Research article
Interleukin-4 activates the PI3K/AKT signaling to promote apoptosis and inhibit the proliferation of granulosa cells

https://doi.org/10.1016/j.yexcr.2021.113002Get rights and content

Abstract

The inflammatory microenvironment has been demonstrated to play a role in folliculogenesis, ovulation and premature ovarian failure (POF), as well as infertility. In this study, we aimed to explore the role of inflammation in modulating growth and apoptosis in granulosa cells (GCs), the main components of ovarian follicles. ELISA was used to analyze the levels of inflammatory factors (IL-1β, IL-4, IL-6 and IL-10) in follicular fluid samples and GCs derived from POF patients and healthy normal individuals. CCK-8, flow cytometry and TUNEL assays were used to assess the effect of IL-4 on GC growth and apoptosis. Western blotting was used to examine the effect of IL-4 on the activation of PI3K/Akt, Erk1/2 and Jnk signaling. The results showed that IL-4, IL-1β and IL-6 levels were increased in follicular fluid samples and GCs derived from POF patients compared with those from healthy individuals. GC growth was weakened when cells were treated with IL-4, while apoptosis was increased. In addition, IL-4 increased the level of p-Akt/Akt in GCs. In addition, LY294002, an inhibitor of PI3K, abolished the effect of IL-4 by inhibiting GC growth and promoting apoptosis. In summary, this study demonstrated that IL-4 levels were increased in POF samples and that IL-4 could inhibit GC growth and induce GC apoptosis by activating PI3K/Akt signaling.

Introduction

Premature ovarian failure (POF) is a disease of ovarian function dysregulation that occurs in women less than 40 years. POF is characterized by the premature loss of ovarian follicles or folliculogenesis arrest, low levels of hormones, high levels of gonadotropins, and even infertility [1,2]. The incidence of POF is approximately 1% in women aged <40 years and 0.1% in women aged <30 years [3], and this disease can lead to anxiety, depression, hot flashes, osteoporosis and sexual dysfunction [4,5].

Granulosa cells (GCs), which are the main cell type in ovarian follicles, play crucial roles in the regulation of follicular development. GCs are closely linked to oocytes, and these cell types are interdependent and inseparable. GCs supply the main nutrients for oocyte maturation, while oocytes modulate GC proliferation and differentiation [6]. GC apoptosis is the central link in the initiation of follicular atresia [7,8]. Thus, exploring the mechanisms underlying GC apoptosis is critical for the management of POF.

Evidence has demonstrated that follicle dysfunction is related to the inflammatory microenvironment of the ovary [[9], [10], [11]]. Inflammation is closely associated with folliculogenesis and ovulation, the dysregulation of which can cause impaired oocyte quality and POF, thereafter resulting in infertility [12,13]. Our previous study demonstrated a vital role of inflammation in GC apoptosis and provided new insight into developing a new class of anti-inflammatory therapeutic agents to delay POF [14]. Inflammatory cytokines are generated throughout folliculogenesis and participate in ovulation induction. Interleukin (IL)-18 has been reported to be associated with the response to ovarian stimulation and is the reason for successful pregnancy following in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) [15]. IL-18 induces the production of IL-1β and tumor necrosis factor (TNF)-α, which are necessary for follicular growth and oocyte maturation, and women with unexplained infertility have reduced levels of IL-18 [12]. IL1-RII and IL-4 expression was increased in GCs from cattle follicles with different persistence times, which may contribute to follicular persistence and ovulation failure in cattle with follicular cysts [16]. Additionally, inflammation can trigger the generation of reactive oxygen species (ROS), resulting in excessive oxidative stress, which can significantly induce GC apoptosis [17,18]. However, the direct role of inflammatory factors in GC apoptosis and the molecular mechanisms remain unclear.

This study was carried out to explore the effect of inflammation on GC apoptosis and uncover the underlying mechanisms. First, we compared the expression levels of proinflammatory cytokines (IL-1β and IL-6) and other cytokines (IL-4 and IL-10) in follicular fluid from POF patients and healthy individuals. Next, we assessed the role of IL-4, which showed the largest difference between POF patients and healthy individuals among the 4 analyzed factors, on GC apoptosis and viability. Finally, we assessed whether PI3K/Akt, Erk1/2 or Jnk signaling was involved in IL-4-mediated GC apoptosis.

