1. Introduction
Preeclampsia, a hypertensive disorder of pregnancy, is one of the leading causes of maternal and fetal morbidity and mortality. The disease is a cause of serious consequences, including impairments of multiple organ systems (central nervous, hepatic, pulmonary, renal, and hematologic systems) and the leading cause of maternal mortality in both developed and developing countries. Moreover, fetal complications include placental abruption, intrauterine growth restriction, premature delivery, and intrauterine fetal death [
1,
2,
3].
The etiology of preeclampsia remains not fully understood [
4,
5]. Preeclampsia likely begins at implantation with superficial invasion of placenta vessels leading to excessive release of physiological antiangiogenic factors [
6]. According to this theory, abnormal spiral artery remodeling in early pregnancy causes placental hypoxia. The ischemic placenta releases into the maternal circulation large amounts of soluble factors, such as reactive oxygen species, pro-inflammatory cytokines, and anti-angiogenic factors, which lead to the clinical manifestations of the disease [
7,
8].
It is known that in normal pregnancy, systemic inflammation, oxidative stress, and alterations in levels of angiogenic factors and vascular reactivity are observed, but this process is exacerbated in preeclampsia with associated breakdown of compensatory mechanisms, eventually leading to placental and vascular dysfunction. One hypothesis concerns placental endothelial cell dysfunction [
4,
9]. Changes in endometrial levels of many angiogenic growth factors have been observed in pregnancy-induced hypertension (PIH), including vascular endothelial growth factor A (VEGF-A) and placental growth factor (PIGF) [
10,
11].
Preeclampsia is also characterized by excessive and progressive activation of the immune system along with increases in proinflammatory cytokines and antiangiogenic factors in the fetoplacental unit andan increase in vascular endothelium in pregnant women. A single, major underlying mechanism of preeclampsia is yet to be identified. Inflammation is an active process regulated by various mediators that control key cellular events to restore tissue homeostasis. Impaired resolution of inflammation probably plays a vital role in the development of chronic inflammatory diseases, and preeclampsia is believed to be one of them [
12]. It is suggested that preeclamptic women display an exaggerated inflammatory response in the course of pregnancy due to unbalanced regulation of innate and adaptive immune responses [
13].
Much attention is paid to the use of antihypertensive drugs from different pharmacological groups in pregnancy [
14]. Drug treatment options in preeclampsia are limited because some antihypertensive drugs, such as angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor (AT1) antagonists (sartans), have shown teratogenic effects on the fetus [
15]. Methyldopa belongs to the group of drugs acting on the central nervous system to evoke depression in the cardiovascular system [
16]. Numerous reports have confirmed the great usefulness of this drug for reducing blood pressure in pregnant women, both in preeclamptic women and in women with chronic hypertension [
16]. The reduction of blood pressure in pregnant women with hypertension using methyldopa can have a significant positive impact on the uteroplacental circulation, which improves the provision of oxygen and nutrients to the developing fetus [
17,
18,
19]. This drug remains a mainstay of preeclampsia treatment mostly due to reported uteroplacental perfusion stability and fetal hemodynamics [
20,
21]. However, treatment of preeclampsia remains sometimes challenging, since methyldopa has some adverse side effects, such as hepatotoxicity [
22], and may be hard to tolerate due to causing dizziness, depression, or headache [
23].
