补体激活在急性肝衰竭SPHK1活化中机制研究

Our previously study found that the enzyme sphingosine kinase 1(SphK1) play an important role in the pathogenesis of acute liver failure,and is an up-stream regulator of HMGB1.Complement activation in murine hepatitis virus strain 3(MHV-3) induced acute liver failure. Pretrantment with Cobra venom factor (CVF,15U/mouse) to deplete complement components abolished MHV-3 induced up-regulation of SphK1 in mice,and complement activation may be one of the factor on SphK1 activation in acute liver failure.In the present study,we investigate the potential role of complement activation in MHV-3 and D-GalN/LPS induced animal model and acute liver failure patients,and which pathway play an important role in the pathogenesis of acute liver failure.Examine the association of complement activation and up-regulation of SphK1 in acute failure,and to explore the mechanism of SphK1 up-regulation through C5a and its receptor C5L2 and CD88 interaction.Ateempt to elucidate complement activition and which pathway is essential for progression of acute liver failure and SphK1 up-regulation, and provide potential novel therphies to treat acute liver failure and other liver inflammatory disorders.

在前期工作发现SphK1在急性肝衰竭发病中具有重要作用，是HMGB1上游调控分子。MHV-3感染小鼠急性肝衰竭模型存在补体激活，CVF清除补体下调SphK1水平，补体激活可能是SphK1活化的始动因素的基础上，进一步研究补体激活在MHV-3感染和D-GalN/LPS诱导急性肝衰竭小鼠以及急性肝衰竭患者发病中的作用和急性肝衰竭补体激活途径，急性肝衰竭补体激活与Sphk上调的关系，探究急性肝衰竭肝脏C5a通过C5L2和CD88受体调控SphK1的机制。进一步弄清补体激活及其途径在肝衰竭发生和调控Sphk1的机制，为肝衰竭的治疗提供新的干预靶点。

近年来发现，补体激活在急性肝衰竭(ALF)中发挥重要作用。我们的前期研究发现SphK1在ALF发病中具有重要作用，ALF存在补体激活，CVF 清除补体下调SphK1，进一步研究补体激活上调SphK1的机制。.本项目建立和优化D-GalN/LPS和对乙酰氨基酚ALF两种小鼠模型，上述模型和慢加亚急性肝衰竭早期患者补体高度激活，表现为血浆C3水平降低、补体活化产物C3a和C5a水平升高、肝脏C3aR和C5aR的mRNA水平增加。CVF预处理部分清除补体显著降低血清C3a、C5a水平和肝脏C3aR、C5aR的mRNA表达，显著降低血清ALT/AST、炎症因子（TNF-a、IL-β、IL-6）和HMGB1水平，减轻肝脏炎症坏死和HMGB1肝细胞质移位，提高生存率和促进急性肝衰竭肝细胞再生。.补体经典途径的激活物和成分免疫复合物水平和C1q水平在ALF小鼠和慢加亚急性肝衰竭患者无明显变化。补体旁路激活途径抑制剂CR2-FH预处理降低急性肝衰竭12h血清C3a和C5a峰值水平、C3aR和C5aR的mRNA表达和肝脏补体C3b沉积，降低血清ALT/AST、炎症因子、HMGB1、肝脏炎症坏死和死亡率。伴随补体激活，肝脏SphK1表达升高，CVF清楚补体下调肝脏SphK1表达。.SphK1基因敲除降低PBMC和腹腔巨噬细胞LPS刺激的C5L2表达，而对CD88表达无影响，表明SphK1对LPS刺激小鼠巨噬细胞或PBMC的C5L2表达具有重要作用。阻断C5a/C5aR 通路降低D-GalN/LPS 诱导ALF的肝损伤和提高生存率，下调D-GalN/LPS诱导ALF肝脏和C5a刺激巨噬细胞的SphK1表达。C5a/C5aR 通路诱导巨噬细胞SphK1表达通过p38 MAPK 活化。.ALF小鼠肝脏iRhom2高表达，siRNA下调iRhom2表达保护ALF肝损伤。C5aRa阻断C5a/C5aR通路下调ALF和C5a刺激腹腔巨噬细胞iRhom2表达，C5a/C5aR 通路诱导的iRhom2表达通过p38-MAPK活化。.综上所述，补体激活特别是旁路途径激活上调SphK1在ALF发病具有重要作用，补体活化产物C5a通过C5a/C5aR通路激活p38-MAPK磷酸化上调SphK1和iRhom2表达,为ALF治疗提供新的干预靶点。