Background
Probiotic foods are ubiquitous in our daily lives, and the number of people taking probiotics is increasing. Pregnant women in particular, attach great importance to nutritional supplements during pregnancy. Therefore, many pregnant women voluntarily take certain probiotics supplements, such as golden bifid and acidophilus milk, during pregnancy. The use of probiotics in the United States and Canada ranges from 1.3 to 3.6%, whereas the probability of using probiotics in pregnant women in the Netherlands has risen to 13.7% [
1]. The International Scientific Association for Probiotics and Prebiotics (ISAPP) defines probiotics as live microorganisms that can induce a health benefit on the host if administered in adequate amounts. Generally, Lactobacillus and Bifidobacterium are widely used [
2]. However, metabolic byproducts, dead microorganisms or other nonviable products based on microorganisms are not probiotics. Currently, golden bifid (i.e.,
live combined Bifidobacterium and Lactobacillus tablets) is voluntarily purchased and taken by pregnant women as a nonprescription drug. Moreover, recent research has shown that probiotics serve to mediate certain biological effects, including diminishing free radical injury, inflammation, and tumor progression and regulating lipid metabolism [
3‐
6].
Autophagy is an evolutionary adaptive response of eukaryotic cells that moves senescent cells and organelles to lysosomes for degradation and recycling and ultimately maintains homeostasis of the intracellular environment [
7,
8]. The placenta, a unique organ formed during pregnancy, is an important survival organ for material exchange between the fetus and mother. It can provide nutrition, gas exchange, and immune and metabolic support for developing fetuses. Studies have shown that the basal level of placental trophoblast autophagy plays an important role in the whole pregnancy process of embryo implantation and placental vascular recasting [
9,
10]. If autophagy is excessive, it will change from a protective mechanism to a damaging mechanism, hindering the metabolic functioning of placental trophoblasts and leading to certain placental-derived diseases, such as preeclampsia and fetal growth restriction [
11,
12]. Moreover, LC3 (microtubule associated protein l light chain3) and Beclin1 are commonly used to detect autophagy and further reflect the metabolic function of the placenta [
13,
14]. Recent studies have suggested that probiotics can mildly regulate macrophages to enhance the immune response to foreign invaders, the mechanism underlying which is the enhancement of macrophage autophagy to defend against infection by probiotics [
15]. Lactobacillus acidophilus and
Bacillus clausii are potent activators of innate immune responses in the murine macrophage cell line RAW264.7 [
16]. Probiotics mediate the immunostimulatory activity by interacting with both microorganism-associated molecular patterns and Toll-like receptors, which also participate in the stimulation of autophagy in macrophages. Despite this evidence, only a few studies have explored the regulation of autophagy by probiotics [
17‐
19]. In view of the scarcity of information regarding the role of probiotics in mediating autophagy in humans, especially the effects of probiotics on placental autophagy in pregnant women during pregnancy, the present study aimed to investigate the changes caused by orally supplemented probiotics in the expression of the autophagy-related proteins LC3 and Beclin1 that reflect the metabolic functions of the placenta.
In this study, the pregnant women in the probiotic group voluntarily took a golden bifid supplement at 32 weeks of gestation. The aim of this research was to investigate the effect of probiotics on placental autophagy and to provide novel insight into probiotic-mediated placental protection.
Discussion
Autophagy plays an important role in the regulation of placental function and the maintenance of normal pregnancy by participating in eukaryotic cell self-metabolism and in the establishment of utero-placental circulation [
10]. Our results indicate that the supplementation of probiotics can impose a slight but not significant influence on placental autophagy, and paradoxically, the influence is controversial. At the protein level, there was increased expression of LC3, but the expression of Beclin1 was decreased (Fig.
3). Moreover, no significant difference was found in LC3 mRNA between the two groups (Fig.
4), whereas the Beclin1 mRNA expression in the probiotic group was significantly lower than that in the control group (
P < 0.05, Fig.
