Use of mitochondrial acid 5 in preparing medicine for treating reproductive organ damage caused by diabetes

Abstract

The present application proposes the use of mitochondrial acid 5 in the preparation of drugs for treating reproductive organ damage caused by diabetes, which relates to the field of biomedicine technology. The present application proves through experiments that MA‑5, as a synthetic indole‑3‑acetic acid derivative, provides a new treatment idea for testicular damage caused by diabetes, which not only helps to improve reproductive function, but also may provide a new strategy for the overall treatment of diabetes. In addition, as a natural plant hormone, MA‑5 has high safety and acceptability and may become a new alternative therapy with clinical application prospects. Although the long-term administration of MA‑5 does not significantly affect blood sugar levels, its alleviating effect on testicular damage shows that MA‑5, as a long-term treatment method, can continuously improve reproductive dysfunction caused by diabetes 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 mitochondrial acid 5 in the preparation of a drug for treating reproductive organ damage caused by diabetes. Background Art

[0002] Diabetes mellitus (DM) is a significant risk factor for male reproductive system damage. This includes neuroendocrine dysfunction, spermatogenesis, and ejaculation disorders, all of which are closely linked to oxidative stress disturbances. Research suggests that reducing oxidative stress can help mitigate diabetic-induced testicular damage. However, current treatments primarily focus on controlling blood sugar and improving overall health, and effective treatments for diabetic-induced testicular damage remain lacking.

[0003] Some of the shortcomings of the existing technology are: 1. At present, the treatment of reproductive organ damage caused by diabetes mainly relies on the use of diabetes drugs, and there is a lack of targeted drugs. 2. The current treatment options lack comprehensive strategies, especially those that can simultaneously regulate multiple pathogenic mechanisms (such as apoptosis, oxidative stress and autophagy). Testicular damage caused by diabetes is a complex multifactorial process, and a single treatment is often difficult to effectively solve the problem. 3. Although some drugs such as antioxidants and anti-inflammatory drugs can alleviate testicular damage, testicular damage caused by diabetes is a stubborn chronic disease like diabetes, and long-term use of such drugs will produce greater side effects. In addition, traditional anti-diabetic drugs are usually not able to effectively reduce reproductive system damage in the short term, and long-term use may lead to drug resistance or other negative reactions. 4. Testicular damage caused by diabetes is often accompanied by oxidative stress, apoptosis and autophagy, but existing treatments are still insufficient to intervene in these cell death pathways. Summary of the Invention

[0004] The purpose of this application is to provide the use of mitochondrial acid 5 in the preparation of a drug for treating reproductive organ damage caused by diabetes. It discloses mitochondrial acid 5 (MA-5) as a potential treatment option. Through its comprehensive therapeutic effects such as antioxidant, anti-apoptotic and autophagy activation, it shortens the treatment cycle, reduces the harm of long-term medication, and improves the intervention effect on key cell death pathways, thereby achieving effective treatment goals.

[0005] In order to solve the above technical problems, the technical solutions adopted in this application are:

[0006] The invention relates to a use of mitochondrial acid 5 in the preparation of a drug for treating reproductive organ damage caused by diabetes. The drug is in an oral dosage form and further comprises a pharmaceutically acceptable excipient.

[0007] Using mitochondrial acid 5 (MA-5) to inhibit cellular oxidative stress to improve diabetes-related damage, especially for testicular protection, has several significant advantages:

[0008] 1. Diabetic testicular damage can lead to changes in testicular tissue morphology and function, such as sparse arrangement of seminiferous tubules, separation of spermatogenic cells from the stroma, a significant decrease in the number and disorder of the spermatogenic epithelium, the presence of sloughed spermatogenic cells in the seminiferous tubules, and a significant decrease in spermatogenesis. Treatment with MA-5 reduced blood glucose levels and testicular mass and organ coefficient in mice. This study demonstrates for the first time that MA-5 treatment of diabetes can improve these testicular tissue changes.

[0009] 2. Studies both domestically and internationally have shown that hyperglycemia-induced reproductive system damage may be related to overactive oxidative stress. The findings in diabetic mice in this study are consistent with previous reports, showing abnormalities in SOD and MDA oxidative stress markers. Excessive oxidative stress can generate large amounts of reactive oxygen species (ROS). The accumulation of ROS can lead to apoptosis, which manifests as mitochondrial cytochrome c release in response to cellular stress or apoptotic signals. As an apoptosis-inducing factor, cytochrome c can form apoptosomes with Apaf-1, Pro-Caspase-9, and ATP / dATP, recruiting and activating Caspase-3, which in turn triggers a cascade of caspases, leading to apoptosis. In this study, diabetic mice also underwent apoptosis, with elevated expression of apoptotic proteins (BAX, Cleaved-Caspase-3, and Cleaved-Caspase-9) and decreased expression of the anti-apoptotic protein (BCL-2). Treatment with MA-5 reversed the expression of these apoptosis-related proteins in a dose-dependent manner, indicating that MA-5 can alleviate diabetic testicular damage by improving apoptosis.

