RNA (Ribonucleic Acid) molecule, nano-liposome and application of RNA molecule in preparation of product for treating testis injury and / or testis aging

Abstract

The invention discloses an RNA (Ribonucleic Acid) molecule, a nano-liposome and application of the RNA molecule to preparation of a product for treating testis injury and / or testis aging. The nucleotide sequence of the RNA molecule is as shown in SEQ ID NO. 1. The RNA molecule and the nano-liposome loaded with the RNA molecule provided by the invention show an excellent anti-inflammatory function and a remarkable targeting property in vivo, and most of the RNA molecule and the nano-liposome loaded with the RNA molecule can be taken by testis macrophages, so that the treatment effect is effectively improved, the repair and regeneration of testis tissues are promoted, the spermatogenic ability of testis is remarkably improved, the normal reproductive endocrine environment is recovered, and the curative effect is good. The testosterone level is improved. The invention provides a brand new technical strategy for treating testis injury, improving testis aging and improving male fertility, and is expected to play an important role in future clinical application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to RNA molecules, nanoliposomes, and their use in the preparation of products for treating testicular injury and / or testicular aging. Background Technology

[0002] Testicular torsion is a common urological emergency, most frequently seen in adolescents. Its typical symptom is testicular torsion along the spermatic cord axis, leading to obstructed blood supply. Ischemia-reperfusion injury triggers a severe inflammatory response, and without timely treatment, it can result in testicular tissue necrosis and permanent damage. During ischemia-reperfusion, oxidative stress occurs, producing a large number of reactive oxygen species that damage testicular cells and affect endocrine function. Testosterone synthesis and secretion may be inhibited, leading to impaired spermatogenesis. Furthermore, with age, testicular aging is an inevitable physiological phenomenon, manifested as a decrease in the number of germ cells and declining spermatogenesis, hypothalamic-pituitary-testicular axis dysfunction, and decreased testosterone levels. Both of these conditions severely impact male reproductive function and quality of life. Currently, clinical treatment for testicular torsion primarily involves surgical repositioning to restore blood supply, but effective treatments are lacking for existing testicular damage and subsequent spermatogenic dysfunction. For testicular aging, testosterone replacement therapy is a common approach, but it carries risks such as cardiovascular disease and stimulation of prostate hyperplasia.

[0003] The development of molecular biology has brought new hope for the treatment of testicular diseases and the improvement of male reproductive function. Nanoliposomes, as a novel drug delivery system, have shown great clinical translational potential in the biomedical field. They possess excellent biocompatibility and biodegradability, protecting encapsulated drugs from enzymatic degradation and other factors in vivo. Their small size allows them to effectively penetrate biological barriers, achieving targeted drug delivery. Nanoliposomes can also serve as effective carriers for gene therapy agents, making them one of the most promising gene therapy methods. Furthermore, the surface of nanoliposomes can be functionalized to enhance their interaction with specific cell types, improving drug targeting and efficacy. The development of nanomaterials holds promise for overcoming the limitations of traditional treatment methods, providing new ideas and strategies for the treatment of testicular diseases. Summary of the Invention

[0004] To address the shortcomings of existing traditional treatments in addressing male reproductive dysfunction caused by testicular torsion and testicular aging, this invention provides innovative methods based on molecular therapy and nanoliposome materials to more effectively improve male reproductive function. The invention also provides RNA molecules, nanoliposomes, and their applications in the preparation of products for treating testicular injury and / or testicular aging.

[0005] The first objective of this invention is to provide an RNA molecule.

[0006] A second objective of this invention is to provide the use of the said RNA molecule in the preparation of products for treating testicular injury.

[0007] A third objective of this invention is to provide the use of the said RNA molecule in the preparation of products that improve testicular aging.

[0008] A fourth object of the present invention is to provide the use of the said RNA molecule in the preparation of products that enhance male fertility.

[0009] A fifth object of the present invention is to provide the use of the said RNA molecule in the preparation of products that enhance the spermatogenic capacity, testosterone synthesis capacity and / or testosterone secretion capacity of testicular tissue.

[0010] The sixth objective of this invention is to provide a nanoliposome.