One possibility of obtaining a stronger antihypertensive effect with possible reduction of side effects is combined therapy [
24]. This kind of therapy shows a greater chance of obtaining a hypotensive response in a pathogenically complex disease such as preeclampsia, due to different mechanisms of drug action, greater potency of the hypotensive effect due to synergistic effects, and the possibility of using drugs in doses that give the lowest possible likelihood of adverse effects. Therefore, the search for new drugs for use in combined therapy is necessary. One source of new drugs is herbal plants, which include known plant materials with proven antihypertensive effects [
25]. A growing number of studies indicate the cardioprotective effects of flavonoids—natural polyphenolic compounds commonly found in fruits, vegetables, and beverages. It is well known that such flavonoids as quercetin, apigenin, and chrysin are found in many medicinal plants, for example, buckwheat (
Fagopyrum esculentum Moench), hawthorn (
Crataegus spp.), and passionfruit (
Passiflora spp.) [
26]. Baicalein and scutellarin are characteristic of the genus
Scutlaria (for example, in Baikal skullcap (
Scutellaria baicalensis Georgi), although they were also found in other plants: baicalein in Indian trumpetflower (
Oroxylum indicum) [
27] and thyme (
Thymus vulgaris L.) [
28]; scutellarin in Erigeron breviscapus [
29]. It is known that quercetin, a flavonoid well known for its antihypertensive action, may be considered a prototype for a safe antihypertensive drug [
2]. Moreover, other flavonoids such as quercetin [
30], apigenin [
31], chrysin [
32], baicalein [
33,
34], and scutellarin [
35] exhibit vasoprotective properties, and many other activities, such as anti-oxidation via several pathways, anti-inflammation, anti-ischemia, cardioprotection, and anti-hypertension [
36]. These flavonoids have not demonstrated teratogenic or abortive effects, so they are generally recognized as safe [
37,
38].
Although the flavonoids possess antihypertensive activities via various mechanisms of action [
2,
36], so far, data on their effects in pregnancy-induced hypertension are generally unavailable. To address this problem, in this study we decided to investigate the effects of the combined administration of each of the tested flavonoids with methyldopa in vitro—using cells that were derived from human trophoblasts (JEG-3 cells) and human umbilical vein endothelial cells (HUVEC)—on the expression of selected markers responsible for inflammation (tumor necrosis factor α—TNF-α; interleukin 1—IL-1β; interleukin 6—IL-6) and vascular effects (hypoxia-inducible factor 1α—HIF-1α; placental growth factor—PIGF; transforming growth factor β—TGF-β;vascular endothelial growth factor—VEGF) at the mRNA and protein levels.
3. Discussion
Although it is known that in vitro tests will not exactly match the processes occurring in the entire tissue or body, the use of models based on the cells of the obtained target tissues allows one to approximate the phenomena occurring in vivo [
39]. Therefore, the studies were carried out using human JEG-3 cell trophoblasts and human umbilical vein endothelial cells (HUVEC). JEG-3 cells were used as a model for the investigation of changes in placenta trophoblasts according to other studies [
39,
40,
41], whereas HUVEC were used as a model for the investigation of changes in endothelial cells [
42].
In the first stage, the influences of methyldopa and the tested flavonoids on the cytotoxicity of compounds were measured using an evaluation of overall survival rate with the MTT technique [
43]. It was found that all compounds act similarly in the range of 20–100 μmol, and in practice no significant differences in overall survival rate were observed between the concentrations of all compounds, although for both cell systems used (JEG-3 and HUVEC) when analyzed together (main effect versus dose), the lowest cytotoxicity was noted at 40 μmol. Therefore, for further research, this concentration was used both for substances administered separately and in combination with methyldopa. Detailed understanding of the pathophysiology of the inflammatory process in preeclampsia is still a subject of research [
44,
45]. It is known that TNF-α is a central regulator of inflammation, and this cytokine plays a crucial role in causing inflammation predominantly by means of T lymphocytes. It is also associated with inflammatory mechanisms related to implantation, placentation, and pregnancy outcome, since overproduction of TNF-α may lead to such events as recurrent pregnancy loss, early and severe pre-eclampsia, and recurrent implantation failure syndrome [
46]. In our study it was found that all the compounds tested, i.e., methyldopa and all flavonoids, reduced the expression of TNF-α at both the mRNA and protein levels to similar extents in JEG-3 cells. In these cells the combined administration any flavonoid together with methyldopa showed stronger decreases in TNF-α mRNA expression and protein presence, and the strongest effect was found for methyldopa + scutellarin. On the other hand, in HUVEC cells all the compounds tested, i.e., methyldopa and all flavonoids, increased mRNA TNF-α expression to a similar extent, whereas their combined treatment with methyldopa lowered the expression and led to values similar to the control. At the protein level, less visible effects were noted, although combined administration also produced control-like effects. It is known that TNF-α inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression, and methyldopa reversed TNF α-induced inflammation and increased eNOS expression in vitro [
47]; our effects ofmethyldopa may be consistent with these observations.
