连翘苷对β-羟丁酸所致奶牛皱胃平滑肌氧化应激的干预机制

Displacement of Abomasum (DA) is a typical gastric disorder disease, it is generally believed that atony and expansion are the important reasons of DA. Our groups previously reported that β-hydroxybutyric acid (β-HB) is able to cause oxidative stress in bovine abomasum smooth muscle cell (BSMC) , in turn, can lead to abomasum smooth muscle relaxation, however, the target genes are unknown. The present project aims to screen the target genes and signaling pathways of β-HB-induced oxidative stress, to the previously found oxidation/antioxidant genes (Nrf2, Foxo3a Mt2, LCN2, Lemd3, HBP1) as the clue, based on the RNA-sequencing, real time RT-PCR, Western-blot technology and others molecular biology methods from the levels of experimental animals and primary culture BSMCs. Meanwhile, phillyrin will be added to study the protective mechanisms on β-HB-induced oxidative stress through detecting the changes of target genes, protein and characteristic index related to oxidative stress in the BSMC, the intervention mechanisms of phillyrin against β-HB-induced oxidative stress are evaluated from the gene and protein aspects. The purpose is to reveal the etiology of DA and develop theoretical basis for the prevention and treatment of DA drugs.

皱胃变位（DA）的病因和致病机制是目前研究的热点，认为皱胃弛缓扩张是引发皱胃变位的重要原因。项目组前期研究表明β-羟丁酸(β-HB)能够引起皱胃平滑肌细胞（BSMC）氧化应激，进而可导致皱胃平滑肌弛缓，然而其损伤的靶基因尚不完全清楚。本研究拟运用β-HB刺激BSMC和初生犊奶牛，通过高通量测序技术RNA-Seq，以先前发现的氧化/抗氧化基因（Nrf2，Foxo3a，Mt2，LCN2，Lemd3，HBP1等）为线索，寻找β-HB作用的靶基因和信号通路，运用Real-time RT-PCR从基因水平验证RNA-Seq的结果，运用Western-blot等验证基因表达产物及其下游分子特定变化。添加天然抗氧化活性成分连翘苷，检测BSMC中靶基因、靶蛋白及相关氧化应激特征指标的变化，从基因和蛋白水平阐明连翘苷对β-HB所致细胞氧化应激的干预机制。为揭示皱胃变位的病因和研发防治DA的药物提供理论依据。

奶牛皱胃变位(Displaced abomasums, DA)是围产期奶牛常见疾病，该病的发生主要与皱胃扩张、皱胃弛缓、神经元调节紊乱等因素相关，因治疗费用高和淘汰率高给奶牛养殖业带来巨大的经济损失。本项目研究了DA的病因和致病机制这一研究的热点和难点问题。本项目研究发现连翘苷能明显提高体外培养的皱胃平滑肌细胞活性，巨噬细胞作为机体重要免疫细胞，也参与皱胃迟缓这一过程；借助荧光定量PCR和Western-blot等相关分子生物学手段，通过实验细胞模型和实验动物从基因、分子及蛋白水平分析并筛选β-HB致氧化应激的靶基因，研究发现连翘苷对β-羟丁酸诱导牛巨噬细胞氧化和抗氧化相关指标 ROS、CAT、MDA 和 SOD 有明显的抑制作用；荧光定量结果显示，连翘苷能够明显抑制β-HB 诱导的 IL-6 和 IL-1β含量升高，且呈剂量依赖式；蛋白免疫印迹发现连翘苷对β-羟丁酸诱导牛巨噬细胞 Nrf2 信号通路关键蛋白、MAPK 信号通路均有明显的干预作用。本项目组研究表明β-羟丁酸(β-HB)能够引起奶牛皱胃平滑肌细胞氧化应激，然后从基因变化和靶基因角度研究了连翘苷、黄连素等中药单体对β-HB诱导的氧化应激性损伤的干预机制。并利用连翘苷、黄连素的抗氧化作用，通过细胞中相关氧化应激特征指标、靶基因及亚显微结构变化等，从DNA、基因和蛋白水平阐明连翘苷对β-HB所致细胞氧化应激的干预机制，以此寻找减少β-HB对奶牛皱胃平滑肌损害的新途径，最终为DA的防治提供理论依据。