Abstract
Damage to lamellar keratinocytes, an essential cellular component of the epidermal layer of hoof tissue, can have a detrimental effect on hoof health and the overall production value of dairy cows. We isolated and cultured cow lamellar keratinocytes using the Dispase II and collagenase methods. We purified them by differential digestion and differential velocity adherent methods at each passaging and identified them by keratin 14 immunofluorescence. We established an in vitro model of inflammation in laminar keratinocytes using LPS and investigated whether chicoric acid protects against inflammatory responses by inhibiting the activation of the TLR4/MAPK/NF-κB signaling pathway. The results showed that cow lamellar keratinocytes were successfully isolated and cultured by Dispase II combined with the collagenase method. In the in vitro inflammation model established by LPS, the Chicoric acid decreased the concentration of inflammatory mediators (TNF-α, IL-1β, and IL-6), down-regulated the mRNA expression of TLR4 and MyD88 (P < 0.01), down-regulated the expression of TLR4, MyD88, p-ERK, p-p38, IKKβ, p-p65, p-p50 (P < 0.05), and increased the IκBα protein expression (P < 0.05). In conclusion, Chicoric acid successfully protected cow lamellar keratinocytes from LPS-induced inflammatory responses by modulating the TLR4/MAPK/NF-κB signaling pathway and downregulating inflammatory mediators.