[0011] A seventh object of the present invention is to provide a method for preparing the nanoliposomes.

[0012] An eighth object of the present invention is to provide the use of the nanoliposomes in the preparation of products for treating testicular injury.

[0013] A ninth object of the present invention is to provide the use of the nanoliposomes in the preparation of products that improve testicular aging.

[0014] The tenth object of the present invention is to provide the use of the nanoliposomes in the preparation of products that enhance male fertility.

[0015] To achieve the above objectives, the present invention is implemented through the following solution: An RNA molecule whose nucleotide sequence is shown in SEQ ID NO.1.

[0016] The application of the RNA molecule in the preparation of products for treating testicular injury.

[0017] Preferably, the testicular injury includes testicular torsion.

[0018] Preferably, the testicular injury includes testicular ischemia-reperfusion.

[0019] The application of the RNA molecule in the preparation of products that improve testicular aging.

[0020] The application of the RNA molecule in the preparation of products that enhance male fertility.

[0021] The application of the RNA molecule in the preparation of products that enhance the spermatogenic capacity, testosterone synthesis capacity, and / or testosterone secretion capacity of testicular tissue.

[0022] A nanoliposome containing the aforementioned RNA molecule, namely the RNA-NPs of this invention.

[0023] A method for preparing the nanoliposomes includes the following steps: encapsulating the RNA molecules into the nanoliposomes by a double emulsion solvent evaporation method.

[0024] Preferably, the method includes the following steps: adding an aqueous solution of the RNA molecule, DOTAP, and PEG. 5k -b-PLGA 11k After mixing with chloroform, emulsify thoroughly to remove the chloroform, and you will get the product.

[0025] More preferably, the method includes the following steps: mixing the aqueous solution of the RNA molecule, the DOTAP, and the PEG. 5k -b-PLGA 11k The trichloromethane is subjected to an ice bath and ultrasonic treatment to fully emulsify it. The trichloromethane is then removed by rotary evaporation to obtain the final product.

[0026] More preferably, the method includes the following steps: mixing the aqueous solution of the RNA molecule, the DOTAP, and the PEG. 5k -b-PLGA 11k The chloroform is emulsified by ultrasonication in an ice bath, and then water is added and emulsified again by ultrasonication in an ice bath to form a water-in-oil-in-water emulsion; the chloroform in the emulsion is removed by rotary evaporation to obtain the final product.

[0027] More preferably, the method includes the following steps: mixing the aqueous solution of the RNA molecule, the DOTAP, and the PEG. 5k -b-PLGA 11k The chloroform is emulsified by ultrasonication in an ice bath, and then water is added and emulsified again by ultrasonication in an ice bath to form a water-in-oil-in-water emulsion; the chloroform in the emulsion is removed by rotary evaporation, and then freeze-dried to obtain the final product.

[0028] More preferably, the conditions for ultrasound include: power of 60W to 100W and time of 0.5min to 5min.

[0029] More preferably, the conditions for the ultrasound include: power of 80W and duration of 1min.

[0030] More preferably, the RNA molecule, the DOTAP, and the PEG 5k -b-PLGA 11k The mass-to-volume ratio of the chloroform is (0.1-1) mg: (0.2-20) mg: (5-500) mg: (0.5-5) mL.

[0031] More preferably, the RNA molecule, the DOTAP, and the PEG 5k -b-PLGA 11k The mass-to-volume ratio of the trichloromethane is 0.4 mg: 2.0 mg: 50 mg: 1 mL.

[0032] More preferably, the PEG 5k -b-PLGA 11k It was prepared by ring-opening polymerization.

[0033] More preferably, the PEG 5k -b-PLGA 11k The preparation method includes the following steps: polyethylene glycol 5000, D,L-lactide, glycolide and stannous isooctanoate are fully polymerized in nitrogen, and the resulting reaction product is first dissolved in chloroform and then precipitated in diethyl ether to obtain the product.

[0034] More preferably, the PEG 5k -b-PLGA 11k The preparation method includes the following steps: polyethylene glycol 5000, D,L-lactide, glycolide and stannous isooctanoate are fully polymerized in nitrogen. The resulting reaction product is purified under vacuum, dissolved in chloroform and then precipitated in diethyl ether. After further purification under vacuum, the product is obtained.

