Medicament for treating ischemia-reperfusion injury and use thereof

Abstract

This invention relates to a drug for treating ischemia-reperfusion injury and its application. The drug activates the CCL2-chemokine receptor 2 signaling axis, triggering the downstream G protein αi2 subunit-mediated phosphatidylinositol 3-kinase / protein kinase B signaling pathway. This, in turn, synergistically regulates the antioxidant defense system driven by nuclear factor erythroid 2-related factor 2 and the B-cell lymphoma 2-mediated anti-apoptotic pathway, thereby treating the ischemia-reperfusion injury. The results of this study establish a novel therapeutic strategy for testicular ischemia-reperfusion injury based on engineered exosomes, and simultaneously reveal the previously undiscovered cytoprotective role of the CCL2 / CCR2 signaling axis in non-immune cells.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and biotechnology, specifically to the application of mesenchymal stem cell exosomes with upregulated CCL2 protein expression in the preparation of drugs for treating testicular ischemia-reperfusion injury and the drugs thereof. Background Technology

[0002] Testicular torsion is a common urological emergency, predominantly affecting adolescents and young men. Spermatic cord torsion obstructs testicular blood supply, leading to ischemic injury. While surgical reduction can restore testicular perfusion, it can induce secondary tissue damage, known as ischemia-reperfusion injury, which often exceeds the initial ischemia. Clinical studies have confirmed that testicular ischemia lasting more than 6 hours results in a survival rate of less than 50%, and for every additional hour of pre-hospital delay, the risk of orchiectomy increases by 4%, highlighting the clinical urgency of this condition. The pathophysiological mechanisms of injury after testicular torsion reduction are complex, primarily involving reactive oxygen species bursts, inflammatory cascade disorder, and blood-testis barrier disruption. Ultimately, this can lead to testicular atrophy in 12%–62% of cases, and approximately one-third of patients develop oligospermia, elevated antisperm antibodies, and other complications, severely impairing fertility. Despite the significant clinical importance of this disease, there are currently no targeted therapies to alleviate testicular reperfusion injury, maintain spermatogenesis, or improve long-term fertility outcomes after torsion reduction. Therefore, developing novel strategies to combat testicular ischemia-reperfusion injury has become an urgent clinical problem to be solved.

[0003] Mesenchymal stem cell-derived exosomes (MSC-Exos), especially human umbilical cord mesenchymal stem cell exosomes, have emerged as highly promising therapeutic carriers as nanoscale vesicles. Rich in bioactive substances such as proteins, nucleic acids, and lipids, they serve as important mediators of intercellular communication. Due to their low immunogenicity, ease of storage, and ability to deliver active cargo across cells, MSC-derived exosomes are considered a potential alternative to live cell therapy. Existing studies suggest that ordinary MSC exosomes possess antioxidant, anti-inflammatory, and anti-apoptotic effects to some extent; however, issues such as high component heterogeneity, unstable efficacy, and insufficient targeting and effect consistency limit their translational application in testicular I / R injury scenarios. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a drug for treating ischemia-reperfusion injury. The drug activates the CCL2-chemokine receptor 2 signaling axis, triggering the downstream G protein αi2 subunit-mediated phosphatidylinositol 3-kinase / protein kinase B signaling pathway, thereby synergistically regulating the antioxidant defense system driven by nuclear factor erythroid 2-related factor 2 and the anti-apoptotic pathway mediated by B-cell lymphoma 2, thereby treating the ischemia-reperfusion injury.

[0005] In one embodiment, the treatment of ischemia-reperfusion injury is testicular ischemia-reperfusion injury.

[0006] In one embodiment, the drug activates the CCR2 / GNAI2 / PI3K-Akt signaling axis in the Sertoli cells of the testis, synergistically regulating the Nrf2-driven antioxidant defense system and the Bcl-2-mediated anti-apoptotic effect, thereby treating the testicular ischemia-reperfusion injury.

[0007] In one embodiment, the drug is a mesenchymal stem cell exosome with upregulated CCL2 protein expression.

[0008] In one embodiment, the mesenchymal stem cell exosomes are hydrogen sulfide-treated mesenchymal stem cell exosomes.

[0009] In one embodiment, the mesenchymal stem cell exosomes are obtained by pretreatment with sodium hydrosulfide at concentrations of 1 μmol / L to 400 μmol / L.

[0010] In one embodiment, the mesenchymal stem cell exosomes are obtained by pretreatment with 200 μmol / L sodium hydrosulfide.

[0011] In one embodiment, the present invention provides a pharmaceutical composition for treating ischemia-reperfusion injury, the pharmaceutical composition comprising the aforementioned drug.

[0012] In one embodiment, the present invention provides the use of CCR2 protein in the preparation of drugs for treating ischemia-reperfusion injury.

[0013] In one embodiment, the CCR2 protein is derived from mesenchymal stem cell exosomes.

[0014] This study systematically prepared and identified H2S-Exos, and evaluated its therapeutic effect on testicular ischemia-reperfusion injury using an in vitro hypoxia-reoxygenation model supported by TM4 cells and an in vivo mouse testicular torsion model. The aim was to elucidate its potential molecular mechanisms, with a focus on screening for hydrogen sulfide-induced exosomal proteomic alterations. The study found that hydrogen sulfide pretreatment resulted in high abundance of chemokine ligand 2 (CCL2) expression in MSC-Exos. CCL2-rich H2S-Exos activated the CCL2-chemokine receptor 2 (CCR2) signaling axis, triggering the downstream G protein αi2 subunit (GNAI2)-mediated phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway, thereby synergistically regulating the Nrf2-driven antioxidant defense system and the Bcl-2-mediated anti-apoptotic pathway. The results of this study establish a novel treatment strategy for testicular ischemia-reperfusion injury based on engineered exosomes, and also reveal the cytoprotective role of the CCL2 / CCR2 signaling axis in non-immune cells that has not yet been discovered.

