Application of spermidine in preparation of agent for resisting mitochondrial oxidative damage and copper death damage

Abstract

The invention discloses application of spermidine in preparation of an agent for resisting mitochondrial oxidative damage and copper death damage, and relates to the technical field of biology. The invention provides an application of spermidine in preparing an agent for resisting mitochondrial oxidative damage and copper death damage, and further provides an application of spermidine in inhibiting copper death induced by copper sulfate and relieving iron-sulfur cluster protein loss and ester acylation protein aggregation induced by copper accumulation. The spermidine can obviously relieve cell viability decline induced by copper sulfate, reverse ROS and MDA level increase and mitochondrial membrane potential decline induced by copper sulfate and relieve iron-sulfur cluster protein loss and ester acylation protein aggregation induced by copper accumulation, and has obvious effects of inhibiting mitochondrial oxidative damage and copper death. The invention provides a method for effectively relieving copper death of ovarian granular cells, and solves the problem that the existing antioxidant and cytoprotective agent for resisting mitochondrial oxidative damage generate side effects in the use process.

Description

Technical Field

[0001] The invention relates to the field of biological technology, and in particular to application of spermidine in preparing an agent for resisting mitochondrial oxidative damage and copper death damage. Background Art

[0002] Mitochondria are essential in energy metabolism, regulating ROS production and cell death. However, mitochondria are susceptible to oxidative damage in certain pathological processes. Mitochondrial oxidative damage refers to the damage to the structure and function of mitochondria due to excessive production of free radicals such as reactive oxygen species during mitochondrial energy metabolism due to various factors, which exceeds the body's antioxidant capacity. This damage affects the normal function of mitochondria and reduces energy production efficiency.

[0003] Copper death is a new type of programmed cell death. It is caused by the excessive accumulation of copper ions in cells, which directly combines with the acylated components in the tricarboxylic acid cycle of mitochondrial respiration, resulting in the aggregation of acylated proteins and the loss of iron-sulfur cluster proteins, inducing protein toxic stress and ultimately leading to cell death.

[0004] At present, although some methods have been used to combat mitochondrial oxidative stress damage, many existing antioxidants and cytoprotectants may produce side effects during use, such as interference with other normal physiological processes, toxic reactions, etc., and there are currently no reports of drugs that can combat mitochondrial oxidative damage and copper death damage agents. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides the use of spermidine in the preparation of an agent for resisting mitochondrial oxidative damage and copper death damage. Spermidine has potential effects in resisting mitochondrial oxidative damage and copper death damage, and effectively solves the problem of side effects generated by existing antioxidants and cell protectants used to resist mitochondrial oxidative stress damage during use.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: to provide the use of spermidine in the preparation of an agent for resisting mitochondrial oxidative damage and copper death damage.

[0007] Furthermore, spermidine is used in the preparation of an agent for resisting mitochondrial oxidative damage and copper death damage in ovarian granulosa cells.

[0008] Furthermore, the concentration of spermidine in the agent against mitochondrial oxidative damage and copper death damage is 35-45 μmol / L.

[0009] Furthermore, the concentration of spermidine in the agent against mitochondrial oxidative damage and copper death damage is 40 μmol / L.

[0010] Furthermore, the mitochondrial oxidative damage is mitochondrial oxidative damage induced by a copper sulfate solution at a concentration of 450-550 μmol / L.

[0011] Furthermore, the mitochondrial oxidative damage is mitochondrial oxidative damage induced by a copper sulfate solution with a concentration of 500 μmol / L.

[0012] Furthermore, copper death is copper death induced by a copper sulfate solution with a concentration of 450-550 μmol / L.

[0013] Furthermore, copper death is copper death induced by a copper sulfate solution with a concentration of 500 μmol / L.

[0014] Furthermore, the application is an application in at least one of the following (1)-(3):

[0015] (1) Inhibit copper sulfate-induced copper death;

[0016] (2) Alleviate the loss of iron-sulfur cluster proteins induced by copper accumulation;

[0017] (3) Alleviate the aggregation of acylated proteins induced by copper accumulation.

[0018] An agent for resisting mitochondrial oxidative damage and copper death damage comprises spermidine and pharmaceutically acceptable excipients.