[0010] 3. Excessive oxidative stress can also lead to ferroptosis, which manifests as abnormal iron levels, dense mitochondrial membranes, and reduced or absent mitochondrial cristae. This study found abnormal mitochondrial morphology near the sperm of diabetic mice, manifested by smaller mitochondria, reduced or even absent mitochondrial cristae, and ruptured and swollen mitochondrial outer membranes. After treatment with MA-5, mitochondrial cristae near sperm gradually became clearer, returning to normal size and morphology. This may help improve diabetic testicular damage.

[0011] 4. Furthermore, ferroptosis is also manifested by decreased expression of GPX4 and GSH. GPX4, a key factor, plays a crucial role in processes such as spermatogenesis. GPX4 consists of three types: mGPx4, which is primarily transported to mitochondria; nGPx4, which is primarily localized to the nucleolus; and cGPx4, which is primarily distributed in the cytoplasm and nucleus. Studies have found that the expression of mGPX4 and nGPX4 mRNA in testicular tissue is significantly higher than in other tissues. In particular, during spermatogenesis, the expression of mGPx4 and nGPx4 mRNA in the testis is significantly induced. Nrf2 can manipulate downstream antioxidant genes (such as GPX4) to influence ferroptosis. This evidence suggests that ferroptosis may play a key role in testicular damage. Experiments have shown that Nrf2 and GPX4 expression is decreased in the DM group. However, treatment with MA-5 at varying concentrations has been shown to increase Nrf2 and GPX4 expression. By reducing ferroptosis, MA5 may not only inhibit diabetes-induced excessive cellular oxidative stress but also promote functional recovery of testicular cells and improve fertility.

[0012] 5. The study found that VADC1 is mainly located in TM4 cells, VDAC2 is mainly located in late spermatocytes and spermatids, and VDAC3 is located in all types of testicular cells, especially in the testicular interstitial cells. In the late stage of sperm maturation, VDACs undergo necessary modification and folding and are finally expressed in mature sperm, especially in flagella. At the same time, VDAC-mediated ATP binding and transport provide essential energy for sperm forward movement. VDAC is located in the acrosomal region as a pore protein and regulates Cl - VDACs transport ions such as Ca[2+] and participate in the complex acrosome reaction. This suggests that abnormal expression of VDACs may be a key factor in testicular tissue disease. Experiments have shown elevated expression of VDAC1 and VDAC3 in the DM group. Treatment with MA-5 at varying concentrations can reduce VDAC1 and VDAC3 levels, effectively and targetedly improving diabetic testicular damage.

[0013] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0014] This application has demonstrated through experiments that MA-5, as a synthetic indole-3-acetic acid derivative, provides a new therapeutic approach for testicular damage caused by diabetes, which not only helps improve reproductive function, but also may provide a new strategy for the overall treatment of diabetes. In addition, as a natural plant hormone, MA-5 has high safety and acceptability and may become a new alternative therapy with clinical application prospects. Although long-term administration of MA-5 does not significantly affect blood sugar levels, its alleviating effect on testicular damage indicates that MA-5, as a long-term treatment method, can continuously improve reproductive dysfunction caused by diabetes and has potential clinical advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. 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 relevant drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 This is a comparison chart of the changes in body mass of diabetic mice in different experimental groups in the examples of this application;

[0017] Figure 2 This is a comparison chart of the effects of MA-5 on blood sugar in diabetic mice in the examples of this application;

[0018] Figure 3 This is a comparison chart of the effects of MA-5 on blood lipids in diabetic mice in the examples of this application;

[0019] Figure 4 The changes in reproductive capacity of diabetic mice in different experimental groups in the examples of this application;

[0020] Figure 5 This is a diagram showing the effect of MA-5 on testicular structural damage in diabetic mice in the examples of this application;

[0021] Figure 6 This is an electron micrograph of the effect of MA-5 on the structural damage of mitochondria in the testes of diabetic mice in the examples of this application;

[0022] Figure 7 This is a comparison chart of the effects of MA-5 on oxidative stress levels in mouse testicular tissue in the examples of this application;

[0023] Figure 8 This is a comparison chart of the effects of MA-5 on apoptosis proteins in diabetic mice in the examples of this application;

[0024] Figure 9This is a comparison chart of the effects of MA-5 on the expression of ferroptosis proteins in diabetic mice in the examples of this application. DETAILED DESCRIPTION

[0025] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0026] The invention relates to a use of mitochondrial acid 5 in the preparation of a drug for treating reproductive organ damage caused by diabetes. The drug is in an oral dosage form and further comprises a pharmaceutically acceptable excipient.

