Chinese herbal medicine for miscarriage affects decidual micro-environment and fetal growth
- Under a Creative Commons license
Open Access
Highlights
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- Spontaneous abortion decidua displays a pro-inflammatory micro-environment.
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- JLFC01 inhibits inflammation in first trimester decidual cells.
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- JLFC01 suppresses inflammation in SA/IUGR-prone mouse placentae.
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- JLFC01 may improve gestation by enhancing angiogenesis at fetal–maternal interface.
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- JLFC01 also promotes fetal growth and potentially modulates fetal programming.
Abstract
Introduction
Intrauterine
growth restriction complicates 5–10% of pregnancies. This study aims to
test the hypothesis that Chinese herbal formula, JLFC01, affects
pregnancy and fetal development by modulating the pro-inflammatory
decidual micro-environment.
Methods
Human
decidua from gestational age-matched elective terminations or
incomplete/missed abortion was immunostained using anti-CD68 + anti-CD86
or anti-CD163 antibodies. qRT-PCR and Luminex assay measured the
effects of JLFC01 on IL-1β- or TNF-α-induced cytokine expression in
first trimester decidual cells and on an established spontaneous
abortion/intrauterine growth restriction (SA/IUGR)-prone mouse
placentae. The effect of JLFC01 on human endometrial endothelial cell
angiogenesis was evaluated by average area, length and numbers of
branching points of tube formation. Food intake, litter size, fetal
weight, placental weight and resorption rate were recorded in
SA/IUGR-prone mouse treated with JLFC01. qRT-PCR, Western blot and
immunohistochemistry assessed the expression of mouse placental IGF-I
and IGF-IR.
Results
In
spontaneous abortion, numbers of decidual macrophages expressing CD86
and CD163 are increased and decreased, respectively. JLFC01 reduces
IL-1β- or TNF-α-induced GM-CSF, M-CSF, C–C motif ligand 2 (CCL2),
interferon-γ-inducible protein-10 (IP-10), CCL5 and IL-8 production in
first trimester decidual cells. JLFC01 suppresses the activity of IL-1β-
or TNF-α-treated first trimester decidual cells in enhancing
macrophage-inhibited angiogenesis. In SA/IUGR-prone mice, JLFC01
increases maternal food intake, litter size, fetal and placental weight,
and reduces fetal resorption rate. JLFC01 induces IGF-I and IGF-IR
expression and inhibits M-CSF, CCL2, CCL5, CCL11, CCL3 and G-CSF
expression in the placentae.
Discussion
JLFC01 improves gestation by inhibiting decidual inflammation, enhancing angiogenesis and promoting fetal growth.
Keywords
- Chinese herbal medicine;
- Decidual cells;
- IUGR;
- Macrophage;
- Spontaneous abortion
Abbreviations
- CCL, C–C motif ligand;
- CHM, Chinese herbal medicine;
- CM, conditioned media;
- EVTs, extravillous trophoblasts;
- FTDCs, first trimester decidual cells;
- GA, gestational age;
- GD, gestational day;
- HEEC, human endometrial endothelial cell;
- IP-10, interferon-γ-inducible protein-10;
- IRB, institutional review board;
- IUGR, intrauterine growth restriction;
- NK, natural killer;
- OSU, The Ohio State University;
- SA, spontaneous abortion
1. Introduction
Spontaneous
abortion (SA) complicates 15% of human pregnancies. Although
chromosomal anomalies account for more than 50% of SA, abnormal fetal
development, maternal systemic disorders and environmental insults
contribute to its incidence [1].
The mechanisms causing SA remain unknown thereby precluding the
development of effective prevention and treatment. Various attempts
including immunological approaches [2], [3], [4], [5] and [6]
have not significantly improved the SA-associated live birth rate.
