Application of antioxidant in preparation of medicine for treating skeletal muscle fat infiltration and fibrosis after rotator cuff injury

Abstract

The invention discloses application of an antioxidant in preparation of a medicine for treating fat infiltration and fibrosis related diseases after rotator cuff injury, and belongs to the technical field of medicines. SD rat rotator cuff injury model research finds that vitamin C and N-acetylcysteine have dual core effects after rotator cuff injury: 1, muscle fiber atrophy, fibrosis hyperplasia and local inflammatory response of skeletal muscle after injury are improved, and muscle fat infiltration is remarkably inhibited; 2, the anti-oxidation defense capability of the body is enhanced, the expression of antioxidant genes such as superoxide dismutase (SOD1, SOD2 and SOD3) and peroxide reductase (PRDX5) is promoted, the activity of antioxidant enzyme is improved, and the oxidative stress level is effectively relieved. The antioxidant provided by the invention can simultaneously improve fat infiltration, fibrosis, muscle atrophy and local chronic inflammation after rotator cuff injury and regulate oxidative stress, is expected to reduce the risk of postoperative retearing and promote functional recovery, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to the application of antioxidants vitamin C and N-acetylcysteine in the preparation of a medicament for treating a skeletal muscle fat infiltration and fibrosis related condition after rotator cuff injury. BACKGROUND

[0002] Rotator cuff injury is one of the common shoulder diseases in clinic, and the incidence rate significantly increases with age, with a prevalence rate of up to 25% to 30% in people over 60 years old. After rotator cuff injury, the affected skeletal muscle tissue often has two core pathological changes of fat infiltration and fibrosis. Muscle fat infiltration refers to the abnormal invasion and accumulation of fat cells in the muscle tissue, filling between muscle fibers and around muscle bundles. Muscle fibrosis refers to the replacement of part of the muscle tissue by excessive extracellular matrix deposition and scar tissue. These two pathological changes are the key reasons for the high postoperative re-tear rate (up to 20% to 94%) and poor functional recovery.

[0003] Current clinical treatment methods have obvious limitations: surgical repair (such as arthroscopic rotator cuff repair ARCR) can only reconstruct the anatomical structure of the tendon, but cannot reverse the already formed muscle fat infiltration and fibrosis; conservative treatment (non-steroidal anti-inflammatory drugs, physical therapy, etc.) can only relieve pain to a certain extent, and has limited effect on the intervention of skeletal muscle pathological degeneration. Existing researches have confirmed that oxidative stress is the core mechanism driving fat infiltration and fibrosis after rotator cuff injury - the body produces excessive reactive oxygen species (ROS) after injury, which exceeds the clearance capacity of the antioxidant system. On the one hand, excessive ROS induces tendon stem cell apoptosis and damages muscle fiber structure; on the other hand, ROS activates adipogenic differentiation pathways (such as PPARy / CEBPa signal axis) and fibrogenic pathways (such as TGF-beta mediated collagen over-deposition), ultimately leading to loss of skeletal muscle function.

[0004] Although some studies suggest that antioxidants may play a role in tendon repair after rotator cuff repair, the existing technology still has the following gaps in this field: first, the synergistic improvement effect of specific antioxidants (such as vitamin C and N-acetylcysteine) on skeletal muscle fat infiltration and fibrosis after rotator cuff injury has not been determined; second, there is a lack of specific drug administration schemes (including dosage form, dosage, administration cycle, etc.) for the above pathological processes; third, the recovery effect of antioxidants on the biomechanical properties of injured skeletal muscle (which is directly related to the risk of postoperative re-tear) has not been systematically verified. Therefore, developing a new type of therapeutic drug based on antioxidants that can simultaneously inhibit fat infiltration and fibrosis has important clinical value for improving the prognosis of patients with rotator cuff injury. SUMMARY

