Application of mitochondrial acid 5 in preparation of medicine for treating reproductive organ injury caused by diabetes mellitus

Abstract

The invention provides application of mitochondrial acid 5 in preparation of a medicine for treating reproductive organ injury caused by diabetes mellitus, and relates to the technical field of biological medicine. Experiments prove that as an artificially synthesized indole-3-acetic acid derivative, MA-5 provides a brand new treatment thought for testis injury caused by diabetes mellitus, not only is beneficial to improvement of reproductive function, but also possibly provides a new strategy for overall treatment of diabetes mellitus. Besides, as a natural plant hormone, MA-5 has relatively high safety and acceptability, and possibly becomes a new alternative therapy with a clinical application prospect. Although the long-term administration of the MA-5 does not significantly influence the blood glucose level, the alleviation effect of the MA-5 on testis injury shows that the MA-5 as a treatment means for long-term use 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 an important risk factor for male reproductive system damage. It includes neuroendocrine regulation disorders, spermatogenesis and ejaculation dysfunction, which are closely related to oxidative stress disorders. Studies have shown that reducing oxidative stress can help alleviate testicular damage caused by diabetes. However, current treatment technologies mainly focus on controlling blood sugar and improving overall health, and there is still a lack of effective treatments for testicular damage caused by diabetes.

[0003] Some 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 relieve 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 effective in reducing 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 the present 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 can shorten the treatment cycle, reduce the harm of long-term medication, improve the intervention effect on key cell death pathways, and achieve effective treatment goals.

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

[0006] The invention discloses a use of mitochondrial acid 5 in preparing a medicine for treating reproductive organ damage caused by diabetes. The medicine is an oral dosage form and further comprises pharmaceutically acceptable excipients.

[0007] Using mitochondrial acid 5 (MA-5) to inhibit cellular oxidative stress to improve diabetes-related damage, especially for the protection of the testicles, 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 matrix, a significant decrease in the number of spermatogenic epithelial layers, disordered arrangement, detached spermatogenic cells in the seminiferous tubules, and a significant decrease in sperm production. After treatment with MA-5, blood sugar levels decreased and the testicular mass and organ coefficient of mice decreased. This experiment is the first to prove that MA-5 treatment of diabetes can improve the above-mentioned testicular tissue lesions.

[0009] 2. Studies at home and abroad have shown that reproductive system damage caused by hyperglycemia may be related to over-activated oxidative stress response. The performance of diabetic mice in this experiment is consistent with previous literature reports, showing abnormal SOD and MDA oxidative stress indicators. Excessive oxidative stress can produce a large number of reactive oxygen free radicals (ROS), and the accumulation of ROS can lead to cell apoptosis. Cell apoptosis is manifested as follows: cell stress response or apoptosis signal can cause the release of mitochondrial cytochrome c. As an apoptosis inducing factor, it can form apoptotic bodies with Apaf-1, Pro-Caspase-9, ATP / dATP, recruit and activate Caspase-3, and then trigger the Caspase cascade reaction, leading to cell apoptosis. In this experiment, diabetic mice also underwent cell apoptosis, with increased expression of apoptotic proteins (BAX, Cleaved-Caspase-3, Cleaved-Caspase-9) and decreased expression of anti-apoptotic proteins (BCL-2). After treatment with MA-5, the expression of the above apoptosis-related proteins was reversed in a dose-dependent manner, indicating that MA-5 can alleviate diabetic testicular damage by improving cell apoptosis.

[0010] 3. Excessive oxidative stress can also lead to ferroptosis, which can be manifested as abnormal iron ion levels, dense mitochondrial membranes, and reduced or disappeared mitochondrial cristae. This experimental study found that the mitochondrial morphology near the sperm of diabetic mice was abnormal, manifested as smaller mitochondria, reduced or even disappeared mitochondrial cristae, and ruptured and swollen mitochondrial outer membranes. After treatment with MA-5, it can be seen that the mitochondrial cristae near the sperm gradually became clear, and the size and morphology returned to normal. It is beneficial to improve diabetic testicular damage.