[0035] More preferably, the mass ratio of the polyethylene glycol 5000, the D,L-lactide, the glycolide and the stannous isooctanoate is (1-10) g: (1-10) g: (1-10) g: (1-50) g.

[0036] More preferably, the mass ratio of polyethylene glycol 5000, D,L-lactide, glycolide, and stannous isooctanoate is 3.09g:4.45g:3.58g:12.5g.

[0037] More preferably, the polymerization reaction conditions include heating at 100℃~200℃ for 1h~10h.

[0038] More preferably, the conditions for the polymerization reaction include heating at 150°C for 6 hours.

[0039] The application of the nanoliposomes in the preparation of products for treating testicular injury.

[0040] Preferably, the testicular injury includes testicular torsion.

[0041] Preferably, the testicular injury includes testicular ischemia-reperfusion.

[0042] Application of the nanoliposomes in the preparation of products that improve testicular aging.

[0043] The application of the nanoliposomes in the preparation of products that enhance male reproductive capacity.

[0044] The nanoliposomes are used in the preparation of products that enhance the spermatogenic capacity, testosterone synthesis capacity, and / or testosterone secretion capacity of testicular tissue.

[0045] Compared with the prior art, the present invention has the following beneficial effects: The RNA molecules and RNA-loaded nanoliposomes provided by this invention exhibit excellent anti-inflammatory functions and significant targeting in vivo. Most of them can be taken up by testicular macrophages, effectively improving therapeutic effects, promoting testicular tissue repair and regeneration, significantly improving testicular spermatogenesis, restoring a normal reproductive endocrine environment, and increasing testosterone levels. This provides a novel technical strategy for treating testicular injury, improving testicular aging, and enhancing male fertility, and is expected to play an important role in future clinical applications. Attached Figure Description

[0046] Figure 1 The preparation and characterization of RNA-loaded nanoliposomes in Example 1 are shown in Figure 1. A is a schematic diagram of the preparation method; B is a transmission electron microscopy (TEM) image of RNA-NPs; and C is the particle size distribution of RNA-NPs.

[0047] Figure 2 A represents the in vitro anti-inflammatory function of the RNA-loaded nanoliposomes in Example 1; B represents the efficiency of Cy5-RNA-NPs uptake by macrophages in vitro as detected by flow cytometry; C represents the expression levels of NLRP3, ASC, pro-casp1, pro-IL1β, GAPDH, cl-Casp1, and cl-IL 1β in macrophages treated with RNA-NPs as detected by Western blotting; and D represents the grayscale analysis results of B.

[0048] Figure 3 This is a schematic diagram of the process for treating the testicular torsion model in Example 2.

[0049] Figure 4 Gross images of testicular tissue from mice in the Sham, PBS, NC-NPs, and RNA-NPs groups 28 days after injection in Example 2.

[0050] Figure 5The following is an in vivo tracing of the RNA-loaded nanoliposomes 28 days after injection in Example 2: A is a tracing image of Cy5-RNA-NPs in testicular tissue; B is the result of counting and statistically analyzing the Cy5-positive cell rate in macrophages, neutrophils, spermatogenic cells, Sertoli cells, myoid cells, and interstitial cells of testicular tissue. Figure 6 The table shows the inflammatory signaling pathways in the testicular tissues of mice in the Sham, PBS, NC-NPs, and RNA-NPs groups after injection for 1, 3, and 7 days in Example 2. A shows the expression levels of NLRP3, ASC, pro-casp1, pro-IL1β, GAPDH, cl-Casp1, and cl-IL 1β in the testicular tissue treated with RNA-NPs, detected by Western Blot. B shows the grayscale analysis results of NLRP3 expression in A.

[0051] Figure 7 The results of sperm analysis, histological analysis, and testosterone measurement in mice of the Sham group, PBS group, NC-NPs group, and RNA-NPs group in Example 2 are shown below. A shows the sperm concentration of each group of mice 28 days after injection; B shows the sperm motility of each group of mice 28 days after injection; C shows the serum testosterone level of each group of mice 28 days after injection; D shows HE staining images of testicular tissue of each group of mice 1, 3, 7, and 28 days after injection; E shows the statistical results of the Johnsen score based on D, with a maximum score of 10 points and a minimum score of 1 point, N=6. Statistical data are expressed as mean ± standard error, ns represents no statistical difference, * P<0.05, **P<0.01, ***P<0.001.