[0005] The purpose of this invention is to provide the application of antioxidants in the preparation of drugs for treating skeletal muscle fat infiltration and fibrosis after rotator cuff injury, specifically to determine the effective role of specific antioxidants (such as vitamin C and N-acetylcysteine) in inhibiting the aforementioned pathological changes, and the corresponding dosing regimens. Based on a rotator cuff injury model established by the inventors in SD rats, this invention confirms the significant ameliorative effects of vitamin C and N-acetylcysteine ​​on skeletal muscle fat infiltration and fibrosis after rotator cuff injury, and proposes specific dosage forms, dosages, and treatment cycles to reduce the risk of postoperative re-tear.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The application of antioxidants in the preparation of drugs for treating skeletal muscle fat infiltration and fibrosis after rotator cuff injury, wherein the antioxidant is one or both of vitamin C (L-ascorbic acid) and N-acetylcysteine ​​(NAC). Preferably, vitamin C and N-acetylcysteine ​​are used, covering different antioxidant pathways (e.g., vitamin C preferentially upregulates SOD1 / SOD2, and NAC preferentially upregulates SOD3 / PRDX5), synergistically enhancing the antioxidant defense effect.

[0008] Optionally, the drug may also include other pharmaceutically acceptable antioxidants, such as vitamin E, glutathione, resveratrol, etc., as alternative or adjunctive active ingredients to provide additional benefits against different oxidative stress pathways.

[0009] The drug is prepared into a clinically acceptable formulation using conventional pharmaceutical methods. The formulation is selected from granules, capsules, tablets, suspensions, emulsions, solutions, or syrups.

[0010] The drug is preferably prepared as an oral formulation (non-invasive and convenient administration, suitable for long-term use). Specific dosage forms may include oral solutions, tablets, capsules, or granules; among these, oral solutions are the most preferred dosage form (rapid absorption, easy and precise dosage control, and good efficacy demonstrated in animal studies). One or more pharmaceutically acceptable excipients may be added to the formulation, such as: sterile purified water as the solvent (for preparing oral solutions); stabilizers such as sodium bicarbonate (to adjust pH to maintain the stability of vitamin C); fillers such as microcrystalline cellulose (for tablet or capsule formation); and sweeteners such as steviol glycosides (to improve taste and enhance oral compliance).

[0011] In a preferred embodiment, the vitamin C concentration in the oral solution is 0.8%–1.2% (preferably about 1.0%), corresponding to an adult daily dose of about 500–1000 mg (this dose is calculated based on a 1% solution in rat experiments: a 200 g rat administered 10 mL of the solution daily by gavage is equivalent to about 500 mg of vitamin C daily for a 60 kg adult). The N-acetylcysteine ​​concentration in the oral solution is 0.15%–0.2% (preferably about 0.17%), corresponding to an adult daily dose of about 300–600 mg (similarly calculated: a 200 g rat administered 10 mL of a 0.17% solution daily by gavage is equivalent to about 300 mg of NAC daily for a 60 kg adult). Administration begins one week before the rotator cuff injury model is established and continues for at least eight weeks after model establishment. This administration period covers the key processes of skeletal muscle pathological changes following rotator cuff injury, helping to inhibit the vicious cycle of "oxidative stress–inflammatory response–pathological degeneration" at an early stage.

[0012] The antioxidants exert their therapeutic effects through a multi-target mechanism, including but not limited to:

[0013] (1) Enhance antioxidant defense: Upregulate the gene and protein expression of superoxide dismutase (SOD1, SOD2, SOD3) and peroxide reductase 5 (PRDX5), increase SOD enzyme activity in tissues, and reduce the carbonyl content of protein, a marker of oxidative damage, thereby clearing excess ROS and reducing the damage of oxidative stress to tissues.

[0014] (2) Inhibit fat infiltration: Downregulate the expression of adipogenic differentiation-related genes (such as PPARγ, CEBPα, FABP4, Leptin, Adipoq), reduce the formation of lipid droplets in myofibrils (Perilipin-1 positive lipid droplet signal is significantly reduced), block the abnormal differentiation of fibroadipocytes (FAPs) into adipocytes, thereby reducing fat infiltration in skeletal muscle.

