A novel perspective on di-hexyl phthalate (2-ethylhexyl)-induced reproductive toxicity in females: Lipopolysaccharide synergizes with mono-2-ethylhexyl ester to cause inflammatory apoptosis rather than autophagy in ovarian granulosa cells

Di-hexyl phthalate (2-ethylhexyl) (DEHP) has been confirmed to cause female reproductive toxicity in humans and model Animals by affecting the survival of ovarian granulosa cells (GCs), but the interrelationships between DEHP's on Autophagy, Apoptosis, and inflammation in GCs are not clear. Our previous study demonstrated that DEHP exposure resulted in the disturbance of intestinal flora associated with serum LPS release, which in turn led to impaired ovarian function. LPS has also been shown to determine cell fate by modulating cellular Autophagy, Apoptosis, and inflammation. Therefore, this study investigated the role and link between LPS and Autophagy, Apoptosis, and inflammation of GCs in DEHP-induced ovarian injury. Here, we constructed an in vivo injury model by continuous gavage of 0-1500 mg/kg of DEHP in female mice for 30 days and an in vitro injury model by treatment of human ovarian granulosa cells (KGN) cells with mono-2- ethylhexyl ester (MEHP, an active metabolite of DEHP in vivo). In addition, the expression of relevant pathway molecules was detected by immunohistochemistry, immunofluorescence, qRT-PCR, and Western blotting after the addition of the Autophagy Inhibitor 3-methyladenine (3-MA), the Apoptosis inhibitor Z-VAD- FMK and the NF-κB Inhibitor BAY11-7082. The current study found that Autophagy and Apoptosis were significantly activated in GCs of DEHP-induced atretic follicles in vivo and found that MEHP-induced KGN cells Autophagy and Apoptosis were independent and potentially cytotoxic of each other in vitro. Further studies confirmed that DEHP exposure resulted in LPS release from the intestinal tract and entering the ovary, thereby participating in DEHP-induced inflammation of GCs. In addition, we found that exogenous LPS synergized with MEHP could activate the NF-κB signaling pathway to induce inflammation and Apoptosis of GCs in a relatively prolonged exposure condition. Meanwhile, inhibition of inflammatory activation could rescue Apoptosis and estrogen secretion function of GCs induced by MEHP combined with LPS. These results indicated that the increased LPS influenced by DEHP might cooperate with MEHP to induce inflammatory Apoptosis of GCs, an important cause of ovarian injury in mice.

Apoptosis; Autophagy; Di-hexyl phthalate (2-ethylhexyl); Inflammation; NF-κB pathway; Ovarian Granulosa Cells.