[0052] Figure 8 The results of the co-cage experiment of mice in the Sham group, PBS group, NC-NPs group and RNA-NPs group in Example 2 are shown in Figure A; A is a photo of newborn mice in each group at the end of the co-cage experiment; B is the statistical result of the number of newborn mice in each group at the end of the co-cage experiment.

[0053] Figure 9 The results of sperm analysis, histological analysis, and testosterone measurement of mice in the PBS group, NC-NPs group, and RNA-NPs group after 1 month of injection in Example 3 are shown. A represents the sperm concentration of each group of mice; B represents the sperm motility of each group of mice; C represents the serum testosterone level of each group of mice; ns represents no statistical difference, ***P<0.001.

[0054] Figure 10The histological evaluation results of mice in the PBS group, NC-NPs group and RNA-NPs group after 1 month of injection in Example 3 are shown in Figure 3. A shows the HE staining and immunofluorescence staining images of testicular tissue of mice in each group 28 days after treatment. B shows the statistical results of the number of PNA positive cells in A. C shows the statistical results of the number of CYP11A1 positive cells in A. ns represents no statistical difference, ***P<0.001.

[0055] Figure 11 The results of the co-cage experiment of mice in the PBS group, NC-NPs group and RNA-NPs group after 1 month of injection in Example 3 are shown in Figure A. A photo of newborn mice in each group at the end of the co-cage experiment is shown in Figure B. The number of newborn mice in each group at the end of the co-cage experiment is shown in Figure B. ns represents no statistical difference, ***P<0.001. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified. In the following embodiments, all experimental data are expressed as mean ± standard error (SEM). Appropriate statistical methods, such as t-tests or analysis of variance (ANOVA), are used to assess differences between groups; a p-value less than 0.05 is considered statistically significant.

[0057] Example 1: Preparation, characterization, cellular uptake, and in vitro functional identification of RNA-loaded nanoliposomes 1. Synthesis of RNA molecules An RNA molecule with the nucleotide sequence 5'-GGATGGCTTTGATGAGCTA-3' (SEQ ID NO.1) was synthesized by Ribo Biotech, and a Cy5-labeled RNA molecule (SEQ ID NO.1) was also synthesized.

[0058] 2. Preparation of RNA-loaded nanoliposomes (1) PEG 5k -b-PLGA 11k Preparation Diblock copolymer PEG was synthesized using ring-opening polymerization. 5k -b-PLGA 11k The specific steps are as follows: Add 3.09g of polyethylene glycol 5000 (PEG) 5kAdd the monomers to a three-necked flask and dry under vacuum for 4 hours. Then add 4.45 g of D,L-lactide and 3.58 g of glycolide, and continue drying for another 4 hours. Then add 12.5 g of stannous isooctanoate, and heat to 150 °C under nitrogen protection for 6 hours to obtain the crude product. After the reaction is complete, place the flask containing the crude product under vacuum at 150 °C for 30 minutes to remove unreacted monomers, obtaining the copolymer. Then, dissolve the obtained copolymer in chloroform and precipitate it in 100 mL of diethyl ether. Finally, remove the diethyl ether by vacuum drying to obtain the final product PEG. 5K -b-PLGA 11k .

