Application of E3 ubiquitin ligase RLIM gene inhibitor in preparation of medicine for treating ovarian aging or injury

Abstract

The invention provides an application of an E3 ubiquitin ligase RLIM gene inhibitor in preparation of a medicine for treating ovarian aging or injury. Experiments find that the expression level of the RLIM gene in the ovary is positively correlated with the aging degree, and the expression of the RLIM gene is remarkably improved along with age increase. In-vivo and in-vitro experiments show that ovarian granular cell senescence can be effectively delayed and ovarian functions can be improved by inhibiting RLIM expression, and particularly, the proportion of atresia follicles is reduced, the number of healthy follicles is increased, and serum AMH and E2 levels are remarkably improved. The pharmaceutical composition provided by the invention comprises a siRNA inhibitor (preferably, siRLIM-2) of specific targeting RLIM and a nano-liposome carrier, and efficient targeting delivery can be realized through tail vein injection. The treatment scheme has no toxic effect on important organs, is high in safety, and provides a new effective means for treatment of ovarian aging and related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of E3 ubiquitin ligase RLIM gene inhibitors in the preparation of drugs for treating ovarian aging or damage. Background Technology

[0002] Currently, the aging population is becoming increasingly prominent. Organ aging due to old age can cause serious damage to the body, including an increased risk of organ fibrosis, metabolic diseases, and tumors. The ovary is a vital female reproductive organ, playing a crucial role in regulating endocrine function and maintaining fertility. Ovarian function typically begins to decline around age 35, gradually diminishes after age 37, and is depleted by age 50. Ovarian aging is mainly characterized by a decrease in the number and quality of follicles, which can lead to hormonal imbalances, reduced fertility, lower reproductive quality, and ultimately, menopause and infertility. Furthermore, ovarian aging can lead to a series of age-related diseases, including cardiovascular disease, osteoporosis, neurodegenerative diseases, and diabetes. Therefore, exploring the mechanisms of ovarian aging and finding effective prevention and treatment strategies are critical issues that urgently need to be addressed in clinical practice.

[0003] Cell senescence is the cornerstone of organ aging. Granulosa cells, as important components of the ovary, experience a decline in function, which is a significant cause of ovarian aging. Therefore, research on ovarian granulosa cell senescence is of great importance.

[0004] Therefore, there is an urgent clinical need to find drugs to treat ovarian aging or damage. Summary of the Invention

[0005] The purpose of this invention is to provide the application of E3 ubiquitin ligase RLIM gene inhibitors in the preparation of drugs for treating ovarian aging or damage. The RLIM gene inhibitors provided by this invention have multiple advantages such as significant efficacy, clear targeting, high safety, and superior delivery efficiency, providing a new and effective approach for the treatment of ovarian aging and related diseases, and have important clinical application value and industrialization prospects.

[0006] To achieve the aforementioned objective, the present invention adopts the following technical solution: In a first aspect of the invention, the use of an E3 ubiquitin ligase RLIM gene inhibitor in the preparation of a medicament for treating ovarian aging or damage is provided.

[0007] Furthermore, the E3 ubiquitin ligase RLIM gene inhibitor includes siRLIM-1, siRLIM-2, and siRLIM-3, with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0008] Furthermore, the treatment for ovarian aging or damage includes improving the number of follicles and hormone levels.

[0009] In a second aspect of the invention, a pharmaceutical composition is provided comprising an E3 ubiquitin ligase RLIM gene inhibitor.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the excipients are selected from one of the following: fillers, disintegrants, binders, lubricants, sweeteners, or colorants.

[0012] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections, and dispersants.

[0013] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention provides the application of an E3 ubiquitin ligase RLIM gene inhibitor in the preparation of drugs for treating ovarian aging or damage. Through systematic in vitro and in vivo experiments, it provides an innovative treatment regimen based on the E3 ubiquitin ligase RLIM gene inhibitor, which has the following significant beneficial effects: 1. Significant therapeutic effect Experiments have shown that inhibiting RLIM expression can effectively improve ovarian function. For example... Figure 7 As shown, downregulation of RLIM in vivo significantly reduced the proportion of atretic follicles and increased the number of healthy follicles. Simultaneously, serum AMH and E2 levels were significantly elevated, indicating substantial improvement in ovarian function. All three siRNA sequences obtained through screening showed good downregulation effects, with siRLIM-2 showing particularly significant effects, providing optimal candidate sequences for drug development and demonstrating clear industrialization potential.

