Application of dihydroartemisinin in preparation of medicine for preventing, delaying and / or treating ovarian senescence and related diseases thereof

Abstract

The invention relates to an application of dihydroartemisinin in preparation of medicines for preventing, delaying and / or treating ovarian senescence and related diseases thereof. It is found for the first time that dihydroartemisinin can effectively prevent, delay and / or treat ovarian senescence, including ovarian fibrosis and age-related fertility decline caused by ovarian fibrosis. Dihydroartemisinin significantly improves the ovary structure and function through the multi-target synergistic effect of regulating reproductive endocrine, inhibiting senescence-related phenotypes, resisting ovarian fibrosis and the like, specifically, the oestrus cycle is recovered, the ovary weight is increased, and the ovulation number and the litter size are increased. Moreover, the invention also proves that dihydroartemisinin has the potential of preventing fertility decline in a middle-aged model, is good in safety, does not cause injury to main visceral organs and reproductive organs, has normal liver and kidney function indexes, and provides a new strategy for clinical intervention of ovarian senescence.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology. Specifically, this invention relates to the use of dihydroartemisinin in the preparation of medicaments for the prevention, delay, and / or treatment of ovarian aging and related diseases. Background Technology

[0002] As the first organ in the female reproductive system to experience functional decline, the ovary's physiological aging process typically accelerates significantly after age 30, manifesting as a decrease in the number and quality of oocytes, leading to an irreversible decline in female fertility with age. With the global trend of women delaying childbearing age, age-related infertility has become a major clinical challenge in reproductive medicine. The molecular mechanisms of ovarian aging are extremely complex, involving the interaction of multiple signaling pathways, including telomere depletion, oxidative stress imbalance, accumulation of advanced glycation end products (AGEs), DNA damage accumulation, chronic low-grade inflammation, and mitochondrial dysfunction. However, current clinical practice lacks rigorously validated and safe interventions that can effectively delay or even reverse ovarian function decline.

[0003] Notably, recent studies have revealed that ovarian aging is often accompanied by tissue fibrosis. Significantly increased collagen fiber deposition has been observed in the ovarian tissue of postmenopausal women and animal models of reproductive aging, suggesting that fibrosis may be one of the important pathological features of declining ovarian function. Fibrosis is essentially caused by the continuous activation of fibroblasts, leading to excessive deposition of extracellular matrix and abnormal proliferation of connective tissue, thereby disrupting the normal structure and function of organs. In organs such as the lungs and liver, fibrosis has been shown to cause progressive sexual dysfunction and even organ failure; however, research on fibrosis in the field of ovarian aging is still in its early stages.

[0004] Currently, the main approved antifibrotic drugs globally include pirfenidone and nintedanib. While both can slow the progression of fibrosis in some organs (such as the lungs) by inhibiting inflammatory signaling pathways, their efficacy primarily lies in delaying disease progression rather than reversing existing fibrotic lesions. Furthermore, their long-term safety and organ specificity remain significant limitations. Moreover, the mechanisms and pathological characteristics of fibrosis differ significantly across organs. For example, cardiac fibrosis mainly involves factors such as pressure load and ischemic injury, exhibiting primarily reactive fibrosis; while ovarian fibrosis is a progressive pathological change resulting from the combined effects of chronic inflammation, oxidative stress, and hormonal changes during aging. The significant differences in cell origin, signaling pathways, and microenvironment regulation among different organs make it difficult to directly apply antifibrotic strategies effective for one organ to others. To date, no clinically specific drugs for ovarian fibrosis have been developed.

[0005] Therefore, developing a drug that can effectively prevent, delay, or even reverse ovarian aging is not only an urgent need to address the increasingly serious age-related fertility problems worldwide, but also a major breakthrough to fill the current gap in anti-fibrotic treatment in the reproductive system. Summary of the Invention

[0006] To address the above problems, the purpose of this invention is to provide a drug for preventing, delaying, and / or treating ovarian aging and related diseases. The inventors have discovered that dihydroartemisinin (DHA) can effectively prevent, delay, and / or treat ovarian aging, including ovarian fibrosis and the resulting age-related decline in fertility. Furthermore, it has been demonstrated in a middle-aged model to have the potential to prevent fertility decline, with good safety profile and no damage to major organs or reproductive organs, providing a new strategy for clinical intervention in ovarian aging. Therefore, this invention provides the use of dihydroartemisinin in the preparation of a drug for preventing, delaying, and / or treating ovarian aging and related diseases.

[0007] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0008] This invention provides the use of dihydroartemisinin or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention, delay and / or treatment of ovarian aging and related diseases.

[0009] According to some embodiments of the present invention, the dihydroartemisinin or a pharmaceutically acceptable salt thereof is the sole active ingredient in the drug.

[0010] According to some embodiments of the present invention, the medicament further comprises other active ingredients for preventing, delaying and / or treating ovarian aging and related diseases.

[0011] According to some embodiments of the present invention, the ovarian aging is a natural decline in ovarian function associated with aging.

