Use of a polypeptide of trichodesma spp. in the preparation of a medicament for the treatment of postmenopausal urogenital syndrome

Abstract

The application discloses application of a polypeptide of Rumex madaio in preparation of a medicine for treating postmenopausal urogenital syndrome, and belongs to the field of biological medicine. The polypeptide of Rumex madaio has a molecular weight of 200-3000 Da and contains a characteristic peptide segment IIRSPEPEVKILVDRD derived from A0A4D6DHD9 protein. The application proves through a GSM model of castrated rats that the polypeptide of Rumex madaio can, without increasing the serum estradiol level, up-regulate the phosphorylation levels of p-AKT1(Ser473) and p-ERa(Ser118) in a non-ligand-dependent manner, activate the AKT / ERa signal pathway, realize the synergistic repair of four organs of the uterus, the vagina, the bladder and the urethra, and repair the barrier function of the bladder epithelium. The application first discloses a non-estrogen-dependent GSM treatment mechanism of the polypeptide of Rumex madaio, can avoid hormone-related risks, and is suitable for hormone-intolerant people.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of herbaceous plant polypeptides in the preparation of drugs for treating postmenopausal urogenital syndrome. Background Technology

[0002] Genitourinary syndrome of menopause (GSM) is a chronic progressive disease prevalent in postmenopausal women. Its core cause is the synergistic degeneration of the structure and function of multiple organs in the urogenital system due to estrogen deficiency. Clinical manifestations include vulvar and vaginal symptoms (dryness, itching, burning, pain), sexual dysfunction, and urinary symptoms (urinary urgency, frequency, dysuria, difficulty urinating, recurrent lower urinary tract infections), which seriously affect the quality of life of patients [1-3]. According to statistics, there are more than 230 million postmenopausal women in my country. The prevalence of GSM in women one year after menopause is 64.7%, and it is as high as 84.2% six years after menopause [4]. However, the diagnosis rate is only about 20%, and the treatment rate is only 24% [5-6]. With the aging of the population, the prevention and treatment of this disease has become a public health challenge that needs to be addressed in the field of women's reproductive health in my country.

[0003] Currently, clinical intervention for GSM faces a dual limitation.

[0004] First, the limitations of hormone replacement therapy (HRT). Although HRT is the standard treatment, it has strict contraindications (such as a history of hormone-sensitive tumors, active thrombotic diseases, etc.), and long-term use may increase the risk of breast hyperplasia, thrombosis, etc., thus limiting its clinical application.

[0005] Second, the limitations of phytoestrogens intervention. The intervention effects of phytoestrogens such as soy isoflavone (SI) are weak and unstable. Their effects still depend on estrogen-like effects and cannot avoid hormone-related risks. They are also not suitable for people with hormone contraindications[7].

[0006] Furthermore, a pathogenesis model based solely on "estrogen deficiency" cannot fully explain the complex clinical manifestations of GSM. Although low estrogen levels and changes in the vaginal microenvironment are common characteristics of postmenopausal women, not all individuals develop GSM symptoms [3]; simply restoring estrogen levels cannot guarantee symptom relief [8-9]. This suggests that its pathological mechanism also involves factors such as local microenvironment imbalance, disordered immune inflammatory regulation, and decreased tissue repair capacity.

[0007] Rumex patientia L. × Rumex tianshanicus A. Los was recognized as a new food ingredient in 2021. Currently, there are publicly available patents related to Rumex patientia peptides, such as CN117683839A (An active Rumex patientia peptide and its preparation method and functional product) and CN115368434A (An active Rumex patientia peptide with high SOD content and its preparation method), etc. These patents all involve preparation processes or the field of nutritional and health foods (such as pancreatic lipase inhibition, anti-glycation, antioxidant, sleep aid, and probiotic promotion activities). No patents or non-patent literature reports have been found regarding the use of Rumex patientia peptides for the treatment of GSM or any urogenital system diseases. Further searching revealed that although some prior art documents are related to this application, none of them constitute a technological inspiration for this invention. For example, CN122272694A discloses a pharmaceutical composition containing *Gnaphalium affine* for treating gynecological diseases such as polycystic ovary syndrome (PCOS), but the composition is a compound extract of the whole *Gnaphalium affine* herb and the leaves of *Trichosanthes kirilowii* and *Sapindus mukorossi*, and does not involve *Gnaphalium affine* peptides, let alone the treatment of GSM; WO2012145809A1 discloses phytoestrogens (such as soy isoflavones) for treating postmenopausal lower genital tract diseases, but its mechanism of action relies on the classical estrogen receptor activation pathway, which is fundamentally different from the non-estrogen-dependent AKT / ERα phosphorylation activation mechanism of this invention; CN1039458 54B discloses a single protein polypeptide with a molecular weight of approximately 32.5 kDa isolated from yam (Dioscorea) for increasing serum estrogen levels. Its source plant (yam), molecular weight (approximately 32.5 kDa, far exceeding 200-3000 Da), and mechanism of action (increasing serum E2) are completely different from those of this invention. WO2023213141A1 discloses a polypeptide targeting the ovaries, but its polypeptide contains a specific amino acid sequence of the metabolin family and is used to treat ovarian diseases such as polycystic ovary syndrome. It does not involve polypeptides derived from edible herbs, nor does it involve GSM treatment. In summary, none of the above-mentioned prior art discloses or implies the use of a mixture of 200-3000 Da polypeptides derived from edible herbs for GSM treatment via a non-estrogen-dependent pathway, and therefore does not constitute a technical revelation for this invention.

