复方阿胶浆协同化疗的减毒增效作用及其机制实验研究
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摘要
恶性肿瘤的综合治疗已成为当今抗肿瘤临床治疗领域的公认模式。随着中医药抗肿瘤研究的不断深入,研究方法的逐年更新,各种中药制剂在恶性肿瘤综合治疗中所发挥的作用已经受到国内普遍关注。近年来中药治疗恶性肿瘤的临床及实验研究颇多,众多研究提示了中药在肿瘤患者改善临床症状、延长带瘤生存期、提高生活质量、配合放化疗增效减毒方面具有明显的中医特色与优势。例如,西黄丸、复方斑蝥胶囊、参莲胶囊、平消胶囊、金龙胶囊、参一胶囊、威麦宁胶囊、紫龙金片、紫金龙胶囊、复方苦参注射液、消癌平注射液、艾迪注射液、康艾注射液、康莱特注射液等具有抗肿瘤作用,已经在临床得到了普遍应用；放化疗辅助药如贞芪扶正颗粒、健脾益肾颗粒、百令胶囊、益中生血胶囊、参芪扶正注射液等在增效减毒方面发挥着积极作用。此外,针对中药抗肿瘤的机制研究也取得了不少成果,该领域研究主要围绕对肿瘤细胞增殖、分化、凋亡、癌基因及抑癌基因的控制及免疫调节等方面,然而针对放化疗增效减毒的临床研究仍存在目标性欠缺等问题,基础研究深入不够,现有的文献也不足以回答目前医学界普遍关注“中药增效是否增毒,减毒是否减效”临床问题。因此,有必要从本领域的基础研究来辅助解决临床实际问题。1研究目的
建立lewis肺癌移植瘤模型,以复方阿胶浆、环磷酰胺(Cyclophosphamide,CTX)为干预药物,从影响肿瘤抑制率与舒缓外周血象、调节免疫功能、促进细胞凋亡、抗血管生成、影响周期蛋白CyclinD1以及粘附分子CD44的表达几个方面研究复方阿胶浆的增效减毒作用及其相关机制,为复方阿胶浆在肿瘤辅助治疗中的进一步推广提供基础研究根据。2研究方法
lewis肺癌瘤株接种于C57BL/6小鼠腋下,建构移植瘤模型。第3天按拉丁表法随机分为模型对照组、CTX组、复方阿胶浆组(中剂量)、复方阿胶浆大、中、小剂量+环磷酰胺组(联合用药1、2、3),共观察13天。第7、13天取血检测血常规；实验前及实验后每4天给小鼠称重并用游标卡尺测量肿瘤体积,绘制体重变化及肿瘤生长曲线。实验结束后,取脾、胸腺称重并计算脏器指数,采用MTT法检测脾淋巴细胞增殖能力；取瘤块称重并计算抑瘤率；免疫组化染色法分别检测血管内皮生长因子VEGF、凋亡蛋白Bcl-2/Bax、周期蛋白CyclinD1、粘附分子CD44表达。同期进行另一组实验,分组、给药方法同前,观察各组小鼠的生存期。
3研究结果
3.1复方阿胶浆对化疗药物的减毒作用研究
3.1.1复方阿胶浆对化疗药物血液学毒性的影响
给药后第7天,CTX组与模型对照组及其它给药组比较WBC数均显著降低(p<0.01),联合给药各组WBC数均高于CTX组(p<0.05～0.01),且与模型对照组相比无显著差异(p>0.05)。实验各组RBC、HGB、PLT数与模型对照组比较无显著差异(p>0.05)。
给药后第13天,CTX组及联合给药各组WBC均高于模型对照组(p<0.01)；CTX组及联合给药3组RBC、HGB数低于模型对照组(p<0.01),联合给药2组较CTX组RBC、HGB明显升高(p<0.05),且与模型对照组无显著性差异(p>0.05)。各组血小板数无明显差异(p>0.05)。
3.1.2复方阿胶浆对免疫功能的调节
复方阿胶浆及联合用药各组脾脏指数均显著高于模型对照组(p<0.01)；与CTX组比较,联合用药1、2组脾脏指数明显升高(p<0.05)。与模型对照组比较,复方阿胶浆组能够增加小鼠的胸腺指数(p<0.01)。复方阿胶浆及联合用药2组脾淋巴细胞增殖能力均高于模型对照组(p<0.05)。
3.2复方阿胶浆对化疗药物的增效作用及其机制研究
给药各组的瘤重均较模型对照组明显减轻(p<0.05),复方阿胶浆组抑瘤率约25.88%；联合用药后抑瘤率分别提高了9.78%(联合给药1组)、7.79%(联合给药2组),经统计学检验,联合用药各组与CTX组比较瘤重无明显差异(p>0.05),但结合肿瘤体积变化曲线可推测复方阿胶浆有提高CTX抑瘤率的趋势。
CTX、联合用药1、2组与模型对照组比较可明显下调VEGF表达(p<0.01),其中联合用药1、2组与CTX组比较无明显差异(p>0.05)；复方阿胶浆组对VEGF表达无明显影响(p>0.05)。给药各组与模型对照组比较均能明显下调Bcl-2表达(p<0.01),其中联合用药1组与CTX组相比,下调作用更明显(p<0.01)。给药各组与模型对照组比较Bax表达水平升高(p<0.05),其中联合用药2组与CTX组比较升高最为明显(p<0.01)。复方阿胶浆组、联合用药1组与模型对照组比较能明显下调CyclinD1的表达水平(p<0.05),而联合用药各组与CTX组比较无显著性差异(p>0.05)。复方阿胶浆组、CTX组、联合用药1、2组与模型对照组比较均能下调CD44的表达(p<0.05),其中联合用药各组与CTX组比较无显著性差异(p>0.05)。
4结论
