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  2. Activation of the p62-Keap1-Nrf2 pathway protects against oxidative stress and excessive autophagy in ovarian granulosa cells to attenuate DEHP-induced ovarian impairment in mice

Activation of the p62-Keap1-Nrf2 pathway protects against oxidative stress and excessive autophagy in ovarian granulosa cells to attenuate DEHP-induced ovarian impairment in mice

  • Ecotoxicol Environ Saf. 2023 Sep 28:265:115534. doi: 10.1016/j.ecoenv.2023.115534.
Bo Xu 1 Tiantian He 1 Hong Yang 1 Wenjie Dai 1 Ling Liu 1 Xiaoqian Ma 1 Jiaxue Ma 1 Guoqin Yang 1 Rui Si 1 Xing Du 1 Xufeng Fu 2 Xiuying Pei 3
Affiliations

Affiliations

  • 1 School of Basic Medical Science, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Key Laboratory of Reproduction and Genetics of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan 750004, China.
  • 2 School of Basic Medical Science, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Key Laboratory of Reproduction and Genetics of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan 750004, China. Electronic address: fuxufeng100@163.com.
  • 3 School of Basic Medical Science, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Key Laboratory of Reproduction and Genetics of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan 750004, China. Electronic address: peixiuying@163.com.
Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is widely used in various plastics but has been demonstrated to cause female reproductive toxicity. However, the exact mechanism underlying the ovarian damage induced by DEHP remains unclear. In this study, DEHP was administered orally to 5-week-old female mice for 30 days at doses of 0, 250, 500, and 1000 mg/kg/day. The findings demonstrated that DEHP exposure disrupted ovarian function and follicular development as well as induced oxidative stress and Autophagy in ovarian granulosa cells (GCs). Further, 200 µM mono-(2-ethylhexyl) phthalate (MEHP), the primary metabolite of DEHP in vivo, induced Autophagy in both human ovarian granulosa cells line (KGN) and mouse primary GCs within 24 h in vitro. However, it did not affect the p62-dependent Autophagy flux. Furthermore, MEHP-induced Autophagy was inhibited by the Autophagy Inhibitor 3-MA and exacerbated by the Autophagy activator rapamycin, indicating that MEHP induces excessive Autophagy in GCs. Subsequently, we found that MEHP-induced autophagic cell death was primarily attributed to oxidative damage from elevated intracellular ROS levels. Meanwhile, MEHP exposure induced nuclear translocation of erythroid-derived factor 2-related factor (Nrf2), a key regulator of antioxidant activity resulting in activating antioxidant effects. Interestingly, we also found that MEHP-induced increase in p62 competitively binds Keap1, thereby facilitating nuclear translocation of Nrf2 and establishing a positive feedback loop in antioxidant regulation. Therefore, this study demonstrated that inhibition of Nrf2 could aggravate oxidative damage and enhance excessive Autophagy caused by MEHP, while activation of Nrf2 could reverse the trend. These findings have also been reinforced in studies of cultured ovaries in vitro. Our study suggests that the p62-Keap1-Nrf2 pathway may serve as a potential protective mechanism against DEHP-induced oxidative stress and excessive Autophagy in mouse GCs.

Keywords

Di-(2-ethylhexyl) phthalate; Excessive autophagy; Female reproductive toxicity; Ovarian granulosa cells; Oxidative stress; P62-Keap1-Nrf2 pathway.

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