Zuang Li , Yuewei Lin , Yuxin Zou , Yunyi Liang , Lihua Zeng , Yixuan Wang , Yucheng Li , Yun Zong , Yuying Zhang , Yunling Zheng , Yixuan Cui , Liuqian Huang , Zhuoting Chen , Xinyi Pan , Ling Zhu
{"title":"左归丸通过Notch1/Nrf2通路改善卵巢干细胞的干性、调控细胞周期和减少凋亡,从而改善化疗诱导的卵巢衰老","authors":"Zuang Li , Yuewei Lin , Yuxin Zou , Yunyi Liang , Lihua Zeng , Yixuan Wang , Yucheng Li , Yun Zong , Yuying Zhang , Yunling Zheng , Yixuan Cui , Liuqian Huang , Zhuoting Chen , Xinyi Pan , Ling Zhu","doi":"10.1016/j.jep.2024.119105","DOIUrl":null,"url":null,"abstract":"<div><h3>Ethnopharmacological relevance</h3><div>Zuogui Pills (ZGP) is a classic traditional Chinese herbal formula originating from the Ming Dynasty. It has been widely used in the treatment of kidney deficiency-related diseases, including ovarian aging.</div></div><div><h3>Aim of the study</h3><div>To investigate the effects and potential mechanisms of ZGP on ovarian aging induced by the chemotherapeutic agent cyclophosphamide (CTX), as well as its impact on the therapeutic target, oogonial stem cells (OSCs), involving the Notch1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway.</div></div><div><h3>Materials and methods</h3><div>This study utilized High-Performance Liquid Chromatography (HPLC) to analyze the active components of Zuogui Pills (ZGP). In <em>vivo</em> experiments involved the establishment of an ovarian aging model in female rats through intraperitoneal injection of CTX, followed by an 8-week treatment with ZGP and dehydroepiandrosterone (DHEA). The Notch pathway inhibitor DAPT was administered via intraperitoneal injection, followed by ZGP intervention to validate its therapeutic effects. Transcriptomic sequencing was used to analyze the differential genes before and after ZGP treatment of CTX-induced ovarian aging, and KEGG and GO analyses were applied to assess the changes in relevant signaling pathways and biological processes. In <em>vitro</em> experiments included the extraction, separation, and purification of ovarian germ stem cells, followed by transfection with a Notch1 overexpression plasmid. The CTX active component 4-Hydroxycyclophosphamide (4HC) was used for model intervention, and ZGP, DHEA-containing serum, and DAPT were applied to intervene with the oogonial stem cells. The effects of CTX modeling, the therapeutic efficacy of ZGP, and the general condition of the rats were observed. H&E staining was employed to assess ovarian morphology and follicle counting at various stages. Serum hormone levels were measured using ELISA, while qPCR, Western blot, flow cytometry, immunofluorescence, and IHC were utilized to analyze the expression of the Notch1/Nrf2 pathway, cell cycle proteins, and stemness-related indicators. Flow cytometry, TUNEL fluorescence, and CCK8 assays were conducted to evaluate changes in cell cycle composition, apoptosis, and proliferation. Finally, ChIP-qPCR was employed to validate the transcriptional regulation of the target gene <em>NFE2L2</em> by Notch1.</div></div><div><h3>Results</h3><div>ZGP improved serum sex hormones in ovarian aging rats, enhanced ovarian index, and optimized ovarian and uterine morphology, as well as follicle quantity composition. After transcriptome sequencing, KEGG analysis enriched the Notch signaling pathway and cell cycle, while GO analysis highlighted enrichment in the Notch pathway and stem cell population maintenance. Various experiments validated that ZGP significantly improved the expression of cell cycle-related proteins Cyclin D1 (CCND1), Cyclin E1 (CCNE1), cyclin-dependent kinase inhibitor 1a (CDKN1A), stemness markers Mouse Vasa Homolog (MVH), Octamer-binding Transcription Factor 4 (Oct4), Fragilis, 5-Bromo-2′-deoxyuridine (BrdU), as well as Notch1 and Nrf2 in aging ovarian tissues and OSCs. Additionally, ZGP promoted the proliferation of 4HC-damaged OSCs, optimized OSCs cell cycle composition, reduced G<sub>0</sub>/G<sub>1</sub> phase arrest, and decreased early and late apoptosis. ZGP could reverse the detrimental effects on stemness and cell cycle of OSCs caused by blocking the Notch pathway. Furthermore, ZGP may activate the regulation of its target gene <em>NFE2L2</em> by upregulating Notch1 expression in OSCs, thereby exerting therapeutic effects.</div></div><div><h3>Conclusion</h3><div>ZGP protects ovarian function in CTX-induced ovarian aging rats by regulating the Notch1/Nrf2 pathway. It restores serum sex hormone levels, maintains normal follicle development, promotes the proliferation of aged OSCs, optimizes the cell cycle, reduces apoptosis, and preserves stemness, thereby alleviating ovarian aging.</div></div>","PeriodicalId":15761,"journal":{"name":"Journal of ethnopharmacology","volume":"339 ","pages":"Article 119105"},"PeriodicalIF":4.8000,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Zuogui pills ameliorate chemotherapy-induced ovarian aging by improving stemness, regulating cell cycle and reducing apoptosis of oogonial stem cells via the Notch1/Nrf2 pathway\",\"authors\":\"Zuang Li , Yuewei Lin , Yuxin Zou , Yunyi Liang , Lihua Zeng , Yixuan Wang , Yucheng Li , Yun Zong , Yuying Zhang , Yunling Zheng , Yixuan Cui , Liuqian Huang , Zhuoting Chen , Xinyi Pan , Ling Zhu\",\"doi\":\"10.1016/j.jep.2024.119105\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Ethnopharmacological relevance</h3><div>Zuogui Pills (ZGP) is a classic traditional Chinese herbal formula originating from the Ming Dynasty. It has been widely used in the treatment of kidney deficiency-related diseases, including ovarian aging.