[0015] This study rigorously validated the hierarchical regulatory relationship of this signaling pathway through pharmacological inhibition experiments: the CCR2 antagonist BMS-CCR2 completely eliminated the protective effect of H2S-Exos, confirming that the binding of CCL2 to CCR2 is a necessary condition for initiating downstream signal transduction; the PI3K inhibitor apelelis and the Akt inhibitor MK2206 specifically blocked their respective downstream signaling cascades without affecting upstream molecules, confirming the linear regulatory relationship of CCL2 / CCR2→GNAI2→PI3K→Akt→(Nrf2 / HO-1 & Bcl-2). Notably, supplementing untreated exosomes with exogenous CCL2 replicated the effects of H2S-Exos, while supplementing H2S-Exos with exogenous CCL2 further enhanced pathway activation, confirming that high abundance expression of CCL2 is a necessary and sufficient condition for enhanced therapeutic effects of H2S-Exos. Immunohistochemical verification of testicular tissue further confirmed the above results. In mice treated with H2S-Exos, the expression of GNA12, phosphorylated PI3K, phosphorylated Akt, HO-1 and Bcl-2 in the supporting cells was significantly activated, while the expression of Keap1 was significantly suppressed. Moreover, HO-1 was only located in the supporting cells, suggesting that the supporting cells are the main target cells of H2S-Exos-mediated antioxidant protection.

[0016] An interesting finding of this study is that sperm morphology and function exhibit a disjointed response pattern after testicular ischemia-reperfusion injury. During the acute injury phase, sperm morphology remained largely unchanged, but motility was significantly impaired, suggesting functional damage to sperm membrane integrity or energy metabolism. This separation of structure and function underscores the importance of incorporating functional indicators into reproductive injury research. H2S-Exos intervention partially maintained sperm motility, indicating that exosome-mediated metabolic support may have unique advantages in maintaining sperm function under stress. Sperm survival is highly dependent on efficient energy metabolism, and ischemia-reperfusion injury disrupts mitochondrial adenosine triphosphate (ATP) production, leading to impaired sperm motility. The improving trend in sperm motility after H2S-Exos treatment suggests that it may maintain sperm function by enhancing mitochondrial bioenergetics or improving cellular metabolic adaptation under hypoxic conditions.

[0017] This study confirms that hydrogen sulfide pretreatment of mesenchymal stem cell exosomes can enhance the therapeutic effect of mesenchymal stem cell exosomes on testicular ischemia-reperfusion injury by increasing the abundance of CCL2 expression; H2S-Exos maintains the structural and functional integrity of the testis by activating the CCR2 / GNAI2 / PI3K-Akt signaling axis in Sertoli cells and synergistically regulating Nrf2-mediated antioxidant response and Bcl-2-mediated anti-apoptotic effect. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are representative Western blot images of CCL2 expression in untreated exosomes and H2S-Exos; Figure 2 This is a quantitative analysis of CCL2 protein levels using GAPDH as an internal control. Data are expressed as mean ± standard deviation (n=3), ***P<0.001; Figure 3 This is a representative fluorescence microscopy image of intracellular reactive oxygen species levels detected by the dichlorofluorescein diacetate probe. Scale bar: 100 μm. Figure 4 This is a graph showing the quantitative analysis results of reactive oxygen species fluorescence intensity; data are expressed as mean ± standard deviation (n=3), *P<0.05, **P<0.01; Figure 5This is a graph showing the superoxide dismutase activity in cell lysates. Data are expressed as mean ± standard deviation (n=3), *P<0.05, **P<0.01; Figure 6 This is a graph showing the malondialdehyde (MDA) content in cell lysates. Data are expressed as mean ± standard deviation (n=3), *P<0.05, **P<0.01; Figure 7 This is a Johansson score chart showing the integrity of seminiferous tubules and spermatogenic function. Each sample was evaluated by two blinded pathologists, assessing at least 50 seminiferous tubules. Data are expressed as mean ± standard deviation (n=8). *P<0.05, **P<0.01, ***P<0.001 Figure 8 This is a graph showing the superoxide dismutase activity in testicular tissue homogenate; data are expressed as mean ± standard deviation (n=8), *P<0.05, **P<0.01, ***P<0.001 Figure 9 This is a graph showing the malondialdehyde (MDA) content in testicular tissue homogenate; data are expressed as mean ± standard deviation (n=8), *P<0.05, **P<0.01, ***P<0.001 Figure 10 This is a schematic diagram illustrating the molecular mechanism by which H2S-Exos alleviates testicular ischemia-reperfusion injury. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] I. Materials and Methods

[0022] 1. Preparation, isolation, identification, and optimization of preparation parameters of exosomes 1.1 Preparation of untreated exosomes and hydrogen sulfide pretreated exosomes

[0023] Fourth-generation human umbilical cord mesenchymal stem cells were passaged to the sixth generation, and processed when the cell confluence reached 90%. The hydrogen sulfide pretreated group was cultured in medium containing different concentrations of sodium hydrosulfide (1, 200, 400, 1000 μmol / L) for 24 hours, followed by replacement with fresh ordinary medium; the untreated exosome control group was cultured in ordinary medium throughout. Cell supernatants were collected and mixed 24 and 48 hours after medium replacement, and subjected to gradient centrifugation to remove viable cells and cell debris. The supernatant was concentrated using a pre-washed 100 kDa ultrafiltration tube and then filtered through a 0.22 μm filter membrane. The protein concentration of the exosome preparation was determined by the dicaprylic acid (BCA) method. After adding protease inhibitors, the samples were aliquoted and stored at -80°C.

[0024] 1.2 Identification of untreated exosomes and hydrogen sulfide pretreated exosomes According to the guidelines of the International Society for Extracellular Vesicles, the isolated vesicles were identified to confirm their exosome properties, specifically including: ① detection of the expression of exosome markers CD9, CD63, CD81 and Alix by Western blotting; ② observation of vesicle morphology by transmission electron microscopy (Hitachi HT7800, Japan); ③ detection of vesicle particle size and concentration by nanoparticle tracking analysis technology (Malvin NanoSight NS300, UK); and sterility, mycoplasma and endotoxin tests of the prepared product.

[0025] 1.3 Determination of hydrogen sulfide content The methylene blue method was used to detect hydrogen sulfide levels at key stages of exosome preparation. Sodium hydrosulfide stock solution, conditioned medium after cell pretreatment, cell supernatant, and exosome suspensions before and after ultrafiltration were mixed with zinc acetate, N,N-dimethyl-p-phenylenediamine, and ferric chloride, respectively. Absorbance was measured at 665 nm, and hydrogen sulfide concentration was calculated based on a standard curve.

