Method for regulating and controlling oxidative stress induced sheep ovarian granular cell apoptosis by using allicin

Abstract

The invention relates to the technical field of ovarian granule cell apoptosis, and discloses a method for regulating oxidative stress induced sheep ovarian granule cell apoptosis by using allicin, and the method comprises the following steps: firstly preparing a culture solution, then preparing an allicin stock solution, and identifying sheep ovarian granule cells; the method comprises the following steps: firstly, measuring the activity of ovarian granular cells after independent treatment of H2O2 and allicin, determining the optimal treatment time and concentration of H2O2 and allicin, then carrying out co-treatment of H2O2 and allicin, detecting the apoptosis of the granular cells by using flow cytometry, detecting the expression of apoptosis-related genes by using real-time fluorescent quantitative PCR, detecting the secretion of steroid hormones by using ELISA, and carrying out data analysis. The invention solves the problems that in the prior art, an effective regulation and control method for ovarian granular cell apoptosis induced by oxidative stress is lacked, and traditional medicines possibly have large side effects, unobvious effects and the like.

Description

Technical Field

[0001] The invention relates to the technical field of ovarian granulosa cell apoptosis, and in particular to a method for regulating sheep ovarian granulosa cell apoptosis induced by oxidative stress by utilizing allicin. Background Art

[0002] Granulosa cells (GCs) are the largest cell group that makes up the follicles and play a key role in the development, maturation and atresia of follicles. Ovarian granulosa cell apoptosis is the main cause of mammalian follicular atresia, and oxidative stress is the main factor that causes granulosa cell damage and ultimately apoptosis. Therefore, exploring how to alleviate oxidative stress-induced ovarian granulosa cell apoptosis is crucial to improving the reproductive capacity of female animals. Allicin is a natural active substance found in garlic and is widely used in the field of animal husbandry. It is necessary to design a method to use allicin to regulate oxidative stress-induced ovarian granulosa cell apoptosis.

[0003] In the prior art, there is a lack of effective methods for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells, and traditional drugs may have problems such as large side effects and unclear effects. Summary of the invention

[0004] The purpose of the present invention is to provide a method for regulating ovarian granulosa cell apoptosis induced by oxidative stress using allicin, so as to achieve the purpose of solving the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for regulating ovarian granulosa cell apoptosis induced by oxidative stress using allicin, comprising the following steps:

[0006] S1: First, the culture solution is prepared, and then the allicin stock solution is prepared;

[0007] S2: Identification of ovine ovarian granulosa cells;

[0008] S3: Determine the viability of ovarian granulosa cells after H2O2 and allicin treatment alone to determine the optimal treatment time and concentration of H2O2 and allicin;

[0009] S4: Flow cytometry to detect granulocyte apoptosis;

[0010] S5: Extraction of total RNA from cells, followed by reverse transcription of RNA samples;

[0011] S6: Real-time fluorescence quantitative PCR detection of apoptosis-related gene expression;

[0012] S7: Measurement of steroid hormone secretion;

[0013] S8: Analyze the detected data.

[0014] Preferably, the culture medium in S1 is prepared by mixing 89% DMEM / F12 culture medium, 10% FBS and 1% penicillin-streptomycin mixture, mixing them evenly, wrapping the centrifuge tube with sealing film, and storing them in a 4°C refrigerator for later use.

[0015] Preferably, the S1 allicin stock solution is prepared by using a pipette to absorb 4.38 μL of allicin with a molecular weight of 162.27 and dissolving it in 1 mL of dimethyl sulfoxide (DMSO) in an ultra-clean workbench to obtain a concentrated stock solution with a concentration of 30 mM, and then dispensing it into centrifuge tubes, sealing them with sealing film, and storing them in a -20°C refrigerator.

[0016] Preferably, the detection of granulocyte apoptosis in S4 is based on the instructions of the AnnexinV-FITC apoptosis detection kit (Tongren Chemical), and the granulocytes in the control group, H2O2 group and Allicin+H2O2 group are collected for treatment, and then the cell apoptosis rate is detected by flow cytometry.

[0017] Preferably, the specific steps of reverse transcription treatment of the RNA sample in S5 are to prepare the mixed solution listed in Table 1 in a centrifuge tube free of RNase contamination to achieve effective removal of genomic DNA, then directly add 4 μL of 5×HiScript qRT SuperMix reagent to the above reaction system, and ensure sufficient mixing, incubate at 37°C for 15 minutes, and then treat at 85°C for 5 seconds. The cDNA product obtained after the reverse transcription reaction is completed should be properly stored in a -20°C refrigerator for subsequent experiments.