[0008] In summary, the existing technologies have the following shortcomings: (1) There is a lack of safe and effective non-hormone-dependent GSM treatment drugs; (2) The medicinal uses of plant polypeptides have not been fully developed, and their application in urogenital diseases is a blank area; (3) Existing research on plant polypeptides in the field of tissue repair is mostly limited to phenotypic observation, and there is a lack of systematic research on their non-estrogen-dependent deep molecular mechanisms. Summary of the Invention

[0009] To address the shortcomings of existing technologies, such as the lack of safe and effective non-hormone-dependent GSM treatments, insufficient development of medicinal applications for plant peptides, and a lack of research on the tissue repair mechanisms of plant peptides, this invention is the first to use plant peptides derived from plant leaves for the treatment of postmenopausal urogenital syndrome. It reveals that these peptides exert a multi-organ synergistic repair effect through a non-estrogen-dependent AKT / ERα signaling pathway. A GSM treatment strategy with "non-estrogen-dependent ERα phosphorylation activation, AKT / ERα signaling pathway mediation, and four-organ synergistic repair" as its core innovation is proposed, aiming to provide safe and effective GSM treatments for individuals with hormone-restricted conditions.

[0010] In one aspect of the invention, the invention provides the use of herbaceous plant polypeptides in the preparation of a medicament for treating postmenopausal urogenital syndrome.

[0011] In some embodiments of the present invention, the herbicide polypeptide is derived from the whole plant of *Rumex patientia* L. × *Rumex tianshanicus* A. Los, and has a molecular weight of 200-3000 Da. The herbicide polypeptide can be prepared by enzymatic hydrolysis-ultrafiltration methods known in the art, for example, by enzymatic hydrolysis with acidic and neutral proteases followed by membrane treatment, as described in CN115368434B.

[0012] In some embodiments of the present invention, the leafy herb polypeptide comprises the IIRSPEPEVKILVDRD characteristic peptide (Score = 816.59, mass deviation 1.2 ppm) derived from the A0A4D6DHD9 protein. This characteristic peptide can serve as a chemical identification marker for the leafy herb polypeptide product, used for product quality control and batch-to-batch consistency confirmation. A0A4D6DHD9 is a predicted protein derived from leafy herbs, and currently there is no experimental verification information on its biological function in public databases. The present invention does not rely on the specific function of this protein, but rather uses this characteristic peptide as a chemical identification marker for the leafy herb polypeptide product.

[0013] In some embodiments of the present invention, the herbicide peptides exert their effects through a non-estrogen-dependent pathway. This non-estrogen-dependent pathway includes upregulating estrogen receptor α (ERα) phosphorylation levels and upregulating protein kinase B (AKT) phosphorylation levels, thereby exerting a repair effect by activating the AKT / ERα signaling pathway. Specifically, in a castrated rat GSM model, serum estradiol (E2) levels after herbicide peptide intervention showed no statistically significant difference compared to the model group (P>0.05), but the expression of p-AKT1(Ser473) / AKT1 and p-ERα(Ser118) / ERα proteins in uterine tissue was significantly increased (P<0.01), and the expression of both phosphorylated proteins in bladder tissue also significantly increased (P<0.05), suggesting that the herbicide peptides activate the AKT / ERα signaling pathway in a non-ligand-dependent manner.

[0014] In some embodiments of the present invention, the herbaceous plant polypeptide can achieve synergistic repair of multiple organs of the urogenital system, including the uterus, vagina, bladder, and urethra, without relying on the restoration of systemic estrogen levels. In uterine tissue, after intervention with the herbaceous plant polypeptide, the number of endometrial glands increased, glandular lumens expanded, interstitial blood vessel distribution recovered synchronously, collagen fiber content increased significantly, and distribution became more uniform. The herbaceous plant polypeptide induced selective aggregation of ERα in the nuclei of endometrial epithelial cells, exhibiting an "epithelial-targeted nuclear aggregation" characteristic, while ERα expression in interstitial cells did not show significant recovery. In vaginal tissue, the herbaceous plant polypeptide induced high aggregation of ERα in the nuclei of cells in the middle and superficial layers of the vaginal epithelium, consistent with the "epithelial-targeted regulation" pattern in uterine tissue. In bladder tissue, after intervention with the herbaceous plant polypeptide, the transitional epithelial cells of the bladder were tightly arranged, the epithelial continuity was intact, the boundary between the submucosa and muscle layer was clear, and ERα expression showed no significant difference from the castration group. In urethral tissue, after intervention with the herbaceous plant polypeptide, the morphology of the urethral lumen approached that of the sham-operated group, and the boundaries of each layer were clear.

[0015] In some embodiments of the present invention, the herbaceous plant peptide can repair the bladder epithelial barrier function. Specifically, after intervention with the herbaceous plant peptide, the proportion of moderate to severe proteinuria was significantly reduced, the ZO-1 immunofluorescence signal was restored to a continuous linear distribution, and CD11B / MPO double staining showed a significant reduction in neutrophil and monocyte / macrophage infiltration, indicating that the herbaceous plant peptide achieves systematic reconstruction of the bladder epithelial barrier by simultaneously inhibiting inflammatory cell infiltration and repairing tight junctions.

[0016] In some embodiments of the present invention, the herbicide peptides exhibit differentiated regulatory mechanisms on the uterus and bladder. Transcriptomic analysis revealed that the repair effects of the herbicide peptides on the uterus focus on the phagosome pathway (NES=1.52, nominal p=0.002) and the set of genes regulating immune responses (NES=1.42, nominal p=0.003); the repair effects on the bladder focus on complement and coagulation cascades (NES=2.32, padj=0.01), ECM-receptor interactions (NES=2.21, padj=0.02), and the PI3K-Akt signaling pathway (NES=2.05, padj=0.03). No enrichment of estrogen receptor-related gene sets was observed in either tissue, confirming its estrogen-free regulatory pattern at the genome-wide level.

[0017] In some embodiments of the present invention, the effective dose of the herbicide polypeptide, calculated in a castrated rat model, is 100-400 mg / kg / day, preferably 200-400 mg / kg / day, and more preferably 400 mg / kg / day. According to the body surface area conversion method recommended in the FDA 2005 industry guidance and NMPA / CDE guidelines (HED = animal dose × Km_animal / Km_human, where rat Km = 6 and human Km = 37), the corresponding human equivalent dose (HED) for the above rat dose is 16.2-64.8 mg / kg / day (approximately 972-3888 mg / day for a 60 kg adult). The actual human dosing regimen needs to be further determined in subsequent clinical trials.

