Efficacy and mechanism of Peony pods extract for treating premature ovarian failure: UPLC-Q-TOF/MS method combined with network pharmacology and experimental validation

Efficacy and mechanism of Peony pods extract for treating premature ovarian failure: UPLC-Q-TOF/MS method combined with network pharmacology and experimental validation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy and mechanism of Peony pods extract for treating premature ovarian failure: UPLC-Q-TOF/MS method combined with network pharmacology and experimental validation Cailian Fan, Haili Shan, Xining Geng, Hanghang Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6025082/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Peony pods, as a byproduct of peony seeds, are often discarded. This study aimed to explore the effect and mechanism of Peony pods extract (PPE) for treating premature ovarian failure (POF) by UPLC-Q-TOF/MS method combined with network pharmacology and in vivo experimental validation. Firstly, a total of 26 components of PPE were detected by UPLC-Q-TOF/MS analysis. Subsequently, cisplatin was used to construct the POF model, and PPE treatment could significantly improve the ovarian index, estrous cycle and structure of ovarian tissues, increase the serum levels of AMH and estradiol E 2 , and reduce the serum levels of FSH and LH in POF mice. Then, network pharmacology analysis was performed and found that multiple signaling pathways were involved in improving POF of PPE, including PI3K/AKT, Ras and Rap1 signaling pathways. To further verification, related proteins were detected by WB, and the expression levels of Bax, p-PI3K, and p-AKT were decreased, while the expression levels of Bcl-2, HO-1, and Nrf-2 were increased after PPE administration. In summary, PPE could exert therapeutic effect on POF by reducing cell apoptosis and improving hormone levels, showing great potential for application in functional food. Peony pods extract premature ovarian failure network pharmacology UPLC-Q-TOF/MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Premature ovarian failure (POF) is a common gynecological disease that occurs in women under the age of 40, which is characterized by a decrease in estrogen, an increase in gonadotropins, and ovarian failure [ 1 , 2 ] . The patient mainly presents with clinical symptoms such as amenorrhea, breast atrophy, hot flashes, and osteoporosis [ 1 ] . According to the epidemiological survey, the incidence rate of POF in women before the age of 40 is 1/100, before the age of 30 is 1/1000, before the age of 20 is 1/10000, and the incidence rate is increasing year by year with the intensification of social competition [ 3 ] . POF is recognized by the World Health Organization as one of the difficult gynecological diseases. At present, the main treatment method for POF is estrogen and progesterone replacement therapy, but it is often accompanied by adverse reactions such as osteoporosis and cardiovascular diseases [ 4 ] . In addition, long-term management is required after the diagnosis of POF, which seriously affects women's physical and mental health [ 5 ] . Therefore, it is urgent to seek better and less adverse treatment methods for treating POF. POF is a heterogeneous disease with a complex pathogenesis. Reports have shown that approximately 20–30% of POF patients have family inheritance [ 6 ] . The number of follicles in the ovaries and the rate of follicular atresia determine the duration of ovarian failure. Mutations and deletions in many genes can lead to POF [ 7 ] . Recent studies have found that POF is mainly associated with X chromosome abnormalities, autosomal abnormalities, and autoimmune diseases [ 8 , 9 ] . Autoimmune diseases produce some related antibodies due to immune hyperfunction, which can bind to ovarian specific antigens, resulting in antigen antibody reactions that disrupt ovarian function [ 10 ] . In addition, iatrogenic factors such as radiotherapy, chemotherapy, and gynecological surgery can often lead to POF [ 11 ] . Currently, approximately 25% of POF are related to iatrogenic factors [ 12 ] . Many chemotherapy drugs can cause primitive follicles to stop developing, reduce the number of follicles, thicken the ovarian capsule, and cause interstitial fibrosis [ 13 ] . In addition to the above factors, unhealthy lifestyle habits and psychological stress can cause ovulation disorders and early menopausal symptoms in women by affecting the normal regulation of the pituitary ovarian axis or directly affecting the secretion of the ovaries, leading to ovarian dysfunction [ 14 ] . Although there have been many studies on the pathogenesis of premature ovarian failure, none of them can fully explain the pathophysiological changes and clinical characteristics of POF. Therefore, it is necessary to further explore the mechanism of POF. Peony ( Paeonia suffrutiosa Andr.) belongs to the Ranunculaceae family and is first recorded in the "Shennong Classic of Materia Medica" [ 15 ] . It has a planting history of more than two thousand years in China [ 15 ] . Reports have displayed that flowers and the root bark of peony contain abundant flavonoids, which have physiological and pharmacological activities such as antioxidant, anti-inflammatory, antibacterial, anti-tumor, and anti-cancer, and are widely used in the pharmaceutical industry [ 16 – 18 ] . In addition, peony seeds contain a high amount of unsaturated fatty acids, which are considered a good source of edible oil [ 19 ] . Peony pods, as the residual pods after extracting peony seeds for oil, are often discarded as waste [ 20 ] . In recent years, studies have found that peony pods contain various types of compounds such as volatile oils, organic acids, and polysaccharides [ 21 , 22 ] . Pharmacological studies have shown that peony pods extract has anti-oxidant, anti-bacterial, and anti-inflammation activities [ 22 , 23 ] . Above research preliminarily indicates that peony pods have value for development and utilization. Unfortunately, the total annual production of peony pods exceeds 50,000 tons in China, all of which are discarded as waste, resulting in serious waste [ 20 ] . Therefore, it is necessary to conduct systematic research on the components and pharmacological activities of PPE to provide reference for further development and utilization of PPE. Based on this, UPLC-Q-TOF-MS/MS technology was used to analyze the components of PPE, and network pharmacology combined with in vivo experiments was performed to investigate the effect and mechanism of PPE in improving POF. Material and methods Reagents The primary antibodies against p-AKT (AF3262), AKT (AF0836), p-PI3K (AF3241), PI3K (AF6241), Nrf-2 (AF0639), and HO-1 (AF5393) were obtained from Affinity Biosciences (Jiangsu, China). The primary antibodies against β-tubulin (GB122667-100) and the secondary antibody against rabbit and mouse (GB23303/GB23301) were purchased from Wuhan Servicebio Technology Co., Ltd (Wuhan, China). The ECL system for western blotting substrate detection and BCA protein quantification kit were obtained from New Cell & Molecular Biotech Co., Ltd (Jiangsu, China). AMH, FSH, LH and E 2 test kits were obtained from Jiangsu Meimian Industrial Co., Ltd (Jiangsu, China). SOD, CAT, GSH-Px and MDA test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Ethanol was obtained from Damao Chemical Reagent Factory (Tianjin, China). Preparation of PPE Peony pods were collected from the Peony Breeding Base of Pingdingshan University and Pingdingshan Academy of Agricultural Sciences, and were identified by Dr. Xining Geng of Pingdingshan University. A specimen (PDSU-GZ109) was collected in Henan Engineering Research Center of Funiu Mountain’s Medicinal Resources Utilization and Molecular Medicine. Dried peony pods (60.0 kg) were added to 60% ethanol at a mass/volume ratio of 1:10, stirred to ensure uniform mixing. The mixture was heated and refluxed for extraction for 2 h, twice. After extraction, the supernatant was concentrated using a rotary evaporator, and finally freeze-dried to obtain 10.3 kg of PPE (17.28%). PPE was stored in a -20 ℃ freezer for future use. UPLC-Q-TOF/MS analysis 1.0 g of PPE was accurately weighed and added to a conical flask with a stopper. Then 50 mL of 60% methanol-water solution was added. After ultrasonic extraction for 30 min, the solution was centrifuged at a speed of 13,200 rpm for 10 min. The supernatant of PPE was obtained and analyzed by UPLC-Q-TOF/MS. UPLC analysis condition: Waters ACQUITYTM BEH C18 (2.1 × 50 mm, 1.7 µm) chromatographic column; Column temperature: 35°C; Flow rate: 0.4 mL/min; Mobile phase A: water (containing 0.1% formic acid), mobile phase B: acetonitrile (containing 0.1% formic acid). Elution gradient: 0-1.5 min, 5–15% B; 1.5–7.5 min, 15–40% B; 7.5–10.0 min, 40–45% B; 10.0–13.0 min, 45–55% B; 13.0–16.0 min, 55–90% B; 16.0–17.0 min, 90%-100% B; 17.0–18.0 min, 100%-100% B; 18.0–19.0 min, 100%-2%B; 19.0–20.0 min, 2% -2% B. The injection volume was 2 µL. Mass spectrum condition: the capillary voltage of ion source is 3.0 kV in positive ion mode. The positive ion mode of the cone hole voltage was 40.0 V, and the secondary cone hole voltage was 4.0 V. The ion source temperature is 100.0 ℃, the desolvation gas temperature was 300.0 ℃, the reverse blowing gas flow rate was 50.0 L/h, the desolvation gas flow rate was 800.0 L/h, and the collision gas was argon gas. In MSE mode, the low-energy channel collision energy was 5.0 eV, and the high-energy channel collision energy was 20.0–50.0 eV. Correct the mass axis with sodium formate solution and the mass accuracy with leucine enkephalin as the internal standard (positive ion mode m/z 556.2771). The LockSprayTM flow rate was 5.0 µL/min.The data was collected using the Waters Masslynx 4.1 mass spectrometry control platform, and the total ion chromatogram of the analyzed samples was analyzed in MSE mode. Network pharmacology analysis Firstly, Swiss Target ( http://www.swisstargetprediction.ch/ ) was used to obtain potential targets for chemical composition in PPE. Secondly, POF related human targets were gained by the OMIM ( https://omim.org/ ), GeneCards ( https://www.genecards.org/ ), and Drugbank ( https://www.drugbank.ca/ ) databases. After removing duplicate values of disease targets, Venny 2.1.0 was used to obtain the common target information of "PPE-POF". Meanwhile, the relevant information was edited into "drug-component", "target-gene" and other related files in the table. Then, cytoscape 3.10.2 software was used to construct an "effective ingredient target" network for PPE treatment of POF. Subsequently, the protein-protein interaction networks (PPI) was obtained from STRING. Finally, gene ontology (GO) and kyoto encylopaedia of genes and genomes (KEGG) analysis were enriched using DAVID ( http://david.abcc.ncifcrf.gov/ ). GO function analysis histogram was plotted by an online platform for data analysis and visualization ( https://www.bioinformatics.com.cn/ ). Animal experiments 70 female C57 mice (6–8 weeks, 22–25 g) were purchased from Huaxing Experimental Animal Farm in Huiji District, Zhengzhou City. All animals were fed a chow diet and distilled water, and were maintained on a regular cycle (12-h light/dark) at room temperature (24 ± 2°C) and 60–70% humidity. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Pingdingshan University (project approval number: PDSU-20240012). 70 mice were adaptively fed for one week. After one week, vaginal exfoliated cell smears of all mice were observed under microscope for 8 consecutive days. Mice with normal estrous cycles (calculated based on two estrous cycles) were selected for the experiment. After screening, 62 mice with normal estrous cycles were obtained for the experiment. 62 mice were randomly divided into the control group (n = 10), model group (n = 12), PPE-L group (50 mg/kg/d PPE, n = 10), PPE-M group (200 mg/kg/d PPE, n = 10), PPE-H group (400 mg/kg/d PPE, n = 10), and HRT group (0.3 mg/kg/d estradiol valerate and 1.3 mg/kg/d medroxyprogesterone acetate, n = 10). The control group was given an equal volume of physiological saline, while other mice were orally administered to each group at 9:00 am every day for the first 7 days. On the 8th day, cisplatin (3 mg/kg/day) was continuously injected intraperitoneally for 5 days. During 28 days of continuous gavage, vaginal exfoliated cells from mice were collected at 3:00 pm every day, and cells were observed under a light microscopy with a CCD camera (Olympus CKX53, Tokyo, Japan). Sample collection After the last administration, mice were only given water treatment. After 16 h, blood was collected from the eye sockets, centrifuged at 1,500 g for 15 min to separate the serum. The serum was transferred to the labeled EP tubes and stored in a -20℃ freezer for future use. After serum collection, the mice were euthanized by cervical dislocation with anesthesia. Then, bilateral ovaries were separated, and washed with physiological saline. The surface moisture was absorbed by filter paper and weighed on an electronic balance. The left ovaries of all mice were stored in a -80 ℃ freezer for future use, and the right ovaries of all mice were fixed in 4% paraformaldehyde solution for pathological observation. Histological staining of ovarian tissues Ovarian tissues were collected and fixed in 4% paraformaldehyde solution after mice was sacrificed. After fixed, ovarian tissues were embedded in paraffin and cut into 4- µ m thick slices for morphological and pathological evaluations. Tissue sections were stained with hematoxylin and eosin (H&E) and examined by a light microscopy with a CCD camera (Olympus CKX53, Tokyo, Japan).The number of follicles at each level was counted by two independent experienced technicians using the recognized criteria developed by Pedersen and Peters for follicular staging [ 24 ] . Biochemical marker analysis The separated serum was extracted from the EP tube and the levels of E 2 , FSH, LH and AMH in each group of serum were detected according to the instructions of corresponding ELISA kits. In addition, SOD, CAT, GSH-Px, and MDA in each group of ovarian tissues were measured following the instructions of corresponding test kits. Western blot analysis The left ovarian tissues were ground and added to lysis solution to extract total protein. 30 µg protein from each group of mice was used for western blot analysis as follows: the proteins were separated by 10% polyacrylamide gel electrophoresis at 80V, 20 min, then 100V, 60 min. After that, the gels were transferred to PVDF membrane under 250 mA conditions for 150 minutes, then sealed it with skim milk for 2h and incubated the primary antibody overnight. Next, the membranes were washed and incubated using the secondary antibody at room temperature for 2h. Finally, the targeted proteins were observed by adding chemiluminescence solution in a dark room. Statistical analyses All results were expressed as Mean ± SD. The significance of difference was calculated by a one-way ANOVA with Turkey's test, and p < 0.05 were considered significant. The quantitative analysis of proteins was performed by Image J software. Results Chemical components in PPE were detected by UPLC-Q-TOF/MS analysis The chemical components of PPE were identified based on their relative retention times (t R ), excimer ion peaks, secondary fragments, and other information. As displayed in Fig. 1 and Table S1 , a total of 26 components of PPE were identified by UPLC-Q-TOF/MS analysis. PPE improved ovarian function in cisplatin‑induced POF To evaluate whether PPE could prevent cisplatin-induced ovarian injury in vivo , the body weight, ovarian indices, estrous cycle and follicle count of mice were determined in our study. As displayed in Fig. 2 A and B , the weight, ovarian index of the model group in mice were significantly lower than those of the control group ( p < 0.01). After PPE and HRT treatment, the weight, ovarian indices of the PPE-M, PPE-H and HRT group were obviously improved ( p < 0.05). Vaginal smears were subsequently obtained to assess the estrous cycle of the mice to reflect ovarian function. As shown in Fig. 2 C and D , mice in the control group presented regular estrous cycles, and irregular estrous cycles were observed in the mice in the model group. While PPE and HRT treatment altered estrous cycle disorders in the mice. Furthermore, the development of follicles in the ovarian tissues of mice in each group was observed by HE staining (Fig. 2 E). Compared with the control group, most of the follicles in the model group were degenerated, which had irregular shapes, collapsed transparent bands, loose cells. After PPE and HRT intervention, the ovaries of mice in PPE-H, PPE-M and HRT group showed an increase in mature and secondary follicles, with multiple layers of tightly arranged granulosa cells, forming irregular cavities between cells filled with follicular fluid. In order to more accurately reflect follicular development, statistical analysis of the number of follicles was performed. Date in Fig. 2 F and G revealed that the model group exhibited a significant decrease in the number of antral follicles and corpora lutea at all stages ( p < 0.01), while the PPE and HRT treatment group showed an increase in the number of antral follicles and corpora lutea ( p < 0.05). These results showed that PPE has the ability to improved ovarian function in cisplatin-induced POF. PPE ameliorated hormone disorder in cisplatin‑induced POF Serum hormone levels are important indicators reflecting ovarian function in clinical practice [ 25 ] . Therefore, levels of E 2 , AMH, LH and FSH in the serum of mice were detected by Elisa kits. As displayed in Fig. 3 , the serum E 2 and AMH levels in the model group were significantly reduced ( p < 0.01), and levels of LH and FSH were was significantly increased in the model group compared with the control group ( p < 0.01). After oral administration of PPE, both PPE-M and PPE-H group were able to increase serum E 2 and AMH levels and reduce LH and FSH levels in POF mice ( p 0.05). Above result indicated that PPE could effectively improve the hormone imbalance in cisplatin-induced POF. PPE alleviated cisplatin-induced oxidative stress injury Research has confirmed that oxidative stress is closely related to POF [ 26 ] . Oxidative stress can mediate cell apoptosis through different pathways. As is well known, cisplatin can induce oxidative stress and trigger cell apoptosis by forming ROS such as hydroxyl radicals and superoxide [ 27 ] . Therefore, four indicators (activities of SOD, GSH-Px, CAT and MDA), which could reflect the level of oxidative stress, were determined using different kits [ 28 ] . Data in Fig. 4 revealed that cisplatin treatment significantly decreased the levels of SOD, CAT and GSH-Px, and increased the content of MDA ( p < 0.01) in mouse ovaries. Whereas the levels of SOD, CAT and GSH-Px were increased, and the content of MDA was decreased in mouse ovaries after PPE and HRT intervention ( p < 0.05). This result indicated that PPE could effectively alleviate cisplatin-induced oxidative stress damage in POF mice. Network construction, GO function and KEGG pathway enrichment analyzes Swiss Target online target prediction was conducted on the chemical components of PPE, and a total of 415 relevant targets were obtained ( Table S2 ). Additionally, 635 targets for POF were obtained from OMIM, GeneCards, and Drugbank databases ( Table S3 ). The intersection of PPE component targets and POF targets was performed by a Venn diagram (Fig. 5 A), which obtained 62 common target genes. To visualize the relationships