removed. This finding indicates that detraining is not merely a passive reversal of training adaptation but involves active transcriptional remodeling aimed at “quieting” specific signaling and developmental pathways.

[0073] In addition to long-term adaptation modules, gene programs for acute and transient responses to exercise stimuli were also captured, reflecting the immediate physiological demands of physical activity. The expression pattern of Cluster 4 perfectly matched the exercise protocol: low expression at baseline, sharp upregulation after training, and rapid recovery to baseline after training cessation (Figures 4A and 4B). This "immediate" expression pattern suggests its direct involvement in the execution of muscle work and adaptation.

[0074] The functional notes clearly show that module 4 is highly enriched with the core components of the muscle contraction mechanism (Figure 4C). Figure 10 The analysis focused primarily on GO and KEGG items related to muscle structure and function, including "muscle system processes," "muscle cell differentiation," "muscle contraction," "muscle organ development," "striated muscle cell development," "sarcomeres," "actin binding," "muscle structural components," "motor proteins," and "actin cytoskeleton regulation."

[0075] The PPI network of this module exhibits a tightly interwoven structure of myofibril building blocks and dyneins. Core genes include:

[0076] Actn1 (α-actin): The core component of actin filaments, forming the myofibril scaffold, and interacting with myosin to drive contraction;

[0077] Myh2 (myosin heavy chain): a molecular dynamic protein that drives silkiness;

[0078] Ttn (tipenis): A giant molecular spring that provides elasticity;

[0079] Ckm (creatine kinase): Catalyzes creatine phosphorylation to rapidly regenerate ATP and maintain muscle contraction;

[0080] Tnni2 and Tnnc2 (troponin): Downstream proteins regulated by calcium, they synergistically regulate fast-twitch muscle fiber differentiation and maintain the muscle contraction-relaxation cycle.

[0081] These core genes are mostly executive factors of muscle-specific contractile mechanisms. Therefore, this module represents the core transcriptional blueprint for the instantaneous synthesis and strengthening of the muscle "machine" under mechanical load.

[0082] Driving this device requires synchronous upregulation of the cellular energy production system. Module 5 (another transient upregulation module) captures this process. Functional analysis clearly defines Module 5 as the mitochondrial bioenergetics module (…). Figure 11 It is highly enriched with genes related to "cellular respiration", "oxidative phosphorylation", "mitochondrial respiratory chain complex I assembly", "mitochondrial protein complex", "respiratory chain complex", and "thermoproduction". The proteins encoded by these genes constitute the core subunits of the electron transport chain complex, which are jointly responsible for aerobic ATP generation (the core genes of the module include Ndufa12, Ndufb10, Ndufa5, and Uqcr11).

[0083] Therefore, the synergistic transient upregulation of the structural module (module 4) and the energy module (module 5) provides a complete systemic explanation of the acute response of skeletal muscle to exercise: synchronous transcription of genes related to "engine" and "fuel supply".

[0084] This application also reveals a more complex and nuanced role in exercise-mediated immune regulation, challenging the simplistic "pro-inflammatory / anti-inflammatory" dichotomy. The study identified adaptive immune module 6 (Cluster 6), similar to the acute structural and metabolic modules, which is transiently and reversibly downregulated during exercise. Figure 6 , Figure 12 ).

[0085] Functional analysis revealed that this module was enriched for processes such as leukocyte-mediated immunity and adaptive immune response. Crucially, it also enriched epigenetic regulatory factors, including SAGA complex components (key histone acetyltransferase complexes). The co-regulation of immune effector genes and chromatin-modifying enzymes suggests a preparatory mechanism for rapid transcriptional activation of immune-related gene loci.

[0086] This application proposes an acute downregulation-induced enhanced "immune readiness" or surveillance state. In this model, exercise puts the immune system into a more alert and efficient response standby mode, enabling it to more effectively respond to pathogen threats. This "readiness state" is strictly dependent on exercise stimulation, and returns to baseline after training stops, thereby avoiding the metabolic depletion and autoimmune risks associated with chronic immune activation.

[0087] To translate transcriptomic findings into function-related physiological outcomes, this application further investigates whether the aforementioned immune regulation alters host susceptibility to related viruses. Based on known associations between motility, immunity, and metabolic factors, this application focuses on a group of host gene expression changes that mediate key regulation following common arbovirus invasion of host cells or infection.

[0088] The reasons for selecting these functional genes are: arboviruses have a very high probability of reaching muscle tissue after entering the body; some viruses (such as Chikungunya virus) specifically target muscles and joints; in addition, various cells (especially white blood cells) circulate between muscles and skin.

