黄药子抑制胃癌生长转移的实验研究
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摘要
胃癌是全世界范围内发病率较高的恶性肿瘤之一,2008年全球新发胃癌患者近99万例,占全部新发癌症病例8%,死亡近74万,占全部死亡病例10%。目前,在世界范围内GC占肿瘤死亡率的第2位,仅次于肺癌。流行病学调查统计,我国胃癌的发病率2000年居第三位,2004～2007年上升至第二位。2010年中国卫生统计年鉴报告,我国胃癌年死亡率为23.10/10万,是世界平均水平的2倍多。胃癌已成为严重威胁人民群众身体健康、阻碍社会经济发展的重大疾病。目前手术根治是胃癌的主要治疗方式,对于早期胃癌患者术后5年生存率较高,可达90%以上,但绝大部分胃癌在确诊时已处于进展期,丧失手术根治可能,5年生存率仅为11%-40%。西医针对中晚期胃癌主要有根治性或姑息性手术、放化疗及免疫治疗等治疗手段,然而全球范围的中晚期胃癌的西医疗效并不理想,那么,中医药在治疗中晚期胃癌的治疗中就有了相当的发挥余地。因此,继续寻找特异性强、高效低毒的抗肿瘤药物,具有较强的临床和现实意义。
近年来,中医药在胃癌的预防及治疗中发挥着较突出的作用,体外多项研究已证实中药抗肿瘤效果确切。临床实践表明,单独应用中药或中药复方治疗胃癌疗效确切,同时能减轻放化疗的毒副作用。黄药子为薯截科植物黄独(Dioscorea bulbifera L.)的块茎,其性寒凉,味苦,具有凉血、消瘿、降火、解毒的作用。黄药子在临床应用广泛,主治多种病症。既往的文献报道,中药黄药子具有强大的抗肿瘤作用,临床报道运用含有黄药子的黄白胃癌汤、抗癌乙片等治疗胃癌、贲门癌和食道癌疗效确切。还有报道称黄药子对肝癌、乳腺癌、宫颈癌、原发性支气管肺癌、淋巴瘤等也有一定疗效。相关研究表明中药抗肿瘤机制包括：抑制原癌基因的表达,促进抑癌基因的表达;调控癌变组织周围环境；诱导癌细胞凋亡；降低胃癌细胞侵袭和转移能力等。但有关黄药子治疗胃癌的具体机制尚不清楚。
为探讨上述问题,本研究拟通过观察黄药子对体外培养的胃癌细胞株的增殖、侵袭及转移等恶性生物学行为的影响,初步探讨其作用的分子机制；并建立裸鼠胃癌皮下移植瘤动物模型,观察黄药子对荷瘤鼠的生长抑制的影响。本研究对黄药子治疗胃癌提供科学的理论和实验依据,同时对丰富中医抗肿瘤理论,促进中医临床转化具有重要意义。
目的
1.通过观察黄药子对体外胃癌细胞株SGC-7901氧化还原能力、生长增殖及转移能力的影响,探讨黄药子抑制胃癌生长转移的分子机制。
2.通过观察黄药子对胃癌动物模型肿瘤生长的影响,探讨黄药子的抑瘤效果。
方法
1.体外培养胃癌细胞,黄药子使用乙醇提取,制成不同浓度溶液。
2.分光光度法检测不同浓度的黄药子乙醇提取物对胃癌细胞羟基自由基和DPPH自由基清除率。
3. ABTS快速试验检测不同浓度的黄药子醇提取物对胃癌细胞的抗氧化能力及还原能力。
4.MTT法检测不同浓度的黄药子醇提取物对胃癌细胞的增殖影响。5.流式细胞术检测不同浓度的黄药子醇提取物对胃癌细胞凋亡的影响。
6. Transwell侵袭性检测不同浓度的黄药子醇提取物对胃癌细胞侵袭性的影响。
7.建立胃癌裸鼠皮下移植瘤,观察黄药子醇提取物对肿瘤生长的影响。
8. ELISA检测胃癌裸鼠外周血中炎性细胞因子的表达情况,观察黄药子醇提取物对炎性细胞因子浓度的影响。
所有数据采用SPSS13.0统计软件进行统计。检验显著性水准取α=0.05,以P<0.05为差异有统计学意义。
结果
1分光光度法测定结果表明：不同浓度的黄药子乙醇提取物具有一定的羟基自由基和DPPH自由基清除率。三种提取物中,浓度2mg/ml,70%乙醇提取物的DPPH自由基清除能力最强,清除率为55.2%；80%乙醇提取物的OH自由基清除能力最高,清除率为51.2%；70%和90%乙醇提取物对OH自由基清除率分别为38.7%和39.6%。
2还原能力的测试结果显示：黄药子醇提取物具有较强的还原能力。70%的乙醇提取物还原能力为每1克提取物相当于28.1μmol Fe2+,80%的乙醇提取物还原能力为每1克提取物相当于49.3μmol Fe2+,90%的乙醇提取物还原能力为每1克提取物相当于35.2μmol Fe2+,其中80%的乙醇提取物具有最强的还原能力。
3ABTS快速检测试验结果表明：黄药子醇提取物具有较强的抗氧化能力。80%7,醇提取物的清除能力最强,接近72%。70%和90%乙醇提取物的清除率分别为55%和45%。随着时间的推移和浓度增大总抗氧化能力逐渐增加。最低浓度为0.2mg/ml时,其清除率为8%。最高浓度为2mg/ml,清除率为52%。在4min、7min、12min,浓度为0.2、0.4、1.0mg/ml时,清除率稳步上升。当浓度分别为1.4、1.8、2mg/ml时,自由基清除率变化均不明显,般在55%和67%之间波动。
4MTT法细胞增殖实验结果显示：不同浓度乙醇黄药子提取物溶液均能抑制胃癌SGC-7901细胞的增殖。其中,黄药子70%乙醇提取物溶液0.5、1、2mg/ml抑制胃癌SGC-7901细胞增殖率分别为34.5%、41.2%和45.6%；黄药子80%乙醇提取物溶液0.5、1、2mg/ml抑制胃癌SGC-7901细胞增殖率分别为41.3%、55.8%和62.3%；黄药子90%乙醇提取物溶液0.5、1、2mg/ml抑制胃癌SGC-7901细胞增殖率分别为28.7%、32.4%和34.9%；以2mg/ml的黄药子80%乙醇提取物的抑制率最高,达62.3%。
5流式细胞仪细胞凋亡检测结果显示：培养48h后,黄药子醇提取物可明显促进SGC-7901细胞的凋亡,而对照组SGC-7901细胞只出现少量的凋亡。0.5、1和2mg/ml黄药子70%乙醇提取物处理的SGC-7901细胞凋亡率从空白对照组的(3.81±1.56)%分别上升至(5.36±1.85)%、(10.31±2.46)%(P<0.05)和(18.54±3.26)%(P<0.01)；0.5、1和2mg/ml黄药子80%乙醇提取物处理的SGC-7901细胞凋亡率分别为(6.62±1.15)%、(14.27±2.28)%(P<0.05)和(25.07±4.21)%(P<0.01),并呈一定浓度依赖性。0.5、1和2mg/ml黄药子90%乙醇提取物处理的SGC-7901细胞凋亡率分别为(5.65±1.63)%、(11.81±2.75)%(P<0.05)和(20.32±3.54)%(P<0.01)。
