α-苦瓜素经LRP1受体介导的JNK信号通路诱导肝细胞L02早期凋亡的机制研究

Study on the Mechanism of α-momordicin Inducing Early Apoptosis of Hepatocyte L02 via LRP1-mediated JNK Signaling Pathway

目的:探讨α-苦瓜素诱导肝细胞L02(简称"L02细胞")早期凋亡的信号通路。方法:制备并纯化α-苦瓜素。采用流式细胞术检测0(阴性对照)、160、80μg/mLα-苦瓜素作用L02细胞2～8 h后的细胞凋亡率。小干扰RNA(siRNA)沉默低密度脂蛋白受体相关蛋白1(LRP1)得到LRP1-siRNA细胞,然后采用液相芯片分析术检测α-苦瓜素(160μg/mL)分别作用L02细胞(正常组)和LRP1-siRNA细胞(沉默组)0、0.25、0.5、1、2 h后c-Jun氨基末端激酶(JNK)信号通路中促调蛋白磷酸化JNK(p-JNK)、磷酸化凋亡前体蛋白(p-Bad)、胱天蛋白酶9(Caspase-9)及抑调蛋白磷酸化蛋白53(p-p53)、磷酸化蛋白激酶B(p-Akt)、磷酸化B淋巴细胞瘤2(p-Bcl-2)、Caspase-8的表达情况,分析α-苦瓜素对L02细胞JNK信号通路的作用。结果:制备并纯化得到α-苦瓜素(纯度>97%);与阴性对照组比较,160μg/mLα-苦瓜素作用8 h时,可显著诱导细胞早期凋亡(P<0.05),80μg/mLα-苦瓜素诱导的早期凋亡不明显(P>0.05);与0 h比较,作用后0.25 h促调蛋白p-JNK、p-Bad和Caspase-9表达量显著增加(P<0.05),而抑调蛋白pp53、p-Akt、p-Bcl-2和Caspase-8表达量无变化;与正常组相比,沉默组各时间点的p-JNK、p-Bad和Caspase-9的表达均显著减少(P<0.05)。结论:α-苦瓜素可能通过LRP1受体介导JNK信号通路诱导肝细胞凋亡,为揭示其肝毒性提供了参考。

OBJECTIVE：To explore the signaling pathway of α-momordicin inducing early apoptosis of hepatocyte L02（called “the L02 cell”for short）. METHODS：α-momordicin was prepared and purified. Flow cytometry was used to detect the apoptotic rate of the L02 cell after treated with 0（negative control）,160 and 80 μg/mL of α-momordicin for 2-8 h. LRP1-siRNA cells were obtained by small interfering RNA（siRNA）silencing low density lipoprotein receptor-related protein 1（LRP1）. The expression of proapoptotic protein p-JNK,p-Bad,Caspase-9,inhibitor of apoptosis protein p-p53,p-Akt,p-Bcl-2 and active Caspase-8 in the L02 cell （normal group） and LRP1-siRNA （silence group） after treated with α-momordicin for 0,0.25,0.5,1,2 h were determined by liquid biochip analysis in c-Jun N-terminal kinase （JNK） signaling pathway. Effects of α-momordicin on JNK signaling pathway were analyzed. RESULTS：α-momordicin could be prepared and purified （purity〉97%）. Compared with negative control group,160 μg/mL α-momordicin could significantly induced early apoptosis of the cell after treated for 8 h（P〈0.05）;early apoptosis of the cell induced by 80 μ g/mL α-momordicin was not obvious （P〉0.05）. Compared with 0 h,the expression of p-JNK,p-Bad and Caspase-9 were increased significantly and even reached the peak value 0.25 h after medication （P〈0.05）,while the expression of p-p53,p-Akt,p-Bcl-2 and Caspase-8 had no change. Compared with normal group,the expression of p-JNK,p-Bad and Caspase-9 of silence group were decreased significantly at different time points （P〈0.05）. CONCLUSIONS：α-momordicin can induce the apoptosis of hepatocytes via LRP1 receptor mediated JNK signaling pathway,and will provide the reference for its hepatotoxicity.