4.1成功制备了小鼠lewis肺癌移植瘤模型。CTX给药后第7天小鼠白细胞数较其它各组明显减低,给药后第13天RBC、HGB数较其它各组明显降低。复方阿胶浆可以有效恢复WBC、RBC、HGB计数,可能会促进骨髓造血功能。
4.2复方阿胶浆可以增加荷瘤小鼠的脾脏、胸腺指数,促进脾淋巴细胞的增殖功能,推测其可能对免疫系统有调节作用。
4.3复方阿胶浆、复方阿胶浆联合CTX均有抗肿瘤作用,复方阿胶浆联合CTX无明显增效性,二药的抗肿瘤效应可能与下调凋亡相关蛋白Bcl-2、上调Bax蛋白表达及下调周期素蛋白CyclinD1、粘附分子CD44的表达有关
总结：复方阿胶浆对化疗药物具有减毒作用,体现在两个方面：①对化疗药物CTX引起的血象下降有保护作用；②能够增强机体免疫功能。在减毒的同时,还能发挥抗肿瘤效应,不仅不降低化疗药物的疗效,在一定程度上可能有增效的趋势。
Comprehensive treatment of malignant tumors has become a model in anti-tumor therapy. With the progress on the study of traditional Chinese medicine in the treatment of cancer and the updates annually in research methods, various formulations of Chinese medicine integrated in the treatment of malignant tumors have got much attention from scholars in our country and abroad. There are lots of clinical and experimental research in the treatment of malignant tumors with Chinese medicine in recent years, which suggest the unique advantages and certain efficacy of Chinese medicine in alleviating clinical symptoms, prolonging survival time with tumor, improving the quality of life, toxicity reducing and efficacy enhancing of chemotherapy. Many Chinese patent drugs have been listed, such as Xihuang pills, Fufang Banmao capsule, Shenlian capsule, Pingxiao capsule, Jinlong capsule, Shenyi capsule, Weimaining, Zilongjin, Zijinlong capsule, Compound Kushen injection, Xiaoaiping injection, Aidi injection, Kang ai injection, Kanglaite injection and Chemoradiotherapy adjuvants such as Zhenqifuzheng granule, Jianpiyishen Granules, Bailing capsule, Yizhong shengxue capsule, and Shenqi Fuzheng Injection. Besides that, the study in the anti-tumor mechanism of traditional Chinese medicine has also made lots of achievements, which mainly centered on the tumor cell proliferation, differentiation, apoptosis, oncogenes and tumor suppressor genes, and immune regulation. However, the experimental study on toxicity reducing has not deep enough, and especially the research on toxicity decreasing but not effect reducing with both anti-tumor effects of traditional Chinese medicine is still a little. So it is necessary to actively carry out basic experimental research in this field.