It is known that IL-1 is a possible mediator of maternal endothelial dysfunction in preeclampsia [
48,
49], and aberrant IL-1β levels were shown to be associated with a variety of gestational diseases, such as preeclampsia, preterm labor, and spontaneous abortion [
50]. It is known that IL-1 is elevated in maternal blood from women with pre-eclampsia [
51]. We observed that all the compounds tested, i.e., methyldopa and all flavonoids, lowered the mRNA IL-1β expression, and every combined flavonoid and methyldopa treatment showed changes in the same direction in JEG-3 cells. However, at the protein level the effects were slight and not significant, but the administrations of flavonoids together with methyldopa showed stronger increases in expression; in particular, the combination of methyldopa + baicalein produced a very strong effect. In HUVEC cells it was found that all the compounds tested, i.e., methyldopa and all flavonoids, increased mRNA expression, and flavonoids’combined treatment with methyldopa strengthened this effect. In contrast, flavonoids together with methyldopa mostly showed mostly slight decreases in the value of IL-1β at the protein level, but none of the differences in the methyldopa ratio were statistically significant. These somewhat surprising and opposite results obtained depending on the type of cells and the measurement of mRNA or protein levels are difficult to explain; however, in studies on women different results were obtained. For example, onset of labor results in elevations in amniotic fluid levels of IL-1β that are similar in preeclamptic pregnancy to those observed in normal pregnancy [
52]—though this contrasts with the findings of Stallmach [
53]. It is proposed that these apparent contradictions between studies using immunological detection of cytokines and bioactivity studies may reflect changes in cytokine inhibitory binding proteins during preeclamptic pregnancy [
54].
Numerous reports indicate that the plasma of preeclamptic patients contains elevated levels of IL-6, a multifunctional cytokine that regulates, among other things, the acute phase reaction and modulates both pro- and anti-inflammatory events, and may play roles in the pathogenesis of preeclampsia by serving as a source of a key circulating factor that promotes systemic maternal endothelial cell dysfunction [
55]. Additionally, while many of the functions of IL-6 have not been explained yet, it is assumed that IL-6 is a good biomarker for adverse pregnancies [
56]. In this study it was found that all tested compounds, i.e., methyldopa and all flavonoids, increased mRNA IL-6 expression, but every combined administration of a flavonoid together with methyldopa showed a strong contrary inhibitory effect when compared to the value for methyldopa in JEG-3 cells. At the protein level, no significant changes were found for either individual substances or their combinations with methyldopa. In HUVEC cells we observed similar effects at the mRNA level as for JEG-3 cells. However, at the protein level all compounds were found to increase IL-6, but after their combined administration, there were inhibitory effects for all flavonoids compared to the value for methyldopa. Summarizing these results, it can be stated that even if methyldopa and individual flavonoids increased the activity of the formation of this cytokine, the inhibitory effect was generally observed after combined administrations of flavonoids with this drug, which may have a positive effect.
A number of growth factors/cytokines with angiogenic properties have been actively studied in the context of preeclampsia, including HIF-1α, PIGF, TGF-β, and VEGF.
