维康颗粒对肿瘤相关性疲劳小鼠的影响及机制研究
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摘要
背景
近年来,随着乳腺癌的诊疗技术的进步以及人们自我保健意识的加强,乳腺癌患者的死亡率正逐渐下降,生活中经历过或正在经历乳腺癌的人们越来越多。肿瘤相关性疲劳(cancer related fatigue, CRF)的产生与癌症或癌症的治疗相关,并且干扰患者正常生活,极大地影响了癌症患者的生活质量。因此,如何提高这部分日益增多的人群的生活质量就变得十分重要。
目前,CRF越来越引起国内外学者的关注,但CRF发生的病理生理学机制仍然不是很清楚。现代西医学临床上仍缺乏有效的干预手段,这为中医药干预CRF提供了展示的机遇。CRF的治疗要立足于肿瘤疾病本身,中医认为正气不足是肿瘤发生的内在根本原因,气血阴阳失衡、脏腑功能紊乱是它的病理基础,其基本病理变化除正气亏虚外,更有气滞、血瘀、痰结、湿聚、热毒等相互纠结,日久积滞而成有形之肿块。CRF是在肿瘤的基础上形成的。因此,可认为CRF是一种病因明确,治疗多变,以虚损为主,夹有实邪的病证,病理属性总属本虚标实；正气不足,毒瘀内存为其基本病机。因此中医药干预CRF在尽可能消除疲劳相关原因之外,还应强调中医的扶正与祛邪相辅相成,辨证论治,重视整体调节,立足于补养脾肾,行气祛瘀。中医理论指出：“脾为后天之本,气血生化之源,主肌肉四肢”、“肝藏血,肝主筋,为罢极之本”、“肾为先天之本,生命之根,主骨生髓,为作强之官,伎巧出焉”。故认为疲劳与中医肺、脾、肝、肾功能失调密切相关。在此理论基础上,本课题组研制出防治疲劳的中药复方——维康颗粒,从肺、脾、肝、肾着手,具有滋补肝肾、健脾润肺、益气养阴之功效,从而通过多种途径改善疲劳,提高人的生活质量。
因此,本论文从主补肝脾肾,兼以理气祛瘀着手,选择维康颗粒,用其在乳腺癌小鼠模型上进行干预CRF的相关实验研究,以期为临床运用维康颗粒干预CRF提供实验依据,并揭示其作用机制。
目的
通过建立乳腺癌小鼠CRF模型,评价自制中药复方维康颗粒对由肿瘤本身及肿瘤化疗所引起的乳腺癌小鼠CRF的作用,并明确中药复方维康颗粒缓解CRF的代谢组学机制及缓解化疗引起的相关性疲劳的基因组学机制。
方法
1.维康颗粒缓解CRF的代谢组学机制：
实验前,将所有雌性BALB/c小鼠置于游泳箱适应性游泳训练2天,5min/天,水温25℃±1℃。剔除游泳时间过长和过短的小鼠以及过于兴奋和过于安静的小鼠。然后,随机抽取10只小鼠作为正常对照(NS, po,20ml/kg, qd)组,剩余小鼠为待制备乳腺癌小鼠CRF模型组。将1×105cells/ml的4T1乳腺癌细胞接种于小鼠右侧腹壁第4对乳房脂肪垫区域的皮下,制备乳腺癌小鼠CRF模型。当瘤块长到50mm3～100mm3(接种后第7天)的时候,按瘤体积随机分为4组,每组10只,分别为肿瘤对照(NS, po,20ml/kg, qd)组、紫杉醇(PTX, ip,10mg/kg, q2d)组、维康颗粒(WKKL, po,6g/kg, qd)组、维康颗粒+紫杉醇(WKKL, po,6g/kg, qd; PTX, ip,10mg/kg, q2d)组。上述5组小鼠分别进行以下几个方面的实验：
(1)维康颗粒对荷瘤小鼠的一般情况的影响：观察实验过程中各组小鼠的体重、精神状态、活动情况、饮食以及皮毛外观变化。每周对上述小鼠的一般情况进行一次评价。
(2)维康颗粒对荷瘤小鼠的行为学影响：给药的第12天对所有小鼠进行旷场实验。主要观察小鼠在实验箱内的总运动距离、外周格运动距离、中央格运动距离、理毛次数、大便粒数、爬壁次数。于末次给药后30min,观察小鼠6min内的悬尾不动时间。
(3)维康颗粒的对荷瘤小鼠的生存时间的影响：末次药后,不再给予任何干预措施,动态观察荷瘤小鼠生存时间,并计算各组小鼠的中位生存时间、平均生存时间及生命延长率。
(4)维康颗粒对荷瘤小鼠的抗疲劳作用：于开始给药前、给药后的第10天、第20天,用7%体重的铅坠系于小鼠尾部,将小鼠放入水温25℃±1℃的游泳箱中进行力竭游泳测试,并记录力竭游泳时间。
(5)维康颗粒对荷瘤小鼠肿瘤生长的抑制作用：另取上述4组荷瘤小鼠,各组小鼠自接受治疗开始,每3天测量肿瘤体积。在末次给药1小时后,摘眼球采血,分离血浆,置-80℃保存,用于代谢组学研究。并剥离瘤块并称重,计算抑瘤率。
(6)维康颗粒对荷瘤小鼠脏器指数的影响：取前一实验中分离并称重的脾脏、胸腺、肾上腺等器官,按对应的每只小鼠的体重计算脏器指数。
