纳米二氧化硅经JNK/c-Jun应激信号通路诱导心肌收缩功能障碍及其分子机制研究

Our previous study found that silica nanoparticles could activate the JNK/c-Jun signaling pathway and induce the upregulation of tissue factor (TF) which could impact the cardiac contraction function, but the mechanism was still not clear. This study is aimed to investigate the influence of silica nanoparticles on myocardial contraction function and its related mechanisms. The myocardial injury was dected in SD rats and zebrafish after exposed to silica nanoparticles. The cardiac contraction function in rats was anlazed by small animal doppler ultrasound and cardiac magnetic resonance. The immunofluorescence assay was performed to detect the ventricular contraction function. Myocardial elasticity imaging, sarcomere length contraction and the intensity of myocardial contraction were measured to analyze the effect of silica nanoparticles on myocardial contract function. The co-expression of tissue factor (TF), PAR1 and actin protein were detected by qRT-PCR and Western blot, and the JNK/c-Jun and TF/PAR1 signaling pathways were measured by immunofluorescence and in situ hybridization technique. Using the reverse genetics techniques, the invovled signaling pathways was further verified. Using lentiviruse for preparing JNK overexpression and knock-down cell culture system, and TF siRNA to analyze the changes in JNK/c-Jun and TF/PAR1 signaling pathways; and verified the regulatory mechanisms of JNK/c-Jun and TF/PAR1 signaling pathways in silica nanoparticles-induced myocardial contraction dysfunction.

课题组前期发现：纳米SiO2可激活JNK/c-Jun应激信号通路，引起参与调控心肌收缩功能的组织因子（TF）表达上调，但纳米SiO2对心肌收缩功能的影响及调控机制不清。本项目拟深入研究纳米SiO2对心肌收缩功能的影响及其分子机制。体内采用大鼠和斑马鱼模型研究纳米SiO2心肌损伤作用；小动物超声及磁共振分析心脏收缩功能的改变，免疫荧光检测心室收缩功能，通过心肌弹性成像、肌小节收缩长度和心肌收缩强度分析心肌收缩功能改变；原位杂交及免疫荧光检测心肌收缩蛋白表达及其与TF、PAR1共表达；qRT-PCR及Western blot检测JNK/c-Jun和TF/PAR1信号通路基因和蛋白表达水平，反向遗传学技术验证上述信号通路基因时空表达改变。体外采用心肌细胞构建JNK过表达和干扰体系，siRNA沉默TF，进一步验证JNK/c-Jun和TF/PAR1信号通路在纳米SiO2致心肌收缩功能障碍的调控机制。

纳米二氧化硅被广泛应用于生物医药领域，从而增加了人群暴露和健康风险。越来多的证据表明，纳米二氧化硅可能对心血管系统造成不良影响，然而，目前国内外关于纳米二氧化硅心脏毒性相关的研究报道十分匮乏。因此，本研究的目的是探讨纳米二氧化硅对心脏功能的影响及其分子机制。通过超声心动图、超微结构和组织病理学分析发现，SiNPs可引起心肌收缩功能障碍，并伴有心肌结构损伤、肌节段紊乱、间质水肿和心肌细胞凋亡。血清和心脏组织中的心肌酶和炎症因子水平均显著升高，同时伴随着心脏氧化损伤水平升高。SiNPs可显著上调炎症和收缩相关蛋白JNK、p-JNK、c-Jun、TF和PAR1的表达水平。SiNPs可激活JNK/TF/PAR1信号通路的mRNA和蛋白水平，而JNK敲减后，SiNPs引起的心肌毒性显著减弱。此外，纳米二氧化硅可上调心肌肥厚相关特殊标志物ANP、BNP、β-MHC和焦亡相关蛋白的表达，如NLRP3、Cleaved-Caspase-1、GSDMD、IL-18以及Cleaved-IL-1β。体外研究也证实了纳米二氧化硅可增加心肌细胞内ROS水平，激活NLRP3/Caspase-1/GSDMD信号通路。而转染si-NLRP3或加入Caspase-1抑制剂VX-765、NADPH氧化酶抑制剂VAS2870处理的心肌细胞对纳米二氧化硅诱导的心肌细胞焦亡和心肌肥厚均有抑制作用。综上所述，本研究初步阐明了纳米二氧化硅致心肌收缩功能障碍的分子机制：纳米二氧化硅通过激活JNK/c-Jun/TF信号通路引起肌动蛋白表达下调，激活心肌炎症反应，造成心输出量和心室收缩功能下降，进而导致心脏功能障碍。在此基础上，进一步发现，纳米二氧化硅通过激活ROS/NLRP3/Caspase-1信号通路，引起心脏组织氧化应激和炎症反应，导致心肌线粒体损伤，最终造成心肌组织焦亡和心肌肥厚。本研究可为纳米二氧化硅毒理学安全性评价提供科学依据。