摘要
目的:观察七叶灵方对紫杉醇耐药肺癌细胞A549/Taxol增殖和凋亡的影响。方法:将10只SD大鼠随机分为中药组和空白组,每组5只。中药组大鼠灌胃给予18 g/kg七叶灵方药液,每日2次,连续给药3 d。空白组大鼠灌胃给予等体积0.9%NaCl溶液。分别制备含药血清和空白血清。采用浓度梯度诱导法构建紫杉醇耐药肺癌细胞株A549/Taxol。①将细胞分为空白组及不同浓度(5%、10%、20%)含药血清组,各组分别给予空白血清和相应浓度含药血清干预。药物干预前及细胞培养24、48 h后,CCK8法检测各组细胞增殖情况,筛选含药血清干预的最佳浓度。②将细胞分为空白组(常规培养基)、含药血清组(筛选的最佳浓度含药血清)、紫杉醇组(2 mg/L紫杉醇)、含药血清+紫杉醇组(最佳浓度含药血清+2 mg/L紫杉醇),各组予以相应干预。干预前及细胞培养24、48 h后,CCK8法检测各组细胞增殖情况。细胞培养48 h后,流式细胞术检测各组细胞凋亡情况,Western blot检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)、剪切型Caspase-3(cleaved Caspase-3)、聚腺苷酸二磷酸核糖转移酶(PARP)、剪切型PARP (cleaved PARP)的蛋白表达。结果:①与干预前相比,各浓度含药血清作用24、48 h后,A549/Taxol细胞的增殖抑制率显著升高(P<0.05,P<0.01),具有浓度/时间依赖性。选取20%含药血清进行后续实验。②细胞培养48 h后,紫杉醇组的细胞增殖抑制率明显高于含药血清组(P<0.05),含药血清+紫杉醇组对耐药细胞的生长抑制作用明显强于紫杉醇组(P<0.01);与空白组相比,含药血清组、紫杉醇组及含药血清+紫杉醇组的细胞凋亡率均明显升高(P<0.01),且含药血清+紫杉醇组的细胞凋亡率高于紫杉醇组(P<0.01)。③与空白组相比,紫杉醇组、含药血清组和含药血清+紫杉醇组细胞Bcl-2、Caspase-3、PARP的蛋白表达量显著降低(P<0.01),Bax、cleaved Caspase-3、cleaved PARP的蛋白表达量显著上调(P<0.01)。与紫杉醇组相比,含药血清+紫杉醇组细胞Bcl-2、Caspase-3、PARP的蛋白表达量明显下降(P<0.01),Bax、cleaved Caspase-3、cleaved PARP的蛋白表达量显著增加(P<0.05,P<0.01)。结论:七叶灵方含药血清可抑制紫杉醇耐药肺癌细胞A549/Taxol的增殖,诱导细胞凋亡,且与紫杉醇联合应用具有一定的协同效应,其机制可能与调控Bcl-2/Caspase-3/PARP通路有关。
Objective:To observe the effects of Qiyeling Decoction on the proliferation and apoptosis of paclitaxel-resistant lung cancer cell line A549/Taxol. Methods:Ten SD rats were randomly divided into the Chinese medicine group and blank group, 5 rats in each group. The rats in the Chinese medicine group were treated with Qiyeling Decoction at dose of 18 g/kg by intragastric administration, twice a day for 3 days. The rats in the blank group were treated with 0.9% NaCl solution at equivalent volume. The drug-containing serum and blank serum were prepared respectively. Paclitaxel-resistant human lung cancer cell line A549/Taxol was established by concentration gradient induction. ①The cells were divided into the blank group and drug-containing serum groups with different concentrations(5%, 10% and 20%). Each group was treated with the blank serum or drug-containing serum at corresponding concentrations. Before drug intervention and after cell culture for 24 and 48 hours, the cell proliferation of each group was detected by CCK8 assay, and the best concentration of drug-containing serum was selected. ②The cells were divided into the blank group(conventional culture medium), drug-containing serum group(drug-containing serum at the best concentration), paclitaxel group(2 mg/L paclitaxel), drug-containing serum + paclitaxel group(drug-containing serum at the best concentration + 2 mg/L paclitaxel). Each group was treated with the corresponding intervention. Before intervention and after cell culture for 24 and 48 hours, the cell proliferation of each group was detected by CCK8 assay. After cell culture for 48 hours, the cell apoptosis was detected by flow cytometry, and the protein expressions of B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax), cysteinyl aspartate specific proteinase-3(Caspase-3), cleaved Caspase-3, poly ADP-ribose polymerase(PARP) and cleaved PARP were detected by Western blot. Results:①Compared with that before the intervention, after treatment with the drug-containing serum at different concentrations for 24 and 48 hours, the proliferation inhibition rate of A549/Taxol cells was increased significantly(P<0.05, P<0.01), in a concentration-and time-dependent manner. The follow-up experiment was carried out with 20% drug-containing serum. ②After cell culture for 48 hours, the inhibition rate of cell proliferation in the paclitaxel group was significantly higher than that in the drug-containing serum group(P<0.05), and the inhibition effect of the drug-containing serum + paclitaxel group on the growth of drug-resistant cells was significantly stronger than that of the paclitaxel group(P<0.01). Compared with the blank group, the cell apoptosis rate of the drug-containing serum group, paclitaxel group and drug-containing serum + paclitaxel group was significantly increased(P<0.01), and the cell apoptosis rate of the drug-containing serum + paclitaxel group was higher than that of the paclitaxel group(P<0.01). ③Compared with the blank group, the protein expressions of Bcl-2, Caspase-3 and PARP in the paclitaxel group, drug-containing serum group and drug-containing serum + paclitaxel group were decreased significantly(P<0.01), and the protein expressions of Bax, cleaved Caspase-3 and cleaved PARP were increased significantly(P<0.01). Compared with the paclitaxel group, the protein expressions of Bcl-2, Caspase-3 and PARP were significantly decreased in the drug-containing serum + paclitaxel group(P<0.01), and the protein expressions of Bax, cleaved Caspase-3 and cleaved PARP were increased significantly(P<0.05, P<0.01). Conclusion:Qiyeling Decoction can inhibit the proliferation of paclitaxel-resistant lung cancer cells A549/Taxol, and induce the cell apoptosis, which shows certain synergistic effect with paclitaxel. Its mechanism may be related to the regulation of Bcl-2/Caspase-3/PARP pathway.
作者
蔡霄月
杨晓华
张洲姬
张铭
CAI Xiaoyue;YANG Xiaohua;ZHANG Zhouji;ZHANG Ming(Department of Integrated Traditional Chinese and Western Medicine,Shanghai Chest Hospital,Shanghai Jiao Tong University,Shanghai 200030,China;Central Laboratory,Shanghai Chest Hospital,Shanghai Jiao Tong University,Shanghai 200030,China)
出处
《上海中医药大学学报》
CAS
2020年第2期41-47,共7页
Academic Journal of Shanghai University of Traditional Chinese Medicine
基金
国家自然科学基金资助项目(81573893)
上海市科委科研计划项目(16401933400)
上海市卫计委中医药科研专项基金项目(2016JP004)。