[0012] According to some embodiments of the present invention, the prevention, delay, and / or treatment of ovarian aging is selected from one or two of the following:

[0013] (1) Prevention, delay and / or treatment of ovarian fibrosis;

[0014] (2) Prevent, delay and / or treat age-related decline in female fertility.

[0015] Preferably, the prevention, delay, and / or treatment of ovarian fibrosis manifests as a reduction in collagen deposition in ovarian tissue.

[0016] Preferably, the prevention, delay, and / or treatment of age-related fertility decline in women manifests as one or more of the following:

[0017] (a) Increase ovarian weight;

[0018] (b) Increase the number of ovulations;

[0019] (c) Increase the number of live births;

[0020] (d) Increase the proportion of individuals capable of reproduction;

[0021] (e) Restore regular estrous cycles.

[0022] According to some embodiments of the present invention, the drug further comprises pharmaceutically acceptable excipients.

[0023] Preferably, the pharmaceutically acceptable excipient is selected from one or more of the following: carrier, surfactant, preservative, antioxidant, hardener, thickener, and absorption enhancer.

[0024] According to some embodiments of the present invention, the dosage form of the drug is selected from one or more of suppositories, tablets, capsules, granules, drops, and injections, preferably injections. In one specific embodiment of the present invention, the drug is a dosage form suitable for intraperitoneal injection.

[0025] The present invention has at least the following beneficial effects:

[0026] 1. This invention is the first to discover a new use of DHA in the field of reproductive aging. Specifically, this invention is the first to apply DHA to the prevention, delay and / or treatment of ovarian aging, expanding the clinical application scope of this compound. Systematic animal experiments have confirmed that DHA has multiple effects on improving ovarian structure and function, specifically: (1) restoring the regularity of the estrous cycle: significantly improving the disordered estrous cycle in aging individuals, making the cycle regularity close to that of young individuals; (2) increasing ovarian weight and reserve function: effectively reversing age-related ovarian atrophy and improving ovarian reserve capacity; (3) improving fertility: significantly increasing the number of ovulations, average litter size and the proportion of fertile individuals.

[0027] 2. This invention elucidates the molecular mechanism by which DHA improves ovarian aging through multi-target synergistic effects, mainly including the following pathways: (1) Regulating reproductive endocrine balance: increasing serum anti-Müllerian hormone and estradiol levels; decreasing serum follicle-stimulating hormone and luteinizing hormone levels; (2) Inhibiting aging-related phenotypes: downregulating the expression of aging-related secretory phenotype factors (interleukin-1β, interleukin-6), alleviating the chronic inflammatory microenvironment; reducing the expression of cellular aging markers (P53, P21), inhibiting the aging process of ovarian tissue cells; (3) Anti-ovarian fibrosis: significantly inhibiting the expression of key fibrosis factors (Col3a1, TGF-β1, α-SMA, Vimentin) at the transcriptional and protein levels; reducing collagen deposition in ovarian tissue, and improving ovarian tissue structure.

[0028] 3. This invention discovers and demonstrates the preventive potential of DHA against age-related fertility decline. In a middle-aged animal model, DHA intervention effectively prevented estrous cycle disorders, decreased ovarian reserve, and reduced fertility that occur with age. This finding reflects the preventive intervention value of DHA in delaying aging and provides experimental evidence for early clinical intervention.

[0029] 4. This invention demonstrates the good safety profile of dihydroartemisinin under experimental conditions. Within the experimental period and dosage range, DHA exhibits good safety characteristics, mainly as follows: (1) No toxicity to major organs: the morphology of the heart, liver, lungs, and kidneys is normal, and no obvious pathological changes are observed; (2) Good safety in reproductive organs: no tumor formation or abnormal proliferation is observed in the ovaries and uterus; (3) Normal liver and kidney function: the levels of serum creatinine, aspartate aminotransferase, and alanine aminotransferase are all within the normal range. Attached Figure Description

[0030] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0031] Figure 1 The experimental results show that DHA improves estrous cycle and ovarian weight in aged mice; among them, young group: young control group, old group: old control group, DHA-low dose: low-dose DHA group (30 mg / kg), DHA-high dose: high-dose DHA group (60 mg / kg); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; (A) Schematic diagram of the experimental protocol for DHA intervention in aged mice; (B) Schematic diagram of changes in estrous cycle in mice in each group after 60 days of continuous DHA intervention; (C) The proportion of interestrus and estrus in the total estrous cycle in each group of mice; (D and E) Statistical analysis of ovarian weight in each group of mice.

[0032] Figure 2The experimental results show that DHA improves fertility in aged mice; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; (A and B) Statistical analysis of the number of pups in each group of mice; (C) Statistical analysis of the proportion of fertile (successfully giving birth) female mice in each group of mice; (D and E) Statistical analysis of the number of ovulations in each group of mice.