6Transwell侵袭性检测结果显示：人胃癌SGC-7901细胞分别与0.5、1、2mg/ml80%黄药子醇提取物溶液共培养24小时后,细胞侵袭数目与空白对照组相比,呈降低趋势。高倍镜下对穿过下室面的细胞数进行计数,对照组为94.36±9.51个,0.5mg/ml黄药子80%乙醇提取物组为67.52±7.48个,1mg/ml黄药子80%乙醇提取物组为51.24±6.51个,2mg/ml80%黄药子醇提取物组为38.59±5.43个,与对照组比较,0.5、1、2mg/ml80%黄药子醇提取物可以抑制人胃癌细胞株SGC-7901的侵袭(P<0.05),且呈剂量依赖关系。
7建立裸鼠胃癌皮下移植瘤模型情况
将瘤块植入裸鼠皮下后,生长至第7天,形成局部可测量瘤灶,造模成功率100%；皮下肿瘤前期生长较缓,外观呈球形或半球形,初起可活动,日渐变硬,肿瘤大小不一,观察期内各组裸鼠均未出现明显消瘦及体重下降。8黄药子醇提取物对荷瘤裸鼠肿瘤体积的影响
治疗结束后,黄药子高剂量组瘤体积小于模型组(P<0.05),5-FU组和黄药子低剂量组瘤体积与模型对照组比较无统计学差异(P>0.05),黄药子高剂量组与低剂量组及5-FU组比较,差异均无统计学意义(P>0.05),5-FU组和黄药子低、高剂量组三者对瘤体积的抑制率分别为25%、17.86%和39.29%。9黄药子醇提取物对荷瘤裸鼠肿瘤质量的影响
与模型对照组比较,5-FU组和黄药子高剂量组裸鼠移植瘤质量显著下降(P<0.05),黄药子不同剂量组之间瘤质量比较差异无统计学意义(P>0.05),高低剂量黄药子组瘤质量与5-FU组比较,差异亦无统计学意义(P>0.05),5-FU组和黄药子低、高剂量组对荷瘤裸鼠瘤质量的抑制率分别为37.5%、25%和43.75%。10黄药子醇提取物对裸鼠皮下移植瘤外周血血清IL-8水平的影响
与模型对照组比较,5-FU组和黄药子组裸鼠血清IL-8水平显著下降(P<0.05)；黄药子高剂量组血清IL-8水平明显低于低剂量组(P<0.05)。11.黄药子醇提取物对裸鼠皮下移植瘤外周血血清sICAM-1水平的影响
与模型对照组比较,5-FU组和黄药子组裸鼠血清sICAM-1水平显著下降(P<0.05)；黄药子高剂量组血清sICAM-1水平明显低于低剂量组(P<0.05)；黄药子高剂量组血清sICAM-1水平明显低于5-FU组(P<0.05)。
结论
1.黄药子醇提取物对人胃癌SGC-7901细胞具有一定的自由基清除能力和较强的抗氧化还原能力；
2.黄药子醇提取物能抑制人胃癌SGC-7901细胞的增殖,并诱导人胃癌SGC-7901细胞的凋亡;
3.黄药子醇提取物能降低人胃癌SGC-7901细胞的侵袭性；
4.黄药子醇提取物能体内抑制人胃癌裸鼠皮下移植瘤的生长,可能与下调侵袭转移相关炎性因子(IL-8和sICAM-1有一定关系。
Gastric cancer is one of common malignant tumors in worldwide. The global new cases of primary gastric cancer are990000in2008, accounting for8%of all new cancer cases. And740000were died, accounting for10%of all cases of death. At present, GC tumor mortality is accounted for the second, only followed by lung cancer in the world. According to epidemiology statistics, China's gastric cancer incidence rate ranked third in2000, and has risen to second from2004to2007. T he total incidence shows a steady trend. According to the China health statistics annual report in2010, the annual mortality rate of gastric cancer in China was23.10/10million, which was more than2times of the world average.Gastric cancer has become a major disease that threatens the people's health, and hinders economic and social development. At present the major treatment of gastric cancer is surgical operation. The patients with early gastric cancer postoperative5years survival rate is high, which may reach above90%. But the vast majority of gastric cancer is advanced at the time of diagnosis, and has lost the chance of radical operation.5year survival rate of it's is only11%-40%. Western medicine for advanced gastric cancer is radical or palliative operation, radiotherapy and chemotherapy immunotherapy treatment. However, western medicine curative effect of worldwide advanced gastric cancer is not ideal. Thus treatment of traditional Chinese medicine has a considerable role. Therefore, it has strong clinical and practical significance to further look for strong specificity and high efficiency low toxicity of antitumor drugs.