[0011] 4. In addition, ferroptosis is also manifested as a decrease in the expression of GPX4 and GSH. At the same time, the key factor GPX4 plays a key role in spermatogenesis and other processes. There are three types of GPx4, which are divided into mGPx4, which is mainly transported to mitochondria, nGPx4, which is mainly located in the nucleolus, and cGPx4, which is mainly distributed in the cytoplasm and nucleus. The study found that the expression of mGPX4 mRNA and nGPX4 mRNA in testicular tissue was significantly higher than that in other tissues. Especially during spermatogenesis, the expression of mGPx4 mRNA and nGPx4 mRNA in the testis was significantly induced. Nrf2 can manipulate downstream antioxidant genes (such as GPX4) to affect ferroptosis. These evidences suggest that ferroptosis may play an important role in testicular damage. The experiment found that the expression of Nrf2 and GPX4 in the DM group was reduced. After intervention with different concentrations of MA-5, it was found that MA-5 could increase the expression of Nrf2 and GPX4. MA5 can not only inhibit the excessive oxidative stress of cells caused by diabetes by reducing the occurrence of ferroptosis, but also promote the 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 testicular interstitial cells. In the late stage of sperm maturation, VDACs undergo necessary modifications 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 acrosome region as a pore protein, which regulates Cl - , Ca[2+] and other ion transport, and participate in the complex acrosome reaction. It is confirmed that abnormal expression of VDACs may become an important factor in testicular tissue diseases. The experiment found that the expression of VDAC1 and VDAC3 in the DM group was increased, and the content of VDAC1 and VDAC3 could be reduced after intervention with different concentrations of MA-5. It effectively improves diabetic testicular damage in a targeted manner.

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

[0014] This 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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.

[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 effect 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 microscopic image of the effect of MA-5 on the structural damage of testicular mitochondria in diabetic mice in the examples of this application;

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

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

[0024] Fig. 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] 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 the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0026] The invention discloses a use of mitochondrial acid 5 in preparing a medicine for treating reproductive organ damage caused by diabetes. The medicine is an oral dosage form and further comprises pharmaceutically acceptable excipients.

[0027] The present 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 (16-18g) mice were divided into normal group and model group after adaptive feeding for one week. The mice in normal group were fed with ordinary feed, and the diabetic mice were fed with high-sugar and high-fat feed for six weeks. Then, streptozotocin (STZ) was injected intraperitoneally on the 1st and 5th day, and the mice in normal group were given equal volume of citric acid-sodium citrate buffer, and drinking water was not restricted. Blood was collected from the tail of mice at 3d and 7d after STZ injection for random blood glucose measurement. The model was considered to be successfully established if both random blood glucose levels were ≥16.7mmol / L. The mice were randomly divided into six groups: normal control group, diabetic model group, MA-5 low-dose group, MA-5 medium-dose group, MA-5 high-dose group, and MA-5 high-dose normal control group. The normal control group and MA-5 high-dose control group continued to be fed with ordinary feed, and the mice in other groups were still fed with high-sugar and high-fat feed, and were gavaged with different concentrations of MA-5 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 in 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 experiments on sperm motility and sperm count. Part of the testicular tissue was cut and immersed in Bouin fixative, and HE staining was used to examine the sections 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, which was subsequently used to observe the morphological changes of the ultrastructure in the testicular tissue under an electron microscope. The remaining part was cooled in liquid nitrogen in time. After processing, it was stored in a -80°C refrigerator for other project detection.

[0032] Determination of mouse serum total cholesterol (TC) content: Total cholesterol is an important indicator for detecting blood lipids. Dilute the mouse serum with physiological saline to the linear range of the test kit, follow the operating steps specified in the instructions, and measure the absorbance of each well with an enzyme-labeled instrument. Calculate according to the formula.

[0033] Determination of mouse serum triglyceride (TG) content: Triglyceride is another important indicator for detecting blood lipids. Dilute the mouse serum with physiological saline to the linear range of the test kit, follow the operating steps specified in the instructions, and measure the absorbance of each well with an enzyme-labeled instrument. Calculate according to the formula.