between components and genes, the intersecting target data were imported into Cytoscape 3.10.2 software to construct a “component-target" network map of PPE for the treatment of POF ( Fig. 5 B, Table S4 ). Then, PPI network was constructed by importing 62 intersecting genes into STRING, the results of which were shown in Fig. 5 C and Table S5 . The PPI network consisted of 262 nodes and 876 edges, with an average node degree of 28.3. Subsequently, the result of PPI was imported into Cytoscape 3.10.2 software and analyzed the degree values of the targets using Network Analyzer in Cytoscape software. A total of 38 targets with degree values higher than the average were identified as the core targets for the treatment of POF with PPE. The top 10 target proteins with degree values were EGFR, AKT1, ESR1, STAT3, ERBB2, IL6, TNF, KDR, MTOR, and CASP3 (Fig. 5 D, Table S6 ). Finally, GO analysis and KEGG enrichment analysis were performed on the core targets of PPE treatment for POF using the DAVID platform. Through GO enrichment analysis (Fig. 5 E, Table S7 ), we found that there were a total of 108 items related to biological processes in GO enrichment analysis, which were related to phosphorylation, MAPK cascade reaction, cell proliferation, signal transduction. Meanwhile, there were 59 entries related to molecular functions, including protein binding, growth factor activity, enzyme binding, and estrogen activity. Additionally, There were 39 entries for cellular components, involving mitochondria, cell membranes, macromolecular complexes, endoplasmic reticulum. Among them, the targets of GO enrichment mainly included protein binding, positive regulation of gene expression, bidirectional regulation of cell proliferation, estrogen related reactions and mitochondrial activity. KEGG enrichment analysis showed that PPE may exert its protective effect against POF through resistance to EGFR tyrosine kinase inhibitors, endocrine resistance, PI3K/AKT signaling pathway, Ras signaling pathway and Rap1 signaling pathway (Fig. 5 F, Table S8 ). PPE regulated the PI3K/AKT and Nrf-2/HO-1 signaling pathway to alleviate cisplatin-induced POF Based on the KEGG pathway information obtained from the above network pharmacology analysis, we estimated that PPE may exert its effects through the PI3K/AKT signaling pathway and Nrf-2/HO-1 signaling pathway. Western blot was used to detect the expression levels of relevant proteins in ovarian tissues to validate the predicted pathways. As shown in Fig. 6 , the relative expression levels of Bax, p-PI3K, and p-AKT in the model group showed a certain degree of increase, while the relative expression levels of Bcl-2, HO-1, and Nrf-2 were significantly decreased compared with the control group, all of which were significant ( p < 0.01). After PPE treatment, the relative expression levels of Bax, p-PI3K, and p-AKT proteins showed a decreasing trend, and the relative expression levels of Bcl-2, HO-1, and Nrf-2 were significantly increased ( p < 0.01). These findings showed that PPE could alleviate cisplatin-induced POF by inhibiting the PI3K/AKT signaling pathway and activating the Nrf-2/HO-1 signaling pathway. Discussion Research has shown that the incidence of POF is increasing year by year worldwide, and POF has gradually become younger, which has become one of the medical problems [ 3 ] . It not only seriously affects the quality of life, but also brings huge psychological burden in women. At present, hormone replacement therapy is widely used in clinical practice [ 29 ] . Although this method can alleviate patients' symptoms, it will produce many toxic and side effects in the long term and increase the risk of stroke, breast cancer, cholecystitis and other diseases [ 29 ] . Therefore, it is very important to explore new therapeutic drugs. In this study, we found that PPE could significantly increase the ovarian organ index, serum levels of AMH and E 2 , reduce the level of LH and FSH, and improve estrous cycle disorders and follicular development in cisplatin-induced POF mice, indicating that PPE has the potential to improve and treat POF. At present, the common methods for constructing POF model include knocking out corresponding gene targets, damaging immune or metabolic functions, and using chemotherapy drugs or radiation [ 30 ] . Among them, chemotherapy drug is commonly used to construct POF model used for evaluating the efficacy and investigating the mechanism of POF due to its repeatability, short time consumption, and high success rate [ 31 ] . The commonly used chemotherapy drugs include cisplatin, cyclophosphamide, and epirubicin [ 31 ] . In this study, intraperitoneal injection of cisplatin was used to construct POF model mice. The experimental results showed that the ovarian index, serum E 2 and AMH levels were obviously decreased, while serum FSH and LH levels and blocked follicles were significantly increased treated by cisplatin, indicating that intraperitoneal injection of cisplatin could induce hormone secretion imbalance, disrupt ovarian morphology, and cause follicle development impairment, which was consistent with literature [ 32 ] . After PPE intervention, the levels of FSH and LH in the serum of mice were significantly reduced, while the levels of E 2 and AMH were significantly increased. In addition, there was an increase in mature follicles in the ovaries, suggesting that PPE could improve ovarian function in POF mice. Chinese herbal extracts have the characteristics of multi-component, multi-target, and multi pathway effects, which possess unique advantages in preventing and treating chronic and complex diseases [ 33 ] . Network pharmacology can construct biological network models such as drug-target network and gene-disease network, which can predict the potential targets and mechanisms of Chinese herbal extracts [ 34 ] . Therefore, network pharmacology is often used to investigate the material basis and mechanism of traditional Chinese medicine. In our study, result from network pharmacology demonstrated that the main active ingredients of PPE included agustidienoid, asiatic acid, luteolin, apigenin, quillaic acid, etc. These compounds were triterpenoids and flavonoids, respectively, and had antioxidant, anti-inflammatory, anti-apoptotic, and immune enhancing effects [ 35 – 38 ] . Furthermore, the top 10 targets ranked from 65 target information of PPE were EGFR, AKT1, ESR1, STAT3, ERBB2, IL6, TNF, KDR, MTOR, CASP3. These target proteins played a crucial role in the treatment of POF by PPE. Among them, EGFR can activate and promote angiogenesis, and has a regulatory effect on follicular maturation and apoptosis in the ovary [ 39 , 40 ] . AKT1, MTOR, and CASP3 genes are crucial in the occurrence and development of POF [ 41 ] . These targets are key factors in regulating DNA damage and apoptosis pathways. When cellular DNA is damaged, these genes activate downstream pro apoptotic genes, inducing a cascade reaction of apoptosis and ultimately leading to a decline in ovarian function [ 42 ] . ESR1 can directly act on follicles by upregulating estrogen levels, regulating their growth and excretion [ 43 ] . IL-6 and TNF can block the secretion of progesterone and LH receptors by ovarian granulosa cells, accelerating the production of blocked follicles [ 44 ] . Above all, it could be inferred that PPE could treat POF probably by reducing cell apoptosis, anti-inflammatory, and regulating estrogen receptors to improve the growth and development of follicles. Furthermore, KEGG enrichment analysis showed that PPE mainly improved POF through multiple pathways such as EGFR tyrosine kinase inhibitor resistance, endocrine resistance, PI3K/AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway, etc. Among these pathways, the PI3K/Akt signaling pathway has been confirmed to play an important role in the progression of POF [ 45 ] . The activation of various cytokines on PI3K leads to the production of PIP3, which in turn phosphorylates Akt [ 45 ] . After activation, this pathway can regulate the secretion of E 2 and progesterone, promote granulosa cell maturation, and participate in their proliferation and differentiation, which plays a significant role in the development of follicles [ 46 ] . Research has confirmed that overactivation of PI3K/Akt signaling pathway can accelerate premature development and apoptosis of primordial follicles in POF, while administration of traditional Chinese medicine monomers or extracts can effectively inhibit the activation of this signaling pathway to alleviate POF [ 47 ] . Quercetin, a polyphenol belonging to the class of flavonoids, could reduce the levels of phosphorylation and mRNA expression of PI3K, Akt, FOXO3a in POF rats to inhibit follicular atresia and granulosa cell apoptosis [ 48 ] . Polysaccharide extract from Angelica could significantly reduce the levels of p-Akt and FOXO3 protein expressions in POF mice to inhibit the progression of POF [ 49 ] . In our study, PPE could not only reduce the expressions of p-PI3K, p-Akt, and Bax proteins, but also increase the expression of Bcl-2 protein in the ovarian tissue of cisplatin-induced POF mice, suggesting that PPE could exert therapeutic effects on POF by inhibiting the PI3K/AKT signaling pathway. More and more evidence suggests that oxidative stress damage plays a critical role in chemotherapy-induced premature ovarian failure [ 26 ] . Cisplatin, a first-line chemotherapy drug, is commonly used to construct ovarian injury models [ 50 ] . It can induce oxidative stress and trigger cell apoptosis by forming ROS such as hydroxyl radicals and superoxide [ 50 ] . In this study, PPE intervention could significantly decrease the content of MDA, and increase levels of SOD, CAT and GSH-Px, indicating that PPE could alleviate oxidative stress damage in cisplatin-induced