[0018] In some embodiments of the present invention, the medicament is an oral or topical formulation. The oral formulation includes tablets, capsules, granules, or oral liquids; the topical formulation includes vaginal gels, vaginal suppositories, ointments, or creams. The formulation contains edible leafy polypeptides and pharmaceutically acceptable excipients. The types of excipients and the formulation form can be determined according to clinical needs and conventional techniques in the art.

[0019] In another aspect, the present invention provides the application of the above-mentioned herbaceous plant polypeptide in the preparation of a medicament for treating postmenopausal urogenital syndrome, wherein the postmenopausal urogenital syndrome includes one or more of vulvovaginal atrophy, bladder dysfunction, and urethral tissue damage. Specifically, the herbaceous plant polypeptide can be used to prepare a medicament for treating vulvovaginal dryness, itching, burning, and pain in postmenopausal women; it can be used to prepare a medicament for treating urinary urgency, frequency, dysuria, difficulty urinating, and recurrent lower urinary tract infections in postmenopausal women; and it can also be used to prepare a medicament for improving sexual dysfunction in postmenopausal women.

[0020] In some specific embodiments of the present invention, castrated female SD rats (6-8 weeks old, bilateral ovariectomy) were used as a GSM model. After successful modeling, they were continuously administered herbicide peptides (100, 200, 400 mg / kg / day) by gavage for 4 weeks. The results showed that there was no significant difference in serum E2 levels compared with the castrated group (P>0.05). The uterine coefficient was significantly increased in both the medium-dose (200 mg / kg) and high-dose (400 mg / kg) groups (P<0.01, P<0.001). The expression of p-AKT1(Ser473) / AKT1 and p-ERα(Ser118) / ERα proteins in uterine tissue was significantly increased (P<0.01), and the expression of the two phosphorylated proteins in bladder tissue also significantly recovered (P<0.05). ERα selectively accumulated in the nuclei of epithelial cells in uterine and vaginal tissues. The morphology of bladder and urethral tissues was significantly improved. Bladder ZO-1 tight junctions were restored and inflammatory infiltration was reduced. The above results indicate that the herbicide peptides exert a multi-organ synergistic repair effect through the estrogen-free AKT / ERα signaling pathway.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to apply edible herb polypeptides to the treatment of GSM. In the prior art, edible herb polypeptides are only involved in the preparation process or the field of nutritional health food (such as pancreatic lipase inhibition, anti-glycation, anti-oxidation, sleep aid, and promotion of probiotics). This invention is the first to apply it to the treatment of urogenital system diseases, filling the technological gap in the development of the medical applications of edible herb polypeptides.

[0022] (2) This invention reveals for the first time that phytoestrogens exert their repair function by activating the AKT / ERα signaling pathway in a non-ligand-dependent manner. Traditional phytoestrogens (such as soy isoflavones) activate ERα via ligand-receptor binding and require an increase in circulating estrogen levels. This invention found that phytoestrogens exert their function by upregulating the phosphorylation levels of p-AKT1 (Ser473) and p-ERα (Ser118) without altering serum E2 levels, thus mechanistically avoiding hormone-related risks. Serum E2 detection and Western blot results corroborate each other, jointly confirming the non-ligand-dependent activation mode.

[0023] (3) A unique regulatory pattern of ERα “epithelial-targeted nuclear aggregation” induced by phytoestrogen peptides was discovered for the first time. Traditional hormone replacement therapy and phytoestrogen therapy both show a holistic recovery of estrogen signaling; this invention is the first to observe that phytoestrogen peptides selectively restore ERα expression in the nuclei of uterine and vaginal epithelial cells, while there are no significant changes in stromal cells, and ERα expression in bladder tissue is not restored at all. This differential receptor regulation pattern is unprecedented in the field and reflects the unique characteristics of non-estrogen-dependent pathways.

[0024] (4) For the first time, the synergistic repair of four organs—uterus, vagina, bladder, and urethra—is achieved in GSM treatment. Existing non-hormonal therapies (such as vaginal moisturizers and lubricants) can only temporarily relieve local symptoms and cannot achieve systemic repair of multiple organs. This invention provides a novel solution for the multi-organ systemic treatment of GSM by repairing four urogenital organs simultaneously through a non-estrogen-dependent pathway.

[0025] (5) For the first time, the differential regulatory mechanism of phytoestrogen peptides on the uterus and bladder was elucidated at the transcriptome level. In the uterus, the phagosome pathway and immune response regulation were activated, while in the bladder, complement and coagulation cascades, ECM-receptor interactions, and the PI3K-Akt signaling pathway were enriched. No estrogen response genes were enriched in either tissue, providing mechanistic evidence for "non-estrogen dependence" at the whole genome level.

[0026] (6) This invention is the first to discover that phytoesophageal peptides can repair the bladder epithelial barrier function. Current GSM treatments mainly focus on vaginal symptoms and pay insufficient attention to bladder dysfunction. This invention confirms the systematic reconstruction of the bladder epithelial barrier through multiple dimensions such as urinary protein quantification, ZO-1 immunofluorescence, and CD11B / MPO immunofluorescence, providing experimental evidence for the treatment of urinary symptoms of GSM.

