Effect Of Bushen Tiaojing Formula On LIF And Its Receptor in Regulating Granulosa Cell Proliferation And Steroid Hormone Secretion in Aged Mice
Apr 27, 2026
Authors:
Li Kun, Zhang Shuancheng, Sun Ying, Song Yajing, Ji Chuanyuan, Xiong Wenyan, Du Huilan, Ma Yucong
Received: 2025-11-26
Online-first publication date: 2026-04-08
Recommended citation format:
Li Kun, Zhang Shuancheng, Sun Ying, Song Yajing, Ji Chuanyuan, Xiong Wenyan, Du Huilan, Ma Yucong. Effect of Bushen Tiaojing Formula on LIF and Its Receptor in Regulating Granulosa Cell Proliferation and Steroid Hormone Secretion in Aged Mice [J/OL]. Journal of Beijing University of Traditional Chinese Medicine.
CNKI link (as provided): https://link.cnki.net/urlid/11.3574.R.20260407.1733.006
Author Information / Funding
Li Kun: male, master's student.
Corresponding author: Ma Yucong, female, PhD, assistant researcher. Research focus: TCM prevention and treatment of reproductive regulatory-disorder diseases. E-mail: mayucong@hebcm.edu.cn
Funding:
National Natural Science Foundation of China (No. 82405455); Hebei Natural Science Foundation (No. H2022423331); Hebei Administration of Traditional Chinese Medicine Research Projects (No. 2023122, 2023124); Hebei Higher Education Science and Technology Research Project (No. QN2024039); Basic Scientific Research Business Fund Special Project for Hebei Provincial Universities (No. TDZR2024001).
Abstract
Objective:
To investigate the mechanism by which Bushen Tiaojing Formula promotes ovarian granulosa cell proliferation and steroid hormone synthesis in aged mice.
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Methods:
Twenty 7–8-week-old female ICR mice were used as the control group (intragastric administration of distilled water). Eighty 9–10-month-old female mice were randomly assigned (random numbers generated by SPSS 27.0) to four groups: model group (intragastric distilled water), Bushen Tiaojing Formula low-dose group [intragastric Bushen Tiaojing Formula (25.6 g/kg)], Bushen Tiaojing Formula high-dose group [intragastric Bushen Tiaojing Formula (51.2 g/kg)], and leukemia inhibitory factor (LIF) inhibitor group [intragastric Bushen Tiaojing Formula (51.2 g/kg) + intraperitoneal injection of EC330 (0.5 mg/kg)]. Each group received gavage or intraperitoneal injection once daily for 30 days. Vaginal smear cytology was used to observe estrous cycle changes. ELISA was used to measure sex hormone levels. RT-PCR and Western blotting were used to detect mRNA and protein expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1 (Cyclin D1), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 family 19A1 (CYP19A1), LIF, and leukemia inhibitory factor receptor (LIFR) in granulosa cells. Immunohistochemistry was used to detect LIF and LIFR expression.
Results:
Compared with the control group, mice in the model group showed disordered estrous cycles and prolonged cycle length (P < 0.05), decreased estradiol (E2) and progesterone (P4) levels (P < 0.05), increased follicle-stimulating hormone (FSH), luteinizing hormone (LH), and FSH/LH ratio (P < 0.05), and decreased mRNA and protein expression levels of PCNA, Cyclin D1, StAR, 3β-HSD, CYP19A1, LIF, and LIFR in granulosa cells (P < 0.05). Compared with the model group, the Bushen Tiaojing Formula groups had more regular estrous cycles and shorter cycle length (P < 0.05), increased E2 and P4 levels (P < 0.05), decreased FSH, LH, and FSH/LH ratio (P < 0.05), and increased mRNA and protein expression levels of PCNA, Cyclin D1, StAR, 3β-HSD, CYP19A1, LIF, and LIFR in granulosa cells (P < 0.05). Compared with the high-dose Bushen Tiaojing Formula group, the LIF inhibitor group showed disordered estrous cycles and prolonged cycle length (P < 0.05), decreased E2 and P4 levels (P < 0.05), increased FSH, LH, and FSH/LH ratio (P < 0.05), and decreased mRNA and protein expression levels of PCNA, Cyclin D1, StAR, 3β-HSD, CYP19A1, and LIFR (P < 0.05).
Conclusion:
Bushen Tiaojing Formula may upregulate LIF/LIFR, thereby mediating granulosa cell proliferation and steroid hormone synthesis, improving ovarian reserve function, and promoting follicular development.
