Embryo formation is a complex process of synergistic action between embryo and mother. The successful implantation of human embryo, placental formation, embryo growth and development are closely related to the proliferation, differentiation and invasion function of trophoblast. The normal proliferation and biological function of trophoblast are prerequisites for successful pregnancy[10].
HTR-8/SVneo cell line is considered the closest study model to trophoblast cells derived from early human pregnancy (8 ~ 10 weeks) [11]. The HTR-8/SVneo human trophoblast cell line applied in the present study is more similar to primary trophoblasts and normal human physiological conditions. Therefore, we established an in vitro model of oxidative stress by exposing the human trophoblast cell line HTR-8/SVneo cells to H2O2.
The in-depth study on the regulation of trophoblast oxidative stress and autophagy will be beneficial to elucidate the pathogenesis of a class of primary trophoblastic diseases such as abortion, preeclampsia, fetal growth restriction, and provide new therapeutic targets for the treatment of these diseases.
It is becoming increasingly clear that autophagy and oxidative stress are interrelated activities that are essential for fetal development and parturition. Further investigations of their interrelationships at different gestational stages will lead to an improved understanding of the mechanisms by which alterations in their interactions contribute to placental pathology, pregnancy disorders, and premature delivery.
Autophagy is a lysosomal-mediated intracellular degradation system that serves as a primary cell maintenance mechanism via which unnecessary or dysfunctional cell components are degraded [12]. Many experimental and clinical studies have shown that oxidative stress is closely related to autophagy [13–15]. Oxidative stress products are ROS, H2O2 and other reactive oxygen species clusters, and a large proportion of ROS can promote oxidative stress-induced cell damage and apoptosis [16].
In early pregnancy, autophagy induction preserves trophoblast function in the low oxygen and placental nutrient environment. Inadequate regulation of the ROS-autophagy axis leads to abnormal autophagy activity and contributes to the development of preeclampsia and intrauterine growth restriction. ROS-autophagy interactions are altered at the end of gestation and participate in the initiation of parturition at term.
ROS has been copiously reported as early inducers of autophagy. Thus, while ROS results in autophagy induction to maintain trophoblast viability and activity in the first trimester, its increased intensity near term coupled with activation of pro-inflammatory mediators and release of danger signals results in its contribution to the inhibition of autophagy near term. Excessive levels of inadequately opposed oxidative stress and increased or impaired autophagy are significant contributors to pregnancy complications. The increased induction of autophagy in association likely contributes to the development of a restricted growth fetus. These findings strongly indicate that elevated oxidative stress increases autophagy in preeclamptic placentas[17, 18]. The dysregulation of autophagy activity contributes to the pathology of multiple diseases[18–25].
There is also evidence [26, 27] that autophagy and oxidative stress play an essential role in pregnancy [28]. Under normal circumstances[29–32], trophoblast autophagy is suppressed at a basal level. In hypoxic, oxidative stress, infection, hormone stimulation, and other conditions, the autophagy level is much improved. In the early stage of normal pregnancy, autophagy caused by placental hypoxia also produces certain reactive oxygen species and increases oxidative stress [33].
Intracellular ROS levels, especially H2O2, are significantly elevated in starvation environments, inducing autophagy [34, 35]. These oxidation conditions are necessary for autophagy to occur, so the use of antioxidants inhibits the autophagy-lysosome formation and subsequent protein degradation [36, 37].
Therefore, it is crucial to study the effects of oxidative stress and autophagy on trophoblast biological function. Studies [38] have shown that excessive autophagy will lead to HTR-8/SVneo invasion and angiogenesis disorders, indicating that autophagy is essential for human extra-villi's normal function trophoblast. This study provides a new idea for trophoblast autophagy pathogenesis and pregnancy-related diseases under oxidative stress.
The autophagy activity, growth arrest, and apoptosis of cells are enhanced under induction by H2O2 [39].
LC3 is an essential indicator of the severity of autophagy, it appears in two forms in the cytoplasm: LC3I and LC3II. When autophagy occurs, LC3I is converted to LC3II. Therefore, LC3II was used as a molecular marker of autophagy, and the level of LC3II was used to reflect the degree of autophagy [40]. Besides, Beclin-1, the first mammalian autophagy protein identified as a novel Bcl-2-interacting protein. It plays a vital role in the autophagic process's critical step, namely, autophagosome formation [41]. Subsequent studies have demonstrated that this landmark protein is essential for autophagy[42]. Activation of the kinase activity of the Beclin-1 promotes autophagosome maturation [43].
