Elsevier

Journal of Ethnopharmacology

Volume 262, 15 November 2020, 112993
Journal of Ethnopharmacology

Dingkun Pill replenishes diminished ovarian reserve through the PI3K/AKT/mTOR signaling pathway in TWP-induced mice

https://doi.org/10.1016/j.jep.2020.112993Get rights and content

Abstract

Ethnopharmacological relevance

Diminished ovarian reserve (DOR) can lead to poor fertility and shorten the reproductive lifespan of females. The Dingkun Pill (DKP), a traditional Chinese-patented medication, has been an integral part of traditional Chinese medicinal treatment for the management of gynecological diseases for centuries. Relevant clinical studies have shown that DKP is able to protect against DOR, however, its mechanism of action is not yet fully elucidated.

Study goals

This study was conducted with the aim of exploring the impact of tripterygium wilfordii polyglycosidium (TWP) on the PI3K/AKT/mTOR pathway in the context of the pathophysiology of DOR and the mechanism of action of DKP.

Materials and methods

Eighty female balb/c mice with regular estrous cycles were assigned to Blank, Model, DKP and hormone replacement therapy (HRT) groups in a random manner. With the exception of the Blank group, mice in the other groups were exposed to 40 mg/kg/d TWP suspension for 30 days to DOR induction. Following this, either DKP or hormones were orally administrated to determine their effect on disease progression. During the experiment, changes in body weight and the estrous cycles of the mice were observed. Post treatment, serum sample anti-mullerian hormone (AMH), estradiol (E2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were quantified using enzyme-linked immunosorbent assay (ELISA). The mice were then sacrificed in order to harvest their ovaries for hematoxylin and eosin (HE) staining. This process allowed for the assessment of ovarian morphology and follicular quantification. Apoptotic ovarian cells of the ovary were assessed using TUNEL technique, while Caspase-3 and Cytochrome C (Cyt C) expressions of the ovary were examined through immunohistochemistry (IHC). Western blotting analysis was used to quantify levels of Bax, Bcl-2, Caspase-3, Cyt C, mTOR, P-mTOR, AKT, P-AKT, P-PI3K and PI3K proteins, while mRNA levels of Bax, Bcl-2, PI3K, AKT and mTOR were measured in ovarian tissue using RT-PCR.

Results

The findings revealed that DKP was able to improve levels of serum hormones and promote the recovery of the estrous cycle. DKP augmented the total amount of primordial follicles while reducing the number of follicles that were atretic follicles. The apoptosis index of growing follicles and Bax, Cyt C and Caspase-3 expressions decreased, while the Bcl-2: Bax ratio increased. DKP suppressed levels of phosphorylation and the mRNA expressions of mTOR, AKT and PI3K.

Conclusions

It was demonstrated that DKP was able to increase ovarian reserves through inhibition of the PI3K/AKT/mTOR signaling pathway, which lead to the suppression of primordial follicle activity and a reduction in levels of apoptosis of early growing follicles. This highlights its potentially beneficial role for the treatment of DOR.

Introduction

Diminished ovarian reserve (DOR) is a subfertility condition in which the number of ovarian primordial follicles is decreased (Cohen et al., 2015; Tal and Seifer, 2017). It is one of the most common reasons of poor ovarian response (Devine et al., 2015). For most mammals, the primordial follicle pool is established prior to birth, with its development stopping shortly after birth (Zhang et al., 2018a). Activation of only a minor percentage of primordial follicles occurs throughout follicular growth, with 99.9% of follicles developing atresia at various stages. The remaining primordial follicle pool is then reflective of the female's reproductive lifespan.

In these processes, apoptosis is the main mechanism of germ cell clearance (Worku et al., 2017). Several models of DOR have been documented to induce high rates of ovarian cell apoptosis (Tan et al., 2019). Genetic knockout mice models of abnormal primordial follicle activation showed failure of ovarian maturation, with large numbers of germ cells undergoing apoptosis (Jiang et al., 2016). Apoptosis is controlled by molecules of the B-cell lymphoma/leukemia-2 (Bcl-2) family, which include pro-apoptotic protein Bcl-2 associated X protein (Bax) and anti-apoptotic protein Bcl-2. When cells are stimulated, Bax moves from the cytoplasm into mitochondria and changes mitochondrial permeability, resulting in cytoplasmic mitochondrial Cytochrome C (Cyt C) release, which in turn activates Caspase-3 and culminates in apoptosis (Chang et al., 2014).

