Effects Of Ghrelin On β-casein Synthesis In BMECs Of Lactating Cows

Milk protein is the main substance to determine the nutritional quality of cow’s milk. Milk protein content is related to milk quality and consumer health.Milk protein content is also an important symbol of dairy industry core competitiveness. Acylated ghrelin(AG) and unacylated ghrelin(UAG) are two major circulating peptides. In addition to stimulation of GH secretion, food uptake, inflammation, energy metabolism, AG was reported to regulate the synthesis the β-casein(CSN2) in mammary epithelial cells(MECs) and mammary tissues. However, the specific mechanism is unclear. UAG is far more abundant in bovine plasma than AG. In contrast to AG, the regulating action of UAG in the expression of CSN2 remains poorly understood. Mastitis disrupts normal milk secretion from MECs. However, the mechanism is litter known. Thus, we investigated the effect of AG and UAG on the expression of CSN2 in bovine mammary epithelial cells(BMECs), mammary gland tissue and LPS-induced mastitis model.The BMECs were separated by tissue explants adherent method, and the expression of CK-18 was detected by FCW and IF in order to ensure the cells purity. The milk proteins, AG and GHSR1 a were expressed in BMECs. The cell growth curve was a typical "S" type. These results demonstrated that cultured BMECs can be used in subsequent experiment.Next, the BMECs were treated with increasing concentrations of AG or UAG(0-100 ng/m L) for different time points. The results showed that both AG and UAG increase the expression of CSN2 in a dose-dependent manner in BMECs. Increased expression of CSN2 was blocked by the antagonists of GHSR1a([D-Lys3]-GHRP-6) and Gαs subunit(NF449) in BMECs. In addition,both AG and UAG activated AKT, ERK1/2 and JNK pathways were inhibited by the antagonists of GHSR1 a and Gαs subunit. Blockade of AKT, ERK1/2and JNK pathways prevented the expression of CSN2 induced by AG or UAG.In addition, we found that both AG and UAG cause cell proliferation through identical signaling pathways. Finally, our results showed that AG or UAG improve the expression of CSN2 in cultured mammary explants. Taken together, these results demonstrate that both AG and UAG facilitate the expression of CSN2 in a GHSR1a-dependent manner in BMECs and cultured mammary explants.Finally, the effect and mechanism of AG and UAG on the expression of CSN2 in LPS-induced mastitis model were evaluated. The results showed that LPS remarkablely increases the expression of pro-inflammatory factors,whereas inhibits the expression of CSN2 compared with NT group. We further found that AG or UAG suppressed the expression of pro-inflammatory factor in LPS-induced BMECs. AG or UAG play an anti-inflammatory effect via ERK1/2 and AKT signal pathways in a GHSR1 a and Gαs dependent manner in LPS-induced BMECs. Both AG and UAG increase the expression of CSN2,whereas blocking ERK1/2 and AKT signal pathways inhibit AG- or UAG-induced expression of CSN2 in LPS-induced BMECs. LPS improves the expression of GHSR1 a. [D-Lys3]-GHRP-6 and NF449 suppressed the phosphorylation of ERK1/2 and AKT signal pathways and the expression of CSN2 in LPS-induced BEMCs. Both AG and UAG increased the expression of CSN2 by inhibiting LPS-induced apoptosis via p53/Bax/Bcl-2 pathways. Taken together, our results demonstrate that both AG and UAG suppressed the expression of inflammation factors and increased the expression of CSN2 in a GHSR1a-dependent manner in LPS-induced mastitis model. In addition, AG and UAG increased the expression of CSN2 by inhibiting LPS-induced apoptosis via p53/Bax/Bcl-2 pathways.In conclusion, AG and UAG act on AKT, ERK1/2 or JNK pathways to facilitate the expression of CSN2 in a GHSR1a-dependent manner in BMECs and cultured mammary explants. AG and UAG suppressed the production ofinflammation factors and increased the expression of CSN2 via GHSR1 adependent ERK1/2 and AKT signal pathways. Finally, both AG and UAG increased the expression of CSN2 by inhibiting LPS-induced apoptosis via p53/Bax/Bcl-2 pathways. These results will provide scientific basis to further study the regulatory mechanism of protein synthesis of cow’s milk and the treatment of mastitis and improve the nutritional quality of cow’s milk.