[0015] (3) Reduce fibrosis: Reduce abnormal deposition of collagen fibers in damaged muscles (Masson staining and Sirius red staining indicate a decrease in the proportion of collagen area), improve the arrangement of collagen fibers, and inhibit excessive activation of myofibroblasts, thereby alleviating the degree of muscle fibrosis.

[0016] (4) Relieve inflammatory response: Reduce the aggregation of inflammatory cells at the site of injury (reduce the number of CD45 positive cells), inhibit the release of pro-inflammatory factors such as TNF-α and IL-1β, improve the skeletal muscle microenvironment, and avoid the chain reaction of amplified inflammation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Dual Pathological Intervention: Animal model experiments have for the first time clearly demonstrated that vitamin C and N-acetylcysteine ​​can significantly and simultaneously inhibit both fatty infiltration and fibrosis in skeletal muscle after rotator cuff injury, overcoming the shortcomings of existing treatments that are "single-target intervention with limited effects." Under antioxidant intervention, the area of ​​fatty infiltration and Perilipin-1 positive lipid droplets stained with Oil Red O in the supraspinatus muscle tissue were significantly reduced, and Masson and Sirius red staining showed a reduction in collagen deposition, proving that this invention can simultaneously improve fatty infiltration and fibrosis. This drug treatment can address preoperative rotator cuff injuries, delaying or even avoiding surgical intervention, and can also be used for postoperative recovery of severe rotator cuff injuries, reducing the risk of re-tear.

[0019] (2) Clear clinical translational value: The dosage, formulation, and dosing cycle proposed in this invention are all based on animal experimental verification results, which can directly provide guidance for clinical drug use. The vitamin C and N-acetylcysteine ​​used are both marketed safe drugs, and their safety has been verified in clinical practice, reducing the risk of translating the regimen into clinical application.

[0020] (3) Improved biomechanical properties: Mechanical tests in animal experiments demonstrated that intervention with antioxidants significantly improved the mechanical strength indices of the supraspinatus-tendon complex in rats with rotator cuff injuries: the maximum failure load increased by approximately 1.2–1.3 times, stiffness by approximately 1.3–1.8 times, and failure energy by approximately 1.5–2 times. This improvement in muscle tissue mechanical properties indicates a significant reduction in the risk of postoperative re-tear and contributes to long-term functional recovery.

[0021] (4) Convenient and safe administration: The treatment regimen is administered orally, requiring no invasive procedures, resulting in high patient compliance. No significant toxic side effects were found with long-term use of Vitamin C and NAC; in rat experiments, long-term administration did not cause abnormal changes in animal weight or damage to major organs, indicating that the regimen of this invention has good safety.

[0022] (5) Clear and controllable mechanism: This invention exerts its therapeutic effect by specifically regulating the three-level pathological pathway of "oxidative stress-adipogenesis-fibrosis", with a clear and well-defined target. This provides a basis for further optimization of dosage and potential combination therapy, and is conducive to monitoring and controlling the efficacy. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the surgical modeling process for the acute rotator cuff injury model in SD rats in Example 1. A—Locating the surgical area; B—Incising the skin and bluntly dissecting the subcutaneous tissue; C—Exposing the supraspinatus muscle; D—Severing the supraspinatus tendon to create a rotator cuff injury; E—Suturing the deltoid muscle to cover the wound; F—Suturing the skin incision to complete the model establishment.

[0024] Figure 2HE staining results of supraspinatus muscle tissue from rats in each group at 2, 4 and 8 weeks post-surgery in Example 1.

[0025] Figure 3 This study compares the inflammatory cell infiltration in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks post-surgery (CD45 immunofluorescence staining).

[0026] Figure 4 The results of Masson staining of supraspinatus muscle tissue in each group of rats in Example 1 at 2, 4 and 8 weeks after surgery are shown.

[0027] Figure 5 The results of Sirius red staining of supraspinatus muscle tissue in each group of rats in Example 1 at 2, 4 and 8 weeks after surgery are shown.

