Medicinal Plants for the Treatment of Mental Diseases in Pregnancy: An In Vitro Safety Assessment

Pregnancy is a critical period for medical care, during which the well-being of woman and fetus must be considered. This is particularly relevant in managing non-psychotic mental disorders since treatment with central nervous system-active drugs and untreated NMDs may have negative effects. Some well-known herbal preparations (phytopharmaceuticals), including St. Johnʼs wort, California poppy, valerian, lavender, and hops, possess antidepressant, sedative, anxiolytic, or antidepressant properties and could be used to treat mental diseases such as depression, restlessness, and anxiety in pregnancy. Our goal was to assess their safety in vitro, focusing on cytotoxicity, induction of apoptosis, genotoxicity, and effects on metabolic properties and differentiation in cells widely used as a placental cell model (BeWo b30 placenta choriocarcinoma cells). The lavender essential oil was inconspicuous in all experiments and showed no detrimental effects. At low-to-high concentrations, no extract markedly affected the chosen safety parameters. At an artificially high concentration of 100 µg/mL, extracts from St. Johnʼs wort, California poppy, valerian, and hops had minimal cytotoxic effects. None of the extracts resulted in genotoxic effects or altered glucose consumption or lactate production, nor did they induce or inhibit BeWo b30 cell differentiation. This study suggests that all tested preparations from St. Johnʼs wort, California poppy, valerian, lavender, and hops, in concentrations up to 30 µg/mL, do not possess any cytotoxic or genotoxic potential and do not compromise placental cell viability, metabolic activity, and differentiation. Empirical and clinical studies during pregnancy are needed to support these in vitro data.

Chemical Induction of Trophoblast Hypoxia by Cobalt Chloride Leads to Increased Expression of DDIT3

Choriocarcinoma cells BeWo b30 are used to model human placental trophoblast hypoxia using cobalt (II) chloride and hydroxyquinoline derivative (HD) as chemical inducers of hypoxia-inducible factor (HIF). In this study, it was shown that both substances activate the hypoxic pathway and the epithelial–mesenchymal transition and inhibit the pathways of cell proliferation. However, CoCl2 caused activation of the apoptosis pathway, increased the activity of effector caspases 3 and 7, and increased the expression of the unfolded protein response target DDIT3. The mTORC1 pathway was activated upon exposition to CoCl2, while HD suppressed this pathway, as it happens during real trophoblast hypoxia. Thus, effect of CoCl2 on BeWo cells can be a model of severe hypoxia with activation of apoptosis, while HD mimics moderate hypoxia.

Uroven' mikroRNK miR-374 povyshaetsya v kletkah BeWo b30 pri gipoksii

Vozdejstvie gipoksii na kletki trofoblasta v processe formirovaniya placenty cheloveka mozhet privodit' k razvitiyu ryada patologij beremennosti, takih kak preeklampsiya i zaderzhka rosta ploda. Patogenez dannyh sostoyanij ne do konca raskryt i mozhet byt' svyazan s izmeneniem ekspressii v trofoblaste ryada genov i regulyatornyh molekul, vklyuchaya mikroRNK. Cel'yu dannogo issledovaniya bylo izuchit' profili mikroRNK i ekspressii sootvetstvuyushchih genov-mishenej v modeli gipoksii trofoblasta. Modelirovanie trofoblasta provodili s ispol'zovaniem kletochnoj linii horiokarcinomy cheloveka BeWo b30. V kachestve induktorov gipoksicheskogo otveta ispol'zovali hlorid kobal'ta (CoCl2) i proizvodnoe oksihinolina. Analizirovali uroven' mRNK i mikroRNK s pomoshch'yu sekvenirovaniya sleduyushchego pokoleniya (NGS) s podtverzhdeniem ekspressii otdel'nyh genov PCR. Analizirovali vtorichnuyu strukturu mRNK gena-misheni znachimo izmenivshihsya mikroRNK i vozmozhnuyu konkurenciyu za mesta svyazyvaniya. Izmenenie ekspressii klyuchevyh genov otveta na gipoksiyu podtverdilo relevantnost' CoCl2 i proizvodnogo oksihinolina v kachestve induktorov gipoksii. Vyyavleno povyshenie urovnya mikroRNK semejstva miR-374 pri indukcii gipoksicheskogo puti v modeli trofoblasta. Nablyudaemye izmeneniya soprovozhdalis' snizheniem ekspressii mRNK gena FOXM1, kotoraya sluzhit mishen'yu dlya hsa-miR-374a-5p i hsa-miR-374b-5p. Dannye mikroRNK mogut konkurirovat' za mesta svyazyvaniya v mRNK FOXM1 s hsa-miR-21-5p. Uchastie gena FOXM1 v regulyacii invazivnogo potenciala kletok pozvolyaet predpolozhit' rol' mikroRNK miR-374 i FOXM1 v patogeneze narusheniya invazii trofoblasta pri formirovanii placenty kak predposylki k razvitiyu zaderzhki rosta ploda i preeklampsii.