[0018] Preferably, the step of detecting apoptosis-related gene expression by real-time fluorescence quantitative PCR in S6 comprises designing and synthesizing specific primers for GAPDH, Bax and Bcl-2, using cDNA as a template, using Bio-Rad's CFX96 system for reaction, GAPDH as an internal reference, and performing the reaction according to 2 -ΔΔCt The relative expression of the target gene was calculated by the RT-qPCR reaction system (10 μL), including: 5 μL 2×M5HiPerSYBR Permix EsTaq, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 1 μL template cDNA, and 3 μL ddH2O.

[0019] Preferably, the steroid hormone secretion determination in S7 is the determination of the secretion levels of E2 and P4 in the supernatant of the granulosa cells of the control group, the H2O2 group and the Allicin+H2O2 group.

[0020] Preferably, the data analysis in S8 is expressed as mean ± standard error (mean ± SEM), and graphed using Graphpad Prism8. One-Way ANOVA and t-test are used to analyze the significance of the differences in the experimental data. When P < 0.05, it is statistically significant, * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

[0021] The present invention provides a method for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin. The method has the following beneficial effects:

[0022] (1) The present invention first uses H2O2 to construct an oxidative stress model of sheep ovarian granulosa cells. The results show that 400μM H2O2 treatment of granulosa cells for 4 hours can induce apoptosis of sheep follicular granulosa cells. At this concentration, the cell viability is significantly reduced, and the cell viability reaches 52.7%. The morphology of the follicular granulosa cells also changes slightly, but most cells can still grow attached to the wall, indicating that the cells have reached a state of oxidative stress at this concentration. Due to differences in species, culture conditions, treatment methods, etc., the present invention determines that 400μM H2O2 treatment of granulosa cells for 4 hours is the optimal condition for inducing oxidative stress in sheep ovarian granulosa cells, which solves the problem of lack of conditions and specific parameters for constructing an oxidative stress model of sheep ovarian granulosa cells in the prior art.

[0023] (2) The present invention alleviates the apoptosis of ovarian granulosa cells caused by oxidative stress by adding allicin. The results show that compared with the control group, the apoptosis rate and the expression of pro-apoptotic genes in the granulosa cells treated with H2O2 were significantly increased (P < 0.01); and it is worth noting that compared with the H2O2 group, the apoptosis rate and the expression of pro-apoptotic genes in the cells treated with allicin were significantly reduced (P < 0.01). In addition, compared with the control group, the secretory function of the granulosa cells in the H2O2 group was abnormal, resulting in an increase in the secretion level of estradiol (E2) (P < 0.01) and a decrease in the secretion level of progesterone (P4); and after the addition of allicin, the secretion levels of E2 and P4 were restored (P < 0.05). The present invention determines that allicin can alleviate the apoptosis and secretory function disorder of sheep ovarian granulosa cells induced by oxidative stress, and solves the problems in the prior art that there is a lack of effective regulation methods for the apoptosis of sheep ovarian granulosa cells induced by oxidative stress, and that traditional drugs may have large side effects and unclear effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the reaction system of Table 1 of the present invention;

[0025] Figure 2 This is an immunofluorescence identification diagram of sheep ovarian granulosa cells of the present invention;

[0026] Figure 3 This is a schematic diagram of the effect of different concentrations of H2O2 on the activity of sheep ovarian granulosa cells in the present invention.

[0027] Figure 4 Schematic diagram of the effect of different concentrations of allicin on the activity of sheep ovarian granulosa cells;

[0028] Figure 5 The figure is a schematic diagram showing the effect of allicin and H2O2 on the apoptosis rate of sheep ovarian granulosa cells according to the present invention.

[0029] Figure 6 The figure is a schematic diagram showing the effect of allicin of the present invention on the expression of apoptosis-related genes in sheep ovarian granulosa cells.

[0030] Figure 7 Schematic diagram of the effect of allicin of the present invention on the secretion of E2 and P4 in ovarian granulosa cells treated with H2O2. DETAILED DESCRIPTION

[0031] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described with reference to the accompanying drawings.