[0028] Figure 6 The results of comparing the SOD antioxidant enzyme activity in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks after surgery in Example 1 (WST-8 colorimetric method). **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0029] Figure 7 This is a comparison of protein carbonyl content in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks post-surgery in Example 1. **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0030] Figure 8 The results show the immunofluorescence of ROS content in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks after surgery in Example 1.

[0031] Figure 9 This is a comparison of the expression levels of oxidative stress-related genes (antioxidant genes such as SOD1 / 2 / 3 and PRDX5) in the supraspinatus muscle tissue of rats in different groups at 2, 4, and 8 weeks after surgery (RT-PCR analysis). **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0032] Figure 10 The figures show the Oil Red O staining results of the degree of fat infiltration in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks after surgery, as well as the area of ​​lipid deposition in the muscle. **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0033] Figure 11 The images show the immunofluorescence results of Perilipin-1, a marker of fatty infiltration, in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks post-surgery, and the distribution of Perilipin-1-positive lipid droplets in the muscle. **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0034] Figure 12 The immunohistochemical results of adipogenic transcription factor CEBPα in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks after surgery in Example 1, and the differences in CEBPα positive expression. **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0035] Figure 13 The immunohistochemical results and changes in PPARγ expression levels in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks post-surgery are shown in Example 1. **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0036] Figure 14 This section compares the expression levels of other lipid metabolism-related gene genes (Leptin, Adipoq, etc.) in the supraspinatus muscle tissue of rats in each group at 2, 4, and 8 weeks post-surgery. **** indicates a highly significant difference compared to the control group (P < 0.0001).

[0037] Figure 15 This section compares the changes in body weight and the rate of wet weight loss of the supraspinatus muscle in rats at 2, 4, and 8 weeks post-surgery in each group of rats in Example 2. Where: a indicates no significant difference between the 2-week and 4-week groups; b indicates a significant difference between the two marked groups (P < 0.05); **** indicates an extremely significant difference compared to the control group (P < 0.0001).

[0038] Figure 16 This is a schematic diagram of the biomechanical tensile test of the rat supraspinatus muscle-tendon complex in Example 2. The supraspinatus tendon was fixed between the fixtures of the material testing machine using the Krackow suture method. An initial preload and a constant tensile speed were set, and the mechanical parameters at the time of tendon rupture were measured.

[0039] Figure 17 The results show the maximum failure load of the supraspinatus muscle-tendon complex in each group of rats at 4 and 8 weeks post-surgery in Example 2. * indicates a significant difference compared to the control group (P < 0.05); ** indicates an extremely significant difference (P < 0.01).

[0040] Figure 18 The results show the stiffness of the supraspinatus muscle-tendon complex in each group of rats at 4 and 8 weeks post-surgery in Example 2. ** indicates a highly significant difference compared to the control group (P < 0.01).

[0041] Figure 19 The results show the failure energy of the supraspinatus muscle-tendon complex in each group of rats at 4 and 8 weeks post-surgery in Example 2. ** indicates a highly significant difference compared to the control group (P < 0.01); *** indicates a highly significant difference (P < 0.001). Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art, the experimental methods are conventional methods, and the materials and reagents used are all commercially available.

[0043] Example 1: Experimental study on the inhibition of skeletal muscle fat infiltration and fibrosis after rotator cuff injury by vitamin C and N-acetylcysteine

[0044] 1. Establishment of an animal model of rotator cuff injury: Seventy-two healthy male SD rats (SPF grade, weighing approximately 200g) were randomly divided into four groups (n≈18 per group): an injury model control group (control or control group), a vitamin C treatment group (VC group or V group), and an N-acetylcysteine ​​treatment group (NAC group or N group). From one week before rotator cuff injury surgery until sacrifice, rats in groups V and N were continuously administered the corresponding antioxidant solution by gavage daily (group V received 1% vitamin C solution, and group N received 0.17% N-acetylcysteine ​​solution). Rats in the injury model control group were administered an equal volume of purified water. All rats were fed normally and were fasted and deprived of water for 12 hours before surgery.

