血液净化治疗多脏器衰竭犬对血浆磷脂代谢与凋亡信号途径的影响及分子机制研究

Multiple organ dysfunction syndrome (MODS) is the leading cause of morbility and mortality in critical illness. It is reported that continuous blood purification (CBP) provided better haemodynamic stability and increased survival in clinical trial. Abnormal membrane-lipid microenvironment induced changes of plasma phospholipid metabolism is closely related to the endoplasmic reticulum(ER) stress mediated apoptosis signal pathway in MODS. A standard dog MODS model was established by hemorrhagic shock plus resuscitation and endotoxiemia, these dogs were randomly assigned to either treatment with continuous veno-venous hemodiafiltration(CVVHDF group, n=8) or spontaneous course (MODS group, n=8). In order to investigate the effects of CVVHDF on the endoplasmic reticulum stress mediated apoptosis signal pathway and plasma phospholipid metabolism profile in dogs with MODS, we detect the percentage of apoptosis rate in multiple organs, test CHOP、JNK、caspase-12 mRNA and protein levels of ER stress, analyze of plasma phospholipid metabolism profile. Further exploration of the relationship between the changes of plasma phospholipid metabolism profile and ER stress mediated apoptosis signal pathway in MODS, and molecular mechanisms of CVVHDF ameliorate apoptotic signaling pathway can provide new therapeutic targets for the prevention and treatment of MODS.

多器官功能障碍综合征(MODS)病情凶险、病死率高。连续性血液净化(CBP)治疗MODS确有疗效,可降低MODS病死率。MODS病程中膜脂微环境改变所致的血浆磷脂代谢变化与内质网应激介导细胞凋亡信号途径的异常表达密切相关。本研究建立犬MODS模型,随机分为对照组和治疗组，治疗组行连续性血液透析滤过(CVVHDF)治疗。观察CVVHDF治疗MODS犬中对内质网应激介导细胞凋亡重要信号蛋白表达及血浆磷脂代谢的影响：1.脏器组织细胞凋亡率；2.内质网应激标志蛋白基因CHOP、JNK及 caspase-12mRNA表达；3.内质网应激标志蛋白CHOP、JNK及caspase-12蛋白表达；4.血浆磷脂代谢轮廓分析。揭示MODS病变过程中血浆磷脂代谢变化与内质网应激介导细胞凋亡重要信号途径异常表达之间的相互关系，及CVVHDF减轻机体细胞凋亡的分子作用机制，为防治MODS所致细胞凋亡提供新的治疗靶点

目的：观察CVVHDF治疗MODS犬中对内质网应激介导细胞凋亡重要信号蛋白表达及血浆磷脂代谢的影响，揭示MODS病变过程中血浆磷脂代谢变化与内质网应激介导细胞凋亡重要信号途径异常表达之间的相互关系，及CVVHDF减轻机体细胞凋亡的分子作用机制。方法：14只Beagle犬采用失血性休克+复苏再灌注+内毒素血症建立MODS模型，随机分为CVVHDF组(n=8)和MODS组(n=6)，CVVHDF组在内毒素注射完毕后给于CVVHDF治疗24小时，MODS组不给CVVHDF治疗。观察CVVHDF治疗MODS犬中对内质网应激介导细胞凋亡重要信号蛋白表达及血浆磷脂代谢的影响。结果：①生命体征及器官功能指标：CVVHDF治疗后平均动脉压和心率基本保持平稳、体温下降、尿量增加，心功能、肺功能、肾功能指标明显改善。②肝脏、肾脏、肺脏组织病理学改变：CVVHDF组器官组织病理提示组织损伤明显，经CVVHDF治疗后组织损害明显减轻。③器官组织细胞凋亡：与MODS组相比，CVVHDF组肝脏、肾脏、肺脏细胞凋亡明显降低。④内质网应激介导细胞凋亡重要信号途径检测：与MODS组相比，CVVHDF组肝脏组织中JNK2 mRNA基因表达明显升高，在肺脏组织中表达明显降低；CVVHDF组肾、肺组织中caspase12 mRNA基因表达明显降低。CVVHDF组目的基因CHOP蛋白含量水平在肝脏、肾脏组织中显著升高，JNK2蛋白含量水平在肝脏组织细胞无明显改变，caspase12蛋白表达在肾脏组织细胞无明显改变。⑤代谢组学分析鉴定差异代谢产物：与MODS组相比，CVVHDF组治疗后硫酸对甲酚、乳酸、柠檬酸、马尿酸、胆碱、LPEs和MAGs等代谢产物水平升高，而肉碱和PC38:4水平降低。结论: CVVHDF治疗通过改变促炎因子与抗炎因子平衡来调节免疫内环境稳态，减轻内质网应激所触发的内质网凋亡信号通路，降低磷脂酶活性，抑制组织器官损伤从而减缓MODS进展。