[0032] Example 1: A preferred embodiment of the method provided by the present invention for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin is as follows: Figures 1 to 7 A method for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin comprises the following steps:

[0033] S1: First, the culture solution is prepared, and then the allicin stock solution is prepared;

[0034] S2: Identification of sheep ovarian granulosa cells. After digesting the cells with trypsin, convert them into single cell suspensions; inoculate the cell suspension in a 35mm cell culture dish, and then culture in a 37°C, 5% CO2 cell culture incubator for 24 hours. When the cell density reaches about 70%, wash it 3 times with PBS, 5 minutes each time; fix the cells with 4% paraformaldehyde solution at room temperature for 15 minutes. Subsequently, discard the paraformaldehyde solution, and rinse the cells three times with PBS buffer on a decolorization shaker, each time for 5 minutes. Next, add 1mL of 0.5% TritonX-100 permeabilization solution to the cells and incubate them at 4°C for 10 minutes. After the permeabilization treatment, rinse the cells again with PBS buffer for 5 minutes three times on a decolorization shaker. Then, add 1mL of 2% bovine serum albumin solution to the cells and incubate at 37°C for 30min to achieve cell blocking. After blocking, discard the blocking solution directly without additional washing. Then dilute the primary antibody (FSHR rabbit polyclonal antibody) at a ratio of 1:200 and incubate the diluted primary antibody at 4°C overnight. After removing the primary antibody solution, continue to use PBS buffer on a decolorizing shaker to fully rinse the cells three times for 5min. Thereafter, dilute the goat anti-rabbit IgG secondary antibody at a ratio of 1:200 under light-proof conditions and add it to the cells. Incubate at 37°C for 2h. After the secondary antibody incubation is completed, the light-proof operation is also performed, and PBS buffer is used for three 5min rinses. Next, add DAPI dye solution with a concentration of 0.05μg / mL to the cells and incubate for 10min at room temperature and in a light-proof environment for staining. Finally, use PBS buffer to rinse the cells three times for 5min on a decolorizing shaker, then add 1mL of PBS buffer to each well, and immediately observe the image under a fluorescence microscope;

[0035] S3: The viability of ovarian granulosa cells after H2O2 and allicin treatment was measured to determine the optimal treatment time and concentration of H2O2 and allicin, and the density of passaged sheep ovarian granulosa cells was adjusted to 1×10 4 / ml, spread into a 96-well cell culture plate, and add H2O2 and allicin when the cell density reaches about 80%; H2O2 sets 6 concentration gradients (0, 100, 200, 400, 600, 800μM) and 2 treatment times (4h and 24h), and allicin sets 10 concentration gradients (0, 5, 10, 20, 30, 40, 50, 60, 70, 80μM) and 2 treatment times (24h and 48h), each treatment is repeated 5 times, and the wells with only complete culture medium added are set as blank controls, and then 10μL of CCK-8 working solution is added to each well, and then it is placed in an incubator for 3h incubation. After the culture process is completed, the sample needs to be properly wrapped with tin foil to avoid light influence, and then transferred to the microplate reader for detection. The specific operation was to measure the absorbance of the cells at a wavelength of 450nm, and calculate the cell viability according to the instructions. Then, the viability of the ovarian granulosa cells after co-treatment with H2O2 and allicin was determined. The density of the passaged sheep ovarian granulosa cells was adjusted to 1×10 4 / ml, spread into a 96-well cell culture plate, and when the cell density reaches about 80%, select 5, 10, 20, and 30μM allicin for 20h, then add the optimal H2O2 concentration to treat the cells for 4h, repeat 5 times per well, and then add 10μL of CCK-8 working solution to each well, and then incubate in an incubator for 3h. After the culture is completed, wrap it in tin foil to avoid light and take it to the microplate reader, detect the absorbance of the cells at 450nm, and calculate the cell viability according to the instructions to determine the optimal treatment concentration of allicin;

[0036] S4: Flow cytometry to detect granulocyte apoptosis;

[0037] S5: Extraction of total RNA from cells, followed by reverse transcription of RNA samples. The specific steps for RNA extraction are as follows: Before the experiment begins, make sure to start the centrifuge in advance and adjust the temperature to 4°C. At the same time, pre-cool chloroform, isopropanol and 75% ethanol to ensure that the RNA extraction is carried out under ice bath conditions. First, discard the culture medium and wash the sample 2-3 times with 1×PBS solution.