Keywords:
Bushen Tiaojing Formula; leukemia inhibitory factor; granulosa cells; steroid hormones; mice
Introduction
With the implementation of China's "three-child policy" and the trend toward delayed childbearing, the population of older infertile women continues to expand. Age is a decisive factor affecting reproductive capacity. In older women, follicle number and oocyte quality decline significantly, pregnancy and live-birth rates decrease, and miscarriage rates increase markedly [1]. Even with assisted reproductive technologies such as artificial insemination and in vitro fertilization–embryo transfer, clinical pregnancy rates in older women remain far lower than those in younger women. Therefore, delaying the decline of fertility in older women has become a key issue in the context of fertility policy.
Steroid hormone levels are commonly used as important indicators of ovarian function and are closely related to follicular development and oocyte maturation [2]. Estradiol (E2) is an important steroid hormone that promotes the development of female reproductive organs; it is secreted by ovarian granulosa cells, and its level reflects the differentiation capacity of granulosa cells [3]. Progesterone (P4) is one of the key steroid hormones regulating complex physiological processes in women, and regulation of oocyte maturation and release, menstrual cycle changes, embryo implantation and decidualization, and uterine growth all depend on P4 [4]. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) promote granulosa cell proliferation, follicular development, and oocyte release; their ratio (FSH/LH) is often used to reflect ovarian age [5]. E2 can regulate FSH and LH levels, thereby promoting granulosa cell proliferation and follicular development [6].
Within granulosa cells, steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 family 19A1 (CYP19A1) are key enzymes for steroid hormone synthesis and jointly participate in the steroidogenesis process [3,7]. Proliferating cell nuclear antigen (PCNA) and cyclin D1 (Cyclin D1) are key markers of cell proliferation, regulating DNA replication and repair and cell-cycle changes [8]. Leukemia inhibitory factor (LIF) is widely expressed in oocytes, granulosa cells, and follicular fluid and is an important factor promoting cell proliferation and follicular development [9].
Our team's prior clinical research indicates that Bushen Tiaojing Formula is derived (modified) from Wuzi Yanzong Pill combined with Yangjing Zhongyu Decoction, based on the TCM theory that "the kidney governs reproduction." It has the effects of tonifying the kidney and replenishing essence, nourishing blood, and regulating menstruation. Previous clinical research confirmed that Bushen Tiaojing Formula can promote follicular development and improve embryo quality and pregnancy rate in infertile patients [10–11]. Animal experiments have shown that Bushen Tiaojing Formula can upregulate ovarian LIF protein expression in mice [12]. However, whether Bushen Tiaojing Formula can promote granulosa cell proliferation and steroid hormone secretion by regulating LIF expression remains unclear. This study aims to explore the effects and regulatory mechanism of Bushen Tiaojing Formula on granulosa cell proliferation and hormone secretion in early-aging mice, providing new therapeutic targets and experimental evidence for improving fertility in older women via the kidney-tonifying method and enriching the connotation of the TCM theory that "the kidney governs reproduction."
1. Materials
1.1 Animals
A total of 100 female SPF-grade ICR mice were used: 20 mice aged 7–8 weeks (body weight 23–27 g) and 80 mice aged 9–10 months (body weight 35–40 g). All mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2021-0006. They were housed at the Experimental Animal Center of Hebei University of Chinese Medicine, use license number: SYXK (Hebei) 2022-010, under a 12 h/12 h light–dark cycle, with free access to food and water, and acclimated for 7 days.
1.2 Ethical review
This study was approved by the Experimental Animal Ethics Committee of Hebei University of Chinese Medicine, approval number: DWLL202203105.
1.3 Main drugs and reagents
Bushen Tiaojing Formula consisted of:
Prepared Rehmannia root (Shu Di Huang) 20 g, Angelica sinensis (Dang Gui) 9 g, White peony root (Bai Shao) 9 g, Chinese yam (Shan Yao) 12 g, Cornus officinalis (Shan Zhu Yu) 15 g, Goji berry (Gou Qi Zi) 12 g, Glossy privet fruit (Nv Zhen Zi) 9 g, Epimedium (Yin Yang Huo) 10 g, Placenta Hominis (Zi He Che) 10 g, Raspberry fruit (Fu Pen Zi) 10 g, Dodder seed (Tu Si Zi) 12 g, Cyperus (Xiang Fu) 6 g. All decoction pieces were purchased from Shijiazhuang Lerentang Chinese Medicine Co., Ltd. After soaking for 30 min, the herbs were decocted with water, concentrated, and centrifuged to prepare decoctions containing crude drug at 2.7 g/mL and 5.4 g/mL, respectively, stored at 4°C for later use [13].
