Korean J Physiol Pharmacol.  2023 Mar;27(2):167-176. 10.4196/kjpp.2023.27.2.167.

Rehmannioside D mitigates disease progression in rats with experimental-induced diminished ovarian reserve via Forkhead Box O1/KLOTHO axis

• Liang Y
• Wang H
• Chen J
• Chen L
• Chen X

• KMID: 2539571
• DOI: http://doi.org/10.4196/kjpp.2023.27.2.167

Abstract

This study aims to explore the impact of Rehmannioside D (RD) on ovarian functions of rats with diminished ovarian reserve (DOR) and its underlying mechanisms of action. A single injection of cyclophosphamide was performed to establish a DOR rat model, and fourteen days after the injection, the rats were intragastrically administrated with RD for two weeks. Rat estrus cycles were tested using vaginal smears. Ovarian tissues were histologically evaluated, the number of primordial, mature, and atretic follicles was calculated, and the apoptotic rate of granulosa cells. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E₂ ) levels were determined by ELISA assays. Protein levels of Forkhead Box O1 (FOXO1), KLOTHO, Bcl-2, and Bax were investigated in ovarian tissues of DOR rats. The binding between FOXO1 and KLOTHO was verified by ChIP assay. High-dose administration of RD into DOR rats improved their estrus cycles, increased ovarian index, enhanced the number of primordial and mature follicles, reduced the number of atretic follicle number, and ovarian granulosa cell apoptosis in addition to inhibiting FSH and LH levels and upregulating E₂ expression. FOXO1 and KLOTHO were significantly suppressed in DOR rats. FOXO1 knockdown partially suppressed the protective effects of RD on DOR rats, and KLOTHO overexpression could restore RD-induced blockade of DOR development despite knocking down FOXO1. FOXO1 antibody enriched KLOTHO promoter, and the binding between them was reduced in DOR group compared to that in sham group. RD improved ovarian functions in DOR rats and diminished granulosa cell apoptosis via the FOXO1/KLOTHO axis.

Keyword

Forkhead Box O1; KLOTHO; Ovarian reserve; Rehmannioside D

Figure

• 

  Fig. 1 High-dose administration of RD improved ovarian functions and decreased apoptosis of granulosa cells in DOR rats. (A) Vaginal smear was used to investigate estrous cycles of rats. (B) Ovarian index. (C) H&E staining of ovary, and the number of primordial, mature, and atretic follicles. (D–F) ELISA assay was used to determine the levels of FSH (D), LH (E), and E2 (F). (G) Cell apoptosis in rat ovaries was tested by TUNEL staining. (H) The expression of Bcl-2 and Bax was measured by Western blotting. Values are presented as mean ± SD. DOR, diminished ovarian reserve; RD, Rehmannioside D; FSH, Follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol. n = 10. *p < 0.05.

• 

  Fig. 2 RD treatment relieved DOR progression in rats by upregulating FOXO1. (A, B) qRT-PCR (A) and Western blotting (B) were implemented to investigate the expression of FOXO1 in ovarian tissues. DOR rats were injected with sh-FOXO1 or its negative control and administrated with a high dose of RD for 2 weeks. (C, D) qRT-PCR (C) and Western blotting (D) were used to determine FOXO1 in ovarian tissues. (E) The estrous cycles of rats were measured using vaginal smears. (F) Ovarian index. (G) Ovarian H&E staining, and the number of primordial, mature, and atretic follicles. (H–J) The levels of FSH (H), LH (I), and E2 (J) were examined by ELISA assay. (K) Cell apoptosis in rat ovaries was investigated with TUNEL staining. (L) Western blotting was used to test the expression of Bcl-2 and Bax. Values are presented as mean ± SD. DOR, diminished ovarian reserve; RD, Rehmannioside D; FSH, Follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; FOXO1, Forkhead Box O1. n = 10. *p < 0.05.

• 

  Fig. 3 FOXO1 enhanced KLOTHO transcription by binding to the KLOTHO promoter. (A, B) The expression of KLOTHO in ovarian tissues was measured by qRT-PCR (A) and Western blotting (B). (C) The binding site of FOXO1 and the KLOTHO promoter was predicted by Jaspar database. (D) ChIP assay was used to detect the binding of FOXO1 to the KLOTHO promoter. (E, F) qRT-PCR (E) and Western blotting (F) were used to investigate the expression of KLOTHO in rat ovarian tissues. Values are presented as mean ± SD. DOR, diminished ovarian reserve; RD, Rehmannioside D; FOXO1, Forkhead Box O1; ChIP, Chromatin immunoprecipitation. n = 10. *p < 0.05.

• 

  Fig. 4 RD restored ovarian functions in DOR rats through the FOXO1/KLOTHO axis. DOR rats were injected with sh-FOXO1, LV-KLOTHO, and their negative controls, in addition to intervention with a high dose RD for 2 weeks. (A, B) The expression of KLOTHO in ovarian tissues was determined by qRT-PCR (A) and Western blotting (B). (C) Vaginal smear test was used to measure the estrous cycles of the rats. (D) Ovarian index. (E) Ovarian H&E staining, and quantitative histograms of primordial, mature, and atretic follicles. (F–H) The levels of FSH (F), LH (G), and E2 (H) were tested by ELISA assay. (I) Ovarian TUNEL staining was used to detect cell apoptosis. (J) The expression of Bcl-2 and Bax was investigated using Western blotting. Values are presented as mean ± SD. DOR, diminished ovarian reserve; RD, Rehmannioside D; FSH, Follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; FOXO1, Forkhead Box O1. n = 10. *p < 0.05.

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