[0027] This application is about the research of mitochondrial acid 5 in treating reproductive organ damage caused by diabetes, including the following steps:

[0028] 1. Establishment of animal model

[0029] SPF male C57BL / 6J mice (16-18 g) were acclimated for one week and then divided into a normal group and a model group. The normal group mice were fed a normal diet, while the diabetic mice were fed a high-sugar, high-fat diet for six weeks. Streptozotocin (STZ) was intraperitoneally injected on days 1 and 5, respectively. The normal group mice were given an equal volume of citric acid-sodium citrate buffer, with no water restriction. Blood samples were collected from the tail of the mice for random blood glucose measurements on days 3 and 7 after STZ injection. Successful modeling was considered when both random blood glucose levels were ≥16.7 mmol / L. The mice were then randomly divided into six groups: a normal control group, a diabetic model group, a low-dose MA-5 group, a medium-dose MA-5 group, a high-dose MA-5 group, and a high-dose MA-5 normal control group. The normal control and high-dose MA-5 control groups continued to be fed a normal diet, while the other groups continued to be fed a high-sugar, high-fat diet and were treated with varying concentrations of MA-5 by gavage for 8 weeks.

[0030] 2. Physiological index measurement

[0031] Blood samples were collected from the tail vein to measure blood glucose levels. At the end of the experiment, the mice were anesthetized and blood was collected from the eyeballs. The blood was collected using a clean EP tube, placed in a 4°C refrigerator overnight, and the supernatant was collected by low-temperature centrifugation. After blood was collected from the eyeballs, the mice were killed by cervical dislocation. The testicles and epididymal tissues were quickly opened and removed, and other useless tissues adhered to them were peeled off. The tail of the epididymis was selected for sperm motility and sperm count experiments. Part of the testicular tissue was cut and immersed in Bouin's fixative, and used for HE staining. The sections were examined under a microscope (Leica DM2000, Germany) to observe the morphological and pathological changes of the testicular tissue. Another part of the testicular tissue was cut and immersed in electron microscopy fluid, and then used for electron microscopy to observe the morphological changes of the ultrastructure in the testicular tissue. The remaining part was promptly cooled in liquid nitrogen. After processing, it was stored in a -80°C refrigerator for other tests.

[0032] Determination of mouse serum total cholesterol (TC): Total cholesterol is an important indicator of blood lipids. Dilute mouse serum with normal saline to within the linear range of the test kit. Follow the instructions and measure the absorbance of each well with a microplate reader. Calculate the value according to the formula.

[0033] Mouse serum triglyceride (TG) content determination: Triglycerides are another important indicator of blood lipids. Dilute mouse serum with normal saline to within the linear range of the test kit. Follow the instructions and measure the absorbance of each well with a microplate reader. Calculate according to the formula.

[0034] 3. Molecular biology analysis

[0035] Western blot analysis revealed significantly increased expression of apoptotic proteins (cleaved-Caspase-3, cleaved-Caspase-9, BAX) and decreased expression of the anti-apoptotic protein (BCL-2) in the DM group compared with the control group. Furthermore, the BAX / BCL-2 ratio was significantly elevated in DM, with a significant decrease beginning in the low-dose MA-5 group, demonstrating the inhibitory effect of MA-5 on apoptosis. MA-5 treatment reversed the expression of these apoptosis-related proteins in a dose-dependent manner, suggesting that MA-5 can improve cell apoptosis. Further studies revealed that GPX4 is a key protein in the development of ferroptosis. VDACs are voltage-dependent ion channels, and erastin-induced ferroptosis can affect their permeability. Compared with the control group, Nrf2 and GPX4 expression was significantly decreased in the DM group. Furthermore, these expressions gradually increased after treatment with different concentrations of MA-5, with significant differences observed in the high-dose group. However, compared with the control group, the expression of VDAC1 and VDAC3 in the DM group was significantly elevated. After treatment with different concentrations of MA-5, these levels were significantly reduced in the medium-dose group. Ferroptosis is involved in the development and progression of diabetic testicular damage, and MA-5 can increase the expression of Nrf2 and GPX4 while also reducing the levels of VDAC1 and VDAC3. Therefore, MA5 can reduce the occurrence of ferroptosis and improve cellular oxidative stress.

[0036] Superoxide dismutase (SOD) activity assay in mouse testicular tissue: SOD is the primary scavenger of intracellular free radicals and a reliable indicator of the body's antioxidant capacity. Cryopreserved testicular tissue was used for SOD assay. Sample preparation was as follows: Accurately weigh the testicles according to a weight (g): volume (mL) ratio of 1:9. Testicles of appropriate size were cut on ice and weighed. A corresponding volume of physiological saline was added and ground to prepare a tissue homogenate. The homogenate was then centrifuged at 4°C, 4000 rpm, for 10 minutes. The supernatant was aspirated for analysis.