LIF inhibitor: EC330 (Shanghai Yuanye Bio-Technology Co., Ltd.; batch no. S84285).
Isoflurane (Shenzhen RWD Life Science Co., Ltd.; batch no. 20231201).
ELISA kits for E2, P4, FSH, and LH (Wuhan Huamei Bioengineering Co., Ltd.; batch nos. CSB-E07280m, CSB-E05104m, CSB-E06871m, CSB-E12770m).
Methylene blue staining solution; BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.; batch nos. G1300, PC0020).
Antibodies and molecular reagents including StAR, LIFR, RNA extraction and reverse transcription kits, and Green qPCR Master Mix (Wuhan Servicebio Technology Co., Ltd.; batch nos. GB111430, GB111476, G3013, G3030, G3337, G3326).
Antibodies for PCNA, Cyclin D1, LIF, Tubulin, GAPDH; HRP secondary antibodies (Proteintech, USA; batch nos. 60097-1-Ig, 60186-1-Ig, 26757-1-AP, 66240-1-Ig, 60004-1-Ig, SA00001-1, SA00001-2).
3β-HSD antibody (Beijing Bioss Biotechnology Co., Ltd.; batch no. bs-3906R).
CYP19A1 antibody (Absin Bioscience Inc., Shanghai; batch no. abs136404).
SuperKine ECL ultra-sensitive chemiluminescence reagent (Apexbio/Ya Ke Yin Biotechnology; batch no. BMU102-CN).
1.4 Main instruments and equipment
VMR small-animal anesthesia machine (MIDMARK, USA); 5424R centrifuge (Eppendorf, Germany); VersaMax microplate reader (Molecular Devices, USA); RM2245 microtome, EG11508 embedding station, CM1950 cryostat (Leica, Germany); ND2000/2000C spectrophotometer and QuantStudio 5 real-time PCR system (Thermo Fisher Scientific, USA); ECLIPSE Ci-L Plus microscope (Nikon, Japan); Mini-PROTEAN Tetra electrophoresis system and Trans-Blot SD semi-dry transfer system (Bio-Rad, China); WIX-miniBLOT transfer tank (Wix Technology, Beijing); Touch Imager chemiluminescent imaging system (Yibote Life Science, Shanghai).
2. Methods
2.1 Modeling and grouping
Female mice aged 9–10 months are considered equivalent to women aged 35–40 years [14]. At this age, mice show ovarian functional decline, reduced fertility, disordered or prolonged estrous cycles, and ovarian index, anti-Müllerian hormone, follicle number, pregnancy rate, and fertility rate are all significantly lower than in 2-month-old mice [15]. In this study, 9–10-month-old female mice were used as the model, and 7–8-week-old mice served as normal controls. Using random numbers generated in SPSS 27.0, the 9–10-month-old mice were divided into four groups: model group, Bushen Tiaojing Formula low-dose group, Bushen Tiaojing Formula high-dose group, and LIF inhibitor group, with 20 mice per group.
2.2 Administration
Administration was performed daily at 9:00 a.m.
Control and model groups received an equal volume of distilled water by gavage.
Low-dose and high-dose groups received Bushen Tiaojing Formula by gavage at 25.6 g/(kg·d) and 51.2 g/(kg·d), respectively.
The LIF inhibitor group received Bushen Tiaojing Formula [51.2 g/(kg·d)] by gavage, followed immediately by intraperitoneal injection of EC330 [0.5 mg/(kg·d)].
All treatments were given once daily for 30 days.
2.3 Sample collection
Twenty-four hours after the final administration, mice were anesthetized by continuous inhalation of isoflurane and blood was collected via the orbital sinus. Blood was left at room temperature for 2 h, centrifuged at 3,500 r/min (radius 8.4 cm) for 15 min, and the supernatant was collected and stored frozen. Mice were sacrificed by cervical dislocation, and both ovaries were rapidly removed. For each group, ovaries from 5 mice were randomly fixed in 4% paraformaldehyde for 48 h, paraffin-embedded, and sectioned for immunohistochemistry [16]. Ovaries from the remaining mice were punctured under a stereomicroscope with a 1 mL syringe needle to release and collect granulosa cells [17]. Cells were washed with PBS, centrifuged, snap-frozen in liquid nitrogen, and stored at −80°C.
