A distinct mechanism of senescence activation in amnion epithelial cells by infection, inflammation, and oxidative stress

Abstract

Problem: We investigated p38MAPK activation-induced fetal membrane cell senescence in response to inflammation (tumour necrosis factor-alpha [TNF-α]) and infection (lipopolysaccharide [LPS]), factors associated with spontaneous preterm birth.

Method of study: Primary amnion epithelial cells (AECs) were exposed to TNF-α, 50 ng/mL and LPS, 100 ng/mL. Cigarette smoke extract (CSE), a known OS inducer, was used as positive control. AECs were cotreated with the antioxidant N-acetyl cysteine (NAC) and p38MAPK inhibitor SB203580 to determine the effect of OS and p38MAPK. Western blot analysis was performed for active (Phospho-p38MAPK) and total p38MAPK. Senescence was determined by flow cytometry, and culture supernatants were tested for IL-6 using ELISA.

Results: TNF-α, but not LPS, increased p38MAPK activation compared to untreated cells (P = .01). The number of senescent cells and senescence-associated IL-6 was higher in both TNF-α and LPS-treated cells compared to control (P = .001, P = .01, respectively). Antioxidant NAC inhibited p38MAPK activation by TNF-α. p38MAPK inhibitor SB203580 reduced the development of senescence and IL-6 by TNF-α and LPS. CSE treatment validated our current data.

Conclusion: TNF-α caused OS-mediated p38MAPK induction, senescence, and IL-6 increase from AECs. LPS also induced senescence and IL-6 increase. Inflammatory and infectious factors may cause premature fetal cell senescence contributing to preterm birth pathophysiology.

Keywords: fetal membranes; interleukin-6; lipopolysaccharide; p38MAPK; tumour necrosis factor-alpha.

Figure 1. p38MAPK activation

(A) Amnion cells exposed to TNF-α demonstrated activation of p38MAPK. NAC inhibited this activation, confirming an OS-mediated effect. (B) LPS treatment did not activate p38MAPK to statistical significance. NAC decreased this effect; however, it did not reach statistical significance. (C) Amnion cells exposed to CSE had the highest activation of p38MAPK. NAC inhibited this activation, confirming an OS-mediated effect.

Figure 2. Senescence activation

(A) C12FDG fluorescence was increased in TNF-α treated amnion cells. SB203580 co-treatment inhibited this change, confirming this p38-mediated effect. (B) LPS treatment also increased C12FDG fluorescence to a lesser degree, which was also inhibited by SB203580 co-treatment. (C) Amnion cells exposed to CSE had the largest increase in C12FDG fluorescence. SB203580 inhibited change, confirming a p38-mediated effect. (D) Senescence-associated β-Galactosidase staining was shown in TNF-α, LPS and CSE treated amnion cells, confirming the C12FDG fluorescence results.

Figure 3. Senescence-associated inflammatory cytokine IL-6

TNF-α treated amnion cells increased the inflammatory cytokine IL-6 when compared to untreated cells. This effect was down-regulated when co-treated with SB203580, confirming a p-38 mediated effect. LPS treatment also increased IL-6, to a lesser degree. This effect was reduced with SB203580 co-treatment. Amnion cells exposed to CSE increased the inflammatory cytokine IL-6; however, not significantly. Co-treatment with SB203580 decreased IL-6 production.