The TGF-β superfamily includes inhibins, activins, bone morphogenic peptides, and growth and differentiation factors. It has been established that TGF-β is one of the cytokines with expression in macrophages and epithelial tissue [
57], for example, in asthmatic epithelium [
58], and moreover, it is associated with preeclampsia risk [
54,
59]. It is also well known that the increased TGF β-1 level may lead to preeclampsia [
60,
61]. In this study, the effect of administering all substances was similar and did not significantly affect mRNA TGF-β expression in relation to the control group, but combined treatment using flavonoids with methyldopa lowered the expression in relation to the methyldopa group in JEG-3 cells. Similarly, at the protein level the administration of flavonoids together with methyldopa showed a general inhibitory effect on the action of the flavonoids alone, and the strongest inhibitory effect was noted for methyldopa + quercetin when compared to methyldopa alone. In HUVEC cells, especially after treatments of different flavonoids combined with methyldopa, at both the mRNA and protein levels, a general inhibitory effect in relation to the action of methyldopa alone was also found. Summarizing the above, the combined treatments of flavonoids and methyldopa lowered both TGF-β mRNA and its protein level in both types of cells, which is a positive sign for their possible use in preeclampsia.
The VEGF family, in particular, has been of great interest, due to its known association with hypertension and nephropathy, and its role as a biomarker of endothelial dysfunction, platelet activation and tissue hypoxia [
62]. These angiogenic factors are also potent mediators of the inflammatory response, and they augment inflammatory symptoms in patients with preeclampsia [
45]. VEGF as a proangiogenic factor needs consideration as a biomarker associated with endothelial cell damage in pregnancy with severe preeclampsia [
63]. However, in patients, the VEGF level in the PIH group was significantly lower than in the pregnancy group at advanced pregnancy, and the VEGF level significantly and gradually decreased with PIH aggravation; therefore, its role is not simple [
64]. In this study we found that all the tested compounds, i.e., methyldopa and all flavonoids, act in a similar way, significantly increasing mRNA VEGF expression in JEG-3 cells, but every combination of flavonoids significantly lowered the expression in relation to the methyldopa group, showing an inhibitory effect, which was most apparent for the combinations methyldopa + quercetin and methyldopa + chrysin. At the protein level, generally the flavonoids and methyldopa decreased the level of VEGF, and the administration of methyldopa with quercetin also showed a strong inhibitory effect in relation to the action of methyldopa alone. Moreover, similar results were shown in HUVEC cells, and the strongest inhibitory effect in relation to methyldopa was obtained for the combination of methyldopa and quercetin at both the mRNA and protein levels. Since methyldopa decreased the VEGF level in severe preeclampsia patients by 10% at the dose of 250 mg and by 57% at the dose of 500 mg [
63], it can be assumed that administering methyldopa together with flavonoids, especially quercetin, will allow lower doses of the drug to be used, which may be beneficial in reducing the risk of side effects from methyldopa.
PIGF is a VEGF related molecule that is expressed at high levels by trophoblast cells in the placenta [
62] and it is known to play an important role in the pathophysiology of preeclampsia [
65]. It is known that the rise in plasma PIGF levels observed in normal pregnancies is significantly attenuated in pregnancies complicated by preeclampsia [
66]. In our study, methyldopa and all flavonoids acted at a similar level by increasing mRNA PIGF expression, but only methyldopa and apigenin showed statistically significant increases in JEG-3 cells. Their combined treatment did not produce significant differences in relation to the values for methyldopa, but at the protein level the administration of methyldopa with all flavonoids increased the level of PIGF in relation to the effect of methyldopa. In HUVEC, methyldopa and all flavonoids increased mRNA expression similarly, and the strongest effect was noted for methyldopa and scutellarin. On the other hand, every combination of flavonoids significantly lowered the expression in relation to the methyldopa group, and the strongest inhibitory effect was found for the combination methyldopa + quercetin. At the protein level, similar effects were found. It could be stated that, while the effect of individual compounds is positive and increases the expression and level of this factor, the combined administration of methyldopa with flavonoids is rather unfavorable. Nevertheless, the effect of methyldopa on PIGF is not entirely clear, since there are even some data suggesting that methyldopa may have a specific effect on placental and/or endothelial cell function in preeclampsia patients, altering angiogenic proteins; however, the drug did not change the level of PIGF in women with preeclampsia [
67].