[0019] The present invention has the following beneficial effects:

[0020] 1. The research of the present invention shows that spermidine has a significant effect of protecting porcine follicular granulosa cells from mitochondrial oxidative damage. The inventors found through research that spermidine can significantly alleviate the decrease in cell activity induced by copper sulfate, reverse the increase in ROS and MDA levels and the decrease in mitochondrial membrane potential induced by copper sulfate, improve cell activity, alleviate the loss of iron-sulfur cluster proteins and esterification protein aggregation induced by copper accumulation, and has a significant effect of inhibiting mitochondrial oxidative damage and copper death.

[0021] 2. The present invention develops a new use of spermidine as a treatment for mitochondrial oxidative damage stress and copper death in pig follicular granulosa cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of the activity of granulosa cells of pig ovarian follicles under different treatments are shown in the figure;

[0023] Figure 2 This is the result diagram of iron-sulfur cluster protein expression under different treatments;

[0024] Figure 3 The fluorescence intensity diagram of iron-sulfur cluster protein under different treatments;

[0025] Figure 4Quantification of iron-sulfur cluster protein expression under different treatments;

[0026] Figure 5 The results of acylated protein expression under different treatments are shown;

[0027] Figure 6 The results of acylated protein expression under different treatments are shown;

[0028] Figure 7 The figure is the quantitative graph of acylated protein expression under different treatments;

[0029] Figure 8 The fluorescence intensity of acylated proteins under different treatments;

[0030] Fig. 9 The results of cellular ROS levels under different treatments;

[0031] Fig.10 This is the MDA quantification result diagram under different treatments;

[0032] Fig.11 The diagrams show the mitochondrial membrane potential and mitochondrial fluorescence labeling results under different treatments;

[0033] Fig.12 The diagrams show the mitochondrial membrane potential and mitochondrial fluorescence labeling results under different treatments;

[0034] Fig.13 The fluorescence intensity ratio results under different treatments are shown in Figure 2.

[0035] Fig.14 Quantification of fluorescence intensity under different treatments;

[0036] Fig.15 This is the expression result diagram of copper ion content under different treatments;

[0037] Fig.16 This is the expression result diagram of pyruvate content under different treatments;

[0038] Fig.17 This is the expression result of α-ketoglutarate content under different treatments. DETAILED DESCRIPTION

[0039] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0040] Main reagents: FBS (Vivacell, 2250340); DMEM / F12 1:1basic (Hyclone, SH30023.01); Enhanced CCK-8 kit (Biyuntian, C0042); PBS phosphate buffer powder (Solarbio, P1010); Penicillin-streptomycin-gentamicin mixed solution (Solarbio, P1010); Rapid transfer solution (Biyuntian, P0575-10L); Electrophoresis solution (Biyuntian, P0014B); Precision Plus Protein TM Dual Color Standards (Bio-Rad, 1610374); 30% gel preparation solution (Solerabol, A1010); Ultra-sensitive ECL chemiluminescence kit (Biyuntian, P0018AM); Wb primary antibody dilution (Biyuntian, P0023A); Wb secondary antibody dilution (Biyuntian, P0023D); β-actin (abclonal, AC026, 1 / 1000); β-tubulin (abclonal, AC008, 1 / 1000); horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (Biyuntian, A0208, 1 / 1000) FDX1 (Abmart, T510671, 1 / 2500); LIAS (Abmart, PH2927, 1 / 1000); DLAT (abclonal, A14530, 1 / 1000); ACO-2 (abclonal, A4524, 1 / 1000); Anti-Lipoic Acid (abcam, ab58724, 1:6000); QuickBlock TM Immunofluorescence staining primary antibody diluent (Biyuntian, P0262); QuickBlock TM Immunofluorescence staining secondary antibody diluent (Biyuntian, P0265); QuickBlock TM Immunofluorescence staining blocking solution (Biyuntian, P0620); DAPI staining solution (Biyuntian, C1005); Cell copper (Cu) colorimetric test kit (Elabscience, E-BC-K775-M); Enhanced mitochondrial membrane potential detection kit (Biyuntian, C2003S); Reactive oxygen detection kit (Biyuntian, S0033S); Malondialdehyde (MDA) assay kit (Nanjing Jiancheng, A003-1-2). α-Ketoglutarate detection kit (Solerbo, BC5425); Pyruvate detection kit (Solerbo, BC2205).