[0026] 1.4 Optimization of Exosome Preparation Parameters 1.4.1 Optimization of Sodium Hydrosulfide Pretreatment Concentration

[0027] To determine the optimal sodium hydrosulfide concentration for mesenchymal stem cell pretreatment, a TM4 cell hypoxia-reoxygenation injury model was first constructed according to the method described in Section 2.2.2. At the start of reoxygenation, untreated exosomes or H2S-Exos (both at a concentration of 5 × 10⁻⁶ μmol / L) were prepared from mesenchymal stem cells pretreated with different concentrations of sodium hydrosulfide (1, 200, 400, and 1000 μmol / L). 8Cells were treated with NaHS (particles / mL) for 24 hours, and cell viability was detected using a cell counting kit-8 (CCK-8). The results were verified by observing cell morphology. It should be noted that sodium hydrosulfide itself is not hydrogen sulfide, but it continuously releases active hydrogen sulfide molecules in the system to exert its effect. For the sake of simplicity and alignment of the mechanism, the NaHS donor treatment group is uniformly referred to as the hydrogen sulfide treatment group. 1.4.2 Optimization of exosome intervention dosage

[0028] The optimal therapeutic dose of H2S-Exos was investigated by pretreatment with the optimal sodium hydrosulfide concentration. At the start of reoxygenation, four different particle concentrations of H2S-Exos (2.5 × 10⁻⁶) were used. 8 5×10 8 10×10 8 20×10 8 TM4 cells subjected to hypoxia-reoxygenation injury were treated with particles / mL for 24 hours. The optimal intervention dose was selected based on the cell viability and cell morphology recovery as detected by the CCK-8 assay.

[0029] 1.5 Proteomic analysis of exosomal protein expression To systematically evaluate the effect of hydrogen sulfide pretreatment on exosome contents, a comparative proteomics analysis was performed on untreated exosomes and H2S-Exos prepared by pretreatment with an optimized concentration (200 μmol / L) of sodium hydrosulfide, and differentially expressed proteins were verified by Western blotting.

[0030] 1.5.1 Sample Preparation Exosomal proteins were dissolved in a buffer solution containing 1% sodium deoxycholate, 10 mmol / L tris(2-carboxyethyl)phosphine, and 40 mmol / L chloroacetamide (pH 8.5), and the protein concentration was determined by the dioctanine method. The sodium deoxycholate concentration was diluted to 0.5%, and trypsin was added at a protein-to-protein ratio of 1:50. The proteins were then digested overnight at 37°C. The resulting peptides were acidified with 1% trifluoroacetic acid and desalted using an SDB-RPS solid-phase extraction column.

[0031] 1.5.2 Mass Spectrometry Analysis The peptides were resuspended in 0.1% formic acid solution and analyzed by liquid chromatography-tandem mass spectrometry using a Thermo Fisher Vanquish Neo ultra-high performance liquid chromatography system in tandem with an Orbitrap Astral mass spectrometer (nanospray ion source). The ion source voltage was set to 1800 V, the ion transfer tube temperature to 280 °C, and the data-independent acquisition (DIA) mode was used with a normalized collision energy of 25% and a default charge number of 2. The peptides were separated using a self-made column (15 cm × 150 μm inner diameter, C18 stationary phase, 100 Å pore size, 1.5 μm particle size) with a gradient elution time of 22 min. Mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was an 80% acetonitrile solution containing 0.1% formic acid. The first-level mass spectrometry parameters for data acquisition independence are: Orbitrap resolution 240,000 (mass-to-charge ratio at 200), scan range mass-to-charge ratio 380,980, maximum injection time 5 ms, and automatic gain control target value 500%; the second-level mass spectrometry parameters are: isolation window 2Th, high-energy collision dissociation collision energy 25%, precursor ion scan range mass-to-charge ratio 380,980, daughter ion scan range mass-to-charge ratio 150~2000, maximum injection time 3 ms, and automatic gain control target value 500%.

[0032] 1.5.3 Mass Spectrometry Data Analysis Raw data were processed using DIA-NN software (v2.1.0) in library-free mode, and the human UniProt database (20,422 annotated entries) was searched. Search parameters: trypsin / P digestion, allowing one missed cleavage site, a maximum of two variable modifications per peptide, peptide length of 552 amino acids, precursor ion charge number of 16, precursor ion mass-to-charge ratio range of 380-980, daughter ion mass-to-charge ratio range of 150-2000; the false discovery rate at both peptide and protein levels was controlled to be within 1%.

[0033] 1.5.4 Principal Component Analysis Principal component analysis was used to assess the overall differences in protein expression profiles between the untreated exosome group and the H2S-Exos group. The log-2 transformed protein intensity data were centered and standardized using R language stats (version 4.3.1) before analysis.

[0034] 1.5.5 Screening for Differentially Expressed Proteins Differentially expressed proteins in exosomes were screened based on a fold change > 1.5 and a p < 0.05. Fisher's exact test was used to assess the significance of pathway enrichment. Proteins with a fold change of |log²| > 1 and a p < 2 were defined as significantly differentially expressed proteins.

[0035] 1.5.6 Western blot analysis to verify high abundance of CCL2 expression Exosomal proteins were lysed using RIPA lysis buffer, and protein concentration was determined by the dioctanine method. 40 μg of protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After incubation with CCL2 primary antibody, protein expression was detected.

[0036] 1.5.7 GO / KEGG Enrichment Analysis Using the clusterProfiler package (v4.0) in R, gene ontology (GO, including biological processes, cellular components, and molecular functions) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on upregulated proteins. The significance threshold was set as P < 0.05 after Benjamini-Hochberg correction. The enrichment results were visualized using the ggplot2 package (v3.4.0).

[0037] 2. Cell culture and hypoxia-reoxygenation model construction

[0038] Mouse support cell line TM4 was cultured on DuPont modified Eagle medium / nutrient mixture F-12 (1:1), with 2.5% fetal bovine serum, 5% horse serum and 1% penicillin-streptomycin antibiotics added to the medium; the cells were cultured in a constant temperature incubator at 37°C and 5% carbon dioxide.

[0039] 2.2 Construction of Hypoxia-Reoxygenation Model Following previous research methods, a hypoxia-reoxygenation model of TM4 cells was constructed by inducing chemical hypoxia with cobalt chloride. Cells were exposed to different concentrations of cobalt chloride (0, 62.5, 125, 250, and 500 μmol / L) for 24 hours to establish the hypoxia model, followed by reoxygenation for 24 hours using normal complete culture medium. Cell viability was assessed using the CCK-8 assay, and the protein level of hypoxia-inducible factor 1α (HIF-1α) in the supernatant was detected using enzyme-linked immunosorbent assay (ELISA). Based on a comprehensive evaluation of cell viability, HIF-1α expression, and cell morphology, the optimal cobalt chloride concentration for subsequent experiments was determined.