[0037] Malondialdehyde (MDA) content in mouse testicular tissue: MDA is cytotoxic and can cause downstream interactions with many biomolecules. Therefore, MDA is often used as an indicator of lipid peroxidation. Take 10% tissue homogenate and perform the MDA test using the instructions provided with the kit.

[0038] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0039] Example

[0040] 1. Establishment of animal model

[0041] SPF male C57BL / 6J (16-18 g) mice were divided into a normal group and a model group after adaptive feeding for one week. The normal group mice were fed with ordinary feed, and the diabetic mice were fed with high-sugar and high-fat feed for six weeks. Then, they were intraperitoneally injected with 100 mg·kg of streptozotocin (STZ) solution on the first and fifth days respectively. -1 ·d -1 (Before use, use 0.1mmol / L citric acid-sodium citrate buffer solution at pH 4.4 to prepare a 1% concentration) and inject twice. The normal group of mice was given an equal volume of citric acid-sodium citrate buffer solution and had no restriction on drinking water.

[0042] On days 3 and 7 after STZ injection, random blood glucose was measured by taking blood samples from the tail of the mice. If the random blood glucose levels were ≥16.7 mmol / L on both occasions, the model was considered established successfully. The mice that did not form a model were eliminated and randomly divided into six groups: normal control group (Control group, n=15), diabetic model group (DM group, n=15), MA-5 low-dose group (DM+25 mg·kg -1 MA-5 group, n = 15), MA-5 medium dose group (DM + 50 mg kg -1 MA-5 group, n = 15), MA-5 high-dose group (DM + 100 mg kg -1 MA-5 group, n = 15) and MA-5 high-dose normal control group (Control + 100 mg·kg -1 MA-5 group, n=15). The control group and the MA-5 high-dose control group continued to be fed with ordinary feed, while the mice in the other groups were still fed with a high-sugar and high-fat feed and simultaneously gavaged with different concentrations of MA-5 for 8 weeks.

[0043] 2. Physiological index measurement

[0044] 2.1 Effects of MA-5 on the physiological state of diabetic mice

[0045] During the modeling period, the mice were weighed at a fixed time every day, and their body shape and hair characteristics were observed. The mice in the Control group grew and developed normally, with smooth and shiny hair, good mental state, and lively spirits. The mice in the DM group were in poor spirits, with dry and dull hair and slow reactions. In the later stage, they had decreased appetite, increased water intake, increased urination, and sticky and loose stools. After intervention with different concentrations of MA-5, the mice's mental state, hair texture, appetite, and excrement all improved.

[0046] Effects of 2.2MA-5 on body weight in diabetic mice

[0047] Changes in body mass of diabetic mice in each group Figure 1 As shown, Figure 1Middle: normal control group (Control group); diabetic model group (DM group); MA-5 low-dose group (DM+25 mg / kg MA-5 group); MA-5 medium-dose group (DM+50 mg / kg MA-5 group); MA-5 high-dose group (DM+100 mg / kg MA-5 group); MA-5 high-dose control group (Control+100 mg / kg MA-5 group): n=10-13; compared with the Con group, *P<0.05; compared with the DM group, #P<0.05.

[0048] The body weight of mice in each group showed an increasing trend during the feeding stage. After modeling, the body weight of mice in the DM group increased most significantly compared with the Control group. The body weight changes of mice after MA-5 administration showed a decrease in body weight, but there was no significant difference compared with the DM group. At the same time, we also observed that the body weight of mice in the normal drug administration group did not increase significantly, and their body weight decreased compared with the Control group. Figure 1 A (Weight changes of diabetic mice at different time points). The testicles of the mice were weighed after sampling. The experiment found that the testicle weight of the DM group mice was slightly increased compared with the control group. After treatment with MA-5, the testicle weight was reduced. Figure 1 B (The effect of MA-5 on the testicular weight of diabetic mice) shows a significant decrease at high doses (P<0.05). At the same time, the testicular organ coefficient (testicular weight / mouse body weight × 100%) of the DM group mice was significantly increased compared with the control group (P<0.05), while it was significantly decreased in the low-dose MA-5 group. Figure 1 C (Effects of MA-5 on organ coefficients in diabetic mice).

[0049] 2.3 Effect of MA-5 on blood glucose in diabetic mice

[0050] Effects of MA-5 on blood glucose in diabetic mice Figure 2 As shown, Figure 2 Middle A: Changes in blood glucose at different time points in diabetic mice; B: Effects of MA-5 on intraperitoneal glucose tolerance in diabetic mice; C: AUC; n = 8-13; compared with the Control group, *P < 0.05; compared with the DM group, #P < 0.05;

[0051] The DM group mice showed a continuous increase in blood glucose (P<0.05). After intervention with different concentrations of MA-5, it was observed that the blood glucose of the treated group mice showed a downward trend, and the low and high doses could significantly reduce their blood glucose levels (P<0.05). Figure 2 As shown in A.

