Application of trehalose in preparation of medicine for improving reproductive organ injury caused by diabetes mellitus

Abstract

The invention provides application of trehalose in preparation of a medicine for improving reproductive organ injury caused by diabetes mellitus, and relates to the technical field of biological medicine. As an innovative autophagy activator, trehalose provides a new treatment thought for testis injury caused by diabetes mellitus, has the advantages of small side effect, high safety and the like, and is expected to become an effective replacement therapy in clinical application. Although the long-term administration of the trehalose does not significantly influence the blood glucose level, the relieving effect of the trehalose on testis injury shows that the trehalose can continuously improve reproductive dysfunction caused by diabetes mellitus as a treatment means for long-term use, and has potential clinical advantages.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular, to the use of trehalose in the preparation of a drug for improving reproductive organ damage caused by diabetes. Background Art

[0002] Diabetes mellitus (DM) is an important risk factor for male reproductive system damage. Including testicular damage and decreased sperm motility, these damages are closely related to the disorder of autophagy function. However, current treatment technologies mainly focus on controlling blood sugar and improving overall health. There is still a lack of effective solutions for the specific treatment of testicular damage caused by diabetes. The following are some shortcomings of existing technologies: 1. At present, most of the damage to diabetic reproductive organs is alleviated by diabetes drugs, and there is a lack of targeted drugs. Therefore, it is urgent to develop more targeted therapeutic drugs that directly act on the mechanism of reproductive organ damage caused by diabetes, such as regulating oxidative stress, inhibiting inflammatory response, restoring autophagy levels or improving microvascular lesions. New strategies to improve the treatment effect, reduce the burden on patients, and improve reproductive function. 2. Although some drugs such as antioxidants and anti-inflammatory drugs can alleviate testicular damage, they may have side effects and affect the health of patients. In addition, traditional anti-diabetic drugs usually fail to effectively reduce reproductive system damage, and long-term use may lead to drug resistance or other negative reactions. 3. Testicular damage caused by diabetes is often accompanied by cell apoptosis and pyroptosis, but existing treatments are still insufficient in intervening in these cell death pathways. Most treatments lack the ability to simultaneously inhibit apoptosis, pyroptosis, and regulate autophagy, thus limiting the therapeutic efficacy.

[0003] Therefore, from the perspective of long-term drug safety, the current treatment options for testicular damage caused by diabetes need to be improved. Emphasis should be placed on developing more targeted drugs to reduce side effects, improve treatment efficacy, and effectively intervene in cell death pathways to protect patients' reproductive health.

[0004] For testicular damage caused by diabetes, existing treatments mostly focus on controlling blood sugar levels, but there is a lack of effective intervention measures for testicular damage. Summary of the invention

[0005] The main purpose of the present invention is to provide the use of trehalose in the preparation of a drug for improving reproductive organ damage caused by diabetes. The principle is to use trehalose as a treatment method through its antioxidant, anti-apoptotic and autophagy activating effects, thereby improving testicular damage caused by diabetes. This not only helps to reveal the potential of trehalose in biomedicine, but also provides a new idea for clinical treatment.

[0006] In order to solve the above technical problems, the technical solution adopted in this application is:

[0007] The present application provides a use of trehalose in preparing a drug for improving reproductive organ damage caused by diabetes.

[0008] Trehalose (TLS) has shown several significant advantages in treating testicular damage caused by diabetes, especially in terms of the defects and shortcomings of current technologies. TLS has the following important features:

[0009] 1. Targeted drugs: TLS is currently the first targeted drug specifically for testicular damage caused by diabetes. It can activate autophagy, restore the autophagic function in the testicular cells of diabetic mice, reduce the accumulation of damaged cells, improve the energy state and metabolism of cells, and avoid further expansion of cell death. In addition, the restoration of autophagy helps to alleviate the testicular cell damage caused by diabetes, which can effectively slow down the decline of testicular function and improve fertility.

[0010] 2. Small side effects: Compared with conventional anti-diabetic drugs and other treatments, TLS has relatively small side effects. Many traditional drugs may cause drug resistance or other negative reactions in long-term use, while TLS, as a natural food additive, has high safety and acceptability. This provides patients with a safer treatment option and reduces the risks of long-term medication.