</div></div><div><h3>Aim of the study</h3><div>To investigate the effects and potential mechanisms of ZGP on ovarian aging induced by the chemotherapeutic agent cyclophosphamide (CTX), as well as its impact on the therapeutic target, oogonial stem cells (OSCs), involving the Notch1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway.</div></div><div><h3>Materials and methods</h3><div>This study utilized High-Performance Liquid Chromatography (HPLC) to analyze the active components of Zuogui Pills (ZGP). In <em>vivo</em> experiments involved the establishment of an ovarian aging model in female rats through intraperitoneal injection of CTX, followed by an 8-week treatment with ZGP and dehydroepiandrosterone (DHEA). The Notch pathway inhibitor DAPT was administered via intraperitoneal injection, followed by ZGP intervention to validate its therapeutic effects. Transcriptomic sequencing was used to analyze the differential genes before and after ZGP treatment of CTX-induced ovarian aging, and KEGG and GO analyses were applied to assess the changes in relevant signaling pathways and biological processes. In <em>vitro</em> experiments included the extraction, separation, and purification of ovarian germ stem cells, followed by transfection with a Notch1 overexpression plasmid. The CTX active component 4-Hydroxycyclophosphamide (4HC) was used for model intervention, and ZGP, DHEA-containing serum, and DAPT were applied to intervene with the oogonial stem cells. The effects of CTX modeling, the therapeutic efficacy of ZGP, and the general condition of the rats were observed. H&E staining was employed to assess ovarian morphology and follicle counting at various stages. Serum hormone levels were measured using ELISA, while qPCR, Western blot, flow cytometry, immunofluorescence, and IHC were utilized to analyze the expression of the Notch1/Nrf2 pathway, cell cycle proteins, and stemness-related indicators. Flow cytometry, TUNEL fluorescence, and CCK8 assays were conducted to evaluate changes in cell cycle composition, apoptosis, and proliferation. Finally, ChIP-qPCR was employed to validate the transcriptional regulation of the target gene <em>NFE2L2</em> by Notch1.</div></div><div><h3>Results</h3><div>ZGP improved serum sex hormones in ovarian aging rats, enhanced ovarian index, and optimized ovarian and uterine morphology, as well as follicle quantity composition. After transcriptome sequencing, KEGG analysis enriched the Notch signaling pathway and cell cycle, while GO analysis highlighted enrichment in the Notch pathway and stem cell population maintenance. Various experiments validated that ZGP significantly improved the expression of cell cycle-related proteins Cyclin D1 (CCND1), Cyclin E1 (CCNE1), cyclin-dependent kinase inhibitor 1a (CDKN1A), stemness markers Mouse Vasa Homolog (MVH), Octamer-binding Transcription Factor 4 (Oct4), Fragilis, 5-Bromo-2′-deoxyuridine (BrdU), as well as Notch1 and Nrf2 in aging ovarian tissues and OSCs. Additionally, ZGP promoted the proliferation of 4HC-damaged OSCs, optimized OSCs cell cycle composition, reduced G<sub>0</sub>/G<sub>1</sub> phase arrest, and decreased early and late apoptosis. ZGP could reverse the detrimental effects on stemness and cell cycle of OSCs caused by blocking the Notch pathway. Furthermore, ZGP may activate the regulation of its target gene <em>NFE2L2</em> by upregulating Notch1 expression in OSCs, thereby exerting therapeutic effects.</div></div><div><h3>Conclusion</h3><div>ZGP protects ovarian function in CTX-induced ovarian aging rats by regulating the Notch1/Nrf2 pathway. It restores serum sex hormone levels, maintains normal follicle development, promotes the proliferation of aged OSCs, optimizes the cell cycle, reduces apoptosis, and preserves stemness, thereby alleviating ovarian aging.</div></div>\",\"PeriodicalId\":15761,\"journal\":{\"name\":\"Journal of ethnopharmacology\",\"volume\":\"339 \",\"pages\":\"Article 119105\"},\"PeriodicalIF\":4.8000,\"publicationDate\":\"2024-11-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of ethnopharmacology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0378874124014041\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, MEDICINAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of ethnopharmacology","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0378874124014041","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MEDICINAL","Score":null,"Total":0}
Zuogui pills ameliorate chemotherapy-induced ovarian aging by improving stemness, regulating cell cycle and reducing apoptosis of oogonial stem cells via the Notch1/Nrf2 pathway
Ethnopharmacological relevance
Zuogui Pills (ZGP) is a classic traditional Chinese herbal formula originating from the Ming Dynasty. It has been widely used in the treatment of kidney deficiency-related diseases, including ovarian aging.