[0033] Figure 3 The experimental results show that DHA reduces the expression of ovarian aging-related biomarkers and improves hormonal imbalances in aged mice; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; (AD) represent the detection results of anti-Müllerian hormone, follicle-stimulating hormone, luteinizing hormone, and estradiol levels in the serum of mice in each group; (E and F) represent the detection results of the levels of inflammatory factors interleukin-1β and interleukin-6 in the serum of mice in each group; (G and H) represent the relative mRNA expression levels of cellular aging biomarkers P53 and P21 in the ovarian tissue of mice in each group, as detected by real-time quantitative PCR.

[0034] Figure 4 The experimental results show that DHA improves ovarian fibrosis in aged mice; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; (AD) are the relative mRNA expression levels of fibrosis-related factors type III collagen (Col3a1), transforming growth factor-β1 (TGF-β1), vimentin, and α-smooth muscle actin (α-SMA) in the ovarian tissues of mice in each group, as detected by real-time quantitative PCR; (E) shows the collagen fiber deposition in the ovarian tissues of mice in each group, as observed by Masson staining; (F and G) show the protein expression levels of TGF-β1 and α-SMA in the ovarian tissues of mice in each group, as detected by immunohistochemical staining.

[0035] Figure 5 The experimental results show that DHA prevents age-related fertility decline in middle-aged mice; among them, young group: young control group, middle-aged group: middle-aged control group, DHA-low dose: low-dose DHA group (30 mg / kg), DHA-high dose: high-dose DHA group (60 mg / kg); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; (A and D) ovarian weight statistics of mice in each group; (B, E and F) number of pups and proportion of fertile female mice in each group; (C) schematic diagram of estrous cycle changes of mice in each group after 60 days of continuous DHA intervention; (G) proportion of interestrus and estrus in the total estrous cycle of mice in each group.

[0036] Figure 6 The results show the safety assessment of DHA intervention; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns indicates no significance; (A) Hematoxylin-eosin (HE) staining results of heart, liver, lung and kidney tissues of mice in each group after 60 days of continuous DHA intervention; (B) Comparison of morphological images of ovarian and uterine tissues of mice in each group; (CE) Detection results of the concentration levels of renal function indicators creatinine, aspartate aminotransferase and alanine aminotransferase in the serum of mice in each group. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0038] Unless otherwise specified, the methods or conditions used in the following examples were performed according to conventional methods disclosed in the art. Unless otherwise stated, all reagents or instruments used in the following examples are commercially available products.

[0039] Experimental animals: C57BL / 6J mice.

[0040] Animal housing conditions: Mice were housed in an SPF-grade animal room at a temperature of 22±2℃ and a relative humidity of 50±10%, with a 12-hour light / 12-hour dark cycle, and free access to food and water.

[0041] Statistical analysis methods: Experimental data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using GraphPadPrism software. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0042] Example 1 - Effects of DHA on estrous cycle and ovarian weight in aged mice

[0043] 1. Experimental Grouping and Procedures

[0044] To evaluate the intervention effect of DHA on ovarian aging-related functions, this embodiment selected 2-month-old (young control group) and 12-month-old (aged mouse) female mice as experimental subjects.

[0045] Aged mice were randomly divided into three groups: a low-dose DHA group (30 mg / kg), a high-dose DHA group (60 mg / kg), and a saline control group (i.e., the aged control group), with a sample size of no less than 5 mice in each group. All drugs were administered via intraperitoneal injection once daily for 60 consecutive days. The young control group received no drug intervention and was simply fed synchronously.

[0046] 2. Detection Indicators and Methods

[0047] (1) Monitoring of estrous cycles

[0048] After 60 days of continuous DHA intervention, vaginal smears were collected daily at the same time to monitor the estrous cycle changes of each mouse for a total of 14 days. During sampling, the vaginal cavity was gently rinsed with sterile saline using a pipette. The rinsing solution was placed on a glass slide, allowed to air dry, and then stained with Wright's stain (Solarbio, G1040) for cytological observation under a light microscope. The estrous cycle stages were determined based on the main cell types: proestrus was dominated by round, nucleated epithelial cells; estrus was dominated by keratinized squamous epithelial cells; diaestrus showed a mixture of epithelial cells and leukocytes; and metestrus was dominated by leukocytes with a small number of keratinized epithelial cells.

[0049] (2) Ovarian weight measurement

[0050] After 60 days of continuous DHA intervention, mice in each group were sacrificed, and ovarian tissue was rapidly collected. The surrounding adipose tissue was carefully dissected, and the wet weight of each ovary was measured using a precision analytical balance, with the results recorded to two decimal places. Simultaneously, morphological photographs were taken of the ovarian samples from each group to observe and compare their gross morphological differences.

