基于PI3K/AKT及Myc信号通路调控探讨益气除痰法治疗弥漫大B细胞淋巴瘤的机制研究

Diffused large B-cell lymphoma ( DLBCL) represents the largest group of non Non-Hodgkin’s lymphoma. In the theory of traditional Chinese medicine (TCM), this tumor was usually characterized by vital energy asthenia and phlegm stagnancy. Therefore, we prescribed Benefiting Vital Energy and Eliminating Phlegm of TCM for patients with this tumor and achieved satisfactory clinical efficacy. Preliminary cytological experiments found that Benefiting Vital Energy and Eliminating Phlegm of TCM can inhibit the activity of DLBCL, but the specific mechanism is not clear.Our previous research revealed the high protein expression of p110α and its downstream Foxp1 protein ,which indicated the possible activation of PI3K/AKT and downstream Myc signaling pathway.We also detected inhibited pAKT expression and increased anoikis in lung cancer cells treated with Benefiting Vital Energy and Eliminating Phlegm of TCM. Thus we hypothesized that Benefiting Vital Energy and Eliminating Phlegm of TCM could regulate vital proteins in the PI3K and Myc signaling pathway to suppress the growth of lymphoma cells. The purposes of this study were to observe the anti-tumor activity and chemotherapy sensitization effect of Benefiting Vital Energy and Eliminating Phlegm of TCM in vitro and in vivo, to investigate its effect on cell apoptosis and cell cycles and to detect its role in regulating the vital proteins expression including p110α,pAKT, PTEN, Myc, miR-34a and Foxp1. As a result, we could screen for target proteins of Benefiting VitaI Energy and Eliminating Phlegm of TCM in treating DLBCL. We hoped to provide a theoretical foundation of individual and integrated traditional and western medicine treatment of DLBCL on the basis of this study.

弥漫大B细胞淋巴瘤（DLBCL）是最常见的非霍奇金淋巴瘤，气虚痰结是DLBCL基本病机之一。我科采用益气除痰法拟方治疗该肿瘤取得良好疗效。实验发现益气除痰方可以抑制DLBCL的活性，但具体机制不清。我们前期工作提示DLBCL中存在p110α及下游Foxp1蛋白的过度表达，提示PI3K/AKT通路及下游Myc通路存在激活，并发现益气除痰方可以抑制pAKT表达并诱导肺癌细胞凋亡。因此我们推测益气除痰方可通过调控PI3K及Myc信号通路中的关键分子来抑制淋巴瘤生长。本研究拟观察益气除痰方对于DLBCL细胞和移植瘤模型的抑瘤活性及化疗增敏活性，明确该方对淋巴瘤细胞凋亡及增殖周期的影响，并检测该方对PI3K及Myc信号通路中的关键分子p110α、pAKT、PTEN、Myc、miR-34a及Foxp1的调控作用，从中筛选益气除痰方的靶蛋白。本研究将为DLBCL的中西医结合及个体化治疗提供新的理论基础。

弥漫大B细胞淋巴瘤（DLBCL）是最常见的非霍奇金淋巴瘤，气虚痰结是DLBCL基本病机之一。我科采用益气除痰法拟方治疗DLBCL取得良好疗效，但具体机制不清。本研究发现益气除痰方对DLBCL细胞系OCI-ly10和SUDHL-6的抑制作用均具有浓度和时间依赖性。高浓度中药与低浓度和高浓度阿霉素联合使用具有协同抗肿瘤作用。益气除痰方可以诱导DLBCL细胞发生凋亡，机制如下：一、益气除痰方可以上调PTEN的表达，下调PI3K/PTEN/AKT信号通路蛋白包括p110а、pAKT、pPRAS40、pGSK和pS6蛋白表达水平从而促进凋亡。二、Myc/miR-34a/ Foxp1通路可以调控DLBCL细胞系SUDHL-6的凋亡，抑制cmyc可以上调miR-34a表达，从而下调转录因子Foxp1水平，进而促进相应的凋亡指标Bax和PARP上调，Bcl2下调，从而诱导凋亡。益气除痰方可以使miR-34a表达上调，FOXP1表达下调，凋亡因子Bax，PARP表达上调；Bcl2，PCNA表达下调，从而促进细胞凋亡。体内实验采用ly10细胞建立DLBCL小鼠移植瘤模型，对照、中药、阿霉素组和联合组四组荷瘤鼠移植瘤体积变化存在显著性差异，中药、阿霉素组和联合组移植瘤体积均较对照组小。四组荷瘤鼠移植瘤瘤重存在显著性差异，中药、阿霉素组和联合组移植瘤瘤重均较对照组轻。中药、阿霉素组和联合组的抑瘤率分别为26.4%，31.2%和47.3%。中药组与对照组相比，pAKT，pPRAS40，pS6，pGSK四种蛋白的表达均呈现下降的趋势。本项目的完成为DLBCL的个体化治疗及中西医结合治疗提供新靶点，包括PI3K/PTEN/AKT及Myc/miR-34a/ Foxp1信号通路，今后将致力于在临床实践中推广以上靶点的检测，为临床上精准使用益气除痰方打下基础，更好的为DLBCL患者造福。