In recent years, traditional Chinese medicine in the prevention and treatment of gastric cancer plays a more prominent role. And the studies in vitro have confirmed that Chinese medicine anti-tumor effect is exact. Clinical practice shows that separate application of traditional Chinese medicine or Chinese medicine compound treatment of gastric cancer has curative effect, at the same time, that can reduce the side effects of radiation and chemotherapy. Rhizoma dioscoreae bulbiferae is the tuber of potato plants Dioscorea bulbifera1. It is cold and bitter taste,and it has the role of cooling blood, eliminating gall, sending fire and detoxification. Rhizoma dioscoreae bulbiferae is widely used in clinical. It mainly treat a variety of disorders. Past literature reported that Chinese medicine rhizoma dioscoreae bulbiferae has powerful antitumor effect. Clinical reports that using huangbai stomach soup containing rhizoma dioscoreae bulbiferae to treat gastric cancer, cardia cancer and esophagus cancer have curative effect. There have reports that rhizoma dioscoreae bulbiferae was used to treat liver cancer, breast cancer, cervical cancer, primary bronchial lung cancer, lymphoma, etc. It also has certain curative effect. Related studies have shown that Chinese medicine anti-tumor mechanisms include inhibiting the expression of proto-oncogenes, promoting the expression of tumor suppressor genes, regulating of cancerous tissue surrounding environment, inducing cancer cell apoptosis, reducing the ability of gastric cancer cell invasion and metastasis. But the concrete mechanism of rhizoma dioscoreae bulbiferae is not clear for the treatment of gastric cancer. To study the above problems, this study proposed by observing the effect of malignant biological behavior of dioscoreae bulbiferae on cultured in vitro. And preliminary explore the molecular mechanism of its action and establish the animal model of nude mouse stomach subcutaneous transplantation tumor dioscoreae bulbiferae. Then we observe the effect of rhizoma dioscoreae bulbiferae on a tumor-burdened rat growth inhibition. This study of rhizoma dioscoreae bulbiferae can provide scientific theoretical and experimental basis for treatment of cancer of the stomach, and can enrich anti-tumor theory at the same time. It is of great significance to promote the traditional Chinese medicine clinical transformation.
Objective
1To observe of the effect of redox ability, proliferation and metastasis by dioscorea bulbifera on gastric cancer SGC-7901cells in vitro, and to investigate the molecular mechanism of dioscoreae bulbiferae inhibition of gastric cancer growth and metastasis.
2To observe the effect of the anti-tumor effect of dioscorea bulbifera by investigating the tumor growth in animal model of gastric cancer.
Method
1Gastric cancer cell was cultured in vitro, and dioscorea bulbifera was extracted by ethanol to make of different concentration solution.
2We use the spectrophotometric to detect the clearance rate of hydroxyl radical and DPPH radical of alcohol extraction of dioscorea bulbifera of different concentration on gastric cancer cell.
3We use ABTS rapid test to detect the antioxidant activity of extracts of different concentrations of alcohol extraction of dioscorea bulbifera on gastric cancer cells and reduction ability.
4We use MTT method to detect the effect of proliferation of different concentrations of alcohol extraction of dioscorea bulbifera on gastric cancer cells.
5We use the flow cytometry to detect the effect of different concentrations on the apoptosis of gastric cancer cells.
6We use the Transwell invasion to detect the effect of different concentrations of Dioscorea bulbifera alcohol effect on the invasion of gastric cancer cells.
7We established the gastric carcinoma transplanted subcutaneously in nude mice and observed the effects of ethanol extract of Dioscorea bulbifera L on tumor growth.
8We use the ELISA to detect the expression of the inflammatory cytokines in peripheral blood of gastric cancer in nude mice. We observed the effects of inflammatory cytokine concentrations of ethanol extract of Dioscorea bulbifera L.
All data used SPSS13statistical software. The significant level of α=0.05, with P＜0.05as the difference has statistical significance. Result
1Spectrophotometry
Different concentrations of rhizoma dioscoreae bulbiferae ethanol extracts has certain hydroxyl radicals and DPPH free radical clearance. In the three kinds of extracts, the DPPH radical scavenging capacity with concentration of2mg/ml,70%ethanol extract was strongest. It's clearance ratewhich was55.2%. The OH radical scavenging capacity of80%ethanol extract was the highest. It's clearance rate was51.2%. And the OH radical scavenging capacity of70%and90%ethanol extracts were38.7%and39.6%respectively.
2Reducing power test
Rhizoma dioscoreae bulbiferae ethanol extracts have stronger reducing power. The reducing power of70%ethanol extract was28.1μmol Fe2+, The reducing power of80%ethanol extract was equival to49.3μmol Fe2+per1g, the reducing power of90%rhizoma dioscoreae bulbiferae ethanol extracts was equival to35.2μmol Fe2+per1g.80%ethanol extract has the strongest reducing
power.