[0014] 2. Excellent safety Importantly, the RLIM overexpression experiment showed that although targeting RLIM could significantly affect ovarian function, it had no adverse effects on the function of important organs (heart, liver, spleen, lungs, and kidneys) in mice, and blood biochemical indicators were all within the normal range, demonstrating that the treatment regimen has good safety. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1The study investigated the changes in E3 ubiquitin ligase RLIM in ovarian tissues of mice at different ages. A. Immunohistochemistry of RLIM in ovarian tissues of young, middle-aged, and old mice; B. Quantitative results of immunohistochemistry; C. Trends in RLIM protein levels in ovarian tissues of mice at three weeks of age; D. Trends in RLIM mRNA levels. NS indicates no statistically significant difference.

[0017] Figure 2 The study investigated how overexpression of RLIM in the ovaries leads to decreased ovarian function. A. Tracing of the adenovirus vector ZsGreen in frozen sections of ovarian tissue; B. Hematoxylin & Eosin (HE) staining of paraffin sections of mouse ovarian tissue, identifying primary follicles (PF), secondary follicles (SF), antral follicles (ANF), and atretic follicles (ATF); C. Ratio of healthy follicles to atretic follicles; D. Serum levels of anti-Müllerian hormone (AMH), estrogen (E2), progesterone (PROG), and follicle-stimulating hormone (FSH). NS indicates no statistically significant difference.

[0018] Figure 3 To investigate how overexpression of RLIM in the ovaries accelerates granulosa cell senescence. A. Changes in RLIM and cellular senescence marker protein levels in ovarian tissues of two groups of mice; B. β-gal staining of mouse ovaries; C. Immunohistochemistry of RLIM, P21, and P16 in mouse ovarian tissues. .

[0019] Figure 4 To assess the general condition and organ function of mice overexpressing RLIM in the ovary. A. Changes in body weight in both groups of mice; B. Weight of vital organs in mice; C. Blood biochemical levels in mice, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase 1 (LDH1), creatinine (CREA), and blood urea nitrogen (BUN). NS indicates no statistically significant difference.

[0020] Figure 5 To investigate changes in cellular senescence following RLIM overexpression in granulosa cells. A. RLIM lentiviral transfection of granulosa cells; B. Protein changes in cellular senescence markers after RLIM upregulation in granulosa cells; C. Changes in the mRNA levels of P21 and P16 after RLIM overexpression; D. Cellular β-gal staining and statistical analysis. NS indicates no statistically significant difference.

[0021] Figure 6To downregulate changes in cellular senescence markers in granulosa cells after RLIM. A. Downregulate protein levels of cellular senescence-related markers in granulosa cells after RLIM; B. γH2AX immunofluorescence assay after RLIM knockdown; C. EdU cell proliferation assay after RLIM knockdown. NS indicates no statistically significant difference.

[0022] Figure 7 To investigate changes in ovarian function in mice after downregulation of RLIM. A. Changes in RLIM protein levels in ovarian tissues of both groups of mice; B. Changes in mRNA levels; C. HE staining of paraffin sections of mouse ovarian tissue; D. Follicle count; E. Serum AMH and E2 levels. NS indicates no statistically significant difference. Detailed Implementation

[0023] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0024] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0026] The application of the E3 ubiquitin ligase RLIM gene inhibitor of this application in the preparation of drugs for treating ovarian aging or damage will be described in detail below with reference to embodiments and experimental data.

[0027] Example 1: Validation of the age-related correlation between RLIM expression and ovarian aging I. Experimental Objective To verify the correlation between RLIM expression levels and ovarian aging.

[0028] II. Experimental Methods 1. Experimental subjects Beijing Vital River Co., Ltd. purchased 12-week, 36-week, and 48-week female mice and collected ovarian tissue for paraffin sectioning, protein and mRNA extraction.

