木鳖子-淫羊藿有效部位筛选与其组方抗肺癌作用机制初探

Screening Effective Sites of Momordicae Semen-Epimedii Folium and Anti-lung Cancer Mechanism of Its Prescription

目的:初步确认木鳖子-淫羊藿抗肺癌有效部位,研究其作用机制。方法:在前期研究的基础上,优化木鳖子-淫羊藿的提取方法,并在药效指导下进行有效部位的筛选。通过D101型大孔树脂吸附、洗脱得到木鳖子和淫羊藿不同的乙醇洗脱部位和水部位;采用噻唑蓝(MTT)比色法检测药物总提物和不同洗脱部位对多种肿瘤细胞株增殖的影响,筛选最佳洗脱部位以及肿瘤敏感株;流式细胞术检测木鳖子-淫羊藿洗脱部位对A549细胞内活性氧(ROS)及凋亡的影响;蛋白免疫印迹法(Western blot)检测A549细胞内凋亡相关蛋白肿瘤蛋白53(p53)、B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)、胱天蛋白酶-3(Caspase-3)、Caspase-9蛋白的表达水平。结果:木鳖子对A549细胞的增殖抑制效果优于木鳖子仁,半数抑制浓度(IC50)分别为(86.83±2.88)、(95.10±18.13)mg·L^(-1);淫羊藿总提物作用于A549抗增殖的IC50为(4.71±0.81)mg·L^(-1);木鳖子与淫羊藿总提物的大孔树脂40%、60%、80%乙醇和无水乙醇洗脱部位抑制A549增殖的IC50分别为(45.32±4.38)、(14.95±0.73)、(17.07±1.76)、(14.46±2.35)、(51.7±2.26)、(12.37±0.67)、(20.29±0.93)、(3.43±0.91)mg·L^(-1);与正常组比较,木鳖子-淫羊藿1∶1组方抑制A549增殖具有时间依赖性和浓度依赖性;与正常组比较,50 mg·L^(-1)木鳖子-淫羊藿组方药物显著增加A549细胞内ROS表达(P<0.01);与正常组比较,12.5、25、50 mg·L^(-1)木鳖子-淫羊藿组方药物可显著增加A549细胞凋亡(P<0.01);与正常组比较,25、50 mg·L^(-1)木鳖子-淫羊藿组方药物显著增加A549细胞内p53的表达(P<0.01);与正常组比较,12.5、25、50 mg·L^(-1)木鳖子-淫羊藿组方药物可显著增加Bax的表达(P<0.01);与正常组比较,50 mg·L^(-1)木鳖子-淫羊藿组方药物可显著降低Caspase-3、Caspase-9的表达(P<0.01)。结论:木鳖子的抗肿瘤增殖作用较木鳖子仁更强;木鳖子和淫羊藿抗肿瘤活性物质主要集中在60%乙醇~无水乙醇洗脱部位;在配比研究中,木鳖子和淫羊藿1∶1组方对A549细胞抗增值效果较好,其机制可能与增加A549细胞内ROS的生成,进而提高促凋亡蛋白p53、Bax的表达,最终导致Caspase-3和Caspase-9的激活而诱导细胞内源性凋亡有关。

Objective:To preliminarily confirm the effective anti-lung cancer sites of Momordicae Semen and Epimedii Folium and study their mechanism of action.Method:On the basis of preliminary research,the extraction method of Momordicae Semen and Epimedii Folium was optimized and the effective parts were screened under the guidance of pharmacological effects.Different ethanol elution and water elution sites of Momordicae Semen and Epimedii Folium were obtained through adsorption and elution with D101 macroporous resin.The methylthiazolyldiphenyl-tetrazolium bromide(MTT)colorimetric assay was used to detect the effects of total drug extracts and different elution sites on the proliferation of various tumor cell lines,and to screen for the optimal elution site and tumor sensitive strains.Flow cytometry was used to detect the effect of the elution sites of Momordicae Semen and Epimedii Folium on intracellular reactive oxygen species(ROS)and apoptosis in A549 cells.Western blot was used to compare the expressions of tumor protein 53(p53),Bcl-2-associated X protein(Bax),cysteinyl aspartate specific proteinase-3 and 9(Caspase-3 and Caspase-9)proteins in A549 cells.Result:The inhibitory effect of Momordicae Semen on the proliferation of A549 cells was better than the kernel of Momordicae Semen,with 50%inhibitory concentration(IC50)being(86.83±2.88)mg·L^(-1) and(95.10±18.13)mg·L^(-1),respectively.The effect of total extracts of Epimedii Folium on A549 anti proliferation IC50 value was(4.71±0.81)mg·L^(-1).The IC50 values of the 40%,60%,and 80%ethanol and anhydrous ethanol eluted macroporous resins of the total extracts of Momordicae Semen and Epimedii Folium inhibiting A549 proliferation were(45.32±4.38)、(14.95±0.73)、(17.07±1.76)、(14.46±2.35)、(51.7±2.26)、(12.37±0.67)、(20.29±0.93)、and(3.43±0.91)mg·L^(-1),respectively.Compared with the normal group,the 1∶1 combination of Momordicae Semen and Epimedii Folium inhibited A549 cell proliferation in a time-dependent and concentration-dependent manner.Compared with the normal group,50 mg·L^(-1) of the combination of Momordicae Semen and Epimedii Folium significantly increased intracellular ROS expression(P<0.01).Compared with the normal group,12.5,25,50 mg·L^(-1) of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of A549 cell apoptosis(P<0.01).Compared with the normal group,25,50 mg·L^(-1) of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of p53 in A549 cells(P<0.01).Compared with the normal group,12.5,25,50 mg·L^(-1) of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of Bax(P<0.01).Compared with the normal group,50 mg·L^(-1) of the combination of Momordicae Semen and Epimedii Folium significantly reduced the expressions of Caspase-3 and Caspase-9(P<0.01).Conclusion:The antitumor effect of Momordicae Semen is better than that of the kernel of Momordicae Semen.The anti-tumor substances of Momordicae Semen and Epimedii Folium mainly concentrate in the 60%ethanol to anhydrous ethanol elution site.A549 cells are sensitive to the 1∶1 combination of Momordicae Semen and Epimedii Folium,which can effectively inhibit the cell proliferation.The mechanism may be related to increasing the generation of ROS in A549 cells,promoting their apoptosis,increasing the expressions of apoptotic proteins such as p53 and Bax,and reducing the expressions of Caspase-3 and Caspase-9.