[0038] 1 mL of TRIzol lysis buffer needs to be added to each well, and then the solution is repeatedly and thoroughly mixed with a pipette. Note that new pipette tips are replaced between different processing steps to ensure no cross contamination. After adding the lysis buffer, place the sample on ice and keep it for 5 minutes to achieve sufficient cell lysis. After the lysis is completed, the lysis buffer is immediately transferred to a centrifuge tube.

[0039] Add 200 μL of chloroform to the centrifuge tube and mix thoroughly using an oscillator. Next, place the mixed centrifuge tube in an ice bath for 5 minutes. Thereafter, transfer it to a centrifuge and set the speed to 12,000 × g for 15 minutes.

[0040] Transfer the supernatant to a new centrifuge tube, add 500 μL of isopropanol, and mix by gently inverting. Then, place the mixture on ice for 10 minutes, and finally place it in a centrifuge, set the speed to 12,000 × g, and centrifuge for 10 minutes.

[0041] Gently pour off the supernatant. Then, add 1 mL of 75% ethanol to the container for washing. After gently blowing and mixing the solution thoroughly, put the container into a centrifuge and set the speed to 7500×g for 5 minutes.

[0042] During the operation, we noticed a small amount of white precipitate at the bottom of the tube. First, the supernatant needs to be gently poured off and the remaining liquid attached to the wall of the tube needs to be sucked up with a pipette. Next, the test tube is placed on ice for 5 minutes to dry. Finally, 20 μL of DEPC water is added to dissolve the precipitate.

[0043] The concentration and purity of the extracted cell RNA were measured. RNA samples with a purity, i.e., an OD value (OD value is 260 / 280) between 1.8 and 2.0, can be used for subsequent experiments, and the samples are stored for subsequent experiments.

[0044] The extracted RNA samples can be directly reverse transcribed or stored in a -80°C freezer for a long time.

[0045] S6: Real-time fluorescence quantitative PCR detection of apoptosis-related gene expression. The steps of real-time fluorescence quantitative PCR detection of apoptosis-related gene expression include designing and synthesizing specific primers for GAPDH, Bax and Bcl-2, using cDNA as a template, using Bio-Rad's CFX96 system for reaction, GAPDH as an internal reference, and following 2 -ΔΔCt The relative expression of the target gene was calculated by the method. The RT-qPCR reaction system (10 μL) included: 5 μL 2×M5HiPerSYBR Permix EsTaq, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 1 μL template cDNA, and 3 μL ddH2O;

[0046] S7: determination of steroid hormone secretion, namely, determination of E2 and P4 secretion levels in the supernatant of granulosa cells in the control group, H2O2 group and Allicin+H2O2 group;

[0047] S8: Analyze the detected data.

[0048] The culture medium in S1 is prepared by mixing 89% DMEM / F12 culture medium, 10% FBS and 1% penicillin-streptomycin mixture, mixing them evenly, wrapping the centrifuge tube with sealing film, and storing it in a 4°C refrigerator for use.

[0049] The S1 allicin stock solution was prepared by using a pipette to dissolve 4.38 μL of allicin with a molecular weight of 162.27 in 1 mL of dimethyl sulfoxide (DMSO) in an ultra-clean workbench to obtain a concentrated stock solution with a concentration of 30 mM, which was then dispensed into centrifuge tubes, sealed with sealing film, and stored in a -20°C refrigerator.

[0050] The detection of granulosa cell apoptosis in S4 was based on the instructions of Annexin V, FITC apoptosis detection kit (Tongren Chemical). Granulosa cells from the control group, H2O2 group and Allicin+H2O2 group were collected and processed, and then the cell apoptosis rate was detected by flow cytometry.

[0051] The specific steps of reverse transcription treatment of RNA samples in S5 are to prepare the mixed solution listed in the table in a centrifuge tube without RNase contamination to achieve effective removal of genomic DNA, and then in the above reaction system (such as Figure 1 Directly add 4 μL of 5x HiScript qRT SuperMix reagent to the reaction system in the reaction tube and ensure that it is fully mixed. Incubate at 37°C for 15 min, then treat at 85°C for 5 s. After the reverse transcription reaction is completed, the cDNA product obtained should be properly stored in a -20°C refrigerator for subsequent experiments.

[0052] Determination of steroid hormone secretion in S6 The secretion levels of E2 and P4 in the supernatant of granulosa cells of the control group, H2O2 group and Allicin+H2O2 group were determined.