[0029] 2. Experimental Methods 2.1 Immunohistochemistry 1) Baking: Baking paraffin slices in an oven at 65℃ for 1.5-2 hours; 2) Dewaxing and hydration: Immediately immerse the sections in environmentally friendly dewaxing solution I for 20 minutes, then immerse them in dewaxing solution II for 20 minutes, anhydrous and 95% ethanol for 10 minutes each, 80% and 75% ethanol for 5 minutes each, and finally rinse them in double-distilled water for 5 minutes. 3) Wash with PBS solution for 5 minutes, repeat 3 times; 4) Antigen retrieval: Dilute 20x EDTA antigen retrieval solution (pH 9.0) with double-distilled water to 1x, pour it into the slide container, microwave on high for 10 minutes, then put the slides into the boiling antigen retrieval solution and boil on medium heat twice for 7 minutes each time. Pay attention to whether the liquid has decreased and ensure that it completely covers the slide tissue, and add more as needed. 5) After cooling to room temperature, wash with PBS solution for 5 minutes, repeat 3 times; 6) Removal of catalase: Dilute 30% hydrogen peroxide with PBS to 3%, add it dropwise to the tissue to ensure complete tissue coverage, place it in a humidified chamber, and incubate at 37°C in a biochemical incubator for 30 minutes to remove catalase. 7) Remove 3% hydrogen peroxide, wash with PBS solution for 5 minutes, repeat 3 times; 8) Blocking of non-specific sites: 10% goat serum was added to the tissue to block non-specific sites, and the tissue was placed in a humidified box and incubated in a biochemical incubator at 37°C for 1 hour. 9) Primary antibody incubation: Add an appropriate amount of primary antibody to the tissue and incubate overnight at 4°C; 10) The next day, remove the humidified chamber and allow it to warm for 1 hour, then wash with PBS solution for 5 minutes. Repeat this process 3 times. 11) Secondary antibody incubation: Add an appropriate amount of HRP secondary antibody and incubate at 37℃ in a biochemical incubator for 1 hour; 12) Wash with PBS solution for 5 minutes, repeat 3 times; 13) Color development: Develop the color with DAB colorimetric solution, then rinse with running water in double-distilled water for 5 minutes; 14) Staining the nucleus: Stain with hematoxylin for 5 minutes, stop with double distilled water, stain with differentiation solution for 30 seconds, stain with blue solution for 30 seconds, stop with double distilled water, and observe under a microscope; 15) Dehydration: The slices were placed in 75% ethanol and 80% ethanol for 5 minutes each, and in 95% ethanol and anhydrous ethanol for 10 minutes each. 16) Mounting: After the sections are dried in a fume hood, they are mounted with neutral resin and observed under a microscope.

[0030] 2.2 Protein Extraction 1) Tissue grinding and lysis: The tissue was placed in a grinding tube, and an appropriate amount of RIPA lysis buffer, PMSF, phosphatase inhibitor and grinding beads were added. The tissue was ground on a grinder at 4°C, set to 60Hz, 180 seconds, with a pause every 20 seconds to observe the degree of tissue grinding and whether further grinding was necessary. 2) If it is a cell, add an appropriate amount of RIPA lysis buffer, PMSF and phosphatase inhibitor; 3) After mixing thoroughly by blowing, pyrolyze on ice for 30 minutes, mixing once every 5 minutes to ensure complete pyrolysis; 4) Ultrasonic lysis: Intensity 30-40Hz, operate on ice, pause for 7-8 seconds for each tissue lysis, repeat 8-10 times, lyse cells for 3-4 seconds each time, repeat 8-10 times, continue lysis on ice for 30 minutes. 5) Centrifuge at 12,000 rpm, 4°C for 25 minutes; 6) Collect the supernatant and record its volume; 7) Protein concentration determination by BCA quantification: Prepare BCA kit solution A and solution B at a ratio of 1:50. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard to the enzyme-labeled tubes sequentially, and add 1 μL to each experimental group. Add PBS to each tube to a final volume of 20 μL. Add 200 μL of the A / B mixture and mix well. Incubate at 37°C for 30 minutes. Read the OD value at 562 nm using an enzyme-labeled reader. 8) Plot a standard curve based on the OD values, and calculate the concentration and protein loading amount for each group; 9) Add 1 / 4 volume of loading buffer according to the protein volume of each group, mix well and seal. First, boil the water on high heat, then place the protein sample on a floating plate and put it in boiling water. Cook on medium heat for 10 minutes to denature it. 10) After centrifuging the cooked egg whites, store them in a refrigerator at -80°C.

[0031] 2.3 Western blotting of proteins 1) Add the prepared electrophoresis buffer to the electrophoresis tank and vertically pull out the comb teeth; 2) Remove the sample from the -80℃ freezer, mix it with a vortex mixer and centrifuge briefly, then add the protein marker and sample in sequence; 3) After adding sufficient electrophoresis buffer, connect the power supply, adjust the voltage to 60V, and perform constant voltage electrophoresis for 2.5-3 hours; 4) Prepare the transfer buffer in advance and keep it on ice; 5) Place the sponge, 5 layers of filter paper, the cut glue, the PVDF membrane (pre-activated in methanol and double-distilled water and equilibrated in the transfer solution) on the black side of the transfer clamp, then place the sponge, the transfer clamp, and the black glue and white membrane. 6) Place the transfer clamp into the transfer tank, black to black, white to red, adjust the current to 280mA, and transfer the film on ice for 90 minutes under constant current. 7) Place the membrane in 5% skim milk and seal it on a shaker at room temperature for 1 hour; 8) Prepare primary antibody with 5% BSA, cut the membrane according to the molecular weight of each indicator, place the band in the antibody incubation box, add 2-3 ml of antibody, and incubate overnight at 4°C; 9) After removing the band, warm it to room temperature, recover the primary antibody, and wash three times with TBST for 10 minutes each time; 10) Dilute HRP rabbit or mouse secondary antibody with TBST, add it to the band, and incubate at 37°C for 1 hour; 11) Recover the secondary antibody, wash three times with TBST for 10 minutes each time; 12) Prepare ECL exposure solution at a 1:1 ratio, incubate the bands, and then expose them; 13) Image Lab analyzes images.

