Zuogui Wan alleviated radiation-induced premature ovarian failure and ferroptosis via miR-21-5p/DNMT3b

Abstract

Background

The number of patients with radiation-induced premature ovarian failure (POF) has increased rapidly. In this research, we investigated the therapeutic effects and molecular mechanisms of the classic TCM prescription Zuogui Wan (ZGW) in radiation-induced POF.

Methods

SD rats and ovarian granulosa cells were exposed to γ-radiation with or without ZGW treatment. The morphological alterations in the ovaries, cell death, and mitochondrial damage were identified through H&E staining, Hoechst/PI staining and TEM, respectively. Serum hormone levels were quantified using ELISA. Western blot was employed to assess the protein expression levels of GPX4 and DNMT3b. The RNA expression levels of miR-21 and DNMT3b were determined by PCR. Fe²⁺, MDA and GSH content were detected using the corresponding assay kit. FerroOrange was used for Fe²⁺ visualization.

Results

Radiation-induced POF and ferroptosis have been observed both in vivo and in vitro. ZGW strongly inhibited the increase in intracellular Fe²⁺ and MDA levels, as well as the decrease in estradiol, AMH, GSH, and GPX4 levels induced by radiation, exerting effects of anti-POF and anti-ferroptosis. miR-21-5p targeted DNMT3b and suppressed its expression. Inhibition of miR-21-5p or overexpression of DNMT3b counteracted the protective effect of ZGW.

Conclusion

ZGW alleviated radiation-induced POF and ferroptosis through the miR-21-5p/DNMT3b axis.

Introduction

Medical factors such as surgery, radiotherapy, and chemotherapy account for 50%-70% of the total incidence rate of premature ovarian failure (POF), leading to reproductive disorders in women under 40 years old [1, 2]. Ovarian follicles are the basic functional units of the ovary, consisting of a centrally located oocyte, surrounded by granulosa cells, and outer layers of thecal cells [3, 4]. Granulosa cells proliferate in developing follicles, regulate meiosis, and promote oocyte development [5]. The death of oocytes and granulosa cells, as well as an increase in the number of atretic follicles, are the main manifestations of POF [6].

Programmed cell death (PCD) plays a fundamental role in organismal development. Abnormal regulation of PCD is observed in various human diseases. Biomolecules, radiation, and other factors can trigger pathological changes in cells, thereby inducing PCD. Reactive oxygen species (ROS) generated by radiation activate the mitochondrial intrinsic apoptosis pathway, leading to ATP synthesis disorders, decreases in mitochondrial membrane potential (MMP), and defects in mitochondrial electron transport chains [7]. Elevated cell apoptosis rate is associated with the increasing radiation dose [8]. Oxidative stress, DNA damage, and endoplasmic reticulum (ER) stress induction induced by radiation result in cell autophagy [9]. Radiation triggers an increase in free iron and lipid metabolism enzymes (e.g., ACSL4), which then synergistically initiate high-level lipid peroxidation and ferroptosis [10]. Overall, the studies above suggested radiation as an exogenous inducer of cell death.

Ferroptosis is a well-studied PCD, mainly induced by iron-dependent excessive accumulation of lipid peroxidation [11, 12]. In the past few years, researchers have successively reported the important impacts of ferroptosis on the occurrence and development of ovarian dysfunctions [13]. Iron overload-induced ferroptosis reduces the quality of oocytes by increasing oxidative stress and mitochondrial dysfunction [14]. Ferroptosis-related pathways and genes also regulate the growth and secretory function of granulosa cells, thereby affecting the development and maturation of oocytes [15].

Radiation-induced damage is referred to as ‘heat evil’ (Pathogenic heat) in the diagnosis of traditional Chinese medicine (TCM), and it diminishes ‘qi’ (Vital energy) and hurts ‘yin’ (The cooling/calming/substantive aspect of the body). The ‘heat evil’ can easily cause injury to the ‘kidney jing’ (Kidney essence), which is regarded as the foundation of vitality. As the radiation exposure time increases, kidney essence deficiency syndrome (manifesting as sexual dysfunction and reproductive dysfunction) appears sequentially. Zuogui Wan (ZGW) is a kidney tonifying prescription formulated by Zhang Jingyue, a renowned physician in ancient China [16]. It has the functions of nourishing ‘qi’ and ‘yin’, replenishing essence and kidney [17], and has good therapeutic effects on gynecological diseases (e.g., estrogen deficiency, POF, irregular menstruation) and aging-related diseases [18]. Study have shown that ZGW regulates the mitochondria-dependent apoptosis in the follicles, improves ovarian ultrastructure, and has preventive and therapeutic effects on POF [19]. In chemotherapy-induced POF, ZGW reduces the concentration of follicle-stimulating hormone (FSH), increases the number of antral follicles, ovarian volume and mean diameter [20].

Numerous TCM formulas, such as Bu-Shen-Ning-Xin decoction and Chaihu-Shugan-San, exert therapeutic effects by modulating microRNA (miRNA) expression. For instance, they have been shown to ameliorate conditions like premature ovarian insufficiency and post-stroke depression through specific miRNA-related pathways [21, 22]. miRNA is a group of non-coding RNA composed of 20–22 nucleotides. It mainly binds to the 3’ untranslated region (3’-UTR) of the target gene, inducing transcriptional inhibition or mRNA degradation, thereby regulating physiological processes such as cell death and inflammation [23]. The expression level of miR-21 is correlated with the radiation dose [24–26]. A study reported that the upregulated expression of miR-21-3p facilitates ferroptosis by enhancing lipid peroxidation [27], while another study suggested that downregulating miR-21-3p promotes ferroptosis [28]. The role of miR-21-3p in ferroptosis is disease-specific. By targeting different downstream genes, it can have opposing effects, either promoting or suppressing ferroptosis. miR-21-5p inhibits ferroptosis through the AKT/mTOR pathway, leading to alterations in the levels of GPX4, GSH, ROS, and Fe²⁺ [29].

