Preterm birth is most frequently associated with infection in the literature. An increase in vaginal pathogen colonization is considered the primary possible mechanism behind infection-associated preterm birth [22] and LPS is frequently used in experimental animal models that mimic infection-mediated preterm birth [23]. Likewise, the placenta consists of multiple zones and cell populations, which can generate different responses [24]. Therefore, in evaluating protein localizations and cellular expression statuses, different markers were used to assess the status of cells, placental zones, and changes in the cervix and uterine tissues. Even though fetal placental membranes offer physical protection against LPS and microbial agents [25], it has been shown in the literature that vaginal pathogens can reach the amniotic membrane [26].

The placenta, a vital maternal–fetal interface, plays a key role in numerous physiological functions such as bidirectional molecular transport, endocrine regulation, immune defense, and maintaining maternal–fetal tolerance. Necroptosis has been identified to have a dual function within placental biology. While necroptotic signaling is essential for proper placental development and fetal organ formation, its dysregulation is implicated in the development of pregnancy-related disorders. Recent studies have associated abnormal necroptosis with the pathogenesis of significant pregnancy complications, including preeclampsia, fetal growth restriction, recurrent miscarriage, and gestational diabetes mellitus [27]. In our study, no significant difference was observed in RIPK3 expression and protein levels in the placenta, while NEC-1 application did not cause an effect on RIPK3. However, MLKL expression and protein levels decreased in the placenta after LPS application. Although slight expression was observed in LZ in the placenta after NEC-1 application, no significant increase was observed. NEC-1 could not reverse the effect of LPS at the protein level in the L + N group. The results may suggest that LPS directly suppresses MLKL and prevents necroptosis. In addition, although not mediated by RIPK3, the fact that MLKL did not increase after LPS application in the placenta suggests that a different apoptotic or necroptotic pathway other than canonical necroptosis may be activated. Namely;

Although NEC-1 is used to determine necroptosis and target RIP1 kinase activity in a wide variety of pathological cell death events [18], not only does it inhibit RIP1, but it also diminishes RIP3 expression and phosphorylation, thereby hindering the formation of the RIP1-RIP3 complex by attenuating their interaction, as shown in many disease contexts [18, 28]. In addition, the literature has reported that NEC-1 inhibits necroptosis and is associated with apoptosis inhibition [29]. In scenarios where apoptosis is mediated through RIP1, NEC-1 can inhibit RIPK1 activity and complex IIa formation, thus blocking cell death. However, if apoptosis occurs independently of RIP1, NEC-1 has no effect. Another viewpoint proposes that inhibition of necroptosis by NEC-1 may lead to a shift toward apoptotic cell death [18, 30]. Therefore, the fact that NEC-1 does not have an inhibitory effect on RIPK3 in the placenta suggests that the axis has shifted to an apoptosis-independent direction from NEC-1 and that the protein level may have decreased due to the disruption of the necrosome structure and insufficient phosphorylation of MLKL due to inadequate formation of the RIP-RIP3 complex.

Our literature defines premature rupture of membranes (PROM) as the tearing of fetal membranes before birth contractions begin. This event can lead to spontaneous labor but also increases the risk of intra-amniotic infection and placental abruption. The fetal membranes are composed of two layers: the inner side amnion and the outer side chorion, which is attached to the decidua of the endometrium. As a result, the amnion is sensitive to alterations in the amniotic cavity, while the chorion is crucial for sustaining immune tolerance at the maternal–fetal boundary. Selective apoptosis drives membrane remodeling toward the end of pregnancy, leading to membrane weakening. Inflammatory processes can activate proteases within the fetal membranes, compromising their integrity and increasing susceptibility to rupture. While intrauterine infection frequently accompanies membrane rupture, it remains uncertain whether infection is a cause or consequence of the rupture [31,32,33].

As a remarkable result, the depletion of nuclear MLKL expression observed immunohistochemically in the amniotic membrane in the LPS-treated group suggested that MLKL may be a protein associated with membrane rupture observed in preterm birth. Phosphorylation of MLKL is associated with ATP depletion and plasma membrane disruption. It can be thought that MLKL, which carries out membrane translocation after oligomerization following RIPK3-mediated phosphorylation, is depleted by disrupting membrane integrity [27] and aggravating the inflammatory responses of surrounding cells [34]. However, this process, which is related to the amniotic membrane, needs to be investigated more.

Necroptosis is involved in the pathogenesis of bacterial infections. Limiting these infections and generating a balanced response is vital for survival [35]. At the right time, the destruction of the epithelium, which acts as a barrier for bacteria, can facilitate bacterial progression [36]. Necroptosis is a possible route to overcome the epithelial barrier. In some cases, necroptosis can limit excessive infection [37]. The dual effect of necroptosis may be related to the type of bacteria and the severity of the infection, and more studies are needed to determine how the balance is achieved in cases where we can talk about a beneficial or harmful effect [38].

