Role of the GalNAc-galectin pathway in the healing of premature rupture of membranes

Abstract

Background: Premature rupture of the membranes (PROM) is a key cause of preterm birth and represents a major cause of neonatal mortality and morbidity. Natural products N-acetyl-d-galactosamine (GalNAc), which are basic building blocks of important polysaccharides in biological cells or tissues, such as chitin, glycoproteins, and glycolipids, may improve possible effects of wound healing.

Methods: An in vitro inflammation and oxidative stress model was constructed using tumor necrosis-α (TNF-α) and lipopolysaccharide (LPS) action on WISH cells. Human amniotic epithelial cells (hAECs) were primarily cultured by digestion to construct a wound model. The effects of GalNAc on anti-inflammatory and anti-oxidative stress, migration and proliferation, epithelial-mesenchymal transition (EMT), glycosaminoglycan (GAG)/hyaluronic acid (HA) production, and protein kinase B (Akt) pathway in hAECs and WISH cells were analyzed using the DCFH-DA fluorescent probe, ELISA, CCK-8, scratch, transwell migration, and western blot to determine the mechanism by which GalNAc promotes amniotic wound healing.

Results: GalNAc decreased IL-6 expression in TNF-α-stimulated WISH cells and ROS expression in LPS-stimulated WISH cells (P < 0.05). GalNAc promoted the expression of Gal-1 and Gal-3 with anti-inflammatory and anti-oxidative stress effects. GalNAc promoted the migration of hAECs (50% vs. 80%) and WISH cells through the Akt signaling pathway, EMT reached the point of promoting fetal membrane healing, and GalNAc did not affect the activity of hAECs and WISH cells (P > 0.05). GalNAc upregulated the expression of sGAG in WISH cells (P < 0.05) but did not affect HA levels (P > 0.05).

Conclusions: GalNAc might be a potential target for the prevention and treatment of PROM through the galectin pathway, including (i) inflammation; (ii) epithelial-mesenchymal transition; (iii) proliferation and migration; and (iv) regression, remodeling, and healing.

Keywords: Galectin; Healing; N-acetyl-d-galactosamine; PROM.

Fig. 1

Inhibition of TNF-α-induced inflammatory response and LPS-induced oxidative stress by GalNAc. (A-B) GalNAc pretreatment reduces the expression of inflammatory factors in TNF-α-stimulated WISH cells. (C-D) GalNAc pretreatment reduces the expression of ROS in LPS-stimulated WISH cells. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Fig. 2

GalNAc inhibits TNF-α-stimulated inflammatory factor expression in WISH cells via galectins. (A) Protein level expression of Gal-1 in cell supernatant. (B) Protein level expression of Gal-3 in cell supernatant. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Fig. 3

GalNAc-stimulated WISH cells EMT-MET. (A) Vimentin and CK-19 protein concentrations in WISH cells (from protein blotting analysis). (B) Vimentin and CK-19 expression analysis in WISH cells based on protein blotting results. (C) Immunofluorescence detection of Vimentin expression in WISH cells under 100X microscopy. (D) Analysis of Vimentin expression in WISH cells based on immunofluorescence results. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Fig. 4

Effect of GalNAc on the activity of hAECs and WISH cells. (A) CCK-8 for detection of WISH cell activity. (B) PCNA protein concentration in WISH cells (from protein blot analysis). (C) PCNA expression analysis in WISH based on protein blotting results. (D) CCK-8 for detection of hAEC activity. (E) PCNA protein concentration in hAECs (from protein blot analysis). (F) PCNA expression analysis in hAECs based on protein blotting results. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Fig. 5

GalNAc promotes migration of hAEC and WISH cells. (A-B) Effect of GalNAc on WISH cell migration by transwell migration assay under 40X microscopy. (C-D) Detection of the effect of GalNAc on the migration of hAECs by scratch assay under 40X microscopy. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Fig. 6

GalNAc promotes cell migration by targeting the Akt pathway. (A) Akt total and phosphorylated protein concentrations in WISH cells (from protein blotting analysis). (B) Akt and pAkt expression analysis in WISH cells based on protein blotting results. (C-D) Intervention using Akt inhibitors and activators to observe the migratory capacity of cells by transwell migration assay under 40X microscopy. *<0.05, **<0.01, ***<0.001, ****<0.0001

Fig. 7

GalNAc induces sulfated glycosaminoglycan production. (A) GAG expression level in cell supernatant. (B) HA expression level in cell supernatant. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001