2.4 Outcome measurements
2.4.1 Vaginal smear cytology for estrous cycle observation
Vaginal exfoliated cells were collected daily at 10:00 a.m., smeared on slides, and estrous cycles were observed [16].
2.4.2 ELISA for serum sex hormone levels
After thawing serum at room temperature, samples were centrifuged. Reagents were prepared, samples added, incubated, washed, developed, and absorbance values measured strictly according to the ELISA kit instructions. Standard curves were plotted to calculate E2, P4, FSH, and LH concentrations.
2.4.3 RT-PCR for granulosa-cell mRNA expression
Total RNA was extracted from granulosa cells according to the kit instructions and reverse-transcribed to cDNA. PCR reaction mixtures were prepared and amplified using a real-time PCR system under the following conditions: 95°C pre-denaturation 30 s; 95°C denaturation 15 s; 60°C annealing/extension 30 s; 40 cycles. Relative mRNA levels were calculated by the 2−ΔΔCt2^{-\Delta\Delta Ct}2−ΔΔCt method. Primers were synthesized by Wuhan Servicebio Technology Co., Ltd. Primer sequences are shown in Table 1.
Table 1. Primer sequences
PCNA: Forward 5′-GTCGGGTGAATTTGCACGTA-3′; Reverse 5′-CTCTATGGTTACCGCCTCCTC-3′ (175 bp)
Cyclin D1: Forward 5′-AGGCGGATGAGAACAAGCAG-3′; Reverse 5′-AAGAAAGTGCGTTGTGCGGTA-3′ (191 bp)
StAR: Forward 5′-GGGCATACTCAACAACCAGGAA-3′; Reverse 5′-CTTGACATTTGGGTTCCACTCTC-3′ (196 bp)
3β-HSD: Forward 5′-TAAGGGTATTCTGTGTGTTACTGGC-3′; Reverse 5′-AGCAGGAAGGCAAGCCAGTAG-3′ (270 bp)
CYP19A1: Forward 5′-CTGGACACCTCTAACACGCTCTT-3′; Reverse 5′-GGCATCTTTCAAATCCTTGACA-3′ (177 bp)
LIF: Forward 5′-GGAGCTGTATCGGATGGTCG-3′; Reverse 5′-GTACTTGTTGCACAGACGGC-3′ (169 bp)
LIFR: Forward 5′-TGAGGAATGCCACAATCAGAGG-3′; Reverse 5′-TGTCGCTTCTCATCACTCCACC-3′ (148 bp)
GAPDH: Forward 5′-CCTCGTCCCGTAGACAAAATG-3′; Reverse 5′-TGAGGTCAATGAAGGGGTCGT-3′ (133 bp)
2.4.4 Western blotting for protein expression in granulosa cells
Granulosa cells were lysed in lysis buffer containing protease inhibitors and homogenized, then centrifuged at 4°C, 12,000×g for 10 min to obtain total protein. Protein concentration was measured using the BCA kit. Samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked at room temperature for 2 h and incubated with primary antibodies overnight at 4°C: PCNA (1:5,000), Cyclin D1 (1:5,000), StAR (1:5,000), 3β-HSD (1:800), CYP19A1 (1:1,000), LIF (1:4,000), LIFR (1:4,000). The next day, membranes were washed with TBST three times, incubated with secondary antibodies (1:6,000) at room temperature for 1 h, washed again three times, and visualized by chemiluminescence. Images were captured and quantified using ImageJ.
2.4.5 Immunohistochemistry for LIF and LIFR in ovaries (expression and localization)
Ovarian tissues fixed in 4% paraformaldehyde for 48 h were dehydrated through graded xylene and ethanol, cleared, embedded in paraffin, and sectioned at 5 μm. After deparaffinization, antigen retrieval, and blocking with 5% bovine serum albumin, sections were incubated overnight with primary antibodies against LIF and LIFR (1:100), followed by secondary antibody incubation (1:500), DAB development, hematoxylin counterstaining, mounting, and observation under a light microscope. ImageJ was used for positive area analysis.
2.5 Statistical analysis
SPSS 27.0 was used. Data consistent with normal distribution were expressed as mean ± standard deviation (xˉ±s\bar{x} \pm sxˉ±s). One-way ANOVA was used for comparisons among multiple groups. For pairwise comparisons, LSD was used when variances were equal; Dunnett's T3 was used when variances were unequal. P < 0.05 indicated statistical significance.