1 Objective
In this study, lewis lung cancer xenograft model were made, and treated with Compound Ejiao slurry (CEJS) and chemotherapy drug cyclophosphamide (CTX). Explore the influence of CEJS on the toxic and side effects of chemotherapy agents, from the peripheral blood and immune functions associated with the toxicity of chemotherapy, especially for the protection of blood, to the tumor inhibition rate and anti-tumor mechanism. Observe the anti-tumor effect of the drug and further seek the possible anti-tumor mechanism for CEJS in order to provide new experimental basis and scientific guidance for the clinical application of CEJS.
2 Methods
Lewis lung cancer xenograft model were made by armpit injection using lewis cells of lung cancer. C57BL/6 mice were randomly divided into model group, CTX group, CEJS group(medium dosage), CEJS high, medium, low-dosage plus CTX groups(combination group 1,2,3) according to latin table method. Thirteen days were observed. The measurement of blood routine were carried out on day 7th, 13th. The weight of mice and tumor volume were measured every four days before the experiment and after. Drew the curve of weight changes and tumor growth. The volume were measured with a vernier caliper. Calculated thymus and spleen index, and tested lymphocyte proliferation function with MTT method after the experiment. Removed the tumor and calculated the inhibitory rate. The expression of vascular endothelial growth factor VEGF, apoptosis protein Bcl-2/Bax, CyclinD1, and adhesion molecules CD44 were detected by immuno-histochemistry. Compared the survival rate of mice in each group.
3 Results
3.1 Study on the attenuation effect of CEJS on chemotherapy drug
3.1.1 Effects of CEJS on hematological toxicity of chemotherapy drug
On the 7th day after treatment, the CTX group declined more obviously, compared the numerical value of WBC with other groups (p<0.05～0.01).The WBC amount of each combination group rised significantly than CTX group (p<0.05～0.01). There were no significant difference between the experimental groups and model group in the number of RBC, HGB, and PLT (p>0.05). On the 13th day after treatment, the WBC amount of CTX and every combination group increased more rapidly compared with the model group (p<0.01). Compared the numerical value of RBC and HGB with the model group, the CTX and combination group 3 were lower significantly (p<0.01). The numerical value of RBC and HGB increased in combination group 2 compared with CTX group (p<0.05). Each group had no significant difference in platelet count (p>0.05).
3.1.2 CEJS on the regulation of immune function
The spleen index in the CEJS group and combination groups increased significantly compared with the control group (p<0.01). The spleen index in combination group 1 and 2 raised significantly compared with the CTX group (p<0.05).The thymus gland index in CEJS increased significantly (p<0.01). The proliferation function of spleen cells in CEJS and combination group 2 were stronger than the control and CTX group (p<0.05).
3.2 Study on the synergy effect of CEJS and the mechanism of anti-tumor effect
Compared with model group, administration could reduce the tumor weight in each group (p<0.05). CEJS had an inhibition rate of 50%. The combination administration groups had no significant difference between CTX group (p>0.05). Inhibition rate after combination therapy were increased by 9.78%(combination group 1),7.79%(combination group 2). There was a trend of inhibition rates of CTX increasing by CEJS, with the same results from tumor volume curve.
The expression level of VEGF could be significantly down-regulated in CTX or combination group 1 and 2, compared with the control group (p<0.05). CEJS group had no significant difference with the control (p>0.05). The expression in combination group 1 and 2 were not significantly different with the CTX group (p>0.05). The expression level of Bcl-2 could be significantly down-regulated and the level of bax could be significantly up-regulated in each experimental group, compared with the control group (p<0.01～0.05). Compared with the CTX group, the expression of Bcl-2 were obviously lower in combination group 1 (p<0.05). The expression of bax were significantly higher in combination group 2, compared with the CTX group (p<0.01). CEJS and combination group 1 resulted in more significant suppression of the expression of CyclinDl when compared with the model group (p<0.05). The expression of CD44 in CTX group, CEJS group, combination groupl,2 were lower than the model group (p<0.05).
4 Conclusions
4.1 Successfully prepared lewis lung cancer xenograft model in mice. White cell amounts in CTX group significantly reduced compared with other groups on the 7th day after administration. RBC and HGB amounts in CTX group significantly reduced compared with other groups on the 13th after administration. CEJS could effectively recover WBC, RBC, HGB count and promote the bone marrow hematopoietic function.