[0041] Main instruments: constant temperature cell culture incubator (Thermo, USA); multifunctional microplate reader (Thermo, USA); -80℃ ultra-low temperature refrigerator (Thermo, USA); gel imaging system (Bio-Rad, USA); fluorescence inverted microscope (Olympus, USA); vertical electrophoresis instrument (Bio-Rad, USA); membrane transfer instrument (Bio-Rad, USA).

[0042] Experimental groups: Porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate separately or in combination and divided into control group, copper sulfate (CuSO4) group, spermidine (SPD) group, and spermidine + copper sulfate (SPD + CuSO4) group.

[0043] Example 1 Cell Isolation, Culture and Treatment

[0044] The isolation and culture of primary porcine granulosa cells specifically include: porcine ovaries are obtained from a local slaughterhouse and stored in a 37°C phosphate buffer solution (Solarbio, Beijing, China) containing 1% double antibodies (penicillin and streptomycin), and transported back to the laboratory within 2 hours after collection; the collected porcine ovaries are cleaned with a 37°C PBS solution containing 1% triple antibodies, the follicular fluid is extracted into a 37°C complete culture medium with a 1mL syringe in a clean bench, the cell suspension is filtered with a 40μm cell strainer, and after collecting the filtrate, the centrifuge parameters are set to 1000g for 10min to obtain a cell precipitate, the cells are washed twice with a 37°C PBS solution containing 1% triple antibodies, and then the complete culture medium is added to resuspend the cells to an appropriate density for plating, and the cell culture plate is placed in a 37°C, 5vt% CO2 incubator. The medium is changed once every 24 hours and cultured until the cell density reaches 70%, and then the granulosa cells are processed.

[0045] Example 2 Cell activity detection

[0046] The cells were inoculated and cultured in a 96-well plate. When the density reached 70%, the porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination. After the treatment, the culture medium was discarded, and the cells were washed twice with a 37°C PBS solution containing 1% triple antibody. 10% CCK8 solution was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. The absorbance at 450nm was measured with an enzyme marker and then the cell activity was calculated. The cell activity results of the porcine ovarian follicle granulosa cells after being treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination, are shown in FIG. Figure 1 shown.

[0047] Depend on Figure 1It can be seen that the cell activity was 54% after treatment with 500 μmol / L copper sulfate, but after combined treatment with spermidine, the cell activity recovered to 66%, indicating that spermidine can partially alleviate the decrease in cell activity caused by CuSO4.

[0048] Example 3 Cell protein expression

[0049] The cells were inoculated and cultured in a 6-well plate. When the density reached 80%, the porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination. After the treatment, the culture medium was discarded, and the cells were washed twice with 4°C PBS for 30 seconds each time. 120 μL RIPA was evenly added to each cell culture dish and placed in a 4°C refrigerator for lysis for 15 minutes. The lysed granulosa cells were scraped off with a cell scraper and placed in a 1.5 mL EP tube. The cells were fully broken using a cell disruptor. After centrifugation at 15000 r / min for 10 minutes at 4°C in a high-speed refrigerated centrifuge, the supernatant was taken and placed in a new EP tube. The protein concentration was normalized after the BCA method was used to detect the protein concentration. The protein was separated by 12% SDS-PAGE separation gel and 5% After separation by SDS-PAGE concentrated gel, the membrane was transferred to a PVDF membrane and transferred at a constant flow rate of 400 mA for 25 minutes. The PVDF membrane was blocked with 5% skim milk for 2 hours, and the PVDF membrane was incubated with primary antibodies such as β-actin, β-tubulin, FDX1, ACO-2, LIAS, CTR1, DLAT, and Lip overnight. The PVDF membrane was washed 3 times with TBST for 10 minutes each time. The PVDF membrane was incubated with secondary antibodies (goat anti-rabbit) for 2 hours, and after washing 3 times with TBST, the PVDF membrane was soaked with supersensitive ECL chemiluminescent solution and placed in the sample placement area of ​​the chemiluminescent imaging system for development and imaging.

