南蛇藤茎提取物齐墩果烷型三萜化合物对胃癌细胞BGC-823增殖和凋亡的作用

Extracted from Nansheteng(Celastrus orbiculatus) Inhibits Proliferation and Apoptosis of Gastric Cancer BGC-823 Cells

目的探讨南蛇藤茎提取物齐墩果烷型三萜化合物对人胃癌BGC-823细胞增殖和凋亡的影响,以期为进一步开发南蛇藤多萜成为抗肿瘤药物奠定更加坚实的理论和实验基础。方法实验分为空白组,南蛇藤茎提取物齐墩果烷型三萜化合物组(28-Hydroxy-3-oxoolean-12-en-29-oic acid, 10、20、40、80、160μmol·L;)。细胞计数试剂盒(cell count kit, CCK-8)检测南蛇藤茎提取物齐墩果烷型三萜化合物对BGC-823细胞增殖能力的影响,并确定南蛇藤茎提取物齐墩果烷型三萜化合物的用药浓度,高内涵成像进一步观察南蛇藤茎提取物齐墩果烷型三萜化合物的对BGC-823细胞分裂增殖能力的影响,线粒体膜电位检测试剂盒检测南蛇藤提取物齐墩果烷型三萜化合物对BGC-823细胞线粒体膜电位水平的影响,磷脂酰丝氨酸蛋白抗体-异硫氰基荧光素/碘化丙啶(Annexin V-fluoresceine isothiocyanate/Propidium Iodide, Annexin V-FITC/PI)法检测南蛇藤提取物齐墩果烷型三萜化合物对BGC-823细胞凋亡的影响,免疫印迹法(Western Blot)检测南蛇藤茎提取物齐墩果烷型三萜化合物对BGC-823细胞相关凋亡蛋白的表达水平。结果与空白组比较,随着南蛇藤茎提取物齐墩果烷型三萜化合物用药浓度的升高,BGC-823细胞增殖分裂能力逐渐降低(P<0.01),根据CCK-8实验结果计算出半抑制浓度(IC_(50)),筛选出新的南蛇藤茎提取物齐墩果烷型三萜化合物组(28-Hydroxy-3-oxoolean-12-en-29-oic acid, 40、80、160μmol·L^(-1))。该三萜化合物还能够使线粒体膜电位水平下降,诱导细胞凋亡(P<0.05),同时抑制B细胞淋巴瘤/白血病-2(B-cell lymphoma-2,Bcl-2)蛋白表达(P<0.01),促进Bcl-2相关X蛋白(BCL2-Associated X, Bax)和剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved cysteinyl aspartate specific proteinase-3,Cleaved Caspase-3)蛋白表达(P<0.01)。结论南蛇藤茎提取物齐墩果烷型三萜化合物能够抑制胃癌BGC-823细胞分裂和增殖,诱导细胞凋亡,该过程可能与降低线粒体膜电位水平,同时下调Bcl-2蛋白表达,上调Bax和Cleaved Caspase-3蛋白表达有关。

Objective To investigate the effects of oleanolane triterpenes extracted from Nansheteng(Celastrus orbiculatus)(28-hydroxy-3-oxoolean-12-en-29-oic acid) on the proliferation and apoptosis of human gastric cancer BGC-823 cells. Methods The experiment was divided into blank group, 28-hydroxy-3-oxoolean-12-en-29-oic acid groups(10,20,40,80,160 μmol·L^(-1)). Cell count kit(CCK 8) detected the effect of 28-hydroxy-3-oxoolean-12-en-29-oic acid on BGC-823 cell proliferation, and the drug concentration was determined. High intension imaging method was used to observe the effect on the cell division. The mitochondrial membrane potential detection kit was used to determine the effect on mitochondrial membrane potential of BGC-823 cells. Annexin V-FITC/PI method was used to detect the apoptosis. Western Blot was used to detect the expressions of apoptosis related proteins of BGC-823 cells. Results With the increase of the concentration of 28-hydroxy-3-oxoolean-12-en-29-oic acid, the proliferation and division of BGC-823 cells decreased gradually(P<0.01). Based on the results of CCK-8 experiment, the semi-inhibitory concentration(IC_(50)) was calculated, and 28-hydroxy-3-oxoolean-12-en-29-oic acid groups(40,80,160 μmol·L;) were selected. 28-hydroxy-3-oxoolean-12-en-29-oic acid can also decrease the mitochondrial membrane and potentialinduce apoptosis(P<0.05), inhibit the expression of Bcl-2 protein(P<0.01), and promote the expressions of Bax and Cleaved caspase-3 proteins(P<0.01). Conclusion 28-hydroxy-3-oxoolean-12-en-29-oic acid can inhibit BGC-823 cell division and proliferation and induce apoptosis, which may be related to decreasing the mitochondrial membrane, down-regulation of Bcl-2 protein expression and up-regulation of expressions of Bax and Cleaved caspase-3 protein.