糖皮质激素经P-糖蛋白途径阻止HBV相关急性肝衰竭发生的分子机制

The mortality of patients with acute liver failure (ALF) is extremely high. Glucocorticosteroid is only one drug that has been confirmed to be effective in preventing the development of ALF. However, the mechanism of glucocorticosteroid treatment is not clear. Published data showed that the improvement of hepatoprotection was very important to prevent death of hepatocytes and the development of ALF, and cytoprotection associated P-glycoprotein (P-gp) played an important role in prevent death of tumor cells. Our previous results showed that enhanced expressionf of P-gp was observed in the hepatocytes of patients after receiving glucocorticosteroid therapy. So, the following hypothesis is proposed: glucocorticosteroid is able to prevent the development of ALF associated with HBV through the pathway of P-gp mediated hepatoprotection. Firstly, P-gp mediated hepatoprotection will be confirmed to be a key mechanism of glucocorticosteroid preventing death of hepatocytes and the development of ALF among patients with chronic HBV infection, and in murine model of ALF, and in cultured hepatocytes. Secondly, different protein kinase inhibitors and RNA interference are used to study the roles of protein kinase C (PKC)/NF-κB, phosphatidylinositol 3-kinase (PI3K) and human pregnane X receptor (PXR) pathway in involving P-gp expression of L02 cell lines induced by glucocorticosteroid. Finally, a eukaryotic expression vector containing cDNA from human wild-type or mutant-type multidrug resistance 1 (MDR1) is stably transfected into L02 cell lines to study the effect of P-gp on intracellular reactive oxygen species (ROS), mitochondrial membrane permeability transition (MPT) and caspase activation of L02 cell lines. The results of our study mentioned above will be helpful to clarify the molecular mechanism for glucocorticosteroid preventing the development of HBV associated acute liver failure via P-glycoprotein pathway, and to provide some evidences for glucocorticosteroid to be used in treating acute pre-liver failure (pre-ALF) associated with HBV.

急性肝衰竭(ALF)病死率高，糖皮质激素(GC)是唯一能有效阻止ALF发生的药物，但治疗机制未明。增强肝细胞保护是阻止肝细胞死亡与ALF发生的关键，P-糖蛋白(P-gp)介导的细胞保护在阻止肿瘤细胞死亡中起重要作用，我们前期研究显示GC治疗后肝细胞增强表达P-gp,由此推测：GC可能经P-gp途径阻止HBV相关ALF的发生。本研究拟先在HBV感染者、鼠ALF模型和培养细胞中证实增强P-gp介导的肝细胞保护是GC阻止肝细胞死亡与ALF发生的关键途径；然后，采用蛋白酶抑制剂和RNA干扰技术，探讨PKC/NF-kB、PI3K与PXR信号途径在GC诱导L02细胞P-gp表达中的作用；最后，将野生或突变型人MDR1 cDNA稳定转染L02细胞，探讨P-gp对L02细胞ROS、MPT与Caspase激活的影响，从而阐明GC经P-gp途径阻止ALF发生的分子机制，为用GC治疗急性肝衰竭前期提供实验依据。

急性肝衰竭(ALF)病死率高，糖皮质激素是唯一可能有效阻止ALF发生的药物、但疗效尚存争议，其原因主要是糖皮质激素的治疗机制尚未阐明。为了阐明糖皮质激素经P-糖蛋白(P-gp)途径阻止HBV相关急性肝衰竭发生的分子机制，我们进行了下列研究：①P-gp在糖皮质激素阻止HBV相关急性肝衰竭发生中的作用；②P-gp介导的肝细胞保护在糖皮质激素阻止鼠急性肝衰竭发生中的作用；③P-gp在糖皮质激素阻止肝细胞死亡中的作用；④糖皮质激素在调控L02细胞P-gp表达中的作用与分子机制。结果显示：①轻度CHB患者肝组织P-gp水平显著低于pre-ACLF和ACLF患者，但pre-ACLF患者肝组织P-gp水平显著高于ACLF患者；②糖皮质激素治疗组肝衰竭患者的存活率显著高于未用激素的肝衰竭患者；③在170例HBV相关pre-ACLF患者中，糖皮质激素治疗组的肝衰竭发生率与存活率分别为8.9%和96.4%，而非激素治疗组的肝衰竭发生率与存活率分别为70.2%和52.6%，两者间相差非常显著；④地塞米松治疗后患者肝细胞P-gp表达水平显于高于治疗前，而地塞米松治疗后患者肝细胞凋亡较治疗前显著减少；⑤在GalN/LPS所致的急性肝衰竭鼠模型中，糖皮质激素治疗组鼠的存活率显著高于非激素治疗组，糖皮质激素治疗组鼠肝细胞P-gp表达水平显著高于非激素治疗组鼠，糖皮质激素治疗组鼠肝细胞凋亡数量显著少于非激素治疗组鼠；⑥地塞米松处理组L02细胞的P-gp蛋白与mRNA表达水平显著高于未用地塞米松处理的L02细胞，地塞米松与TRAIL联合处理组L02细胞的凋亡水平显著低于单用TRAIL处理的L02细胞，地塞米松、TRAIL与P-gp抑制剂Tariquidar联合处理组L02细胞的凋亡水平显著高于地塞米松与TRAIL联合处理组L02细胞；⑦联合应用地塞米松与Akt抑制剂LY294002处理的L02细胞P-gp蛋白与 mRNA表达水平较单独应用地塞米松组显著降低（P＜0.05），联合应用地塞米松与NF-kB抑制剂PDTC处理的L02细胞P-gp蛋白与 mRNA表达水平较单独应用地塞米松组显著降低（P＜0.05），转染针对人PXR siRNA的L02细胞P-gp蛋白与 mRNA表达水平较转染非针对人PXR siRNA的L02细胞显著降低（P＜0.01）。上述结果提示，糖皮质激素通过激活PI3K/Akt、NF