3Rapid detection of ABTS
Rhizoma dioscoreae bulbiferae ethanol extracts had stronger antioxidant capacity. The removal of the80%ethanol extract was highest, which was close to72%. The clearance rate in70%and90%ethanol extracts of was55%and45%respectively. And the total antioxidant capacity increased gradually with the time going and the concentration increasing. When the lowest concentration was0.2mg/ml, the clearance rate was8%. When the highest concentration was2mg/ml, the clearance rate was52%. When4min,7min and12min, the concentration was0.2mg/m,0.4mg/m and1.0mg/ml. The clearance rate has risen steadily. When the concentrations were1.4mg/ml,1.8mg/ml and2mg/ml, the changes of free radical clearance were not obvious, that were generally between55%and67%.
4Detection cell proliferation by MTT method
Different concentrations of rhizoma dioscoreae bulbiferae ethanol extracts solution could inhibit gastric cancer SGC-7901cells' proliferation.70%rhizoma dioscoreae bulbiferae ethanol extracts solution with0.5mg/ml1mg/ml and2mg/ml could inhibit gastric cancer SGC-7901cell proliferation rate, there are34.5%,41.2%and34.5%respectively. The cell proliferation rate in inhibits gastric cancer SGC-7901of80%rhizoma dioscoreae bulbiferae ethanol extracts solution were0.5mg/ml、1mg/ml、2mg/ml, they were41.3%、55.8%and62.3%respectively. In90%rhizoma dioscoreae bulbiferae ethanol extracts solution with0.5mg/m、1mg/m、2mg/ml, the cell proliferation rate to inhibit gastric cancer SGC-7901were28.7%,32.4%and28.7%respectively. The inhibition rate of80%rhizoma dioscoreae bulbiferae ethanol extracts had the highest of62.3%.
5Flow cytometry instrument to detect cell apoptosis
After culture48h, rhizoma dioscoreae bulbiferae ethanol extracts could obviously promote the SGC-7901cells apoptosis, and the control group only had a small amount of SGC-7901cells apoptosis.
The cell apoptosis rate in70%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、lmg/m and2mg/ml were rised from the blank control group (3.81±1.56)%respectively up to (5.36±1.85)%,(10.31±2.46)%(P＜0.05, and (18.54±3.26)%(P＜0.01);0.5,1and2mg/ml airpotato yam80%ethanol extract of SGC-7901cell apoptosis rate respectively (6.62±1.15)%,(14.27±2.28)%(P＜0.05) and (25.07±4.21)%(P＜0.01), and a certain concentration dependence.0.5,1and2mg/ml airpotato yam90%ethanol extract processing of SGC-7901cell apoptosis rate (5.65±1.63)%,respectively(11.81±2.75)%(P＜0.05) and (20.32±3.54)%(P＜0.01).
6Transwell invasive testing
Human gastric cancer SGC-7901cells were cultured24hours respectively with80%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、1mg/m and2mg/ml. Compared with the blank control group, the cell invasion number showed a trend of decrease. We count the cells number under through the room at high magnification, the control group was94.36±9.51,80%rhizoma dioscoreae bulbiferae ethanol extracts with0.5mg/ml were67.52±7.48.80%rhizoma dioscoreae bulbiferae ethanol extracts with1mg/ml were51.24±6.51.80%rhizoma dioscoreae bulbiferae ethanol extracts with2mg/ml were38.59±5.43. Compared with control group,80%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、1mg/m and2mg/ml can inhibit gastric cancer cell line (P＜0.05). There was a dose-response relationship.
7Subcutaneous transplantation tumor model of human gastric cancer in nude mice
The tumors were implanted subcutaneously in nude mice, up to seventh days, the formation of local measurable tumor, model success rate of100%; subcutaneous tumor growth is slow, the appearance of spherical or hemispherical, early active, gradually hardens, tumor size is not a group, the observation period nude mice showed no obvious weight loss and weight loss.
8Effect of dioscorea bulbifera L alcohol extracts on tumor volume
After the end of treatment, tumor volume of high dose group is smaller than that of the model group (P＜0.05), the volume of5-FU group and low dose group and model group showed no statistical difference (P＞0.05), compared with5-FU group, high-dose group and low dose group, there were no significant differences (P＞0.05),5-FU group and low, high dose group three inhibition on the tumor volume was respectively25%,17.86%and39.29%.
9Inhibition of tumor weight rate by dioscorea bulbifera alcohol extract
Compared with the model group,5-FU group and high dose group of dioscorea bulbifera nude mice transplanted tumor mass was significantly decreased (P＜0.05), between different dose groups of Dioscorea bulbifera1tumor weight had no significant difference (P＞0.05), low dose of group tumor mass compared with5-FU group, the difference had no statistical significance (P>0.05),5-FU group and Dioscorea bulbifera low, high dose group of nude mice tumor mass was respectively37.5%,25%and43.75%.
10IL-8level of dioscorea bulbifera alcohol extract on tumor peripheral blood serum
Compared with the model group, serum IL-8levels decreased significantly in5-FU group and dioscorea bulbifera group (P<0.05); the serum IL-8levels in high dose group were significantly lower than those of low dose group (P＜0.05).
11sICAM-1level of serum level of dioscorea bulbifera alcohol extract on tumor peripheral blood serum
Compared with the model group, serum sICAM-1levels decreased significantly in5-FU group and dioscorea bulbifera group(P<0.05); serum sIC AM-1levels in high dose group were significantly lower than those of the low dose (P＜0.05). Conclusion
1Rhizoma dioscoreae bulbiferae alcohol extract has the certain ability of free radicals scavenging,reduction and antioxidant on human gastric cancer cell line.
