白头翁总皂苷碱水解产物的抗肿瘤作用及机制研究
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摘要
目的：前期工作提示白头翁总皂苷具有明显的体内外抗肿瘤作用，其碱水解产物体外抗肿瘤作用优于白头翁总皂苷。本论文将系统探讨白头翁总皂苷碱水解产物（PAHS）的抗肿瘤作用及其可能的机制。
方法：采用HPLC-ELSD法对PAHS中8种含量较高的皂苷进行测定；采用MTT及集落形成实验检测白头翁总皂苷碱水解产物对人肝癌SMMC-7721以及人肺癌A549的增殖抑制作用，同时采用Giemsa染色观察PAHS对肿瘤细胞形态的改变；在体内，建立小鼠肉瘤S180、小鼠肝癌H22、C57小鼠Lewis肺癌模型，取荷瘤小鼠血液进行血液检查并对小鼠肿瘤组织进行病理检查；在机制方面，采用hoechest33342染色观察SMMC-7721凋亡细胞形态变化；通过流式细胞仪检测PAHS对7721及A549细胞凋亡及周期的影响；采用western blot法检测线粒体通路和PI3K通路内重要信号分子的表达。
结果：HPLC结果表明，白头翁总皂苷碱水解产物中8种皂苷单体（白头翁皂苷D、常春藤皂苷元3-O-β-D-吡喃葡萄糖基-(1→4)-α-L-吡喃阿拉伯糖苷、白头翁皂苷、hederacolchiside A1、白头翁皂苷F、齐墩果酸3-O-β-D-吡喃葡萄糖基-(1→4)-β-D-吡喃葡萄糖基-(1→3)-α-L-吡喃鼠李糖基-(1→2)-α-L-吡喃阿拉伯糖苷、齐墩果酸3-O-β-D-吡喃葡萄糖基-(1→3)-α-L-吡喃鼠李糖基-(1→2)-α-L-吡喃阿拉）含量的总和超过85%； MTT及集落形成实验表明PAHS (0.80-25.00μg/ml)可以时间和剂量依赖性的抑制SMMC-7721及A549细胞的增殖，抑制7721细胞集落的形成；体内试验表明白头翁总皂苷碱水解产物(50，100，200mg/kg)可以抑制小鼠肉瘤，小鼠肝癌及肺癌的生长且具有剂量依赖性，在给药剂量200mg/kg时三种模型的抑制率分别达到49.8%、43.7%、70.6%，HE染色表明PAHS组(50，100，200mg/kg)小鼠肿瘤组织均有大面积坏死及凋亡细胞的出现。电镜切片检查发现，荷肝癌小鼠及荷Lewis肺癌小鼠肿瘤组织均出现染色质集聚成团、边集等明显细胞凋亡特征;与CTX组相比，PAHS处理组小鼠的脾系数、胸腺系数均有提高，提示PAHS的免疫抑制作用较小。机制研究表明，PAHS (6.25，12.50，25.00μg/ml)可以诱导SMMC-7721及A549细胞凋亡，同时可以将SMMC-7721细胞周期阻滞在S期，将A549细胞生长阻滞在G0/G1期。western blot实验结果表明白头翁总皂苷碱水解产物可以上调Cyt-C， CleavedCaspase-3，Bax等蛋白的表达，同时下调PI3Kp85、p-Akt、p-mTOR、p-p70S6K等相关蛋白的表达。
结论：白头翁总皂苷碱水解产物具有抗肺癌和肝癌的作用，其机制可能与调节PI3K/AKT/mTOR信号通路引起的细胞凋亡有关。
Aim: In our previous study, we found that total saponins from Pulsatilla chinensisinhibited the growth of tumors in vivo and in virtro. Compared with total saponins, Alkalihydrolysate of total saponins had better antitumor effect. In this study, we will investigateantitumor effect and mechanisms of alkali hydrolysate of total saponins from Pulsatillachinensis.
Methods: The HPLC method was used to determination the contents of eightsaponins in alkali hydrolysate of total saponins from Pulsatilla chinensis; In vitro, MTTand clone formation test were used to examine the growth of human carcinomaSMMC-7721and A549. Additionally, Giemsa staining was also used to observe themorphology of tumour cells; In vivo, ICR mice bearing H22and S180cells were used, C57mice transplanted with Lewis tumor cells were also used. Blood extracted from tumourbearing mice was used to make the blood examination. Tumor tissue was stained withhematoxylin and eosin, and then observed the change of cell morphology; Ultrastructure oftumor tissue was examined with transmission electron microscope; Flow cytometry wasused to detect the cell cycle and apoptosis of tumor cells; Hoechst33258assay kit wereperformed to observe the morphology of apoptosis cells; Important signal molecules ofmitochondrial pathway and PI3K pathway were detected by western blot.
