Effects of Huluan decotion (护卵汤) on cyclophosphamide-induced autoimmune premature ovarian failure in murine models

doi:10.19852/j.cnki.jtcm.20240927.001

Abstract

Abstract:

OBJECTIVES: To investigate the therapeutic effect of Huluan decotion (护卵汤, HLD) on cyclophosphamide-induced premature ovarian failure (POF) in mice and its regulatory mechanisms.

METHODS: Female BALB/c mice were administered cyclophosphamide and administered received different doses of HLD for 28 d. Levels of sex hormone, such as estradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the sera, were assessed using enzyme-linked immunosorbent assay (ELISA). Follicular structure variances were observed through hematoxylin and eosin (HE) staining, while Forkhead box L2 (FOXL2) expression were analyzed via immune-ohistochemical staining. The primary mechanism of POF were investigated through Western blot analysis.

RESULTS: E2 levels decreased, and FSH and LH levels increased in POF model mice, but these trends were reversed with HLD or premarin administration, the expressions of WNT family member 4 (Wnt4), β-Catenin and FOXL2 were downregulated in POF model mice, whereas high expression levels were observed in control mice and other groups.

CONCLUSION: HLD effectively treats POF induced with cyclophosphamide in mice by enhancing expressions of Wnt4, β-Catenin and FOXL2.

Key words: primary ovarian insufficiency, prescriptions, cyclophosphamide, wnt4 protein, beta catenin, signal transduction, Huluan decotion

FENG Guiling, ZHOU Xiaolin, SHEN Chengwan, LI Panxiao, ABULIZI ·Abudula. Effects of Huluan decotion (护卵汤) on cyclophosphamide-induced autoimmune premature ovarian failure in murine models[J]. Journal of Traditional Chinese Medicine, 2025, 45(2): 266-271.

Figures/Tables 5

Table 1 Serum levels of FSH, LH, and E2 in each group

Group	n	FSH (ng/mL)	LH (IU/L)	E₂(pd/mL)	AMH (ng/mL)
Con	10	12.52±2.55^a	2.86±0.58^a	1166.55±214.55^a	0.28±0.04^a
Mol	10	31.10±5.28^b	5.02±0.86^b	698.80±156.32^b	0.08±0.03^b
HLD-L	10	17.36±3.67^a	3.55±0.48^c	704.89±156.04	0.13±0.05^a
HLD-M	10	16.15±4.22^a	3.46±0.56^a	960.10±173.28^a	0.23±0.04^a
HLD-H	10	11.21±3.76^a	3.13±0.68^a	965.31±148.67^a	0.24±0.04^a
P	10	20.90±4.16^a	3.48±0.74^a	902.81±187.33^a	0.24±0.06^a

Figure 1 HE stained ovarian sections of each group A: control group; B: model group; C: premarin group; D: low of HLD groups; E: moderate of HLD groups; F: high dose of HLD group. Mice in the model, premarin, and HLD groups were administered a loading dose of CTX (100 mg·kg-1·d-1) followed by daily injections for 2 weeks, and then, positive group treated with premarin (0.03 mg/kg) once daily for 4 weeks. Control and model group received the same volume of distilled water, low (2 g/kg), moderate (5 g/kg, the dose used in vivo in human studies), and high (10 g/kg) dose of HLD groups treated by gastrogavage once daily for 4 weeks. Stained with HE, the numbers of primary and secondary follicles increased significantly compared with those in the model group. HLD: Huluan decotion; CTX: cyclophosphamide; HE: hematoxylin-eosin. Magnification × 200. Scale bar: 200 μm.

Figure 2 Comparison of the number of follicles at different levels in each group of mice Con: control group; Mol: model group; P: premarin group; L-HLD, M-HLD and H-HLD represent low, moderate, and high dose of Huluan decotion groups, respectively. Mice in the model, premarin, and HLD groups were administered a loading dose of CTX (100 mg·kg-1·d-1) followed by daily injections for 2 weeks, and then, positive group treated with premarin (0.03 mg/kg) once daily for 4 weeks. Control and model group received the same volume of distilled water, low (2 g/kg), moderate (5 g/kg, the dose used in vivo in human studies), and high (10 g/kg) dose of HLD groups treated by gastrogavage once daily for 4 weeks. The numbers of primary and secondary follicles in the Mol group decreased significantly compared with those in the con group, while treatment with HLD or premarin, the numbers of primary and secondary follicles increased significantly compared with those in the model group. HLD: Huluan decotion; CTX: cyclophosphamide. Data are presented as the mean ± standard deviation using one-way analysis of variance (n = 10). aP < 0.01 vs group Control; bP < 0.01 vs group MOL; cP < 0.05 vs group Mol.