Section snippets

Follicular fluid samples

Follicular fluid samples from 20 POF patients and 10 healthy control individuals were obtained from Tianjin Central Hospital of Obstetrics and Gynecology (Tianjin, China) between March 2021 and July 2021. Basal information is shown in Table 1. Experiments involving human samples were carried out according to the Declaration of Helsinki and received the approval of the Institutional Review Board of Tianjin Central Hospital of Obstetrics and Gynecology (No. 2021ZXFC01). Informed written consent

IL-1β, IL-4, and IL-6 levels are increased in follicular fluid samples from POF patients

To investigate the effect of inflammation on POF pathogenesis, we first assessed the levels of IL-1β, IL-4, IL-6 and IL-10 in follicular fluid samples from POF patients (n = 20) and healthy individuals (n = 10). The results demonstrated that the concentrations of IL-1β, IL-4 and IL-6 were significantly elevated in the follicular fluid samples from POF patients compared with those from healthy individuals, while the IL-10 level showed no obvious difference between the two groups, as measured by

Discussion

Accumulating evidence shows that inflammatory processes are involved in ovarian folliculogenesis and ovulation and have negative effects on ovarian follicular dynamics [12,21]. The level of inflammatory factors, high sensitivity C-reactive protein (hsCRP), IL-1β, and IL-18, were significantly increased in PCOS [22]. Resveratrol treatment significantly lowered proinflammatory factors (TNF-α and IL-6) and increased the concentration of IL-10, an anti-inflammatory factor, in the ovaries, and

Conclusions

This study reveals that IL-4 level was increased in patients with POF as compared with healthy individuals. Also, we demonstrate IL-4 can inhibit GC growth and induce GC apoptosis by activating PI3K/Akt signaling. We will verify the experimental data set by using distinct GCs derived from healthy individuals in future to support the model proposed. Collectively, this study enriches the understanding of the role of inflammation in the pathogenesis of POF.

Credit author statement

Ying Han: Conceptualization, Methodology, Writing – original draft, Conceptualization, Funding acquisition, Revising. Ruqiang Yao: Resources, Investigation, Writing-review&editing, Supervision. Zexin Yang: Validation, Investigation, Formal analysis, Data curation. Shuang Li: Validation, Software. Wenjia Meng: Formal analysis. Yinfeng Zhang: Investigation, Data curation. Yunshan Zhang: Project administration. Haining Luo: Conceptualization, Writing – review & editing, Supervision, Funding

Ethics approval

This study was approved by the Institutional Review Board of Tianjin Central Hospital of Gynecology Obstetrics [1] (No. 2021ZXFC01).

Funding

This study was supported by the Open Fund of Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation (Nos. 2021XH01 and 2021XHY04) and the National Natural Science Foundation of China (Grant No. 81803927).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References (37)

  • V. Stearns et al.

    Breast cancer treatment and ovarian failure: risk factors and emerging genetic determinants

    Nat. Rev. Cancer

    (2006)
  • K. Bahrehbar et al.

    Human embryonic stem cell-derived mesenchymal stem cells improved premature ovarian failure

    World J. Stem Cell.

    (2020)
  • S. Wang et al.

    Acupuncture reduces apoptosis of granulosa cells in rats with premature ovarian failure via restoring the PI3K/Akt signaling pathway

    Int. J. Mol. Sci.

    (2019)
  • F. Matsuda-Minehata et al.

    The regulation of ovarian granulosa cell death by pro- and anti-apoptotic molecules

    J. Reprod. Dev.

    (2006)
  • X. Du et al.

    NORFA, long intergenic noncoding RNA, maintains sow fertility by inhibiting granulosa cell death

    Commun Biol

    (2020)
  • M. Ebrahimi et al.

    The role of autoimmunity in premature ovarian failure

    Iran. J. Reproductive Med.

    (2015)
  • A. Agita et al.

    Inflammation, immunity, and hypertension

    Acta Med. Indones.

    (2017)
  • C.E. Boots et al.

    Inflammation and human ovarian follicular dynamics

    Semin. Reprod. Med.

    (2015)
  • Cited by (6)

    1

    These authors contributed equally to this study.

    View full text