Levels of miR-374 increase in BeWo b30 cells exposed to hypoxia

In humans, trophoblast hypoxia during placental development can be a cause of serious pregnancy complications, such as preeclampsia and fetal growth restriction. The pathogenesis of these conditions is not fully clear and may be associated with changed expression of some genes and regulatory molecules, including miRNA, in trophoblast cells. The aim of this study was to analyze miRNA profiles and measure the expression of their target genes in a model of trophoblast hypoxia. Human choriocarcinoma BeWo b30 cells were used as a trophoblast model. Hypoxia was induced by cobalt chloride (CoCl2) and an oxyquinoline derivative. MRNA and miRNA expression profiles were evaluated by means of next generation sequencing (NGS); the expression of individual genes was analyzed by PCR. We studied the secondary structure of mRNAs of target genes for those miRNAs whose expression had changed significantly and analyzed potential competition between these miRNAs for the binding site. The observed changes in the expression of the key genes involved in the response to hypoxia confirmed the feasibility of using CoCl2 and the oxyquinoline derivative as hypoxia inducers. The analysis revealed an increase in miR-374 levels following the activation of the hypoxia pathway in our trophoblast model. The changes were accompanied by a reduction in FOXM1 mRNA expression; this mRNA is a target for hsa-miR-374a-5p and hsa-miR374b-5p, which can compete with hsa-miR-21-5p for the binding sites on FOXM1 mRNA. The involvement of FOXM1 in the regulation of the invasive cell potential suggests the role of miR-374 and FOXM1 in the pathogenesis of disrupted trophoblast invasion during placental development as predisposing for fetal growth restriction and preeclampsia.

Inspired by the human placenta: a novel 3D bioprinted membrane system to create barrier models

Barrier organ models need a scaffold structure to create a two compartment culture. Technical filter membranes used most often as scaffolds may impact cell behaviour and present a barrier themselves, ultimately limiting transferability of test results. In this work we present an alternative for technical filter membrane systems: a 3D bioprinted biological membrane in 24 well format. The biological membrane, based on extracellular matrix (ECM), is highly permeable and presents a natural 3D environment for cell culture. Inspired by the human placenta we established a coculture of a trophoblast-derived cell line (BeWo b30), together with primary placental fibroblasts within the biological membrane (simulating villous stroma) and primary human placental endothelial cells—representing three cellular components of the human placental villus. All cell types maintained their cell type specific marker expression after two weeks of coculture on the biological membrane. In permeability assays the trophoblast layer developed a barrier on the biological membrane, which was even more pronounced when cocultured with fibroblasts. In this work we present a filter membrane free scaffold, we characterize its properties and assess its suitability for cell culture and barrier models. Further we show a novel placenta inspired model in a complex bioprinted coculture. In the absence of an artificial filter membrane, we demonstrate barrier architecture and functionality.