Numerous studies have demonstrated that miRNAs play a critical role in the pathogenesis of POF [30, 31]. The Akt/mTOR signaling pathway is a well-established regulator of ovarian reserve and is implicated in POF [32, 33]. Notably, specific miRNAs, such as miR-497-3p and placental exosome-derived miR-10a-5p, have been shown to promote POF by targeting the PI3K/AKT/mTOR and TrkB/Akt/mTOR pathways, respectively [34, 35]. Given this established link between miRNA dysregulation and Akt/mTOR signaling in POF, and considering that miR-21-5p is a known modulator of this pathway, we hypothesize that miR-21-5p may also be involved in the pathogenesis of POF. Our previous research demonstrated that ZGW triggers the expression of miR-21-5p in radiation-exposed cells, but the functional and specific mechanisms remain elusive. In this study, we investigated whether ZGW can alleviate radiation-induced POF and its molecular mechanisms.

Materials and methods

Experimental animals and groups

Three-week-old immature female Sprague-Dawley (SD) rats, weighing between 40 g and 50 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Rats were kept in rooms with controlled humidity, temperature (25 °C) and a 12-hour light/dark cycle. Free access to food and water was provided. In this study, sixty SD rats were randomly assigned to 6 equal groups. The first stage of the study involved two groups, which were labeled as the control group and the irradiation (IR) group, respectively. γ-radiation-induced ovarian injury was established using Gammacell 40 Exactor with two Cesium (137CS) source (Atomic Energy of Canadian Inc., Mississauga, Canada). Rats in the IR group were given γ-rays to whole body at the dose of 3.2 Gy with a dose rate of 0.46 Gy/min [36, 37]. The second stage of the study divided rats into two groups: the IR group and the IR + ZGW group. Both groups received γ-radiation exposure at 3.2 Gy. Rats in the IR + ZGW group were orally administered ZGW at the dose of 1.6 g/kg once a day for 12 consecutive weeks before γ-radiation treatment [38]. The last two groups were classified into the NC-inhibitors group and the miR-21-5p-inhibitors group for the third stage of the study. Rats were transfected with NC-inhibitors and miR-21-5p-inhibitors plasmids, respectively, then followed by ZGW and radiation treatment. For specific experimental details, please refer to Table 1. Animal experiments were approved by the Animal Ethics Committee of Shenzhen Hospital (Futian) of Guangzhou University of Chinese Medicine and conducted in accordance with ARRIVE guidelines. Animal suffering was minimized.

Table 1.

Animal experimental timelines

Part 1: Model Establishment
Time	Control	IR
Day 1	-	One-shot irradiation (3.2 Gy)
Day 14	Detection
Part 2: Treatment Evaluation
Time	IR	IR + ZGW
Day 1–84	Saline (p.o., once daily)	ZGW (1.6 g/kg, p.o., once daily)
Day 71	One-shot irradiation (3.2 Gy)
Day 84	Detection
Part 3: Mechanism Exploration
Time	NC-inhibitors	miR-21-5p-inhibitors
Day 1, 31, 61, 91	NC-inhibitors (0.01 nmol, i.p.)	miR-21-5p-inhibitors (0.01 nmol, i.p.)
Day 31–114	ZGW (1.6 g/kg, p.o., once daily)
Day 101	One-shot irradiation (3.2 Gy)
Day 115	Detection

Isolation, culture and treatment of rat ovarian granulosa cells

The rats were euthanized to collect their ovaries. The ovaries were washed with PBS containing 1% (w/v) penicillin-streptomycin for three times. The adipose tissue and the envelope surrounding the ovary were then removed. Under a stereoscopic microscope, the follicle was punctured with a 30-gauge needle, and granulosa cells were collected. The granulosa cell suspension was filtered using a 200-mesh cell sieve and washed with PBS. After centrifugation, cells were resuspended in DMEM/F12 medium (11320033, Gibco, Carlsbad, USA) (10% FBS and 1% (w/v) penicillin-streptomycin) and cultured in an incubator (37 °C, 5% CO₂) for further experimentation. Firstly, cells were divided into six groups and irradiated with a series of doses: 0, 2, 4, 6, 8, 10 Gy, at a dose rate of 0.46 Gy/min. The dose of 6 Gy was chosen for further investigation. Secondly, cells in the IR + ZGW-L group, IR + ZGW-M group, and IR + ZGW-H group were pre-treated with 0.2, 0.4, and 0.8 mg/ml of ZGW for 24 h before exposure to 6 Gy radiation, respectively. And 0.8 mg/ml ZGW was used for subsequent experiments. Then, four groups of cells were transfected with NC-mimics, miR-21-5p-mimics, NC-inhibitors, and miR-21-5p-inhibitors plasmids, respectively. All four groups of cells received treatment with 0.8 mg/ml of ZGW and 6 Gy of radiation. Finally, the cells were divided into the following groups: NC-inhibitors + OE-NC, miR-21-5p-inhibitors, OE-DNMT3b, miR-21-5p-inhibitors + OE-DNMT3b. The corresponding transfection operation was performed before treatment with 0.8 mg/ml of ZGW and exposure to 6 Gy of radiation.