4.2 CEJS could increase the spleen and thymus index of tumor-bearing mice, and promote proliferation of spleen lymphocytes, suggesting that it may have a moderating effect on immunity.
4.3 CEJS could not improve the effects of chemotherapy obviously, but it itself had some anti-tumor effect, which may be associated with down-regulation of apoptosis-related protein Bcl-2, Bax and down-regulation of cyclin protein CyclinD1, and adhesion CD44 molecules.
Summary:CEJS had toxic-reducing effects of chemotherapy, reflected in two aspects:①Protected the decreased blood caused by CTX.②Enhanced immune function. Reduced the damage of bone marrow hematopoietic function, also played an anti-tumor effect. CEJS did not affect the efficacy of chemotherapy drugs only, but also had a synergistic trend to a certain extent.
建立lewis肺癌移植瘤模型,以复方阿胶浆、环磷酰胺(Cyclophosphamide,CTX)为干预药物,从影响肿瘤抑制率与舒缓外周血象、调节免疫功能、促进细胞凋亡、抗血管生成、影响周期蛋白CyclinD1以及粘附分子CD44的表达几个方面研究复方阿胶浆的增效减毒作用及其相关机制,为复方阿胶浆在肿瘤辅助治疗中的进一步推广提供基础研究根据。2研究方法
lewis肺癌瘤株接种于C57BL/6小鼠腋下,建构移植瘤模型。第3天按拉丁表法随机分为模型对照组、CTX组、复方阿胶浆组(中剂量)、复方阿胶浆大、中、小剂量+环磷酰胺组(联合用药1、2、3),共观察13天。第7、13天取血检测血常规；实验前及实验后每4天给小鼠称重并用游标卡尺测量肿瘤体积,绘制体重变化及肿瘤生长曲线。实验结束后,取脾、胸腺称重并计算脏器指数,采用MTT法检测脾淋巴细胞增殖能力；取瘤块称重并计算抑瘤率；免疫组化染色法分别检测血管内皮生长因子VEGF、凋亡蛋白Bcl-2/Bax、周期蛋白CyclinD1、粘附分子CD44表达。同期进行另一组实验,分组、给药方法同前,观察各组小鼠的生存期。
3研究结果
3.1复方阿胶浆对化疗药物的减毒作用研究
3.1.1复方阿胶浆对化疗药物血液学毒性的影响
给药后第7天,CTX组与模型对照组及其它给药组比较WBC数均显著降低(p<0.01),联合给药各组WBC数均高于CTX组(p<0.05～0.01),且与模型对照组相比无显著差异(p>0.05)。实验各组RBC、HGB、PLT数与模型对照组比较无显著差异(p>0.05)。
给药后第13天,CTX组及联合给药各组WBC均高于模型对照组(p<0.01)；CTX组及联合给药3组RBC、HGB数低于模型对照组(p<0.01),联合给药2组较CTX组RBC、HGB明显升高(p<0.05),且与模型对照组无显著性差异(p>0.05)。各组血小板数无明显差异(p>0.05)。
3.1.2复方阿胶浆对免疫功能的调节
复方阿胶浆及联合用药各组脾脏指数均显著高于模型对照组(p<0.01)；与CTX组比较,联合用药1、2组脾脏指数明显升高(p<0.05)。与模型对照组比较,复方阿胶浆组能够增加小鼠的胸腺指数(p<0.01)。复方阿胶浆及联合用药2组脾淋巴细胞增殖能力均高于模型对照组(p<0.05)。
3.2复方阿胶浆对化疗药物的增效作用及其机制研究
给药各组的瘤重均较模型对照组明显减轻(p<0.05),复方阿胶浆组抑瘤率约25.88%；联合用药后抑瘤率分别提高了9.78%(联合给药1组)、7.79%(联合给药2组),经统计学检验,联合用药各组与CTX组比较瘤重无明显差异(p>0.05),但结合肿瘤体积变化曲线可推测复方阿胶浆有提高CTX抑瘤率的趋势。
CTX、联合用药1、2组与模型对照组比较可明显下调VEGF表达(p<0.01),其中联合用药1、2组与CTX组比较无明显差异(p>0.05)；复方阿胶浆组对VEGF表达无明显影响(p>0.05)。给药各组与模型对照组比较均能明显下调Bcl-2表达(p<0.01),其中联合用药1组与CTX组相比,下调作用更明显(p<0.01)。给药各组与模型对照组比较Bax表达水平升高(p<0.05),其中联合用药2组与CTX组比较升高最为明显(p<0.01)。复方阿胶浆组、联合用药1组与模型对照组比较能明显下调CyclinD1的表达水平(p<0.05),而联合用药各组与CTX组比较无显著性差异(p>0.05)。复方阿胶浆组、CTX组、联合用药1、2组与模型对照组比较均能下调CD44的表达(p<0.05),其中联合用药各组与CTX组比较无显著性差异(p>0.05)。
4结论
4.1成功制备了小鼠lewis肺癌移植瘤模型。CTX给药后第7天小鼠白细胞数较其它各组明显减低,给药后第13天RBC、HGB数较其它各组明显降低。复方阿胶浆可以有效恢复WBC、RBC、HGB计数,可能会促进骨髓造血功能。
4.2复方阿胶浆可以增加荷瘤小鼠的脾脏、胸腺指数,促进脾淋巴细胞的增殖功能,推测其可能对免疫系统有调节作用。
4.3复方阿胶浆、复方阿胶浆联合CTX均有抗肿瘤作用,复方阿胶浆联合CTX无明显增效性,二药的抗肿瘤效应可能与下调凋亡相关蛋白Bcl-2、上调Bax蛋白表达及下调周期素蛋白CyclinD1、粘附分子CD44的表达有关