2Rhizoma dioscoreae bulbiferae alcohol extract can inhibit proliferation and induced apoptosis ofhuman gastric cancer SGC-7901cell.
3Rhizoma dioscoreae bulbiferae alcohol extract can reduce the invasiveness of human gastric cancer SGC-7901cell line.
4Rhizoma dioscoreae bulbiferae alcohol extract can inhibit the growth of subcutaneous xenograf in gastric cancernude mice, and its possible mechanism of invasion and metastasis was related to downregulation of inflammatory factors (IL-8and sICAM-1).
近年来,中医药在胃癌的预防及治疗中发挥着较突出的作用,体外多项研究已证实中药抗肿瘤效果确切。临床实践表明,单独应用中药或中药复方治疗胃癌疗效确切,同时能减轻放化疗的毒副作用。黄药子为薯截科植物黄独(Dioscorea bulbifera L.)的块茎,其性寒凉,味苦,具有凉血、消瘿、降火、解毒的作用。黄药子在临床应用广泛,主治多种病症。既往的文献报道,中药黄药子具有强大的抗肿瘤作用,临床报道运用含有黄药子的黄白胃癌汤、抗癌乙片等治疗胃癌、贲门癌和食道癌疗效确切。还有报道称黄药子对肝癌、乳腺癌、宫颈癌、原发性支气管肺癌、淋巴瘤等也有一定疗效。相关研究表明中药抗肿瘤机制包括：抑制原癌基因的表达,促进抑癌基因的表达;调控癌变组织周围环境；诱导癌细胞凋亡；降低胃癌细胞侵袭和转移能力等。但有关黄药子治疗胃癌的具体机制尚不清楚。
为探讨上述问题,本研究拟通过观察黄药子对体外培养的胃癌细胞株的增殖、侵袭及转移等恶性生物学行为的影响,初步探讨其作用的分子机制；并建立裸鼠胃癌皮下移植瘤动物模型,观察黄药子对荷瘤鼠的生长抑制的影响。本研究对黄药子治疗胃癌提供科学的理论和实验依据,同时对丰富中医抗肿瘤理论,促进中医临床转化具有重要意义。
目的
1.通过观察黄药子对体外胃癌细胞株SGC-7901氧化还原能力、生长增殖及转移能力的影响,探讨黄药子抑制胃癌生长转移的分子机制。
2.通过观察黄药子对胃癌动物模型肿瘤生长的影响,探讨黄药子的抑瘤效果。
方法
1.体外培养胃癌细胞,黄药子使用乙醇提取,制成不同浓度溶液。
2.分光光度法检测不同浓度的黄药子乙醇提取物对胃癌细胞羟基自由基和DPPH自由基清除率。
3. ABTS快速试验检测不同浓度的黄药子醇提取物对胃癌细胞的抗氧化能力及还原能力。
4.MTT法检测不同浓度的黄药子醇提取物对胃癌细胞的增殖影响。5.流式细胞术检测不同浓度的黄药子醇提取物对胃癌细胞凋亡的影响。
6. Transwell侵袭性检测不同浓度的黄药子醇提取物对胃癌细胞侵袭性的影响。
7.建立胃癌裸鼠皮下移植瘤,观察黄药子醇提取物对肿瘤生长的影响。
8. ELISA检测胃癌裸鼠外周血中炎性细胞因子的表达情况,观察黄药子醇提取物对炎性细胞因子浓度的影响。
所有数据采用SPSS13.0统计软件进行统计。检验显著性水准取α=0.05,以P<0.05为差异有统计学意义。
结果
1分光光度法测定结果表明：不同浓度的黄药子乙醇提取物具有一定的羟基自由基和DPPH自由基清除率。三种提取物中,浓度2mg/ml,70%乙醇提取物的DPPH自由基清除能力最强,清除率为55.2%；80%乙醇提取物的OH自由基清除能力最高,清除率为51.2%；70%和90%乙醇提取物对OH自由基清除率分别为38.7%和39.6%。
2还原能力的测试结果显示：黄药子醇提取物具有较强的还原能力。70%的乙醇提取物还原能力为每1克提取物相当于28.1μmol Fe2+,80%的乙醇提取物还原能力为每1克提取物相当于49.3μmol Fe2+,90%的乙醇提取物还原能力为每1克提取物相当于35.2μmol Fe2+,其中80%的乙醇提取物具有最强的还原能力。
3ABTS快速检测试验结果表明：黄药子醇提取物具有较强的抗氧化能力。80%7,醇提取物的清除能力最强,接近72%。70%和90%乙醇提取物的清除率分别为55%和45%。随着时间的推移和浓度增大总抗氧化能力逐渐增加。最低浓度为0.2mg/ml时,其清除率为8%。最高浓度为2mg/ml,清除率为52%。在4min、7min、12min,浓度为0.2、0.4、1.0mg/ml时,清除率稳步上升。当浓度分别为1.4、1.8、2mg/ml时,自由基清除率变化均不明显,般在55%和67%之间波动。
4MTT法细胞增殖实验结果显示：不同浓度乙醇黄药子提取物溶液均能抑制胃癌SGC-7901细胞的增殖。其中,黄药子70%乙醇提取物溶液0.5、1、2mg/ml抑制胃癌SGC-7901细胞增殖率分别为34.5%、41.2%和45.6%；黄药子80%乙醇提取物溶液0.5、1、2mg/ml抑制胃癌SGC-7901细胞增殖率分别为41.3%、55.8%和62.3%；黄药子90%乙醇提取物溶液0.5、1、2mg/ml抑制胃癌SGC-7901细胞增殖率分别为28.7%、32.4%和34.9%；以2mg/ml的黄药子80%乙醇提取物的抑制率最高,达62.3%。
5流式细胞仪细胞凋亡检测结果显示：培养48h后,黄药子醇提取物可明显促进SGC-7901细胞的凋亡,而对照组SGC-7901细胞只出现少量的凋亡。0.5、1和2mg/ml黄药子70%乙醇提取物处理的SGC-7901细胞凋亡率从空白对照组的(3.81±1.56)%分别上升至(5.36±1.85)%、(10.31±2.46)%(P<0.05)和(18.54±3.26)%(P<0.01)；0.5、1和2mg/ml黄药子80%乙醇提取物处理的SGC-7901细胞凋亡率分别为(6.62±1.15)%、(14.27±2.28)%(P<0.05)和(25.07±4.21)%(P<0.01),并呈一定浓度依赖性。0.5、1和2mg/ml黄药子90%乙醇提取物处理的SGC-7901细胞凋亡率分别为(5.65±1.63)%、(11.81±2.75)%(P<0.05)和(20.32±3.54)%(P<0.01)。