Results: The total content of eight saponins (pulsatilla saponin D, hederagenin3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranoside, pulsatilla saponin A,hederacolchiside A1, pulsatilla saponin F, oleanolic acid3-O-β-D-glucopyranosyl-(1
→4)-β-D-gluco-pyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside, oleanolic acid3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-
arabinopyranoside, oleanolic acid3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopy-ranoside) in alkali hydrolysate of total saponins from Pulsatilla chinensis was more than85%. In vitro, PAHS (0.80-25.00μg/ml) exhibited an inhibited effect on SMMC-7721andA549cells growth in concentration and time-dependent manners by MTT and cloneformation test; In vivo, PAHS (50，100，200mg/kg) could inhibit the tumor weight of H22,S180and Lewis in concentration manners, when treated with200mg/kg, the inhibitionrate were49.8%,43.7%and70.6%. The HE staining of all three tumors were presentedwith wide ranges of necrosis tissue. The results of transmission electronic microscopeshowed that PAHS (50，100，200mg/kg) groups exhibited remarkable ultrastructuralcharacteristics of apoptotic programmed cell death such as nuclei degenerated, cytoplasmshrinkage, chromatin condensated, and decreases of cell volume. Compared with CTXgroup, the number of WBC, spleen index, thymus index were imprpove. These resultsindicated that PAHS had little toxicity. PAHS (6.25,12.50,25.00μg/ml) could induceapoptosis on SMMC-7721and A549cells. Additionally, SMMC-7721cells were arrestedin S phase and A549cells in G0/G1phase. Western blotting showed that PAHS could upregulate the expression of Cyt-C、Cleaved Caspase-3、Bax, and down regulate theexpression of PI3Kp85、p-Akt、p-mTOR、p-p70S6K.
Conclusions: PAHS could inhibit the growth of liver and lung cancer, the mechanismmight be that PAHS induce apoptosis through PI3K/AKT/mTOR pathway.
方法：采用HPLC-ELSD法对PAHS中8种含量较高的皂苷进行测定；采用MTT及集落形成实验检测白头翁总皂苷碱水解产物对人肝癌SMMC-7721以及人肺癌A549的增殖抑制作用，同时采用Giemsa染色观察PAHS对肿瘤细胞形态的改变；在体内，建立小鼠肉瘤S180、小鼠肝癌H22、C57小鼠Lewis肺癌模型，取荷瘤小鼠血液进行血液检查并对小鼠肿瘤组织进行病理检查；在机制方面，采用hoechest33342染色观察SMMC-7721凋亡细胞形态变化；通过流式细胞仪检测PAHS对7721及A549细胞凋亡及周期的影响；采用western blot法检测线粒体通路和PI3K通路内重要信号分子的表达。
结果：HPLC结果表明，白头翁总皂苷碱水解产物中8种皂苷单体（白头翁皂苷D、常春藤皂苷元3-O-β-D-吡喃葡萄糖基-(1→4)-α-L-吡喃阿拉伯糖苷、白头翁皂苷、hederacolchiside A1、白头翁皂苷F、齐墩果酸3-O-β-D-吡喃葡萄糖基-(1→4)-β-D-吡喃葡萄糖基-(1→3)-α-L-吡喃鼠李糖基-(1→2)-α-L-吡喃阿拉伯糖苷、齐墩果酸3-O-β-D-吡喃葡萄糖基-(1→3)-α-L-吡喃鼠李糖基-(1→2)-α-L-吡喃阿拉）含量的总和超过85%； MTT及集落形成实验表明PAHS (0.80-25.00μg/ml)可以时间和剂量依赖性的抑制SMMC-7721及A549细胞的增殖，抑制7721细胞集落的形成；体内试验表明白头翁总皂苷碱水解产物(50，100，200mg/kg)可以抑制小鼠肉瘤，小鼠肝癌及肺癌的生长且具有剂量依赖性，在给药剂量200mg/kg时三种模型的抑制率分别达到49.8%、43.7%、70.6%，HE染色表明PAHS组(50，100，200mg/kg)小鼠肿瘤组织均有大面积坏死及凋亡细胞的出现。电镜切片检查发现，荷肝癌小鼠及荷Lewis肺癌小鼠肿瘤组织均出现染色质集聚成团、边集等明显细胞凋亡特征;与CTX组相比，PAHS处理组小鼠的脾系数、胸腺系数均有提高，提示PAHS的免疫抑制作用较小。机制研究表明，PAHS (6.25，12.50，25.00μg/ml)可以诱导SMMC-7721及A549细胞凋亡，同时可以将SMMC-7721细胞周期阻滞在S期，将A549细胞生长阻滞在G0/G1期。western blot实验结果表明白头翁总皂苷碱水解产物可以上调Cyt-C， CleavedCaspase-3，Bax等蛋白的表达，同时下调PI3Kp85、p-Akt、p-mTOR、p-p70S6K等相关蛋白的表达。
结论：白头翁总皂苷碱水解产物具有抗肺癌和肝癌的作用，其机制可能与调节PI3K/AKT/mTOR信号通路引起的细胞凋亡有关。