总结：复方阿胶浆对化疗药物具有减毒作用,体现在两个方面：①对化疗药物CTX引起的血象下降有保护作用；②能够增强机体免疫功能。在减毒的同时,还能发挥抗肿瘤效应,不仅不降低化疗药物的疗效,在一定程度上可能有增效的趋势。
Comprehensive treatment of malignant tumors has become a model in anti-tumor therapy. With the progress on the study of traditional Chinese medicine in the treatment of cancer and the updates annually in research methods, various formulations of Chinese medicine integrated in the treatment of malignant tumors have got much attention from scholars in our country and abroad. There are lots of clinical and experimental research in the treatment of malignant tumors with Chinese medicine in recent years, which suggest the unique advantages and certain efficacy of Chinese medicine in alleviating clinical symptoms, prolonging survival time with tumor, improving the quality of life, toxicity reducing and efficacy enhancing of chemotherapy. Many Chinese patent drugs have been listed, such as Xihuang pills, Fufang Banmao capsule, Shenlian capsule, Pingxiao capsule, Jinlong capsule, Shenyi capsule, Weimaining, Zilongjin, Zijinlong capsule, Compound Kushen injection, Xiaoaiping injection, Aidi injection, Kang ai injection, Kanglaite injection and Chemoradiotherapy adjuvants such as Zhenqifuzheng granule, Jianpiyishen Granules, Bailing capsule, Yizhong shengxue capsule, and Shenqi Fuzheng Injection. Besides that, the study in the anti-tumor mechanism of traditional Chinese medicine has also made lots of achievements, which mainly centered on the tumor cell proliferation, differentiation, apoptosis, oncogenes and tumor suppressor genes, and immune regulation. However, the experimental study on toxicity reducing has not deep enough, and especially the research on toxicity decreasing but not effect reducing with both anti-tumor effects of traditional Chinese medicine is still a little. So it is necessary to actively carry out basic experimental research in this field.
1 Objective
In this study, lewis lung cancer xenograft model were made, and treated with Compound Ejiao slurry (CEJS) and chemotherapy drug cyclophosphamide (CTX). Explore the influence of CEJS on the toxic and side effects of chemotherapy agents, from the peripheral blood and immune functions associated with the toxicity of chemotherapy, especially for the protection of blood, to the tumor inhibition rate and anti-tumor mechanism. Observe the anti-tumor effect of the drug and further seek the possible anti-tumor mechanism for CEJS in order to provide new experimental basis and scientific guidance for the clinical application of CEJS.