POF. In addition, Nrf-2 is currently considered the most important endogenous antioxidant protein [ 51 ] . Research has shown that Nrf-2 can activate downstream factors, such as NQO1 and HO-1, to clear ROS through sustained enzymatic reactions [ 52 ] . Our findings showed that PPE treatment increased the relative expression levels of HO-1 and Nrf-2, suggesting that PPE could alleviate cisplatin-induced oxidative stress by activating the Nrf-2/HO-1 signaling pathway. Conclusion In this study, we found that PPE could alleviated cisplatin-induced POF by improving hormone levels, enhance antioxidant capacity and alleviate cell apoptosis. Mechanistically, PPE prevented cisplatin-induced POF by inhibiting the PI3K/AKT signaling pathway and activating the Nrf-2/HO-1 signaling pathway. Our study provides preliminary evidence that PPE has the potential to be developed as a dietary supplement for improving POF, which is beneficial for further development and utilization of PPE. Abbreviations POF Premature ovarian failure UPLC-Q-TOF/MS Ultrahigh performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry PPE Peony pods extract KEGG Kyoto encyclopedia of genes and genomes PI3K Phosphatidylinositol 3-kinase AKT Protein kinase B Ras Rat sarcoma Rap1 RAS-associated protein 1 AMH Anti-mullerian hormone E 2 Estradiol FSH Follicle stimulating hormone LH Luteinizing hormone SOD Superoxide dismutase GSH-Px Glutathione peroxidase MDA Malondialdehyde Bax Bcl-2 associated X protein Bcl-2 B-cell lymphoma/leukemia 2 Nrf-2 Nuclear factor erythroid 2-related factor 2 HO-1 Heme oxygenase 1 PPI Protein-protein interaction networks GO Gene ontology CAT Catalase HRT Hormone replacement therapy Declarations Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary. Ethics Statement The study received ethical approval from the Institutional Animal Care and Use Committee (IACUC) of Pingdingshan University (project approval number: PDSU-20240012). All participants signed an informed consent form. Author contributions Wang H.H. designed this study. Fan C.L., Wang H.H., Geng X.N. and Shan H.L. performed the experiment, analyzed all data, wrote and revised the paper. Acknowledgements We are grateful to Dr. Xining Geng for identifying Peony Pods and the Henan Engineering Research Center of Funiu Mountain’s Medicinal Resources Utilization and Molecular Medicine for providing experimental instruments and equipment. Competing interests The authors declare no conflicts of interest. Funding This work was supported by the Scientific Research Foundation for the introduction of talent of Pingdingshan University (No. PXY-BSQD-2022040, PXY-BSQD-2024025, PXY-BSQD-202109 ) and Henan scientific and technological research projects (No. 232102310460). 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(2) Methyl gallate. (3) Albiflorin. (4) Paeoniflorin. (5) Ellagic acid. (6) Di-O-galloyl-2,3-(S)-hexahydroxydiphenoylscyllo-quercitol II. (7) Nasutin C. (8) Luteolin. (9) Polyporusterone B. (10) Apigenin. (11) Scolopianate A. (12) Suffruticosol A. (13) Cannabidiolic acid. (14) Hecogenin acetate. (15) Angustidienolide. (16) Quillaic acid. (17) Asiatic acid. (18) 30-Norhederagenin. (19) Hederagenin. (20) Ethyl(22E)-3,3-[1,2-ethanediylbis(oxy)]cholesta-4,22-dien-24-oate. (21) 11-Oxo-oleanolic acid. (22) Dihydrocelastrol. (23) Oleanolic acid. (24) Ursolic acid. (25) 3-oxo-olean-12-en-28-oic acid. (26) 3-oxo-urs-12-en-28- oic acid.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/18ff395768ec2af9ad47dcfc.png"},{"id":77622768,"identity":"babbf2d0-482e-482e-883e-777fdf889a7f","added_by":"auto","created_at":"2025-03-03 16:10:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1262191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPE improved ovarian function in cisplatin-induced POF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): Body weight of mice after treatment in each group (n=9-10). (B): ovarian index of mice after treatment in each group (n=9-10). (C): Representative images of HE staining of mice at various stages of the estrous cycle (n=3), Scale bars, 50\u0026nbsp;µm. (D): Percentage of estrous cycle in each stage. (E): HE staining of the ovaries in each group. Scale bars, 100\u0026nbsp;µm. (F): Ratio of number of antral follicles in mice (n=3). (G): Ratio of number of corpora lutea in mice (n=3). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the model group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/08e14ed28053a8dee4eef2a8.png"},{"id":77621114,"identity":"70ad21b0-8783-4427-99e1-85a0479171ab","added_by":"auto","created_at":"2025-03-03 15:44:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1680438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPE ameliorated hormone disorder in cisplatin-induced POF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): Serum E\u003csub\u003e2\u003c/sub\u003e of mice after treatment in each group (n=6). (B): Serum AMH of mice after treatment in each group (n=6). (C): Serum LH of mice after treatment in each group (n=6). (D): Serum FSH of mice after treatment in each group (n=6). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the model group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/894ec83a0012ff801f67253d.png"},{"id":77621119,"identity":"a42e918e-9fe4-480f-a211-34fed75636a9","added_by":"auto","created_at":"2025-03-03 15:44:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1097211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPE alleviated oxidative stress injury in cisplatin-induced POF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp; : SOD level in ovarian tissue after treatment in each group (n=6). (B): CAT level in ovarian tissue after treatment in each group (n=6). (C): GSH-Px level in ovarian tissue after treatment in each group (n=6). (D): MDA level in ovarian tissue after treatment in each group (n=6). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the model group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/4286d0960b5cddb3bbeb2b5b.png"},{"id":77622255,"identity":"50abffbd-d0c8-4696-bab4-46f2dc12cea9","added_by":"auto","created_at":"2025-03-03 16:00:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1250716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork pharmacology of PPE protecting against POF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Veen diagrams demonstrate of common targets by PPE and POF. B: Component-target network of PPE. C: protein–protein interaction (PPI) network. D:38 targets with degree values higher than the average. E: Gene Ontology (GO) analysis of PPE. F: Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of PPE.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/429a9d82d3949149ae313eba.png"},{"id":77621115,"identity":"de62ce98-fa98-43bf-bd3e-293570441641","added_by":"auto","created_at":"2025-03-03 15:44:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2516051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPE regulated the PI3K/AKT and Nrf-2/HO-1 signaling pathway to alleviate cisplatin-induced POF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): Detection of Bcl-2, Bax, HO-1, Nrf-2, AKT, p-AKT, PI3K and p-PI3K protein expression levels in ovarian tissues by WB in each group (n=3). (B): Quantitative analysis of Bcl-2/Bax protein level. (C): Quantitative analysis of HO-1/β-tubulin protein level. (D): Quantitative analysis of p-AKT/AKT protein level. (E): Quantitative analysis of p-PI3K/PI3K protein level. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus the model group.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/61d1bfc229683b9ff0e1b2ac.png"},{"id":77888563,"identity":"4e6f6ede-3b75-4866-89bb-500541fb6d05","added_by":"auto","created_at":"2025-03-06 13:33:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7588575,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/deee3d77-063e-4b55-a8e8-c6eb857260b8.pdf"},{"id":77621595,"identity":"22664a57-0edb-4105-9667-cfab4ef94f20","added_by":"auto","created_at":"2025-03-03 15:52:52","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4964031,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.rar","url":"https://assets-eu.researchsquare.com/files/rs-6025082/v1/bb58ffbf520ffd03ba0c5f7b.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy and mechanism of Peony pods extract for treating premature ovarian failure: UPLC-Q-TOF/MS method combined with network pharmacology and experimental validation","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePremature ovarian failure (POF) is a common gynecological disease that occurs in women under the age of 40, which is characterized by a decrease in estrogen, an increase in gonadotropins, and ovarian failure\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The patient mainly presents with clinical symptoms such as amenorrhea, breast atrophy, hot flashes, and osteoporosis\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. According to the epidemiological survey, the incidence rate of POF in women before the age of 40 is 1/100, before the age of 30 is 1/1000, before the age of 20 is 1/10000, and the incidence rate is increasing year by year with the intensification of social competition\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. POF is recognized by the World Health Organization as one of the difficult gynecological diseases. At present, the main treatment method for POF is estrogen and progesterone replacement therapy, but it is often accompanied by adverse reactions such as osteoporosis and cardiovascular diseases\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In addition, long-term management is required after the diagnosis of POF, which seriously affects women's physical and mental health\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is urgent to seek better and less adverse treatment methods for treating POF.