3. Results
3.1 Changes in estrous cycles
The control group (reproductive-age mice) showed regular estrous cycles. Compared with the control group, before administration, the four early-aging mouse groups had prolonged estrous cycles (P < 0.05). After administration, compared with the control group, the model group showed disordered and prolonged cycles; compared with the model group, both the low- and high-dose Bushen Tiaojing Formula groups showed more regular cycles and shortened cycle length, and the high-dose group's cycle length was closer to that of the reproductive-age control group. Compared with the high-dose Bushen Tiaojing Formula group, the LIF inhibitor group showed prolonged and disordered cycles. See Table 2 and Figure 1.
Table 2. Comparison of estrous cycles (days; xˉ±s\bar{x} \pm sxˉ±s; n = 20)
Control: before 5.22±0.86; after 5.21±0.81
Model: before 8.53±2.11*; after 9.59±2.56*
Bushen Tiaojing Formula low-dose (25.6 g/kg): before 8.56±1.96*; after 7.16±1.31#
Bushen Tiaojing Formula high-dose (51.2 g/kg): before 8.63±1.79*; after 6.04±1.01#
LIF inhibitor group (51.2 g/kg + 0.5 mg/kg): before 8.56±1.65*; after 8.59±1.73△
F: 12.014 (before), 19.954 (after); P < 0.001 (before), P < 0.001 (after)
Notes: *P < 0.05 vs control; #P < 0.05 vs model; △P < 0.05 vs high-dose group.
(P: proestrus; E: estrus; M: metestrus; D: diestrus)
3.2 Sex hormone levels
Sex hormone levels reflect ovarian function. Compared with the control group, the model group had decreased E2 and P4 and increased FSH, LH, and FSH/LH. Compared with the model group, the Bushen Tiaojing Formula groups showed improvement (P < 0.05). Compared with the low-dose group, the high-dose group showed higher E2 and lower FSH and FSH/LH (P < 0.05). Compared with the high-dose group, the LIF inhibitor group showed lower E2 and P4 and higher FSH, LH, and FSH/LH (P < 0.05). See Table 3.
Table 3. Comparison of sex hormone levels (xˉ±s\bar{x} \pm sxˉ±s; n = 16)
Control: E2 48.13±4.49 ng/L; P4 1.31±0.28 μg/L; FSH 8.15±1.48 IU/L; LH 7.69±1.82 IU/L; FSH/LH 1.11±0.30
Model: E2 32.91±6.88*; P4 0.82±0.16*; FSH 21.59±3.24*; LH 12.02±2.67*; FSH/LH 1.84±0.32*
Low-dose (25.6 g/kg): E2 41.44±3.91#; P4 1.14±0.18#; FSH 14.97±3.83#; LH 10.47±1.83#; FSH/LH 1.46±0.40#
High-dose (51.2 g/kg): E2 46.36±4.57#□; P4 1.22±0.23#; FSH 10.10±1.87#□; LH 9.15±1.77#; FSH/LH 1.14±0.30#□
LIF inhibitor (51.2 g/kg + 0.5 mg/kg): E2 34.77±6.29△; P4 0.92±0.19△; FSH 18.16±2.64△; LH 11.05±2.26△; FSH/LH 1.70±0.36△
F: 25.654, 15.042, 65.341, 10.390, 14.849; P < 0.001 for all
Notes: *P < 0.05 vs control; #P < 0.05 vs model; □P < 0.05 vs low-dose; △P < 0.05 vs high-dose.
3.3 PCNA and Cyclin D1 protein and mRNA expression in granulosa cells
Compared with the control group, the model group showed decreased PCNA and Cyclin D1 protein and mRNA (P < 0.05). Compared with the model group, both low- and high-dose Bushen Tiaojing Formula groups showed increased PCNA and Cyclin D1 protein and mRNA (P < 0.05), with the high-dose group higher than the low-dose group (P < 0.05). Compared with the high-dose group, the LIF inhibitor group showed decreased PCNA and Cyclin D1 protein and mRNA (P < 0.05). See Figure 2 and Tables 4–5.
Table 4. PCNA and Cyclin D1 protein expression (xˉ±s\bar{x} \pm sxˉ±s; n = 3)
Control: PCNA/Tubulin 1.00±0.09; Cyclin D1/Tubulin 1.00±0.08
Model: 0.48±0.14*; 0.41±0.15*
Low-dose: 0.81±0.06#; 0.76±0.19#
High-dose: 0.95±0.11#□; 1.07±0.23#□
LIF inhibitor: 0.62±0.03△; 0.56±0.14△
F: 28.553; 8.491; P < 0.001; P = 0.003
Notes: * vs control; # vs model; □ vs low-dose; △ vs high-dose.
