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Sodium salicylate reduced transcript abundance of hypoxia-associated genes in MAC-T cells.

L. K. Mamedova



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Sodium salicylate reduced transcript abundance of hypoxia-associated genes in MAC-T cells.
C. M. Ylioja, T. H. Swartz, L. K. Mamedova*, B. J. Bradford. Kansas State University Manhattan, KS.

Hypoxia is an oxygen deficiency commonly found in growing tissues and is speculated to occur in the rapidly developing mammary gland in peripartum dairy cattle. Low oxygen concentrations can activate hypoxia-inducible factor-1 (HIF-1), which increases transcription of genes involved in angiogenesis (VEGF) and glucose transport (GLUT1). The mRNA stability of these genes is positively regulated by AUF1. In our previous research, postpartum administration of sodium salicylate (SS) increased whole lactation milk yield in multiparous cows but tended to reduce milk yield in primiparous. Because rapid mammary tissue development likely occurs in cows approaching first lactation, we hypothesized that SS inhibited the activation of HIF-1α and decreased transcription of downstream targets. MAC-T cells were treated with SS (100 μM) or control media before incubation under either hypoxic (1% O2) or normoxic conditions for 12 h. Additionally, cells were transfected with either HIF1α siRNA or a scrambled siRNA negative control 48 h before hypoxia treatments. HIF1α, GLUT1, VEGF, and AUF1 were quantified using the 2-ΔCt method and normalized to the internal control gene NENF. Transcript abundance was assessed using a linear mixed model with the fixed effects of SS, hypoxia, and siRNA and all 2- and 3-way interaction terms, and the random effect of plate nested within hypoxia. SS tended to decrease HIF1α as compared with untreated cells (P = 0.09). For GLUT1, SS treatment interacted with hypoxia (P = 0.05), as SS reduced GLUT1 when MAC-T cells were cultured in normoxic conditions (P < 0.01), however, no effect of SS was found in hypoxia-treated cells (P = 0.39). Regardless of oxygen status, SS reduced VEGF (P = 0.04) and AUF1 (P = 0.04) relative to untreated cells. Hypoxia increased GLUT1 (P = 0.01), yet no effect was identified on VEGF (P = 0.45) or AUF1 (P = 0.22). siRNA knocked down HIF1α (P < 0.01), but no effect was found on GLUT1 (P = 0.98), VEGF (P = 0.99), or AUF1 (P = 0.62). In conclusion, SS reduced transcript abundance of genes involved with mammary gland development, but generally did not interact with oxygen status.

Keywords: hypoxia, NSAID, mammary gland development.