[0050] The expression of iron-sulfur cluster proteins in porcine ovarian follicle granulosa cells treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination, is shown in Figure 2 Figure 2 The fluorescence intensity results of iron-sulfur cluster proteins are shown in Figure 3 As shown; the quantification results of iron-sulfur cluster protein expression are shown Figure 4 As shown, Figure 4 In the figure, from top to bottom and from left to right, the results of FDX1 / β-actin, POLD1 / β-actin, ACO-2 / β-actin and LIAS / β-actin are shown in Figure 2. The expression of acylated proteins in porcine ovarian follicle granulosa cells treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination, is shown in Figure 2. Figure 5-Figure 6 The quantification results of acylated protein expression are shown in Figure 7 As shown, Figure 7In the figure, from top to bottom and from left to right, they are the results of DLAT / β-actin, Lip-DLAT / β-actin, HSP70 / β-actin and CTR1 / β-actin.

[0051] The cells were inoculated and cultured in a 24-well plate. When the density reached 80%, the porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination. After the treatment, the culture medium was discarded, and the cells were washed with PBS at 37°C for 30 seconds each time. After fixation with 4% paraformaldehyde for 15 minutes, the cells were washed with PBS for 3 times for 10 seconds each time. After permeabilization with 0.5% TritonX100 for 10 minutes, the cells were washed with PBS for 3 times for 10 seconds each time. The cells were blocked with immunostaining blocking solution at room temperature for 60 minutes. After incubation with the primary antibody Lip in a 4°C refrigerator overnight, the cells were washed with PBS for 3 times for 3 minutes each time. After incubation with the fluorescent secondary antibody Cy3 at room temperature in the dark for 1 hour, the cells were washed with PBS for 3 minutes each time. After incubation with DAPI in the dark for 5 minutes, the cells were washed with PBS for 3 minutes each time. Images were collected under a fluorescent inverted microscope. Results are shown in Figure 2. Figure 8 shown.

[0052] Depend on Figure 2-Figure 4 It can be seen that after treatment with 500 μmol / L CuSO4, the expression level of FDX1 protein increased by 2.6 times, and the expression levels of iron-sulfur cluster protein POLD1, ACO-2, and LIAS protein decreased by 2.6 times, 1.4 times, and 1.3 times, respectively. Spermidine can significantly inhibit the up-regulation of FDX1 expression and the down-regulation of ACO-2 and LIAS expression induced by CuSO4.

[0053] Depend on Figure 5-Figure 8 It can be seen that after treatment with 500μmol / L CuSO4, the expression of DLAT protein decreased by 1.3 times, and the expression of Lip-DLAT and HSP70 proteins increased by 1.3 times and 11 times, respectively. Spermidine can significantly inhibit the upregulation of HSP70 expression induced by CuSO4, but has no effect on DLAT and Lip-DLAT. The results of immunofluorescence detection show that after treatment with 500μmol / L copper sulfate, the immunofluorescence intensity of Lip-DLAT increased significantly, while spermidine combined treatment had no effect on the increase in Lip-DLAT fluorescence intensity caused by copper sulfate.

[0054] Example 4 Detection of Cellular ROS and MDA Levels

[0055] The cells were inoculated and cultured in a 24-well plate. When the density reached 80%, the porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination. After the treatment, the culture medium was discarded, and the cells were washed with PBS at 37°C for 30 seconds each time. 500 μL of 10 μmol / L DCFH-DA was added and incubated in a cell culture incubator at 37°C for 20 minutes. The cells were mixed by inversion every few minutes, and the cells were washed with serum-free cell culture medium for 3 times for 10 seconds each time. Images were collected under a fluorescence inverted microscope. The results are shown in FIG. Fig. 9 shown.

[0056] The cells were inoculated and cultured in a 6-well plate. When the density reached 80%, the porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate separately or in combination. After the treatment, the culture medium was discarded and the cells were washed 3 times with 37°C PBS for 30 seconds each time. The cell samples were collected and added with 120 mL IP lysis solution. The cells were scraped off with a cell scraper and the tip of the pipette was repeatedly blown to lyse the cells. Centrifuge at 12,000 rpm for 10 minutes in a refrigerated centrifuge, the supernatant protein was aspirated, and the protein concentration was determined according to the instructions of the BCA kit. The samples, standards and lysis solution were added to the PCR tube as planned, and the MDA detection working solution was added, mixed, centrifuged instantaneously, and heated at 100°C for 15 minutes. Cool to room temperature, centrifuge at 1000g for 15 minutes, take 200 μL of supernatant, add to a 96-well plate, and detect the absorbance at 532nm with an enzyme reader. The results are as follows. Fig.10 shown.