\u003c/p\u003e \u003cp\u003ePOF is a heterogeneous disease with a complex pathogenesis. Reports have shown that approximately 20\u0026ndash;30% of POF patients have family inheritance\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The number of follicles in the ovaries and the rate of follicular atresia determine the duration of ovarian failure. Mutations and deletions in many genes can lead to POF\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Recent studies have found that POF is mainly associated with X chromosome abnormalities, autosomal abnormalities, and autoimmune diseases\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Autoimmune diseases produce some related antibodies due to immune hyperfunction, which can bind to ovarian specific antigens, resulting in antigen antibody reactions that disrupt ovarian function\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In addition, iatrogenic factors such as radiotherapy, chemotherapy, and gynecological surgery can often lead to POF\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Currently, approximately 25% of POF are related to iatrogenic factors\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Many chemotherapy drugs can cause primitive follicles to stop developing, reduce the number of follicles, thicken the ovarian capsule, and cause interstitial fibrosis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In addition to the above factors, unhealthy lifestyle habits and psychological stress can cause ovulation disorders and early menopausal symptoms in women by affecting the normal regulation of the pituitary ovarian axis or directly affecting the secretion of the ovaries, leading to ovarian dysfunction\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Although there have been many studies on the pathogenesis of premature ovarian failure, none of them can fully explain the pathophysiological changes and clinical characteristics of POF. Therefore, it is necessary to further explore the mechanism of POF.\u003c/p\u003e \u003cp\u003ePeony (\u003cem\u003ePaeonia suffrutiosa\u003c/em\u003e Andr.) belongs to the Ranunculaceae family and is first recorded in the \"Shennong Classic of Materia Medica\"\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. It has a planting history of more than two thousand years in China\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Reports have displayed that flowers and the root bark of peony contain abundant flavonoids, which have physiological and pharmacological activities such as antioxidant, anti-inflammatory, antibacterial, anti-tumor, and anti-cancer, and are widely used in the pharmaceutical industry\u003csup\u003e[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. In addition, peony seeds contain a high amount of unsaturated fatty acids, which are considered a good source of edible oil\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Peony pods, as the residual pods after extracting peony seeds for oil, are often discarded as waste\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In recent years, studies have found that peony pods contain various types of compounds such as volatile oils, organic acids, and polysaccharides\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Pharmacological studies have shown that peony pods extract has anti-oxidant, anti-bacterial, and anti-inflammation activities\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Above research preliminarily indicates that peony pods have value for development and utilization. Unfortunately, the total annual production of peony pods exceeds 50,000 tons in China, all of which are discarded as waste, resulting in serious waste\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is necessary to conduct systematic research on the components and pharmacological activities of PPE to provide reference for further development and utilization of PPE.\u003c/p\u003e \u003cp\u003eBased on this, UPLC-Q-TOF-MS/MS technology was used to analyze the components of PPE, and network pharmacology combined with \u003cem\u003ein vivo\u003c/em\u003e experiments was performed to investigate the effect and mechanism of PPE in improving POF.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eThe primary antibodies against p-AKT (AF3262), AKT (AF0836), p-PI3K (AF3241), PI3K (AF6241), Nrf-2 (AF0639), and HO-1 (AF5393) were obtained from Affinity Biosciences (Jiangsu, China). The primary antibodies against β-tubulin (GB122667-100) and the secondary antibody against rabbit and mouse (GB23303/GB23301) were purchased from Wuhan Servicebio Technology Co., Ltd (Wuhan, China). The ECL system for western blotting substrate detection and BCA protein quantification kit were obtained from New Cell \u0026amp; Molecular Biotech Co., Ltd (Jiangsu, China). AMH, FSH, LH and E\u003csub\u003e2\u003c/sub\u003e test kits were obtained from Jiangsu Meimian Industrial Co., Ltd (Jiangsu, China). SOD, CAT, GSH-Px and MDA test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Ethanol was obtained from Damao Chemical Reagent Factory (Tianjin, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of PPE\u003c/h3\u003e\n\u003cp\u003ePeony pods were collected from the Peony Breeding Base of Pingdingshan University and Pingdingshan Academy of Agricultural Sciences, and were identified by Dr. Xining Geng of Pingdingshan University. A specimen (PDSU-GZ109) was collected in Henan Engineering Research Center of Funiu Mountain\u0026rsquo;s Medicinal Resources Utilization and Molecular Medicine.\u003c/p\u003e \u003cp\u003eDried peony pods (60.0 kg) were added to 60% ethanol at a mass/volume ratio of 1:10, stirred to ensure uniform mixing. The mixture was heated and refluxed for extraction for 2 h, twice. After extraction, the supernatant was concentrated using a rotary evaporator, and finally freeze-dried to obtain 10.3 kg of PPE (17.28%). PPE was stored in a -20 ℃ freezer for future use.\u003c/p\u003e\n\u003ch3\u003eUPLC-Q-TOF/MS analysis\u003c/h3\u003e\n\u003cp\u003e1.0 g of PPE was accurately weighed and added to a conical flask with a stopper. Then 50 mL of 60% methanol-water solution was added. After ultrasonic extraction for 30 min, the solution was centrifuged at a speed of 13,200 rpm for 10 min. The supernatant of PPE was obtained and analyzed by UPLC-Q-TOF/MS.\u003c/p\u003e \u003cp\u003eUPLC analysis condition: Waters ACQUITYTM BEH C18 (2.1 \u0026times; 50 mm, 1.7 \u0026micro;m) chromatographic column; Column temperature: 35\u0026deg;C; Flow rate: 0.4 mL/min; Mobile phase A: water (containing 0.1% formic acid), mobile phase B: acetonitrile (containing 0.1% formic acid). Elution gradient: 0-1.5 min, 5\u0026ndash;15% B; 1.5\u0026ndash;7.5 min, 15\u0026ndash;40% B; 7.5\u0026ndash;10.0 min, 40\u0026ndash;45% B; 10.0\u0026ndash;13.0 min, 45\u0026ndash;55% B; 13.0\u0026ndash;16.0 min, 55\u0026ndash;90% B; 16.0\u0026ndash;17.0 min, 90%-100% B; 17.0\u0026ndash;18.0 min, 100%-100% B; 18.0\u0026ndash;19.0 min, 100%-2%B; 19.0\u0026ndash;20.0 min, 2% -2% B. The injection volume was 2 \u0026micro;L.\u003c/p\u003e \u003cp\u003eMass spectrum condition: the capillary voltage of ion source is 3.0 kV in positive ion mode. The positive ion mode of the cone hole voltage was 40.0 V, and the secondary cone hole voltage was 4.0 V. The ion source temperature is 100.0 ℃, the desolvation gas temperature was 300.0 ℃, the reverse blowing gas flow rate was 50.0 L/h, the desolvation gas flow rate was 800.0 L/h, and the collision gas was argon gas. In MSE mode, the low-energy channel collision energy was 5.0 eV, and the high-energy channel collision energy was 20.0\u0026ndash;50.0 eV. Correct the mass axis with sodium formate solution and the mass accuracy with leucine enkephalin as the internal standard (positive ion mode m/z 556.2771). The LockSprayTM flow rate was 5.0 \u0026micro;L/min.The data was collected using the Waters Masslynx 4.1 mass spectrometry control platform, and the total ion chromatogram of the analyzed samples was analyzed in MSE mode.\u003c/p\u003e\n\u003ch3\u003eNetwork pharmacology analysis\u003c/h3\u003e\n\u003cp\u003eFirstly, Swiss Target (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to obtain potential targets for chemical composition in PPE. Secondly, POF related human targets were gained by the OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Drugbank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.drugbank.ca/\u003c/span\u003e\u003cspan address=\"https://www.drugbank.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases. After removing duplicate values of disease targets, Venny 2.1.0 was used to obtain the common target information of \"PPE-POF\". Meanwhile, the relevant information was edited into \"drug-component\", \"target-gene\" and other related files in the table. Then, cytoscape 3.10.2 software was used to construct an \"effective ingredient target\" network for PPE treatment of POF. Subsequently, the protein-protein interaction networks (PPI) was obtained from STRING. Finally, gene ontology (GO) and kyoto encylopaedia of genes and genomes (KEGG) analysis were enriched using DAVID (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://david.abcc.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"http://david.abcc.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). GO function analysis histogram was plotted by an online platform for data analysis and visualization (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eAnimal experiments\u003c/h3\u003e\n\u003cp\u003e70 female C57 mice (6\u0026ndash;8 weeks, 22\u0026ndash;25 g) were purchased from Huaxing Experimental Animal Farm in Huiji District, Zhengzhou City. All animals were fed a chow diet and distilled water, and were maintained on a regular cycle (12-h light/dark) at room temperature (24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and 60\u0026ndash;70% humidity. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Pingdingshan University (project approval number: PDSU-20240012).\u003c/p\u003e \u003cp\u003e70 mice were adaptively fed for one week. After one week, vaginal exfoliated cell smears of all mice were observed under microscope for 8 consecutive days. Mice with normal estrous cycles (calculated based on two estrous cycles) were selected for the experiment. After screening, 62 mice with normal estrous cycles were obtained for the experiment. 62 mice were randomly divided into the control group (n\u0026thinsp;=\u0026thinsp;10), model group (n\u0026thinsp;=\u0026thinsp;12), PPE-L group (50 mg/kg/d PPE, n\u0026thinsp;=\u0026thinsp;10), PPE-M group (200 mg/kg/d PPE, n\u0026thinsp;=\u0026thinsp;10), PPE-H group (400 mg/kg/d PPE, n\u0026thinsp;=\u0026thinsp;10), and HRT group (0.3 mg/kg/d estradiol valerate and 1.3 mg/kg/d medroxyprogesterone acetate, n\u0026thinsp;=\u0026thinsp;10). The control group was given an equal volume of physiological saline, while other mice were orally administered to each group at 9:00 am every day for the first 7 days. On the 8th day, cisplatin (3 mg/kg/day) was continuously injected intraperitoneally for 5 days. During 28 days of continuous gavage, vaginal exfoliated cells from mice were collected at 3:00 pm every day, and cells were observed under a light microscopy with a CCD camera (Olympus CKX53, Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eAfter the last administration, mice were only given water treatment. After 16 h, blood was collected from the eye sockets, centrifuged at 1,500 g for 15 min to separate the serum. The serum was transferred to the labeled EP tubes and stored in a -20℃ freezer for future use.\u003c/p\u003e \u003cp\u003eAfter serum collection, the mice were euthanized by cervical dislocation with anesthesia. Then, bilateral ovaries were separated, and washed with physiological saline. The surface moisture was absorbed by filter paper and weighed on an electronic balance. The left ovaries of all mice were stored in a -80 ℃ freezer for future use, and the right ovaries of all mice were fixed in 4% paraformaldehyde solution for pathological observation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistological staining of ovarian tissues\u003c/h3\u003e\n\u003cp\u003eOvarian tissues were collected and fixed in 4% paraformaldehyde solution after mice was sacrificed. After fixed, ovarian tissues were embedded in paraffin and cut into 4-\u003cem\u003e\u0026micro;\u003c/em\u003em thick slices for morphological and pathological evaluations. Tissue sections were stained with hematoxylin and eosin (H\u0026amp;E) and examined by a light microscopy with a CCD camera (Olympus CKX53, Tokyo, Japan).The number of follicles at each level was counted by two independent experienced technicians using the recognized criteria developed by Pedersen and Peters for follicular staging\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eBiochemical marker analysis\u003c/h3\u003e\n\u003cp\u003eThe separated serum was extracted from the EP tube and the levels of E\u003csub\u003e2\u003c/sub\u003e, FSH, LH and AMH in each group of serum were detected according to the instructions of corresponding ELISA kits. In addition, SOD, CAT, GSH-Px, and MDA in each group of ovarian tissues were measured following the instructions of corresponding test kits.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe left ovarian tissues were ground and added to lysis solution to extract total protein. 30 \u0026micro;g protein from each group of mice was used for western blot analysis as follows: the proteins were separated by 10% polyacrylamide gel electrophoresis at 80V, 20 min, then 100V, 60 min. After that, the gels were transferred to PVDF membrane under 250 mA conditions for 150 minutes, then sealed it with skim milk for 2h and incubated the primary antibody overnight. Next, the membranes were washed and incubated using the secondary antibody at room temperature for 2h. Finally, the targeted proteins were observed by adding chemiluminescence solution in a dark room.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll results were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The significance of difference was calculated by a one-way ANOVA with Turkey's test, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. The quantitative analysis of proteins was performed by Image J software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChemical components in PPE were detected by UPLC-Q-TOF/MS analysis\u003c/h2\u003e \u003cp\u003eThe chemical components of PPE were identified based on their relative retention times (t\u003csub\u003eR\u003c/sub\u003e), excimer ion peaks, secondary fragments, and other information. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e, a total of 26 components of PPE were identified by UPLC-Q-TOF/MS analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePPE improved ovarian function in cisplatin‑induced POF\u003c/h2\u003e \u003cp\u003eTo evaluate whether PPE could prevent cisplatin-induced ovarian injury \u003cem\u003ein vivo\u003c/em\u003e, the body weight, ovarian indices, estrous cycle and follicle count of mice were determined in our study. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cb\u003eB\u003c/b\u003e, the weight, ovarian index of the model group in mice were significantly lower than those of the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After PPE and HRT treatment, the weight, ovarian indices of the PPE-M, PPE-H and HRT group were obviously improved (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Vaginal smears were subsequently obtained to assess the estrous cycle of the mice to reflect ovarian function. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cb\u003eD\u003c/b\u003e, mice in the control group presented regular estrous cycles, and irregular estrous cycles were observed in the mice in the model group. While PPE and HRT treatment altered estrous cycle disorders in the mice. Furthermore, the development of follicles in the ovarian tissues of mice in each group was observed by HE staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Compared with the control group, most of the follicles in the model group were degenerated, which had irregular shapes, collapsed transparent bands, loose cells. After PPE and HRT intervention, the ovaries of mice in PPE-H, PPE-M and HRT group showed an increase in mature and secondary follicles, with multiple layers of tightly arranged granulosa cells, forming irregular cavities between cells filled with follicular fluid. In order to more accurately reflect follicular development, statistical analysis of the number of follicles was performed. Date in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cb\u003eG\u003c/b\u003e revealed that the model group exhibited a significant decrease in the number of antral follicles and corpora lutea at all stages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the PPE and HRT treatment group showed an increase in the number of antral follicles and corpora lutea (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results showed that PPE has the ability to improved ovarian function in cisplatin-induced POF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePPE ameliorated hormone disorder in cisplatin‑induced POF\u003c/h2\u003e \u003cp\u003eSerum hormone levels are important indicators reflecting ovarian function in clinical practice\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Therefore, levels of E\u003csub\u003e2\u003c/sub\u003e, AMH, LH and FSH in the serum of mice were detected by Elisa kits. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the serum E\u003csub\u003e2\u003c/sub\u003e and AMH levels in the model group were significantly reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and levels of LH and FSH were was significantly increased in the model group compared with the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After oral administration of PPE, both PPE-M and PPE-H group were able to increase serum E\u003csub\u003e2\u003c/sub\u003e and AMH levels and reduce LH and FSH levels in POF mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Moreover, there was no significant difference between PPE-H group and the positive drug group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Above result indicated that PPE could effectively improve the hormone imbalance in cisplatin-induced POF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePPE alleviated cisplatin-induced oxidative stress injury\u003c/h2\u003e \u003cp\u003eResearch has confirmed that oxidative stress is closely related to POF\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Oxidative stress can mediate cell apoptosis through different pathways. As is well known, cisplatin can induce oxidative stress and trigger cell apoptosis by forming ROS such as hydroxyl radicals and superoxide\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Therefore, four indicators (activities of SOD, GSH-Px, CAT and MDA), which could reflect the level of oxidative stress, were determined using different kits\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Data in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e revealed that cisplatin treatment significantly decreased the levels of SOD, CAT and GSH-Px, and increased the content of MDA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in mouse ovaries. Whereas the levels of SOD, CAT and GSH-Px were increased, and the content of MDA was decreased in mouse ovaries after PPE and HRT intervention (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This result indicated that PPE could effectively alleviate cisplatin-induced oxidative stress damage in POF mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNetwork construction, GO function and KEGG pathway enrichment analyzes\u003c/h2\u003e \u003cp\u003eSwiss Target online target prediction was conducted on the chemical components of PPE, and a total of 415 relevant targets were obtained (\u003cb\u003eTable S2\u003c/b\u003e). Additionally, 635 targets for POF were obtained from OMIM, GeneCards, and Drugbank databases (\u003cb\u003eTable S3\u003c/b\u003e). The intersection of PPE component targets and POF targets was performed by a Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), which obtained 62 common target genes. To visualize the relationships between components and genes, the intersecting target data were imported into Cytoscape 3.10.2 software to construct a \u0026ldquo;component-target\" network map of PPE for the treatment of POF \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eTable S4\u003c/b\u003e). Then, PPI network was constructed by importing 62 intersecting genes into STRING, the results of which were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cb\u003eTable S5\u003c/b\u003e. The PPI network consisted of 262 nodes and 876 edges, with an average node degree of 28.3. Subsequently, the result of PPI was imported into Cytoscape 3.10.2 software and analyzed the degree values of the targets using Network Analyzer in Cytoscape software. A total of 38 targets with degree values higher than the average were identified as the core targets for the treatment of POF with PPE. The top 10 target proteins with degree values were EGFR, AKT1, ESR1, STAT3, ERBB2, IL6, TNF, KDR, MTOR, and CASP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cb\u003eTable S6\u003c/b\u003e). Finally, GO analysis and KEGG enrichment analysis were performed on the core targets of PPE treatment for POF using the DAVID platform. Through GO enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cb\u003eTable S7\u003c/b\u003e), we found that there were a total of 108 items related to biological processes in GO enrichment analysis, which were related to phosphorylation, MAPK cascade reaction, cell proliferation, signal transduction. Meanwhile, there were 59 entries related to molecular functions, including protein binding, growth factor activity, enzyme binding, and estrogen activity. Additionally, There were 39 entries for cellular components, involving mitochondria, cell membranes, macromolecular complexes, endoplasmic reticulum. Among them, the targets of GO enrichment mainly included protein binding, positive regulation of gene expression, bidirectional regulation of cell proliferation, estrogen related reactions and mitochondrial activity. KEGG enrichment analysis showed that PPE may exert its protective effect against POF through resistance to EGFR tyrosine kinase inhibitors, endocrine resistance, PI3K/AKT signaling pathway, Ras signaling pathway and Rap1 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, \u003cb\u003eTable S8\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePPE regulated the PI3K/AKT and Nrf-2/HO-1 signaling pathway to alleviate cisplatin-induced POF\u003c/h2\u003e \u003cp\u003eBased on the KEGG pathway information obtained from the above network pharmacology analysis, we estimated that PPE may exert its effects through the PI3K/AKT signaling pathway and Nrf-2/HO-1 signaling pathway. Western blot was used to detect the expression levels of relevant proteins in ovarian tissues to validate the predicted pathways. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the relative expression levels of Bax, p-PI3K, and p-AKT in the model group showed a certain degree of increase, while the relative expression levels of Bcl-2, HO-1, and Nrf-2 were significantly decreased compared with the control group, all of which were significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After PPE treatment, the relative expression levels of Bax, p-PI3K, and p-AKT proteins showed a decreasing trend, and the relative expression levels of Bcl-2, HO-1, and Nrf-2 were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These findings showed that PPE could alleviate cisplatin-induced POF by inhibiting the PI3K/AKT signaling pathway and activating the Nrf-2/HO-1 signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eResearch has shown that the incidence of POF is increasing year by year worldwide, and POF has gradually become younger, which has become one of the medical problems\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. It not only seriously affects the quality of life, but also brings huge psychological burden in women. At present, hormone replacement therapy is widely used in clinical practice\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Although this method can alleviate patients' symptoms, it will produce many toxic and side effects in the long term and increase the risk of stroke, breast cancer, cholecystitis and other diseases\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is very important to explore new therapeutic drugs. In this study, we found that PPE could significantly increase the ovarian organ index, serum levels of AMH and E\u003csub\u003e2\u003c/sub\u003e, reduce the level of LH and FSH, and improve estrous cycle disorders and follicular development in cisplatin-induced POF mice, indicating that PPE has the potential to improve and treat POF.\u003c/p\u003e \u003cp\u003eAt present, the common methods for constructing POF model include knocking out corresponding gene targets, damaging immune or metabolic functions, and using chemotherapy drugs or radiation\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Among them, chemotherapy drug is commonly used to construct POF model used for evaluating the efficacy and investigating the mechanism of POF due to its repeatability, short time consumption, and high success rate\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The commonly used chemotherapy drugs include cisplatin, cyclophosphamide, and epirubicin\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In this study, intraperitoneal injection of cisplatin was used to construct POF model mice. The experimental results showed that the ovarian index, serum E\u003csub\u003e2\u003c/sub\u003e and AMH levels were obviously decreased, while serum FSH and LH levels and blocked follicles were significantly increased treated by cisplatin, indicating that intraperitoneal injection of cisplatin could induce hormone secretion imbalance, disrupt ovarian morphology, and cause follicle development impairment, which was consistent with literature\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. After PPE intervention, the levels of FSH and LH in the serum of mice were significantly reduced, while the levels of E\u003csub\u003e2\u003c/sub\u003e and AMH were significantly increased. In addition, there was an increase in mature follicles in the ovaries, suggesting that PPE could improve ovarian function in POF mice.\u003c/p\u003e \u003cp\u003eChinese herbal extracts have the characteristics of multi-component, multi-target, and multi pathway effects, which possess unique advantages in preventing and treating chronic and complex diseases\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Network pharmacology can construct biological network models such as drug-target network and gene-disease network, which can predict the potential targets and mechanisms of Chinese herbal extracts\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Therefore, network pharmacology is often used to investigate the material basis and mechanism of traditional Chinese medicine. In our study, result from network pharmacology demonstrated that the main active ingredients of PPE included agustidienoid, asiatic acid, luteolin, apigenin, quillaic acid, etc. These compounds were triterpenoids and flavonoids, respectively, and had antioxidant, anti-inflammatory, anti-apoptotic, and immune enhancing effects\u003csup\u003e[\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the top 10 targets ranked from 65 target information of PPE were EGFR, AKT1, ESR1, STAT3, ERBB2, IL6, TNF, KDR, MTOR, CASP3. These target proteins played a crucial role in the treatment of POF by PPE. Among them, EGFR can activate and promote angiogenesis, and has a regulatory effect on follicular maturation and apoptosis in the ovary\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. AKT1, MTOR, and CASP3 genes are crucial in the occurrence and development of POF\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. These targets are key factors in regulating DNA damage and apoptosis pathways. When cellular DNA is damaged, these genes activate downstream pro apoptotic genes, inducing a cascade reaction of apoptosis and ultimately leading to a decline in ovarian function\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. ESR1 can directly act on follicles by upregulating estrogen levels, regulating their growth and excretion\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. IL-6 and TNF can block the secretion of progesterone and LH receptors by ovarian granulosa cells, accelerating the production of blocked follicles\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Above all, it could be inferred that PPE could treat POF probably by reducing cell apoptosis, anti-inflammatory, and regulating estrogen receptors to improve the growth and development of follicles.