4). The reason underlying the lack of significance may be that the two methods are focus on the mRNA level and protein level (i.e., transcription and translation, respectively), where there is posttranscriptional processing after transcription, degradation of the transcription products, translation, posttranslational processing and modification. The sensitivity of RT-PCR for detecting mRNA is very high, so there may be discrepancies in the detection results of the above two methods. Moreover, epigenetic mechanisms such as DNA methylation may also play an important role in the results. Thus, orally supplemented probiotics might decrease the levels of the autophagy-related protein Beclin1 mRNA in the placenta.
However, the autophagy phenomenon of placental trophoblast cells is affected by various factors, such as delivery mode, nutrient supply and pregnancy pathology. Recently, it has been reported that the mode of delivery directly affects placental oxidative stress and autophagy [
20]. Since normal pregnancy in spontaneous vaginal delivery can intermittently affect the blood flow of the placenta and mechanical stress caused by muscle contraction, placental autophagy activity may be enhanced. However, Signorelli et al. showed that placental autophagy activity was significantly reduced compared with that of cesarean section [
21]. Furthermore, Doulaveris reported that the inhibition of autophagy is associated with spontaneous vaginal delivery, and may be caused by preoperative fasting and the use of anesthetic drugs [
22]. Some scholars have shown that the use of local anesthetics in rabbits can promote the activation of the autophagy signaling pathway, thereby enhancing autophagy [
23]. Preoperative fasting will lower the levels of glucose and amino acids and increase the level of autophagy to ensure energy supply and normal metabolism [
24].
Studies have shown that the expression of the autophagy proteins LC3 and Beclin1 in pregnant women with preeclampsia is higher than that in normal pregnant women [
11]. However, some scholars have not found significant differences between preeclamptic patients and normal pregnant women by comparing the placental autophagy-related pathway genes [
25]. Moreover, What’s more, by collecting the placental tissue of 61 pregnant women who underwent elective cesarean section, Hung T et al. found that the expression of autophagy-related proteins LC3 and Beclin1 in the placental tissues of pregnant women with intrauterine growth restriction was higher than that of normal pregnant women [
26]. The pathological process of preeclampsia is accompanied by impaired functioning of the placental trophoblast cells, insufficient remodeling of the uterine spiral arteries, and oxidative damage. Fetal growth restriction may also have pathological processes causing placental dysfunction. It is therefore possible that placental autophagy activity is increased to further help trophoblasts adapt to these pathological changes to maintain bioenergy homeostasis and clear damaged organelles. Pregnant women with pathological pregnancy will have increased expression of autophagy-associated proteins in the placenta. Moreover, although we strove to maintain the homogeneity of the subjects, there were still some potential confounding factors that could interfere with the results, such as food consumption, alcohol consumption, and physical activity. Studies have shown that there is a special “channel” for oral supplementation. Accumulating evidence suggests that supplementation with probiotics can induce immune modulation and thus prevent certain intestinal diseases [
27,
28]. Moreover, Wu et al. showed through Western blot and confocal laser scanning analysis [
29] that the probiotic
Bacillus amyloliquefaciens SC06 induces autophagy to protect against pathogens in macrophages in a dose- and time-dependent manner. Moreover, Lin et al. reported that Bifidobacterium can initiate autophagy activation in intestinal epithelial cells [
19]. Although the specific mechanism is unknown, probiotics can regulate autophagy in the placentas of normal pregnant women. This may have a positive effect on the fetus and infant or provide a clinical basis for probiotic use in pathological pregnancy, which needs to be further explored.
To our knowledge, the study has strengths. This is the first study investigating the influence of oral probiotic supplementation on placental autophagy. The placental tissue included in this study was obtained from normal spontaneous delivery, excluding differences caused by cesarean section. However, immunofluorescence staining was not performed, without which we could not better evaluate the microscopic difference of the placenta after supplementation with probiotics.
Acknowledgments
We gratefully acknowledge the following investigators who participated in this multicenter study: Zhe Li, TYE KIAN DENG, Yuyi Chen, Tong Lu, Zonglin He, Juan Zhou, and Xiaomin Xiao.
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