[0052] Sustained high blood sugar levels can cause glucose metabolism disorders in the body. The intraperitoneal glucose tolerance test detects the body's speed and ability to clear glucose, and is used to determine the glucose metabolism disorders in diabetic mice. In the experiment, we found that the blood sugar levels of diabetic mice were significantly higher than those of the Control group. The blood sugar levels of the six groups of mice reached a peak at 15 minutes, and the blood sugar levels of each group began to decrease 60 minutes after intraperitoneal injection. At 120 minutes, the DM group was still at a high blood sugar level. The blood sugar levels of the treatment groups all decreased, but no significant differences were observed, indicating that MA-5 can reduce the glucose metabolism disorders caused by DM. Figure 2 B.

[0053] The area under the curve (AUC) represents the bioavailability of the drug and can reflect the degree of drug absorption in the body. The area under the curve within AUC (0-120min) can reflect the amount of drug absorbed into the blood circulation. The AUC of DM mice increased significantly (P<0.05). At the same time, it was observed that the high-dose MA-5 group could significantly reduce its change (P<0.05). Figure 2 As shown in C.

[0054] Effects of 2.4MA-5 on blood lipids in diabetic mice

[0055] Determination of mouse serum total cholesterol (TC): Total cholesterol is an important indicator of blood lipids. Dilute mouse serum with normal saline to within the linear range of the test kit. Follow the steps in Table 1 and measure the absorbance of each well with a microplate reader. Calculate the value according to the formula.

[0056] Table 1

[0057]

[0058]

[0059] Mouse serum triglyceride (TG) content determination: Triglycerides are another important indicator of blood lipids. Dilute mouse serum with normal saline to within the linear range of the test kit. Follow the steps shown in Table 2 and measure the absorbance of each well with a microplate reader. Calculate the value according to the formula.

[0060] Table 2

[0061]

[0062] Effects of MA-5 on blood lipids in diabetic mice Figure 3 As shown, Figure 3 A: Total cholesterol (TC) determination in mice; B: Triglyceride (TG) determination in mice; n=8-10; compared with the Control group, *P<0.05; compared with the DM group, #P<0.05;

[0063] Triglyceride (TG) and total cholesterol (TC) are important indicators for blood lipid detection, and their expression is increased in diabetic models. Figure 3 It can be seen that the TC and TG levels in the DM group were significantly higher than those in the control group. After intervention with MA-5, their blood lipid levels were significantly improved, and in the high-dose MA-5 group, the TC and TG levels were significantly reduced.

[0064] Effects of 2.5MA-5 on reproductive capacity of diabetic mice

[0065] Sperm function assay: The left epididymal tail of the mouse was dissected out and placed in 2 mL of 37°C sterile saline, minced and mixed to prepare a sperm suspension. 0.05 mL of the suspension was pipetted onto a cell counting plate and observed under a fixed-magnification microscope. The total number of sperm in the five central squares, the number of sperm with normal sperm morphology and motility, and the total number of sperm were recorded. The results were calculated using the following formula: Sperm motility percentage = total number of sperm with normal motility × 100%.

[0066] The changes in reproductive capacity of diabetic mice in each experimental group are as follows Figure 4 As shown; Figure 4 A: sperm count; B: sperm motility; n = 10; compared with the Control group, *P < 0.05; compared with the DM group, #P < 0.05;

[0067] Diabetic testicular damage can lead to reproductive dysfunction in mice. Figure 4 We found that compared with the Control group, the sperm motility and total number in the testicles of the DM group mice were significantly reduced (P<0.05), resulting in a decrease in sperm quality; after oral administration of different concentrations of MA-5, an upward trend was shown, with a significant increase in sperm concentration in the medium-dose MA-5 group and a significant improvement in sperm motility in the low-dose MA-5 group.

[0068] Effects of 6MA-5 on testicular structural damage in diabetic mice

[0069] HE staining was carried out by baking in a 60°C constant temperature drying oven for 30 minutes. The slides were then observed to have become transparent. The HE operation steps were as shown in Table 3:

[0070] Table 3

[0071]

[0072]

[0073]

[0074] After the above operations were completed, the microstructural changes of the testicular tissue were observed under a light microscope.