[0011] 3. High safety: TLS does not significantly affect blood sugar levels during long-term administration, which indicates that it will not have a negative impact on blood sugar control while improving testicular damage. This feature makes TLS a suitable treatment for long-term use, which helps to continuously improve reproductive dysfunction caused by diabetes.

[0012] 4. Multiple protection mechanisms: TLS provides multiple protection mechanisms by activating autophagy, inhibiting apoptosis and pyroptosis. It not only reduces cell death, but also improves cell energy status and metabolism. This comprehensive protective effect makes TLS excellent in reducing testicular damage and provides patients with a wider range of therapeutic effects.

[0013] 5. Improve reproductive function and microenvironment: TLS significantly improved the testicular structure of diabetic mice, alleviated the problems of seminiferous tubule atrophy and spermatogenesis stagnation. In addition, it restored the blood-testis barrier function, ensuring the normal growth and maturation of sperm and other germ cells. These effects not only help improve fertility, but may also improve overall reproductive health.

[0014] 6. Systemic protection potential: In addition to protecting the testicles, studies have shown that TLS may also protect other tissue damage caused by diabetes (such as the heart, kidneys, nervous system, etc.). This systemic protection ability makes TLS show new application prospects in the overall treatment of diabetes.

[0015] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0016] Trehalose in this application, as an innovative autophagy activator, not only provides a new treatment idea for testicular damage caused by diabetes, but also has the advantages of small side effects and high safety, and is expected to become an effective alternative therapy in clinical applications. Although the long-term administration of TLS did not significantly affect blood sugar levels, its alleviating effect on testicular damage shows that TLS, as a long-term treatment, can continuously improve reproductive dysfunction caused by diabetes and has potential clinical advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Figure 2: Autophagy in the testis of diabetic mice evaluated by MDC staining and WB experiments; (A) is a representative image of Beclin-1, LC3, P62 and ATG5 expression at 12, 16, 20 and 24 weeks; (B) is the quantification of Beclin-1, LC3, P62 and ATG5 levels at 12, 16, 20 and 24 weeks; (C) is a representative fluorescence image of monodansylcadaverine (MDC) staining at 12, 16, 20 and 24 weeks.

[0019] Figure 2 The figures are histological examinations of mouse testicles; (A) is a representative image of blood cell staining; (B) is a representative fluorescence image of biotin tracing studies.

[0020] Figure 3 Figure 2 shows the TUNEL staining and WB detection of each experimental group; (A) is a representative image of terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP end labeling (TUNEL) detection; (B) is a representative gel band of Bax, Bcl-2 and cleaved caspase-3; (C) is a quantitative analysis of Bax, Bcl-2 and cleaved caspase-3 and the ratio of Bax / Bcl-2; (D) is a representative gel band of NLRP3, caspase-1, IL-1β and IL-18. (E) is a quantitative analysis of NLRP3, caspase-1, IL-1β and IL-18.

[0021] Figure 4The figures are TEM, MDC staining and WB analysis of mouse testicular cells in different experimental groups; (A) and (B) are representative images of transmission electron microscopy; (C) is a representative fluorescence image of monodansylcadaverine (MDC) staining; (D) is a representative gel band of Beclin-1, LC3, p62 and ATG5; (E) is a quantitative analysis of Beclin-1, LC3, p62 and ATG5.

[0022] Figure 5 The figure is a comparison chart of protein expression content quantitative analysis by WB detection; (A) is the representative gel band of PK2, PKR1 and PKR2; (B) is the quantification of PK2, PKR1 and PKR2.

[0023] Figure 6 Molecular docking and immunohistochemical staining images; (A) is the molecular docking analysis between TLS and Akt1; (B) is the representative image of p-Akt1 and Akt1 immunohistochemical staining; (C) is the representative gel band of p-Akt, Akt, p-Gsk3β and Gsk3β; (D) is the quantitative analysis of p-Akt, Akt, p-Gsk3β, Gsk3β, p-Akt / Akt and p-Gsk3β / Gsk3β. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If no specific conditions are specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0026] The present invention uses Western blot, immunohistochemistry, fluorescent labeling and other techniques to detect the expression level of autophagy-related markers (such as LC3, p62, etc.) to evaluate the activation effect of trehalose on the autophagy process. The morphological changes of testicular tissue are evaluated by HE staining, other histological staining and measurement of organ coefficients and reproductive capacity, and the effects of trehalose treatment on testicular structure and function are observed.

