牛磺酸抑制AS肉鸡右心肥大过程中calpains介导细胞凋亡作用的研究

Ascites Syndrome (AS) is the main reason for the death of broilers, and it could cause serious economic loss to the poultry industry. Inhibition of the right ventricular hypertrophy is an effective measures to prevent and cure AS. Our previous studies proved that taurine could prevent myocardial hypertrophy by decreasing the expression of μ-calpain and cell apoptosis during the process of ascites syndrome (AS) in broilers, which indicates the effect of taurine on AS has close relationship with calpains mediated cell apoptosis, however the mechanisms of which is still unclear. The present study aims to explore the inhibition effects of taurine on right ventricular hypertrophy and myocardial cell apoptosis through inducing broiler AS by low temperature, durning which the relevant index of right ventricular hypertrophy and cell apoptosis were monitored. In order to research the inhibition mechanism of taurine on the calpains mediated cell apoptosis, the free intracellular ca2+ concentration and the activity of calpains were detected by flow cytometry and fluorescence spectrophotometry, the gene and protein expressions of calpains and the downstream of calpains mediated cell apoptosis pathways were detected by real-time quantitative PCR and Western blot. And then through cell culture in vitro, the prevention effect of taurine on myocardial hypertrophy was further verified by detecting the expression changes of gene and protein on the downstream of calpains mediated cell apoptosis pathways. Finally, the role of taurine on calpains mediated cell apoptosis pathways in the process of right ventricular hypertrophy and the inhibition mechanisms of taurine on the pathways would be elucidated. The results will provide a reference for the application of taurine on the prevention and treatment of AS.

肉鸡腹水综合征(Ascites Syndrome，AS)是导致肉鸡死亡的主要原因之一，可造成严重的经济损失，抑制右心肥大是防治AS的有效手段。项目组前期研究发现，牛磺酸可通过降低肉鸡心肌μ-calpain表达和细胞凋亡，抑制右心肥大并降低AS发病率，提示其作用与抑制calpains介导的细胞凋亡密切相关，但确切机制尚不明确。本项目拟通过低温诱导肉鸡腹水症模型，结合体外心肌细胞培养，采用免疫组化技术和TUNEL法检测右心肥大和心肌细胞凋亡情况，明确牛磺酸对AS右心肥大和心肌细胞凋亡的抑制作用；采用流式细胞术、荧光分光光度法、QPCR、Western-blot等技术检测心肌细胞 [Ca2+]i、calpains活性、calpains介导的凋亡通路中相关因子表达量的变化，阐明牛磺酸对AS右心肥大过程中calpains介导细胞凋亡的抑制作用及其机理，为应用牛磺酸防治AS提供科学的理论依据。

右心肥大是腹水综合症(AS)患鸡的一个典型特征，有效抑制右心肥大是防治AS发生的途径之一。项目组前期研究发现，牛磺酸可通过降低肉鸡心肌μ-calpain表达和细胞凋亡，抑制右心肥大并降低AS发病率，但具体机制尚不清楚。本项目通过低温诱导肉鸡腹水症模型，结合体外心肌细胞培养，采用荧光分光光度法、Q-PCR、Western-blot等技术检测心肌细胞 线粒体膜电位(MMP)、calpains及其介导的凋亡通路中相关因子表达量的变化，对牛磺酸参与AS肉鸡右心肥大过程中calpains介导细胞凋亡的抑制作用及其机理进行了初步研究。首先利用环境低温诱导肉鸡AS的发生，试验结果表明：牛磺酸显著降低了肉鸡腹水病程中右心肥大指数、心肌组织中calpains活性和心肌细胞凋亡指数，增加MMP至正常水平，保护了心肌线粒体结构完整，显著下调右心室肌中μ-calpain、AIF、cytochrome-c、caspase-9、 caspase-3基因和蛋白的表达水平，同时上调calpastatin基因和bcl-2/bax基因及蛋白的表达水平。以上结果说明牛磺酸可通过调节μ-calpain介导的caspase依赖和caspase非依赖的凋亡途径起到抑制心肌细胞凋亡的作用，尤其是牛磺酸对μ-calpain/cytochrome-c介导的线粒体凋亡途径表现出明显的下调作用。接着在体外原代鸡胚心肌细胞培养试验中，利用环境缺氧诱导心肌细胞凋亡，同时在培养液中加入calpains 的特异性抑制剂PD150606。结果表明，牛磺酸显著抑制了缺氧环境下心肌细胞的凋亡情况，降低了缺氧心肌细胞μ-calpain、cytochrome c、procaspase-9的蛋白表达水平，增加了Bcl-2和 procaspase-3的蛋白表达水平，表现出了和PD15060相似的作用，证实牛磺酸可通过下调μ-calpain引发的线粒体凋亡途径起到抑制心肌细胞凋亡的作用。综上，本研究为应用牛磺酸防治AS提供了科学的理论依据。 项目资助发表SCI收录论文2篇，核心论文1篇，待发表1篇。培养博士生1名和硕士生2名，其中2名已经取得硕士学位。项目投入经费20万，由于本人2017年10月至2018年10月去美国访学，版面费及劳务费一直未报；剩余的测试化验加工费及材料费3.4万元将继续用于本研究后续支出。