[0075] Take the testicles and immerse them in electron microscopy fixative in a 4°C refrigerator overnight. After removal, perform the soaking, embedding, and curing reactions according to the procedures in Tables 4-6. The soaking steps are shown in Table 4:

[0076] Table 4

[0077] Reagents Reaction time 50% ethanol 15min 70% ethanol 15min 80% ethanol 15min 90% ethanol 15min 95% ethanol 15min Anhydrous ethanol 15min 100% acetone 15min

[0078] The testicular tissue after the final 100% acetone soaking was subjected to embedding reaction, and the operation sequence is shown in Table 5:

[0079] Table 5

[0080] Reagents Reaction time Reaction temperature Acetone + embedding solution (2:1) 3h Room temperature Acetone + embedding solution (1:2) overnight Room temperature embedding fluid 2h 37℃

[0081] The embedded testicular tissue was subjected to a curing reaction, and the order of the curing reaction is shown in Table 6:

[0082] Table 6

[0083] Reaction time Reaction temperature overnight 37℃ 12h 45℃ 24h 60℃

[0084] The immersed testicular tissue was cut into 60 nm thin sections using an ultramicrotome, and then stained with 3% uranyl acetate-lead citrate and observed using an electron microscope.

[0085] Effects of MA-5 on testicular structural damage in diabetic mice Figure 5 HE staining is an important method for observing tissue pathological changes. Figure 5 HE microscopic observations showed that the testes of the control group mice had intact seminiferous tubules, which were tightly arranged, with clear and smooth boundaries between the basement membrane and the supporting membrane. Furthermore, the supporting cells and sperm in the seminiferous tubules were abundant and neatly arranged. The seminiferous tubules of the DM group mice were damaged to varying degrees, with sparse arrangement and separation of spermatogenic cells from the basement membrane. The number of spermatogenic epithelial layers was significantly reduced, with disordered arrangement. Desquamated spermatogenic cells were observed in the seminiferous tubules, and spermatogenesis was significantly reduced. After treatment with MA-5, damage to the seminiferous epithelium and seminiferous tubules was significantly alleviated, as evidenced by an increase in the number of spermatogenic epithelial layers, orderly arrangement, and gradually tighter adhesion to the basement membrane. This increased spermatogenesis and the gradual recovery of spermatogenic function were also observed.

[0086] Effects of MA-5 on testicular mitochondrial structural damage in diabetic mice Figure 6Figure 2 shows a representative transmission electron micrograph (TEM) image at magnification = 3000× / 8000×. Arrows point to mitochondria. Electron microscopy can be used to observe ultrastructure. We used TEM to observe the morphology of mitochondria near sperm. Mitochondria in the control group had clear cristae, intact membranes, and normal size. In contrast, mitochondria in the DM group became smaller and rounder, with fewer or even absent cristae. Mitochondria also showed swelling, even forming "plate-like" mitochondria. With increasing MA-5 concentrations, mitochondrial morphology gradually recovered. At the medium dose of MA-5, the mitochondrial cristae near sperm gradually became clearer, with pronounced wrinkles.

[0087] 3. Molecular biology analysis

[0088] Effects of 3.1MA-5 on oxidative stress levels in mouse testicular tissue

[0089] Determination of superoxide dismutase (SOD) activity in mouse testicular tissue: SOD is the main substance for scavenging free radicals in cells and is a reliable indicator for measuring the body's antioxidant capacity.

[0090] Frozen testicular tissue was collected for SOD assay. Sample preparation was as follows: After accurate weighing, the sample was adjusted to a weight (g): volume (mL) ratio of 1:9. Testicles of appropriate size were cut on ice and weighed, and the corresponding volume of saline was added. Grind the sample to create a tissue homogenate. The sample was then centrifuged at 4°C, 4000 rpm for 10 minutes, and the supernatant was removed for analysis. Reagent configuration is shown in Table 7:

[0091] Table 7

[0092] Reagents Solution preparation SOD substrate application solution Substrate stock solution: buffer = 1:200 Enzyme working solution Enzyme stock solution: enzyme diluent = 1:10

[0093] Take the supernatant and operate according to the instructions of the SOD test kit. The specific steps are shown in Table 8:

[0094] Table 8

[0095]

[0096] The calculation formula is as follows:

[0097]

[0098]

[0099] Determination of malondialdehyde (MDA) content in mouse testicular tissue:

[0100] MDA is cytotoxic and can cause downstream interactions with many biomolecules. Therefore, MDA is often used as an indicator to measure the degree of lipid peroxidation. The preparation liquid of the detection kit is shown in Table 9 and Table 10:

[0101] Table 9

[0102] Reagents Mass / Volume Reagent 2 1.2mL distilled water 34mL

[0103] Table 10

[0104] Reagents Mass / Volume Reagent 3 1 piece distilled water 60mL glacial acetic acid 60mL

[0105] Take 10% tissue homogenate and operate according to the instructions of the MDA test kit. The specific operating steps are as shown in Table 11:

[0106] Table 11

[0107]

[0108]

[0109] Vortex mix, then heat in a constant temperature metal bath at 95°C for 40 min, centrifuge at 4000 rpm in a water-cooled centrifuge for 10 min, aspirate the supernatant, add it to a 96-well plate, protect from light, and measure the OD value of each tube at a wavelength of 532 nm using a microplate reader.