[0032] 2.4 RNA extraction, reverse transcription, and real-time quantitative PCR 1) Place the tissue in a grinding tube, add 200 μL of Trizol and RNase-free grinding beads, seal the tube and grind it on a grinder at 4°C. Set the grinder to 60 Hz for 180 seconds, pausing every 20 seconds to observe the degree of tissue grinding and whether further grinding is needed. Then add 800 μL of Trizol and lyse on ice for 30 minutes. 2) Add 1 ml of Trizol to the collected cells and lyse them on ice for 30 minutes; 3) Then add 200 μL of chloroform, mix by inverting, and let stand on ice for 30 minutes; 4) Centrifuge at 4℃, 12,000 rpm for 20 minutes; 5) After centrifugation, the liquid separates into three layers: the bottom layer is a red organic layer, the middle layer is a white protein layer, and the top layer is a transparent RNA layer. 6) Aspirate the upper transparent layer, avoiding the middle and lower layers, then add 500 μL of isopropanol to precipitate the RNA and incubate overnight at -20°C. 7) The next day, remove the RNA and allow it to warm up for at least 1 hour, and cool the centrifuge to 4°C beforehand; 8) Prepare 75% ethanol by mixing anhydrous ethanol and DEPC water in a 3:1 ratio; 9) Centrifuge at 4℃, 12,000 rpm for 20 minutes; 10) After centrifugation, white flocculent precipitate can be seen on the bottom and side walls of the tube. Discard the supernatant, add 1 ml of prepared 75% ethanol to each tube, wash the precipitate, centrifuge at 4°C and 12,000 rpm for 20 minutes, and repeat the operation twice. 11) After rinsing twice, aspirate the supernatant and invert the EP tube to allow any remaining liquid at the bottom to drain out; 12) Add an appropriate amount of DEPC to water to resuspend, mix thoroughly by blowing and stirring to ensure complete dissolution, and measure the concentration; 13) Reverse transcription Table 1-16 Microliter System

[0033] 42℃ for 2 minutes, then maintain at 4℃.

[0034] Table 2 - 20 μL System

[0035] 52℃ for 15 minutes, 85℃ for 5 seconds, 4℃ for maintenance. Store the product at -20℃.

[0036] 14) Real-time quantitative PCR 14.1 Prepare the following reaction system in a qRT-PCR tube. Table 3 - Reaction systems prepared in qRT-PCR tubes:

[0037] 14.2 After mixing, perform qRT-PCR reaction.

[0038] 14.3 Bio-Rad software analysis results.

[0039] Table 4 - Primer Sequences

[0040] III. Experimental Results The expression levels of RLIM in the ovarian tissues of young, middle-aged, and aged mice were detected. The results showed that the expression of RLIM in mouse ovaries increased with age. Figure 1 A, B). Protein detection results also showed that the expression of RLIM in mouse ovaries gradually increased with age. Figure 1 C). mRNA results showed that the expression level of RLIM in the middle-aged and elderly groups was significantly higher than that in the young group (C). Figure 1 D). The above experimental results indicate differences in RLIM expression in the ovaries of mice of different ages.

[0041] Example 2: Effects of overexpression of ovarian RLIM on ovarian function and granulosa cell senescence in mice I. Experimental Objective To further confirm that RLIM regulates granulosa cell senescence, we injected the AdV-mRLIM vector into the mouse ovarian cyst to achieve RLIM overexpression in the ovary and verified the damaging effect of RLIM overexpression on ovarian function.

[0042] II. Experimental Methods 1. Intraovarian cyst injection of adenovirus in mice 1) After weighing the mice, they were anesthetized by intraperitoneal injection of 4% chloral hydrate solution at a dose of 10 μL / g; 2) Remove the hair from the lower back, especially around the ovaries on both sides. Place the mouse back-up on a PE glove and place it under a stereomicroscope. Position the mouse properly and cover its head with gauze to avoid damaging its vision. 3) Disinfect the incision area with 75% alcohol, lift the skin of the mouse's kidney area with ophthalmic forceps, and make an incision of about 0.5 cm on the inside of the back with ophthalmic scissors. Then cut the peritoneum deeper, taking care to avoid blood vessels and thick muscle layers to avoid bleeding and difficulty in finding the location. 4) Locate the ovarian fat pad, and gently remove the ovary, fallopian tube, and fat pad using micro-forceps, taking care to keep the ovary moist; 5) Use a microsyringe (33 gauge, G) to draw 4 μL of empty or target gene adenovirus. Use micro forceps to grasp the loose area between the ovary and fallopian tube and insert it into the ovarian surface membrane. Inject slowly until the ovarian sac is seen to be filled. After injection, leave it for 2 minutes to reduce fluid leakage. Be careful not to puncture the membrane. 6) Return the ovary to its original position in the abdominal cavity. After confirming there is no bleeding, suture the peritoneum with 6-0 absorbable sutures and the skin with 4-0 sutures. Disinfect with povidone-iodine after suturing, place the mouse on a heating pad for anesthesia and recovery, and carefully observe the mouse's respiratory rate. Once fully awake, return the mouse to its cage.