[0027] In order to achieve the above purpose, the specific technical solution of this patent is implemented as follows:

[0028] 1. Establishment of animal model:

[0029] SPF male C57BL / 6J mice weighing 22±2 g and aged 5-6 weeks were used for experimental animal selection. The mice were divided into two groups: control group (Con) and diabetes-induced group (DM). Mice in the DM group were intraperitoneally injected with streptozotocin (STZ) for five consecutive days, while mice in the control group were injected with an equal amount of saline. After intraperitoneal injection, blood samples were collected through the tail vein to measure blood glucose levels. Mice with blood glucose levels reaching a certain threshold were considered diabetic model mice (DM group), and testicular tissues were collected four times at different time points 24 weeks after the diagnosis of diabetes. In addition, four groups were randomly selected from another group of mice for further treatment: control group (Con), diabetes-induced group (DM), Con-TLS group and DM-TLS group. Con-TLS group and DM-TLS group were intraperitoneally injected with TLS solution for two consecutive days every four weeks for the next 24 weeks, and TLS was added to drinking water.

[0030] 2. Physiological index measurement:

[0031] Blood samples were collected through the tail vein to measure blood glucose levels. After the experimental procedures, the mice were anesthetized, and their testicles were removed and weighed to calculate the organ coefficient. The fresh epididymal tail of the mice was mixed with normal saline at 37°C to prepare a sperm suspension. Sperm motility was determined by the following formula: number of active sperm / total sperm number × 100%. Histological examination was performed to observe the structural changes of the seminiferous tubules. After immersion in paraformaldehyde for 24 h, the testicles were immersed in paraffin and sectioned. After hematoxylin and eosin (HE) staining and fixation with neutral glue, the sections were examined under a microscope (Leica DM2000, Germany).

[0032] 3. Molecular Biology Analysis

[0033] Immunohistochemistry p-Akt1 (S473) and Akt1 antibodies (1:400, Wanlai Bio, China) were incubated with dewaxed tissue sections overnight. After washing with PBS, the sections were incubated with biotinylated horse anti-rabbit immunoglobulin G (IgG) (1:200, Wuhan Saiweier Biotechnology Co., Ltd., China) for 30 min, then incubated with avidin-biotin-peroxidase complex using the ABC kit and finally stained with tetrahydrochloride (DAB). The expression of p-Akt1 and Akt1 in the tissues was observed and photographed using a microscope (BX53, Olympus, Japan). Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and MDC staining were performed.

[0034] Western blot (WB) assay showed that the proportion of TUNEL-positive cells in DM group mice was significantly higher than that in control group (Con), while the number of TUNEL-positive cells in DM-TLS group mice was less, indicating that TLS treatment reduced cell apoptosis. In addition, the level of anti-apoptotic protein Bcl-2 was significantly decreased in DM group, while the levels of pro-apoptotic markers cleaved caspase-3 and Bax were significantly increased, indicating that the apoptotic pathway was activated. After TLS treatment, the level of Bcl-2 in DM-TLS group mice increased slightly, while the levels of cleaved caspase-3 and Bax and the Bax / Bcl-2 ratio decreased significantly, showing the inhibitory effect of TLS on apoptosis. Further analysis found that the levels of NLRP3, caspase-1, IL-1β and IL-18 were significantly increased in DM group mice, while these levels were inhibited by TLS treatment in DM-TLS mice. These results indicate that TLS promotes the survival of testicular cells by inhibiting DM-induced apoptosis and pyroptosis.

[0035] 4. Signaling pathway research

[0036] The regulatory effect of TLS on PK2 / PKR signaling pathway and Akt / Gsk3 signaling pathway was studied, and its protective mechanism in testicular injury was analyzed. The binding ability of TLS to Akt1 was verified by molecular docking experiments, and its mechanism of action was further explored.

[0037] The study found that the expression of PK2 and PKR2 was significantly decreased, while the expression of PKR1 was significantly increased in the testes of diabetic (DM) mice. Compared with the control group (Con), TLS treatment significantly increased the expression of PK2 and PKR2 and decreased the expression of PKR1 in DM mice. This suggests that TLS can regulate the PK2 / PKR signaling pathway, thereby providing protection against DM-induced testicular damage.