[0110] The calculation formula is as follows:

[0111]

[0112] Effects of MA-5 on oxidative stress levels in mouse testicular tissue Figure 7 shown. Figure 7 A: SOD content determination; B: MDA content determination; n = 8-10; compared with the Control group, *P < 0.05;

[0113] Abnormal oxidative stress levels are closely related to the occurrence of diabetic testicular damage. Figure 7 As shown in the results, compared with the control group, the SOD activity in the testes of the DM mice was significantly decreased, while the MDA content was significantly increased, with significant differences (P<0.05). Compared with the DM group, the SOD activity of the mice treated with different concentrations of MA-5 increased, which improved the condition to a certain extent. At the same time, the MDA content showed a downward trend after treatment, with a significant difference in the high-dose MA-5 group.

[0114] 3.2WB detection of germ cell apoptosis and ferroptosis in diabetic mice

[0115] Western blot (WB) detection:

[0116] 1. Protein Extraction: Weigh the tissue pellet using an electronic balance. Add freshly prepared tissue lysis buffer (RIPA: Cocktail: PMSF: Na₃VO₄ = 100:1:1:1) to 1 mg of tissue. Grind thoroughly on ice. Pipette the entire tissue suspension into a labeled EP tube. Centrifuge at 4°C, 12,000 rpm for 15 min. Aspirate the supernatant.

[0117] 2. Protein Concentration: Determine sample concentration using the BCA protein concentration assay kit. Dissolve the extracted supernatant sample and protein standards at low temperature. Dilute the protein standards to 1 mg / mL with ultrapure water. Add the protein standards to a 96-well plate according to the concentration ratio (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg / mL). Make up to 20 μL with ultrapure water. Reserve one replica well for each protein standard in the 96-well plate. Take 2 μL of the sample supernatant and add 18 μL of double-distilled water to make up to 20 μL (i.e., each sample is diluted 10-fold for testing; the final protein concentration calculation requires multiplying back the dilution factor). Reserve one replica well for each sample well. Calculate the required number of wells and add 200 μL of the prepared BCA working solution (Solution A:Solution B = 50:1) to each well. After completing the above steps, heat the 96-well plate in a 37°C thermostated metal bath for 30 minutes. Measure the absorbance of each well at 562 nm using a microplate reader. The standard protein sample is used to calculate the standard curve. Substitute the measured OD value of the sample into the standard curve to calculate the sample concentration. Take an appropriate amount of supernatant, mix thoroughly with the lysate and 5× protein loading buffer, and heat in a thermostated metal bath at 97°C for 5-10 minutes to denature. After cooling, store in a -20°C refrigerator until needed.

[0118] 3. Preparation of Separating Gel and Stacking Gel

[0119] Rinse the glass plates with ultrapure water and air dry. Clamp the two plates horizontally and place them in a gel rack. Place the gel rack on a level surface. Prepare the separation gel according to the molecular weight of the target protein. Add the gel preparation reagents sequentially according to the SDS-PAGE preparation sequence. Mix thoroughly by pipetting, avoiding excessive foaming. Once prepared, slowly add the separation gel from one side of the glass plate to the interlayer of the sealed gel rack to the desired height. Slowly add anhydrous ethanol from one side of the glass plate to flatten the gel surface.

[0120] After the separation gel has completely solidified, remove the anhydrous ethanol and blot any remaining residue with filter paper, ensuring that the anhydrous ethanol is completely removed while also preventing the entry of foreign matter. Prepare a 5% stacking gel according to the gel preparation recipe (e.g., 2 pieces: 4ml of water, 1ml of 30% acrylamide, pH 6.81ml, 0.08ml of 10% SDS, 0.06ml of 10% AP, 8μL of TEMED). Similarly, after adding each reagent, pipette and mix thoroughly. After mixing, fill the interlayer with a pipette and quickly insert the accompanying comb into the stacking gel, still avoiding the formation of bubbles. Once the stacking gel is completely solidified, remove it and set aside.

[0121] 4. Electrophoresis: For this experiment, the testicular tissue sample was loaded with approximately 50 μg of protein. Rainbow markers were added to each well, with a loading volume of approximately 1.5 μL. Sample loading was performed accurately to avoid sample overflow. After loading, check the electrophoresis instrument for any abnormalities, adjust the instrument voltage to 60V, and begin electrophoresis. Observe for bubbles at the bottom of the electrophoresis plate and for changes in the liquid between the two plates. Once the protein sample has been electrophoresed below the contact surface of the separating gel and the rainbow markers have separated, adjust the instrument voltage to 90V. Stop electrophoresis when the desired height is reached.