[0034] 3. Molecular biology analysis

[0035] Western blot (WB) detection showed that compared with the Control group, the expression of apoptotic proteins (Cleaved-Caspase-3, Cleaved-Caspase-9, BAX) in the DM group was significantly increased, while the expression of anti-apoptotic proteins (BCL-2) was decreased. At the same time, we also observed that the BAX / BCL-2 ratio of DM was significantly increased, and it had a significant decrease effect starting from the low-dose group of MA-5, showing the inhibitory effect of MA5 on apoptosis. After treatment with MA-5, the expression of the above apoptosis-related proteins was reversed in a dose-dependent manner, indicating that MA-5 can improve cell apoptosis. Further studies found that GPX4 is a key protein for the occurrence and development of ferroptosis, and VDACs is a voltage-dependent ion channel. Erastin-induced ferroptosis can affect its permeability. Compared with the Control group, the expression of Nrf2 and GPX4 in the DM group was significantly reduced. At the same time, it gradually increased after treatment with different concentrations of MA-5, and there was a significant difference in the high-dose group. However, compared with the Control group, the expression of VDAC1 and VDAC3 in the DM group was significantly increased, and after treatment with different concentrations of MA-5, it was significantly reduced in the medium-dose group. Ferroptosis is involved in the development of diabetic testicular damage, and MA-5 can increase the expression of Nrf2 and GPX4, and can also reduce the content of VDAC1 and VDAC3, so MA5 can reduce the occurrence of ferroptosis and improve the condition of cellular oxidative stress.

[0036] Determination of superoxide dismutase (SOD) activity in mouse testicular tissue: SOD is the main substance that removes free radicals in cells and is a reliable indicator of the body's antioxidant capacity. Take frozen testicular tissue for SOD determination. Sample processing is as follows: After accurate weighing, follow the weight (g): volume (mL) = 1:9. Cut the testicles of appropriate size on ice, add the corresponding volume of physiological saline, grind to make tissue homogenate. Then centrifuge at 4℃, 4000rpm for 10min, and aspirate the supernatant for measurement.

[0037] Determination of malondialdehyde (MDA) content in mouse testis tissue: MDA is cytotoxic and can cause downstream interactions of many biological molecules, so MDA is often used as an indicator to measure the degree of lipid peroxidation. Take 10% tissue homogenate and operate according to the instructions of the MDA test kit.

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

[0039] Example

[0040] 1. Establishment of animal model

[0041] SPF male C57BL / 6J (16-18g) mice were divided into a normal group and a model group after adaptive feeding for one week. The mice in the normal group 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, pH 4.4, to prepare a 1% concentration for use) and inject twice. The normal group of mice was given an equal volume of citric acid-sodium citrate buffer solution, and drinking water was not restricted.

[0042] The random blood glucose was measured by taking blood samples from the tail of mice at 3d and 7d after STZ injection. The model was considered to be established successfully if the random blood glucose was ≥16.7mmol / L at both times. The mice without 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+25mg·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 The control group and the MA-5 high-dose control group continued to be fed with ordinary feed, and the mice in other groups were still fed with high-sugar and high-fat feed, and were 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 to observe their body shape and hair characteristics. The mice in the Control group grew and developed normally, with smooth and moist 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 improved.

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

[0047] Changes of body mass in 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+25mg / kg MA-5 group); MA-5 medium-dose group (DM+50mg / kg MA-5 group); MA-5 high-dose group (DM+100mg / kg MA-5 group); MA-5 high-dose control group (Control+100mg / kg MA-5 group): n=10-13; compared with Con group, *P<0.05; compared with DM group, #P<0.05.

[0048] The body weight of mice in each group increased 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 testicle weight of mice was weighed after sampling. The experiment found that compared with the control group, the testicle weight of mice in the DM group increased slightly, and after treatment with MA-5, the testicle weight decreased. 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), and it was significantly decreased in the MA-5 low dose group, as shown in Figure 2. Figure 1 C (Effect of MA-5 on organ coefficients of 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 A: Changes of blood glucose in diabetic mice at different time points; B: Effect 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 there was no significant difference. This shows 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 changes (P<0.05). Figure 2 As shown in C.

