基于AMPK-HMGR信号通路研究菊粉肠道代谢产物对产蛋鸡胆固醇合成的调节作用

The eggs with lower-cholesterol content are valuable for peoples’ health. Our recent study showed that dietary supplementation with inulin to laying hens reduced cholesterol content in eggs by 18.8%, but the mechanism of the regulation is not clearly. It was reported that AMP-activated protein kinase (AMPK) is a target of some drugs. AMPK decreases the cholesterol synthesis of body through phosphorylating the key enzyme activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in cholesterol synthesis. Whether the short chain fatty acids (SCFA), which are the fermentation product of inulin in laying hens intestinal, could activate AMPK, and the reduction of egg cholesterol by inulin is acted through the AMPK-HMGR pathway are worth to study. Molecular biology and cell biology technology will be used in animal, microbe and cell culture experiments and conduct to study the effects of inulin on the key indexes in liver about AMPK-HMGR pathway, liver cholesterol synthesis, and egg cholesterol deposition of laying hens. The content of VLDLy in serum, which is the key indicator in cholesterol transport, and the expression of oocyte vitellogenesis receptor (OVR) in ovary, which is the key indicator in cholesterol deposition will also be determined to build a bridge between the initial signaling pathway and the final egg cholesterol deposition. The final purpose of this study is to provide the theory basis for the regulation of inulin in cholesterol synthesis of laying hens and production of egg with lower-cholesterol content.

降低鸡蛋胆固醇含量对于改善人类健康状况有实际意义。课题组研究发现，在蛋鸡日粮中添加菊粉可降低鸡蛋胆固醇含量18.8%，但具体调控机制尚不明确。据报道，腺苷酸激活蛋白激酶（AMPK）是多种药物的作用靶点，其能通过磷酸化胆固醇合成关键酶β-羟基-β-甲基-戊二酸单酰辅酶A还原酶（HMGR）降低机体胆固醇合成。而菊粉在蛋鸡肠道中的代谢产物短链脂肪酸（SCFA）能否激活AMPK，进而通过AMPK-HMGR信号通路发挥其降胆固醇作用值得探讨。采用分子生物学、细胞生物学技术通过动物试验、微生物和细胞培养试验研究菊粉对肝脏AMPK-HMGR通路关键指标、肝脏胆固醇合成以及鸡蛋胆固醇沉积的影响，同时测定血液胆固醇转运关键指标载脂蛋白VLDLy含量及卵巢胆固醇沉积关键指标卵母细胞卵黄生成受体（OVR）表达量，为初始信号通路和最终鸡蛋胆固醇沉积架起桥梁，为菊粉调控蛋鸡胆固醇合成和低胆固醇鸡蛋的生产提供依据。

降低鸡蛋胆固醇含量对于改善人类健康状况有实际意义。课题组研究发现，在蛋鸡日粮中添加菊粉可降低鸡蛋胆固醇含量，但具体调控机制尚不明确。据报道，腺苷酸激活蛋白激酶（AMPK）是多种药物的作用靶点，其能通过磷酸化胆固醇合成关键酶β-羟基-β-甲基-戊二酸单酰辅酶A还原酶（HMGR）降低机体胆固醇合成。而菊粉在蛋鸡肠道中的代谢产物短链脂肪酸（SCFA）能否激活AMPK，进而通过AMPK-HMGR信号通路发挥其降胆固醇作用值得探讨。采用分子生物学、细胞生物学技术通过动物试验、微生物和细胞培养试验研究菊粉对肝脏AMPK-HMGR通路关键指标、肝脏胆固醇合成以及鸡蛋胆固醇沉积的影响，同时测定血液胆固醇转运关键指标载脂蛋白VLDLy含量及卵巢胆固醇沉积关键指标卵母细胞卵黄生成受体表达量，为初始信号通路和最终鸡蛋胆固醇沉积架起桥梁。选择38周龄海兰褐壳蛋鸡360只产蛋鸡随机分为5个处理，每个处理6个重复，每个重复12只。5个处理分别为在基础日粮中添加0（对照）、0.5、1.0、1.5和2.0%的菊粉。试验期56天。结果表明：与对照组相比，饲粮添加菊粉（1.0和1.5%）可显著提高平均蛋重。1.5%菊粉组可降低料蛋比，提高产蛋率。饲粮添加菊粉可增加蛋鸡盲肠内容物乙酸、丙酸、丁酸的含量，显著降低盲肠内容物pH。饲粮中添加菊粉可提高蛋鸡盲肠Bacteroidales_S24-7_ group相对丰度。56天，各添加组显著降低蛋鸡肝脏极低密度脂蛋白胆固醇含量。56天，1.0、1.5、2.0%的菊粉组显著降低鸡蛋胆固醇含量；1.0和1.5%菊粉组显著增加粪便胆汁酸含量；1.5%的菊粉组蛋鸡肝脏AMP含量和AMP/ATP显著提高；1.5%的菊粉组显著增加蛋鸡肝脏AMPK活性，显著降低蛋鸡肝脏HMGR活性。产蛋鸡肝细胞培养试验表明，添加乙酸、丙酸的细胞培养组胆固醇合成量较低，AMPK活性较强、HMGR活性较低，说明菊粉发酵产物SCFA可影响肝细胞胆固醇合成。以上结果说明菊粉的添加可能通过影响蛋鸡肠道代谢产物SCFA的产量，进一步激活AMPK，提高AMPK活性，降低HMGR活性，进而调控鸡蛋胆固醇含量。本研究可为菊粉调控蛋鸡胆固醇合成提供理论依据。