ZGW preparation

ZGW was an ancient prescription made of adhesive Rehmannia (24 g), Rhizoma Dioscoreae (12 g), Barbary Wolfberry fruit (12 g), Cornus officinalis (12 g), China dodder (12 g), Colla Cornus Cervi (12 g), tortoise shell glue (12 g), and medicinal Cyathula root (9 g), which was purchased from Beijing Tongrentang Pharmaceutical Factory (State Food and Drug Administration approval number: Z11020735). ZGW was dissolved in sterile saline for rats and DMEM/F12 medium for ovarian granulosa cells administration. Ultrasonication assisted in drug dissolution. The drug was prepared weekly, and the prepared solution was aliquoted and stored at -20 °C after preparation.

Histopathological observation and ovarian follicle counting

Ovarian tissues were collected after treatment and underwent a series of procedures, including rinsing with saline, fixation in 4% paraformaldehyde, dehydration through a series of ethanol, and embedding in paraffin. The tissues were then sliced into sections with a thickness of 4 μm. Subsequently, the sections were dewaxed in xylene and rehydrated through a descending ethanol series to distilled water. Prepared sections were stained with Hematoxylin and Eosin (H&E) (HB190108, Yeasen, Shanghai, China) for pathological and morphological observation. For staining, nuclei were stained with hematoxylin for 5 min and were washed in running tap water for bluing. Cytoplasmic staining was performed using eosin solution for 10–30 s and sections were washed with color-enhancing solution for 1–2 s. Finally, the stained sections were dehydrated, cleared in xylene, and mounted with mounting medium. The histopathological examination and ovarian follicles counting were conducted using a microscope (Olympus, Tokyo, Japan). Primordial follicles are the smallest in size, characterized by a single layer of granulosa cells and the absence of a zona pellucida. The appearance of the zona pellucida marks the transition to the primary follicle stage, while secondary follicles are distinguished by a clearly visible antral cavity. Atretic follicles exhibit characteristic features such as pyknotic nuclei, apoptotic cells, and structural disintegration or disorganization.

Hoechst/Propidium Iodide (PI) staining

Cell viability in ovarian tissues was assessed by staining with Hoechst 33,342 (C1022, Beyotime, Shanghai, China) and PI (ST511, Beyotime). Briefly, fresh, unfixed frozen Sect.  (5 μm) were incubated with a mixture of Hoechst 33,342 (5 µg/ml) and PI (10 µg/ml) in PBS for 10 min at room temperature in the dark. After washing with PBS, the sections were mounted with an anti-fade mounting medium (P0126, Beyotime) and immediately imaged using a fluorescence microscope (Olympus).

ELISA assay of serum estradiol and AMH levels

Rats were completely anesthetized for tissue collection. The abdominal cavity was opened, and the blood samples (1.5-2 ml) were collected using needles. Blood samples were placed at room temperature for 30 min, followed by centrifugation at 3000 rpm at 4 °C for 15 min. Levels of estradiol and AMH in serum were determined using commercial assay kits (Rat Estradiol ELISA Kit and Rat AMH ELISA kit) (E-OSEL-R0001 and E-EL-R3022, Elabscience, Wuhan, China) according to the instructions.

Iron detection

FerroOrange (F374, Dojindo Laboratories, Kumamoto, Japan) was used to detect intracellular Fe²⁺ content according to the manufacturer’s manuals. The ovarian granulosa cells were incubated with 1 µM FerroOrange in Hank’s balanced salt solution (HBSS) for 30 min at 37 °C, and observed by confocal laser scanning microscopy (Olympus, Tokyo, Japan). The concentration of iron in the tissues was measured using inductively coupled plasma mass spectrometer (ICP-MS) (Thermo Fisher Scientific, Waltham, USA).

GSH detection

Intracellular GSH levels were evaluated using a commercial GSH assay kit (S0053, Beyotime, Shanghai, China) following the manufacturer’s instructions. GSH was assayed in fresh tissue and cell samples. To prevent oxidation, the procedure was performed as rapidly as possible with pre-cooled reagents that were prepared beforehand. For tissue samples, protein removal reagent M was added followed by homogenization. For cell pellets, the pellets were resuspended in reagent M and subjected to two cycles of rapid freezing in liquid nitrogen and thawing at 37 °C. The processed tissue or cell lysates were then incubated at 4 °C for 10 min and centrifuged at 10,000 × g for 10 min at 4 °C. The resulting supernatant was collected and mixed with protein removal reagent M and the GSH assay working solution. After a 5-minute incubation at room temperature, NADPH solution was added to the mixture. The absorbance at 412 nm was measured immediately. The GSH concentration in the samples was calculated based on a standard curve.

Western blotting

Total protein was extracted from tissues or cells using RIPA (P0013B, Beyotime, Shanghai, China), and the concentrations were measured with the BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). After boiling and denaturation, the extracted proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% Bovine Serum Albumin (BSA) for 1 h, then incubated with primary antibodies (GPX4, MA5-32827; DNMT3b, MA5-32058; β-Actin, PA1-183; GAPDH, PA1-987) (Invitrogen, Carlsbad, USA). After incubation, the membranes were washed with TBST and then incubated with secondary antibodies (31460, Invitrogen). The bands were visualized using an enhanced chemiluminescence kit (Bio-Rad Laboratories, Hercules, USA). Finally, the proteins were detected using an Amersham Imager 600 (GE, Boston, USA).