[0045] like Figure 1 As shown, the surgery was performed under sterile conditions: Anesthesia was administered via intraperitoneal injection of 3% sodium pentobarbital solution (1.0–1.5 mL / kg). After adequate anesthesia, the rats were fixed in a lateral decubitus position. The hair on the left shoulder area was shaved, and the skin was disinfected with povidone-iodine. A longitudinal incision of approximately 0.5 cm was made at the left acromion landmark, and the subcutaneous tissue was bluntly dissected to expose the deltoid muscle. A small incision was made along the posterior fiber direction of the deltoid muscle to expose the attachment of the supraspinatus tendon. Using small curved forceps, the supraspinatus muscle was passed through the scapular spine from above, and 4-0 nylon suture was passed between the supraspinatus muscle and the scapular spine and ligated proximally to fix the tendon. The supraspinatus tendon was then cut at the greater tubercle of the humerus (without suture repair), creating a complete rotator cuff tear model. After rinsing the surgical field with saline, the deltoid muscle and skin incision were sutured in layers without implanting any repair material. Postoperatively, rats in each group were administered the appropriate solution or pure water via gavage according to their group assignment. All animals were allowed free movement and routinely administered antibiotics (penicillin, 30,000 U per animal, intramuscular injection, once daily for 3 consecutive days) to prevent infection. At 2, 4, and 8 weeks postoperatively, some rats from each group were sacrificed (by overdose anesthesia and neck dislocation method), and the entire supraspinatus muscle and its tendon attachment tissue from both shoulders were harvested for subsequent analysis.

[0046] 2. HE staining (hematoxylin-eosin staining): The collected supraspinatus muscle tissue was fixed, dehydrated, and routinely embedded in paraffin. Sections were prepared to a thickness of 5 μm, yielding paraffin sections. After dewaxing in xylene and hydration with graded ethanol, the nuclei were stained with hematoxylin, and the cytoplasm with eosin. After sufficient differentiation and blueing in running water, dehydration with graded ethanol, and clearing with xylene, the sections were mounted with neutral resin. Changes in muscle fiber morphology and nucleus morphology were observed under a light microscope. Results are as follows: Figure 2 As shown.

[0047] 3. Masson Staining: After dewaxing paraffin sections to water, they were mordanted overnight at room temperature with Bouin's solution to improve staining. The sections were then stained sequentially with hematoxylin and Mayer's trichrome staining reagent: first, the nuclei were stained with Weigert hematoxylin solution, then counterstained with Mayer's hematoxylin, followed by differentiation with acidic differentiation solution, thorough rinsing with running water, and then stained sequentially with Ponceau S, acid fuchsin, phosphomolybdic acid solution, and aniline blue solution to develop collagen fibers. Finally, the sections were rapidly dehydrated with anhydrous ethanol, cleared with xylene, and mounted with neutral resin. The staining results showed that collagen fibers appeared blue and muscle fibers appeared red, demonstrating collagen deposition in muscle fibers and used to assess the degree of fibrosis. Results are as follows... Figure 4 As shown, compared with the control group, the amount of muscle tissue fibrosis in groups V and N was significantly reduced.

[0048] 4. Sirius Red Staining: After dewaxing paraffin sections to water, the nuclei were stained with iron hematoxylin solution for 10 minutes and rinsed with running water. Sirius red staining solution was then added and incubated for approximately 1 hour. The sections were then rapidly washed twice with acidified aqueous solution and rinsed with distilled water. After routine dehydration with ethanol and clearing with xylene, the sections were mounted. Under a polarized light microscope, mature collagen fibers appeared red-orange, while immature collagen appeared green. This staining method can help assess the arrangement and maturity of collagen fibers, allowing observation of changes in collagen fiber arrangement and type. Results are as follows: Figure 5 As shown.