Table 5. PCNA and Cyclin D1 mRNA expression (xˉ±s\bar{x} \pm sxˉ±s; n = 6)
Control: PCNA/GAPDH 1.00±0.20; Cyclin D1/GAPDH 1.00±0.20
Model: 0.35±0.08*; 0.39±0.11*
Low-dose: 0.71±0.07#; 0.74±0.05#
High-dose: 0.96±0.18#□; 1.04±0.18#□
LIF inhibitor: 0.43±0.08△; 0.53±0.07△
F: 28.279; 26.567; P < 0.001; P < 0.001
Notes: * vs control; # vs model; □ vs low-dose; △ vs high-dose.
3.4 StAR, 3β-HSD, and CYP19A1 protein and mRNA expression in granulosa cells
Compared with the control group, the model group showed decreased StAR, 3β-HSD, and CYP19A1 protein and mRNA (P < 0.05). Compared with the model group, low- and high-dose Bushen Tiaojing Formula groups showed increased expression (P < 0.05), and the high-dose group was higher than the low-dose group (P < 0.05). Compared with the high-dose group, the LIF inhibitor group showed decreased expression (P < 0.05). See Figure 3 and Tables 6–7.
Table 6. Protein expression (xˉ±s\bar{x} \pm sxˉ±s; n = 3)
Control: StAR/Tubulin 1.00±0.21; 3β-HSD/Tubulin 1.00±0.24; CYP19A1/GAPDH 1.00±0.12
Model: 0.30±0.05*; 0.46±0.11*; 0.56±0.13*
Low-dose: 0.61±0.14#; 0.90±0.27#; 0.86±0.16#
High-dose: 0.90±0.18#□; 0.98±0.29#; 0.98±0.07#
LIF inhibitor: 0.41±0.14△; 0.48±0.15△; 0.61±0.05△
F: 11.647; 4.360; 10.302; P = 0.001; 0.027; 0.001
Notes: * vs control; # vs model; □ vs low-dose; △ vs high-dose.
Table 7. mRNA expression (xˉ±s\bar{x} \pm sxˉ±s; n = 6)
Control: StAR/GAPDH 1.00±0.18; 3β-HSD/GAPDH 1.00±0.15; CYP19A1/GAPDH 1.00±0.11
Model: 0.36±0.07*; 0.48±0.08*; 0.48±0.06*
Low-dose: 0.64±0.06#; 0.73±0.06#; 0.76±0.05#
High-dose: 0.91±0.15#□; 0.92±0.08#□; 0.97±0.14#□
LIF inhibitor: 0.40±0.05△; 0.54±0.07△; 0.55±0.06△
F: 39.032; 35.210; 40.258; P < 0.001 for all
Notes: * vs control; # vs model; □ vs low-dose; △ vs high-dose.
3.5 LIF and LIFR expression in ovaries and antral follicles
LIF and LIFR were expressed in oocytes, granulosa cells, theca cells, and corpus luteum (Figure 4). In larger antral follicles, LIF and LIFR expression in granulosa cells was higher than in small follicles and corpus luteum cells. Antral follicles were selected for comparisons among groups. Compared with the control group, the model group showed lower LIF and LIFR (P < 0.05). Compared with the model group, the low- and high-dose Bushen Tiaojing Formula groups showed increased LIF and LIFR in follicular granulosa cells (P < 0.05), and the high-dose group was higher than the low-dose group (P < 0.05). Compared with the high-dose group, the LIF inhibitor group showed decreased LIFR expression (P < 0.05). See Figure 5 and Table 8.
Table 8. Positive area of LIF and LIFR in follicular granulosa cells (%; xˉ±s\bar{x} \pm sxˉ±s; n = 5)
Control: LIF 19.83±2.33; LIFR 20.74±3.10
Model: LIF 10.43±1.45*; LIFR 9.62±1.15*
Low-dose: LIF 13.33±1.27#; LIFR 13.84±1.69#
High-dose: LIF 17.04±1.66#□; LIFR 19.01±3.53#□
LIF inhibitor: LIF 15.05±1.22; LIFR 11.02±1.54△
F: 23.905; 20.773; P < 0.001; P < 0.001
Notes: * vs control; # vs model; □ vs low-dose; △ vs high-dose.
3.6 LIF and LIFR protein and mRNA expression in granulosa cells
Compared with the control group, the model group had decreased LIF and LIFR protein and mRNA expression (P < 0.05). Compared with the model group, the low- and high-dose Bushen Tiaojing Formula groups showed increased LIF and LIFR protein and mRNA, and the high-dose group was higher than the low-dose group (P < 0.05). Compared with the high-dose group, the LIF inhibitor group showed decreased LIFR protein and mRNA expression (P < 0.05). See Figure 6 and Tables 9–10.
