基于“溶酶体-线粒体轴心”理论探讨人参皂苷Rh2脂肪酸酯衍生物诱发H22细胞凋亡机制

Ginsenoside Rh2 has an anticancer action, however, it was confirmed to have a low bioavailability in vivo and difficult to be used for clinic. In preliminary experiments, we have synthesized Rh2 fatty acid esters and confirmed Rh2 esters had a better bioavailability. Meanwhile, the effects of Rh2 esters on inducing apoptosis of H22 cells were significantly higher than Rh2. So far, the molecular mechanisms of Rh2 ester inducing the apoptosis of H22 cells are still unclear. Previous studies indicated that the permeability of lysosomal membrane played an important role in apoptosis, known as lysosomal pathway of apoptosis. Based on our preliminary studies, we propose that Rh2 esters can induce apoptosis in H22 cells by "lysosomes - mitochondrial axis" signal pathway. In the present study, two-dimensional gel electrophoresis and mass spectrometry are used to screen the related proteins which induce the permeability of lysosomal membrane；The changes of ROS,sphingosine,and pH value during the permeation of lysosmal membrane are analyzed; a laser scanning confocal microscopy is applied to observe the changes of Bax translocation; Thereafter, RNAi technology,western blot, and qRT-PCR are used to illustrate whether cathepsins which released from lysosome could activate apoptogenic protein Bid, Bax, and then cause the release of Cyt C from mitochondria; later, accumulation of Cyt C could trigger the signal pathway of caspases. The study in vitro are also proven by in-vivo experiments. The results of this reseach could provide a theoretical basis for revealing the anticancer mechanism of Rh2 ester.

人参皂苷Rh2具有抗癌作用，然而Rh2被证实在体内生物利用率低，难以发挥功效。前期我们合成Rh2脂肪酸酯并证实其生物利用率显著提高且诱导肝癌H22细胞凋亡能力显著高于Rh2，但其分子机制不明。基于溶酶体在细胞凋亡早期中重要作用，本项目进一步研究"溶酶体-线粒体轴"通路在Rh2酯诱导H22细胞凋亡中作用。拟用双向凝胶电泳和质谱技术筛选Rh2酯诱发溶酶体膜通透增加的相关蛋白、激光共聚焦观察线粒体膜上Bax蛋白转位、测定细胞内ROS、鞘氨醇含量及pH值变化等来分析Rh2酯引发溶酶体膜通透增加的相关因子；利用RNAi、免疫印迹、实时PCR技术探讨溶酶体膜通透增加所释放的组织蛋白酶是否可激活凋亡蛋白Bid、Bax表达，促线粒体释放Cyt C、进而激活Caspases等下游凋亡通路。细胞实验进一步通过体内试验进行验证。本项目研究将阐明Rh2酯诱导H22细胞凋亡分子机制，为Rh2酯抗癌作用提供理论支持。

人参皂苷Rh2具有抗癌作用，然而Rh2被证实在体内生物利用率低，难以发挥功效。前期我们合成Rh2脂肪酸酯并证实其生物利用率显著提高且诱导肝癌H22细胞凋亡能力显著高于Rh2，但其分子机制不明。基于溶酶体在细胞凋亡早期中重要作用，本项目进一步研究“溶酶体-线粒体轴”通路在Rh2酯诱导H22细胞凋亡中作用。研究表明：1）Rh2-O对HepG2细胞生长抑制作用和诱导HepG2细胞凋亡的能力均优于Rh2。Rh2-O处理HepG2细胞24 h的IC50为20.15 μM，约为Rh2的IC50的一半。IC50剂量下的Rh2-O对人正常肝细胞HL-02无显著毒性。2）Rh2-O通过下调细胞内周期蛋白cyclin D3，cyclin E，CDK4，CDK6，p21WAF1/CIP1和p27KIP1的表达水平诱导HepG2细胞发生G1周期阻滞。p38 MAPK磷酸化和Akt失活参与了Rh2-O诱发HepG2细胞G1周期阻滞这一过程；3）Rh2 和Rh2-O可同时通过外在死亡受体信号通路和内在线粒体信号通路激活caspase家族凋亡蛋白，诱导细胞凋亡。溶酶体膜通透化是线粒体功能紊乱的上游通路；4）Rh2-O和Rh2均是通过诱导肿瘤细胞凋亡、改善荷瘤小鼠的免疫系统和抑制肿瘤血管生成来实现抗肿瘤活性。Rh2和Rh2-O均可通过调节Bcl-2家族蛋白的表达水平，并激活caspase家族蛋白来诱导肿瘤细胞凋亡。另一方面，Rh2和Rh2-O通过增加IL-2 和TNF-α水平，T淋巴细胞数量，CD4+/CD8+比例和NK细胞水平来改善荷瘤小鼠的免疫系统；5）Rh2和Rh2-O的体内体外抗肿瘤活性的作用机制相似。Rh2-O的抗肿瘤活性优于Rh2可能是由于Rh2-O具有更高的吸收率。