[0049] 5. Oil Red O Staining: Frozen sections (8–10 μm thick) of freshly collected supraspinatus muscle tissue were prepared. After equilibration at room temperature, the sections were washed with PBS buffer for 10 minutes. Pretreatment with 60% isopropanol for approximately 30 seconds was performed, followed by discarding the isopropanol. The sections were immediately immersed in pre-prepared Oil Red O staining working solution and stained in a sealed container for 15 minutes. The sections were then removed, slightly differentiated with 60% isopropanol, and rinsed with distilled water. The nuclei were then counterstained with hematoxylin for 5 minutes, followed by rinsing with running water. Finally, the sections were mounted with a water-based mounting medium. Oil Red O stains lipids red, and hematoxylin stains the nuclei blue, allowing for quantitative analysis of the area ratio of fatty infiltration (red lipid droplets) within the muscle tissue under a microscope. Results are shown below. Figure 10 As shown.

[0050] 6. Immunohistochemistry (IHC): After routine dewaxing to water, paraffin sections are microwaved in EDTA buffer for antigen retrieval (high temperature 3 minutes + medium temperature 4 minutes + low temperature 4 minutes, then naturally cooled). Sections are incubated with catalase blocking agent at room temperature for 15 minutes to block endogenous peroxidase activity, followed by washing with PBS. Then, non-specific sites are blocked with 5% goat serum at 37°C for 30 minutes. Primary antibodies (such as anti-CEBPα, anti-PPARγ, etc.) are added, and the sections are incubated overnight at 4°C, followed by washing with PBS. The next day, after incubation at 37°C, the corresponding HRP-labeled secondary antibody is added, and the sections are incubated at 37°C for 30 minutes, followed by washing with PBS. DAB chromogenic reagent is used for development; the development time is controlled under a microscope until the target signal appears, at which point the reaction is terminated by rinsing with distilled water. Cell nuclei are counterstained with hematoxylin for 40 seconds, differentiated in running water, and then blued back to normal. After dehydration with graded ethanol and clearing with xylene, the sections are mounted with neutral resin. Primary antibody is omitted for negative control sections. The location and intensity of the brownish-red DAB positive signal were observed under a microscope, and the positive area or optical density was quantitatively calculated using image analysis software. Figure 12 , Figure 13 As shown.

[0051] 7. Immunofluorescence detection of ROS: After equilibration with PBS, frozen sections were fixed in 4% paraformaldehyde fixative for 15 minutes, followed by three washes with PBS. The sections were then incubated with commercial ROS fluorescent probe working solution at 37°C in the dark for 30 minutes to react with intracellular ROS and generate fluorescent products. Unreacted probes were washed away with PBS. Cell nuclei were counterstained with DAPI for 15 minutes, washed thoroughly with PBS, and mounted with an anti-fluorescence quencher. Images were observed and captured under a fluorescence microscope; the intensity of red fluorescence represents the ROS level. Results are as follows: Figure 8 As shown.

[0052] 8. Immunofluorescence detection of inflammatory cells and lipid droplets (CD45 / Perilipin-1): After equilibration with PBS, frozen sections were blocked with 5% sheep serum in a humidified chamber at 37°C for 1 hour. Primary antibodies (anti-CD45 or anti-Perilipin-1, labeled with the total number of inflammatory cells and lipid droplet surface proteins, respectively) were added, and the sections were incubated overnight at 4°C, followed by washing with PBS. After warming to 37°C, fluorescently labeled secondary antibodies (such as FITC or Cy3-labeled secondary antibodies) were added, and the sections were incubated at 37°C for 30 minutes, followed by washing with PBS. Nuclei were stained with DAPI for 15 minutes, and the sections were then mounted. The distribution and number changes of CD45-positive cells (e.g., red) and Perilipin-1-positive lipid droplets (e.g., red) were observed under a confocal fluorescence microscope, and quantitative image analysis was performed. Results are as follows: Figure 3 and 11 As shown.