Aim: In our previous study, we found that total saponins from Pulsatilla chinensisinhibited the growth of tumors in vivo and in virtro. Compared with total saponins, Alkalihydrolysate of total saponins had better antitumor effect. In this study, we will investigateantitumor effect and mechanisms of alkali hydrolysate of total saponins from Pulsatillachinensis.
Methods: The HPLC method was used to determination the contents of eightsaponins in alkali hydrolysate of total saponins from Pulsatilla chinensis; In vitro, MTTand clone formation test were used to examine the growth of human carcinomaSMMC-7721and A549. Additionally, Giemsa staining was also used to observe themorphology of tumour cells; In vivo, ICR mice bearing H22and S180cells were used, C57mice transplanted with Lewis tumor cells were also used. Blood extracted from tumourbearing mice was used to make the blood examination. Tumor tissue was stained withhematoxylin and eosin, and then observed the change of cell morphology; Ultrastructure oftumor tissue was examined with transmission electron microscope; Flow cytometry wasused to detect the cell cycle and apoptosis of tumor cells; Hoechst33258assay kit wereperformed to observe the morphology of apoptosis cells; Important signal molecules ofmitochondrial pathway and PI3K pathway were detected by western blot.
Results: The total content of eight saponins (pulsatilla saponin D, hederagenin3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranoside, pulsatilla saponin A,hederacolchiside A1, pulsatilla saponin F, oleanolic acid3-O-β-D-glucopyranosyl-(1
→4)-β-D-gluco-pyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside, oleanolic acid3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-
arabinopyranoside, oleanolic acid3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopy-ranoside) in alkali hydrolysate of total saponins from Pulsatilla chinensis was more than85%. In vitro, PAHS (0.80-25.00μg/ml) exhibited an inhibited effect on SMMC-7721andA549cells growth in concentration and time-dependent manners by MTT and cloneformation test; In vivo, PAHS (50，100，200mg/kg) could inhibit the tumor weight of H22,S180and Lewis in concentration manners, when treated with200mg/kg, the inhibitionrate were49.8%,43.7%and70.6%. The HE staining of all three tumors were presentedwith wide ranges of necrosis tissue. The results of transmission electronic microscopeshowed that PAHS (50，100，200mg/kg) groups exhibited remarkable ultrastructuralcharacteristics of apoptotic programmed cell death such as nuclei degenerated, cytoplasmshrinkage, chromatin condensated, and decreases of cell volume. Compared with CTXgroup, the number of WBC, spleen index, thymus index were imprpove. These resultsindicated that PAHS had little toxicity. PAHS (6.25,12.50,25.00μg/ml) could induceapoptosis on SMMC-7721and A549cells. Additionally, SMMC-7721cells were arrestedin S phase and A549cells in G0/G1phase. Western blotting showed that PAHS could upregulate the expression of Cyt-C、Cleaved Caspase-3、Bax, and down regulate theexpression of PI3Kp85、p-Akt、p-mTOR、p-p70S6K.
Conclusions: PAHS could inhibit the growth of liver and lung cancer, the mechanismmight be that PAHS induce apoptosis through PI3K/AKT/mTOR pathway.
引文
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