[0040] 3. In vitro experimental design 3.1 Experimental grouping for evaluating the therapeutic effect of H2S-Exos

[0041] To evaluate the protective effect of H2S-Exos against hypoxia-reoxygenation injury, TM4 cells were divided into four groups: control group, hypoxia-reoxygenation group, untreated exosome group, and H2S-Exos group. Control group cells were cultured normally without any treatment; hypoxia-reoxygenation group cells were treated with 250 μmol / L cobalt chloride for 24 hours, followed by reoxygenation for 24 hours; untreated exosome group cells were treated with 5 × 10⁻⁶ exosomes at the start of reoxygenation. 8Untreated exosomes at a concentration of 5 × 10⁶ particles / mL were used to treat hypoxia-reoxygenation-damaged cells for 24 hours; the H₂S-Exos group received 5 × 10⁶ particles / mL at the start of reoxygenation. 8 Cells damaged by hypoxia-reoxygenation were treated with H2S-Exos particles / mL for 24 hours. Cells were collected after 24 hours of treatment for subsequent assays.

[0042] To verify the role of the CCL2 / CCR2 / GNAI2 / PI3K-Akt signaling axis, cells were pretreated for 24 hours with a CCR2 antagonist (BMS-CCR2, 20 nmol / L), a PI3K inhibitor (apelis, 300 nmol / L), or an Akt inhibitor (MK2206, 100 nmol / L) before exosome treatment.

[0043] 3.2 Detection of oxidative stress levels Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Intracellular reactive oxygen species (ROS) levels were detected using a dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. Cells were incubated with 10 μmol / L DCFH-DA at 37°C in the dark for 20 minutes. After washing with phosphate buffer, the cells were imaged using a fluorescence microscope, and the average fluorescence intensity was quantified using ImageJ software (version 1.53, National Institutes of Health).

[0044] 3.3 Apoptosis and cell viability detection Apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate / propidium iodide double staining, and the proportion of apoptotic cells was analyzed using FlowJo software. Cell viability was detected by acridine orange / propidium iodide double staining. Cells were incubated with 1 μg / mL acridine orange and 5 μg / mL propidium iodide for 15 minutes, and the images were taken by fluorescence microscopy. The number of live cells (acidine orange positive, green) and dead cells (propidium iodide positive, red) was quantified using ImageJ software.

[0045] 3.4 Observation of cell ultrastructure using transmission electron microscopy Cells were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated with graded ethanol, and then embedded in epoxy resin. 70 nm ultrathin sections were prepared, stained with uranium acetate and lead citrate, and the ultrastructure of organelles was observed by transmission electron microscopy, with a focus on the morphology of mitochondria and endoplasmic reticulum.

[0046] 3.5 Western blot analysis of protein immunoblotting to verify signaling pathways Total protein was extracted from TM4 cells using RIPA lysis buffer, and protein concentration was determined by the dicaprynic acid method. 30 μg of protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk at room temperature for 1 hour, the membrane was incubated overnight at 4°C with primary antibody. The primary antibody targets included CCR2, GNA12, phosphorylated PI3K, total PI3K, phosphorylated Akt, total Akt, Nrf2, Kelch-like epichlorohydrin-associated protein 1 (Keap1), heme oxygenase 1 (HO-1), glutathione S-transferase (GST), Bcl-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). After washing, horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature for 1 hour. Enhanced chemiluminescence substrate was used for development, and the gray values ​​of protein bands were quantified using ImageJ software. GAPDH was used as an internal control for standardization.

[0047] 4. Establishment of experimental animal and testicular ischemia-reperfusion injury models 4.1 Laboratory Animals

[0048] Male C57BL / 6 mice, aged 9-10 weeks and weighing 20-25g, were purchased from Fuzhou Wu's Laboratory Animal Co., Ltd. Mice were housed in a specific pathogen-free environment at a temperature of 22±2℃, relative humidity of 50%±10%, and a 12-hour light / 12-hour dark cycle. Mice were given free access to standard rodent feed and sterile water. All animal experimental protocols were approved by the Animal Ethics Committee of Huaqiao University (Approval No.: LHJJSQA2024012, Approval Date: September 18, 2024) and followed the 3R principles of animal ethics (Reduce, Replace, Optimize).

[0049] 4.2 Construction of a testicular ischemia-reperfusion injury model A mouse model of testicular ischemia-reperfusion injury was established using methods described in previous studies. Mice were anesthetized with 1.25% tribromoethanol via intraperitoneal injection (0.02 mL / 10 g body weight). A scrotal incision was made to expose the left testis, which was then twisted clockwise 720° and immobilized for 2 hours to establish the ischemia model. Preliminary experiments confirmed that 4 hours of ischemia would lead to severe testicular structural damage, making it impossible to reliably assess the treatment effect; therefore, 2 hours was chosen as the ischemia time. Subsequently, the immobilization was released, the testis was repositioned to restore perfusion, and the scrotum was sutured. Mice in the sham-operated group underwent the same surgical procedure, but without testicular torsion. All mice were sacrificed 24 hours after reperfusion, and samples were collected.

[0050] 5. In vivo experimental design

[0051] 5.1 Experimental grouping and intervention measures Male C57BL / 6 mice were randomly divided into four groups of eight mice each: sham-operated group, ischemia-reperfusion group, untreated exosome group, and H2S-Exos group. In the sham-operated group, only the left testis was exposed without torsion. In the ischemia-reperfusion group, a testicular ischemia-reperfusion injury model was established, and 100 μL of phosphate-buffered saline was injected via the tail vein 30 minutes before reperfusion. In the untreated exosome group, after model establishment, 5 × 10⁻⁶ exosomes were injected via the tail vein 30 minutes before reperfusion. 8 100 μL of phosphate-buffered saline was administered to untreated exosomes; 30 minutes before reperfusion, the H2S-Exos group was intravenously injected with 5 × 10⁻⁶ phosphate buffer. 8 100 μL phosphate-buffered saline solution of H2S-Exos particles. The exosome dosage was adjusted based on the optimal in vitro concentration and mouse blood volume.

[0052] 5.2 Sample Collection Twenty-four hours after reperfusion, mouse blood was collected through the retroorbital venous plexus, and serum was separated by centrifugation for biochemical index detection. Mice were euthanized by cervical dislocation, and the left testis and epididymis were removed. The testis was divided into two parts: one part was fixed in 10% neutral formalin for 24-48 hours for histopathological analysis, and the other part was flash-frozen in liquid nitrogen for oxidative stress index detection and protein extraction. The epididymal tail was processed immediately for sperm quality analysis.