[0051] 3. Experimental Results

[0052] (1) Mice estrous cycle

[0053] After 60 days of continuous DHA intervention, the changes in the estrous cycle of mice in each group were as follows: Figure 1 As shown in Figure B, the young control group exhibited a regular estrous cycle with orderly alternation of each phase (proestrus, estrus, metestrus, and estrus), with a cycle length of approximately 4-5 days, consistent with normal physiological characteristics of mice. The aged control group showed severely disordered estrous cycles, with the cycle fluctuations disappearing. Compared to the aged control group, the low-dose DHA group (DHA-low dose) showed a significant improvement in estrous cycles, with partial recovery of the periodic fluctuations. The high-dose DHA group (DHA-high dose) showed further improvement in the regularity of the estrous cycle, with the cycle length and phase alternation sequence highly similar to the young control group, indicating that DHA can dose-dependently restore the reproductive endocrine rhythm of aged mice.

[0054] The proportions of interestrus and estrus periods in the total estrous cycle for each group of mice are as follows: Figure 1As shown in Figure C, compared with the young control group, the proportion of interestrus and estrus was significantly increased in the aged control group. Compared with the aged control group, the proportion of interestrus and estrus was decreased in the low-dose DHA group, indicating that DHA intervention can improve the reproductive cycle of aged mice; in contrast, the proportion of interestrus and estrus was further decreased in the high-dose DHA group, showing a significant difference from the aged control group but similar to the young control group. The above statistical results indicate that DHA can significantly shorten the abnormally prolonged interestrus and estrus in aged mice, thereby restoring a regular estrous cycle.

[0055] (2) Changes in mouse ovarian weight

[0056] like Figure 1 As shown in Figure D, the ovarian weight of the elderly control group was significantly lower than that of the young control group, indicating that aging leads to ovarian tissue atrophy. Compared with the elderly control group, the ovarian weight of the low-dose DHA group significantly rebounded, and the ovarian weight of the high-dose DHA group further increased, approaching the level of the young control group.

[0057] like Figure 1 As shown in Figure E, the ovarian volume of the elderly control group was significantly reduced, with signs of atrophy visible. The ovarian volume of the low-dose DHA group and the low-dose DHA group significantly recovered, resembling that of the young control group.

[0058] In conclusion, Figure 1 The results showed that, compared with the untreated aged control group, the estrous cycle disorder in DHA-treated aged mice was significantly improved, with mice no longer continuously in the interestrus or estrus phase, and their cycle regularity approaching that of younger mice. Furthermore, statistical analysis of ovarian weight indicated that DHA intervention significantly increased ovarian weight in aged mice and restored their ovarian reserve function, suggesting that DHA has the potential to improve age-related ovarian structural and functional decline.

[0059] Example 2 - Effects of DHA on Fertility in Aged Mice

[0060] 1. Experimental Grouping and Procedures

[0061] Same as Example 1.

[0062] 2. Detection Indicators and Methods

[0063] (1) Statistics on the number of ovulations

[0064] After 60 days of continuous DHA intervention, female mice in each group underwent superovulation treatment. First, pregnant mare serum gonadotropin (PMSG, 500 IU / kg) was injected intraperitoneally, followed by human chorionic gonadotropin (hCG, 500 IU / kg) 48 hours later. 13-16 hours after hCG injection, mice were sacrificed, and the oviducts were rapidly isolated. Cumulooocyte complexes (COCs) were collected from the oviducts. The COCs were placed in EmbryoMax M2 medium (MR-015-D, Sigma-Aldrich, USA) containing hyaluronidase (MR-051-F, Millipore, USA), and cumulus cells were separated using an oral pipette and pipette. The total number of released oocytes was counted.

[0065] (2) Statistics on the number of piglets born

[0066] After 60 days of continuous DHA intervention, the medication was discontinued, and the female mice in each group continued to be housed under normal conditions. Fertility was assessed two months after medication discontinuation. Female mice in each group were housed overnight with male mice of known fertility (female:male = 1:2). The vaginal plug was examined at 9:00 AM the following morning to confirm mating. The day the vaginal plug was observed was defined as day 1 of pregnancy (D1). On D8, the mice were euthanized by cervical dislocation, the uterus was removed, and the number of implantation sites within the uterus was observed and counted.

[0067] 3. Experimental Results

[0068] (1) Statistics on the number of piglets born in each group

[0069] Figure 2 Tables A and 2B present the statistical results of litter size for each group of mice. Compared to the young control group, the litter size of mice in the older control group was significantly lower, indicating that 12-month-old female mice were in a state of obvious reproductive decline. Compared to the older control group, the litter size of mice in the low-dose DHA group and the high-dose DHA group was significantly higher. This statistical difference confirms that DHA has a clear effect on improving fertility.

[0070] (2) Analysis of statistical results of the proportion of fertile female mice

[0071] Analysis of statistical results of the proportion of fertile female mice as follows Figure 2 As shown in Figure C, the proportion of fertile female mice in the aged control group was significantly lower than that in the young control group. Compared with the aged control group, the proportion of fertile female mice in the low-dose DHA group and the high-dose DHA group was significantly higher, indicating that DHA intervention can significantly increase the proportion of fertile individuals in aged female mice, enabling most aged female mice to regain fertility.

