LXRα/CYP7A1信号通路在肉鸡腹水综合征胆固醇分解代谢紊乱中的作用及机制

Ascites syndrome (AS) is a important nutritional and metabolic disease harm for the broiler industry in the world, which is a result affected by multi-factors. The exact pathogenesis of AS remains unknown, mostly considered to be related to pulmonary arterial vascular remodeling, heart failure and oxygen free radical injury.Cholesterol 7α-hydroxylase (CYP7A1) is a rate-limiting enzyme in the classical bile acid synthesis pathway, catalyzing the decomposition of cholesterol to bile acid. It plays an important role in the steady state maintaining of cholesterol and bile acids synthesise. Cholesterol level of AS broilers was significantly higher than normal ones in our preliminary study,indicating there would be a disorder of cholesterol metabolism. So we infer that expression of CYP7A1 influence cholesterol catabolism in AS broilers, which may be a new mechanism of AS. In order to asscertain this hypothesis, the AS model will be replicated. Cholesterol catabolism and expression rules of LXRα/CYP7A1 signal path will be systematacially studied and verified by in vivo and in vitro interference model of chicken hepatocytes. The correlation between them will also be analyzed. The change rules of cholesterol catabolism disorders, effects and mechanisms of LXRα/CYP7A1 on it in AS broilers will be revealed out. The molecular mechanism of AS will be elucidated from a new perspective- cholesterol catabolism disorders, providing new ideas for prophylaxis and treatment of AS.

肉鸡腹水综合征是危害世界肉鸡生产的一种重要营养代谢病，其发生是多因子作用的结果，确切的发病机理至今尚未阐明,多认为与肺动脉血管重构、心脏衰竭、氧自由基损伤有关。胆固醇7α羟化酶（CYP7A1）是胆汁酸经典合成途径的限速酶，催化胆固醇分解为胆汁酸，在维持胆固醇稳态及胆汁酸合成中发挥重要作用。申请人前期研究发现腹水征肉鸡胆固醇显著高于正常鸡，表明胆固醇代谢出现了紊乱。因此，我们推测CYP7A1表达可影响腹水征肉鸡胆固醇分解代谢，这可能是腹水征发生的新机制。为进一步证实该假说，我们将复制肉鸡腹水综合征模型，通过体内、体外肝细胞干扰模型试验，系统研究和验证胆固醇分解代谢、胆固醇分解LXRα/CYP7A1信号通路表达变化规律，分析两者的相关性，探明肉鸡腹水征胆固醇分解代谢紊乱规律及LXRα/CYP7A1在其中的作用及机制，从新的视角揭示肉鸡腹水综合征发生的分子机制，为腹水综合征的防治提供新思路。

本试验旨在研究LXRα/CYP7A1信号通路在肉鸡腹水综合征（AS）胆固醇分解代谢紊乱中的作用。.动物试验：选择1日龄健康罗斯308肉公鸡216只，随机分成正常对照组和低温诱导组，每组6个重复，每个重复18只鸡，试验期为42天。测定全期生产性能及21、28、35和42d的血液生化指标、AS相关经典指标（HCT、AHI等），并检测肝脏总胆固醇（TC）代谢相关基因、蛋白的表达。结果显示：（1）与正常对照组相比，低温诱导组肉鸡的全期日增重显著降低，料重比和腹水征发生率显著提高，HCT和AHI显著升高。（2）21d时，AS组肉鸡血清TC极显著高于对照组。21、35d时，肝脏TC合成、吸收、转运相关基因mRNA水平均差异不显著；21d时，肝脏TC分解相关基因CYP7A1和LXRα mRNA表达及CYP7A1蛋白表达显著降低，而35d时差异不显著。.细胞试验：1、以18胚龄鸡原代肝细胞为素材，进行CoCl2处理以构建鸡肝细胞缺氧模型，CoCl2添加浓度为0μM、50μM、100μM、150μM、200μM。检测缺氧24h后肝细胞存活率及HIF-1α mRNA表达。结果显示：当CoCl2浓度为50μM、100μM、150μM和200μM时，肝细胞存活率均极显著高于对照组，100μM CoCl2可使HIF-1α mRNA表达显著升高。2、以相同素材进行LXRα激动剂T0901317处理以构建鸡肝细胞LXRα激动模型，T0901317添加浓度为0μM、0.01μM、0.1μM、1μM、10μM。检测处理24h后肝细胞LXRα和CYP7A1 mRNA表达。结果显示：添加0.1μM T0901317使肝细胞LXRα和CYP7A1 mRNA表达均显著升高。3、以相同素材进行CoCl2+T0901317处理24h，以缺氧培养（100μM CoCl2）为对照，检测细胞上清TC、肝细胞LXRα和CYP7A1 mRNA及CYP7A1蛋白表达。结果显示：与缺氧组相比，CoCl2+T0901317组细胞上清TC含量降低，肝细胞LXR和CYP7A1 mRNA表达及CYP7A1蛋白表达显著增加。.综上所述，在AS早期，TC代谢变化更为敏感；AS肉鸡TC增加可能是由于TC分解代谢相关基因CYP7A1、LXRα表达下降。细胞试验进一步验证了LXRα/CYP7A1信号通路在AS肉鸡TC代谢紊乱中有作用。