[0043] 2. Hematoxylin & Eosin (HE) staining 1) Baking: Baking paraffin slices in an oven at 65℃ for 1.5-2 hours; 2) Dewaxing and hydration: Immediately immerse the sections in environmentally friendly dewaxing solution I for 20 minutes, then immerse them in dewaxing solution II for 20 minutes, anhydrous and 95% ethanol for 10 minutes each, 80% and 75% ethanol for 5 minutes each, and finally rinse them in double-distilled water for 5 minutes. 3) Staining the nucleus: Stain with hematoxylin solution for 5 minutes, stop with double distilled water, and observe under a microscope; 4) Differentiation: Stain with differentiation solution for 30 seconds, then stop with double distilled water; 5) Blueing: Stain with blueing solution for 30 seconds, then stop with double distilled water and observe under a microscope; 6) Cytoplasmic staining: Stain with freshly changed eosin solution for 20 seconds, then immerse in anhydrous ethanol for 10 seconds and remove to air dry in a fume hood; 7) Mounting: After mounting with neutral resin, observe and photograph under a microscope.

[0044] 3. Follicle count 1) After the mouse ovaries were removed, they were fixed with 4% paraformaldehyde and then sent to Wuhan Pinofi Biotechnology Co., Ltd. for paraffin embedding and sectioning; 2) The ovary was embedded along the largest cross section along the longitudinal axis, and serially sectioned with a tissue thickness of 4 micrometers. Four tissue sections were placed on each slide. 3) When counting follicles, select one slide every 80 micrometers, i.e., select slides 1, 6, 11, 16, etc., for HE staining; 4) Characteristics of follicles at each stage: Primordial Follicle (PMF): An oocyte surrounded by a single layer of flattened granulosa cells; Primary follicle (PF): A single layer of cuboidal granulosa cells surrounding the oocyte; Secondary follicle (SF): The outer ring of the oocyte has two or more layers of cuboidal granulosa cells, and the granulosa cells are arranged in an orderly manner, without sinus cavity; Antral follicle (ANF): The follicle contains a large sinus cavity with a greater number of granulosa cell layers; Atretic follicle (ATF): The oocyte has abnormal morphology, disordered arrangement of granulosa cells, and nuclear pyknosis and karyolysis in both cells.

[0045] 4. Serum sex hormone assay 1) After the intervention, during the interestrus period of the mice, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate solution at a dose of 10 μL / g; 2) Collect blood from the eyeball and place it in a 1.5 ml EP tube. During the process, allow the blood to flow out naturally as much as possible and avoid squeezing the mouse's heart to prevent hemolysis. 3) After incubating at room temperature for at least 30 minutes, centrifuge at 3000 rpm for 15 minutes; 4) Take the serum and place it in a new EP tube, and record the volume. Do not aspirate the lower layer of blood cells. It can be used immediately for hormone detection, or it can be frozen at -80°C for subsequent measurements. Avoid repeated freeze-thaw cycles. 5) The serum taken from the -80℃ freezer was thawed and then centrifuged again; 6) Serum levels of anti-Müller's hormone (AMH), estradiol (E2), progesterone (PROG), and follicle stimulating hormone (FSH) were detected using enzyme-linked immunosorbent assay (ELISA). 7) Before starting the operation, let the reagent kit stand at room temperature for at least 30 minutes; 8) Dilute the 20x concentrated washing solution to 1x with deionized water. Prepare the enzyme conjugate and antibody at a ratio of 10:1 and prepare immediately before use. 9) Sample addition: Set up 1 blank control well without adding anything, set up 2 replicate wells for each of the 6 standards, add 50 μL to each well, and add 50 μL of the sample to be tested to each test well; 10) Except for the blank control wells, add 50 μL of enzyme conjugate mixture to each well, mix well, and then attach the plate to the plate and incubate at 37°C for 1 hour. 11) After discarding the liquid in the well, add 200 μL of washing buffer to each well using a pipette, let it sit for 10 seconds, then discard the liquid. Wrap the microplate with a paper towel and pat it dry. Repeat 3 times to remove as many air bubbles as possible from the well. 12) After washing the plate, add 50 μL of colorimetric solution A to each well, then add 50 μL of colorimetric solution B, mix well, attach the plate, and incubate at 37°C for 15 minutes. 13) Add 50 μL of stop solution to each well, mix well, and read the OD value at 450 nm wavelength using a microplate reader. The measurement should be completed within 10 minutes after adding the stop solution. 14) Plot a standard curve based on the concentration and OD value of the standard, and then calculate the concentration of the sample to be tested.