Aim of the study
To investigate the effects and potential mechanisms of ZGP on ovarian aging induced by the chemotherapeutic agent cyclophosphamide (CTX), as well as its impact on the therapeutic target, oogonial stem cells (OSCs), involving the Notch1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway.
Materials and methods
This study utilized High-Performance Liquid Chromatography (HPLC) to analyze the active components of Zuogui Pills (ZGP). In vivo experiments involved the establishment of an ovarian aging model in female rats through intraperitoneal injection of CTX, followed by an 8-week treatment with ZGP and dehydroepiandrosterone (DHEA). The Notch pathway inhibitor DAPT was administered via intraperitoneal injection, followed by ZGP intervention to validate its therapeutic effects. Transcriptomic sequencing was used to analyze the differential genes before and after ZGP treatment of CTX-induced ovarian aging, and KEGG and GO analyses were applied to assess the changes in relevant signaling pathways and biological processes. In vitro experiments included the extraction, separation, and purification of ovarian germ stem cells, followed by transfection with a Notch1 overexpression plasmid. The CTX active component 4-Hydroxycyclophosphamide (4HC) was used for model intervention, and ZGP, DHEA-containing serum, and DAPT were applied to intervene with the oogonial stem cells. The effects of CTX modeling, the therapeutic efficacy of ZGP, and the general condition of the rats were observed. H&E staining was employed to assess ovarian morphology and follicle counting at various stages. Serum hormone levels were measured using ELISA, while qPCR, Western blot, flow cytometry, immunofluorescence, and IHC were utilized to analyze the expression of the Notch1/Nrf2 pathway, cell cycle proteins, and stemness-related indicators. Flow cytometry, TUNEL fluorescence, and CCK8 assays were conducted to evaluate changes in cell cycle composition, apoptosis, and proliferation. Finally, ChIP-qPCR was employed to validate the transcriptional regulation of the target gene NFE2L2 by Notch1.
Results
ZGP improved serum sex hormones in ovarian aging rats, enhanced ovarian index, and optimized ovarian and uterine morphology, as well as follicle quantity composition. After transcriptome sequencing, KEGG analysis enriched the Notch signaling pathway and cell cycle, while GO analysis highlighted enrichment in the Notch pathway and stem cell population maintenance. Various experiments validated that ZGP significantly improved the expression of cell cycle-related proteins Cyclin D1 (CCND1), Cyclin E1 (CCNE1), cyclin-dependent kinase inhibitor 1a (CDKN1A), stemness markers Mouse Vasa Homolog (MVH), Octamer-binding Transcription Factor 4 (Oct4), Fragilis, 5-Bromo-2′-deoxyuridine (BrdU), as well as Notch1 and Nrf2 in aging ovarian tissues and OSCs. Additionally, ZGP promoted the proliferation of 4HC-damaged OSCs, optimized OSCs cell cycle composition, reduced G0/G1 phase arrest, and decreased early and late apoptosis. ZGP could reverse the detrimental effects on stemness and cell cycle of OSCs caused by blocking the Notch pathway. Furthermore, ZGP may activate the regulation of its target gene NFE2L2 by upregulating Notch1 expression in OSCs, thereby exerting therapeutic effects.
Conclusion
ZGP protects ovarian function in CTX-induced ovarian aging rats by regulating the Notch1/Nrf2 pathway. It restores serum sex hormone levels, maintains normal follicle development, promotes the proliferation of aged OSCs, optimizes the cell cycle, reduces apoptosis, and preserves stemness, thereby alleviating ovarian aging.
期刊介绍:
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about people''s use of plants, fungi, animals, microorganisms and minerals and their biological and pharmacological effects based on the principles established through international conventions. Early people confronted with illness and disease, discovered a wealth of useful therapeutic agents in the plant and animal kingdoms. The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and sometimes recorded in herbals and other texts on materia medica. Many valuable drugs of today (e.g., atropine, ephedrine, tubocurarine, digoxin, reserpine) came into use through the study of indigenous remedies. Chemists continue to use plant-derived drugs (e.g., morphine, taxol, physostigmine, quinidine, emetine) as prototypes in their attempts to develop more effective and less toxic medicinals.