6Transwell侵袭性检测结果显示：人胃癌SGC-7901细胞分别与0.5、1、2mg/ml80%黄药子醇提取物溶液共培养24小时后,细胞侵袭数目与空白对照组相比,呈降低趋势。高倍镜下对穿过下室面的细胞数进行计数,对照组为94.36±9.51个,0.5mg/ml黄药子80%乙醇提取物组为67.52±7.48个,1mg/ml黄药子80%乙醇提取物组为51.24±6.51个,2mg/ml80%黄药子醇提取物组为38.59±5.43个,与对照组比较,0.5、1、2mg/ml80%黄药子醇提取物可以抑制人胃癌细胞株SGC-7901的侵袭(P<0.05),且呈剂量依赖关系。
7建立裸鼠胃癌皮下移植瘤模型情况
将瘤块植入裸鼠皮下后,生长至第7天,形成局部可测量瘤灶,造模成功率100%；皮下肿瘤前期生长较缓,外观呈球形或半球形,初起可活动,日渐变硬,肿瘤大小不一,观察期内各组裸鼠均未出现明显消瘦及体重下降。8黄药子醇提取物对荷瘤裸鼠肿瘤体积的影响
治疗结束后,黄药子高剂量组瘤体积小于模型组(P<0.05),5-FU组和黄药子低剂量组瘤体积与模型对照组比较无统计学差异(P>0.05),黄药子高剂量组与低剂量组及5-FU组比较,差异均无统计学意义(P>0.05),5-FU组和黄药子低、高剂量组三者对瘤体积的抑制率分别为25%、17.86%和39.29%。9黄药子醇提取物对荷瘤裸鼠肿瘤质量的影响
与模型对照组比较,5-FU组和黄药子高剂量组裸鼠移植瘤质量显著下降(P<0.05),黄药子不同剂量组之间瘤质量比较差异无统计学意义(P>0.05),高低剂量黄药子组瘤质量与5-FU组比较,差异亦无统计学意义(P>0.05),5-FU组和黄药子低、高剂量组对荷瘤裸鼠瘤质量的抑制率分别为37.5%、25%和43.75%。10黄药子醇提取物对裸鼠皮下移植瘤外周血血清IL-8水平的影响
与模型对照组比较,5-FU组和黄药子组裸鼠血清IL-8水平显著下降(P<0.05)；黄药子高剂量组血清IL-8水平明显低于低剂量组(P<0.05)。11.黄药子醇提取物对裸鼠皮下移植瘤外周血血清sICAM-1水平的影响
与模型对照组比较,5-FU组和黄药子组裸鼠血清sICAM-1水平显著下降(P<0.05)；黄药子高剂量组血清sICAM-1水平明显低于低剂量组(P<0.05)；黄药子高剂量组血清sICAM-1水平明显低于5-FU组(P<0.05)。
结论
1.黄药子醇提取物对人胃癌SGC-7901细胞具有一定的自由基清除能力和较强的抗氧化还原能力；
2.黄药子醇提取物能抑制人胃癌SGC-7901细胞的增殖,并诱导人胃癌SGC-7901细胞的凋亡;
3.黄药子醇提取物能降低人胃癌SGC-7901细胞的侵袭性；
4.黄药子醇提取物能体内抑制人胃癌裸鼠皮下移植瘤的生长,可能与下调侵袭转移相关炎性因子(IL-8和sICAM-1有一定关系。
Gastric cancer is one of common malignant tumors in worldwide. The global new cases of primary gastric cancer are990000in2008, accounting for8%of all new cancer cases. And740000were died, accounting for10%of all cases of death. At present, GC tumor mortality is accounted for the second, only followed by lung cancer in the world. According to epidemiology statistics, China's gastric cancer incidence rate ranked third in2000, and has risen to second from2004to2007. T he total incidence shows a steady trend. According to the China health statistics annual report in2010, the annual mortality rate of gastric cancer in China was23.10/10million, which was more than2times of the world average.Gastric cancer has become a major disease that threatens the people's health, and hinders economic and social development. At present the major treatment of gastric cancer is surgical operation. The patients with early gastric cancer postoperative5years survival rate is high, which may reach above90%. But the vast majority of gastric cancer is advanced at the time of diagnosis, and has lost the chance of radical operation.5year survival rate of it's is only11%-40%. Western medicine for advanced gastric cancer is radical or palliative operation, radiotherapy and chemotherapy immunotherapy treatment. However, western medicine curative effect of worldwide advanced gastric cancer is not ideal. Thus treatment of traditional Chinese medicine has a considerable role. Therefore, it has strong clinical and practical significance to further look for strong specificity and high efficiency low toxicity of antitumor drugs.