In recent years, many researchers have suggested of the presence of complete phosphatidylinositol 3-kinase (PI3K) signaling systems in granulosa cells and oocytes, which work in tandem to facilitate follicular development, a process that includes initiation and survival of primordial follicles, follicle maturity, ovulation and follicular atresia (Zhang et al., 2018a, Zhang et al., 2018b). PI3K is a lipid kinase that phosphorylates the 3′-OH group on the inositol ring of inositol phospholipids to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3). In order to recruit more proteins, PIP3 binds to kinases containing the pleckstrin homology (PH) domain, which include 3-phosphoinositide-dependent protein kinase-1 (PDK1) and protein kinase B (PKB or AKT) (Zheng et al., 2012). AKT is a major PI3K downstream molecule that is activated via PDK1-mediated phosphorylation (Baek et al., 2017). A number of substrates, such as the tuberous sclerosis complex (TSC1/TSC2), are phosphorylated by AKT (Jang et al., 2016). The highly conserved serine/threonine (Ser/Thr) protein kinase, the mammalian target of rapamycin (mTOR), is expressed during all follicle stages in oocytes as well as granulosa cells (Guo et al., 2018). Mammalian mTOR comprises of dual complexes, the mTOR complex 1 (mTORC1) and 2 (mTORC2), which are distinct in terms of structure and function (Caron et al., 2015; Sulaimanov et al., 2017). Existing literature has demonstrated that TSC1/TSC2 are negative regulators of mTORC1. AKT can inhibit the activity of TSC1/TSC2 through phosphorylation and augmentation of mTORC1 activity. AKT directly activates mTORC1 through the phosphorylation of PRAS40 (Chamcheu et al., 2019). All these processes have been found to be significant for cell survival, differentiation, proliferation and the progression of the cell cycle (Morita et al., 2015; Rabanal-Ruiz et al., 2017).

Several studies conducted on genetically modified mouse models have linked the pathophysiology of premature ovarian failure (POF) and infertility with aberrant PI3K/AKT/mTOR pathways (Liu et al., 2018). The phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a lipid phosphatase that is involved in the conversion of PIP2 to PIP3, is a major down-regulator of PI3K. Targeted knockout of the PTEN gene in mice oocytes of primordial follicles was found to have triggered hyperactivation of PI3K signaling, causing early activation and depletion of mice primordial follicle pools, which manifested as POF and fertility loss (Reddy et al., 2008). Mice models with TSC1/2 gene deletion in oocytes experienced abnormally rapid early activation of primordial follicles as a consequence of elevated levels of mTORC1 activity (Adhikari et al., 2009, 2010). Furthermore, primordial or developing oocytes with negative mTOR expression resulted in mice infertility (Guo et al., 2018). All the above mentioned findings support the critical role of the PI3K/AKT/mTOR pathway in regulating the rate, onset and degree of follicle activation, dormancy, survival and atresia, and are critical determining factor of female fertility.

The Dingkun Pill (DKP), a traditional Chinese-patented medication, is a mix of approximately thirty herbs. In clinical studies, DKP has been shown to be an effective enhancer of ovarian function. Tan et al. demonstrated that DKP can improve menstrual flow, sex hormone levels, increase average ovarian volumes and the numbers of antral follicles in patients with DOR (Tan et al., 2018). Xie et al. reported that adjuvant DKP therapy along with clomiphene treatment was able to efficiently enhance the rate of ovulation and pregnancy in patients with DOR, and was superior to single agent treatment using clomiphene (Xie, 2018). However, the underling mechanisms of these effects are still not known. Therefore, this study ventured to uncover the impact of DKP on the P13K/AKT/mTOR signaling pathway and its subsequent relationship with DOR.

Section snippets

Animals

Eight week old female balb/c mice (n = 100) that were 18–20 g in weight were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate number: SCXK (Beijing) 2017-0033). An SPF grade laboratory animal room was used to house the animals under controlled relative humidity level (45%–70%), temperature (20–24 °C) and photoperiods (12 h dark/light cycled). Food and tap water were given ad libitum. All experimental protocols used were in strict compliance with the Guidelines

General status and change in body weight of mice

Mice in all four groups did not show significant weight change prior to TWP treatment. Post TWP treatment, statistically significant variability was observed between the four groups on the 19th, 22nd and 25th day (P < 0.05), as shown in Fig. 1B. The differences in weight gained did not significantly differ amongst the four groups (Fig. 1C), neither did the ratios of kidney, liver, spleen, thymus and uterus to body weight (Fig. 1D–H). However, the ovarian index decreased significantly in the

Discussion

As an increasing proportion of women in modern society postpone childbearing due to lifestyle, career and financial constraints, DOR has become an increasingly common clinical occurrence. The incidence of DOR ranges from 6% to 64% in infertile women of different ages (Mutlu and Erdem, 2012). Based on current literature, recurrent miscarriages may be associated with DOR (Atasever et al., 2016; Cohen et al., 2017). Furthermore, the occurrence of embryo implantation and the pregnancy rate in young