[0089] As shown in the heatmap in Figure 5, the results are significant: exercise training induces a significant downregulation of the expression of most host invasion or regulatory factors, including viral invasion receptors (such as Havcr1, Vldlr, Hs3st1, Hs3st5, Glce, Cd209a, Cd209b) and genes that promote viral internalization and potential immune regulation (such as Axl, Itgb3, Ccr2, Mrc1) (Figure 5).

[0090] This expression pattern (high expression in resting mice, strong suppression after exercise) is consistent with the genes in Module 2 mentioned above. The exercise-induced downregulation is mostly persistent, although some genes (including Ldlr, Havcr2, and Ncl) show reversible upregulation after training stops (Figure 5).

[0091] These data provide direct molecular evidence for exercise-induced infection protection: exercise creates a more cellular environment less susceptible to arbovirus invasion by systematically reducing the availability of key cellular "docking sites" or "carriers." This finding translates whole-body transcriptome data (resting, continuous exercise, and training stoppage) into a definitive outcome directly related to immune resistance or overall health, confirming that exercise can induce a state of antiviral protection.

[0092] The core finding of this application is the identification of a transcriptomic program (Module 1) that exhibits significant tolerance to the initial period of cessation of training. Despite a partial decline in muscle mass and bone density after cessation, this genetic program remains strongly activated, providing a compelling molecular basis for the concept of "motor memory." Interestingly, this molecular persistence is consistent with phenotypic changes in skeletal microstructure. Micro-CT data show an overall decrease in bone mass during cessation, but the complexity of the trabecular bone network (BS / BV) remains largely intact.

[0093] Functionally, this module is enriched with metabolic and cellular processes, and its encoded proteins are located in mitochondria, consistent with persistent enhancements in cellular bioenergetics and stress resistance. The identification of Hif1a and Sod1 as core nodes in the module network is highly significant: these core regulatory factors are known to coordinate cellular responses to metabolic and oxidative stress, respectively.

[0094] This application proposes that this sustained transcriptional activity provides a molecular "imprint" of previous exercise, enabling muscles to maintain an enhanced state of metabolic readiness. This "transcriptional pre-preparation" may lower the re-adaptation threshold, allowing previously athletic individuals to regain functional benefits more efficiently upon resuming exercise—a hypothesis worthy of further longitudinal studies to verify.

[0095] In stark contrast to this sustained activation, this application found a core leukocyte immune module (module 2) that was potently and persistently suppressed by exercise. This does not suggest a harmful suppression of adaptive immunity, but rather a beneficial calibration reflecting the level of underlying inflammation within muscle tissue. The data in this application point to a more localized mechanism: exercise-induced relief of underlying inflammatory tension within muscle tissue.

[0096] The synergistic downregulation of key immune regulatory factors such as Pax5 and Irf4 suggests that exercise promotes a more resting and anti-inflammatory baseline state of the tissue-resident immune network. Crucially, this application translates this transcriptomic feature into a definitive functional outcome, demonstrating that inhibition of this module is associated with reduced expression of host factors required for arbovirus invasion. This establishes a novel and direct link between exercise-induced immune regulation and enhanced antiviral defense, suggesting that exercise can physically reshape the cellular environment and reduce susceptibility to specific pathogens.

[0097] This application further elucidates the temporal complexity of exercise-induced immune regulation, identifying a second immune-related network (module 6) with unique transient downregulation characteristics. Unlike the persistent resting state of module 2, the genes in module 6 are acutely suppressed during active training and rapidly rebound to baseline after training stops.

[0098] This application argues that this transient silence is not systemic immunosuppression, but rather an acute protective mechanism during periods of high physiological demand. By temporarily downregulating specific immune sensors and epigenetic regulators (such as SAGA complex components within the muscle module), the muscle microenvironment effectively applies an "acute brake" to inflammatory signals, preventing excessive, tissue-damaging inflammatory responses caused by the repetitive mechanical stress and micro-damage inherent in continuous endurance training, and ensuring that cellular resources are preferentially used for structural repair and metabolic adaptation. This temporary brake is released once the repetitive mechanical stimulation during the off-training period disappears.

[0099] In summary, the temporal separation of Modules 2 and 6 reveals a highly sophisticated two-layered regulatory strategy: establishing a persistently reduced inflammatory baseline (Module 2) while simultaneously activating an acute, stress-response buffer system (Module 6) to actively manage acute damage to muscle function.