Table 9. LIF and LIFR protein expression (xˉ±s\bar{x} \pm sxˉ±s; n = 3)
Control: LIF/Tubulin 1.00±0.08; LIFR/Tubulin 1.00±0.20
Model: 0.36±0.08*; 0.39±0.20*
Low-dose: 0.78±0.18#; 0.87±0.08#
High-dose: 1.02±0.05#□; 1.14±0.10#□
LIF inhibitor: 0.89±0.05; 0.60±0.07△
F: 22.168; 13.659; P < 0.001; P < 0.001
Notes: * vs control; # vs model; □ vs low-dose; △ vs high-dose.
Table 10. LIF and LIFR mRNA expression (xˉ±s\bar{x} \pm sxˉ±s; n = 6)
Control: LIF/GAPDH 1.00±0.21; LIFR/GAPDH 1.00±0.17
Model: 0.37±0.04*; 0.32±0.08*
Low-dose: 0.63±0.06#; 0.60±0.07#
High-dose: 0.91±0.16#□; 0.86±0.15#□
LIF inhibitor: 0.76±0.11; 0.41±0.06△
F: 20.876; 37.818; P < 0.001; P < 0.001
Notes: * vs control; # vs model; □ vs low-dose; △ vs high-dose.
4. Discussion
Female fertility begins to decline after age 32 and declines rapidly after age 37 [18], which aligns with the TCM concept of "at the fifth seven (age 35), the Yangming channel declines." In TCM, it is believed that "the kidney stores essence" and that "essence is the foundation of the body." Congenital essence derived from parents is stored in the kidney during fetal life and constitutes the main component of kidney essence. The follicles nurtured by the ovary are a concrete form of reproductive essence and are key to embryonic development; therefore, it is said that "the kidney governs reproduction." In older women, kidney essence and qi begin to decline and "Tiangui" becomes exhausted, leading to insufficient nourishment for normal ovarian and follicular growth and inadequate energy and nutritional support for oocytes; the function of "kidney governing reproduction" declines. Kidney-tonifying therapy is a major clinical method for infertility; it aims to replenish kidney essence and strengthen kidney qi so that Ren and Chong channels are abundant, qi and blood are sufficient and well-regulated, thereby maintaining the ovarian internal environment and ensuring normal follicular development.
Bushen Tiaojing Formula is derived (modified) from Wuzi Yanzong Pill combined with Yangjing Zhongyu Decoction. It consists of prepared Rehmannia root, Angelica sinensis, white peony root, Chinese yam, Cornus officinalis, goji berry, glossy privet fruit, Epimedium, Placenta Hominis, raspberry fruit, dodder seed, and Cyperus. In the formula, prepared Rehmannia nourishes liver and kidney and benefits essence and marrow and serves as the monarch herb; Cornus officinalis, goji berry, and glossy privet fruit nourish liver and kidney, and Chinese yam tonifies the kidney and astringes essence, serving as minister herbs; Epimedium and Placenta Hominis tonify the kidney and benefit essence, and tonify qi and blood; raspberry fruit and dodder seed tonify liver and kidney and secure essence; Angelica sinensis and white peony root nourish blood and regulate menstruation; Cyperus regulates qi and activates blood, serving as assistant herbs. The whole formula tonifies kidney and replenishes essence, nourishes blood, and regulates menstruation, leading to abundant kidney essence and regulated menstruation to support conception.
Observation of estrous cycle changes is an effective method to evaluate reproductive function and the impact of drugs on reproductive function. The estrous cycle in female animals is regulated by secretion levels of hormones such as E2, P4, FSH, and LH [19–20]. In early-aging mice, insufficient or unstable ovarian hormone secretion can lead to prolonged estrous cycles or even absence of a clear cycle. The model group exhibited varying degrees of prolonged and disordered estrous cycles, which is related to reduced E2 and P4 levels and increased FSH/LH ratio causing regulatory disturbances. Compared with the model group, the low- and high-dose Bushen Tiaojing Formula groups had significantly shortened diestrus and more regular cycles, significantly increased E2 and P4, and decreased FSH/LH ratio. These results suggest that Bushen Tiaojing Formula can restore estrous cycles by improving hormone levels in early-aging mice.