Chinese herbal medicine (CHM) has been widely used in Asia for
centuries. Although studies suggest that CMH prevents SA [7],
evidence is insufficient to assess the effectiveness of CHM in treating
SA. First appearing in Chung-Ching Chang's “Synopsis of Golden Chamber”
about 1800 years ago, JLFC01 is derived from a traditional Chinese
formula that successfully treats blood stagnation.
Intrauterine growth restriction (IUGR) complicates 5–10% of pregnancies [8]. Many studies indicate that preeclampsia and IUGR [9]
are initiated in the first trimester as a consequence of insufficient
uteroplacental blood flow to the developing fetal-placental unit and
suggest a similar origin for SA. Blastocyst-derived semi-allogeneic
extravillous trophoblasts (EVTs) traverse the decidua and inner third of
the myometrium and interact with resident decidual cells, decidual
natural killer (NK) cells and macrophages. Invading EVTs transform
uterine spiral arteries into high-capacitance vessels accompanied by
expression of angiogenic factors and microvascular angiogenesis [10]. The resulting increased uterine blood flow to the intervillous space is pivotal for fetal-placental development [9] and [11]. Disturbances of this environment disrupt early fetal development and elicit long-term complications in affected children.
An
aberrant pro-inflammatory decidual micro-environment elicits
preeclampsia, SA and IUGR. In addition to mediating spiral artery
transformation, decidual macrophages are critical modulators of the
immune balance at the fetal–maternal interface. The function,
differentiated state and responsiveness of macrophages are governed by
their micro-environment and proximity to adjacent cells [12] and [13]. Generally, macrophage polarization is divided into classically (M1) and alternatively (M2) activated groups [14].
Besides bridging both innate and adaptive immunity, which defend
against pathogens, decidual macrophages are important mediators of
implantation, placental development and cervical ripening [15]. In early pregnancy, M2 macrophages play a key role in inducing immunotolerance of the fetal semi-allograft [15].
Tight regulation of macrophage trafficking/function plays crucial roles
during placentation. Under physiological steady state, macrophages
isolated from first trimester decidua are polarized toward an
immunotolerant M2 phenotype [16]. By contrast, pro-inflammatory M1 polarization of macrophages is linked to adverse pregnancy outcomes [17].
Our studies indicate that potent pro-inflammatory cytokines, IL-1β and
TNF-α, stimulate human first trimester decidual cells (FTDCs) to secrete
several chemokines that recruit NK cells [18] and monocytes [19], [20] and [21].
The decidual cells then promote macrophage differentiation toward an M1
subtype via the regulation of colony-stimulating factors [13].
The
current study postulates that these decidual M1 macrophages are
integral to the onset of SA and proposes to treat and/or prevent SA by
counteracting the resulting pro-inflammatory decidual milieu. Initial
observations determined that unlike the dominant M2 immunotolerant
macrophage population of normal first trimester human decidua, the
decidual macrophage population accompanying SAs displays a
pro-inflammatory M1 phenotype. Complementing these in situ
observations, the modulating effects of JLFC01 on: 1) IL-1β- or
TNF-α-induced expression of several cytokines by FTDCs; 2) FTDCs in
modulating human endometrial endothelial cell (HEEC)
angiogenesis-inhibiting activity of macrophages were examined. These in vitro
observations were extended to include CBA/J x DBA/2J mice, an
established SA/IUGR-prone model, in which the effects of JLFC01
ingestion were assessed. An aberrant decidual pro-inflammatory
micro-environment [22]
can also interfere with normal fetal growth by disrupting fetal
programming. The insulin-like growth factors (IGFs) and their receptors
are potent regulators of protein turnover, mitogenesis and
differentiation [23] and implicated in fetal-placental development [24].
Abnormal IGF expression, malfunctioning IGF receptors or defective
downstream signaling pathways are proposed to contribute to the
development of IUGR [25].
Thus, the effects of JLFC01 ingestion were compared on placental
expression of mRNA and protein levels of IGFs and their receptors in
SA/IUGR-prone mice.