[0059] (2) Encapsulation of RNA molecules This embodiment uses a dual-emulsion solvent evaporation technique to prepare RNA-loaded liposomes, as follows: Figure 1 As shown in A, the specific steps are as follows: First, 0.4 mg of RNA molecules (SEQ ID NO.1) was dissolved in 50 μL of nuclease-free water to obtain an RNA solution; then 2.0 mg of cationic liposome DOTAP (CAS No.: 132172-61-3) and 50 mg of PEG were added. 5k -b-PLGA 11k 1 mL of chloroform and 50 μL of RNA solution were ultrasonically emulsified at 80 W for 1 min in an ice bath. Then, 10 mL of nuclease-free water was added, and the mixture was ultrasonically emulsified again at 80 W for 1 min in an ice bath to form a water-in-oil-in-water emulsion. The emulsion was transferred to a 50 mL round-bottom flask, and the organic solvent chloroform was removed under reduced pressure using a rotary evaporator. The nuclease-free water was concentrated to a volume of 1 mL to obtain a liquid formulation of RNA-loaded liposomes (denoted as RNA-NPs). For further purification, the liquid formulation of RNA-NPs was freeze-dried to obtain a lyophilized formulation of RNA-NPs. The liquid formulation of RNA-NPs was white, and the lyophilized formulation was a white powder.

[0060] To facilitate observation of macrophage uptake of nanoliposomes, the same method was used, with the only difference being that 0.4 mg of Cy5-labeled RNA molecules (SEQ ID NO.1) were dissolved in 50 μL of nuclease-free water to obtain a Cy5-RNA solution, which was then used with Cy5-RNA solution, DOTAP, and PEG. 5k -b-PLGA 11kLiquid and lyophilized formulations of nanoliposomes (denoted as Cy5-RNA-NPs) loaded with Cy5-labeled RNA molecules (SEQ ID NO.1) were prepared using chloroform. Cy5 is a red fluorescent dye, and Cy5-RNA-NPs emit red fluorescence, which facilitates subsequent detection of the uptake efficiency of nanoliposomes in testicular macrophages using fluorescence microscopy and flow cytometry.

[0061] The same method was used, the only difference being: nuclease-free water, DOTAP, and PEG were used directly. 5k -b-PLGA 11k Liquid and lyophilized formulations of unloaded RNA liposomes (NC-NPs) were prepared using chloroform.

[0062] 3. Characterization of RNA-loaded nanoliposomes (1) Morphology of nanoliposomes The lyophilized RNA-NPs were dispersed in water to form a 0.5 mg / mL aqueous solution. 5 μL of this solution was pipetted onto a copper grid and allowed to air dry for 5 hours. The morphology of the RNA-NPs was observed using a field emission transmission electron microscope (Talos F200X). Figure 1 As shown in B, RNA-NPs are cystic in shape. Cy5-RNA-NPs and NC-NPs, as identified by TEM, exhibit the same morphology as RNA-NPs.

[0063] (2) Particle size determination of nanoliposomes The lyophilized formulation of RNA-NPs was dispersed in water, and the particle size was measured at room temperature (25 °C) using a dynamic light scattering instrument (Zetasizer Nano ZS90). NPs with particle sizes ranging from 0.3 nm to 5.0 μm could be detected within minutes. Generally, a 1.5 ml sample is sufficient for particle size testing, and the sample can be recovered for other tests. Particle size was measured using NIBS technology. Figure 1 As shown in C, most RNA-NPs are uniform in size, with an average particle size of 142 ± 2.3 nm. Cy5-RNA-NPs and NC-NPs were identified as having similar particle size and distribution to RNA-NPs.

[0064] 4. Cellular uptake efficiency of nanoliposomes Mouse bone marrow-derived macrophages (BMDM) were used at 10 5 Cells / well were seeded in 24-well cell culture plates.

[0065] The lyophilized formulation of Cy5-RNA-NPs was added to the culture medium of macrophages at a working concentration of 20 mM, and the macrophages were co-cultured with Cy5-RNA-NPs. This group of cells was designated as the RNA-NPs group. The lyophilized formulation of NC-NPs was added to the culture medium of macrophages at a working concentration of 20 mM, and the macrophages were co-cultured with NC-NPs. This group of cells was designated as the NC-NPs group. 10 μL of PBS was added to the culture medium of macrophages. This group of cells was designated as the PBS group. Macrophages without any external treatment were used as a control group. The cells were cultured for another 2 hours.

[0066] After culture, the four groups of cells were washed with PBS, and the expression of Cy5 in the cells was determined by flow cytometry.

[0067] like Figure 2 As shown in A, flow cytometry analysis revealed that, compared to RNA molecules alone, RNA loaded onto nanoliposomes, including both NC-NPs and RNA-NPs, could be efficiently absorbed by macrophages.