Conclusion

The current investigation highlighted the use of DKP as a protective factor against TWP-induced ovarian damage. This negative effect was mediated by decreasing the levels of phosphorylation of several key proteins in the PI3K/AKT/mTOR signaling pathway, which led to a decrease of primordial follicle activation and inhibition of follicular apoptosis. DKP has the potential to be used as an antidote to counteract TWP-mediated reproductive toxicity in women of a child-bearing age. Although further

Authors’ contributions

Yanxia Chen and Xiaodi Fan conceived and designed the study. Kun Ma and Yanxia Chen performed the experiments. Yuan Yuan and Kaili Wang analyzed the data. Caidie Tian and Min Li drafted the original draft. Yanxia Chen and Xiaodi Fan reviewed and edited the manuscript. All authors read and approved the final manuscript.

Grant support

This work was supported by National Natural Science Foundation of China [81674019, Kun Ma], The Innovative Funding for PhD Students at China Academy of Chinese Medical Sciences [CX201910, Yanxia Chen] and Beijing Natural Science Foundation [7194314, Xiaodi Fan].

Declaration of competing interest

The authors declare that no conflicts of interest exist.

Acknowledgements

The authors acknowledge the Department of Pathology of Xiyuan Hospital, China Academy of Chinese Medical Science for providing helpful advice. Research was supported by grants from National Natural Science Foundation of China (81674019), the Innovative Funding for PhD Students at China Academy of Chinese Medical Sciences (CX201910) and Beijing Natural Science Foundation (7194314).

References (47)

  • S.H. Baek et al.

    Ginkgolic acid inhibits invasion and migration and TGF-β-induced EMT of lung cancer cells through PI3K/Akt/mTOR inactivation

    J. Cell. Physiol.

    (2017)
  • A. Caron et al.

    The roles of mTOR complexes in lipid metabolism

    Annu. Rev. Nutr.

    (2015)
  • L. Chang et al.

    PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways

    Cell Death Dis.

    (2014)
  • Y. Chang et al.

    Egg quality and pregnancy outcome in young infertile women with diminished ovarian reserve

    Med. Sci. Mon.

    (2018)
  • J.C. Chamcheu et al.

    Role and therapeutic targeting of the PI3K/Akt/mTOR signaling pathway in skin cancer: a review of current status and future trends on natural and synthetic agents therapy

    Cells

    (2019)
  • L. Chen et al.

    Effect of clomiphene citrate and Dingkun Dan on ovulation induction and clinical pregnancy of polycystic ovary syndrome

    Zhongguo Zhongyao Zazhi

    (2017)
  • J. Cohen et al.

    Diminished ovarian reserve, premature ovarian failure, poor ovarian responder--a plea for universal definitions

    J. Assist. Reprod. Genet.

    (2015)
  • J. Cohen et al.

    Outcomes of first IVF/ICSI in young women with diminished ovarian reserve

    Minerva Ginecol.

    (2017)
  • K. Devine et al.

    Diminished ovarian reserve in the United States assisted reproductive technology population: diagnostic trends among 181,536 cycles from the society for assisted reproductive technology clinic outcomes reporting system

    Fertil. Steril.

    (2015)
  • G. Gonzalez

    Determining the stage of the estrous cycle in female mice by vaginal smear

    Cold Spring Harb. Protoc.

    (2016)
  • J. Guo et al.

    Oocyte stage-specific effects of mTOR determine granulosa cell fate and oocyte quality in mice

    Proc. Natl. Acad. Sci. U.S.A.

    (2018)
  • J.Y. Huang et al.

    Effect of shen-tonifying traditional Chinese herbs xianzi yizhen capsules on PI3K/AKT signaling pathway of mice with premature ovarian failure

    Chin. J. Integr. Tradit. West. Med.

    (2018)
  • L. Huang et al.

    Traditional Chinese medicine Dingkun Pill facilitates uterine receptivity for implantation in mice

    Biol. Reprod.

    (2019)
  • Cited by (12)

    • Integrated metabolomics and network pharmacology to reveal the therapeutic mechanism of Dingkun Pill on polycystic ovary syndrome

      2022, Journal of Ethnopharmacology
      Citation Excerpt :

      Previous research suggested that DKP could increase the ongoing pregnancy rate for expected poor ovarian response women (Song et al., 2021). Ma et al. demonstrated that DKP was able to increase ovarian reserves by augmenting the total amount of primordial follicle and reducing the number of atretic follicles (Ma et al., 2020). Our previous study showed that triterpenoid saponins, flavonoids, alkaloids, terpenoids and phenylpropanoids were the major components in DKP (Dou et al., 2020).

    View all citing articles on Scopus
    1

    Kun Ma and Yanxia Chen equally contributed to this work.

    View full text