下丘脑黑皮质素系统在脱氧雪腐镰刀菌烯醇诱导厌食中的作用及分子机制

Anorexia caused by DON can break energy homeostasis, and cause serious damage to human and animal health. Currently, anorexia toxicity mechanism of DON mainly focus on peripheral organs. Prior studies and the applicant's previous work all found that DON can improve the expression level of neuropeptides (α-MSH and POMC) in hypothalamic neurons. However, the exact mechanism remained unknown. On the basis of our previous study, at the cellular level, the present project on the one hand, using immunocytochemistry and cell patch clamp electrophysiological techniques to detect the effects on activity of neurons and KATP channel, to clarify the mechanism on α-MSH secretion of DON; on the other hand, study the influence of MAPK→inflammatory cytokines→cytokine receptor→JAK2/STAT3 pathway of DON by using western Blot, inhibitors and siRNA technology, in order to investigate the transcription regulatory mechanism of DON on POMC expression. In vivo, central administration DON and related signaling pathway blockers to detect influence of DON on food intake of mice, C Fos, neuropeptides and signaling pathway proteins expression level in hypothalamus, to further illustrate the central nervous regulating mechanism of anorexia toxicity induced by DON, to provide the basic theoretical basis for DON control and maximum levels set for human and animal health.

脱氧雪腐镰刀菌烯醇（DON）诱导的厌食破坏机体能量稳态，严重危害人畜健康。目前其厌食毒性作用机制研究主要集中在外周靶标。申请者前期研究发现 DON 显著上调下丘脑神经元细胞抑采食神经肽的表达，但其具体机制仍有待阐明。本项目拟在前期研究基础上，在细胞水平，运用免疫细胞化学和细胞膜片钳技术分别检测 DON 对神经元细胞兴奋性及其KATP通道活性的影响，探讨DON影响α-MSH分泌的分子机制；同时运用Western Blot、阻断剂阻断和siRNA干扰等方法探究 DON 对MAPK→炎症性细胞因子/受体→JAK2/STAT3通路的作用，研究 DON 影响POMC表达的转录调控机制；在活体水平，小鼠中枢注射DON及相关通路阻断剂，观察DON 对小鼠采食量、下丘脑中C-Fos、神经肽及信号通路蛋白表达的影响，进而阐明中枢调控DON诱导厌食的作用机制，从而为人畜DON防控策略及安全限量研究提供理论依据

脱氧雪腐镰刀菌烯醇（DON）诱导的厌食破坏机体能量稳态，严重危害人畜健康。目前其厌食毒性作用机制研究主要集中在外周靶标，下丘脑作为调节食欲的主要整合中枢，目前其调节食欲的作用机制还不清楚。本项目分别在细胞和活体水平，运用磷酸化 iTRAQ 定量蛋白质组学、细胞免疫荧光染色、脑片c-Fos免疫荧光染色以及Western Blot等方法检测了 DON 对抑采食神经肽POMC以及相关信号通路蛋白表达的影响。结果表明，在细胞水平，DON可以显著提高下丘脑细胞抑采食神经肽α-MSH的分泌和POMC蛋白的表达。同时，还显著提高了Akt、Erk1/2、FoxO1及 STAT3的磷酸化水平，显著降低了 AMPK 的磷酸化水平。在活体水平，灌胃和中枢给药DON 均可以显著降低小鼠采食量。口服给药1h后，DON在激活POMC 神经元的同时，还显著提高了小鼠下丘脑POMC蛋白的表达以及Akt、Erk1/2、FoxO1及 STAT3的磷酸化水平，显著降低了 AMPK 的磷酸化水平，与细胞水平结果一致。总之，DON 可能通过激活 PI3K/Akt/Erk1/2→FoxO1 和JAK2/ STAT3 通路上调抑采食神经肽 POMC 的表达来诱导厌食。本研究阐明的DON诱导厌食的中枢调控作用机制，可为人畜DON防控策略及安全限量研究提供理论依据。