[0027] In summary, the mechanism of action of this invention is non-estrogen-dependent AKT / ERα phosphorylation activation, without elevated serum E2 levels and without hormone-related risks, thus making it applicable to a wider range of patients, including those with contraindications to hormone therapy. Regarding repair efficacy, this invention achieves synergistic repair of four organs: the uterus, vagina, bladder, and urethra, while existing hormone replacement therapies and phytoestrogens are mainly limited to symptom relief in a single or limited organ. At the molecular regulatory level, this invention works through the immune / phagosome / complement / ECM pathway, fundamentally different from the estrogen-dependent signaling pathway regulation model in existing technologies. In existing technologies, the treatment approach for GSM mainly focuses on supplementing or replacing estrogen (HRT, phytoestrogens, etc.). Based on the teachings of CN122272694A, those skilled in the art might attempt to use *Gastrodia elata* extract for the treatment of gynecological diseases, but this document does not refer to GSM, let alone *Gastrodia elata* polypeptide monomers. Based on the teachings of CN103945854B, those skilled in the art might attempt to use yam polypeptides for urogenital diseases, but this document clearly teaches that its polypeptides exert their effects by increasing serum E2, which is a classic estrogen replacement approach. Those skilled in the art have no motivation to deduce a non-estrogen-dependent pathway that "does not increase E2" from the "increase E2" mechanism in this document. Based on the teachings of WO2012145809A1, those skilled in the art would tend to choose known phytoestrogens (such as soy isoflavones) rather than *Gastrodia elata* polypeptides whose mechanisms of action are completely unknown. Therefore, this invention deviates from the teachings of the prior art in three dimensions: "source selection" (*Gastrodia elata* vs. yam / soybean), "mechanism selection" (non-estrogen-dependent vs. estrogen-dependent), and "product form" (polypeptide mixture vs. single protein or small molecule), and its technical solution is non-obvious. The above findings were not reasonably predictable by those skilled in the art and represent unexpected technical effects, forming the core basis of the inventiveness of this invention. This invention is particularly applicable to individuals with contraindications to hormones (approximately 30-40% of the patient population), solving a long-standing technical problem that has remained unresolved in existing technologies.

[0028] This research and development was conducted based on the research platform of Beijing Hospital and was completed on the basis of the research group's previous research. Attached Figure Description

[0029] Figure 1 Mass spectrometry identification of edible leaf peptides (EDP), serum estradiol (E2) levels, uterine coefficient, and gross morphological images of the uterus and bladder were performed; among these, Figure 1 A represents the LC-MS / MS mass spectrometry identification results of the herbicide peptide; Figure 1 B is a bar chart showing the serum estradiol (E2) levels in rats of each group as detected by ELISA. Figure 1 C is a bar chart of the uterine coefficient (uterine wet weight / body weight ratio) for each group of rats; Figure 1D is a photograph showing the gross morphology of the uterus and bladder tissues of a castrated rat. Figure 2 H&E staining, Masson trichrome staining and ERα immunohistochemical staining images of rat uterine tissue are shown, and the comparison results of four groups are presented: sham operation group, castration model group, leafy green peptide group and soy isoflavone group. Figure 3 H&E staining and ERα immunohistochemical staining images of rat vaginal tissue are shown, displaying the comparative results of four groups: sham-operated group, castration model group, leafy green peptide group, and soy isoflavone group. Figure 4 H&E staining, Masson trichrome staining and ERα immunohistochemical staining images of rat bladder tissue are shown, and the comparison results of four groups are presented: sham-operated group, castration model group, leafy green peptide group and soy isoflavone group. Figure 5 H&E staining and Masson trichrome staining images of rat urethral tissue are shown, and the comparative results of four groups are presented: sham-operated group, castration model group, leafy grass polypeptide group and soy isoflavone group. Figure 6 Images show the urinary protein levels, serum creatinine levels, and immunofluorescence staining of bladder tissue in castrated rats; among them, Figure 6 A represents the semi-quantitative results of urinary protein detection in each group of rats; Figure 6 B is a bar chart showing the serum creatinine (Cre) levels of rats in each group; Figure 6 C shows CD11B / MPO double staining and ZO-1 immunofluorescence staining of bladder tissue; Figure 7 The image shows the Western blot results of the effects of plant extract peptides on AKT1 and ERα phosphorylation in the uterine and bladder tissues of ovariectomized rats; among them... Figure 7 A represents the Western blot bands and quantitative analysis bar charts of p-AKT1(Ser473) / AKT1 and p-ERα(Ser118) / ERα protein expression in uterine tissue; Figure 7 B shows representative Western blot bands and quantitative analysis bar charts of p-AKT1(Ser473) / AKT1 and p-ERα(Ser118) / ERα protein expression in bladder tissue. Figure 8 This is a transcriptome analysis diagram of rat uterine tissue, including principal component analysis (PCA) and GO / KEGG enrichment analysis results. Figure 9 This is a transcriptome analysis of rat bladder tissue, including principal component analysis (PCA) and GO / KEGG enrichment analysis results. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0031] Example 1:

[0032] Sources and identification of polypeptides from leafy green plants.

[0033] The Rumex patientia peptide used in this invention was purchased from Weihai Shideyuan Biotechnology Co., Ltd. (product: Rumex patientia protein peptide). This peptide is an active peptide product obtained from the whole plant of Rumex patientia L. × Rumex tianshanicus A. Los through enzymatic hydrolysis, membrane separation, and other processes. It is a pale yellow to off-white powder that is easily soluble in water.

[0034] To clarify the material basis of the herbaceous peptides used in this invention, LC-MS / MS mass spectrometry identification was performed. After pretreatment including reductive alkylation and desalting, the samples were acquired using high-resolution mass spectrometry. The raw mass spectrometry data were retrieved from the Rumex protein database using Byonic software. The results showed that the molecular weights of the identified peptides were mainly distributed between 200-3000 Da. Figure 1 A) Upon testing, the total protein content of this batch of polypeptide powder was 35.6%, of which peptides with a molecular weight of 200-3000 Da accounted for 91.7% of the total protein (based on GPC peak area). The peptide with the highest score was the IIRSPEPEVKILVDRD sequence (SEQ ID NO.1) derived from the A0A4D6DHD9 protein (Score=816.59, mass deviation 1.2ppm). Its secondary mass spectrum clearly showed the matching of peptide fragments, confirming the accuracy of the peptide sequence (Table 1).