[0038] The study further explored the interaction between TLS and AKT and the activity of the AKT / Gsk3 signaling pathway. Molecular docking results showed that TLS binds to AKT1, AKT2, AKT3, and GSK3, with binding energies of -7.6, -6.0, -6.3, and -5.7 kcal / mol, respectively.

[0039] The experimental results showed that the level of p-Akt1 decreased in DM mice, while TLS treatment significantly upregulated the expression of p-Akt1 in DM-TLS mice. WB analysis showed that the ratio of p-Akt to Akt in DM group mice was significantly reduced, and this change was reversed after TLS treatment.

[0040] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0041] Example

[0042] The animal procedures used in the experiments of this application have been approved by the Experimental Animal Ethics Committee of Hubei University of Science and Technology and comply with the guidelines of the National Institutes of Health (NIH). SPF-grade male C57BL / 6J mice (weight 22 ± 2 g, 5-6 weeks old) were provided by Pengyue Laboratory Animal Breeding (China).

[0043] 1. Animal experiments and grouping

[0044] Control (Con): control group; DM: diabetic model group; Con-TLS: control + TLS treatment group; DM-TLS: diabetes + TLS treatment group.

[0045] 2. Experimental methods

[0046] 2.1 Establishment of mouse diabetes model: 5-6 week-old mice were intraperitoneally injected with STZ (Sigma, Germany) at a dose of 50 mg / kg / d for five consecutive days. -1 On the 7th day after the injection, blood glucose levels were measured by tail vein blood sampling. Mice with blood glucose levels >16.7mmol / L were identified as diabetic model mice (DM group). The control group (Con) mice were injected with an equal amount of normal saline.

[0047] 2.2TLS Processing

[0048] The mice in the Con-TLS and DM-TLS groups received intraperitoneal injection of TLS solution at a dose of 1 mg / g·d-1 every four weeks for two consecutive days, and drank water containing 2% TLS for 24 weeks. The mice in the control group (Con and DM) received an equal amount of normal saline instead of TLS solution.

[0049] 2.3 Sampling time points

[0050] Testicular samples were removed from the mice at 12, 16, 20, and 24 weeks after diabetes diagnosis for subsequent analysis.

[0051] 3. Biotin tracing study

[0052] 3.1 The mouse testicles were injected with EZ-link Sulfo-NHS-LC-biotin; 30 minutes after injection, the mice were killed and the testicles were carefully removed from the abdomen and fixed.

[0053] 3.2 Tissue processing

[0054] The fixed testicular tissue was embedded in paraffin and preserved.

[0055] 3.3 Tissue sections

[0056] The paraffin-embedded testicular tissue was sliced ​​at a thickness of 5 μm.

[0057] 3.4 Fluorescence color development

[0058] The sections were placed in a solution containing Alexa Fluor-labeled streptavidin and incubated at room temperature for 2 hours. The stained sections were observed under a fluorescence microscope (DM2000, Leica, Germany).

[0059] 4. Measurement of organ coefficients and reproductive capacity

[0060] 4.1 Mouse anesthesia and organ extraction

[0061] After the experiment, the mice were anesthetized, and their testicles were removed and weighed.

[0062] 4.2 Preparation of sperm suspension

[0063] Fresh tissue was taken from the epididymis of the mouse tail, and sperm suspension was prepared using 37°C physiological saline.

[0064] 4.3 Sperm motility assessment

[0065] Sperm motility was determined by the following formula: number of active spermatozoa / total number of spermatozoa × 100%.

[0066] The data of blood glucose level (experiment 2.1), testis weight (experiment 4.1), sperm count and sperm motility (experiment 4.3) of STZ-induced DM mice are shown in Table 1.

[0067] Table 1

[0068]

[0069] As can be seen from Table 1, compared with the Con group mice, the blood glucose level of STZ-induced DM mice was higher than that of the control group. Although TLS treatment was able to partially restore blood glucose, it was not completely restored. In addition, we observed that the testicular tissue weight of DM mice was significantly reduced compared with the Con group, and the sperm motility and number were also significantly reduced (P<0.05). Compared with the DM group, TLS treatment did not reverse the decrease in testicular weight and sperm count, but significantly increased the sperm motility of Con-TLS group mice (P<0.05).

