健脾益气摄血方缓解免疫性血小板减少症乏力症状与线粒体功能相关性研究

ITP-related fatigue is one of the key problems to be solved in the treatment of disease. Glucocorticoid has both pros and cons in the treatment due to the symptoms. Clinical research showed that large dose of glucocorticoid can temporarily relieve fatigue, but the symptoms may get even more serious after withdrawl. And chronic fatigue can seriously affect the quality of life and even cause anxiety in ITP patients. Nevertheless, the pathogenesis of fatigue remains unclear. Based on the above, this study intends to carry out relevant research due to ITP-related fatigue, which guided by the theory of traditional Chinese medicine. Main research contents include: ITP mice model will be established by passive immunization method, and then treated by Jianpi Yiqi Shexue Formula. Mitochondrial ultrastructure of liver, spleen, thymus, heart, and calf muscle cells will be observed, mitochondrial respiratory chain complexes (Ⅰ,Ⅱ, Ⅲ, Ⅳ, Ⅴ), mitochondrial enzyme complex Ⅱ subunits (SDHA), mitochondrial matrix protease (ClpP) activity will be detected and also expression level of ClpP mRNA, protein and that of ATP will be tested. Thereby to explore the correlation between ITP-related fatigue and mitochondrial function, and the effect mechanism of Jianpi Yiqi Shexue Formula in ITP-related fatigue as well.

ITP患者乏力症状是该病治疗中亟待解决的关键问题，糖皮质激素在对乏力症状的治疗上有利有弊。临床观察发现，大剂量糖皮质激素可以暂时缓解乏力症状，而减停时乏力症状会更加明显。且长期持续的乏力会严重影响ITP患者生存质量，甚至导致患者情绪低落而发生焦虑事件。但迄今为止，对于导致ITP患者乏力原因尚无明确认识和有效治疗措施。基于上述，本课题以中医理论为指导，围绕ITP乏力进行相关研究。主要研究内容为：通过被动免疫造模法建立ITP小鼠模型，以健脾益气摄血方为干预药物，以模型小鼠脾脏、肝脏、胸腺、心脏、腓肠肌线粒体结构，线粒体呼吸链复合物（Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ）活性，线粒体酶复合物Ⅱ亚基（SDHA）、线粒体基质蛋白酶ClpP及ClpP mRNA与蛋白质，线粒体活性氧（ROS）和ATP等表达水平为观察指标，探讨ITP乏力发生与线粒体功能相关性，并探究健脾益气摄血方改善ITP乏力症状的效应机制。

基于“ITP发病和导致患者乏力症状与线粒体能量代谢失衡密切相关”的理论假说。采用被动免疫造模法构建ITP小鼠模型，以“健脾益气摄血方”为干预药物，选择模型小鼠部分器官与组织。观察各组小鼠活动状态、负重游泳时间、体重与脏器指数、PLT、血小板计数等有效指标，检测线粒体功能相关指标，包括ROS含量、mtDNA相对拷贝数、ATP含量及SDHA、ClpP、LonP1表达。结果：与正常组比较，模型组（p＜0.01）、强的松组（p＜0.05）、健脾益气摄血各剂量组（p＜0.01）小鼠脾脏组织ROS含量增多，脾脏组织线粒体DNA相对拷贝数明显下调（p＜0.01），结肠组织ATP含量明显下调（p＜0.01）；与模型组比较，强的松组、健脾益气摄血各剂量组小鼠脾脏组织ROS含量减少，脾脏组织线粒体DNA相对拷贝数未见统计学差异（p＞0.05），结肠组织ATP含量均显著升高（p＜0.01）；SDHA在ITP小鼠的脾脏、心脏、脑组织中表达升高（p＜0.01），强的松组和健脾益气摄血方高剂量组脾脏SDHA蛋白表达水平下调（p＜0.01），与相应组织的SDHA mRNA转录水平检测结果基本一致；ClpP在ITP小鼠脾脏、心脏和骨骼肌组织中表达明显上调（p＜0.05），各治疗组小鼠脾脏ClpP表达明显下调（p＜0.05），与相应组织的ClpP mRNA转录水平检测结果基本一致；LonP1在ITP小鼠心脏、骨骼肌组织中表达明显上调（p＜0.05），在ITP小鼠脾脏中表达明显下调（p＜0.01），强的松与健脾益气摄血方中剂量可使其表达上调（p＜0.05），与相应组织的LonP1 mRNA转录水平的检测结果基本一致。研究证实了ITP乏力的发生发展与线粒体功能障碍及能量代谢失衡密切相关；调节线粒体关键蛋白酶表达，维护线粒体蛋白质稳态，改善线粒体功能是健脾益气摄血方缓解ITP乏力的关键效应机制。本研究结果进一步丰富了“从脾论治”ITP的理论依据和科学内涵。