[0053] 5.3 Hematoxylin-eosin staining The fixed testicular tissue was graded dehydrated, cleared with xylene, embedded in paraffin, and 4μm serial sections were prepared. Hematoxylin-eosin staining was performed, and the samples were independently evaluated by two blinded pathologists. At least 50 seminiferous tubules were observed in each sample, and spermatogenic function was assessed using the Johnson score (1-10 points).

[0054] 5.4 Immunohistochemical staining 4μm paraffin sections were dewaxed and rehydrated, and antigen retrieval was performed using citrate buffer (pH 6.0). After blocking endogenous peroxidase activity and non-specific binding sites, primary antibody was added and incubated overnight at 4°C. The primary antibody targets included GNA12, phosphorylated PI3K, phosphorylated Akt, Keap1, HO-1, and Bcl-2. After washing, horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature for 1 hour. 3,3'-diaminobenzidine was used for staining, hematoxylin was used for counterstaining, and protein expression levels were assessed after imaging.

[0055] 5.5 Detection of oxidative stress in testicular tissue and analysis of proteins by Western blotting Testicular tissue was added to phosphate buffer at 10% (w / v) to prepare tissue homogenate. The supernatant was collected, and superoxide dismutase activity and malondialdehyde content were detected according to the method in section 2.3.2. Total protein was extracted from frozen testicular tissue using RIPA lysis buffer, and protein concentration was determined by the dicaprin method. 30 μg of protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk at room temperature for 1 hour, caspase-3 and β-actin primary antibody were added and incubated overnight at 4°C. After washing, horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature for 1 hour. Enhanced chemiluminescence substrate development was performed, and the gray values ​​of protein bands were quantified using ImageJ software. β-actin was used as an internal control for standardization.

[0056] 5.6 Sperm quality analysis The epididymal tail was placed in 0.1 mL of phosphate buffer pre-warmed at 37°C, and the sperm were minced to release them. After a short incubation, the sperm morphology was observed using an optical microscope. Forward and non-forward motility sperm were counted in at least 5 random fields of view to assess sperm motility.

[0057] 6. Statistical Analysis All experiments were performed with at least three biological replicates, and experimental data are expressed as mean ± standard deviation. Before comparing multiple groups, the Shapiro-Wilk test was used to verify normality, and the Levene test to verify homogeneity of variance. For normally distributed data with homogeneous variances, one-way ANOVA was used, followed by Tukey's multiple comparison test. For non-normally distributed or heterogeneous variance data, Brown-Forsythe test and Welch ANOVA were used, followed by Dunnett's T3 multiple comparison test. Comparisons between two groups were performed using either unpaired t-tests or Mann-Whitney U tests, depending on the data type. All statistical analyses were performed using GraphPad Prism 9.0 software, and a p-value < 0.05 was considered statistically significant.

[0058] III. Results 1. Hydrogen sulfide pretreatment reprogrammed the proteome of mesenchymal stem cell exosomes and induced high-abundance expression of CCL2.

[0059] 1.2 Hydrogen sulfide pretreatment alters the proteomic composition of exosomes To verify the biosafety and integrity of exosomes pretreated with hydrogen sulfide, their physicochemical properties were systematically identified. Nanoparticle tracking analysis showed that H2S-Exos prepared by pretreatment with different concentrations of sodium hydrosulfide (1, 200, 400, 1000 μmol / L) all met the guidelines of the International Society for Extracellular Vesicles in terms of particle size distribution, particle concentration, and protein content. Western blotting analysis showed that both untreated exosomes and H2S-Exos pretreated with various concentrations of sodium hydrosulfide highly expressed exosome markers CD9, CD63, CD81, and Alix. Transmission electron microscopy revealed that H2S-Exos retained the typical cup-shaped, double-membrane vesicle morphology of exosomes. These results indicate that hydrogen sulfide pretreatment does not alter the basic structure and molecular characteristics of exosomes.

[0060] To rule out the possibility that the protective effect of H2S-Exos originates from direct delivery of hydrogen sulfide, the residual amount of hydrogen sulfide in key stages of exosome preparation was first detected using the methylene blue method. The results showed that hydrogen sulfide was detectable in the sodium hydrosulfide mother liquor, but no residual hydrogen sulfide was detected in any exosome preparation stage, including the conditioned medium after cell pretreatment and the exosome suspensions before and after ultrafiltration. This confirmed that the effect of H2S-Exos is not directly mediated by hydrogen sulfide.

[0061] To analyze hydrogen sulfide-induced proteomic alterations in exosomes, data-independent acquisition mass spectrometry was used to perform whole-proteomic analysis on untreated exosomes and H2S-Exos prepared by pretreatment with 200 μmol / L sodium hydrosulfide. A total of 3232 proteins were identified from six exosome samples (three biological replicates per group). The Pearson correlation coefficients of protein intensities after log-2 transformation ranged from 0.958 to 0.988, indicating good reproducibility of the quantitative results. Principal component analysis showed that the three biological replicates in both groups were tightly clustered, and there was a significant separation between the untreated exosome group and the H2S-Exos group.

[0062] Differential expression analysis showed that, compared with untreated exosomes, 54 proteins were upregulated and 16 proteins were downregulated in H2S-Exos, with CCL2 being one of the most significantly upregulated proteins; Western blotting further confirmed that the expression level of CCL2 in H2S-Exos was significantly higher than that in untreated exosomes. Figure 1 and Figure 2 Ontological enrichment analysis of the upregulated proteins showed that they were mainly enriched in pathways such as transforming growth factor-β signaling pathway and tumor necrosis factor signaling pathway.

[0063] 2. Construction of a cobalt chloride-induced hypoxia-reoxygenation model and optimization of H2S-Exos preparation parameters. 2.1 Optimization of cobalt chloride concentration in the hypoxia-reoxygenation model

[0064] To establish an optimal in vitro hypoxia-reoxygenation model, TM4 cells were exposed to different concentrations of cobalt chloride (0, 62.5, 125, 250, and 500 μmol / L) for 24 hours, followed by reoxygenation for 24 hours. Results showed that cobalt chloride reduced cell viability in a concentration-dependent manner. The cell viability rates in the 62.5, 125, 250, and 500 μmol / L cobalt chloride treatment groups were 78.1%, 69.0%, 54.6%, and 37.6%, respectively, all significantly lower than the control group (P<0.05). The expression of the hypoxia stress marker HIF-1α gradually increased with increasing cobalt chloride concentration, reaching 5.40 pg / mL in the 250 μmol / L group and 6.23 pg / mL in the 500 μmol / L group. Morphological observation showed that cells treated with 250 μmol / L cobalt chloride exhibited significant shrinkage and rounding, with a decrease in adherent cell density of approximately 50%. Taking into account both the effectiveness of hypoxia injury and the cell viability requirements of subsequent experiments, 250 μmol / L was selected as the standard cobalt chloride concentration for constructing the hypoxia-reoxygenation model.

