Research Paper Volume 16, Issue 5 pp 4541—4562
Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice
- 1 School of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, China
- 2 Zhejiang Famous Chinese Medicine Clinic, The Third Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
Received: September 4, 2023 Accepted: January 19, 2024 Published: February 28, 2024
https://doi.org/10.18632/aging.205607How to Cite
Copyright: © 2024 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Ningxin-Tongyu-Zishen formula (NTZF) is a clinical experience formula for the treatment of premature ovarian insufficiency (POI) in traditional Chinese medicine (TCM), and the potential mechanism is unknown. For in vivo experiments, POI mouse models (C57BL/6 mice), were constructed by subcutaneous injection of D-galactose (D-gal, 200 mg/kg). After treatment of NTZF (10.14, 20.27, 40.54 g/kg;) or estradiol valerate (0.15 mg/kg), ovarian function, oxidative stress (OS) and protein expression of Sirt1/p53 were evaluated. For in vitro experiments, H2O2 (200 μM) was used to treat KGN to construct ovarian granulosa cells (OGCs) cell senescence model. Pretreatment with NTZF (1.06 mg/mL) or p53 inhibitor (Pifithrin-α, 1 μM) was performed before induction of senescence, and further evaluated the cell senescence, OS, mRNA and protein expression of Sirt1/p53. In vivo, NTZF improved ovarian function, alleviated OS and Sirt1/p53 signaling abnormalities in POI mice. In vitro experiments showed that NTZF reduced the level of OS and alleviated the senescence of H2O2-induced KGN. In addition, NTZF activated the protein expression of Sirt1, inhibited the mRNA transcription and protein expression of p53 and p21. Alleviating OGCs senescence and protecting ovarian function through Sirt1/p53 is one of the potential mechanisms of NTZF in the treatment of POI.
Abbreviations
NTZF: Ningxin-Tongyu-Zishen formula; POI: premature ovarian insufficiency; POF: premature ovarian failure; HRT: hormonal replacement therapy; TCM: traditional Chinese medicine; OGCs: ovarian granulosa cells; OS: oxidative stress; ROS: reactive oxygen species; D-gal: D-galactose; TIC: total ion chromatography; EV: estradiol valerate; ELISA: enzyme linked immunosorbent assay; FSH: follicle stimulating hormone; LH: luteinizing hormone; E2: estradiol; AMH: anti-Müllerian hormone; FBS: fetal bovine serum; HE: hematoxylin-eosin; GPX: glutathione Peroxidase; SOD: superoxide dismutase; DTNB: 5,5′-Dithiobis-(2-nitrobenzoic acid); IHC: immunohistochemistry; CCK-8: Cell Counting Kit-8; PFT-α: Pifithrin-α; SA-β-gal: senescence-associated β-galactosidase; LC-MS: liquid chromatography-mass spectrometry; IF: immunofluorescence; MFI: mean fluorescence intensity; q-PCR: quantitative polymerase chain reaction; DAPI: 4′,6-diamidino-2-phenylindole; DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; PBS: phosphate buffer saline; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; PCNA: proliferating cell nuclear antigen; γ-H2AX: gamma-histone family member X; Sirt1: sirtuin1; p53: protein 53; p21: protein 21.