In recent years, traditional Chinese medicine in the prevention and treatment of gastric cancer plays a more prominent role. And the studies in vitro have confirmed that Chinese medicine anti-tumor effect is exact. Clinical practice shows that separate application of traditional Chinese medicine or Chinese medicine compound treatment of gastric cancer has curative effect, at the same time, that can reduce the side effects of radiation and chemotherapy. Rhizoma dioscoreae bulbiferae is the tuber of potato plants Dioscorea bulbifera1. It is cold and bitter taste,and it has the role of cooling blood, eliminating gall, sending fire and detoxification. Rhizoma dioscoreae bulbiferae is widely used in clinical. It mainly treat a variety of disorders. Past literature reported that Chinese medicine rhizoma dioscoreae bulbiferae has powerful antitumor effect. Clinical reports that using huangbai stomach soup containing rhizoma dioscoreae bulbiferae to treat gastric cancer, cardia cancer and esophagus cancer have curative effect. There have reports that rhizoma dioscoreae bulbiferae was used to treat liver cancer, breast cancer, cervical cancer, primary bronchial lung cancer, lymphoma, etc. It also has certain curative effect. Related studies have shown that Chinese medicine anti-tumor mechanisms include inhibiting the expression of proto-oncogenes, promoting the expression of tumor suppressor genes, regulating of cancerous tissue surrounding environment, inducing cancer cell apoptosis, reducing the ability of gastric cancer cell invasion and metastasis. But the concrete mechanism of rhizoma dioscoreae bulbiferae is not clear for the treatment of gastric cancer. To study the above problems, this study proposed by observing the effect of malignant biological behavior of dioscoreae bulbiferae on cultured in vitro. And preliminary explore the molecular mechanism of its action and establish the animal model of nude mouse stomach subcutaneous transplantation tumor dioscoreae bulbiferae. Then we observe the effect of rhizoma dioscoreae bulbiferae on a tumor-burdened rat growth inhibition. This study of rhizoma dioscoreae bulbiferae can provide scientific theoretical and experimental basis for treatment of cancer of the stomach, and can enrich anti-tumor theory at the same time. It is of great significance to promote the traditional Chinese medicine clinical transformation.
Objective
1To observe of the effect of redox ability, proliferation and metastasis by dioscorea bulbifera on gastric cancer SGC-7901cells in vitro, and to investigate the molecular mechanism of dioscoreae bulbiferae inhibition of gastric cancer growth and metastasis.
2To observe the effect of the anti-tumor effect of dioscorea bulbifera by investigating the tumor growth in animal model of gastric cancer.
Method
1Gastric cancer cell was cultured in vitro, and dioscorea bulbifera was extracted by ethanol to make of different concentration solution.
2We use the spectrophotometric to detect the clearance rate of hydroxyl radical and DPPH radical of alcohol extraction of dioscorea bulbifera of different concentration on gastric cancer cell.
3We use ABTS rapid test to detect the antioxidant activity of extracts of different concentrations of alcohol extraction of dioscorea bulbifera on gastric cancer cells and reduction ability.
4We use MTT method to detect the effect of proliferation of different concentrations of alcohol extraction of dioscorea bulbifera on gastric cancer cells.
5We use the flow cytometry to detect the effect of different concentrations on the apoptosis of gastric cancer cells.
6We use the Transwell invasion to detect the effect of different concentrations of Dioscorea bulbifera alcohol effect on the invasion of gastric cancer cells.
7We established the gastric carcinoma transplanted subcutaneously in nude mice and observed the effects of ethanol extract of Dioscorea bulbifera L on tumor growth.
8We use the ELISA to detect the expression of the inflammatory cytokines in peripheral blood of gastric cancer in nude mice. We observed the effects of inflammatory cytokine concentrations of ethanol extract of Dioscorea bulbifera L.
All data used SPSS13statistical software. The significant level of α=0.05, with P＜0.05as the difference has statistical significance. Result
1Spectrophotometry
Different concentrations of rhizoma dioscoreae bulbiferae ethanol extracts has certain hydroxyl radicals and DPPH free radical clearance. In the three kinds of extracts, the DPPH radical scavenging capacity with concentration of2mg/ml,70%ethanol extract was strongest. It's clearance ratewhich was55.2%. The OH radical scavenging capacity of80%ethanol extract was the highest. It's clearance rate was51.2%. And the OH radical scavenging capacity of70%and90%ethanol extracts were38.7%and39.6%respectively.
2Reducing power test
Rhizoma dioscoreae bulbiferae ethanol extracts have stronger reducing power. The reducing power of70%ethanol extract was28.1μmol Fe2+, The reducing power of80%ethanol extract was equival to49.3μmol Fe2+per1g, the reducing power of90%rhizoma dioscoreae bulbiferae ethanol extracts was equival to35.2μmol Fe2+per1g.80%ethanol extract has the strongest reducing
power.
3Rapid detection of ABTS
Rhizoma dioscoreae bulbiferae ethanol extracts had stronger antioxidant capacity. The removal of the80%ethanol extract was highest, which was close to72%. The clearance rate in70%and90%ethanol extracts of was55%and45%respectively. And the total antioxidant capacity increased gradually with the time going and the concentration increasing. When the lowest concentration was0.2mg/ml, the clearance rate was8%. When the highest concentration was2mg/ml, the clearance rate was52%. When4min,7min and12min, the concentration was0.2mg/m,0.4mg/m and1.0mg/ml. The clearance rate has risen steadily. When the concentrations were1.4mg/ml,1.8mg/ml and2mg/ml, the changes of free radical clearance were not obvious, that were generally between55%and67%.