[0070] 5. Morphological evaluation

[0071] Paraffin embedding and sectioning techniques were used, combined with Hematoxylin and Eosin (HE) staining, and transmission electron microscopy (TEM) was used to observe the tissue ultrastructure.

[0072] 5.1 Tissue Preparation

[0073] The testicular tissue was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin and sliced; the slices were stained with HE and sealed with neutral gum.

[0074] 5.2TEM sample preparation

[0075] A testicular tissue sample of about 1 mm^3 was taken and fixed with 2.5% glutaraldehyde for 2 hours and then with 1% osmium acid. The sample was treated with acetone 812 and then double stained (uranyl acetate and lead citrate).

[0076] 5.3 Electron microscopy observation

[0077] The samples were examined under a transmission electron microscope (TEM; HT7700, Hitachi, Japan).

[0078] 6. TUNEL assay is used to detect cell apoptosis

[0079] 6.1 Sample preparation

[0080] After deparaffinization, the sections were rehydrated with different concentrations of ethanol.

[0081] The cells were treated with 20 mg / mL proteinase K for 15 min (37° C.) and then treated with 0.1% Triton X-100 for 20 min (room temperature).

[0082] 6.2 Experimental Procedure

[0083] The sections were incubated with a mixture of TdT and dUTP at 37°C for 20 min to label the DNA break ends.

[0084] 6.3 Color development and observation

[0085] 3,3'-Diaminobenzidine (DAB) was used for staining and the sections were observed and photographed under a fluorescence microscope (DM2000, Leica, Germany).

[0086] 7. MDC staining to detect autophagy levels in tissue sections

[0087] 7.1 Main Reagents

[0088] MDC (50 μM): dissolved in DMSO, used as a fluorescent dye to label autophagosomes; PBS (phosphate buffered saline): used to wash slices and remove excess dye.

[0089] 7.2 Experimental procedures

[0090] First, the sections were dewaxed to remove the paraffin in the paraffin-embedded tissue sections so that the dye could penetrate into the tissue; then the sections were rehydrated, and the concentration was gradually reduced with ethanol and treated with water to restore the sections to a water-soluble environment in preparation for subsequent staining. The rehydrated sections were placed at a constant temperature of 37°C and incubated in a 50μM MDC solution for 1 hour. This process allows MDC to penetrate into the tissue and bind to the autophagosomes. Wash with PBS 3 times, 5 minutes each time, to remove unbound MDC and reduce background fluorescence.

[0091] 7.3 Observations

[0092] The sections were observed under a fluorescence microscope (DM2000, Leica, Germany).

[0093] 8. Molecular docking simulation

[0094] 8.1 Protein file preparation

[0095] The protein 3D structure (PDB format) was downloaded from the RCSB PDB database; ligands and water molecules were removed using PyMOL, hydrogen atoms were added, and the structure was saved in PDB format; the PDB file was converted to pdbqt format using Autodock Tools.

[0096] 8.2 Small molecule file preparation

[0097] Download the small molecule two-dimensional structure from the PubChem database; use Sybyl to optimize the small molecule energy structure; use Autodock Tools to convert the optimized file into pdbqt format.

[0098] 8.3 Molecular docking simulation

[0099] AutodockVina was used to set docking parameters (such as grid size and center position), run docking, and obtain binding energy and binding mode of small molecules and proteins. The docking results of TLS and core targets of Akt / Gsk3 pathway are shown in Table 2.

[0100] Table 2

[0101] Target PDB Score(kcal / mol) Akt1 3O96 -7.6 Akt2 3E8D -6.8 Akt3 2X18 -6.3 Csk3β 1Q3D -5.7

[0102] As can be seen from Table 2, in terms of binding energy, TLS showed -7.6 kcal / mol, -6.0 kcal / mol, -6.3 kcal / mol, and -5.7 kcal / mol for Akt1, Akt2, Akt3, and Gsk3β, respectively. The ligand-protein interactions were captured in LigPlot.