[0068] 4. The inhibitory effect of RNA-loaded nanoliposomes on inflammatory responses After culture, protein samples were collected from four groups of cells. Western blotting was used to detect the expression levels of NLRP3 inflammatory pathway-related proteins in macrophages of each group. The antibodies used were as follows: NLRP3 antibody (manufacturer: CST, catalog number: 15101), ASC antibody (manufacturer: CST, catalog number: 67824), pro-casp1 antibody (manufacturer: CST, catalog number: 24232), pro-IL1β antibody (manufacturer: CST, catalog number: 31202), GAPDH antibody (manufacturer: Proteintech, catalog number: 60004), cl-Casp1 antibody (manufacturer: CST, catalog number: 89332), and cl-IL1β antibody (manufacturer: CST, catalog number: 63124).

[0069] like Figure 2 As shown in B and C, compared with the Control group, the expression levels of NLRP3, ASC, pro-casp1, pro-IL1β, cl-Casp1, and cl-IL1β in macrophages of the PBS and NC-NPs groups were significantly increased, while the expression levels of NLRP3, ASC, pro-casp1, pro-IL1β, cl-Casp1, and cl-IL1β in macrophages of the RNA-NPs group were significantly lower than those in the PBS and NC-NPs groups. This indicates that RNA-loaded nanoliposomes have significant anti-inflammatory functions in vitro.

[0070] Example 2: Improvement of reproductive dysfunction caused by testicular torsion by RNA-loaded nanoliposomes 1. Construction of the testicular torsion model Using 12-week-old male C57BL / 6J mice as experimental subjects, a midline incision was made in the scrotum, the right testis was removed, and the left testis was rotated 720 degrees counterclockwise. The testis was fixed with silk sutures for 1 hour and covered with warm, moist gauze. After 1 hour, the torsioned testis was repositioned. Before suturing the incision, the color change of the testis was observed to confirm blood return, thus establishing the testicular torsion model.

[0071] Twelve-week-old male C57BL / 6J mice were used as experimental subjects. A midline incision was made in the scrotum, and the right testis was removed to obtain a sham surgical model, which was designated as the Sham group.

[0072] After the model was constructed, the vital signs of the mice were observed.

[0073] 2. Treatment of testicular torsion model like Figure 3 As shown, after modeling, 10 μL of PBS was injected locally into the left testis of the testicular torsion model, designated as the PBS group; 10 μL of the liquid formulation of NC-NPs prepared in Example 1 was injected locally into the left testis of the testicular torsion model, designated as the NC-NPs group; 10 μL of the liquid formulation of Cy5-RNA-NPs prepared in Example 1 was injected locally into the left testis of the testicular torsion model, designated as the RNA-NPs group; mice in the Sham group did not receive any injection treatment. Each of the above four groups consisted of 24 mice. Testicular tissue was collected from 6 mice in each group at 1, 3, 7, and 28 days after injection for subsequent analysis.

[0074] 3. Evaluation of therapeutic effect (1) General observation 28 days after injection, the testicular tissue of the four groups of mice was as follows: Figure 4 As shown, compared to the Sham group, the PBS group and NC-NPs group mice showed significant testicular atrophy, while the testicular size of the RNA-NPs group mice was basically the same as that of the Sham group. This indicates that RNA-NPs significantly improved the gross morphology of the testes in male mice after testicular torsion injury and restored testicular volume.

[0075] (2) Cellular uptake of RNA-loaded nanoliposomes Twenty-eight days after injection, the expression of Cy5 fluorescence in macrophages, neutrophils, spermatogenic cells, Sertoli cells, myoid cells, and interstitial cells of four groups of mice was detected by immunofluorescence, and the Cy5-positive cell rate was calculated. The antibodies used were as follows: macrophage marker F4 / 80 antibody (manufacturer: CST, catalog number: 30325), neutrophil marker Ly6G antibody (manufacturer: CST, catalog number: 88876), spermatogenic cell DDX4 marker antibody (manufacturer: Abcam, catalog number: ab13840), Sertoli cell marker SOX9 antibody (manufacturer: Abcam, catalog number: ab185966), myoid cell marker αSMA antibody (manufacturer: Abcam, catalog number: ab5694), and interstitial cell marker P450scc antibody (manufacturer: Abcam, catalog number: ab272494).