[0053] 9. Total SOD Activity Assay (WST-8 Method): Fresh supraspinatus muscle tissue was homogenized under ice bath conditions at a ratio of 100 μL SOD sample lysis buffer per 10 mg of tissue. The homogenate was centrifuged at 12000 rpm for 5 minutes at 4℃, and the supernatant was used as the test sample. The total superoxide dismutase activity of each sample was determined using the WST-8 colorimetric method (SOD activity kit). The specific steps included: preparing a mixture of detection buffer, WST-8 chromogenic reagent, and enzyme working solution; adding the sample to the reaction wells of a 96-well plate; incubating for 30 minutes; and measuring the absorbance of each well at 450 nm. The SOD inhibition rate was calculated based on the blank control and converted to enzyme activity units (U / mL). This method affects the colorimetric reaction by inhibiting O2⁻· production, and the final absorbance change reflects the actual activity of SOD enzyme in the sample. Results are as follows: Figure 6 As shown.

[0054] 10. Protein Carbonyl Content Detection: Protein carbonyl content is an indicator of the degree of oxidative damage in tissues. Approximately 200 mg of frozen muscle tissue was homogenized with 2 mL of extraction buffer and centrifuged at 5000 rpm for 10 minutes at 4°C. The supernatant was collected for analysis. The sample supernatant reacted with 2,4-dinitrophenylhydrazine (DNPH) to generate a hydrazone derivative. Unreacted fractions were removed by TCA precipitation, followed by repeated washing of the precipitate three times with an ethanol / ethyl acetate mixture to remove lipids and other interferences. Finally, the precipitate was dissolved in GuHCl solution, and the absorbance (A370) was measured at 370 nm. Using a parallel control without DNPH as a reference, the carbonyl content (nmol / mg) per milligram of sample protein was calculated using a formula, reflecting the level of protein oxidative damage. Results are as follows. Figure 7 As shown.

[0055] 11. RT-PCR Detection of Gene Expression: 100 mg of supraspinatus muscle tissue was used, and total RNA was extracted using TRIzol reagent. The RNA was purified by chloroform separation, isopropanol precipitation, and washing with 75% ethanol. The purified RNA was then dissolved in RNase-free water to determine its purity and concentration. The RNA sample was then treated with a genomic DNA removal reagent, followed by cDNA synthesis: using 1 μg of total RNA as a template, reverse transcription reagent (containing PrimeScript reverse transcriptase, random primers, etc.) was added and reacted at 37℃ for 15 minutes, terminated at 85℃ for 5 seconds, to obtain cDNA. Real-time quantitative PCR (qPCR) was used to detect the mRNA expression level of the target gene. The PCR reaction system consisted of 10 μL of premixed enzyme, primers, and template cDNA. After pre-denaturation at 95℃, the mixture was cycled for amplification (e.g., 95℃ for 30 seconds, 60–65℃ for 30 seconds, 72℃ for 2 minutes, for 40–50 cycles). This experiment designed specific primers for various genes associated with lipid infiltration and oxidative stress, such as: antioxidant enzymes SOD1, SOD2, SOD3, and PRDX5; adipogenic genes PPARγ, CEBPα, and FABP4; and adipokine genes Leptin and Adipoq. β-actin or GAPDH was used as an internal reference gene, and the relative expression levels of each gene were calculated using the 2^-ΔΔCt method. The results were standardized and used for comparisons between different groups and time points. Results are shown below. Figure 9 and 14 As shown.

[0056] Experimental Results: Histological and molecular biological data obtained using the above methods showed that, compared with the untreated injury model control group, both vitamin C and N-acetylcysteine ​​intervention significantly reduced the degree of fatty infiltration and fibrosis in the supraspinatus muscle of rats at 2, 4, and 8 weeks post-surgery. Specifically, the fat area of ​​the supraspinatus muscle tissue stained with Oil Red O in the antioxidant group was significantly smaller than that in the control group, and Perilipin-1 immunofluorescence quantitative analysis showed a decrease in the number of lipid droplets. Masson and Sirius red staining results showed that collagen fiber deposition was reduced and more orderly arranged in the treatment group, and the fibrosis score was lower. Consistent with this, oxidative stress indicators in the antioxidant-treated group were also significantly improved: SOD activity was significantly increased compared with the untreated control group, mRNA expression of antioxidant-related genes SOD1 / 2 / 3 and PRDX5 was upregulated, while protein carbonyl content was significantly lower than that in the control group; at the same time, the expression of adipogenic genes PPARγ and CEBPα was inhibited, and the expression of inflammatory cell infiltration (CD45 positive count) was also reduced compared with the control group. In summary, vitamin C and NAC intervention effectively alleviated fatty infiltration and fibrosis of skeletal muscle after rotator cuff injury, and enhanced antioxidant and anti-inflammatory defenses. These effects were evident even when used as monotherapy, verifying the effectiveness of this invention.