[0100] Finally, the analysis of other transient regulatory modules (modules 4 and 5) provides a complete molecular explanation of the immediate demands of physical activity. The synergistic and immediate upregulation of the sarcomere "structure" module and the mitochondrial "energy" module perfectly demonstrates the tight coupling between cellular structure and metabolic supply required to drive muscle contraction and hypertrophy. The rapid reversal of these programs after training cessation highlights their role as a direct, real-time response to mechanical and metabolic stress, distinct from long-term adaptation programs.

[0101] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for constructing a sports injury assessment model based on musculoskeletal transcriptome features, characterized by: The steps and methods include the following: S1: All animals underwent acclimatization, with the ambient temperature controlled at 22-25℃, relative humidity at 40%-60%, and a light cycle of 12 hours of light and 12 hours of darkness. Mice had free access to food and water. After one week of acclimatization, the animals were randomly divided into a resting control group and an exercise training group. The exercise training group was further divided into a 28-day exercise training group and a 28-day exercise training + 14-day no-training group. S2: The resting control group was kept in the cage and fed normally without exercise. The exercise training group was given exercise intervention according to the preset exercise intensity, duration and frequency. During the exercise intervention, the exercise status and health status were closely observed. S3: Muscle tissue was collected from each group of animals. After fixation with 4% paraformaldehyde, gradient dehydration, and paraffin embedding, paraffin sections were prepared, stained with hematoxylin and eosin (HE), and the morphology of muscle fibers was observed under an optical microscope. The cross-sectional area of ​​the muscle fibers was also measured. S4: After dewaxing, hydration, and antigen retrieval, paraffin sections were incubated with primary antibody at 4°C overnight, then with secondary antibody at room temperature for 30 min. DAB staining was performed, followed by hematoxylin counterstaining, dehydration and clearing, and mounting with neutral resin. The distribution of myofibril types was observed under an optical microscope. S5: Using an animal gait analysis system, animals are placed on the system's transparent walkway and allowed to walk freely. The system automatically collects gait images and data, and analyzes stride length, stride frequency, and time spent in the air using the system's built-in software; endurance tests and maximum speed tests are conducted on the movement capabilities of each group. S6: After fixing the femur of the animal, a CT scan was performed to obtain three-dimensional images. The bone microstructure parameters were analyzed using CTAn software, and single-cell transcriptomics analysis was also performed. S7: Gene validation was conducted. For the immune cell repair-related signaling pathways screened from the transcriptome, specific inhibitors of these pathways were selected and injected intraperitoneally into the exercise group. "Exercise + Inhibitor Group", "Exercise + Solvent Control Group" and "Blank Control Group" were set up. Bone density was detected by CT and the phosphorylation level of Smad1 / 5 / 8 was detected to verify the key role of this pathway in the regulation of bone by exercise. For muscle-related pathways, inhibitor intervention was used. The changes in the cross-sectional area of ​​muscle fibers were observed by HE staining to clarify the pathway regulatory effect and form a complete mechanism chain of "gene-pathway-phenotype". A correlation model of "gait parameters-exercise ability-muscle / bone status" was established.

2. The method for constructing a sports injury assessment model based on musculoskeletal transcriptome features according to claim 1, characterized in that, In step S2, the 28-day exercise training group and the 28-day exercise training + 14-day training stop group undergo moderate-intensity treadmill training; before formal training, a 3-day adaptation period is conducted with training at 10 m / min and 15 min / day; the formal 28-day training plan is 15 m / min and 60 min / day, 7 days a week, with an incline of 10%; after completing the 28-day exercise training + 14-day training stop group, the treadmill exercise is completely stopped, and normal cage activities are resumed for 14 days.

3. The method for constructing a sports injury assessment model based on musculoskeletal transcriptome features according to claim 1, characterized in that, In step S4, the primary and secondary antibodies added are either fast-twitch muscle fiber-specific antibody MyHC-IIb or slow-twitch muscle fiber-specific antibody MyHC-I.

4. The method for constructing a sports injury assessment model based on musculoskeletal transcriptome features according to claim 1, characterized in that, Step S6, single-cell transcriptomics analysis, involved extracting RNA from muscle and bone tissue using the Trizol method, detecting RNA purity with Nanodrop2000, and checking RNA integrity using agarose gel electrophoresis. Qualified RNA samples were subjected to Illumina NovaSeq high-throughput sequencing to obtain raw sequencing data. The raw data underwent quality control to obtain Clean Reads, which were compared with a reference genome to calculate gene expression levels, screen for differentially expressed genes, and perform GO and KEGG enrichment analyses. Canonical correlation analysis was performed using the R language "omicade4" package on the single-cell transcriptome gene expression data, bulk transcriptome differential gene data, and metabolome differential metabolite data to identify cross-omics co-regulatory modules.

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