[0057] Depend on Fig. 9 and Fig.10 It can be seen that after treatment with 500 μmol / L copper sulfate, the levels of ROS and MDA in the cells increased significantly, while co-treatment with spermidine could significantly inhibit the accumulation of ROS and MDA caused by copper sulfate.

[0058] Example 5 JC-1 mitochondrial membrane potential fluorescent probe detection

[0059] The cells were inoculated and cultured in a 24-well plate. When the density reached 80%, the porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate, respectively or in combination. After the treatment, the culture medium was discarded, and the cells were washed with 37°C PBS for 30 seconds each time. JC-1 staining working solution was added and incubated in a 37°C cell culture incubator for 20 minutes. After the incubation at 37°C, the cells were washed with JC-1 staining buffer twice for 10 seconds each time. Images were collected under a fluorescent inverted microscope. The results are shown in the figure. Figure 11-12 The fluorescence intensity ratio results under different conditions are shown in Fig.13 The quantification results of fluorescence intensity are shown in Fig.14 shown.

[0060] Depend on Figure 11-Figure 14It can be seen that after treatment with 500 μmol / L copper sulfate, the mitochondrial red fluorescence / green fluorescence ratio was 1:7.3; after combined treatment with spermidine, the mitochondrial red fluorescence / green fluorescence ratio was 1.94:1, indicating that copper sulfate can significantly cause a decrease in mitochondrial membrane potential, and spermidine can partially alleviate the decrease in mitochondrial membrane potential caused by copper sulfate.

[0061] Example 6 Detection of intracellular copper ion content

[0062] In a preferred embodiment, The cellular copper (Cu) colorimetric test kit for detecting the copper ion content of granulosa cells specifically includes: the cells are inoculated and cultured in a 24-well plate. After the density reaches 80%, the porcine ovarian follicle granulosa cells are treated with 40μmol / L spermidine and 500μmol / L copper sulfate separately or in combination. After the treatment, the culture medium is discarded, the cells are washed with PBS at 37°C for 30 seconds three times, and the reagent four lysis solution is added and placed on ice for lysis for 10 minutes, and then centrifuged at 4°C and 12000r / min for 10 minutes. The supernatant is taken for determination, and part of the supernatant is retained for protein concentration determination. 100μL of the sample to be tested is added to the bottom of the determination well, 50μL of the color developer working solution is added to each well, and the film is covered. Incubate at 37°C for 5 minutes, and the OD value of each well is measured at 580nm on an enzyme reader. The copper ion content of the granulosa cells is calculated based on the standard curve. The porcine ovarian follicle granulosa cells are treated with 40μmol / L spermidine and 500μmol / L copper sulfate separately or in combination. The intracellular copper ion content results are as follows Fig.15 shown.

[0063] Depend on Fig.15 It can be seen that the intracellular copper ion content increased by 5.9 times after treatment with 500 μmol / L copper sulfate, while the intracellular copper ion content decreased to 3.9 times after co-treatment with spermidine, indicating that spermidine can partially alleviate the intracellular copper ion accumulation induced by copper sulfate.

[0064] Example 7 Detection of cellular pyruvate content

[0065] Sample processing: first collect cells into a centrifuge tube, discard the supernatant after centrifugation; add an appropriate volume of extract, and then ultrasonically disrupt (ice bath, power 20% or 200W, ultrasonic for 3s, interval 10s, repeat 30 times), let stand for 30min, 8000g, centrifuge for 10min at room temperature, and take the supernatant for testing; (2) Preheat the spectrophotometer / enzyme reader for more than 30min, adjust the wavelength to 520nm, and zero with distilled water; (3) Preparation of standard: dilute the standard with distilled water to 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0μg / mL; (4) Add 75μL of standard solution or sample and 25μL of reagent 1 to a micro-glass cuvette or 96-well plate, mix well, let stand for 2 minutes, add 125μL of reagent 2, mix well, and measure the absorbance value A at a wavelength of 520nm; (5) Calculation of pyruvate content Establish a standard curve based on the concentration of the standard and the measured value; x is the sodium pyruvate content (μg / mL), and y is the absorbance value. 40μmol / L spermidine and 500μmol / L copper sulfate were used to treat porcine ovarian follicle granulosa cells separately or in combination. The expression results of pyruvate content are shown in the following table. Fig.16 shown.