CCK-8 assay

Rat ovarian granulosa cells were seeded in a 96-well plate at a density of 8000 cells per well and then exposed to different doses of γ-rays. Cell viability was determined by adding 100 µL of DMEM/F12 medium containing 10% CCK-8 reagent (C0037, Beyotime, Shanghai, China) to each well. After a two-hour incubation at 37 °C, the absorbance at 450 nm was measured using a microplate reader (Perkin Elmer, Waltham, USA).

Transmission Electron Microscopy (TEM) assay

For TEM, ovarian granulosa cells were collected after treatment and fixed with 2.5% phosphate-buffered glutaraldehyde (pH 7.4) at room temperature in the dark for 30 min. After dehydration and embedding, ultrathin tissue sections with 1 μm thick were prepared, and image acquisition was performed using a transmission electron microscope (HITACHI, Tokyo, Japan).

Dual-luciferase reporter assay

Bioinformatics prediction website (https://rnasysu.com/encori/) was used to predict the binding sites of miR-21-5p to DNMT3b. The amplified DNMT3b 3’-UTR and mutant DNMT3b 3’-UTR sequence were inserted into the Dual-Luciferase miRNA Target Reporter Vector (E1330, Promega, Madison, USA) to construct DNMT3b-WT (Wild-type) and DNMT3b-MUT (Mutant), respectively. Luciferase reporter vector or mutant vector was co-transfected into cells with NC-mimics or miR-21-5p-mimics using Lipofectamine 2000 (11668030, Invitrogen, Carlsbad, CA). Firefly luciferase and Renilla luciferase activities were detected according to the instructions of the Dual-Glo Luciferase Assay System (E2920, Promega, Madison, USA). Firefly luciferase activity was normalized to Renilla luciferase activity.

miRNA analysis products, plasmid construction

miR-21-5p-mimics and miR-21-5p-inhibitors and negative controls (NC-mimics and NC-inhibitors) (4464066, 4464084, 4464058, 4464076, Thermo Fisher Scientific, Waltham, USA; miR30000790-4-5, miR40000790-4-5, miR3N0000001-4-5, and miR4N0000001-4-5, RiboBio, Guangzhou, China) were transfected into ovarian granulosa cells and rats to either enhance or suppress the function of miR-21-5p. The complementary strand of miR-21-5p acts as an inhibitor of miR-21-5p and competes for the binding site between miR-21-5p and target genes, but it does not reduce the expression level of the miRNA. Plasmid construction of GV141-DNMT3b was performed by GeneChem (Shanghai, China). Cells were transfected with GV141-DNMT3b (OE-DNMT3b) to induce the overexpression of DNMT3b, while an empty GV141 vector (OE-NC) served as an experimental control. All transfection experiments were performed using Lipofectamine 2000 (11668030, Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. For cells, 2.5 µg each of plasmid was diluted in 125 µl Opti-MEM (Gibco, Carlsbad, USA) and then mixed with 125 µl Opti-MEM containing 5 µl of Lipofectamine 2000. The DNA-Lipofectamine complexes were added to the cells, which were then incubated cells for 8 h. Following this incubation, the medium was replaced with fresh medium. After 48 h, cells were harvested for further analysis, and transfection efficiency was assessed by qPCR. For rats, administer via intraperitoneal injection at a dose of 50 nmol/l and a volume of 200 µl.

qPCR assay

miRNA detection kits were all purchased from Thermo Fisher Scientific (Waltham, USA). miRNAs from rat ovarian granulosa cells were isolated using the mirVana miRNA Isolation Kits (AM1561), and cDNA was synthesized with the TaqMan Advanced miRNA cDNA Synthesis Kit (A28007) using 10 ng of total RNA as input. qPCR of rno-miR-21-3p and rno-miR-21-5p was performed using pre-designed TaqMan Advanced miRNA Assays (482951_mir and 482952_mir) and TaqMan Fast Advanced Master Mix (4444557). The expression levels of miRNAs were normalized to U6 (001973). The mRNA expression levels of DNMT3b were also measured using the qPCR method. Total RNA was extracted from the cells using TRIzol (15596026, Life Technologies, Carlsbad, USA), and cDNA was reverse transcribed from 1 µg of total RNA using the Verso cDNA synthesis kit (Thermo Scientific, Waltham, USA). Real-time PCR was carried out using Maxima SYBR Green/ROX PCR Master Mix (K0221, Thermo Scientific, Waltham, USA) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, USA). The thermal cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The DNMT3b expression level was normalized to the level of GAPDH and analyzed using the 2 − ΔΔCt method. Primer sequences were as follows: DNMT3b forward: 5’-ACAACCATTGACTTTGCCGC-3’, and reverse: 5’-CGTTCTCGGCTCTCCTCATC-3’; GAPDH forward: 5’-ATGCCATCACTGCCACTCA-3’, and reverse: 5’-CCTGCTTCACCACCTTCTTG-3’.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10.0 software. All data, obtained from at least three independent biological/technical replicates, were presented as the mean ± standard error of the mean (SEM). The normality of data distribution was assessed using the Kolmogorov-Smirnov test and Q-Q plots. Student’s t-tests and one-way analysis of variance (ANOVA) were used for statistical comparisons among different groups. For one-way ANOVA, Bonferroni’s post hoc test was applied for multiple comparisons. p < 0.05 was considered statistically significant.

