Bryophyllum pinnatum Compounds Inhibit Oxytocin-Induced Signaling Pathways in Human Myometrial Cells

Stefanie Santos, Leonie Zurfluh, Mónica Mennet, Olivier Potterat, Ursula von Mandach, Matthias Hamburger, Ana Paula Simões-Wüst, Stefanie Santos, Leonie Zurfluh, Mónica Mennet, Olivier Potterat, Ursula von Mandach, Matthias Hamburger, Ana Paula Simões-Wüst

Abstract

Bryophyllum pinnatum has been used in the treatment of premature labor, first in anthroposophic hospitals and, recently, in conventional settings as an add-on medication. In vitro work with hTERT human myometrial cells showed that B. pinnatum leaf press juice inhibits the increase of intracellular free calcium concentration induced by oxytocin, a hormone known to play a role in labor. Our aim was to identify fractions/compounds in B. pinnatum press juice that contribute to this inhibitory effect, and to investigate their effect on oxytocin-driven activation of the MAPK cascade. Several fractions/compounds from B. pinnatum press juice led to a concentration-dependent decrease of oxytocin-induced increase of intracellular free calcium concentration, but none of them was as strong as B. pinnatum press juice. However, the combination of a bufadienolide and a flavonoid-enriched fraction was as effective as B. pinnatum press juice, and their combination had a synergistic effect. B. pinnatum press juice inhibited oxytocin-driven activation of MAPKs SAPK/JNK and ERK1/2, an effect also exerted by the bufadienolide-enriched fraction. The effect of B. pinnatum press juice on oxytocin-induced signaling pathways was comparable to that of the oxytocin-receptor antagonist and tocolytic agent atosiban. Our findings further substantiate the use of B. pinnatum press juice preparations in the treatment of preterm labor.

Keywords: Bryophyllum pinnatum; Kalanchoe pinnata; MAPK; cell signaling; intracellular calcium; myometrium cells; oxytocin.

Conflict of interest statement

MM is an employee of Weleda AG, the company that produces the preparations of Bryophyllum pinnatum. APS-W received research funding from Weleda AG during the last 5 years. SS, LZ, MH, UM, and OP declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Santos, Zurfluh, Mennet, Potterat, von Mandach, Hamburger and Simões-Wüst.

Figures

FIGURE 1
FIGURE 1
Concentration-dependent effects of BPJ fractions/compounds on the OT-induced increase of [Ca2+]i. Myometrial hTERT-C3 cells were pre-treated with BPJ, FEF, A-Mix, BEF or BO prior to stimulation with 100 nM of OT (A). Values represent the mean ± SEM of six independent experiments performed in quadruplicate and are expressed as percentage of control; *p < 0.05. In (B), the concentrations of FEF, BEF, A-Mix and BO corresponding to the BPJ concentrations are shown.
FIGURE 2
FIGURE 2
Effect of the combination of BPJ fractions/compounds on the OT-induced rise of [Ca2+]i. Cells were pre-treated with BEF plus FEF (A), or with BEF plus A-Mix (C) prior to stimulation with 100 nM OT. Results of pre-treatment with BPJ and each substance alone are shown in transparent lines (significance symbols regarding comparison to control omitted). Values represent the mean ± SEM of six independent experiments performed in quadruplicate and are expressed as percentage of control; *p < 0.05 compared with control; #p < 0.05 compared to combination. Combination index (CI) values of the combination of BEF with FEF (B) or with A-Mix (C) were calculated from the concentration-response curves. Data were analyzed by the median-effect method. Fa: fraction affected.
FIGURE 3
FIGURE 3
Comparison between the effects of BPJ and atosiban on OT-induced rise of [Ca2+]i in myometrial cell lines. Time-course of OT-induced [Ca2+]i response in hTERT-C3 (A, B) and PHM1-41 (C, D) cells when pre-incubated in the absence (a-d, grey lines) or in the presence of BPJ (A, C) or atosiban (B, D) [Ca2+]i was measured for 4 min before stimulation with 100 nM OT. Data shown are from one representative experiment. Concentration-dependent effect of BPJ (E) or atosiban (F) on the oxytocin-induced [Ca2+]i increase in hTERT-C3 (lighter color) and PHM1-41 (darker color) cells. Values represent themean ± SEM of 4 (PHM1-41) or 6 (hTERT-C3) independent experiments performed in quadruplicate and are expressed as percentage of control; *p < 0.05.
FIGURE 4
FIGURE 4
Effect of BPJ fractions/compounds and atosiban on OT-induced MAPKs phosphorylation. In the time-course experiments, hTERT-C3 cells were pre-treated with or without 20 mg/mL BPJ or 100 nM atosiban for 30 min, before incubation with 100 nM OT for 2, 5, 15, 30, and 45 min (A, C, E). To compare the effects of BPJ and the various fractions/compounds, cells were pre-treated with 20 mg/mL BPJ, 2.20 μg/mL BEF, 17.39 μg/mL FEF, 0.68 μg/mL A-Mix, BEF plus FEF, or BEF plus A-Mix (same concentrations as for single fractions) for 30 min before stimulation with OT for 5 min (B, D, F). Whole cell proteins were subjected to western blot analysis with antibodies against phosphorylated p38 (A, B), SAPK/JNK (C, D) and ERK1/2 (E, F); matching densitometry analyses are depicted bellow the representative blots. Samples from the same experiment were processed in parallel and membranes were probed with GAPDH to confirm equal loading. Blot images are from a representative experiment, and line between bands delineate boundary between the gels that were cropped. Values represent the mean ± SEM of four independent experiments; *p < 0.05 compared with control; #p < 0.05 compared to OT-treated.

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