[0065] 2.2 Optimization of Sodium Hydrosulfide Pretreatment Concentration To determine the optimal sodium hydrosulfide concentration for mesenchymal stem cell pretreatment, TM4 cells subjected to hypoxia-reoxygenation injury were treated with H2S-Exos prepared by pretreatment with different concentrations (1, 200, 400, and 1000 μmol / L) of sodium hydrosulfide. The results showed that H2S-Exos pretreated with 200 μmol / L sodium hydrosulfide significantly improved cell viability, increasing the cell survival rate from 43.85% in the hypoxia-reoxygenation group to 59.48% (P<0.05). This effect was superior to the untreated exosome group (53.62%) and the H2S-Exos groups pretreated with 1 μmol / L (54.63%), 400 μmol / L (51.49%), and 1000 μmol / L (49.50%) sodium hydrosulfide. Morphological analysis confirmed that H2S-Exos pretreated with 200 μmol / L sodium hydrosulfide resulted in optimal cell adhesion and best morphological recovery, characterized by good cell spread and clear outlines. Therefore, 200 μmol / L was chosen as the optimal sodium hydrosulfide concentration for mesenchymal stem cell pretreatment.

[0066] 2.3 Optimization of H2S-Exos intervention dosage H2S-Exos, prepared by pretreatment with 200 μmol / L sodium hydrosulfide, was used to investigate its optimal therapeutic dose for hypoxia-reoxygenation-damaged TM4 cells. Four particle concentrations (2.5 × 10⁻⁶) were set up. 8 5×10 8 10×10 8 20×10 8 Particles / mL). The results showed that 5 × 10 8H2S-Exos particles / mL increased cell viability from 45.50% in the hypoxia-reoxygenation group to 60.72% (P<0.05); higher concentrations (10×10⁻⁶) also increased cell viability. 8 20×10 8 The particle count (particles / mL) did not further improve cell viability, at 57.68% and 58.16%, respectively, compared to 5×10⁻⁶. 8 There was no statistically significant difference in particle size / mL between the groups (P>0.05); morphological observation showed that 5×10 8 The cell spreading and adhesion of the particle / mL group were comparable to those of the high-concentration group, and significantly better than those of the 2.5×10⁻⁶ group. 8 Particles / mL group. Therefore, 5 × 10⁻⁶ was determined. 8 Particles / mL represents the saturation therapeutic dose of H2S-Exos, used in all subsequent experiments.

[0067] 3. H2S-Exos exhibited excellent cytoprotective effects in the hypoxia-reoxygenation model. 3.1 H2S-Exos enhances cell viability and inhibits apoptosis

[0068] Flow cytometry analysis showed that the apoptosis rate in the H2S-Exos group was 51.0%, significantly lower than that in the untreated exosome group (58.2%) and the hypoxia-reoxygenation group (63.3%) (P<0.05). CCK-8 assay showed that the cell viability in the H2S-Exos group reached 63.2%, significantly higher than that in the untreated exosome group (53.1%) and the hypoxia-reoxygenation group (45.5%) (P<0.05). These results indicate that hydrogen sulfide pretreatment can significantly enhance the cytoprotective capacity of mesenchymal stem cell exosomes.

[0069] 3.2 H2S-Exos maintains the integrity of cell morphology and ultrastructure Phase-contrast microscopy revealed that H2S-Exos treatment effectively reversed hypoxia-reoxygenation-induced cellular pathological changes, alleviated cell shrinkage, rounding, and detachment, and restored normal cell spreading morphology, clear outline, and intercellular connections. Transmission electron microscopy further confirmed the protective effect of H2S-Exos, which significantly reduced ultrastructural damage to cells: mitochondria in the hypoxia-reoxygenation group showed significant swelling, cristae breakage or even disappearance, and significant expansion and degranulation of the endoplasmic reticulum; while mitochondria in the H2S-Exos group showed near-normal morphology, clear cristae structure, and tightly packed endoplasmic reticulum without significant expansion.

[0070] 3.3 H2S-Exos enhances the antioxidant capacity of cells Acridine orange / propidium iodide double staining results showed that, compared with the control group, the proportion of live cells (acrimidine orange positive) was significantly increased and the proportion of dead cells (propidium iodide positive) was significantly decreased in the H2S-Exos group (P<0.05). Furthermore, H2S-Exos significantly enhanced the antioxidant capacity of cells; compared with the untreated exosome group, the H2S-Exos group significantly reduced the accumulation of intracellular reactive oxygen species (P<0.05). Figure 3 , Figure 4 ), upregulated superoxide dismutase activity (P<0.05, Figure 5 ), reducing malondialdehyde levels (P<0.05, Figure 6 The above results indicate that H2S-Exos can effectively scavenge reactive oxygen species, enhance the activity of antioxidant enzymes, and reduce lipid peroxidation, thereby exerting excellent cell protection effects.

[0071] 4. H2S-Exos exhibited excellent protective effects against testicular ischemia-reperfusion injury in an in vivo model.

[0072] 4.1 H2S-Exos improves pathological damage to testicular tissue There was no statistically significant difference in body weight among the four groups of mice (P>0.05). Histopathological evaluation by hematoxylin-eosin staining showed that the seminiferous tubules of the sham-operated group were intact, with neatly arranged spermatogenic cells and supporting cells, abundant sperm in the lumen, and normal interstitial tissue. The testes of the mice in the ischemia-reperfusion group showed severe structural damage, manifested as disordered arrangement of spermatogenic cells, shedding of germ cells in the lumen, azoospermia, significant interstitial edema, accompanied by vasodilation, congestion, and hemorrhage. The seminiferous tubule structure and cell arrangement of the untreated exosome group were partially restored, and shedding in the lumen was reduced. The seminiferous tubules of the H2S-Exos group were nearly normal in integrity, with slight cell shedding, sperm visible in the lumen, and only mild interstitial edema. Quantitative analysis of the Johansson score confirmed that H2S-Exos had a significantly better repair effect on testicular structure than untreated exosomes (P<0.05). Figure 7 ).