[0103] 9. IHC detection

[0104] 9.1 Experimental Procedure

[0105] The tissue sections were dewaxed and rehydrated to make them suitable for antibody binding. p-Akt1 (S473) and Akt1 antibodies (Wanleibio, China) were used at a dilution ratio of 1:400 and incubated overnight at 4°C to ensure that the antibodies were fully bound to the target antigen. The sections were washed with PBS to remove the unbound primary antibody and reduce the background signal. Biotinylated anti-rabbit IgG secondary antibody (Servicebio, China) was added at a dilution ratio of 1:200 and incubated at room temperature for 30 minutes. The sections were treated with the ABC kit (Avidin-Biotin-Peroxidase Complex) to amplify the antigen-antibody binding signal. Finally, DAB colorimetric agent (diaminobenzidine tetrachloride) was used to make the target protein show brown signal, and the reaction was stopped and washed with PBS or water.

[0106] 9.2 Results and observations

[0107] The staining results were observed using a BX53 microscope (Olympus, Japan), and the expression and localization of the target protein were recorded and photographed.

[0108] 10. Western blot analysis

[0109] Experimental steps: Testicular tissue homogenate was prepared using RIPA buffer and centrifuged at 12,000 rpm for 15 min at 4°C. Equal amounts of protein were separated by SDS-PAGE, then transferred to a nitrocellulose membrane and detected using antibodies. The membrane was blocked with 0.1% Tween 20 in Tris buffer (TBST) solution and 5% milk, and then incubated at 4°C with antibodies against cleaved caspase-3, Bax, Bcl-2, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 (1:1000, Cell Signaling Technology, USA), sequestosome-1 (P62) (1:500, Wanleibio, China), autophagy-related gene 5 (ATG5), NLRP3, caspase-1, IL-18, IL-1β (1:1000, BiossAntibodies, China), activin 2 (PK2) (1:1000, Abcam, USA), activin receptor 1 (PKR1), activin receptor 2 (PKR2) (1:2000, Santa Cruz Biotechnology, USA), p-Akt, Akt, p-Gsk3β, Gsk3β (1:1000, Cell Signaling Technology, USA), and p-Akt. Signaling Technology, CA, USA) and GAPDH (1: 10000, Proteintech, USA) were incubated overnight. After three TBST washes, the membrane was incubated with secondary antibodies (1: 10000, Jackson Immuno Research Laboratories, USA) at room temperature for 1 hour. The immunoblots were developed using an ECL kit (Pierce Biosciences, USA) and evaluated and quantified using a molecular imager (Bio-Rad Chemi Doc TM XRS+).

[0110] Figure 1The results show the autophagy of diabetic mice testis evaluated by MDC staining and WB experiment (experiments 7 and 10 above). n = 4-5 per group, and the values ​​are expressed as mean ± standard deviation. *P < 0.05 compared with the Con group. Group A and Group B were detected by WB. At 12, 16, 20 and 24 weeks after the onset of DM, the expression levels of autophagy-related proteins Beclin-1, LC3 and ATG5 in the testis of mice in the DM group were significantly lower than those in the Con group, and the P62 content was significantly higher than that in the Con group (P < 0.05), but in the early stage of DM (12 weeks), there was no significant difference in the P62 level between the two groups; Group C was evaluated for autophagy by MDC staining, and the results showed that the fluorescence signal in the Con group was significantly stronger than that in the DM group, indicating that the formation of testicular autophagosomes in diabetic mice at different stages was inhibited. The findings of these results reveal that the reduction of autophagy may play a key role in the pathogenesis of diabetic testicular damage.

[0111] Figure 2 This is a histological examination of the testicles of mice (experiment 3 above), in which group A was stained with hematoxylin and eosin (HE). The arrow indicates the spermatogenic cell layer, and the triangle indicates the spermatogenic process. The image was magnified 400 times, and the scale bar is 20 μm per group (n = 5 per group); it shows that the seminiferous tubules of the DM group mice were atrophied, accompanied by varying degrees of spermatogenesis stagnation, but these effects were partially or significantly offset by TLS treatment. Group B used biotin tracing technology to study the blood-testis barrier function, with white arrows indicating biotin between the ducts and white triangles indicating biotin in the seminiferous tubules. The image was magnified 200 times, and the scale bar is 50 μm (n = 5 per group); the blood-testis barrier function of DM mice in the figure was impaired, as shown by the accumulation of biotin in the seminiferous tubules of DM mice after passing through the Sertoli cell space, and this phenomenon was eliminated by TLS intervention. These findings indicate that TLS is beneficial to the maintenance of fertility by inhibiting the damage to the blood-testis barrier caused by DM.