[0066] Depend on Fig.16 It can be seen that after treatment with 500 μmol / L CuSO4, the pyruvate content decreased by 1.1 times, and spermidine can effectively alleviate the decrease in pyruvate content caused by CuSO4.

[0067] Example 8 Detection of α-ketoglutaric acid content

[0068] (1) Sample processing: first collect cells into a centrifuge tube, discard the supernatant after centrifugation; add an appropriate volume of extract solution, and then disrupt the cells by ultrasonication in an ice bath (power 300w, ultrasonication 3s, interval 7s, total time 3min); centrifuge at 4℃, 12000g for 10min, take 0.8mL of supernatant, and then slowly add 0.15mL of extract solution 2, slowly blow and mix until no bubbles are generated, centrifuge at 4℃, 12000g for 10min, and then take the supernatant for testing; (2) Preheat the UV spectrophotometer / enzyme reader for more than 30min, adjust the wavelength to 340nm, and zero the UV spectrophotometer with distilled water; (3) Add the corresponding reagents in the corresponding wells in sequence according to the operation in Table 1;

[0069] Table 1 Operation table

[0070]

[0071] (4) After fully mixing, measure the absorbance value A1 at 340nm after 20s, and quickly place it in a 37℃ environment for 5min (the microplate reader has a temperature control function that can adjust the temperature to 37℃), take it out, wipe it dry quickly, and measure the absorbance value A2 after 5min and 20s. ΔA measurement = A1 measurement - A2 measurement, ΔA standard = A1 standard - A2 standard, ΔA blank = A1 blank - A2 blank. The blank tube and standard tube only need to be measured 1-2 times.

[0072] Statistical analysis: The experiment was repeated three times, and the results are the mean ± standard deviation. SPSS software (IBM Corporation, NarthCastle, NY, USA) was used for statistical analysis, using one-way analysis of variance (ANOVA) and LSD. Values ​​of P < 0.05 were considered significant. All measurements were repeated at least three times, and the data are presented as the mean ± standard deviation. All figures were drawn by 0rigin software. * indicates significant P < 0.05; ** indicates extremely significant difference P < 0.01; ** indicates extremely significant difference P < 0.001. The expression results of α-ketoglutarate content in porcine ovarian follicle granulosa cells were treated with 40 μmol / L spermidine and 500 μmol / L copper sulfate separately or in combination. Fig.17 shown.

[0073] Depend on Fig.17 It can be seen that after treatment with 500 μmol / L CuSO4, the content of α-ketoglutaric acid decreased by 1.5 times, and spermidine can effectively alleviate the decrease in α-ketoglutaric acid content caused by CuSO4.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of spermidine in the preparation of agents for resisting mitochondrial oxidative damage and copper death damage.

2. The use according to claim 1, characterized in that The spermidine is used in preparing an agent for resisting mitochondrial oxidative damage and copper death damage of ovarian granulosa cells.

3. The use according to claim 1, characterized in that The concentration of spermidine in the agent for resisting mitochondrial oxidative damage and copper death damage is 35-45 μmol / L.

4. The use according to claim 1, characterized in that The mitochondrial oxidative damage is mitochondrial oxidative damage induced by a copper sulfate solution with a concentration of 450-550 μmol / L.

5. The use according to claim 1, characterized in that The copper death is copper death induced by a copper sulfate solution with a concentration of 450-550 μmol / L.

6. The use according to claim 1, characterized in that The application is at least one of the following (1)-(3): (1) Inhibit copper sulfate-induced copper death; (2) Alleviate the loss of iron-sulfur cluster proteins induced by copper accumulation; (3) Alleviate the aggregation of acylated proteins induced by copper accumulation.

7. An agent for resisting mitochondrial oxidative damage and copper death damage, characterized in that: The invention comprises the spermidine as claimed in claim 1 and pharmaceutically acceptable excipients.

Images