[0035] Table 1: Information on Byonic-identified peptides (partial results) (SEQ ID NO.1-5)

[0036] A0A4D6DHD9 is a predicted protein derived from leafy greens, but currently, there is no experimental verification information on its biological function in public databases. This invention does not rely on the specific function of this protein, but rather uses the highest-scoring characteristic peptide as a chemical identification marker for the leafy green polypeptide product, for product quality control and batch-to-batch consistency confirmation.

[0037] The molecular weight distribution of the herbicide peptides was determined by gel permeation chromatography (GPC). The results showed that peptides with a molecular weight of 200-3000 Da accounted for 91.7% of the total peak area, confirming that the molecular weight of the peptide sample was mainly concentrated in the range of 200-3000 Da.

[0038] Example 2:

[0039] The therapeutic effect of herbicide peptides on castrated rat GSM model 2.1 Establishment of animal models SPF-grade female SD rats, 6-8 weeks old and weighing 180-220g, were randomly divided into a sham-operated group and a castration-operated group after one week of acclimatization. Rats in the castration-operated group underwent bilateral ovariectomy (OVX): anesthesia was administered via intraperitoneal injection of sodium pentobarbital (40mg / kg), the skin and abdominal wall were incised along the midline to expose the abdominal cavity, and both ovaries were completely removed. After hemostasis, the incision was sutured layer by layer. In the sham-operated group, only the ovaries were exposed and then sutured without removal.

[0040] Two weeks after the surgery, the effect of castration was confirmed by vaginal smear examination: regular estrous cycles were observed in the sham surgery group, while the vaginal smears of the castration group showed continuous estrus changes, indicating that the oophorectomy was successful.

[0041] 2.2 Grouping and Dosing Rats that successfully developed the model were randomly divided into the following 6 groups, with 6 rats in each group: Sham group: Equal volume of normal saline was administered by gavage once daily for 4 consecutive weeks.

[0042] Castration model group (OVX): Equal volume of physiological saline was administered by gavage once daily for 4 consecutive weeks.

[0043] Low-dose group of edible grass peptides (EDP-L): 100 mg / kg / day of edible grass peptides (calculated based on the weight of peptide powder, the same below), administered by gavage once daily for 4 consecutive weeks.

[0044] Medium-dose group of edible herb polypeptide (EDP-M): edible herb polypeptide 200mg / kg / day, once daily by gavage for 4 consecutive weeks.

[0045] High-dose group of leafy green peptides (EDP-H): leafy green peptides 400mg / kg / day, once daily by gavage for 4 consecutive weeks.

[0046] Positive control group (SI): Soy isoflavones 160 mg / kg / day, administered by gavage once daily for 4 consecutive weeks. This dosage was based on previous literature reports [Song Chenmeng, Zhang Yusen, Cheng Le, et al. Effect of resveratrol combined with soy isoflavones on improving oxidative stress-induced apoptosis in hippocampal tissue of aging rats [J]. Journal of Health Research, 2020, 49(6): 932-937].

[0047] The gavage volume was 10 mL / kg body weight, administered daily between 9:00 and 10:00 AM. Rats were weighed weekly, and the dosage was adjusted accordingly.

[0048] 2.3 Sample Collection Twenty-four hours after the last administration, the weight of rats in each group was measured, and they were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). Whole blood was collected using the abdominal aortic sampling method, and serum was separated and stored at -80°C for later use. After blood collection, the rats were sacrificed, and the uterus, vagina, bladder, and urethra were completely dissected. During uterine tissue dissection, surrounding fat and connective tissue were carefully removed, and the surface liquid was blotted dry with filter paper before immediately weighing the wet weight of the uterus for calculating the uterine coefficient. A portion of each tissue was fixed in 4% paraformaldehyde fixative (for paraffin sectioning and tissue staining), while another portion was rapidly frozen in liquid nitrogen and then transferred to -80°C for storage (for protein extraction and transcriptome sequencing).

[0049] 2.4 Test Items and Results 2.4.1 Uterine coefficient and serum estradiol (E2) detection Uterine coefficient determination: The formula for calculating the uterine coefficient is: Uterine coefficient (mg / g) = Uterine wet weight (mg) / Rat body weight (g). The results of rat body weight, uterine wet weight, and uterine coefficient determination for each group are shown in Table 2.

[0050] Table 2: Body weight, wet uterine weight, uterine coefficient, and serum E2 concentration of rats in each group

[0051] Note: Data are expressed as mean ± SD, n=6. Compared with the OVX group, P<0.05, P<0.01, P<0.001, ****P<0.0001.

[0052] Statistical analysis showed highly significant differences in uterine coefficients among the groups (P<0.001). Compared with the sham surgery group, the castration group had a highly significantly lower uterine coefficient (P<0.001), confirming that castration leads to uterine atrophy. Compared with the castration group, there was no significant difference in uterine coefficient in the low-dose herbal peptide group (100 mg / kg) (P>0.05); the uterine coefficient in the medium-dose herbal peptide group (200 mg / kg) was significantly higher (P<0.01); and the uterine coefficient in the high-dose herbal peptide group (400 mg / kg) was highly significantly higher (P<0.001), and there was no significant difference between the high-dose herbal peptide group and the soy isoflavone group (160 mg / kg) (P=1.000), suggesting that both groups had comparable effects in restoring uterine weight. However, the uterine coefficient in the high-dose herbal peptide group (1.117±0.140 mg / g) was still significantly lower than that in the sham surgery group (2.185±0.001 mg / g, P<0.001), indicating that the high-dose herbal peptide could only partially restore uterine weight and did not reach the normal physiological level. Figure 1 C). Gross morphological photographs of the uterus and bladder are shown. Figure 1 D.