HIF-1α is a transcription factor that is regulated by hypoxia and mediates the effects of hypoxia on gene expression [
68]. It is expressed in the placenta in a gestational-age dependent fashion, with levels being higher in the first trimester and declining as oxygen levels increase later in pregnancy [
54]. Accumulation of HIF-1α is commonly an acute and beneficial response to hypoxia; when chronically elevated, this protein is associated with multiple disease conditions, including preeclampsia [
69]. There are some data indicating that women with preeclampsia are characterized by persistently elevated placental HIF-1α levels [
70,
71]. In this study we found that all the compounds, i.e., methyldopa and all flavonoids, increased mRNA HIF1-α expression, but only for methyldopa was the effect statistically significant in JEG-3 cells; however, the combined administrations of flavonoids with methyldopa lowered the expression in relation to the group from methyldopa, and the strongest inhibitory effects were observed for the combinations of methyldopa + baicalein and methyldopa + chrysin. Similar effects were observed at the protein level, and the strongest inhibitory effect was found for the combination methyldopa + quercetin. In HUVEC, methyldopa and all flavonoids increased HIF-1αmRNA and protein, but as for JEG-3 cells, their combined treatments with methyldopa produced inhibitory effects, leading to values similar to the control. Summarizing the above, it can be stated that even if methyldopa and individual flavonoids increased the activity of the formation of this factor, an inhibitory effect was generally observed after a combined administration of a flavonoid with this drug, which may have a positive effect.
In conclusion, it was found that every combination of a flavonoid and methyldopa in these cells induced a down-regulating effect onevery factor tested, except PIGF, especially at the mRNA level. Since it is known that hypertension generally raises TNF-α, IL-1β, IL-6, HIF-1α, TGF-β, and VEGF mRNA and/or protein levels, the results obtained in the study may provide a positive prognostic factor for such activity in vivo.
4. Materials and Methods
4.1. Materials
Methyldopa, apigenin, baicalein, chrysin, quercetin, and scutellarin were provided by Sigma-Aldrich (Poznan, Poland). Methyldopa was used as a reference substance.
4.2. Cell Lines and Culture Conditions
The primary human umbilical vein endothelial cell line (HUVEC, ATCC CRL1730) and trophoblast-derived human choriocarcinoma cell line (JEG-3, ATCCHTB-36) were obtained from American Type Culture Collection (ATCC). The cell lines were cultured in an incubator at 37 °C with a 5% CO2 atmosphere with 95% humidity. JEG-3 cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Sigma-Aldrich, Poznan, Poland). The medium was enriched with 10% fetal bovine serum (FBS) (Sigma-Aldrich Poznan, Poland) and 0.1% penicillin (100 U/mL)/streptomycin (100 µg/mL) (Sigma-Aldrich, Poznan, Poland). HUVEC were cultured using Vascular Cell Basal Medium and Endothelial Cell Growth KitBBE (ATCC PCS100040).
All flavonoids and methyldopa were separately applied to HUVEC and JEG-3 cells in chosen concentrations (20, 40, and 100 µmol). The concentrations of compounds were selected on the basis of our preliminary experience and similar work [
72]. The compounds were dissolved in dimethyl sulfoxide (DMSO); the content of DMSO was kept below 0.1%, as this concentration was found to be nontoxic to the cell lines.
After selecting the concentration least influencing cell viability, i.e., 40 µmol, in the second part of the experiment all compounds were administered either alone or in combination with methyldopa.