[0073] 4.2 H2S-Exos reduces oxidative stress and apoptosis in testicular tissue Antioxidant capacity assays showed that the superoxide dismutase activity in the testicular tissue of the H2S-Exos group was significantly higher than that of the untreated exosome group and the ischemia-reperfusion group (P<0.05). Figure 8 The malondialdehyde (MDA) level was significantly lower than that of the two groups mentioned above (P<0.05). Figure 9The results indicate that H2S-Exos can alleviate oxidative stress in testicular tissue by enhancing the endogenous antioxidant defense system. In addition, H2S-Exos can significantly inhibit the expression of the apoptosis marker Caspase-3, and its expression level is significantly lower than that of the untreated exosome group and the ischemia-reperfusion group (P<0.05), suggesting that the anti-apoptotic mechanism plays a key role in the testicular protective effect of H2S-Exos.

[0074] 4.3 H2S-Exos improves sperm motility There were no statistically significant differences in sperm morphology among the different groups of mice (P>0.05); however, the proportion of progressively motile sperm in the H2S-Exos group was significantly higher than that in the untreated exosome group and the ischemia-reperfusion group (P<0.05), indicating that the therapeutic effect of H2S-Exos is mainly reflected in the improvement of sperm function rather than morphological repair, further confirming its functional protective effect against testicular ischemia-reperfusion injury.

[0075] 4.4 Biosafety Assessment of H2S-Exos Serum biochemical markers confirmed the biosafety of H2S-Exos. There were no statistically significant differences in liver function indicators (alanine aminotransferase, aspartate aminotransferase) and kidney function indicators (blood urea nitrogen, serum creatinine) among the four groups of mice (P>0.05), indicating that H2S-Exos has good therapeutic safety.

[0076] 5. H2S-Exos activates the CCR2 / GNAI2 / PI3K-Akt signal axes via CCL2.

[0077] 5.1 Proteomics analysis reveals activation of cell protection pathways To investigate the downstream cellular responses to H2S-Exos activation, untreated exosomes or H2S-Exos were applied to TM4 cells under hypoxia-reoxygenation conditions. Data-independent mass spectrometry proteomics analysis was performed on nine biological samples (three replicates per group), identifying a total of 9175 proteins. The Pearson correlation coefficients of protein intensities after log2 transformation ranged from 0.972 to 0.998, confirming excellent reproducibility of the quantitative results, and the coefficient of variation of protein intensities in the replicates was less than 1%. Principal component analysis showed that the biological replicates in each group were tightly clustered, and there was significant separation among the four experimental groups.

[0078] Differential expression analysis showed that, compared with the ischemia-reperfusion control group, the H2S-Exos group had 334 upregulated proteins and 535 downregulated proteins. Among the upregulated proteins were G protein-coupled receptor downstream signal transduction molecules GNAI2, nuclear factor erythroid 2-associated factor 2 (NFE2L2, encoding Nrf2), glutathione S-transferase A1 (GSTA1), heme oxygenase 1 (HMOX1, encoding HO-1), and B-cell lymphoma 2 (BCL2). Conversely, Kelch-like epichlorohydrin-associated protein 1 (KEAP1), a negative regulator of Nrf2, was significantly downregulated. The untreated exosome group also showed a similar expression trend, but with fewer differentially expressed proteins and smaller variations.

[0079] Gene ontology and pathway enrichment analysis of differentially expressed proteins, as described in the Kyoto Encyclopedia of Genetics and Genomics, revealed that H2S-Exos treatment enhanced multiple cell protection processes: cellular components were enriched in nuclear spots, histone acetyltransferase complexes, chromosomal regions, and nucleoribonucleoprotein particles, suggesting the presence of nuclear-centered transcriptional and epigenetic regulation; biological processes were enriched in cellular responses to starvation, hypoxia, negative regulation of apoptosis signaling pathways, responses to hydrogen peroxide, and cellular hypoxia adaptation, indicating a significant enhancement of cellular stress adaptation; among molecular functions, oxidoreductase activity was significantly enriched, consistent with the observed antioxidant effects; pathway analysis showed significant enrichment in cell cycle pathways and the hypoxia-inducible factor 1 signaling pathway. In contrast, the untreated exosome group was only enriched in "response to oxidative stress" and "response to starvation," indicating that hydrogen sulfide pretreatment enables exosomes to initiate a broader and more potent cell protection program.

[0080] 5.2 Western blot verification of the CCL2 / CCR2 / GNAI2 / PI3K-Akt signal axis Western blot analysis of key molecules in this signaling pathway showed that, compared with the untreated exosome group and the ischemia-reperfusion group, H2S-Exos treatment significantly upregulated GNAI2 expression, enhanced PI3K and Akt phosphorylation levels, increased the expression of Bcl-2, Nrf2, HO-1 and GST, and inhibited Keap1 expression.

[0081] To verify the hierarchical regulatory relationship of this pathway, a series of pharmacological inhibition experiments were conducted: supplementing untreated exosomes with exogenous CCL2 replicated the effects of H2S-Exos, upregulating the expression of GNAI2, phosphorylated PI3K, phosphorylated Akt, Bcl-2, Nrf2, HO-1 and GST, and inhibiting Keap1; while supplementing H2S-Exos with exogenous CCL2 further enhanced the above effects, confirming that CCL2 can synergistically enhance H2S-Exos-mediated pathway activation.

[0082] Treatment of cells with the CCR2 antagonist BMS-CCR2 completely eliminated the upregulation of GNAI2 and the phosphorylation of PI3K / Akt, confirming that the binding of CCL2 to CCR2 is a necessary condition for initiating downstream signal transduction. The PI3K inhibitor apelelis specifically inhibited the expression of phosphorylated PI3K and its downstream markers (phosphorylated Akt, Bcl-2, Nrf2, HO-1, GST), but had no effect on GNAI2 expression. The Akt inhibitor MK2206 inhibited the expression of downstream markers, but had no effect on GNAI2 and phosphorylated PI3K. In the H2S-Exos combined with exogenous CCL2 treatment group, inhibitor experiments further confirmed the hierarchical activation mode of this pathway: BMS-CCR2 specifically inhibited the expression of downstream markers and reversed the downregulation of Keap1; apelelis upregulated the expression of upstream GNAI2 while inhibiting downstream effects; MK2206 upregulated the expression of GNAI2 and phosphorylated PI3K, but inhibited the expression of downstream markers.

