Manganese porphyrin-based treatment improves fetal-placental development and protects against oxidative damage and NLRP3 inflammasome activation in a rat maternal hypothyroidism model

Abstract

Oxidative stress (OS) and endoplasmic reticulum stress (ERS) are at the genesis of placental disorders observed in preeclampsia, intrauterine growth restriction, and maternal hypothyroidism. In this regard, cationic manganese porphyrins (MnPs) comprise potent redox-active therapeutics of high antioxidant and anti-inflammatory potential, which have not been evaluated in metabolic gestational diseases yet. This study evaluated the therapeutic potential of two MnPs, [MnTE-2-PyP]⁵⁺ (MnP I) and [MnT(5-Br-3-E-Py)P]5+ (MnP II), in the fetal-placental dysfunction of hypothyroid rats. Hypothyroidism was induced by administration of 6-Propyl-2-thiouracil (PTU) and treatment with MnPs I and II 0.1 mg/kg/day started on the 8th day of gestation (DG). The fetal and placental development, and protein and/or mRNA expression of antioxidant mediators (SOD1, CAT, GPx1), hypoxia (HIF1α), oxidative damage (8-OHdG, MDA), ERS (GRP78 and CHOP), immunological (TNFα, IL-6, IL-10, IL-1β, IL-18, NLRP3, Caspase1, Gasdermin D) and angiogenic (VEGF) were evaluated in the placenta and decidua on the 18th DG using immunohistochemistry and qPCR. ROS and peroxynitrite (PRX) were quantified by fluorometric assay, while enzyme activities of SOD, GST, and catalase were evaluated by colorimetric assay. MnPs I and II increased fetal body mass in hypothyroid rats, and MnP I increased fetal organ mass. MnPs restored the junctional zone morphology in hypothyroid rats and increased placental vascularization. MnPs blocked the increase of OS and ERS mediators caused by hypothyroidism, showing similar levels of expression of HIFα, 8-OHdG, MDA, Gpx1, GRP78, and Chop to the control. Moreover, MnPs I and/or II increased the protein expression of SOD1, Cat, and GPx1 and restored the expression of IL10, Nlrp3, and Caspase1 in the decidua and/or placenta. However, MnPs did not restore the low placental enzyme activity of SOD, CAT, and GST caused by hypothyroidism, while increased the decidual and placental protein expression of TNFα. The results show that treatment with MnPs improves the fetal-placental development and the placental inflammatory state of hypothyroid rats and protects against oxidative stress and reticular stress caused by hypothyroidism at the maternal-fetal interface.

Keywords: Antioxidant; Inflammasome; Metalloporphyrin; Placenta; Rat; Thyroid.

Fig. 1

Plasma levels of free T3 and T4 and reproductive parameters of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A) Free T4. B) Free T3. C) Uterus and placental mass; D) Amniotic fluid mass; E) Number of fetuses; F) Fetal body mass; G) Relative frequency of fetal body mass distribution. H) Fetal liver, kidney, heart, and lung mass (mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; *P < 0.0001; SNK test (A–E); Generalized linear analysis of mixed models followed by Tukey test (F); N = 5–7/group (A–E); N = 22–31/group (F); N = 19–31/group (H)).

Fig. 2

Histomorphometry analysis of the placenta of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A) Photomicrography of the maternal-fetal interface showing the decidua and placenta on 18th DG (hematoxylin and eosin staining; Bar = 500 μm) B) Thickness of the JZ (giant cells + spongiotrophoblast) and PL; C) Percentage of area occupied by glycogen cells, spongiotrophoblasts, and giant cells in the JZ (mean ± SEM; *P < 0.05; **P < 0.01; SNK test; N = 0.01; 6–8/group); D) Percentage of area occupied by maternal vascular sinus, fetal capillary, and fetal mesenchyme/trophoblast of the PL (mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; * * * *P < 0.0001; SNK test; n = 6–8/group). JZ = Junctional zone; LP = Placental labyrinth.

Fig. 3

Expression of HIF1α and Nrf2 at the maternal-fetal interface of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A) Photomicrographs of HIF1α immunolabeling in the decidua, junctional zone, placental labyrinth, and negative control (streptavidin-biotin-peroxidase, contrasted with Harris hematoxylin, Bar = 50 μm). B) Immunostaining area in pixels of HIF1α in the decidua, junctional zone, and placental labyrinth; C) Relative gene expression of Hif1α and Nrf2 in the placenta (mean ± SEM, *P < 0.05; **P < 0.01; SNK test; N = 6–8/group).

Fig. 4

Expression of 8-OHdG and MDA in the decidua and placenta of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A and C) Photomicrographs of the immunostaining of 8-OHdG (A) and MDA (C) in the decidua, junctional zone, and placental labyrinth (streptavidin-biotin-peroxidase, contrasted with Harris hematoxylin, Bar = 50 μm). B and D) Immunostaining area in pixels of 8-OHdG (B) and MDA (D) in the decidua, junctional zone, and placental labyrinth. E) Negative Control (mean ± SEM, **P < 0.01; ***P < 0.001; SNK test; N = 6–7/group).