[0053] Serum E2 detection: Serum E2 levels in rats of each group were detected by ELISA. Compared with the sham-operated group, the serum E2 level in the castration group was significantly lower (P<0.001), confirming the successful establishment of the castration model. There was no statistically significant difference in serum E2 levels between the effective dose groups of the herbicide peptides (200, 400 mg / kg) and the castration group (P>0.05). Figure 1 B) suggests that the herbaceous plant peptides do not depend on the restoration of systemic estrogen levels. After intervention with soy isoflavones (160 mg / kg), serum E2 levels were significantly higher than those in the castration group (P<0.01), suggesting that it may indirectly alter circulating estrogen levels through feedback regulation of the hypothalamus-pituitary-ovarian axis.

[0054] Results analysis: Soy isoflavones exert estrogen-like effects by increasing circulating estrogen levels, while significantly increasing the uterine coefficient, exhibiting a typical estrogen-like mode of action. The high-dose group of edible herb peptides achieved a uterine coefficient recovery comparable to that of soy isoflavones without increasing serum E2 (P=1.000). These two quantitative indicators corroborate each other, jointly confirming that the protective effect of edible herb peptides on the uterus is not mediated through estrogen-like effects, but rather through a non-estrogen-dependent pathway.

[0055] 2.4.2 Histological morphology examination Tissue fixed with 4% paraformaldehyde was embedded in paraffin, sectioned (4μm thickness), and then stained with H&E and Masson trichrome, respectively. The morphology and structure of the tissue were observed under an optical microscope.

[0056] Uterine tissue ( Figure 2 ): H&E staining showed that the sham-operated group had sufficient endometrial thickness, abundant glands with regular branching morphology, full glandular lumens, and dense stromal blood vessels. The castration group had significantly thinner endometrium, a sharp decrease in glandular number, only scattered atrophic glands, and sparse stromal blood vessels, exhibiting typical pathological changes of endometrial atrophy with loss of functional structure. After intervention with the herbaceous plant peptides, although the endometrial thickness did not fully recover to the level of the sham-operated group, the number of glands increased, the glandular lumens expanded, and the stromal blood vessel distribution recovered synchronously. The number of glands in the soy isoflavone group increased compared to the castration group, but the increase was weaker than that in the herbaceous plant peptide group.

[0057] Masson's trichrome staining showed that in the sham-operated group, collagen fibers were evenly distributed and of moderate content, forming a continuous network support structure. In the castration group, collagen fiber content was significantly reduced, distribution was sparse and disordered, and the network support structure was destroyed. After intervention with edible leaf herb peptides, collagen fiber content increased significantly, distribution became more uniform, and the network structure was partially restored. Collagen fiber content in the soy isoflavone group increased slightly, but the increase and evenness of distribution were weaker than in the edible leaf herb peptide group.

[0058] Immunohistochemical staining of ERα showed that in the sham-operated group, ERα was highly expressed in both endometrial epithelial and stromal cells, with abundant positive signal in the epithelial cell nuclei. In the castration group, the overall positive signal of ERα was significantly downregulated. After intervention with the herbicide-containing peptides, a unique "epithelial-targeted aggregation" characteristic was observed: ERα significantly and selectively aggregated in the nuclei of endometrial epithelial cells, while the positive signal in stromal cells remained weak. In the soy isoflavone group, ERα expression recovered somewhat compared to the castration group, but the intensity of the positive signal in the epithelial region was weaker than that in the herbicide-containing peptide group.

[0059] The above results indicate that the high-dose group of edible leaf peptides outperformed soy isoflavones in terms of functional structure repair. The increase in glands and blood vessels can effectively relieve symptoms such as vulvar and vaginal dryness, burning, pain and sexual dysfunction, further demonstrating the unique role of the non-estrogen-dependent pathway.

[0060] vaginal tissue ( Figure 3 ): H&E staining showed that the vaginal epithelium was thinner and the folds were reduced in the castration group. After intervention with physalis peptides, the thickness of the vaginal epithelium recovered and the number of folds increased. Immunohistochemistry of ERα showed that physalis peptides induced a high accumulation of ERα in the nuclei of cells in the middle and superficial layers of the vaginal epithelium, which was highly consistent with the "epithelial-targeted regulation" pattern observed in uterine tissue.

[0061] Bladder tissue ( Figure 4 ): H&E staining showed that in the sham-operated group, the transitional epithelium of the bladder was intact, with neatly arranged cells and a clear boundary between the submucosa and muscle layer. In the castration group, the transitional epithelium of the bladder was thinner, with loosely arranged cells in some areas. After intervention with physalis peptides, the cells of the transitional epithelium of the bladder were tightly arranged, the epithelial continuity was intact, and the boundary between the submucosa and muscle layer was clear.

[0062] Masson's trichrome staining showed that collagen fibers were evenly distributed in the submucosa and intermuscular layer of the bladder in the sham-operated group. In the castration group, collagen fiber content was reduced and the arrangement was loose. After intervention with the herbicide-containing peptides, collagen fiber content significantly increased, and the reticular support structure was partially restored.

[0063] Immunohistochemistry of ERα showed that there was no significant difference in ERα expression between the herbicide group and the castration group, and no trend of expression recovery was observed. This further confirms the non-estrogen-dependent mechanism of herbicide peptides at the receptor level.

[0064] Urethral tissue ( Figure 5 ): H&E staining showed that the urethral lumen in the sham-operated group was regular, the epithelial layer was intact, and the boundaries between the structural layers were clear. The urethral lumen in the castration group was irregular, the epithelial layer was thinner, and the mucosal folds were reduced. After intervention with the herbaceous plant polypeptide, the urethral lumen morphology approached that of the sham-operated group, and the boundaries between the structural layers were clear.

[0065] Masson's trichrome staining showed that collagen fibers in the submucosal layer of the urethra were evenly distributed and neatly arranged in the sham-operated group. Collagen fiber content was reduced and distribution was sparse in the castration group. After intervention with edible leaf herb peptides, the distribution and arrangement of collagen fibers approached those of the sham-operated group, and the integrity of the extracellular matrix was improved.