[0076] like Figure 5 As shown in A and B, more than 95% of Cy5-RNA-NPs were taken up by testicular macrophages, indicating that the RNA-loaded nanoliposomes prepared in this invention have high targeting ability, specifically targeting testicular macrophages and being efficiently taken up by them.

[0077] (3) Status of inflammatory signaling pathways At 1, 3 and 7 days after injection, the expression levels of NLRP3 inflammatory pathway-related proteins in the testicular tissues of the four groups of mice were detected by Western Blot method as described in Example 1.

[0078] like Figure 6 As shown in Figures A and B, at days 1, 3, and 7 after injection, compared with the Sham group, the expression levels of NLRP3, ASC, pro-casp1, pro-IL1β, cl-Casp1, and cl-IL1β in the testicular tissue of mice in the PBS and NC-NPs groups were significantly increased. Conversely, the expression levels of NLRP3, ASC, pro-casp1, pro-IL1β, cl-Casp1, and cl-IL1β in the testicular tissue of mice in the RNA-NPs group were significantly lower than those in the PBS and NC-NPs groups, approaching those in the Sham group. This indicates that RNA-NPs can effectively inhibit the activation of the NLRP3 inflammasome pathway after testicular injury, reduce NLRP3, effectively suppress the inflammatory response, and possess significant anti-inflammatory function.

[0079] (4) Sperm analysis Twenty-eight days after injection, sperm concentration and sperm motility in each group of mice were assessed using a computer-assisted sperm analysis system (CASA). Figure 7As shown in Figures A and B, compared with the PBS group and the NC-NPs group, the sperm count and motility of mice in the RNA-NPs group were significantly improved. This indicates that RNA-NPs significantly improved sperm concentration and motility in male mice after testicular torsion injury.

[0080] (5) Hormone level measurement Serum samples were collected from mice in each group 28 days after injection, and serum testosterone levels were measured using enzyme-linked immunosorbent assay (ELISA). Figure 7 As shown in Figure C, compared with the PBS group and the NC-NPs group, the serum testosterone level in the RNA-NPs group was significantly increased and approached that of the Sham group. This indicates that RNA-NPs restored the ability of male mice to synthesize and secrete testosterone after testicular torsion injury.

[0081] (6) Histological assessment Hematoxylin and eosin (H&E) staining of testicular tissue from mice in each group was performed at 1, 3, 7, and 28 days after injection to assess changes in testicular tissue structure. The Johnsen scoring system was used for quantitative analysis of spermatogenesis. Figure 7 As shown in Figures D and E, compared with the PBS group and the NC-NPs group, the RNA-NPs group showed a significant recovery in both the number and number of spermatogenic cells in the seminiferous tubules of the testis, and the Johnsen score was also significantly improved. This indicates that RNA-NPs improved testicular spermatogenesis in male mice after testicular torsion injury.

[0082] (7) Fertility assessment Following the above procedures, another batch of mice was used for modeling and treatment, resulting in the Sham group, PBS group, NC-NPs group, and RNA-NPs group, with 12 mice in each group. Twenty-eight days after injection, the fertility of each group was assessed using a co-breeding experiment. The four groups of mice were co-breeded with age-matched female mice (female-to-male ratio of 2:1) for two months, and the number of newborn mice was recorded and counted.

[0083] like Figure 8 As shown in Figures A and B, compared with the PBS and NC-NPs groups, the number of newborn mice in the RNA-NPs group after being co-occupied with female mice was significantly increased, approaching the number of newborn mice in the Sham group after being co-occupied with female mice. This indicates that RNA-NPs restored the fertility of male mice after testicular torsion injury.

[0084] Example 3: Improvement of Reproductive Dysfunction Due to Testicular Aging by RNA-Loaded Nanoliposomes 1. Construction of a testicular aging model A 22-month-old (equivalent to about 65 years old in humans) naturally aged C57BL / 6J male mice was used as a model of testicular aging.

