基于内质网钙稳态探讨硒蛋白K在鸡缺硒性骨骼肌卫星细胞增殖分化障碍中的作用

Selenium exerts its biological activity via selenoprotein primarily, and selenium deficiency can cause skeletal muscle damage in chicken. Selenoprotein K (Selk), located in the endoplasmic reticulum, maintains physiological function of skeletal muscle by regulating calcium homeostasis. The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) are the key link in the repair of skeletal muscle injury, however, the role of Selk in proliferation and differentiation of SMSCs induced by selenium deficiency has not yet been elucidated. In the present study, chicken skeletal muscle injury induced by selenium deficiency model and Selk missing and overexpression SMSCs model were established. Simultaneously, Selk missing and overexpression SMSCs model was treated with cytoplasmic Ca2+ chelator and endoplasmic reticulum Ca2+ channel inhibitor. Then, we used immunofluorescence, immunohistochemical, and western blot methods to detect the co-expression of Pax7 and Mrf5, SMSCs proliferation differentiation ability, and the expression of calcium homeostasis, endoplasmic reticulum stress, and apoptosis-related proteins. The cellular morphology, ultrastructure, cytoplasmic and endoplasmic calcium levels of cells were detected by phase contrast microscopy, transmission electron microscopy, and flow cytometry. This present study will reveal the correlation between Selk and SMSCs proliferation and differentiation, and illuminate the molecular mechanism of Selk in proliferation and differentiation disorder of chicken SMSCs induced by selenium deficiency based on endoplasmic reticulum calcium homeostasis. This study will provide important theoretical basis for the mechanism study of skeletal muscle damage caused by selenium deficiency.

硒主要通过硒蛋白发挥其生物学活性，硒缺乏可致鸡的骨骼肌损伤。定位于内质网的硒蛋白K(Selk)通过调控钙稳态维持骨骼肌生理功能。骨骼肌卫星细胞(SMSCs)的增殖分化是骨骼肌损伤修复的关键环节，但Selk在鸡缺硒性SMSCs增殖分化中的具体作用尚不明确。本研究复制鸡缺硒性骨骼肌损伤模型，建立Selk基因沉默及过表达的SMSCs模型并进行胞浆钙螯合剂、内质网钙通道抑制剂处理，采用免疫荧光法、免疫组化法、免疫印迹法检测体内外模型中Pax7与Mrf5的共表达，SMSCs增殖分化能力，钙稳态、内质网应激、凋亡相关蛋白的表达；利用相差显微镜及透射电镜观察体外模型的形态学与超微结构，采用流式细胞术检测其胞浆与内质网钙水平。本课题将明确Selk与SMSCs增殖分化的相关性，从内质网钙稳态的角度阐明Selk在鸡缺硒性SMSCs增殖分化障碍中的分子机制，为缺硒所致骨骼肌损伤的机制研究提供重要的理论依据。

硒蛋白K(SELENOK)是定居于内质网，可调控胞内钙稳态与氧化还原状态的跨膜小分子蛋白质。骨骼肌卫星细胞(SMSCs)是黏附在肌纤维上，位于肌膜和基底膜之间的肌肉干细胞。当骨骼肌受损时，处在静息状态的SMSCs会被激活并进行增殖与分化，最终与受损的肌纤维融合或形成新的肌纤维，完成受损骨骼肌的修复。SELENOK在硒缺乏所致的鸡骨骼肌损伤中发挥重要作用，但其对SMSCs增殖分化中的具体作用机制还尚未阐明。基于此，本项目提出了SELENOK基因沉默导致胞内钙稳态与氧化还原状态失衡，从而抑制SMSCs增殖分化的假设。本项目在应用RNA干扰技术建立SELENOK基因沉默的SMSCs模型基础上，联合Thimerosal、NAC与Oltipraz处理，检测了SMSCs增殖分化、氧化应激、内质网应激与凋亡等相关指标。取得了如下研究结果：(1) SELENOK基因沉默引发SMSCs内质网应激，进而触发凋亡，最终导致SMSCs增殖分化障碍。进一步研究证实，SELENOK基因沉默所致的内质网钙超载是引发内质网应激的重要机制；(2) SELENOK基因沉默引发SMSCs氧化应激也是抑制其增殖分化的重要原因。进一步研究证实，SELENOK基因沉默通过干预Keap1-Nrf2信号通路，抑制了Nrf2入核，进而削弱了Nrf2抗氧化能力，是引发氧化应激的重要机制。本项目明确了SELENOK基因沉默导致SMSCs凋亡，从而抑制其增殖与分化，证实了内质网应激与氧化应激在其中的作用，阐明了钙稳态失衡与Nrf2-ARE信号通路失活在该过程中的具体分子机制，为探讨硒缺乏所致骨骼肌损伤提供重要的理论依据与新颖的研究切入点。