[0066] 2.4.3 Bladder barrier function testing Urine protein detection: Before the last administration, rats were placed alone in metabolic cages to collect urine for 12 hours. Urine protein was detected using urine dry chemistry test strips. The results showed ( Figure 6 In the castration group, the proportion of severe proteinuria reached 66.7% (4 / 6), which was statistically significant compared with the sham-operated group (P<0.001). The high-dose herbal peptide group (400mg / kg) showed significant protective effects, with only one case of mild abnormality (1+). In the soy isoflavone group, the proportion of severe proteinuria was 33.3% (2 / 6), and the protective effect was weaker than that of the high-dose herbal peptide group.

[0067] Serum creatinine detection: There were no significant changes in serum creatinine levels among the groups of rats (P>0.05). Figure 6 B) HE staining of kidney tissue showed no obvious structural abnormalities, suggesting that the abnormal proteinuria originated from damage to the bladder epithelial barrier rather than damage to the kidney's filtration function.

[0068] Immunofluorescence staining: Paraffin sections of bladder tissue were subjected to immunofluorescence staining. Figure 6 C). ZO-1 immunofluorescence showed that in the sham-operated group, the ZO-1 fluorescence signal was distributed in a continuous linear pattern, located at the tight junctions between bladder epithelial cells. In the castration group, the ZO-1 fluorescence signal was significantly weakened and discontinuous. After intervention with foliage peptides, the ZO-1 fluorescence signal recovered to a continuous linear distribution, indicating that the tight junction structure was repaired. CD11B / MPO double staining showed that almost no CD11B was present in the bladder tissue of the sham-operated group. + Monocytes / macrophages and MPO + Neutrophil infiltration. Castration group MPO + Neutrophils and CD11B + Monocyte / macrophage infiltration was significantly increased. Leafy herb peptides significantly inhibited MPO. + Neutrophil infiltration, while reducing CD11B + Mononuclear / macrophage infiltration.

[0069] The above results indicate that the herbicide peptides can effectively alleviate symptoms of urinary tract infections such as urinary urgency, frequency, pain, difficulty urinating, and recurrent lower urinary tract infections by simultaneously inhibiting inflammatory cell infiltration and repairing tight junctions, thereby achieving systematic reconstruction of the bladder epithelial barrier.

[0070] 2.4.4 Transcriptomics Analysis Uterine and bladder tissues were collected from rats in each group, and total RNA was extracted, constructed into libraries, and then subjected to high-throughput sequencing. Differentially expressed genes were analyzed using DESeq2, and gene set enrichment analysis (GSEA) was performed using the Hallmark and GO gene sets from MSigDB.

[0071] Transcriptome results of uterine tissue ( Figure 8 Principal component analysis (PCA) showed a significant aggregation trend between the phytoesophageal peptide group and the sham-operated group, suggesting that the phytoesophageal peptide can restore the expression pattern of the castrated uterus to a near-normal physiological state. GSEA showed that the phytoesophageal peptide group was significantly enriched in the phagosome pathway (NES=1.52, nominal p=0.002) and the immune response regulatory gene set (NES=1.42, nominal p=0.003) in both groups, and no estrogen receptor-related gene set was enriched.

[0072] Bladder tissue transcriptome results ( Figure 9 GSEA analysis showed that the phytoestrogen peptide group was significantly enriched in complement and coagulation cascades (NES=2.32, padj=0.01), ECM-receptor interactions (NES=2.21, padj=0.02), and the PI3K-Akt signaling pathway (NES=2.05, padj=0.03) compared to the castration group. Consistent with the uterus, no enrichment of any estrogen receptor-related gene sets was found in the bladder tissue.

[0073] The above data confirms at the whole genome level that the repair function of phytoesophageal peptides is centered on immune inflammation regulation and ECM remodeling, and is completely independent of estrogen signaling pathways, which is fundamentally different from the estrogen-dependent repair mode of soy isoflavones.

[0074] 2.4.5 Comparison of polypeptide components with different molecular weights To verify the necessity and technical significance of the molecular weight range (200-3000 Da) described in this invention, different molecular weight components of the herbicide peptide (<200 Da, 200-3000 Da, >3000 Da) were prepared, and the effects of each component on the recovery of the uterine coefficient in castrated rats were investigated according to the above method.

[0075] The results showed that the uterine coefficient recovery effect was most significant with the 200-3000 Da fraction (400 mg / kg) (1.117 ± 0.140 mg / g, P < 0.001 vs OVX group); while the uterine coefficient of the <200 Da fraction (400 mg / kg) (0.548 ± 0.095 mg / g) was not significantly different from that of the OVX group (P > 0.05); the uterine coefficient of the >3000 Da fraction (400 mg / kg) (0.621 ± 0.112 mg / g) was also not significantly different from that of the OVX group (P > 0.05). These results indicate that herbaceous plant peptides in the 200-3000 Da molecular weight range are the key active components for achieving the therapeutic effect of GSM, and that molecules with too small or too large molecular weights cannot effectively exert their repair effect.

[0076] Example 3:

[0077] Detection of ERα and AKT1 phosphorylation levels.

[0078] After drug administration, total protein was extracted from the uterus and bladder tissues of rats in each group and quantified using the BCA method. Using β-actin as an internal control, proteins were separated by SDS-PAGE electrophoresis, and after transfer to membranes, they were incubated with anti-ERα antibody, anti-p-ERα (Ser118) antibody, anti-AKT1 antibody, anti-p-AKT1 (Ser473) antibody, and anti-β-actin antibody, respectively. ImageJ software was used for grayscale analysis of the bands, and the p-ERα (Ser118) / total ERα ratio and p-AKT1 (Ser473) / total AKT1 ratio were calculated.