Steroid hormones E2 and P4 regulate follicular development and atresia, granulosa cell proliferation, and oocyte growth [3,4]. In rats with impaired follicular function and development, E2 and P4 levels are significantly lower than in normal rats [21], suggesting impaired synthesis. Ovarian granulosa cells are the central site of steroid hormone synthesis. StAR transfers cholesterol to the outer mitochondrial membrane; 3β-HSD forms a complex with cholesterol side-chain cleavage enzyme and inserts into the inner mitochondrial membrane, catalyzing conversion of pregnenolone into active P4 and androstenedione; androstenedione is converted into E2 by aromatase encoded by CYP19A1 [3,8]. Therefore, StAR, 3β-HSD, and CYP19A1 are positively correlated with E2 and P4 levels [22–23]. This study found that Bushen Tiaojing Formula significantly upregulated StAR, 3β-HSD, and CYP19A1 protein and mRNA expression, which may be one reason it improved steroid hormone levels and restored estrous cycles in early-aging mice.
Granulosa cells in older infertile women exhibit excessive apoptosis, and apoptosis rate is negatively correlated with oocyte quality and pregnancy rate [24]; therefore, promoting granulosa cell proliferation is important for follicular development. PCNA participates in DNA replication and cell proliferation; increased PCNA expression is an early marker of granulosa cell proliferation [25]. Cyclin D1 is an important regulatory protein at the G1/S restriction point in the cell cycle; together they are important regulators of follicular development and atresia [8]. Studies such as Liao Fengjiao's have shown decreased PCNA and Cyclin D1 expression and increased apoptosis in granulosa cells in rat models of ovarian dysfunction and impaired follicular development [21,26]. Compared with the control group, the model group showed significantly decreased PCNA and Cyclin D1 expression, suggesting reduced granulosa cell proliferative capacity in early-aging mice. Kidney-tonifying TCM formulas (e.g., Xin Jia Gui Shen Pill, Bushen Huoxue Formula) have been reported to promote cell proliferation and significantly increase PCNA and Cyclin D1 expression [21,27]. In this study, compared with the model group, Bushen Tiaojing Formula significantly increased PCNA and Cyclin D1 expression in low- and high-dose groups, suggesting it can promote granulosa cell proliferation and thereby promote follicular development.
As a granulosa-cell proliferation factor, LIF can promote the growth of preantral and antral follicles [9]. LIF concentration is higher in larger and mature follicles and significantly lower in atretic follicles [28]. LIF is closely related to E2 and P4 levels and oocyte quality and is an important reference indicator for evaluating oocyte quality [29–30]. Adding LIF to in vitro cultured ovaries and cumulus–oocyte complexes can significantly increase granulosa cell proliferation and PCNA protein expression [31–32]. When LIF binds to its receptor LIFR, it can activate the transcription factor STAT3, thereby inducing transcription of target genes Cyclin D1 and PCNA to promote granulosa cell proliferation [33]. LIF can upregulate StAR and aromatase expression, thereby increasing E2 and P4 levels in the ovary [34–35]. These studies suggest that LIF is a regulatory factor for granulosa cell proliferation and steroid hormone synthesis. In this study, LIF and LIFR expression levels in early-aging mice were significantly decreased, and the downward trend was consistent with granulosa-cell proliferation markers Cyclin D1 and PCNA, steroidogenesis enzymes, and E2 and P4 levels, confirming that LIF can regulate granulosa cell proliferation and steroid hormone synthesis in early-aging mice. Further, Bushen Tiaojing Formula significantly increased LIF and LIFR expression, thereby promoting cell proliferation and steroid hormone expression. After adding a LIF inhibitor, cell proliferation, steroidogenic enzymes, and E2 and P4 levels were significantly decreased, suggesting that Bushen Tiaojing Formula promotes cell proliferation and hormone synthesis by regulating LIF and LIFR.
In summary, Bushen Tiaojing Formula can promote ovarian granulosa cell proliferation and steroid hormone secretion. By upregulating LIF/LIFR expression levels, it increases expression of cell proliferation markers PCNA and Cyclin D1 and steroidogenesis enzymes StAR, 3β-HSD, and CYP19A1, thereby promoting steroid hormone secretion and follicular development. This study provides experimental evidence for improving fertility in older women via kidney-tonifying therapy and enriches the theoretical connotation of "the kidney governs reproduction." However, the mechanism by which kidney-tonifying therapy improves fertility in older women is not fully clear. The specific mechanism of LIF-related signaling pathways in regulating granulosa cell function and follicular development requires further verification through animal experiments and in vitro cell experiments.
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