[0083] The above results fully confirm that the protective effect of H2S-Exos against hypoxia-reoxygenation injury of testicular TM4 cells is mediated by the activation of the CCL2 / CCR2 / GNAI2 / PI3K-Akt signaling pathway, which can synergistically regulate the anti-apoptotic pathway (Bcl-2) and the antioxidant pathway (Nrf2-Keap1-HO-1 / GST), with CCL2 being the key factor in initiating this protective program.

[0084] 6. Immunohistochemistry to verify the expression of key signaling molecules in testicular tissue Histological examination confirmed that the spermatogenic cells in the ischemia-reperfusion injury group were disordered and germ cells detached into the lumen; while the seminiferous tubules in the H2S-Exos group were intact and the spermatogenic cells and supporting cells were neatly arranged. The protective effect was significantly better than that in the untreated exosome group, which was consistent with the results of hematoxylin-eosin staining.

[0085] Immunohistochemical analysis revealed significant differences in protein expression patterns in the testicular tissues of mice across different groups: the expression levels of GNAI2, phosphorylated PI3K, phosphorylated Akt, HO-1, and Bcl-2 in Sertoli cells of the H2S-Exos group were significantly higher than those in the untreated exosome group and the ischemia-reperfusion group, while the expression of Keap1 was significantly suppressed; conversely, the expression of the above protective proteins was significantly downregulated in the ischemia-reperfusion group, with GNAI2, phosphorylated PI3K, phosphorylated Akt, HO-1, and Bcl-2 showing negative or weakly positive expression, while Keap1 showed strong positive expression.

[0086] Notably, there were significant differences in protein expression patterns between spermatogenic cells and Sertoli cells. The expression intensities and trends of phosphorylated PI3K, phosphorylated Akt, HO-1, Bcl-2, and Keap1 differed between the two cell types. HO-1 expression was highly specific, localizing only in Sertoli cells, and the HO-1 expression in the H2S-Exos group was significantly stronger than that in the untreated exosome group and the ischemia-reperfusion group, approaching that of the sham-operated group. Combined with the experimental results demonstrating the potent antioxidant effect of H2S-Exos, this suggests that HO-1 may play a crucial role in the protection of testicular structure.

[0087] 7. Hydrogen sulfide-pretreated exosomes alleviate testicular ischemia-reperfusion injury via CCL2 / CCR2 / GNAI2-mediated PI3K-Akt activation. This study elucidates the molecular mechanism by which H2S-Exos protects against testicular ischemia-reperfusion injury: hydrogen sulfide pretreatment leads to high CCL2 expression in mesenchymal stem cell exosomes, and H2S-Exos delivers CCL2 to target cells in the ischemia-reperfusion microenvironment; CCL2 binds to its homologous receptor CCR2, activating GNAI2 and triggering the PI3K-Akt signaling hub; activated Akt exerts a dual protective effect: firstly, it promotes Nrf2 nuclear translocation, upregulating the expression of antioxidant genes (HO-1, GST); secondly, it stabilizes Bcl-2 expression, inhibiting apoptosis. This novel discovery of the CCL2 / CCR2 / GNAI2 / PI3K-Akt / Nrf2 (Bcl-2) signaling axis reveals the mechanism by which H2S-Exos synergistically antagonizes oxidative stress and apoptosis, providing a new therapeutic strategy for testicular ischemia-reperfusion injury. Figure 10 This is a schematic diagram of the molecular mechanism by which H2S-Exos alleviates testicular ischemia-reperfusion injury: Hydrogen sulfide pretreatment leads to high abundance of CCL2 expression in mesenchymal stem cell exosomes; after H2S-Exos delivers CCL2 to target cells (such as Sertoli cells) in the ischemia-reperfusion microenvironment, CCL2 binds to its homologous receptor CCR2, activating GNAI2 and triggering the PI3K-Akt signaling cascade; activated Akt exerts a dual protective effect: ① promoting Nrf2 nuclear translocation and upregulating the expression of antioxidant genes (HO-1, GST); ② stabilizing Bcl-2 expression and inhibiting apoptosis; this synergistic antioxidant and anti-apoptotic response maintains the structural and functional integrity of the testes; key nodes are marked in red, and protective effects are marked in blue.

[0088] It is understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0089] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A drug for treating ischemia-reperfusion injury, characterized in that, The drug activates the CCL2-chemokine receptor 2 signaling axis, triggering the downstream G protein αi2 subunit-mediated phosphatidylinositol 3-kinase / protein kinase B signaling pathway, thereby synergistically regulating the antioxidant defense system driven by nuclear factor erythroid 2-related factor 2 and the anti-apoptotic pathway mediated by B-cell lymphoma 2, thereby treating the ischemia-reperfusion injury.

2. The drug according to claim 1, characterized in that, The treatment for ischemia-reperfusion injury is testicular ischemia-reperfusion injury.

3. The drug according to claim 2, characterized in that, The drug activates the CCR2 / GNAI2 / PI3K-Akt signaling axis in the Sertoli cells of the testis, synergistically regulating the Nrf2-driven antioxidant defense system and the Bcl-2-mediated anti-apoptotic effect, thereby treating the testicular ischemia-reperfusion injury.

4. The drug according to claim 1, characterized in that, The drug is a mesenchymal stem cell exosome with upregulated CCL2 protein expression.

5. The drug according to claim 4, characterized in that, The mesenchymal stem cell exosomes are hydrogen sulfide-treated mesenchymal stem cell exosomes.

6. The drug according to claim 5, characterized in that, The mesenchymal stem cell exosomes were obtained by pretreatment with sodium hydrosulfide at concentrations ranging from 1 μmol / L to 400 μmol / L.

7. The drug according to claim 6, characterized in that, The mesenchymal stem cell exosomes were obtained by pretreatment with 200 μmol / L sodium hydrosulfide.

8. A pharmaceutical composition for treating ischemia-reperfusion injury, characterized in that, The pharmaceutical composition comprises the drug according to any one of claims 1-7.

9. The composition according to claim 8, characterized in that, The treatment for ischemia-reperfusion injury is testicular ischemia-reperfusion injury.

10. Application of CCR2 protein in the preparation of drugs for treating ischemia-reperfusion injury.

11. The application according to claim 10, characterized in that, The CCR2 protein is derived from mesenchymal stem cell exosomes.

12. The application according to claim 11, characterized in that, The mesenchymal stem cell exosomes are hydrogen sulfide-treated mesenchymal stem cell exosomes.

13. The application according to claim 10, characterized in that, The treatment for ischemia-reperfusion injury is testicular ischemia-reperfusion injury.

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