[0079] Uterine tissue ( Figure 7A): Western blot results showed that, compared with the Sham group, the expression of p-AKT1(Ser473) / AKT1 and p-ERα(Ser118) / ERα proteins in the OVX group was significantly reduced (P<0.01), suggesting that ovariectomy inhibited the activation of AKT and ERα. After treatment with edible leaf peptides, the expression of p-AKT1(Ser473) / AKT1 and p-ERα(Ser118) / ERα was significantly increased, with p-AKT1(Ser473) / AKT1 showing the most significant recovery, which was statistically different from the OVX group (P<0.01). The soy isoflavone group also improved the expression levels of the above proteins, but the overall recovery degree was slightly lower than that of the edible leaf peptide group.

[0080] Bladder tissue ( Figure 7 B): Compared with the Sham group, the expression of p-AKT1(Ser473) / AKT1 (P<0.05) was significantly reduced in the OVX group, and the expression of p-ERα(Ser118) / ERα also showed a decreasing trend. After treatment with leafwort peptides, the expression of both phosphorylated proteins significantly rebounded, and the difference was statistically significant (P<0.05). The soybean isoflavone group also showed a similar improvement effect.

[0081] The above results indicate that the herbaceous plant peptides can reverse the inhibition of AKT1 and ERα activation caused by ovariectomy. This change was not correlated with serum E2 levels (P>0.05), suggesting that it activates ERα in a non-ligand-dependent manner, i.e., by activating the AKT / ERα signaling pathway to improve the function of urogenital tissues. Compared with soy isoflavones, the ERα activation mode of the herbaceous plant peptides is significantly different: soy isoflavones activate ERα in a ligand-receptor-dependent manner and increase circulating E2 levels through feedback regulation; while the herbaceous plant peptides exert a non-ligand-dependent activation effect by upregulating the phosphorylation levels of ERα and AKT (Ser118 and Ser473 sites), without changing serum E2 levels. This difference is the core mechanism that distinguishes this invention from existing phytoestrogen therapy strategies.

[0082] In summary, the advantages of this invention compared with the prior art are shown in Table 3 below.

[0083] Table 3: Comparative Advantages with Existing Technologies

[0084] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

[0085] References [1] Portman DJ, Gass ML; Vulvovaginal Atrophy Terminology ConsensusConference Panel. Genitourinary syndrome of menopause: new terminology for vulvovaginal atrophy from the International Society for the Study of Women'sSexual Health and the North American Menopause Society. Menopause. 2014 Oct;21(10):1063-8. [2] Shen Pinghu. Expert consensus on clinical diagnosis and treatment of menopausal genitourinary syndrome [J]. Chinese Journal of Obstetrics and Gynecology, 2020, 55(10): 659-666. [3] Christmas M, Huguenin A, Iyer S. Clinical Practice Guidelines forManaging Genitourinary Symptoms Associated With Menopause. Clin ObstetGynecol. 2024 Mar 1;67(1):101-114. [4] Palma F, Volpe A, Villa P, et al. Vaginal atrophy of women inpostmenopause. Results from a multicentric observational study: the AGATAstudy. Maturitas, 2016, 83: 40-44. [5] Palma F, Della VE, Cagnacci A. Medical and patient attitude toward vaginal atrophy: the AGATA study. Climacteric, 2016, 19(6): 553-587. [6] Chua Y, Limpaphayom KK, Cheng B, et al. Genitourinary syndrome ofmenopause in five Asian countries: results from the Pan-Asian REVIVE survey. Climacteric, 2017, 20(4): 367-373. [7] Menopause Group, Obstetrics and Gynecology Branch of Chinese Medical Association. Guidelines for Menopause Management and Hormone Therapy in China, 2023 Edition. Chinese Journal of Obstetrics and Gynecology, 2023, 58(01): 4-21. [8] Micks E, Reed SD, Mitchell C. The Postmenopausal VaginalMicrobiome and Genitourinary Syndrome of Menopause. Clin Obstet Gynecol. 2024Mar 1;67(1):79-88. [9] Mitchell CM, Reed SD, Diem S, et al. Efficacy of VaginalEstradiol or Vaginal Moisturizer vs Placebo for Treating PostmenopausalVulvovaginal Symptoms: A Randomized Clinical Trial. JAMA Intern Med. 2018 May1;178(5):681-690.

Claims

1. Application of herbaceous plant polypeptides in the preparation of drugs for treating postmenopausal urogenital syndrome.

2. The application according to claim 1, characterized in that, The molecular weight of the herbaceous plant polypeptide is 200-3000 Da.

3. The application according to claim 1, characterized in that, The herbicide peptides are derived from the whole plant of *Rumexpatientia* L. × *Rumex tianshanicus* A. Los.

4. The application according to claim 1, characterized in that, The herbicide polypeptide contains the characteristic peptide segment shown in SEQ ID NO.1; the amino acid sequence of the characteristic peptide segment is IIRSPEPEVKILVDRD.

5. The application according to claim 1, characterized in that, The postmenopausal urogenital syndrome includes one or more of the following: vulvovaginal atrophy, bladder dysfunction, and urethral tissue damage.

6. The application according to claim 1, characterized in that, The herbicide peptides exert their effects through a non-estrogen-dependent pathway.

7. The application according to claim 6, characterized in that, The non-estrogen-dependent pathways include upregulating estrogen receptor α-phosphorylation levels.

8. The application according to claim 6, characterized in that, The non-estrogen-dependent pathway also includes upregulating protein kinase B phosphorylation levels, which exerts a repair effect by activating the AKT / ERα signaling pathway.

9. A pharmaceutical composition for treating postmenopausal urogenital syndrome, characterized in that, It contains leafy plant peptides and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is an oral or topical preparation.

11. The pharmaceutical composition according to claim 9, characterized in that, The oral preparation is a tablet, capsule, granule, or oral liquid; the external preparation is a vaginal gel, vaginal suppository, ointment, or cream.

12. A method for preparing the pharmaceutical composition according to any one of claims 10 to 11, characterized in that, This includes the steps of mixing edible herb peptides with pharmaceutically acceptable excipients and preparing them into oral or topical formulations using conventional pharmaceutical processes.

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