[0072] (3) Analysis of ovulation count results

[0073] Figure 2Figures D and 2E show the number of ovulations in each group of mice after superovulation treatment. Compared with the young control group, the number of ovulations in the aged control group was significantly decreased, reflecting a severe decline in ovarian reserve. Compared with the aged control group, the number of ovulations in the high-dose DHA group was significantly increased, indicating that DHA intervention can significantly increase the number of ovulations in aged mice.

[0074] In conclusion, Figure 2 The results showed that DHA intervention significantly improved the reproductive function of aged mice. The number of litters in treated aged female mice increased significantly, and the proportion of fertile individuals also increased significantly. Furthermore, the typical phenotype associated with ovarian aging in aged mice—a decrease in the number of ovulations—was effectively reversed after DHA intervention, with the number of ovulations significantly increasing compared to the untreated aged control group.

[0075] Example 3 - Effects of DHA on serum hormones and aging markers in aged mice.

[0076] 1. Experimental Grouping and Procedures

[0077] Same as Example 1.

[0078] 2. Detection Indicators and Methods

[0079] (1) Detection of serum hormone and inflammatory factor levels

[0080] Following 60 days of continuous DHA intervention, blood was collected from the retro-orbital venous plexus of mice within 24 hours of the last administration. Whole blood samples were centrifuged at 4000 r / min for 10 minutes at 4°C to obtain serum. Serum levels of reproductive hormones and inflammatory factors, including anti-Müllerian hormone, estradiol, follicle-stimulating hormone, luteinizing hormone, interleukin-1β, and interleukin-6, were detected using enzyme-linked immunosorbent assay (ELISA). All assays were performed according to the instructions of the corresponding kits (Beijing Beijian Xinyuan Biotechnology Co., Ltd., Beijing, China).

[0081] (2) Real-time quantitative PCR

[0082] Following 60 days of continuous DHA intervention, ovarian tissue samples were collected within 24 hours of the last administration and stored at -80°C. Total RNA was extracted from ovarian tissue using the RNeasy Mini Kit (QIAGEN Sciences, Germantown, MD, USA), strictly following the manufacturer's instructions. Using 2 μg of total RNA as a template, first-strand cDNA was synthesized using the AipScript First-Strand cDNA Synthesis RT-PCR Kit (including OneStep gDNA Removal) reverse transcription kit (i-presci scientific, RT317-02). Real-time quantitative polymerase chain reaction (qPCR) was then performed using the SYBR Green method (i-presci scientific, FP308-02). The expression levels of the target genes were standardized using the housekeeping gene GAPDH as an internal control, and the fold change of P21 and P53 genes relative to the control group was calculated.

[0083] 3. Experimental Results

[0084] like Figure 3 As shown, compared with the young control group, the serum levels of anti-Müllerian hormone (AMH) and estradiol in the aged control group mice were significantly decreased, while the levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were significantly increased, indicating that aging leads to dysfunction of the reproductive endocrine axis. After DHA intervention, the levels of the above hormones showed a significant improvement trend: AMH and estradiol rebounded, while FSH and LH declined. Figure 3 A-3D). Furthermore, serum levels of the inflammatory factors interleukin-1β and interleukin-6 were significantly elevated in the elderly control group, while DHA intervention effectively downregulated their expression (A-3D). Figure 3 E and 3F). qPCR results showed that the mRNA expression levels of cellular senescence markers P53 and P21 in ovarian tissue were significantly upregulated in the elderly control group, but significantly decreased after DHA intervention. Figure 3 These results indicate that DHA can improve ovarian aging by regulating reproductive hormone balance and inhibiting aging-related signals.

[0085] Example 4 - DHA improves ovarian fibrosis in aged mice

[0086] 1. Experimental Grouping and Procedures

[0087] Same as Example 1.

[0088] 2. Detection Indicators and Methods

[0089] (1) Real-time quantitative PCR

[0090] After 60 days of continuous DHA intervention, ovarian tissue samples were collected within 24 hours of the last administration and stored at -80°C. Total RNA extraction, reverse transcription, and qPCR detection methods from the ovarian tissue were the same as in Example 3. Target genes detected included Col3a1, TGF-β1, Vimentin, and α-SMA, which were standardized using GAPDH as an internal reference, and their relative expression levels were calculated.

[0091] (2) Masson staining

[0092] After 60 days of continuous DHA intervention, ovarian tissue samples were collected within 24 hours of the last administration and fixed for 24 hours using 4% paraformaldehyde (4g paraformaldehyde per 100mL). The fixed tissues were then dehydrated, cleared, and embedded in paraffin to form paraffin blocks. After dewaxing the paraffin sections of the ovarian tissue to water, the Masson trichrome staining kit (Solarbio, G1340, Beijing, China) was used. The specific steps included: Weigert iron hematoxylin staining for 5-10 minutes, differentiation in acidic differentiation solution for 5-15 seconds, washing with distilled water followed by Masson blueing solution for 3-5 minutes; staining with Ponceau S and fuchsin for 5-10 minutes; washing with weakly acidic working solution (distilled water: weakly acidic solution = 2:1); differentiation with phosphomolybdic acid solution for 1-2 minutes; washing again with weakly acidic working solution; counterstaining collagen fibers with aniline blue solution for 1-2 minutes; and washing away excess stain with weakly acidic working solution. Finally, the collagen fibers were dehydrated with gradient ethanol, cleared with xylene, and sealed with neutral resin. The deposition of collagen fibers was then observed under an optical microscope.