Results

Radiation induced POF and ferroptosis in rats

To verify the pathological changes in ovaries induced by radiation. Rats were exposed to γ-radiation. The follicles develop through primordial, primary, and secondary stages before acquiring an antral cavity [39]. As shown in Fig. 1A, ovarian follicles with normal oocytes, granulosa cells, and zona pellucida were clearly observed in ovarian tissue sections of the Control group. While the rats subjected to γ-radiation (IR group) showed remarkable morphological changes, including atretic follicles (AF), zona pellucida remnants (yellow circles), and interstitial hyperplasia with vascular hemorrhage (blue arrowheads). Control ovaries exhibited a diverse follicle population, with counts of 10.67 (primordial), 5.33 (preantral), 5.33 (antral), and 12.33 (atretic). Following irradiation, the numbers of primordial, preantral, and antral follicles were significantly reduced to 5.33, 1.33, and 1.67, respectively, whereas the count of atretic follicles increased to 27.33. (Fig. 1B). Estradiol is mainly produced in ovarian follicles, and serum anti-Müllerian hormone (AMH) levels are correlated with the degree of follicle development [40]. The serum hormone level was determined using ELISA. The IR group exhibited significant decreases in both estradiol and AMH levels (Fig. 1C). Hoechst/PI staining was used to determine the cell viability of ovarian tissues. Irradiation increased the red fluorescence in ovarian granulosa cells, suggesting the involvement of necrosis (Fig. 1D). Our previous research has indicated that radiation inhibites the cell viability of oocytes by inducing ferroptosis. Detections were conducted to confirm the involvement of ferroptosis in radiation-induced cell death. In irradiated ovarian tissues, the content of Fe²⁺ and MDA increased, while the levels of GSH and GPX4 decreased (Fig. 1E-H). In summary, radiation induced the accumulation of iron and lipid peroxidation, which synergistically initiate ferroptosis. Ferroptosis may be involved in the mechanism of radiation-induced POF.

Fig. 1.

Radiation induced POF and ferroptosis in rats. A Histological detection (H&E staining). Bar = 200 μm. AF: atretic follicle. Yellow circles: remnants of the zona pellucida. Blue arrowheads: interstitial hyperplasia with vascular hemorrhage. B Morphometric analysis of different ovarian follicle populations (n = 3 per group). C Quantification of serum estradiol and AMH levels (n = 5). D Hoechst/PI staining was used to determine the cell viability of ovarian tissues. Bar = 50 μm. E-G Changes of Fe2⁺ (n = 6 per group), MDA (n = 3 per group), and GSH content (n = 3 per group) in ovarian tissues of irradiated rats. H Western blot of GPX4 in ovarian tissue lysates (n = 3). GAPDH served as the loading control. IR: irradiation

ZGW alleviated radiation-induced POF and ferroptosis in rats

ZGW displayed promising therapeutic effects in radiation-induced POF. The tissue sections of irradiated rats treated with ZGW (IR + ZGW group) showed a normal structure with multiple types of ovarian follicles (Fig. 2A), and ZGW effectively prevented the healthy follicles from transforming into atretic follicles. Compared to the IR group, ZGW treatment significantly increased the counts of primordial (from 6.33 to 12), preantral (from 1.33 to 5), and antral follicles (from 2.33 to 7.33), while significantly decreasing the number of atretic follicles from 28 to 14.3 (Fig. 2B). The serum estradiol and AMH levels significantly elevated after ZGW treatment (Fig. 2C). Meanwhile, ZGW exerted a strong effect to reverse the cell death induced by radiation treatment (Fig. 2D). The increasing iron level and lipid peroxidation were suppressed (Fig. 2E-G), whlie the expression level of GPX4 was upregulated (Fig. 2H). Collectively, ZGW reversed radiation-induced POF and ferroptosis in rats.

Fig. 2.

ZGW alleviated radiation-induced POF and ferroptosis in rats. A Histological detection of ovarian tissue sections (H&E staining). Bar = 200 μm. AF: atretic follicle. Yellow circles: remnants of the zona pellucida. Blue arrowheads: interstitial hyperplasia with vascular hemorrhage. B Morphometric analysis of different ovarian follicle populations (n = 3 per group). C Serum estradiol and AMH levels were detected by ELISA. (n = 5 per group). D Hoechst/PI staining was used to determine the cell viability of ovarian tissues. Bar = 50 μm. E The intracellular Fe2⁺ (n = 6 per group), MDA (n = 3 per group), and GSH (n = 3 per group) content in ovarian tissues. H Western blot of GPX4 in ovarian tissue lysates (n = 3). GAPDH served as the loading control. IR: irradiation; IR+ZGW: irradiation + Zuogui Wan

Radiation-induced ferroptosis in ovarian granulosa cells

Experiments were conducted to determine whether irradiated cells exhibited characteristics of ferroptosis. Ovarian granulosa cells were exposed to various of γ-radiation, and the viability was determined with CCK-8. γ-radiation at doses equal to or greater than 6 Gy showed cytotoxic effects, leading to a decrease in cell viability as the dose increased (Fig. 3A). The content of intracellular Fe²⁺ increased in a dose-dependent manner (Fig. 3B). The upregulation of MDA content and the downregulation of GSH level suggested strong lipid peroxidation (Fig. 3C-D). The expression of GPX4 was also inhibited with the increasing γ-radiation doses (Fig. 3E). Furthermore, ferroptosis is closely related to mitochondrial function [41]. Abnormal mitochondrial morphological changes, including rupture and blistering of the outer mitochondrial membrane, dissolution of cristae, as well as mitochondria shrinkage, were observed after radiation exposure (Fig. 3F).