[0053] The real-time fluorescence quantitative PCR step in S7 includes searching the CDS region sequences of sheep GAPDH, Bax, and Bcl-2 genes in the NCBI database, designing specific primers, using cDNA as a template, and using the Bio-Rad CFXManager3.0 system to perform the reaction. GAPDH is used as an internal reference. -ΔΔCtThe relative expression of the target gene was calculated by RT-qPCR reaction system (10 μL), including 5 μL 2×M5 HiPer SYBR Permix EsTaq, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 1 μL template cDNA, 3 μL ddH2O; the reaction conditions were: 95°C pre-denaturation for 30 s; 95°C for 5 s, annealing temperature for 30 s, and 39 cycles.

[0054] The data analysis in S8 was expressed as mean ± standard error (mean ± SEM) and plotted using Graphpad Prism8. One-Way ANOVA and t-test were used to analyze the significance of the differences in the experimental data. When P < 0.05, it was statistically significant. * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin; characterized in that: The following steps are involved: S1: First, the culture solution is prepared, and then the allicin stock solution is prepared; S2: Identification of ovine ovarian granulosa cells; S3: Determine the viability of ovarian granulosa cells after H2O2 and allicin treatment alone to determine the optimal treatment time and concentration of H2O2 and allicin; S4: Flow cytometry to detect granulocyte apoptosis; S5: Extraction of total RNA from cells, followed by reverse transcription of RNA samples; S6: Real-time fluorescence quantitative PCR detection of apoptosis-related gene expression; S7: Measurement of steroid hormone secretion; S8: Analyze the detected data.

2. The method of regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin according to claim 1, characterized in that: The culture medium in S1 is prepared by mixing 89% DMEM / F12 culture medium, 10% FBS and 1% penicillin-streptomycin mixture, mixing them evenly, wrapping the centrifuge tube with sealing film, and storing it in a refrigerator at 4°C for later use.

3. The method of regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin according to claim 1, characterized in that: The S1 allicin stock solution was prepared by using a pipette to absorb 4.38 μL of allicin with a molecular weight of 162.27 and dissolving it in 1 mL of dimethyl sulfoxide (DMSO) in an ultra-clean workbench to obtain a concentrated stock solution with a concentration of 30 mM, and then dispensed into centrifuge tubes, sealed with sealing film, and stored in a -20°C refrigerator.

4. The method of claim 1, wherein: The detection of granulocyte apoptosis in S4 was carried out according to the instructions of Annexin V-FITC apoptosis detection kit (Tongren Chemical). Granule cells from the control group, H2O2 group and Allicin+H2O2 group were collected for treatment, and the cell apoptosis rate was detected by flow cytometry.

5. The method of regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin according to claim 1, characterized in that: The specific steps of reverse transcription treatment of RNA samples in S5 are as follows: preparing the mixed solution listed in Table 1 in a centrifuge tube free of RNase contamination to achieve effective removal of genomic DNA, then directly adding 4 μL of 5×HiScript qRT Super Mix reagent to the above reaction system, and ensuring sufficient mixing, incubating at 37°C for 15 minutes, and then treating at 85°C for 5 seconds. The cDNA product obtained after the reverse transcription reaction is completed should be properly stored in a -20°C refrigerator for subsequent experimental use.

6. The method of claim 1 for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin, characterized in that: The step of detecting apoptosis-related gene expression by real-time fluorescence quantitative PCR in S6 includes designing and synthesizing specific primers for GAPDH, Bax and Bcl-2, using cDNA as a template, using the CFX96 system of Bio-Rad Company for reaction, and using GAPDH as an internal reference. The relative expression of the target gene was calculated by the RT-qPCR reaction system (10 μL), including: 5 μL 2×M5HiPerSYBR Permix EsTaq, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 1 μL template cDNA, and 3 μL ddH2O.

7. The method of claim 1 for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin, characterized in that: The steroid hormone secretion determination in S7 is the determination of the secretion levels of E2 and P4 in the supernatant of the granulosa cells of the control group, the H2O2 group and the Allicin+H2O2 group.

8. The method of claim 1 for regulating oxidative stress-induced apoptosis of sheep ovarian granulosa cells using allicin, characterized in that: The data analysis in S8 was expressed as mean ± standard error (mean ± SEM), and graphed using Graphpad Prism8. One-Way ANOVA and t-test were used to analyze the significance of the differences in the experimental data. When P < 0.05, it was statistically significant. * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

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