[0046] 5. β-galactosidase staining (SA-β-gal) 1) Before starting the experiment, dilute 10x PBS with deionized water to 1x, and dilute 10x fixative with 1x PBS to 1x. Equilibrate to room temperature before use. 2) Aspirate the culture medium from the six-well plate and wash twice with PBS; 3) Add 1 ml of fixative to each well and fix at room temperature for 15 minutes; 4) Remove the fixative and wash three times with PBS; 5) Prepare working solution: Prepare according to 1 ml system. All solutions should be equilibrated to room temperature before use. X-Gal solution should be heated in a 37°C water bath for at least 1 hour in advance to prevent crystallization. Table 5

[0047] 6) Remove the PBS, add 1 ml of working solution to each well, and incubate overnight in a CO2-free biochemical incubator at 37°C. 7) Observe the cell staining on the second day and stop the procedure if necessary; 8) Aspirate the working solution, wash 3 times with PBS, and then observe and photograph under an inverted white light microscope to count the number of blue cells; 9) If crystals are seen at the bottom of the well plate after the working solution is aspirated, add 75% ethanol and wash for 10-15 minutes to remove the crystals without affecting the blue coloring. 10) If you cannot take pictures immediately, add PBS to the well plate and store it at 4°C.

[0048] 11) When staining frozen sections of tissue, first take the frozen sections out of the -80℃ freezer and let them warm for 30 minutes, then fix them with fixative at room temperature for 10 minutes. Subsequent steps are the same as 4-8.

[0049] III. Experimental Results like Figure 2 As shown, ZsGreen (adenovirus vector tracer) fluorescence was detected in frozen sections of ovarian tissue, indicating that the vector reached the ovarian tissue. Figure 2 A). Compared to the AdV-ZsGreen control group, the AdV-mRLIM group showed a decrease in the proportion of healthy follicles, an increase in the proportion of atretic follicles, a decrease in AMH levels, and a decrease in estrogen and progesterone secretion, indicating a decline in ovarian function. Figure 2 BD).

[0050] β-galactosidase staining and immunohistochemical detection of P21 and P16 results are as follows: Figure 3 As shown, Western blot experiments revealed increased expression of cell senescence markers P21 and P16, and DNA damage marker γH2AX proteins. Figure 3 A). Mouse ovarian β-gal staining results also showed an increase in the number of senescent cells after RLIM overexpression ( Figure 3 B). Immunohistochemical results showed that after overexpression of RLIM in mouse ovaries, the expression of P21 and P16 was significantly increased ( Figure 3 C).

[0051] Changes in general condition and vital organ function in mice after RLIM overexpression in the ovaries: After RLIM overexpression in the ovaries of mice, the body weight and organ weights of the mice were measured. No significant differences were found in body weight, heart, liver, spleen, lung, kidney, and uterus weight between the two groups of mice. Figure 4 A, B). Biochemical indicators from various organs also indicated that elevated RLIM expression in the ovary did not affect the function of vital organs in mice. Figure 4 C).

[0052] In conclusion, overexpression of RLIM exacerbates ovarian aging, which conversely proves the therapeutic value of inhibiting RLIM.

[0053] Example 3: Granulosa cell senescence after in vitro upregulation of RLIM expression I. Experimental Objective To verify the direct effect of regulating RLIM expression on granulocyte senescence.