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

[0055] Determination of mouse serum total cholesterol (TC) content: Total cholesterol is an important indicator for detecting blood lipids. Dilute mouse serum with physiological saline to the linear range of the test kit, follow the operation steps shown in Table 1, and measure the absorbance of each well with an enzyme marker. Calculate according to the formula.

[0056] Table 1

[0057]

[0058]

[0059] Determination of mouse serum triglyceride (TG) content: Triglyceride is another important indicator for detecting blood lipids. Dilute the mouse serum with physiological saline to the linear range of the test kit, follow the operation steps shown in Table 2, and measure the absorbance of each well with an enzyme reader. Calculate 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 of 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, the 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 ability of diabetic mice

[0065] Sperm function assay: The tail of the left epididymis of the mouse was peeled off, placed in 2 mL of 37°C sterile saline, cut into pieces and mixed to make a sperm suspension. 0.05 mL of the suspension was aspirated with a pipette and dropped onto a cell counting plate and observed under a fixed-magnification microscope. At the same time, the total number of sperm in the 5 central squares, the number of sperm with normal sperm morphology and motility, and the total number of all sperm were recorded. Keep a record and calculate the formula as follows: Sperm motility percentage = total number of sperm with normal motility × 100%.

[0066] The changes of reproductive ability 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 cause reproductive dysfunction in mice. Figure 4 We found that compared with the Control group, the sperm motility and total number in the testis of the DM group mice were significantly reduced (P<0.05), resulting in a decrease in sperm quality; after oral gavage treatment with different concentrations of MA-5, an increasing trend was shown, with a significant increase in sperm concentration in the medium-dose MA-5 group, and a significant increase 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 baked in a 60℃ constant temperature drying oven for 30 minutes, and the tissue became transparent. The HE operation steps are 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 testis part and immerse it in the electron microscope fixative in a 4°C refrigerator and fix it overnight. After taking it out, perform immersion, embedding and curing reactions according to the operations in Table 4 to Table 6, wherein the immersion 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 last 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 is 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 slices using an ultra-thin slicer, 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 observation showed that the testes of the mice in the Control group had a complete structure of seminiferous tubules, which were tightly arranged, and the basement membrane and supporting boundary were clear and smooth. At the same time, the supporting cells and sperm in the seminiferous tubules were abundant and neatly arranged. The seminiferous tubules of the mice in the DM group were damaged to varying degrees, the seminiferous tubules were sparsely arranged, and the spermatogenic cells were detached from the basement. The number of layers of the seminiferous epithelium was significantly reduced, the arrangement was disordered, and the detached spermatogenic cells were seen in the seminiferous tubules, and the sperm production was significantly reduced. After treatment with MA-5, the damage to the seminiferous epithelium and seminiferous tubules was significantly alleviated, as shown by the increase in the number of layers of the seminiferous epithelium, orderly arrangement, gradually close combination with the basement membrane, increased sperm production, and gradual recovery of spermatogenic function.

[0086] Effects of MA-5 on the structural damage of mitochondria in the testis of diabetic mice Figure 6As shown, the representative image of the transmission electron microscope is magnification = 3000× / 8000×, and the arrow points to the mitochondria. Electron microscopy can be used to observe ultrastructure. We used electron microscopy to observe the morphological structure of mitochondria near sperm. It can be seen that the mitochondrial cristae of the Control group are clear, the cell membrane is intact, and the size is normal. In the DM group, the mitochondria became smaller and rounder, and the mitochondrial cristae decreased or even disappeared. At the same time, it can also be manifested as swollen mitochondria, and even "plate-like" mitochondria appear. With the increase of MA-5 treatment concentration, the mitochondrial morphology gradually recovered. It can be seen that at the medium dose of MA-5, the mitochondrial cristae near the sperm gradually became clear and the wrinkles were obvious.

