Melatonin modulates SHH/GLI3 signaling and placental angiogenesis to counter acrylamide embryotoxicity

Abstract

Acrylamide (ACR), a prevalent dietary toxicant formed in thermally processed foods via the Maillard reaction, is known to cross the placental barrier. While ACR-induced reproductive and developmental toxicity has been reported, the protective role of melatonin (MTN) via modulation of the SHH/GLI3 signaling pathway remains unclear. Pregnant Balb/c mice were divided into three groups (n = 6/group): control (distilled water), ACR (50 mg/kg/d), and ACR (50 mg/kg/d) + MTN (10 mg/kg/d), treated orally from gestational day (GD) 3.5 to GD 13.5. Placentas and embryos were collected for analysis. Oxidative stress (MDA levels), VEGF expression, and SHH/GLI3 pathway activity were assessed using immunohistochemistry and qRT-PCR. ACR exposure induced significant embryotoxicity, manifested as a 34% reduction in fetal weight (1.60 ± 0.09 g vs. 1.88 ± 0.14 g in controls, p < 0.001) and a 46.2% reduction in fetal crown-rump length (0.7 ± 0.08 cm vs. 1.1 ± 0.1 cm, p <  0.001). MTN co-treatment significantly ameliorated these growth restrictions. IHC analysis revealed that ACR significantly reduced SHH protein expression in the embryonic intestine and liver (p < 0.01), while it increased GLI3 protein levels (p < 0.01). MTN effectively normalized the expression of both proteins. At the molecular level, ACR downregulated SHH expression (p <  0.001) and upregulated GLI3 (p <  0.01), which were reversed by MTN. ACR exposure significantly increased oxidative stress (105% increase in placental MDA, p <  0.001) and reduced placental VEGF expression by 69.3% (p <  0.0001), both of which were significantly mitigated by MTN co-treatment. These integrated findings demonstrate that MTN exerts potent antioxidative and cytoprotective effects by mitigating ACR-induced oxidative stress, restoring SHH/GLI3 protein and gene expression, preserving VEGF-mediated placental angiogenesis, and preventing morphological defects. Our results underscore MTN’s therapeutic potential in counteracting ACR-induced teratogenicity and supporting healthy organogenesis.