[0093] (3) Immunohistochemical staining

[0094] Following 60 days of continuous DHA intervention, ovarian tissue samples were collected within 24 hours of the last administration and fixed with 4% paraformaldehyde for 24 hours. The fixed tissues were then dehydrated, cleared, and embedded in paraffin to form paraffin blocks. After dewaxing and rehydration, paraffin sections of the ovarian tissue underwent antigen heat retrieval using sodium citrate buffer. The retrieval sections were treated with 3% hydrogen peroxide for 10 minutes, washed, and blocked. Primary antibodies TGF-β1 (1:200, Abcam, ab215715) and α-SMA (1:200, Abcam, ab5694) were added, followed by incubation with corresponding species-derived secondary antibodies (1:200, Abcam, ab6721). DAB staining solution (Solarbio, DA1010) was used for development, and hematoxylin counterstaining was applied to the cell nuclei. After staining, the sections were dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, dried, and observed and photographed under a microscope.

[0095] 3. Experimental Results

[0096] (1) DHA inhibits the expression of genes related to ovarian fibrosis in aged mice.

[0097] Figure 4 A-4D shows the mRNA expression levels of fibrosis-related factors in the ovarian tissues of mice in each group, as detected by qPCR.

[0098] Col3a1 ( Figure 4 A): The Col3a1 mRNA expression level in the elderly control group was significantly upregulated compared to the young control group, indicating that aging leads to excessive deposition of extracellular matrix in ovarian cells. Col3a1 expression in the low-dose DHA group was lower than that in the elderly control group, and in the high-dose DHA group it was further significantly lowered to levels close to those in the young control group, showing a clear dose-dependent relationship.

[0099] TGF-β1 ( Figure 4 B): TGF-β1 is a core driver of fibrosis. TGF-β1 expression was significantly higher in the elderly control group than in the younger control group. After DHA intervention, TGF-β1 expression was significantly downregulated, approaching the levels of the younger control group.

[0100] Vimentin Figure 4 C): Vimentin is a marker of fibroblast activation. Vimentin expression was significantly higher in the elderly control group than in the younger control group. After DHA intervention, Vimentin expression decreased significantly, indicating that DHA can inhibit fibroblast activation.

[0101] α-SMA ( Figure 4 D): α-SMA is a specific marker of myofibroblasts, and its expression is directly related to fibrotic activity. α-SMA expression was significantly higher in the elderly control group than in the younger control group. After DHA intervention, α-SMA expression significantly decreased.

[0102] (2) DHA reduces collagen deposition in the ovaries of aged mice.

[0103] Figure 4 E shows the collagen fiber deposition in the ovarian tissue of mice in each group as observed by Masson staining. In the young control group, the ovarian tissue structure was clear with no abnormal deposition; in the older control group, a large amount of diffuse collagen fiber deposition was observed; in the low-dose DHA group, collagen fiber deposition was significantly reduced compared to the older control group; in the high-dose DHA group, collagen fiber deposition was further reduced, and the morphology and structure of the ovarian tissue were significantly improved. These results indicate that DHA intervention can effectively reduce collagen deposition in ovarian tissue.

[0104] (3) DHA inhibits the expression of TGF-β1 and α-SMA proteins in the ovaries of aged mice.

[0105] Figure 4F and 4G show the protein expression levels of TGF-β1 and α-SMA in mouse ovarian tissues of each group as detected by immunohistochemical staining.

[0106] TGF-β1 protein expression ( Figure 4 F): TGF-β1 positive signal was significantly enhanced in the ovarian tissue of the elderly control group. After DHA intervention, the intensity of TGF-β1 positive signal (brownish-yellow signal) was significantly reduced.

[0107] α-SMA protein expression ( Figure 4 G): In the elderly control group, the α-SMA positive signal was significantly enhanced. After DHA intervention, the intensity of the α-SMA positive signal (brownish-yellow signal) was significantly reduced.

[0108] In conclusion, Figure 4 The results showed that DHA intervention significantly inhibited the expression of ovarian fibrosis-related factors in aged mice. qPCR analysis revealed that after DHA intervention, the mRNA levels of Col3a1, TGF-β1, Vimentin, and α-SMA in ovarian tissue were significantly decreased. Masson staining further confirmed that DHA intervention effectively reduced collagen deposition in ovarian tissue. Simultaneously, immunohistochemical analysis showed that the protein expression levels of TGF-β1 and α-SMA also decreased synchronously.