[0087] 3. Molecular biology analysis

[0088] Effects of MA-5 on oxidative stress in mouse testis tissue

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

[0090] Take the frozen testicular tissue and perform SOD determination. Sample processing is as follows: weigh accurately and follow the weight (g): volume (mL) = 1:9. Cut the testicles of appropriate size on ice, add the corresponding volume of physiological saline, grind to make tissue homogenate. Then centrifuge at 4℃, 4000rpm for 10min, and take the supernatant for measurement. The reagent configuration is shown in Table 7:

[0091] Table 7

[0092] Reagents Solution configuration 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 operation 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, so MDA is usually used as an indicator to measure the degree of lipid peroxidation. The test kit preparation liquid 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 follows:

[0106] Table 11

[0107]

[0108]

[0109] Vortex to mix, then heat in a 95°C constant temperature metal bath for 40 min, centrifuge at 4000 rpm in a running water-cooled centrifuge for 10 min, aspirate the supernatant, add it to a 96-well plate, operate in a dark environment, and measure the OD value of each tube with an enzyme marker at a wavelength of 532 nm.

[0110] The calculation formula is as follows:

[0111]

[0112] Effects of MA-5 on oxidative stress levels in testicular tissue of mice 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 It can be seen that compared with the Control group, the SOD activity in the testes of the DM group mice was significantly reduced, and the MDA content was significantly increased, with significant differences (P<0.05); compared with the DM group, the SOD activity of the mice in the MA-5 treatment group with different concentrations increased, which was improved to a certain extent. At the same time, the MDA content showed a downward trend after treatment, and there was 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 pelleted tissue using an electronic balance, add freshly prepared tissue lysis buffer (i.e., RIPA: Cocktail: PMSF: Na3VO4 = 100: 1: 1: 1) according to the ratio of 1 mg tissue to 10 μL lysis buffer. Grind thoroughly on ice. Pipette all the tissue suspension in the grinder into an EP tube, mark it, and centrifuge it at 4°C, 12,000 rpm for 15 minutes, and aspirate the supernatant.

[0117] 2. Protein concentration determination: The BCA protein concentration determination kit is used to determine the sample concentration. The extracted supernatant samples and standard proteins are dissolved in a low temperature environment, and the protein standard is diluted to 1 mg / mL with ultrapure water. Add to the 96-well plate according to the concentration ratio (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg / mL), and make up to 20 μL with ultrapure water. In the 96-well plate, leave one secondary well for each standard protein. Take 2 μL of the sample supernatant, add 18 μL of double distilled water, and make up to 20 μL (that is, each sample concentration is diluted 10 times for detection, and the final calculation of protein concentration needs to be multiplied by the dilution multiple). Each sample well also retains a secondary well. Calculate the number of wells required, and each well needs to add 200 μL of the configured BCA working solution (A solution: B solution = 50:1). After the above operations are completed, the 96-well plate needs to be placed in a 37°C constant temperature metal bath and heated for 30 minutes. The absorbance value of each well is measured at a wavelength of 562nm using an enzyme reader. The standard protein sample is used to calculate the standard curve. After substituting the OD value of the measured sample into the standard curve, the sample concentration is measured. Take an appropriate amount of supernatant, mix it with the lysate and 5× protein loading buffer, and heat it in a constant temperature metal bath at 97°C for denaturation for 5-10 minutes. After cooling, store it in a -20°C refrigerator for later use.

[0118] 3. Preparation of Separation Gel and Concentration Gel

[0119] Wash the glass plate with ultrapure water and blow dry. Clamp the two plates horizontally and put them into the gel rack. Place the gel rack on a horizontal table. The separation gel of the corresponding concentration should be prepared according to the molecular weight of the target protein. When making the gel, add the gel making reagents in sequence according to the SDS-PAGE preparation order, blow and mix, and avoid excessive foam. After preparation, slowly use a pipette to add the separation gel from one side of the glass plate to the closed gel rack interlayer to an appropriate height, and then slowly add anhydrous ethanol from one side of the glass plate to flatten the gel surface.

[0120] After the separation gel is completely solidified, remove the anhydrous ethanol and use filter paper to absorb the remaining residue. Ensure that the anhydrous ethanol is completely removed and avoid foreign matter from entering. According to the gel making formula, make 5% concentrated gel (such as 2 pieces: 4ml of water, 1ml of 30% acrylamide, PH6.81 mL, 0.08mL of 10% SDS, 0.06mL of 10% AP, 8μL of TEMED). It is also necessary to blow and mix with a hose after adding a reagent each time. After mixing, fill the interlayer with a pipette and quickly insert the matching comb into the concentrated gel. It is still necessary to avoid the generation of bubbles. After the concentrated gel is completely solidified, remove it for use.