[0057] Example 2: Effects of Vitamin C and N-acetylcysteine ​​Intervention on Biomechanical Properties of Supraspinatus Muscle After Rotator Cuff Injury

[0058] In this embodiment, the improvement of the supraspinatus-tendon complex strength after injury was evaluated through biomechanical experiments using antioxidant treatment. The experimental animals were grouped and treated as in Example 1. At 4 and 8 weeks post-surgery, rats in each group were sacrificed, and the left supraspinatus muscle along with its tendon-bone attachment was immediately harvested. Figure 16 As shown, the moist-preserved specimen was used for uniaxial tensile mechanical testing: the two ends of the supraspinatus tendon were sutured with high-strength sutures using the Krackow suture method and fixed to the upper and lower clamps of the material testing machine. A preload of 0.1 N was applied to eliminate system relaxation, and then the specimen was stretched at a constant speed of 1 mm / s until complete fracture. Real-time force-displacement data were recorded during the stretching process to obtain parameters such as the maximum failure load (maximum tensile force at tendon rupture, N), total failure energy (energy absorbed during fracture, J), and stiffness (slope of the linear elastic phase, N / mm). The results are as follows: Figure 15 , Figure 17 and Figure 18 As shown.

[0059] Experimental Results: Mechanical testing showed that antioxidant treatment significantly improved the mechanical strength of the muscle-tendon complex after rotator cuff injury. Specifically, at 4 and 8 weeks post-surgery, compared with the untreated control group, the maximum failure load of the supraspinatus muscle in rats in the vitamin C and N groups was significantly increased, with an average increase of approximately 30%–80%; tissue stiffness was significantly enhanced, with an average increase of approximately 30%–80%; and failure energy was also significantly increased, with an average increase of approximately 50%–100% (e.g., ...). Figures 17-19 (As shown). The improved mechanical properties described above indicate that antioxidants help restore the structural strength of damaged muscle tissue and reduce the risk of secondary tearing. This result is consistent with the histological improvement observed in Example 1, further demonstrating that enhancing antioxidant defense to inhibit fatty infiltration and fibrosis can effectively protect and restore the function of rotator cuff muscles.

[0060] It should be noted that the above embodiments are only used to illustrate specific applications of the present invention, and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications or improvements without departing from the concept of the present invention, and these modifications or improvements should also be considered within the scope of protection of the present invention. The scope of protection claimed by the present invention is determined by the appended claims, and the specific embodiments in the specification serve to assist in understanding the principles of the present invention.

Claims

1. The application of antioxidants in the preparation of drugs for treating skeletal muscle fat infiltration and fibrosis after rotator cuff injury, wherein the antioxidant is one or both of vitamin C and N-acetylcysteine.

2. The application according to claim 1, characterized in that, The drug contains one or more pharmaceutically acceptable excipients or excipients.

3. The application according to claim 2, characterized in that, The drug is prepared into a clinically acceptable formulation using conventional pharmaceutical methods.

4. The application according to claim 3, characterized in that, The formulation is selected from granules, capsules, tablets, suspensions, emulsions, solutions, or syrups.

5. The application according to any one of claims 1 to 4, characterized in that, The drug is an oral administration formulation, in which the mass concentration of vitamin C is 0.8% to 1.2% and the mass concentration of N-acetylcysteine ​​is 0.15% to 0.2%, corresponding to a daily dose of approximately 500 to 1000 mg of vitamin C and 300 to 600 mg of N-acetylcysteine ​​for adult patients.

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