[0109] Example 5 - DHA prevents age-related decline in fertility

[0110] 1. Experimental Grouping and Procedures

[0111] To evaluate the preventive effect of DHA on ovarian aging, this study selected 2-month-old (young control group) and 8-month-old (middle-aged model) female mice as experimental subjects.

[0112] Middle-aged mice were randomly divided into three groups: a low-dose DHA group (30 mg / kg), a high-dose DHA group (60 mg / kg), and a saline control group (i.e., the middle-aged control group), with a sample size of no less than 5 mice in each group. All drugs were administered via intraperitoneal injection once daily for 60 consecutive days. The young control group received no drug intervention and was simply fed synchronously.

[0113] 2. Detection Indicators and Methods

[0114] The methods for monitoring estrous cycles and measuring ovarian weight are the same as in Example 1. The methods for counting litters are the same as in Example 2.

[0115] 3. Experimental Results

[0116] (1) Mouse ovarian weight

[0117] Figure 5Figures A and 5D show the statistical results of ovarian weight in each group of mice. The significant difference between the middle-aged control group and the young control group indicates that 8-month-old mice are in the early stages of ovarian function decline. The significant differences between the low-dose DHA group and the high-dose DHA group and the middle-aged control group demonstrate that DHA can effectively maintain ovarian weight and prevent age-related ovarian atrophy. The high-dose DHA group showed better results than the low-dose DHA group, further confirming the dose-dependent nature of DHA.

[0118] (2) Mice estrous cycle

[0119] Figure 5 C illustrates the changes in estrous cycles monitored over 14 consecutive days. The young control group exhibited a regular, fluctuating estrous cycle, with each phase (proestrus, estrus, metestrus, and diaestrus) alternating in an orderly manner, and a cycle length of approximately 4-5 days, consistent with normal physiological characteristics in mice. The middle-aged control group showed estrous cycle irregularities, consistent with a moderate aging phenotype. The low-dose DHA group showed improved estrous cycle regularity. The high-dose DHA group showed further restoration of estrous cycle regularity.

[0120] Figure 5 G represents the proportion of interestrus and estrus periods in the total estrous cycle. Compared to the young control group, the proportion of interestrus and estrus periods was significantly higher in the middle-aged control group. Compared to the middle-aged control group, the proportion of interestrus and estrus periods decreased in the low-dose DHA group, indicating that DHA intervention can improve the reproductive cycle of middle-aged mice; in contrast, the proportion of interestrus and estrus periods further decreased in the high-dose DHA group, showing a significant difference from the middle-aged control group. These statistical results indicate that DHA can effectively prevent age-related estrous cycle disorders.

[0121] (3) Statistics on the number of piglets born in each group

[0122] Figure 5 Figures B and 5F show the litter size of mice in each group after being housed with male mice. Compared to the young control group, the litter size of mice in the middle-aged control group was significantly lower, indicating that reproductive decline begins in 8-month-old female mice. Compared to the middle-aged control group, the litter size of mice in the low-dose DHA group and the high-dose DHA group was significantly higher. This statistical difference confirms that DHA can effectively prevent the decline in fertility with age.

[0123] Figure 5 E shows the proportion of fertile female mice in each group. Compared to the young control group, the proportion of fertile female mice in the middle-aged control group was significantly lower, indicating that a considerable proportion of individuals have lost their fertility by middle age. Compared to the middle-aged control group, the proportion of fertile female mice in the low-dose DHA group and the high-dose DHA group was significantly higher, indicating that DHA can effectively prevent age-related fertility loss.

[0124] In conclusion, Figure 5 The results showed that DHA intervention effectively prevented estrous cycle disorders in middle-aged mice, making their cycle regularity closer to that of younger mice, rather than continuously being in the interestrus or estrus phase. Statistical analysis of ovarian weight indicated that DHA significantly maintained ovarian weight and ovarian reserve function in middle-aged mice. In fertility assessments conducted two months after drug withdrawal, the number of litters and the proportion of fertile individuals in mice treated with DHA intervention were significantly higher than those in the middle-aged control group. These results confirm that DHA can effectively prevent age-related decline in fertility by improving cycle regularity and maintaining ovarian structure and reserve function, demonstrating its potential application value in delaying ovarian aging.

[0125] Example 6 - Safety Assessment After DHA Intervention

[0126] 1. Experimental Grouping and Procedures

[0127] Same as Example 1.

[0128] 2. Detection Indicators and Methods

[0129] (1) Morphological observation of major organs and reproductive organs (HE staining)

[0130] After 60 days of continuous DHA intervention, mice in each group were sacrificed, and heart, liver, lung, kidney, ovary, and uterine tissues were rapidly collected. The tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. After dewaxing and rehydration, the sections were stained with hematoxylin for 1-2 minutes, then blued in tap water for 3 minutes, followed by eosin staining for 30 seconds. After staining, the sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. The morphology and structure of each tissue were then observed under an optical microscope.