2. Methods
2.1. Immunofluorescent staining of decidua for macrophage markers
Decidua
was obtained under Institutional Review Board (IRB) approval at Mackay
Memorial Hospital, Taipei, Taiwan. Gestational age (GA)-matched tissue
was obtained from elective terminations of normal pregnancies between 6
and 12 weeks of gestation without uterine contraction, vaginal bleeding
or evidence of fetal demise. Upon diagnosis of missed/incomplete
abortion, decidual basalis was evacuated within 24 h from patients
without infection or systemic diseases. Serial sections of OCT-embedded
specimens were immunostained with mouse anti-human CD68 (1:25, Dako,
Carpinteria, CA) followed by Rhodamine–conjugated donkey anti-mouse
antibody (1:50, EMD Millipore, Billerica, MA). Sections were then
incubated with rabbit anti-human CD163 (1:250, Sigma–Aldrich, St.
Louise, MO) or CD86 (1:200, GeneTex, Irvine, CA) followed by
corresponding FITC–conjugated secondary antibody (1:100) and
4′,6′-diamidino-2-phenylindole (1:500,000, Sigma–Aldrich). Morphometric
analysis of cell numbers used Axiovision 3.1 software (Carl Zeiss,
Oberkochen, Germany). Five randomly selected fields from each section
(three sections/tissue) were examined. Cell numbers per field (3 × 106 pixel2) were counted and calculated as the mean of 15 fields for each tissue. A total of 15 cases per group were examined.
2.2. Cell isolation and culture
FTDCs were isolated and cultured as previously described [20].
Briefly, decidua from elective termination of 6–12 weeks gestation was
obtained under IRB approval at The Ohio State University (OSU) and Beth
Israel Medical Center, New York, NY. Cells were purified using
Ficoll-Hipaque Plus (GE Healthcare, Piscataway, NJ). CD45 staining
confirmed the absence of leukocytes. Cultured FTDCs were found to be
vimentin-positive and cytokeratin-negative and displayed morphological
changes and enhanced prolactin and plasminogen activator inhibitor-1 as
well as inhibited interstitial collagenase and stromelysin-1 expression
with prolonged tissue factor expression during incubation with a
progestin. Confluent FTDCs were primed with estradiol (10−8 M) + medroxyprogesterone acetate (10−7 M)
for 7d and pre-treated with or without 1.25 μg/ml of a Chinese herbal
formula, JLFC01, for 24 h then incubated with 1 ng/ml of IL-1β or TNF-α
(R&D Systems, Minneapolis, MN) with or without JLFC01. JLFC01 was
manufactured from herbs and processed in stainless steel extractors at a
low temperature (below 100 °C) in order to preserve the activity of
essential ingredients and generate a water decoction. This procedure
follows the sequence described in canonical Chinese medicine book since
the sequence of the herbs processed within a decoction determines the
efficacy of the formula. The extracted liquid is then spray dried to
form a powder. This procedure follows good manufacturing practice (GMP)
guidelines. In addition, both the presence and levels of heavy metal and
microbes are also assessed. Conditioned medium (CM) supernatants were
collected. Monocytes were isolated from peripheral blood of healthy
reproductive age female donors using Ficoll-Hipaque and purified using
anti-CD14-paramagnetic beads according to the manufacturer's
instructions (Miltenyi Biotec, Auburn, CA).
For HEECs [26],
blood vessels in the endometrium obtained from hysterectomy for myomas
was dissected, then, minced and digested with type VII
collagenase/dispase/DNase I followed by filtration through a 70-μm cell
strainer. The cells was labeled with biotinylated UEA-1 (Ulex europaeus)
lectin, and then, separated from non-labeled cells by panning on
activated surface/AIS MicroCELLector flasks coated with streptavidin.
Cells were cultured in EBM-2 medium supplemented with 15% fetal calf
serum.