Fig. 3.

Radiation-induced ferroptosis in ovarian granulosa cells. A CCK-8 assay of the viability of ovarian granulosa cells. B-D Intracellular Fe2⁺ content. Bar = 50 μm. C-D MDA and GSH detection in ovarian granulosa cells. E Western blot of GPX4 in ovarian granulosa cells. β-actin served as the loading control. F TEM images of ovarian granulosa cells. Bar = 500 nm. IR: irradiation

ZGW alleviated radiation-induced ferroptosis in ovarian granulosa cells

To further investigate the mechanisms involved in the therapeutic effect of ZGW, cells were pre-treated with different concentrations of ZGW. Firstly, ZGW suppressed radiation-triggered intracellular iron accumulation (Fig. 4A). Compared to the IR group, the increase in MDA concentration and the decrease in GSH level were reversed by ZGW treatment, suggesting high-level lipid peroxidation induced by radiation was effectively inhibited by ZGW administration (Fig. 4B-C). Meanwhile, ZGW upregulated the expression level of GPX4, indicating the effect of anti-ferroptosis (Fig. 4D). In addition, the TEM image revealed that ZGW prevented ferroptosis in ovarian granulosa cells. As the dosage of ZGW increased, the number of abnormal mitochondria decreased (Fig. 4E).

Fig. 4.

ZGW alleviated radiation-induced ferroptosis in ovarian granulosa cells. A-C Intracellular Fe2⁺, MDA, GSH content detection. Bar = 50 μm. D GPX4 expression in ovarian granulosa cells. GAPDH served as the loading control. E TEM images of γ-radiation and ZGW-treated ovarian granulosa cells. Bar = 500 nm. IR+ZGW-L: irradiation + Zuogui Wan-0.2mg/ml; IR+ZGW-M: irradiation + Zuogui Wan-0.4mg/ml; IR+ZGW-H: irradiation + Zuogui Wan-0.8mg/ml

ZGW alleviated radiation-induced ferroptosis by promoting miR-21-5p expression in ovarian granulosa cells

It is reported that the expression of miR-21 is closely related to radiation dose, and the effects of miR-21-3p and miR-21-5p on ferroptosis vary in different diseases [27–29]. After treatment with γ-radiation and different concentrations of ZGW, the expression levels of miR-21-3p and miR-21-5p were determined using qPCR. ZGW showed no significant effect on miR-21-3p expression but increased miR-21-5p expression, indicating a vital role of miR-21-5p in radiation-induced ferroptosis (Fig. 5A). Ovarian granulosa cells were transfected with miR-21-5p-mimics and miR-21-5p-inhibitors to induce miR-21-5p overexpression and knockdown, respectively, then followed by γ-radiation exposure and ZGW treatment. miR-21-5p-mimics inhibited the intracellular Fe²⁺ and MDA content, and increased GSH and GPX4 level. miR-21-5p-inhibitors exerted completely opposite effects (Fig. 5B-E). As shown in TEM images, cells transfected with miR-21-5p-mimics were in a normal condition, while rupture and blistering of the outer mitochondrial membrane, dissolution of cristae, as well as mitochondria shrinkage were found in the miR-21-5p-inhibitors group (Fig. 5F). In short, inhibiting miR-21-5p expression suppressed the protective effects of ZGW, promoting ferroptosis in ovarian granulosa cells.

Fig. 5.

ZGW alleviated radiation-induced ferroptosis by promoting miR-21-5p expression in ovarian granulosa cells. A The expression levels of miR-21-3p and miR-21-5p were detected by qPCR. B Intracellular Fe2⁺ levels. Bar = 50 μm. C-D Intracellular MDA and GSH levels. E The protein expression levels of GPX4. GAPDH served as the loading control. F Morphological changes of ovarian granulosa cells after transfection were observed using TEM. Bar = 500 nm

miR-21-5p targeting DNMT3b

To explore the potential downstream genes involved in the regulation of miR-21-5p on radiation-induced ferroptosis, the potential target genes were predicted. As shown in Fig. 6A, miR-21-5p had a sequence complementary to the region of DNMT3b, and the sequence was AUAAGCU. The dual luciferase reporter gene assay further confirmed this interaction, demonstrating that overexpression of miR-21-5p overexpression effectively inhibited the luciferase activity of Wild-type (WT) DNMT3b but not Mutant (MUT) (Fig. 6B). The RNA and protein expression levels of DNMT3b in ovarian granulosa cells were suppressed after miR-21-5p overexpression and upregulated after miR-21-5p disruption (Fig. 6C-D). Compared to the IR group, the RNA expression level of DNMT3b decreased with increasing ZGW concentration (Fig. 6E).