[0131] (2) Liver and kidney function indicators

[0132] Blood was collected from the retro-orbital venous plexus of mice within 24 hours of the last administration. Whole blood samples were centrifuged at 4000 r / min for 10 minutes at 4°C to obtain serum. Serum levels of creatinine (renal function indicator) and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (liver function indicators) were detected using enzyme-linked immunosorbent assay (ELISA). All tests were performed according to the instructions of the corresponding kits (Beijing Beijian Xinyuan Biotechnology Co., Ltd., Beijing, China).

[0133] 3. Experimental Results

[0134] (1) Effects of DHA on the morphology of major organs and tissues

[0135] Figure 6A shows the HE staining results of heart, liver, lung, and kidney tissues from mice in each group after 60 days of continuous DHA intervention. The results show that the morphology of the major organs such as the heart, liver, lung, and kidneys of mice remained normal after DHA intervention, and no obvious toxicity-related pathological changes were observed, indicating that DHA has no significant toxic effects on the major organs at the experimental dose.

[0136] (2) Effects of DHA on the morphology of reproductive organ tissues

[0137] Figure 6 B shows a comparison of the histological morphology of the ovaries and uterus in mice from different groups after DHA intervention. The results show that DHA can improve age-related ovarian structural degeneration without causing tumor formation or abnormal proliferation in the uterus and ovaries, indicating that DHA has good reproductive organ safety while improving ovarian function.

[0138] (3) Effects of DHA on liver and kidney function indicators

[0139] Figure 6 C-6E shows the results of detecting the concentration levels of creatinine, aspartate aminotransferase, and alanine aminotransferase in the serum of mice in each group.

[0140] Creatinine ( Figure 6 C): Creatinine is an important indicator reflecting glomerular filtration function. Serum creatinine levels in all groups of mice were within the normal reference range, and there were no significant differences between groups, indicating that DHA intervention did not cause damage to kidney function.

[0141] Aspartate aminotransferase ( Figure 6 D): Aspartate aminotransferase (AST) is mainly found in the mitochondria of hepatocytes and is a sensitive indicator of hepatocyte damage. There were no significant differences in serum AST levels among the groups of mice, indicating that DHA did not cause significant hepatocyte damage.

[0142] Alanine transaminase ( Figure 6 E): Alanine aminotransferase (ALT) is mainly found in the cytoplasm of hepatocytes and is a specific indicator of hepatocyte damage. Serum ALT levels in all groups of mice were within the normal range, with no significant differences between groups, further confirming that DHA has no hepatotoxicity.

[0143] The lack of significant differences in liver and kidney function indicators within the normal range demonstrates the safety of DHA—that is, DHA effectively improves ovarian aging without adversely affecting liver and kidney function.

[0144] In the development and application of natural medicines, systematic safety evaluation is a crucial step. Figure 6The results showed that after DHA intervention, the morphology of the uterus and ovaries in mice remained normal, no signs of tumor formation were observed, and related liver and kidney function indicators were also within the normal range. These results preliminarily indicate that DHA has good safety characteristics under experimental conditions.

[0145] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or similar implementation schemes obtained by those skilled in the art by making some modifications or alterations to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. Use of dihydroartemisinin or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention, delay and / or treatment of ovarian aging and related diseases.

2. The use according to claim 1, wherein, The dihydroartemisinin or a pharmaceutically acceptable salt thereof is the sole active ingredient in the drug.

3. The use according to claim 1, wherein, The drug also contains other active ingredients for the prevention, delay, and / or treatment of ovarian aging and related diseases.

4. The use according to any one of claims 1 to 3, wherein, Ovarian aging is the natural decline in ovarian function associated with aging.

5. The use according to any one of claims 1 to 4, wherein, The prevention, delay, and / or treatment of ovarian aging are selected from one or two of the following: (1) Prevention, delay and / or treatment of ovarian fibrosis; (2) Prevent, delay and / or treat age-related decline in female fertility.

6. The use according to claim 5, wherein, The prevention, delay, and / or treatment of ovarian fibrosis involve reducing collagen deposition in ovarian tissue.

7. The use according to claim 5, wherein, The prevention, delay, and / or treatment of age-related fertility decline in women manifests as one or more of the following: (a) Increase ovarian weight; (b) Increase the number of ovulations; (c) Increase the number of live births; (d) Increase the proportion of individuals capable of reproduction; (e) Restore regular estrous cycles.

8. The use according to any one of claims 1 to 7, wherein, The drug also contains pharmaceutically acceptable excipients.

9. The use according to claim 8, wherein, The pharmaceutically acceptable excipients are selected from one or more of the following: carriers, surfactants, preservatives, antioxidants, hardeners, thickeners, and absorption enhancers.

10. The use according to any one of claims 1 to 9, wherein, The dosage form of the drug is selected from one or more of suppositories, tablets, capsules, granules, drops, and injections, with injections being preferred.

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