Figure 3 Immunohistochemical analysis of the distinct presence of FOXL2 in the oophorons of each group A: control group; B: model group; C: premarin group; D: low dose of HLD group; E: moderate dose of HLD group; F: high dose of HLD group. Mice in the model, premarin, and HLD groups were administered a loading dose of CTX (100 mg·kg-1·d-1) followed by daily injections for 2 weeks, and then, positive group treated with premarin (0.03 mg/kg) once daily for 4 weeks. Control and model group received the same volume of distilled water, low (2 g/kg), moderate (5 g/ kg, the dose used in vivo in human studies), and high (10 g/ kg) dose of HLD groups treated by gastrogavage once daily for 4 weeks. Immunohistochemical staining of ovarian sections unveiled a decrease in FOXL2 expression within the model group, whereas an upsurge was observed in mice treated with HLD-M, HLD-H, and Premarin. HLD: Huluan decotion; CTX: cyclophosphamide; FOXL2: Forkhead Box L2. Magnification × 200. Scale bar: 200 μm.

Figure 4 Expression Wnt4, β- catenin and FOXL2 proteins in the ovaries of mice in each group. A: control group; B: model group; C: premarin group; D: low dose of HLD group; E: moderate dose of HLD group; F: high dose of HLD group. Mice in the model, premarin, and HLD groups were administered a loading dose of CTX (100 mg·kg-1·d-1) followed by daily injections for 2 weeks, and then, positive group treated with premarin (0.03 mg/kg) once daily for 4 weeks. Control and model group received the same volume of distilled water, low (2 g/kg), moderate (5 g/kg, the dose used in vivo in human studies), and high (10 g/kg) dose of HLD groups treated by gastrogavage once daily for 4 weeks. Wnt4, β-catenin, and FOXL2 expression were detected by Western blot analysis. Wnt4, β-catenin, and FOXL2 levels in ovarian tissue decrease in the model group compared to the control group, while administration of HLD or Premarin elevated levels of these proteins. HLD: Huluan decotion; CTX: cyclophosphamide; FOXL2: Forkhead Box L2; Wnt4: WNT family member 4.

References 25.