[0085] 2. Treatment of a testicular aging model 10 μL of PBS was injected into each testis of mice in a testicular aging model group (PBS group); 10 μL of the liquid formulation of NC-NPs prepared in Example 1 was injected into each testis of mice in a testicular aging model group (NC-NPs group); and 10 μL of the liquid formulation of RNA-NPs prepared in Example 1 was injected into each testis of mice in a testicular aging model group (RNA-NPs group). Each of the three groups consisted of 6 mice. Testicular tissue was collected from each group one month after injection for analysis.

[0086] 3. Evaluation of therapeutic effect (1) Sperm analysis Sperm concentration and motility in each group of mice were assessed using a computer-assisted sperm analysis system (CASA). Figure 9 As shown in Figures A and B, one month after injection, compared with the PBS group and the NC-NPs group, the sperm count and motility of mice in the RNA-NPs group were significantly increased. This indicates that RNA-NPs significantly improved sperm concentration and motility in male mice under testicular aging conditions.

[0087] 4. Hormone level measurement Serum samples were collected from each group of mice, and serum testosterone levels were measured using an enzyme-linked immunosorbent assay (ELISA). Figure 9 As shown in Figure C, one month after injection, the serum testosterone level in the RNA-NPs group was significantly higher than that in the PBS group and the NC-NPs group. This indicates that RNA-NPs restored the ability of testicular tissue in male mice to synthesize and secrete testosterone in an aging state.

[0088] 5. Histological assessment Hematoxylin and eosin (H&E) staining was performed on the testicular tissue of mice in each group to assess changes in testicular tissue structure. Immunofluorescence staining was also performed on spermatogenic cells (DDX4 antibody (Abcam, catalog number: ab13840)), spermatids (PNA antibody (Vector LABORATORIES, catalog number: FL-1071)), and Leydig cells (CYP11A1 antibody (Abcam, catalog number: ab272494)) in the testicular tissue of mice in each group, and the number of the three cell types was counted.

[0089] like Figure 10As shown in Figures A through C, one month after injection, compared with the PBS group and the NC-NPs group, the RNA-NPs group showed a significant recovery in both the number and number of spermatogenic cells in the seminiferous tubules of the testis, and a significant increase in the number of DDX4-positive spermatogenic cells, PNA-positive sperm cells, and CYP11A1-positive Leydig cells. This indicates that RNA-NPs increased the number of spermatogenic cells, sperm cells, and Leydig cells in the testes of male mice under aging conditions, thus improving spermatogenic function.

[0090] 6. Fertility assessment Following the above procedures, another batch of 22-month-old naturally aging C57BL / 6J male mice were treated, resulting in PBS, NC-NPs, and RNA-NPs groups, with 12 mice in each group. One month after injection, the fertility of each group was assessed by a co-breeding experiment. Mice in each group were co-breeded with age-matched female mice (female-to-male ratio of 2:1) for two months, and the number of newborn mice was recorded and counted.

[0091] like Figure 11 As shown in Figures A and B, compared with the PBS group and the NC-NPs group, the RNA-NPs group showed a significant increase in the number of newborn mice after being co-occupied with female mice. This indicates that RNA-NPs restored the fertility of male mice in a state of testicular aging.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An RNA molecule, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The use of the RNA molecule of claim 1 in the preparation of a product for treating testicular injury.

3. The use of the RNA molecule of claim 1 in the preparation of products that improve testicular aging.

4. The use of the RNA molecule of claim 1 in the preparation of products that enhance male fertility.

5. The use of the RNA molecule of claim 1 in the preparation of products that enhance the spermatogenic capacity, testosterone synthesis capacity, and / or testosterone secretion capacity of testicular tissue.

6. A nanoliposome, characterized in that, Contains the RNA molecule as described in claim 1.

7. A method for preparing the nanoliposomes of claim 6, characterized in that, Includes the following steps: The RNA molecule described in claim 1 is encapsulated into nanoliposomes using a double emulsion solvent evaporation method.

8. The use of the nanoliposomes of claim 6 in the preparation of products for treating testicular injury.

9. The use of the nanoliposomes of claim 6 in the preparation of products that improve testicular aging.

10. The use of the nanoliposomes of claim 6 in the preparation of products that enhance male reproductive capacity.

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