Each cell line (HUVEC, JEG-3) was treated with the tested compounds for 48 h at 37 °C, with a 5% CO
2 atmosphere. The cell growth was analyzed by counting viable cells in the presence of trypan blue (Sigma-Aldrich, Poznan, Poland) with a Bucker hemocytometer (Sigma-Aldrich Poznan, Poland). To determine the antiproliferative activity for substances studied, we performed the MTT assay adding 10 µL of a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (concentration 5 mg/mL) (Sigma-Aldrich, Poznan, Poland) and then incubating for 4 h at 37 °C. The viable cells were visualized by the development of purple color due to the formation of formazan crystals which were dissolved with 100 µL of isopropyl alcohol at 0.05 N HCl. Next, the absorbance was measured at 570 nm on a microplate reader (Infinite 200, TECAN, Männedorf, Switzerland) using the wavelength of 655 nm as a reference [
72].
4.3. Expression Analysis
The isolation of total cellular RNA was performed according to the manufacturer’s protocol of TriPure Isolation Reagent (Roche, Mannheim, Germany). The synthesis of complementary DNA was performed using the Transcriptor First-Strand Synthesis System (Roche, Mannheim, Germany) according to the manufacturer’s protocol. The obtained transcripts were used directly for the real-time PCR (RT-PCR) or stored at −20 °C.
The mRNA levels of studied genes (PIGF, VEGF, TNF-α, HIF-1α, TGF-β, IL-1β, and IL-6) were analyzed by real-time quantitative PCR using a LightCycler96 Instrument (Roche, Mannheim, Germany) and a LightCycler480 Probes Master kit (Roche, Mannheim, Germany). GAPDH was used as a housekeeping gene for normalization. The PCR program was initiated with an activation at 95 °C for 10 min. Each PCR cycle comprised a denaturation step at 95 °C, an annealing step at a specific temperature, and an extension step at 72 °C. The sequences of the primers were designed using the Oligo 4.0 program (National Biosciences, Colorado Springs, CO, USA,). All oligonucleotide sequences were synthesized by Sigma-Aldrich (Poznan, Poland) and are summarized in
Table 5. The increase in fluorescence of PCR products was monitored and measured and the data were analyzed with the LightCycler96 software.
4.4. ELISA
After 48 h, cell culture supernatants were collected and stored at −30 °C. TGF-β secreted into the medium was detected by the TGF-β-1 Human ELISA Kit (sensitivity: 8.6 pg/mL; Life Technologies, Inc., Carlsbad, CA, USA). The HIF1α Human ELISA Kit (sensitivity: 30 pg/mL; Life Technologies Inc., Carlsbad, CA, USA), Human Placental Growth Factor ELISA Kit (sensitivity: 2 pg/mL; Sigma Aldrich, Poznan, Poland), Human IL-1β ELISA Kit (sensitivity: 0.5 pg/mL; Sigma Aldrich, Poznan, Poland), IL-6 Human ELISA Kit (sensitivity < 1 pg/mL; Life Technologies Inc., Carlsbad, CA, USA), Human VEGF ELISA Kit (sensitivity: 10 pg/mL; Sigma Aldrich, Poznan, Poland), and TNF-α Human ELISA Kit (sensitivity: 0.13 pg/mL; Life Technologies, Inc., Carlsbad, CA, USA) were employed to evaluate the secretions in the cell culture supernatant of these factors according to the manufacturers’ protocols (instructions): 100 µL of each standard and the same of each sample were added to the appropriate well. Wells were covered and incubated at room temperature. The solution was removed and washed several times with PBS. Then, 100 µL of antibody was added to each well. Wells were covered and incubated at room temperature with gentle shaking. Next the wells were washed as described above, and 100 µL of prepared streptavidin solution was added to each well. Wells were covered and incubated at room temperature with gentle shaking. Next the wells were washed again, and substrate reagent was added to each well and then the wells were covered and incubated in the dark at room temperature. The reaction was blocked and the absorbance was measured on a microplate reader (Infinite 200, TECAN). The concentrations of PIGF, VEGF, TNF-α, HIF-1α, TGF-β, IL-1β, and IL-6 were determined by interpolation of the standard curve using linear regression analysis.