1.	Chon Sj, Umair Z, Yoon Ms. Premature ovarian insufficiency: past, present, and puture. Front Cell Dev Biol 2021; 9: 672890.
2.	De Vos M, Devroey P, Fauser Bc. Primary ovarian insufficiency. Lancet 2010; 376: 911- 21. DOI PMID
3.	Zhang Cr, Zhu Wn, Tao W, et al. Moxibustion against cyclophosphamide-induced premature ovarian failure in rats through inhibiting NLRP3-/Caspase-1-/GSDMD-Dependent pyroptosis. Evid Based Complement Alternat Med 2021; 2021: 8874757.
4.	Bellusci G, Mattiello L, Iannizzotto V, et al. Kinase-independent inhibition of cyclophosphamide-induced pathways protects the ovarian reserve and prolongs fertility. Cell Death Dis 2019; 10: 726. DOI PMID
5.	Melekoglu R, Ciftci O, Eraslan S, Cetin A, Basak N. Beneficial effects of curcumin and capsaicin on cyclophosphamide-induced premature ovarian failure in a rat model. J Ovarian Res 2018; 11: 33. DOI PMID
6.	Maas A. Hormone therapy and cardiovascular disease: Benefits and harms. Best Pract Res Clin Endocrinol Metab 2021; 35: 101576.
7.	Farquhar C, Marjoribanks J, Lethaby A, Suckling JA, Lamberts Q. Long term hormone therapy for perimenopausal and postmenopausal women. Cochrane Database Syst Rev 2017; 104: 93-5.
8.	Shareghi-Oskoue O, Aghebati-Maleki L, Yousefi M. Transplantation of human umbilical cord mesenchymal stem cells to treat premature ovarian failure. Stem Cell Res Ther 2021; 12: 454. DOI PMID
9.	Liu X, Song Y, Zhou F, et al. Network and experimental pharmacology on mechanism of Si-Wu-tang improving ovarian function in a mouse model of premature ovarian failure induced by cyclophosphamide. J Ethnopharmacol 2023; 301: 115842.
10.	Zheng S, Ma M, Chen Y, Li M. Effects of quercetin on ovarian function and regulation of the ovarian PI3K/Akt/FoxO3a signalling pathway and oxidative stress in a rat model of cyclophosphamide-induced premature ovarian failure. Basic Clin Pharmacol Toxicol 2022; 130: 240-53.
11.	Guo SJ. Yang Jin's experience of treating infertility and crafts with Shuangbu decoction. Global Tradit Chin Med 2016; 9: 831-3.
12.	Feng GL, Zhang LH, Zhou XL, et al. Efficacy of Bushen Jianpi prescription on autoimmune premature ovarian failure in mice. J Tradit Chin Med 2017; 37: 667-74.
13.	Feng GL, Zhou XL. Effect of Bushen Jianpi recipe on autoimmune premature ovarian failure of mice. J Tradit Chin Med 2016; 57: 71-5.
14.	Zhang XL, Li J, Zhou XL, Chen QL. Effect of Er-Xian decoction on autoimmune premature ovarian failure in mice. J Tradit Chin Med 2021; 41: 725-31.
15.	Wang ZY, Li MZ, Li WJ, Ouyang JF, Gou XJ, Huang Y. Mechanism of action of Daqinjiao decoction in treating cerebral small vessel disease explored using network pharmacology and molecular docking technology. Phytomedicine 2023; 108: 154538.
16.	Myers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB. Methods for quantifying follicular numbers within the mouse ovary. Reproduction 2004; 127: 569-80.
17.	Liu W, Cao JL, Ouyang JF, et al. Chaihu and Longgu Muli decoction demonstrate neuroprotective effects in the rat model of Parkinson's disease with depression by regulating the AMPK/mTOR signaling pathway. Phytomedicine Plus 2023; 3: 100428.
18.	Yang X, Yang L. Current understanding of the genomic abnormities in premature ovarian failure: chance for early diagnosis and management. Front Med (Lausanne) 2023; 10: 1194865.
19.	Han M, Cheng H, Wang J, et al. Abnormal aggregation of myeloid-derived suppressor cells in a mouse model of cyclophosphamide-induced premature ovarian failure. Gynecol Endocrinol 2019; 35: 985-90. DOI PMID
20.	Farhat SA, Jabbari F, Jabbari P, Rezaei N. Targeting signaling pathways involved in primordial follicle growth or dormancy: potential application in prevention of follicular loss and infertility. Expert Opin Biol Ther 2022; 22: 871-81. DOI PMID
21.	Zhang C, Xu X. Advancement in the treatment of diminished ovarian reserve by traditional Chinese and Western medicine. Exp Ther Med 2016; 11: 1173-76. PMID
22.	Han Y, Wang T, Sun S, Zhai Z, Tang S. Cloning of the promoter region of a human gene, FOXL2, and its regulation by STAT3. Mol Med Rep 2017; 16: 2856-62. DOI PMID
23.	Chen H, Chen Q, Zhu Y, et al. MAP3K1 Variant causes hyperactivation of Wnt4/β-Catenin/FOXL2 signaling contributing to 46,XY Disorders/Differences of sex development. Front Genet 2022; 13: 736988.
24.	Gustin SE, Hogg K, Stringer JM, et al. WNT/β-catenin and p27/FOXL2 differentially regulate supporting cell proliferation in the developing ovary. Dev Biol 2016; 412: 250-60. DOI PMID
25.	Zhang P, Li T, Wu X, et al. Oxidative stress and diabetes: antioxidative strategies. Front Med 2020; 14: 583-600.

