热休克蛋白对应激损伤心肌细胞线粒体功能的影响及相关信号通路研究

Heat stress can result in reduction of productivity and even death in chickens. The heart is thought to be the first organ suffering failure in the death of heat stress, and the mitochondrial dysfunction plays an important role in it. Hsp90 is indispensable in the anti-stress injury of cells. Our previous research showed that high expression of Hsp90 could inhibit significantly the heat stress damage and apoptosis of chicken primary myocardial cells, which was associated with Akt, STATs and NF-κB signaling pathways. And heat-stressed apoptosis was performed mainly through the mitochondrial apoptosis pathway. However, the domestic and foreign fields of animal husbandry and veterinary reported little on how the Hsp90 played a regulatory role in it. PKM2 is a newly discovered client protein of Hsp90, and it is not clear whether PKM2 has protective function on mitochondria in the heat-stressed chicken cells. This present study, in the three levels of "cell-organelle-molecule", propose to study whether the expression of Hsp90 play an important role in protecting the mitochondrial function of myocardial cells in heat-stressed chicken and the protection is conducted through the "Hsp90-PKM2-Bcl-2" pathway, and reveal that the regulatory effect of Hsp90 on the protective signaling pathway "Akt-Bax/Bcl-2" and the mitochondrial apoptotic signaling pathway “Cyt-C-Apaf-1-Caspase-9”, with the techniques of siRNA interference、gene over-expression、 co-immunoprecipitation、confocal microscopy、transmission electron microscope, and so on. This study will provide the experimental and theoretical basis for "inducing Hsp90 expression to improve the ability of anti-heat stress in chicken", which has a great practical application prospect.

心脏是热应激鸡死亡过程中首个衰竭器官，主要原因是其线粒体功能损伤。我们前期研究表明，Hsp90高表达显著降低热应激鸡原代心肌细胞损伤与凋亡，且与Akt、STATs和NF-κB等信号通路有关，且其应激凋亡是通过线粒体途径。但国内外畜牧兽医领域对Hsp90在线粒体抗热应激损伤中的分子机制研究很少。PKM2是新近发现的Hsp90伴侣蛋白，其在热应激鸡细胞中是否具有保护线粒体作用，尚不清楚。本研究拟从“细胞-细胞器-分子”三层次，利用siRNA干扰、基因过表达、免疫共沉淀、激光共聚焦、透射电镜等技术研究Hsp90表达对热应激鸡心肌细胞线粒体功能的保护及其是否经过“Hsp90-PKM2-Bcl-2”途径，揭示Hsp90对线粒体保护信号通路“Akt-Bax/Bcl-2”及线粒体凋亡通路“Cyt-C-Apaf-1”调控机制，为“诱导Hsp90表达提高鸡抗热应激损伤能力”提供理论与应用依据。

心脏是热应激鸡死亡过程中首个衰竭器官，主要原因是其线粒体功能损伤。而Hsp90表达能够显著降低热应激鸡原代心肌细胞的损伤与凋亡。本项目重点关注了Hsp90介导的线粒体途径的细胞保护机制。研究发现，热应激可导致鸡心肌细胞线粒体结构与功能受损，且与其热应激时的过氧化水平升高密切相关，而Hsp90在线粒体抵抗其稳态破坏方面发挥重要作用。进一步的机制研究表明，Hsp90可通过自身的线粒体转位调控PKM2-Akt-Bcl-2通路抵抗热应激心肌细胞的线粒体损伤，进而抑制线粒体分子Cyt-C诱导的细胞Caspase途径凋亡机制。此外，我们还明确了中药EGB761可通过诱导心脏微血管内皮细胞分泌Hsp70促进心肌细胞抵抗热应激损伤，且Hsp90可通过PKM2-Akt通路保护热应激心脏血管内皮细胞。从凋亡和自噬的角度揭示了Hsp90以直接互作的方式通过HIF-1α-BNIP3/BNIP3L通路刺激自噬介导的存活和通过PKM2-Akt-HSF-1通路发挥的抗凋亡作用最终保护肾脏免受热应激损伤。最终，我们以口蹄疫病毒（FMDV）为研究对象，证明了Hsp90可通过分子伴侣作用以直接互作的方式促进其靶蛋白的合成与功能发挥。