处于围产期的奶山羊由于血液中钙离子迅速降低极易引发低血钙症,5-羟色胺(5-hydorxytyrptamine,5-HT)可以调节生物体内钙代谢,但具体调节机制尚不明确.该研究通过注射5-羟色氨酸(5-HTP,5-hydroxytryptophan)来增加围产期奶山羊体内的5-HT浓度,采集血液提取RNA后,采用全转录组测序技术筛选鉴定差异表达mRNA、microRNA、LncRNA和CircRNA.通过Metascape对差异表达的基因进行富集分析,发现这些差异表达基因主要参与钙代谢、脂质代谢、免疫反应、细胞凋亡等过程.通过预测转录本之间的结合情况构建LncRNA/CircRNA-microRNA-mRNA的竞争内源性RNA (Competing Endogenous RNA,ceRNA)调控网络,并对网络中的基因进行富集分析发现3个与钙代谢相关的基因,分别是Toll样受体9(Toll Like Receptor 9,TLR9)、缝隙连接蛋白γ2 (Gap Junction Protein Gamma 2,GJC2)和连接素1(Junctophilin1,JPH1),LncRNA MSTRG.1354.2可与TLR9竞争性地结合microRNA NC.030825.1_6027,进而调控TLR9的表达,MSTRG.56601.1和MSTRG.101405.3都可以与GJC2、JPH1竞争性地结合NC.030814.1_19917,进而调控GJC2和JPH1的表达.该研究结果为5-HT调控山羊钙代谢提供了一种全新的分子机制.

Serotonin (5-HT) is a monoamine and it could regulate cell growth by its receptors working on signaling pathways. 5-HTP is the precursor of 5-HT that help 5-HT synthesis. B cell leukemia/lymphoma 3 (Bcl-3) involved in cell death and proliferation through mitogen activated protein kinase (MAPK) pathway. However, there is little information about the effects of MAPK/Bcl-3 on apoptosis of goat mammary gland epithelial cells (GMECs). The aim of this study is to explore the interaction among 5-HTP, MAPK and Bcl-3 in GMEC apoptosis. In this study, 5-HTP treatment decreased cell apoptosis and promoted phosphorylation of ERK1/2 in GMEC. We also found that the activation and inhibition of ERK1/2 could affect GMEC apoptosis. The Annexin V-FITC/PI staining and western blotting results suggested that 5-HTP decreased GMEC apoptosis through ERK1/2 signaling pathway. And the results of RT-qPCR and western blotting demonstrated that both 5-HTP and ERK1/2 positively regulated Bcl-3 expression. Sum up all the results, we could draw the conclusion that 5-HTP decreased GMEC apoptosis through MAPK/ERK/Bcl-3 pathway.

5-Hydroxy-l-tryptophan (5-HTP) is the primary product that converts l-tryptophan into 5-hydroxytryptamine by a rate-limiting enzyme. Our previous study found that 5-HTP could promote the intracellular calcium level in goat mammary epithelial cells (GMECs). Herein, first, dairy goats were injected with 5-HTP or saline daily from 7 days before delivery, and the calcium level in colostrum of 5-HTP-injected goats was significantly higher than that of saline-injected goats. Moreover, miR-99a-3p expression was significantly increased after 5-HTP treatment from transcriptome sequencing analysis and quantitative real-time polymerase chain reaction. In addition, it was found that ATP2B1 is one of the target genes of miR-99a-3p predicted by bioinformatic methods, which plays a crucial role in the maintenance of intracellular calcium homeostasis of mammary epithelial cells. Next, we confirmed that miR-99a-3p could increase the intracellular calcium level via decreasing ATP2B1 in GMECs. Taken together, we draw the conclusion that 5-HTP promotes the calcium level in colostrum possibly by increasing intracellular calcium of mammary epithelial cells induced by the miR-99a-3p/ATP2B1 axis.