[1]	HUANG Haiyang, ZHU Shumin, ZHONG Shaowen, LIU Ying, HOU Shaozhen, GAO Jie, OU Jianzhao, DONG Mingguo, NING Weimin. Fuzheng Xuanfei Huashi prescription (扶正宣肺化湿方) suppresses inflammation in lipopolysaccharide-induced lung injury in mice via toll-like recptor 4/nuclear transcription factor κB and cyclooxygenase-2/prostaglandin E2 pathway [J]. Journal of Traditional Chinese Medicine, 2025, 45(2): 272-280.
[2]	HUANG Jiaen, LUO Qing, DONG Gengting, PENG Weiwen, HE Jianhong, DAI Weibo. Xiahuo Pingwei San (夏藿平胃散) attenuated intestinal inflammation in dextran sulfate sodium-induced ulcerative colitis mice through inhibiting the receptor for advanced glycation end-products signaling pathway [J]. Journal of Traditional Chinese Medicine, 2025, 45(2): 311-325.
[3]	SHI Jinyu, PAN Fuwei, GE Haiya, YANG Zongrui, ZHAN Hongsheng. Mechanism of Qigu capsule (芪骨胶囊) as a treatment for sarcopenia based on network pharmacology and experimental validation [J]. Journal of Traditional Chinese Medicine, 2025, 45(2): 399-407.
[4]	WU Jiaman, TANG Meng, LUO Yu, ZHU Haimin, ZHAO Tianqi, MA Fei, NING Yan. Electroacupuncture enhances the mitophagy of granulosa cells in premature ovarian insufficiency model mice by inactivating the hippo-yes-associated protein/transcriptional co-activator with postsynaptic density protein, drosophila disc large tumor suppressor, and zonula occludens-1 protein binding motif pathway [J]. Journal of Traditional Chinese Medicine, 2025, 45(1): 13-21.
[5]	YAN Kai, WANG Wei, WANG Yan, GAO Huijuan, FENG Xingzhong. Network pharmacology-based study on the mechanism of Tangfukang formula (糖复康方) against type 2 diabetes mellitus [J]. Journal of Traditional Chinese Medicine, 2025, 45(1): 76-88.
[6]	WU Jiaman, NING Yan, TAN Liya, MA Fei, LIN Yanting, ZHUO Yuanyuan. Difference of the gut microbiota of premature ovarian insufficiency in two traditional Chinese syndromes [J]. Journal of Traditional Chinese Medicine, 2025, 45(1): 132-139.
[7]	LUO Kun, ZHONG Yumei, GUO Yanding, ZHANG Linlin, HU Danhui, MA Wenbin, YANG Xin, ZHOU Haiyan. Moxibustion inhibits the macrophage M1 polarization toll-like receptor 4/myeloid differentiation factor 88/nuclear factor kappa B signaling pathway by regulating T-cell immunoglobulin and mucin-containing protein-3 in rheumatoid arthritis [J]. Journal of Traditional Chinese Medicine, 2024, 44(6): 1227-1235.
[8]	ZHANG Guangshun, XU Xiaonan, XU Chuyun, LIAO Guanghui, XU Hao, LOU Zhaohuan, ZHANG Guangji. Actinidia chinensis polysaccharide interferes with the epithelial-mesenchymal transition of gastric cancer by regulating the nuclear transcription factor-κB pathway to inhibit invasion and metastasis [J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 896-905.
[9]	YU Siyun, ZHANG Shiwen, XIA Yu, LIU Xiaoqing, LIU Yajie, FU Jinrong. Network-based pharmacology and experimental validation to explore the mechanism of action of the Jiawei Pentongling formula (加味盆痛灵方) for the treatment of endometriosis-related pain [J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 991-999.
[10]	JIANG Zhaojian, CAI Hongfei, YUAN Cheng, CAO Lin, XU Wendong, HAN Yaming, ZHANG Qin, LI Jing, WANG Qin, LIU Juyan. Ganoderma Lucidum Spore Oil enhances the effect of cyclophosphamide via inhibiting programmed death-1 and prolongs the survival of H22 tumor-bearing mice [J]. Journal of Traditional Chinese Medicine, 2024, 44(4): 652-659.
[11]	ZHU Xuan, LOU An, ZHU Keke, RUAN Mingyu. Effect of Jiedu Huayu decoction (解毒化瘀汤) on oral mucosal Axin and β-catenin expression in oral submucosal fibrosis model rats [J]. Journal of Traditional Chinese Medicine, 2024, 44(4): 688-693.
[12]	GUO Yanding, LUO Kun, ZHANG Linlin, LU Wenting, SHANG Yanan, ZHONG Yumei, HU Danhui, YANG Xin, ZHOU Haiyan. Study on the anti-inflammatory mechanism of moxibustion in rheumatoid arthritis in rats based on phospholipaseA2 signaling inhibition by Annexin 1 [J]. Journal of Traditional Chinese Medicine, 2024, 44(4): 753-761.
[13]	REN Li, HAI Yang, YANG Xue, LUO Xianqin. Yemazhui (Herba Eupatorii Lindleyani) ameliorates lipopolysaccharide-induced acute lung injury via modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flora in rats [J]. Journal of Traditional Chinese Medicine, 2024, 44(2): 303-314.
[14]	LI Liusheng, ZHAO Mingming, CHANG Meiying, SI Yuan, ZHAO Jinning, YANG Bin, ZHANG Yu. Protective effect of modified Huangqi Chifeng decoction (加味黄芪赤风汤) on immunoglobulin A nephropathy through toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B signaling pathway [J]. Journal of Traditional Chinese Medicine, 2024, 44(2): 324-333.
[15]	ZHENG Xueying, GUO Liang, LAI Siyi, LI Fengyue, LIANG Mingli, LIU Wanting, MENG Chun, LIU Guanghui. Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway [J]. Journal of Traditional Chinese Medicine, 2024, 44(2): 268-276.

Home

Online First

Author Center

Reviewer Center

Issues

Announcement

About Us