4Detection cell proliferation by MTT method
Different concentrations of rhizoma dioscoreae bulbiferae ethanol extracts solution could inhibit gastric cancer SGC-7901cells' proliferation.70%rhizoma dioscoreae bulbiferae ethanol extracts solution with0.5mg/ml1mg/ml and2mg/ml could inhibit gastric cancer SGC-7901cell proliferation rate, there are34.5%,41.2%and34.5%respectively. The cell proliferation rate in inhibits gastric cancer SGC-7901of80%rhizoma dioscoreae bulbiferae ethanol extracts solution were0.5mg/ml、1mg/ml、2mg/ml, they were41.3%、55.8%and62.3%respectively. In90%rhizoma dioscoreae bulbiferae ethanol extracts solution with0.5mg/m、1mg/m、2mg/ml, the cell proliferation rate to inhibit gastric cancer SGC-7901were28.7%,32.4%and28.7%respectively. The inhibition rate of80%rhizoma dioscoreae bulbiferae ethanol extracts had the highest of62.3%.
5Flow cytometry instrument to detect cell apoptosis
After culture48h, rhizoma dioscoreae bulbiferae ethanol extracts could obviously promote the SGC-7901cells apoptosis, and the control group only had a small amount of SGC-7901cells apoptosis.
The cell apoptosis rate in70%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、lmg/m and2mg/ml were rised from the blank control group (3.81±1.56)%respectively up to (5.36±1.85)%,(10.31±2.46)%(P＜0.05, and (18.54±3.26)%(P＜0.01);0.5,1and2mg/ml airpotato yam80%ethanol extract of SGC-7901cell apoptosis rate respectively (6.62±1.15)%,(14.27±2.28)%(P＜0.05) and (25.07±4.21)%(P＜0.01), and a certain concentration dependence.0.5,1and2mg/ml airpotato yam90%ethanol extract processing of SGC-7901cell apoptosis rate (5.65±1.63)%,respectively(11.81±2.75)%(P＜0.05) and (20.32±3.54)%(P＜0.01).
6Transwell invasive testing
Human gastric cancer SGC-7901cells were cultured24hours respectively with80%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、1mg/m and2mg/ml. Compared with the blank control group, the cell invasion number showed a trend of decrease. We count the cells number under through the room at high magnification, the control group was94.36±9.51,80%rhizoma dioscoreae bulbiferae ethanol extracts with0.5mg/ml were67.52±7.48.80%rhizoma dioscoreae bulbiferae ethanol extracts with1mg/ml were51.24±6.51.80%rhizoma dioscoreae bulbiferae ethanol extracts with2mg/ml were38.59±5.43. Compared with control group,80%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、1mg/m and2mg/ml can inhibit gastric cancer cell line (P＜0.05). There was a dose-response relationship.
7Subcutaneous transplantation tumor model of human gastric cancer in nude mice
The tumors were implanted subcutaneously in nude mice, up to seventh days, the formation of local measurable tumor, model success rate of100%; subcutaneous tumor growth is slow, the appearance of spherical or hemispherical, early active, gradually hardens, tumor size is not a group, the observation period nude mice showed no obvious weight loss and weight loss.
8Effect of dioscorea bulbifera L alcohol extracts on tumor volume
After the end of treatment, tumor volume of high dose group is smaller than that of the model group (P＜0.05), the volume of5-FU group and low dose group and model group showed no statistical difference (P＞0.05), compared with5-FU group, high-dose group and low dose group, there were no significant differences (P＞0.05),5-FU group and low, high dose group three inhibition on the tumor volume was respectively25%,17.86%and39.29%.
9Inhibition of tumor weight rate by dioscorea bulbifera alcohol extract
Compared with the model group,5-FU group and high dose group of dioscorea bulbifera nude mice transplanted tumor mass was significantly decreased (P＜0.05), between different dose groups of Dioscorea bulbifera1tumor weight had no significant difference (P＞0.05), low dose of group tumor mass compared with5-FU group, the difference had no statistical significance (P>0.05),5-FU group and Dioscorea bulbifera low, high dose group of nude mice tumor mass was respectively37.5%,25%and43.75%.
10IL-8level of dioscorea bulbifera alcohol extract on tumor peripheral blood serum
Compared with the model group, serum IL-8levels decreased significantly in5-FU group and dioscorea bulbifera group (P<0.05); the serum IL-8levels in high dose group were significantly lower than those of low dose group (P＜0.05).
11sICAM-1level of serum level of dioscorea bulbifera alcohol extract on tumor peripheral blood serum
Compared with the model group, serum sICAM-1levels decreased significantly in5-FU group and dioscorea bulbifera group(P<0.05); serum sIC AM-1levels in high dose group were significantly lower than those of the low dose (P＜0.05). Conclusion
1Rhizoma dioscoreae bulbiferae alcohol extract has the certain ability of free radicals scavenging,reduction and antioxidant on human gastric cancer cell line.
2Rhizoma dioscoreae bulbiferae alcohol extract can inhibit proliferation and induced apoptosis ofhuman gastric cancer SGC-7901cell.
3Rhizoma dioscoreae bulbiferae alcohol extract can reduce the invasiveness of human gastric cancer SGC-7901cell line.
4Rhizoma dioscoreae bulbiferae alcohol extract can inhibit the growth of subcutaneous xenograf in gastric cancernude mice, and its possible mechanism of invasion and metastasis was related to downregulation of inflammatory factors (IL-8and sICAM-1).
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