葛根总黄酮诱导NB4细胞自噬及分子机制

The effect of the arsenic trioxide (ATO), as the active ingredient from traditional Chinese medicine white arsenic, on the apoptosis, autophagy and/or differentiation in acute promyelocytic leukemia (APL) has gotten great achievements worldwidely. We have demonstrated that the proteins as BCL-2, JNK, ERK, Beclin1 and the characteristic autophagy related protein LC3-Ⅱ could be up-regulated in NB4 cells in exposure to 10µg/ml PRF 48 hours. Based on the previous studies, furtherly exposed NB4 cells to the lower-dose PRF(<=20μg/ml) w/o ATO(0.25、0.5µM), the autophagic and synergetic effects of PRF combined with lower-dose ATO would be detected by transmission election microscopy, flow cytometry, western blot and RT-PCR. Cell survival and differentiation would be analyzed by cell cycle, CD11 and CD15 using flow cytometry. Combined lower-dose PRF with various inhibitors such as Rapamycin vs mTOR, or 3-MA vs PI3K, or U0126 vs MEK, or SCH7729984 vs ERK to dealing with NB4 cells , the related signaling molecules at the levels of proteins or mRNAs in PI3K/Akt/mTOR and/or MEK/ERK autophagic pathways would be determined to explain the possible molecular mechanism of NB4 cells autophagy induced by lower-dose PRF in vitro.

中药砒霜有效成分三氧化二砷（ATO）诱导急性早幼粒细胞白血病（APL）细胞凋亡、自噬或分化令世人瞩目。课题组前期研究证明10µg/ml 葛根总黄酮（PRF）处理APL细胞株NB4细胞48h，BCL-2、JNK、ERK活化，Beclin1及特征性自噬蛋白LC3-Ⅱ上调。本项目是课题组既往研究的延续，以低剂量PRF(<=20μg/ml)+ATO(0.25、0.5μM)处理NB4细胞，应用透射电镜、流式细胞术、Western blot、RT-PCR等技术验证低剂量PRF诱导NB4细胞自噬，与低浓度ATO的相互作用；细胞周期及CD11b、CD15等了解细胞生存及转归；PRF联合mTOR抑制剂雷帕霉素，PI3K抑制剂3-MA，MEK抑制剂U0126、ERK抑制剂SCH7729984，探讨低剂量PRF诱导NB4细胞自噬与PI3K/Akt/mTOR和/或MEK/ERK自噬途径的关系，阐明可能的分子机制。

葛根总黄酮（PRF）是中药葛根的总黄酮提取物，课题组前期研究证实 PRF对多种白血病细胞株有增殖抑制和诱导凋亡的作用，可能与JNK活化有关，其中以急性早幼粒细胞白血病细胞株NB4细胞最为明显。因此，本研究按国自然标书计划，进一步采用MTT、FCM、Western blot等方法，探讨较低浓度PRF诱导NB4细胞凋亡抑或自噬的作用及可能的分子机制。结果显示10、30、50μg/ml PRF显著抑制NB4细胞增殖，引起NB4细胞凋亡，细胞周期显示G1期比例增加，G2期比例降低。凋亡标志蛋白的检测发现PRF上调caspase-9，cleaved-PARP和cleaved-caspase-3的表达，对Bcl2家族蛋白的检测显示抗凋亡蛋白Bcl-xL和Bcl-2表达降低，促凋亡蛋白Bax表达上调；检测MAPKs家族蛋白发现PRF对p38和ERK表达没有明显影响，但下调JNK蛋白表达，继而检测JNK通路的磷酸化表达显示PRF引起p-JNK，p-c-Jun和p-MKK4表达上调。检测自噬相关蛋白的表达发现10、30、50μg/ml PRF引起NB4细胞的Beclin1和LC3Ⅱ表达上调，而p62表达下调。进一步采用30μg/ml PRF分别联合自噬抑制剂3-MA或自噬激动剂雷帕霉素，发现联合3-MA组cleaved-PARP和cleaved caspase-3较单药组表达增加，而联合雷帕霉素组表达降低。提示PRF干预NB4细胞自噬可能是一种保护机制，但自噬作为一种大分子蛋白降解的主要方式，其表达升高也可能与PML-RARα融合蛋白降解有关，可能与经典的PI3K/Akt/mTOR途径有关。本课题初步阐明了中药葛根有效成分PRF对APL细胞的诱导凋亡和自噬的作用及分子机制，肯定了PRF对APL的治疗中的价值，为PRF的临床试验和应用提供了更为确凿的研究基础和依据。