\u003c/p\u003e \u003cp\u003eFurthermore, KEGG enrichment analysis showed that PPE mainly improved POF through multiple pathways such as EGFR tyrosine kinase inhibitor resistance, endocrine resistance, PI3K/AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway, etc. Among these pathways, the PI3K/Akt signaling pathway has been confirmed to play an important role in the progression of POF\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. The activation of various cytokines on PI3K leads to the production of PIP3, which in turn phosphorylates Akt\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. After activation, this pathway can regulate the secretion of E\u003csub\u003e2\u003c/sub\u003e and progesterone, promote granulosa cell maturation, and participate in their proliferation and differentiation, which plays a significant role in the development of follicles\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Research has confirmed that overactivation of PI3K/Akt signaling pathway can accelerate premature development and apoptosis of primordial follicles in POF, while administration of traditional Chinese medicine monomers or extracts can effectively inhibit the activation of this signaling pathway to alleviate POF\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Quercetin, a polyphenol belonging to the class of flavonoids, could reduce the levels of phosphorylation and mRNA expression of PI3K, Akt, FOXO3a in POF rats to inhibit follicular atresia and granulosa cell apoptosis\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Polysaccharide extract from Angelica could significantly reduce the levels of p-Akt and FOXO3 protein expressions in POF mice to inhibit the progression of POF\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. In our study, PPE could not only reduce the expressions of p-PI3K, p-Akt, and Bax proteins, but also increase the expression of Bcl-2 protein in the ovarian tissue of cisplatin-induced POF mice, suggesting that PPE could exert therapeutic effects on POF by inhibiting the PI3K/AKT signaling pathway.\u003c/p\u003e \u003cp\u003eMore and more evidence suggests that oxidative stress damage plays a critical role in chemotherapy-induced premature ovarian failure\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Cisplatin, a first-line chemotherapy drug, is commonly used to construct ovarian injury models\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. It can induce oxidative stress and trigger cell apoptosis by forming ROS such as hydroxyl radicals and superoxide\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. In this study, PPE intervention could significantly decrease the content of MDA, and increase levels of SOD, CAT and GSH-Px, indicating that PPE could alleviate oxidative stress damage in cisplatin-induced POF. In addition, Nrf-2 is currently considered the most important endogenous antioxidant protein\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Research has shown that Nrf-2 can activate downstream factors, such as NQO1 and HO-1, to clear ROS through sustained enzymatic reactions\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Our findings showed that PPE treatment increased the relative expression levels of HO-1 and Nrf-2, suggesting that PPE could alleviate cisplatin-induced oxidative stress by activating the Nrf-2/HO-1 signaling pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we found that PPE could alleviated cisplatin-induced POF by improving hormone levels, enhance antioxidant capacity and alleviate cell apoptosis. Mechanistically, PPE prevented cisplatin-induced POF by inhibiting the PI3K/AKT signaling pathway and activating the Nrf-2/HO-1 signaling pathway. Our study provides preliminary evidence that PPE has the potential to be developed as a dietary supplement for improving POF, which is beneficial for further development and utilization of PPE.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003ePOF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003ePremature ovarian failure\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eUPLC-Q-TOF/MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eUltrahigh performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003ePPE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003ePeony pods extract\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eKEGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eKyoto encyclopedia of genes and genomes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003ePI3K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003ePhosphatidylinositol 3-kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eAKT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eProtein kinase B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eRas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eRat sarcoma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eRap1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eRAS-associated protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eAMH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eAnti-mullerian hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eEstradiol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eFSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eFollicle stimulating hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eLH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eLuteinizing hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eSuperoxide dismutase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eGSH-Px\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eGlutathione peroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eMalondialdehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eBax\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eBcl-2 associated X protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eBcl-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eB-cell lymphoma/leukemia 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eNrf-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eNuclear factor erythroid 2-related factor 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eHO-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eHeme oxygenase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003ePPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eProtein-protein interaction networks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eGene ontology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eHRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 416px;\"\u003e\n \u003cp\u003eHormone replacement therapy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study received ethical approval from the Institutional Animal Care and Use Committee (IACUC) of Pingdingshan University (project approval number: PDSU-20240012). All participants signed an informed consent form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWang H.H. designed this study. Fan C.L., Wang H.H., Geng X.N. and Shan H.L. performed the experiment, analyzed all data, wrote and revised the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Dr. Xining Geng for identifying Peony Pods and the Henan Engineering Research Center of Funiu Mountain\u0026rsquo;s Medicinal Resources Utilization and Molecular Medicine for providing experimental instruments and equipment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Scientific Research Foundation for the introduction of talent of Pingdingshan University (No. PXY-BSQD-2022040, PXY-BSQD-2024025, PXY-BSQD-202109 ) and Henan scientific and technological research projects (No. 232102310460).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang J, Sun X, Yang ZX, Li SJ, Wang YF, Ren RX, Liu ZY, Yu DH. 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Biogerontology. 2023;24(5):609\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10522-023-10050-1\u003c/span\u003e\u003cspan address=\"10.1007/s10522-023-10050-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Peony pods extract, premature ovarian failure, network pharmacology, UPLC-Q-TOF/MS","lastPublishedDoi":"10.21203/rs.3.rs-6025082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6025082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePeony pods, as a byproduct of peony seeds, are often discarded. This study aimed to explore the effect and mechanism of Peony pods extract (PPE) for treating premature ovarian failure (POF) by UPLC-Q-TOF/MS method combined with network pharmacology and \u003cem\u003ein vivo\u003c/em\u003e experimental validation. Firstly, a total of 26 components of PPE were detected by UPLC-Q-TOF/MS analysis. Subsequently, cisplatin was used to construct the POF model, and PPE treatment could significantly improve the ovarian index, estrous cycle and structure of ovarian tissues, increase the serum levels of AMH and estradiol E\u003csub\u003e2\u003c/sub\u003e, and reduce the serum levels of FSH and LH in POF mice. Then, network pharmacology analysis was performed and found that multiple signaling pathways were involved in improving POF of PPE, including PI3K/AKT, Ras and Rap1 signaling pathways. To further verification, related proteins were detected by WB, and the expression levels of Bax, p-PI3K, and p-AKT were decreased, while the expression levels of Bcl-2, HO-1, and Nrf-2 were increased after PPE administration. In summary, PPE could exert therapeutic effect on POF by reducing cell apoptosis and improving hormone levels, showing great potential for application in functional food.\u003c/p\u003e","manuscriptTitle":"Efficacy and mechanism of Peony pods extract for treating premature ovarian failure: UPLC-Q-TOF/MS method combined with network pharmacology and experimental validation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 15:44:47","doi":"10.21203/rs.3.rs-6025082/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4206a864-eb50-4ff0-bce5-76327bf7f6d9","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-06T13:08:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 15:44:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6025082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6025082","identity":"rs-6025082","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}