Fig. 6.

miR-21-5p targeting DNMT3b. A The inferred binding regions between miR-21-5p and DNMT3b. B Dual-luciferase reporter assay. C-D The gene and protein expression levels of DNMT3b were detected by qPCR and western blot. GAPDH served as the loading control. E The RNA expression level of DNMT3b was detected by qPCR

ZGW alleviated radiation-induced ferroptosis via miR-21-5p/DNMT3b in vitro

Western blot was performed after transfection. For DNMT3b, the NC-inhibitors + OE-NC group showed the lowest expression level, while the expression level in the miR-21-5p-inhibitors group was similar to the OE-DNMT3b group. The miR-21-5p-inhibitors + OE-DNMT3b group had the highest expression level (Fig. 7A). The intracellular iron, MDA content showed the same trend (Fig. 7B-C), while the levels of GSH and GPX4 exhibited an opposite trend (Fig. 7D and A). The abnormal mitochondrial morphological changes induced by γ-radiation were alleviated by ZGW treatment. However, inhibiting miR-21-5p and/or overexpressing DNMT3b destroyed the protective effect of ZGW. This was observed in the miR-21-5p-inhibitors group, OE-DNMT3b group, and miR-21-5p-inhibitors + OE-DNMT3b group (Fig. 7E). Therefore, ZGW reversed radiation-induced ferroptosis through miR-21-5p/DNMT3b in ovarian granulosa cells.

Fig. 7.

ZGW alleviated radiation-induced ferroptosis via miR-21-5p/DNMT3b in vitro. A The protein expression level of DNMT3b was determined by western blot. GAPDH served as the loading control. B Intracellular Fe2⁺ detection. Bar = 50 μm. C Intracellular MDA content assay. D Intracellular GSH content. E The protein expression level of GPX4 was determined by western blot. GAPDH served as the loading control. F Morphological changes of ovarian granulosa cells were showed by TEM. Bar = 500 nm

ZGW alleviated radiation-induced POF and ferroptosis via miR-21-5p/DNMT3b in vivo

Rats were transfected with NC-inhibitors or miR-21-5p-inhibitors plasmids, then followed by radiation and ZGW treatment. Morphological detection of rat ovary sections revealed that miR-21-5p-inhibitors nullified the anti-POF effect of ZGW. Atretic follicles (AF), zona pellucida remnants (yellow circles), and hemorrhage (blue arrowheads) were clearly observed in the miR-21-5p-inhibitors group, and the number of normal follicles significantly decreased. Compared to the NC-inhibitors group, transfection with the miR-21-5p-inhibitors significantly reduced the counts of primordial (from 12.33 to 6.67), preantral (from 4.33 to 1.33), and antral follicles (from 8 to 1.67), while increasing the population of atretic follicles from 12.67 to 24.67 (Fig. 8A-B). Downregulated serum estradiol and AMH levels were determined after miR-21-5p-inhibitors transfection (Fig. 8C). As the proportion of necrotic ovarian granulosa cells increased in the miR-21-5p-inhibitors group (Fig. 8D), the type of cell death was also determined. Here, increased intracellular Fe²⁺ and MDA levels, along with decreased GSH and GPX4 levels, suggested strong lipid peroxidation and the occurrence of ferroptosis (Fig. 8E-I). The RNA expression level of miR-21-5p was shown in Fig. 8H. The downregulation of miR-21-5p promoted DNMT3b expression (Fig. 8I). In conclusion, ZGW demonstrated a strong therapeutic effect through the miR-21-5p/DNMT3b axis in radiation-induced POF model.

Fig. 8.

ZGW alleviated radiation-induced POF and ferroptosis via miR-21-5p/DNMT3b in vivo. A Histological detection (HE staining). Bar = 200 μm. AF: atretic follicle. Yellow circles: remnants of the zona pellucida. Blue arrowheads: interstitial hyperplasia with vascular hemorrhage. B Morphometric analysis of different ovarian follicle populations (n = 3 per goup). C Detection of serum estradiol and AMH (n = 5 per goup). D Cell death was analyzed with Hoechst/PI staining. Bar = 50 μm. E-G The content of Fe2⁺ (n = 6 per goup), MDA (n = 3 per goup), and GSH (n = 3 per goup). H qPCR of miR-21-5p in ovarian tissue lysates. I Western blot of DNMT3b and GPX4 in ovarian tissue lysates (n = 3 biological replicates). GAPDH served as the loading control.

Discussion

Taking high-level doses of radiation or being exposed to it for prolonged periods due to work or accidents, as well as oncologic therapy such as radiotherapy, can have significant impacts on the ovarian function of young women. Worse health outcomes and loss of fertility are major considerations [42]. The incidence rate of POF has increased largely due to the improved survival rate of cancer patients treated with radiotherapy and chemotherapy [43–45].

After excluding pregnancy, patients experiencing amenorrhea for more than 4 months before the age of 40, with almost no follicle development in the ovaries, serum FSH and estradiol levels are measured once a week for 2–4 consecutive weeks. If FSH levels of the above-mentioned patients are elevated (> 40 mIU/ml) and estradiol levels are low (usually < 30 pg/ml), POF can be diagnosed [39]. Due to the sudden onset of POF, patients often exhibit severe perimenopausal syndrome, causing great physical and mental pain. For POF, the main treatment at present is hormone replacement therapy (HRT), which uses estrogen and progesterone to alleviate POF symptoms. High doses of hormones can increase weight, accelerate bone maturation, and affect height, and long-term HRT can increase health risks such as thrombosis, heart attack, stroke, endometrial cancer, and breast cancer [46, 47]. Due to the side effects of HRT, it is difficult for patients to persist in long-term treatment. Slowing down the decline of ovarian function and finding effective methods to reverse POF are currently a topic of great concern.

