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 injury model by continuous gavage of 0–1500 mg/kg of DEHP in female mice for 30 days and an injury model by treatment of human ovarian granulosa cells (KGN) cells with mono-2- ethylhexyl ester (MEHP, an active metabolite of DEHP ). 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 and found that MEHP-induced KGN cells autophagy and apoptosis were independent and potentially cytotoxic of each other . 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.

中文翻译：

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邻苯二甲酸二己酯（2-乙基己基）诱导的女性生殖毒性的新视角：脂多糖与单-2-乙基己酯协同作用，引起卵巢颗粒细胞炎症细胞凋亡而不是自噬

邻苯二甲酸二己酯 (2-乙基己基) (DEHP) 已被证实通过影响卵巢颗粒细胞 (GC) 的存活而对人类和模型动物造成雌性生殖毒性，但 DEHP 对自噬、细胞凋亡和炎症之间的相互关系GC 不清楚。我们之前的研究表明，DEHP 暴露会导致与血清 LPS 释放相关的肠道菌群紊乱，进而导致卵巢功能受损。 LPS 还被证明可以通过调节细胞自噬、细胞凋亡和炎症来决定细胞命运。因此，本研究探讨了LPS与GCs自噬、凋亡和炎症在DEHP诱导的卵巢损伤中的作用和联系。在这里，我们通过对雌性小鼠连续灌胃0-1500 mg/kg DEHP 30天构建了损伤模型，并通过用单2-乙基己酯（MEHP， DEHP 的活性代谢物）。此外，加入自噬抑制剂3-甲基腺嘌呤（3-MA）、凋亡抑制剂Z-VAD-FMK和NF后，通过免疫组化、免疫荧光、qRT-PCR和Western blotting检测相关通路分子的表达。 -κB抑制剂BAY11-7082。目前的研究发现DEHP诱导的闭锁卵泡GC中自噬和凋亡显着激活，并发现MEHP诱导的KGN细胞自噬和凋亡是独立的，并且彼此具有潜在的细胞毒性。进一步的研究证实，DEHP暴露导致LPS从肠道释放并进入卵巢，从而参与DEHP诱导的GC炎症。此外，我们发现外源性LPS与MEHP协同作用，在相对长时间的暴露条件下，可以激活NF-κB信号通路，诱导GCs炎症和凋亡。同时，抑制炎症激活可以挽救MEHP联合LPS诱导的GCs凋亡和雌激素分泌功能。这些结果表明，DEHP影响的LPS增加可能与MEHP协同诱导GCs炎症性凋亡，这是小鼠卵巢损伤的重要原因。