LPS activates necroptosis signaling and enhances the expression of RIPK1, RIPK3, and MLKL, critical mediators of programmed necrotic cell death [39]. In the literature, changes in necroptosis components in different tissues have been evaluated after LPS induction, and some of them have reviewed the effect of NEC-1 on necroptosis proteins. However, no study has been found in the literature evaluating the relationship between necroptosis and preterm birth within the framework of reproductive tissues. When we examine some of these studies that are similar to our research in terms of methodology. In a study evaluating necroptosis components in the hypothalamic axis, 1 mg/kg NEC-1 dissolved in 2% DMSO was administered IP 30 min before the IP injection of LPS, and the subjects were sacrificed 4 h after the injection of LPS (100 μg/kg/IP). NEC-1 has been shown to suppress necroptosis components [40].

In a different study, 1 mg/kg/ip NEC-1 in 5% DMSO was administered to mice together with LPS. When LPS was administered together with a caspase inhibitor, phospho-MLKL (p-MLKL) increased, and a transient interaction was observed between extracellular cold-inducible RNA-binding protein (eCIRP) and MLKL, and eCIRP could exit the cell through pores formed by p-MLKL. NEC-1 suppressed the increase. Within this framework, necroptosis is believed to be a regulated death pathway dependent on MLKL activation, leading to DAMP release. eCIRP, released during necroptosis, further intensifies the septic inflammatory response [41]. In another study, in the sepsis model associated with lung infection, NEC-1 was administered at 20 mg/kg/iv 10 min before the model was established. As a result, MLKL-NLRP3-mediated necroinflammation was significantly upregulated in the lung tissue of septic mice, and this situation could be attenuated by the specific inhibitor NEC-1 [42]. Increased RIPK1, RIPK3, and MLKL activity in LPS-induced thermal hyperalgesia were reversed by NEC-1 [43]. Again, LPS increased p-RIPK1, p-RIPK3, and p-MLKL in acute lung injury, and the necroptotic effect was reversed by NEC-1 [44].

RIPK1 and RIPK3 have been implicated in the development of renal fibrosis after ischemia–reperfusion and kidney injury. NEC-1-treated mice showed reduced kidney damage at 24 h compared to controls [45]. Necrostatin-1 also had a positive effect on cognitive decline by reducing the increases in protein levels of RIPK1, RIPK3, MLKL, and p-MLKL associated with necroptosis in the hippocampal tissue of paclitaxel-treated mice [46]. In another study, It was observed that necroptosis contributed to the formation of damage and increased total and phosphorylated RIP3 and MLKL proteins in deoxynivalenol-induced liver injury. NEC-1 administration inhibited necroptosis and reduced damage [47]. In a different research, it was shown that the expression of RIPK1, RIPK3, and p-MLKL increased in preeclampsia, while pre-treatment with NEC-1 decreased the expression [48].

As seen in the studies mentioned above, RIPK3 and MLKL proteins are upregulated after LPS, and this increase is suppressed after different doses of NEC-1 pretreatment. Our research showed that RIPK3 and MLKL expression increased in the uterine epithelium in the LPS-applied group, and a similar upregulation was observed at the protein level. A parallel increase was also present in the cervix. These increases were significant and, similar to the literature, were suppressed after NEC-1 pretreatment. However, another piece of information that we will add to the literature is that NEC-1 in the uterus may suppress necroptosis activated by the increase in RIPK3 and MLKL after LPS application, probably via MLKL, and in the cervix via RIPK3. Based on the NEC-1 effect seen at the protein level, it has been suggested that NEC-1 does not suppress RIPK3 in the uterus. Thus, the decrease seen in the L + N group is probably achieved by restricting MLKL phosphorylation via RIP1. In the cervix, NEC-1 appears to control necroptosis by specifically suppressing RIPK3. This pathway suggests that a cascade occurs, resulting in the suppression of RIP1 and, therefore, RIPK3 by reducing ROS or the suppression of MLKL and thus limiting necroptosis by reducing RIPK3 conversion directly via RIP1, based on the multifaceted mechanism of action of NEC-1 [18]. This pathway may also inhibit phospho-RIP1-dependent apoptosis and induce caspase 8-dependent apoptosis. In summary, NEC-1 administration in LPS-induced preterm birth suppresses necroptosis at varying levels in reproductive tissues. The hypothesized interactions in tissues are summarized in Fig. 7.

Possible summary of reproductive tissue cross-talk in necroptosis. LPS application upregulates RIPK3 and MLKL proteins in the uterus and cervix, while NEC-1 downregulates these proteins via MLKL in the uterus and RIPK3 in the cervix. LPS application did not change RIPK3 in the placenta but suppressed MLKL. In the amniotic membrane, LPS application depleted MLKL