[0054] II. Experimental Methods 1. Cell model: Human granular cell line COV434 2. Experimental Groups: Overexpression group: Lentiviral transfection with hRLIM Lentiviral construction Shanghai Dianjun Biotechnology customized the pLVX-CMV-3FLAG-EGFP-IRES-puromycin vector to deliver the target gene. Human RLIM (ID: 51132, NM_183353.3, sequence as shown in SEQ ID NO.4) was inserted into the vector to obtain the core plasmid pLVX-CMV-Human RLIM-3FLAG-P2A-EGFP-IRES-puromycin; Seed 293T cells in culture dishes (the number should be determined according to experimental needs). Incubate the cells at 37°C with 5% CO2. Incubate in an incubator for 8-24 hours until the cells adhere to the culture dish and occupy more than 70% of the culture dish area; Take two sterile 1.5 mL centrifuge tubes, add 200 μL of serum-free DMEM to one tube, and add core concentrate to the other tube. Plasmid pLVX-CMV-Human RLIM-3FLAG-P2A-EGFP-IRES-puromycin, viral packaging plasmids psPAX2 and pMD2.G; add 200 μL of serum-free DMEM to another tube, add transfection reagent Lipofectamine 2000, and let stand for 5 minutes; Mix the contents of the two tubes thoroughly, let stand for 15-20 minutes, then add the mixture dropwise to the cell culture dish, gently... Gently shake the petri dish to distribute the liquid evenly, and then place the petri dish in an incubator at 37°C and 5% CO2 for incubation. Infectious lentivirus was harvested 72 hours after transfection, centrifuged at 3500 rpm for 10 minutes at room temperature to remove cell debris, and then... Filter through a 0.45-micron cellulose acetate membrane. Centrifuge at 30,000 rpm for 120 minutes in an ultracentrifuge tube at 4°C. A white viral precipitate will be observed on one side of the tube wall. Discard the supernatant. Invert the centrifuge tube onto sterile absorbent paper to remove any remaining supernatant. Add 80-120 μL of DPBS to each tube, depending on the amount of precipitate. Seal the tube with sealing film and incubate overnight at 4°C to dissolve the precipitate. Aliquot the virus as required and store at -80°C.

[0055] Lentiviral transfection 1) Transfection was performed when the cells were in good condition. After digestion, the cells were seeded into two wells of a 24-well plate, with the cell density controlled at 30-40% (to ensure that the cells could grow for 72 hours). After the cells adhered, they were washed twice with PBS. 300 μL of culture medium was added to each well, followed by 50 μL of lentiviral vector and the target gene, respectively. The plate was then placed in an incubator for transfection. 2) Observe the green fluorescence of cells under a fluorescence microscope every 24 hours for 72 hours; 3) After 72 hours, the cells in the two wells were digested and passaged to 2. Once the cells were confluent, 2 μg / ml puromycin was added to one well (to prevent the concentration of 5 μg / ml from being too high and causing all cells to die), and 5 μg / ml was added to the other well for resistance selection. 4) After 24 hours, the medium was changed, and a large number of cell deaths were observed. Under a fluorescence microscope, the remaining adherent cells were all greenish cells. Subsequently, the medium was continuously changed with puromycin (5 μg / ml) for digestion (prepared fresh for each use), and the cells were passaged, expanded, and cryopreserved. The entire virus transfection process was carried out in a biosafety cabinet. If in a clean bench, the fan must not be turned on.

[0056] 4. Testing indicators: Western Blot: Detection of RLIM, P21, P16, and γH2AX Immunofluorescence: γH2AX focal count EdU proliferation assay: Detecting cell proliferation capacity III. Experimental Results In vitro upregulation of RLIM expression and granulocyte senescence are as follows: Figure 5 As shown, RLIM ( ) was overexpressed in granulosa cells via lentiviral transfection. Figure 5 A) Observe the changes in granulosa cell senescence. Western blot results show that RLIM upregulation in granulosa cells is significant, and after RLIM upregulation, the expression of cellular senescence and DNA damage-related proteins P21, P16, and γH2AX increases. Figure 5B). The mRNA levels of P21 and P16 in granulosa cells also increased after RLIM overexpression, while P16 only showed an increasing trend without statistical difference (Figure 5C). β-gal staining results showed that the number of β-gal-positive cells in the RLIM stable transgenic line was significantly increased, and cellular senescence was obvious ( Figure 5 D).

[0057] Example 4: Evaluation of the therapeutic effect of in vitro downregulation of RLIM I. Experimental Objective To verify the effect of downregulating RLIM in vitro on improving granulocyte senescence.

[0058] II. Experimental Methods: 1. siRNA transfection (1) After the customized siRNA powder was centrifuged at 5 nmol, it was dissolved in 250 μL of DEPC water in a biosafety cabinet to prepare a 20 μm stock solution. After mixing, it was aliquoted and stored at -20℃. A total of 3 siRNAs were customized, and the sequences are as follows. Table 6

[0059] (2) Seed cells in 6-well plates, with cell density controlled at 60-70%, and transfected after cell adhesion; (3) After preparing the system (one well of a 6-well plate), mix thoroughly by blowing and let stand for 5 minutes: Table 7

[0060] Prepare each siRNA according to the system (1 well of a 6-well plate) and mix thoroughly by pipetting. The final siRNA intervention concentration is 50 nm. Table 8

[0061] 5) Add the liquid prepared in step 3 to step 4, mix well by blowing, and let stand for 15 minutes; 6) Aspirate the culture medium from the well plate and add 1750 μL of serum-free culture medium to each well; 7) Add 250 μL of the mixture of 3 and 4 to each well, and add it vertically to each well and mix well. 8) After transfection in an incubator for 6 hours, aspirate the culture medium and add 2 ml of whole serum culture medium; 9) Cells were harvested 24 hours after siRLIM transfection for protein extraction, immunofluorescence and other experiments.