2 Methods
Lewis lung cancer xenograft model were made by armpit injection using lewis cells of lung cancer. C57BL/6 mice were randomly divided into model group, CTX group, CEJS group(medium dosage), CEJS high, medium, low-dosage plus CTX groups(combination group 1,2,3) according to latin table method. Thirteen days were observed. The measurement of blood routine were carried out on day 7th, 13th. The weight of mice and tumor volume were measured every four days before the experiment and after. Drew the curve of weight changes and tumor growth. The volume were measured with a vernier caliper. Calculated thymus and spleen index, and tested lymphocyte proliferation function with MTT method after the experiment. Removed the tumor and calculated the inhibitory rate. The expression of vascular endothelial growth factor VEGF, apoptosis protein Bcl-2/Bax, CyclinD1, and adhesion molecules CD44 were detected by immuno-histochemistry. Compared the survival rate of mice in each group.
3 Results
3.1 Study on the attenuation effect of CEJS on chemotherapy drug
3.1.1 Effects of CEJS on hematological toxicity of chemotherapy drug
On the 7th day after treatment, the CTX group declined more obviously, compared the numerical value of WBC with other groups (p<0.05～0.01).The WBC amount of each combination group rised significantly than CTX group (p<0.05～0.01). There were no significant difference between the experimental groups and model group in the number of RBC, HGB, and PLT (p>0.05). On the 13th day after treatment, the WBC amount of CTX and every combination group increased more rapidly compared with the model group (p<0.01). Compared the numerical value of RBC and HGB with the model group, the CTX and combination group 3 were lower significantly (p<0.01). The numerical value of RBC and HGB increased in combination group 2 compared with CTX group (p<0.05). Each group had no significant difference in platelet count (p>0.05).
3.1.2 CEJS on the regulation of immune function
The spleen index in the CEJS group and combination groups increased significantly compared with the control group (p<0.01). The spleen index in combination group 1 and 2 raised significantly compared with the CTX group (p<0.05).The thymus gland index in CEJS increased significantly (p<0.01). The proliferation function of spleen cells in CEJS and combination group 2 were stronger than the control and CTX group (p<0.05).
3.2 Study on the synergy effect of CEJS and the mechanism of anti-tumor effect
Compared with model group, administration could reduce the tumor weight in each group (p<0.05). CEJS had an inhibition rate of 50%. The combination administration groups had no significant difference between CTX group (p>0.05). Inhibition rate after combination therapy were increased by 9.78%(combination group 1),7.79%(combination group 2). There was a trend of inhibition rates of CTX increasing by CEJS, with the same results from tumor volume curve.
The expression level of VEGF could be significantly down-regulated in CTX or combination group 1 and 2, compared with the control group (p<0.05). CEJS group had no significant difference with the control (p>0.05). The expression in combination group 1 and 2 were not significantly different with the CTX group (p>0.05). The expression level of Bcl-2 could be significantly down-regulated and the level of bax could be significantly up-regulated in each experimental group, compared with the control group (p<0.01～0.05). Compared with the CTX group, the expression of Bcl-2 were obviously lower in combination group 1 (p<0.05). The expression of bax were significantly higher in combination group 2, compared with the CTX group (p<0.01). CEJS and combination group 1 resulted in more significant suppression of the expression of CyclinDl when compared with the model group (p<0.05). The expression of CD44 in CTX group, CEJS group, combination groupl,2 were lower than the model group (p<0.05).
4 Conclusions
4.1 Successfully prepared lewis lung cancer xenograft model in mice. White cell amounts in CTX group significantly reduced compared with other groups on the 7th day after administration. RBC and HGB amounts in CTX group significantly reduced compared with other groups on the 13th after administration. CEJS could effectively recover WBC, RBC, HGB count and promote the bone marrow hematopoietic function.
4.2 CEJS could increase the spleen and thymus index of tumor-bearing mice, and promote proliferation of spleen lymphocytes, suggesting that it may have a moderating effect on immunity.
4.3 CEJS could not improve the effects of chemotherapy obviously, but it itself had some anti-tumor effect, which may be associated with down-regulation of apoptosis-related protein Bcl-2, Bax and down-regulation of cyclin protein CyclinD1, and adhesion CD44 molecules.
Summary:CEJS had toxic-reducing effects of chemotherapy, reflected in two aspects:①Protected the decreased blood caused by CTX.②Enhanced immune function. Reduced the damage of bone marrow hematopoietic function, also played an anti-tumor effect. CEJS did not affect the efficacy of chemotherapy drugs only, but also had a synergistic trend to a certain extent.
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