Results from a previous study indicated LC3 and Beclin-1, two important autophagy-related proteins, found to be significantly overexpressed under H2O2 influence [cite:31]. In consistence with previous studies [44], our findings also showed that Beclin-1, LC3-Ⅱ protein expression was up-regulated in autophagy. The Western blot results obtained in the present study clearly showed that the oxidative stress model of HTR-8/SVneo placental trophoblasts induced autophagy by enhancing autophagy and increasing the level of LC3II (P < 0.01). The protein level of Beclin-1 also increased significantly (P < 0.01), which further indicated that autophagy was enhanced. Compared with the control group, Beclin-1, LC3-Ⅱ protein expression was up-regulated, and laser confocal immunofluorescence results were the same. Meanwhile, the protein expression level of p53 and mTOR was lower than that of the control group. (P < 0.01)
Autophagy is a relatively conservative metabolic pathway that involves many physiological processes and the regulatory mechanisms are very complex. p53 is a significant factor in regulating autophagy.
p53 is an important regulator of autophagy, and its regulation of autophagy is an important part of the stress response of cells stimulated by the external environment [45]. And p53 in the cytoplasm has a negative regulatory effect on autophagy, inhibiting autophagy occurrence [46–50]. p53 proteins can inhibit the activity of AMP-activated protein kinases (AMP-activated protein kinase, AMPK), while AMPK further inhibits the role of mammalian rapamycin target proteins [51]. mTOR activation can inhibit autophagy [46].
mTOR is an atypical serine/threonine-protein kinase receptor for amino acids and ATP, a family member of phosphatidylinositol kinase-associated protein kinase (PIKK).. activation of mTOR enhances phosphorylation of autophagy-related protein complexes and inhibits autophagy. mTOR has a gating effect during autophagy and its activity is critical for autophagosome formation and maturation. It is a negative regulator of autophagy, and many signaling pathways of autophagy go through mTOR pathways [52].
Our findings indicate that H2O2 can induce oxidative stress and enhance the autophagy activity of HTR-8/SVneo placenta trophoblasts. Since activated autophagy persists throughout the process and overlaps with apoptosis, it is harmful to cell growth and development, consistent with previous research findings. Oxidative stress enhances trophoblasts and endothelial cells' autophagy, affecting the invasion of trophoblasts and placental blood vessels' formation. Our study has shown that H2O2 can promote the activation of autophagy in trophoblast cells. However, the mechanism underlying autophagy and apoptosis and a cascade relationship need to be clarified further.
Resveratrol is a natural non-flavone polyphenolic compound with antioxidant, anti-aging and other effects [53, 54]. Resveratrol can modulate mTOR activity. But the protective effect of resveratrol on placental trophoblast was less reported. Hence, the current study took HTR-8-SV/neo cells as the research object to explore the specific mechanism of resveratrol on excessive autophagy induced by oxidative stress in trophoblast cells.
Many pharmacological effects of resveratrol are related to its regulatory effect on autophagy. Currently, resveratrol is an effective autophagy regulator that exerts the role of inducing autophagy or inhibiting autophagy in different cells by regulating autophagy-related signaling pathways[55, 56].
Cell death, preceded by autophagy, plays a positive role in physiologic conditions, depending on the cell environment [57]. We took different major autophagy-related proteins, such as p53, mTOR, Beclin-1 and LC3, which are regulatory proteins of autophagy that participate in the key steps underlying the occurrence of autophagy [58]. As observed by WB and immunofluorescence results, resveratrol treatment can significantly reduce the expression level of Beclin-1 in an oxidative stress model and delays the transformation of LC3I to LC3II (P < 0.01), which is the gold standard of autophagy [59]. Resveratrol treatment can also significantly increase p53 and mTOR expression in an oxidative stress model (P < 0.01). This further proves the down-regulation effect of autophagy.
In conclusion, we have shown that resveratrol treatment can significantly decrease the levels of LC3 and Beclin-1, increase the level of p53 and mTOR autophagy protein in an HTR-8/SVneo human placental trophoblast oxidative stress model. Here we found that resveratrol can significantly reduce the oxidative stress damage of HTR-8/SVneo cells treated with H2O2. Resveratrol may partly play a protective role by activating mTOR/ autophagy and attenuating the cells' autophagy response.
The findings of this study may provide a useful therapeutic target for the pathogenesis of pregnancy-related diseases. Offering new insight into validating the therapeutic potential of resveratrol in the stage of pregnancy. However, it must be emphasized that the precise molecular mechanisms and direct molecular targets of resveratrol regulating autophagy remain unclear, thus requiring further investigation. With this increased knowledge, novel protocols can then be developed to improve pregnancy outcomes.