Fig. 5

Expression and activity of antioxidant enzymes and plasma levels of ROS and peroxynitrite at the maternal-fetal interface of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A, C, E) Photomicrographs of the immunostaining of SOD1 (A), GPx1 (C), and catalase (E) in the decidua, junctional zone, and placental labyrinth (streptavidin-biotin-peroxidase, contrasted with Harris hematoxylin, Bar = 50 μm). B, D, F) Immunostaining area in pixels of SOD1 (B), GPx1 (D), and catalase (F) in the decidua, junctional zone, and placental labyrinth. G) Negative Control. H) Relative gene expression of Sod1, Gpx1, and Cat in the placenta. I) Enzyme activity of SOD, catalase, and GST in the placenta. J) Placental levels of ROS and peroxynitrite (PRX) (mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; SNK test; N = 6–8/group).

Fig. 6

Expression of GRP78 and CHOP at the maternal-fetal interface of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A-C) Photomicrographs of the immunostaining of GRP78 (A) and CHOP (C) in the decidua, junctional zone, and placental labyrinth (streptavidin-biotin-peroxidase, contrasted with Harris hematoxylin, Bar = 50 μm). B-D) Immunostaining area in pixels of GRP78 (B) and CHOP (D) in the decidua, junctional zone, and placental labyrinth; E) Negative Control. F) Relative gene expression of Grp78 and Chop in the placenta (mean ± SEM, *P < 0.05; **P < 0.01; SNK test; N = 6–8/group).

Fig. 7

Expression of VEGF at the maternal-fetal interface of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A) Photomicrographs of the immunostaining of VEGF in the decidua, junctional zone, placental labyrinth, and negative control (streptavidin-biotin-peroxidase, contrasted with Harris hematoxylin, Bar = 50 μm). B) Immunostaining area in pixels of VEGF in the decidua, junctional zone, and placental labyrinth. C) E) Relative gene expression of Vegf in the placenta (mean ± SEM, *P < 0.05; **P < 0.01; N = 6–8/group).

Fig. 8

Expression of TNFα, IL10, IL6, and mediators of the inflammasome-NLRP3 via and pyroptosis at the maternal-fetal interface of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A, C) Photomicrographs of the immunostaining of TNFα (A), and IL10 (C) in the decidua, junctional zone, and placental labyrinth (streptavidin-biotin-peroxidase, contrasted with Harris hematoxylin, Bar = 50 μm). B, D) Immunostaining area in pixels of TNFα (B), and IL10 (D) in the decidua, junctional zone, and placental labyrinth. E) Negative Control. F) Relative gene expression of Tnf, Il6, and Il10 in the placenta. G) Relative gene expression of Nlrp3, Caspase1, Il1β, Il18, and Gasdermin D in the placenta (mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; SNK test; N = 6–8/group).

Fig. 9

PCA analysis on placental and fetal development, antioxidant activity and gene and protein expression at the maternal-fetal interface of control, hypothyroid, and hypothyroid rats treated with [MnTE-2-PyP]⁵⁺(MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺(MnP II). A) Placental development; B) Fetal development; C) Antioxidant enzyme activity and protein expression in the junctional zone (JZ); D) Protein expression in the placental labyrinth (PL); E) Protein expression in the decidua; F) Gene expression in the placenta.

Supplementary Fig. 1

Synthesis route of [MnT(5-Br-3-E-Py)P]⁵⁺.

Supplementary Fig. 2

UV–Vis absorption spectra of H₂T(5-Br-3-Py)P in CH₂Cl₂, H₂T(5-Br-3-E-Py)P⁴⁺ and [MnT(5-Br-3-E-Py)P]⁵⁺ in water.

Supplementary Fig. 3

¹H NMR spectrum recorded at 400 MHz, in CDCl₃, at 25^oC: H₂T(5-Br-3-Py)P NMR ¹H (400 MHz, CDCl₃) δ: 9.38 (s, 4H), 9.18 (s, 4H), 8.91 (s, 8H), 8.70 (s, 4H), −2.90 (br, 2H). The signals marked with (*) denoted solvent impurities.

Supplementary Fig. 4

ESI-MS spectra (on the positive mode) of [MnT(5-Br-3-E-Py)P]⁵⁺ in water.

Supplementary Fig. 5

Reproductive parameters of Wistar rats treated with with [MnTE-2-PyP]⁵⁺ (MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺ (MnP II) on 18th day of pregnancy. A) Uterus and placental mass (g); B) Amniotic fluid mass C) Number of fetuses and D) Fetal body mass (g) (mean ± SEM; SNK test; N = 5–7/group (A-C); N = 47-36/group (D)).

Supplementary Fig. 6

Photomicrographs of the histology of the liver, spleen, lung, heart, brain, and kidneys of Wistar rats treated with [MnTE-2-PyP]⁵⁺ (MnP I) and [MnT(5-Br-3-E-Py)P]⁵⁺ (MnP II) on 18th day of pregnancy. A-C) Liver; D-F) Spleen; G-I) Lung; J-L) Heart; M − O) Brain; P–R) Kidney.