[0062] 2. Cell immunofluorescence 1) Fixation: After aspirating the culture medium from the well plate, wash twice with PBS, add an appropriate amount of 4% paraformaldehyde and fix at room temperature for 20 minutes; 2) Membrane disruption: Remove paraformaldehyde, wash twice with PBS, add 0.1% Triton-X100, and disrupt the membrane at room temperature for 20 minutes; 3) Non-specific site blocking: Remove Triton-X100, wash twice with PBS, add an appropriate amount of 10% goat serum, and incubate at 37°C for 1 hour; 4) Primary antibody incubation: Aspirate goat serum, add an appropriate amount of primary antibody, and incubate overnight at 4°C; 5) The next day, remove the plate and allow it to warm for 1 hour, then wash twice with PBS. 6) Secondary antibody incubation: Add an appropriate amount of fluorescent secondary antibody and incubate at 37℃ in a biochemical incubator for 1 hour; 7) Wash twice with PBS; 8) Staining the nucleus: Stain with DAPI solution at room temperature for 10 minutes; 9) After washing twice with PBS, observe and photograph using an inverted fluorescence microscope.

[0063] 3. EdU staining for cell proliferation (24-well plate) 1) All solutions must be equilibrated to room temperature before use; 2) First, dilute the 10 mm EdU solution with culture medium to prepare a 2x EdU (20 μm) working solution; 3) Then add an equal volume of preheated (37℃) 2x EdU (20μm) working solution to the 24-well plate to make the final concentration 1x EdU (10μm). 4) Incubate at 37℃ for 2 hours; 5) After incubation, remove the culture medium, add 3.7% paraformaldehyde in PBS for fixation, and incubate at room temperature for 15 minutes; 6) Discard the fixative and wash 3 times with BSA Wash Solution (1x, diluted 5x with PBS to 1x); 7) After removing the washing solution, add 0.5% Triton X-100 to break the membrane and incubate at room temperature for 15 minutes; 8) Wash 3 more times with BSA Wash Solution; 9) Prepare the reaction mixture: Table 9

[0064] 10) Add an appropriate amount of reaction mixture to each well and incubate at room temperature in the dark for 30 minutes; 11) Remove the reaction mixture and wash three times with BSA Wash Solution; 12) Stain with DAPI and incubate at room temperature for 10 minutes; 13) After washing three times with BSA Wash Solution, observe and photograph under an inverted fluorescence microscope, and count the number of red fluorescent cells.

[0065] III. Experimental Results Granulocyte senescence after RLIM expression downregulation In the granulosa cell line COV434, RLIM expression was downregulated using siRNA. After determining the knockdown efficiency of RLIM, changes in cellular senescence markers were examined. Western blotting results showed that all three siRLIM sequences had significant downregulation effects. Furthermore, after RLIM downregulation, the expression of cellular senescence markers P21, P16, and HP1γ, as well as the DNA damage marker γH2AX, was significantly reduced. Figure 6 A). Immunofluorescence and EdU cell proliferation assays showed that downregulation of RLIM reduced DNA damage in granulocytes. Figure 6 B), increased cell proliferation ( Figure 6 C).

[0066] Example 5: Effects of RLIM downregulation on mouse ovarian function Based on the downregulation effect of siRLIM on the granulosa cell line COV434, siRLIM-2 was selected for in vivo experiments. Since siRNA is easily degraded in vivo, we encapsulated siRLIM-2 in nanoliposomes, injected it via the tail vein, and then examined ovarian function in mice. The control group received empty liposomes, and the intervention lasted for 4 weeks.

[0067] Protein and mRNA levels in mouse ovaries demonstrate the in vivo downregulation effect of nanoliposome siRLIM-2. Figure 7 Compared with the siNC group, the siRLIM-2 group had fewer atretic follicles and higher serum AMH and E2 levels (A, B). Figure 7 CE).

[0068] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0069] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0070] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of E3 ubiquitin ligase RLIM gene inhibitors in the preparation of drugs for treating ovarian aging or damage.

2. The application according to claim 1, characterized in that, The E3 ubiquitin ligase RLIM gene inhibitors include siRLIM-1, siRLIM-2, and siRLIM-3, with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

3. A pharmaceutical composition, characterized in that, It contains an E3 ubiquitin ligase RLIM gene inhibitor.

4. The pharmaceutical composition according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The excipients are selected from one of the following: fillers, disintegrants, binders, lubricants, sweeteners, or colorants.

6. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections, and dispersants.

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