[0112] Figure 3TLS inhibited testicular apoptosis and pyroptosis caused by diabetes. Group A was stained with TUNEL (experiment 6 above), the image was magnified 200 times, and the scale bar was 50 μm (n = 3 per group); the proportion of TUNEL-positive cells in the DM group was significantly higher than that in the Con group, while the number of TUNEL-positive cells in DM-TLS mice was relatively small; Groups B and C used WB to detect the content of mouse testicular apoptosis-related proteins (experiment 10 above), and the results showed that the apoptosis pathway was activated in the DM group, and the testicular apoptosis of DM-TLS mice was significantly reduced, significantly reducing the levels of caspase-3 and Bax and the Bax / Bcl-2 ratio (P < 0.05); Groups D and E used WB detection to determine whether TLS inhibited NLRP3-mediated cell pyroptosis in DM mice (n = 4 per group). Data are expressed as mean ± standard deviation (SD). *P < 0.05 compared with the Con group, #P < 0.05 compared with the DM group. These results indicate that TLS promotes the survival of testicular cells by inhibiting DM-induced apoptosis and pyroptosis.

[0113] Figure 4 In the diabetic state, TLS can maintain autophagy function, reduce the negative effects of diabetes on the testis, and protect testicular tissue; among them, groups A, B, and C were TEM and MDC staining of testicular cells of mice (experiments 5 and 7 above), and the red arrows marked the autophagosomes; the image of group C was magnified 200 times, and the scale bar was 50μm. The results showed that the number of autophagosomes in DM mice treated with TLS increased significantly; n=4 in each group in group E. Data are expressed as mean ± standard deviation (SD). *P<0.05 compared with the Con group, #P<0.05 compared with the DM group. Group D and Group E were analyzed by WB. TLS significantly increased the levels of LC3 and ATG5 in the DM-TLS group mice and reduced the level of P62 (P<0.05). These results indicate that TLS helps the survival of testicular cells by intervening to slow down the decrease in autophagy levels caused by diabetes.

[0114] Figure 5 In order to investigate the role of TLS in regulating the testicular damage caused by diabetes by regulating the PK2 / PKR signaling pathway, groups A and B were quantitatively analyzed by WB for the expression of PK2, PKR2, and PKR1 (the above experiment 10) (n = 4 per group), and the values ​​were expressed as mean ± SD. *P < 0.05. The results showed that compared with Con mice, the expression of PK2 and PKR2 in the testes of DM mice was significantly reduced, while the expression of PKR1 was significantly increased; TLS administration significantly increased the expression levels of PK2 and PKR2 in DM mice, and reduced the expression of PKR1.

[0115] Figure 6TLS may inhibit DM-induced cell death through the Akt / Gsk3β cascade; Group A shows the docking results of TLS and the core targets of the Akt / Gsk3 pathway by molecular docking (experiment 8 above); Group B images were magnified 400 times, with a scale bar of 20 μm, and immunohistochemistry showed that mice treated with TLS increased the expression of p-Akt1 (experiment 9 above); Group D, n = 4 per group. Data are expressed as mean ± standard deviation (SD). *P<0.05 compared with the Con group, #P<0.05 compared with the DM group. Groups C and D showed by WB analysis that the ratio of activated form of Akt (i.e., phosphorylated Akt, p-Akt) to total Akt protein was significantly reduced in the testicular tissue of DM mice, indicating that diabetes may inhibit the activation of Akt (experiment 10 above). These findings indicate that TLS inhibits DM-induced cell death, possibly through the Akt / Gsk3β cascade pathway.

[0116] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. Use of trehalose in the preparation of drugs for improving reproductive organ damage caused by diabetes.

2. The use of trehalose according to claim 1 in preparing a drug for improving reproductive organ damage caused by diabetes, characterized in that: The dosage form of the drug has been determined to be an oral dosage form.

3. The use of trehalose according to claim 2 in the preparation of a drug for improving reproductive organ damage caused by diabetes, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier.

4. The use of trehalose according to claim 1 in preparing a drug for improving reproductive organ damage caused by diabetes, characterized in that: The trehalose improves testicular damage caused by diabetes by activating autophagic cells.

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