[0121] 4. Electrophoresis: The amount of protein loaded on the testicular tissue sample in this experiment is about 50μg, and the volume of rainbow markers added to both sides of the loading well is about 1.5μL. The sample should be added accurately to avoid sample overflow. After the sample is loaded, check whether there is any abnormality in the electrophoresis instrument connection equipment, and adjust the electrophoresis instrument voltage to 60V to start electrophoresis. It is necessary to observe whether electrophoresis bubbles are generated at the bottom and whether the internal liquid between the two plates changes. After the protein sample is electrophoresed to the contact surface of the separation gel and the rainbow marker is separated, adjust the electrophoresis instrument voltage to 90V. Stop the electrophoresis when the electrophoresis reaches the required height.

[0122] 5. Transfer: Cut the PVDF membrane to a similar size to the gel, remove the glass plate, cut the gel to a suitable size, immerse it in the transfer solution, move the transfer clip, and then immerse the PVDF membrane in the methanol solution to activate it. Absorb the methanol solution on the surface of the PVDF membrane in the transfer solution, transfer it to the gel surface, and fix one end to drive away the bubbles between the gel and the membrane to make them fit tightly. Lock the transfer clip and put it into the transfer tank. Set the transfer conditions: 300mA, 120min. After the transfer is completed, take it out, mark it, and wash it with 1×TBST.

[0123] 6. Blocking: After washing, place the PVDF membrane in 5% skim milk blocking solution and block it on a shaker for 1-2 hours at room temperature. After the block, wash it with 1×TBST. The blocking time and washing times can also be adjusted according to the experimental results.

[0124] 7. Primary antibody incubation: Cut the PVDF membrane as needed and mark it. Place the PVDF membrane in the primary antibody diluent containing antibodies 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) and incubate overnight at 4°C. After primary antibody incubation, remove the membrane and wash it three times in 1×TBST, 5-10 minutes each time.

[0125] 8. Secondary antibody incubation: Place the PVDF membrane in 1×TBST diluted antibody HRP-labeled goat anti-rabbit IgG (1:10000) and HRP-labeled rabbit anti-mouse IgG (1:10000) and incubate on a shaker at room temperature for 1 hour. Then, remove the PVDF membrane and wash it 3 times in 1×TBST, 5-10 minutes each time.

[0126] 9. Development: ECL luminescent liquid A and ECL luminescent liquid B are mixed into super-sensitive luminescent liquid with equal volumes, and the PVDF membrane is evenly immersed in it. The membrane is exposed using a gel imaging system, and its grayscale value is analyzed using Image Lab Software.

[0127] 10. Statistical analysis: All data were expressed as mean ± standard error (x ± SEM) using SPSS26.0 statistical software. Statistical analysis was performed using One-Way ANOVA, and P < 0.05 was considered statistically significant.

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

[0129] Effects of MA-5 on apoptotic proteins in diabetic mice Figure 8 As shown, Figure 8 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 apoptotic proteins (Cleaved-Caspase-3, Cleaved-Caspase-9, BAX) in the DM group was significantly increased compared with the Control group (P<0.05). Figure 8 A, D, E), while the expression of anti-apoptotic protein (BCL-2) decreased (e.g. Figure 8 B), after MA-5 treatment, the expression of apoptotic proteins was significantly decreased (P<0.05), and the expression of anti-apoptotic proteins was increased (P>0.05). At the same time, we also observed that the BAX / BCL-2 ratio of DM was significantly increased, and it had a significant decreasing effect starting from the low-dose MA-5 group (such as 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. Fig. 9 As shown, Fig. 9 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). Fig. 9 A, B), and at the same time, after treatment with different concentrations of MA-5, the expression of VDAC1 and VDAC3 in the DM group was significantly increased (P<0.05) compared with the Control group (as shown in Fig. 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 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 mitochondrial acid 5 in the preparation of a drug for treating reproductive organ damage caused by diabetes.

2. The 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 also contains pharmaceutically acceptable excipients.

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