[0122] 5. Transfer: Cut the PVDF membrane to a similar size as the gel. Remove the glass plate, cut the gel to the appropriate size, immerse it in transfer buffer, and place the transfer cassette. Then, immerse the PVDF membrane in methanol to activate it. Aspirate the methanol solution from the surface of the PVDF membrane in the transfer buffer, transfer it to the gel surface, and secure one end to remove any air bubbles between the gel and membrane, ensuring a tight fit. Secure the transfer cassette and place it in the transfer tank. Set the transfer conditions to 300 mA for 120 min. After transfer, remove the membrane, label it, and wash it with 1× TBST.

[0123] 6. Blocking: Place the cleaned PVDF membrane in 5% skim milk blocking buffer and shake for 1-2 hours at room temperature. Wash with 1× TBST afterward. The blocking time and number of washes can be adjusted based on experimental results.

[0124] 7. Primary Antibody Incubation: Cut the PVDF membrane as needed and label it. Incubate the membrane in primary antibody diluent containing antibodies against BAX (1:1000), BCL-2 (1:1000), Cleaved-Caspase-3 (1:1000), Cleaved-Caspase-9 (1:1000), VDAC1 (1:1000), VDAC3 (1:1000), GPX4 (1:1000), Nrf2 (1:1000), and β-actin (1:10000) overnight at 4°C. After primary antibody incubation, remove the membrane and wash three times in 1× TBST for 5-10 minutes each wash.

[0125] 8. Secondary Antibody Incubation: Place the PVDF membrane in HRP-conjugated goat anti-rabbit IgG (1:10,000) or HRP-conjugated rabbit anti-mouse IgG (1:10,000) diluted in 1× TBST and incubate on a shaker at room temperature for 1 hour. Then, remove the PVDF membrane and wash it three times in 1× TBST, each time for 5-10 minutes.

[0126] 9. Development: Prepare ultra-sensitive luminescent solution by mixing equal volumes of ECL luminescent solution A and ECL luminescent solution B. Immerse the PVDF membrane evenly in the solution, expose the membrane using a gel imaging system, and analyze the grayscale value using Image Lab Software.

[0127] 10. Statistical analysis: All data were expressed as mean ± standard error (x ± SEM) using SPSS 26.0 statistical software. One-way ANOVA was used for statistical analysis, and P < 0.05 was considered statistically significant.

[0128] Apoptosis, also known as programmed cell death, refers to a group of active cell death processes with distinct morphological and biochemical characteristics, caused by various factors under physiological and pathological conditions. This is manifested by abnormal levels of pro-apoptotic and anti-apoptotic proteins.

[0129] Effects of MA-5 on apoptosis proteins in diabetic mice Figure 8 As shown, Figure 8 Middle A: BAX protein expression and protein representative graph; B: BCL-2 protein expression and protein representative graph; C: BAX / BCL-2 ratio; D: Cleaved-Caspase-3 protein expression and protein representative graph; E: Cleaved-Caspase-9 protein expression and protein representative graph; n = 4-5; compared with the control group, *P < 0.05; compared with the DM group, #P < 0.05;

[0130] from Figure 8 It can be seen that the expression of apoptosis proteins (Cleaved-Caspase-3, Cleaved-Caspase-9, BAX) in the DM group was significantly increased compared with the Control group (P<0.05) (e.g. Figure 8 A, D, E), while the expression of anti-apoptotic protein (BCL-2) decreased (e.g. Figure 8 B), after MA-5 treatment, we observed a significant decrease in the expression of apoptotic proteins (P<0.05), and an increase in the expression of anti-apoptotic proteins (P>0.05). At the same time, we also observed a significant increase in the BAX / BCL-2 ratio in DM, and a significant decrease in the MA-5 low-dose group (e.g. Figure 8 C).

[0131] GPX4 is a key protein in the development of ferroptosis. VDACs are voltage-dependent ion channels. Erastin-induced ferroptosis can affect their permeability. Figure 9 As shown, Figure 9 Middle A: Nrf2 protein expression and protein representative graph; B: GPX4 protein expression and protein representative graph; C: VDAC1 protein expression and protein representative graph; D: VDAC3 protein expression and protein representative graph; n = 4-5; compared with the Control group, *P < 0.05; compared with the DM group, #P < 0.05;

[0132] Compared with the Control group, the expressions of Nrf2 and GPX4 in the DM group were significantly decreased (P<0.05). Figure 9 A, B), and at the same time, the expression of VDAC1 and VDAC3 in the DM group was significantly increased compared with the control group (P<0.05). Figure 9 C, D), after treatment with different concentrations of MA-5, the middle dose group showed a significant decrease (P < 0.05).

[0133] 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 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 are within the scope of protection of this application.

Claims

1. Use of mitochondrial acid 5 in the preparation of a drug for treating reproductive organ damage caused by diabetes, characterized in that: The reproductive organ is the testicle.

2. Use of mitochondrial acid 5 according to claim 1 in the preparation of a drug for treating reproductive organ damage caused by diabetes, characterized in that: The medicine is in oral dosage form and further comprises pharmaceutically acceptable excipients.

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