Many TCM formulations have demonstrated efficacy in alleviating POF. For example, Siwu Tang (SWT) has been shown to improve follicular development, promote angiogenesis, and restore ovarian function in a cyclophosphamide (CTX)-induced POF mouse model. In this model, CTX administration led to a significant reduction in the number of follicles at various developmental stages and decreased serum levels of hormones such as estradiol and AMH. These adverse effects were markedly reversed by treatment with SWT [48]. Other TCM formulae, including Danggui Shaoyao San [49], Chen’s peiyuan Tang [50] and Danggui Buxue Decoction [35], have also been reported to exhibit therapeutic potential against POF. ZGW is from Volume 51 of Jingyue Quanshu (a medical book of TCM) [51]. In TCM, it is a commonly used prescription for the treatment of kidney ‘yin’ deficiency, which is commonly used in patients with senile diseases, gynecological diseases, such as osteoporosis, menopausal syndrome, POF, amenorrhea, low menstrual volume, etc. Modern pharmacological studies have confirmed that ZGW can maintain the balance of the immune system, regulate hormone levels, trigger follicle development, promote the development of reproductive organs, and it also plays a role in treating POF and improving perimenopausal syndrome. In our research, we found that radiation induced POF in rats. The ovaries of radiation-exposed rats showed increased atretic follicles, zona pellucida remnants, vascular hemorrhage, and significantly decreased hormone level. Furthermore, we focused on the ovarian granulosa cell death induced by radiation, and the accumulated iron content, lipid peroxidation, and low GPX4 expression level confirmed the induction of ferroptosis. ZGW exhibited strong effects in resisting ferroptosis of ovarian granulosa cells by targeting the downregulation of iron accumulation and lipid peroxidation.

miR-21 has been confirmed to be related to radiation-induced cell death. A study based on differentially expressed ferroptosis genes protein-protein interaction (PPI) network reported that bta-mir-21-5p is associated with ferroptosis genes [45]. In our study, we proposed that miR-21-5p played a vital role in the mechanism of reversing radiation-induced ferroptosis. It was verified that the expression of miR-21-5p was elevated in ZGW-treated irradiated ovarian granulosa cells, and miR-21-5p-mimics further inhibited ferroptosis, while miR-21-5p-inhibitors impaired the protective effect of ZGW.

DNMT3b is an important de novo DNA methyltransferase expressed during early embryonic development. Recent evidence indicates that DNMT3b recruitment is regulated by multiple mechanisms, including chromatin modification, non-coding RNAs, and DNA-binding factors, etc [52]. Radiotherapy efficacy is limited in patients with nasopharyngeal carcinoma (NPC) owing to the elevated DNMT3b level. DNMT3b inhibition leads to cell cycle arrest and apoptosis [53]. Furthermore, inhibiting the expression of miR-21 in MCF-7 cells results in decreased DNMT3b level [54]. In our study, we utilized the dual-luciferase reporter assay to demonstrate the interaction between miR-21-5p and DNMT3b. It was found that miR-21-5p targeted DNMT3b, and the complementary sequence was AUAAGCU. Further experiments indicated that overexpressing DNMT3b promoted radiation-induced ferroptosis after ZGW treatment, suggesting that ZGW reversed radiation-induced ferroptosis via miR-21-5p/DNMT3b axis.

Compared with HRT, ZGW has fewer adverse reactions and better patient compliance, making it a favorable option for clinical application and prevention of POF. However, some limitations may exist in the current research on alleviating POF by ZGW. On the one hand, the animal experiments were conducted under relatively suitable conditions; however, in real life, the dose and frequency of radiation that people receive are often uncertain. On the other hand, the progress of animal experiments cannot fully simulate the effects of human drug therapy. Therefore, more efforts are needed to further investigate the molecular mechanism of ZGW in treating POF in order to lay a solid theoretical foundation for the treatment of POF and other ovarian diseases, and it is necessary to provide more exhaustive scientific evidence.

Abbreviations

POF

Premature Ovarian Failure

ZGW

Zuogui Wan

PCD

Programmed Cell Death

ROS

Reactive Oxygen Species

MMP

Mitochondrial Membrane Potential

FSH

Follicle-stimulating hormone

miRNA

microRN

HBSS

Hank’s Balanced Salt Solution

ICP-MS

Inductively Coupled Plasma Mass Spectrometer

TEM

Transmission Electron Microscopy

HRT

Hormone Replacement Therapy

PPI

Protein-protein Interaction

Authors’ contributions

H.H. conceived and designed the study; Y.L., M.C., M.L. and Z.L. performed the experiments; analyzed the data; Y.L., M.C. and M.L. drafted and revised the manuscript; H.H. supervised the study; all authors reviewed and approved the final version of the manuscript.

Funding

This study was funded by the grants from National Natural Science Foundation of China (No. 82205159); Shenzhen Natural Science Foundation (No. JCYJ20220530150213031); Sanming Project of Medicine in Shenzhen (No. SZZYSM202311011); Guangdong Provincial Medical Science and Technology Research Foundation Project (No. B2025131).

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Animal experiments were approved by the Animal Ethics Committee of Shenzhen Hospital (Futian) of Guangzhou University of Chinese Medicine and conducted in accordance with ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.