(7)维康颗粒对乳腺癌小鼠CRF血浆代谢组学的研究：应用核磁共振氢谱(1H-NMR)技术获取前面实验中小鼠血浆中的原始代谢指纹图谱信息,通过数据采集处理获得各样本数据信息,采用多元数据统计方法(PCA、PLS-DA、 OPLS-DA)模式识别等方法进行数据处理,得到5组小鼠血浆代谢概况,并进行组间两两比较。最后结合模型VIP值、归一化积分值筛选出两两组间小鼠血浆有差异的代谢物(潜在分子标志物),结合数据库和文献进行代谢物鉴定,并分析相关代谢途径变化,揭示乳腺癌小鼠CRF的发生机制、WKKL缓解乳腺癌小鼠CRF的作用机制。
2.维康颗粒缓解化疗引起的相关性疲劳的基因组学机制：
另取25只雌性BALB/c小鼠置于小鼠于游泳箱(高30cm,直径25cm)适应性游泳训练2天,5min/天,水温25℃±1℃。剔除游泳时间过长和过短的小鼠以及过于兴奋和过于安静的小鼠。按游泳时间随机分为3组,每组6只,共18只。分别为正常对照(NS, po,20ml/kg, qd)组、紫杉醇(PTX, ip,10mg/kg, q2d)组、维康颗粒+紫杉醇(WKKL, po,6g/kg, qd; PTX, ip,10mg/kg, q2d)组。上述3组小鼠分别进行以下几个方面的实验：
(1)维康颗粒对化疗小鼠的一般情况的影响：观察实验过程中各组小鼠的体重、精神状态、活动情况、饮食以及皮毛外观变化。每周对上述小鼠的一般情况进行一次评价。
(2)维康颗粒对化疗小鼠的抗疲劳作用：于开始给药前、给药后的第7天、第14天,用7%体重的铅坠系于小鼠尾部,将小鼠放入水温25℃±1℃的游泳箱中进行力竭游泳测试,并记录力竭游泳时间。
(3)维康颗粒对化疗小鼠骨骼肌基因组学的研究：应用基因芯片技术获取前面实验中小鼠骨骼肌中的原始基因信息,应用Affmetrix的GCOSvl.4(genechip operating software versionl.4)对荧光信号的强度和比值进行计算和统计分析,得到各芯片的单张分析结果,对芯片进行质量控制处理。用SAM (significant analysis of microarray)分析法,总体比较PTX组与正常对照组,PTX组与WKKL+PTX组间的基因表达有无显著性差异。结合数据库和文献进行基因成分、功能、进程鉴定,并分析相关通路变化,揭示化疗相关性疲劳的发生机制、WKKL缓解化疗相关性疲劳的作用机制。
结果
1.维康颗粒缓解CRF的代谢组学机制：
(1)维康颗粒对荷瘤小鼠的一般情况的影响：给药干预前,5组小鼠之间的精神状态、活动情况、饮食以及皮毛外观等一般情况未见有显著差别。各组小鼠均活泼好动,皮毛光洁整齐,眼睛有神,对食物敏感,逃避反应快。整个实验期间,NC组小鼠均保持活泼好动、皮毛光洁整齐、对食物敏感、逃避快。与NC组的小鼠比较,其余4组荷瘤小鼠均逐渐出现毛发稀疏、不活泼、行动迟缓和进食减少等现象。实验结束时,4组荷瘤小鼠的一般情况从差到相对较好依次为PTX组、TC组、WKKL+PTX组、WKKL组。干预前,各组小鼠的体重无显著差别(F=0.100,P=0.982)。NC组小鼠的体重逐渐增加。而随着肿瘤的生长,在干预2周和第3周时,TC组小鼠的体重均较NC组显著减轻(P=0.000)、PTX组小鼠的体重均较TC组显著减轻(P<0.05～0.01)、WKKL+PTX组小鼠的体重较PTX组显著增加(P<0.05～0.01)。
(2)维康颗粒对荷瘤小鼠的行为学影响：①旷场实验结果：TC组、PTX组小鼠的5min内的总运动距离(PTC组=0.014、PPTX组=0.003)、外周格运动距离(PTC组=0.000、PPTX组=0.000)较NC组小鼠均显著减少。而WKKL+PTX组小鼠的外周格运动距离较TC组(P=0.006)、PTX组(P=0.044)小鼠显著增加。TC组小鼠较NC组小鼠的爬壁次数显著减少(P=0.039)、理毛次数显著减少(P=0.006)。PTX组小鼠较TC组小鼠的大便粒数显著减少(P=0.000)、爬壁次数显著减少(P=0.003)。WKKL+PTX组小鼠较TC组小鼠的大便粒数显著减少(P=0.000); WKKL+PTX组小鼠的大便粒数较PTX组增加,但是无显著意义。②悬尾实验结果：与NC组小鼠相比,TC组小鼠的不动时间有所延长,但无显著差异(P=0.308)。与TC组小鼠比较,WKKL组(P=0.004)、WKKL+PTX组(P=0.001)小鼠的不动时间显著缩短。
(3)维康颗粒的对荷瘤小鼠的生存时间的影响：与TC组比较,PTX组(P=0.043)、WKKL组(P=0.007)、WKKL+PTX组(P=0.000)小鼠的中位生存时间及平均生存时间均显著延长。与PTX组比较,WKKL+PTX组小鼠的中位生存时间及平均生存时间均显著延长(P=0.039)。与TC组小鼠相比,WKKL+PTX组、WKKL组和PTX组3组小鼠的生命延长率分别为21.61%、13.07%和8.54%。
(4)维康颗粒对荷瘤小鼠的抗疲劳作用：①同一时间点各组小鼠力竭游泳时间的比较：干预前,5组小鼠的力竭游泳时间无显著差异(P=0.992)。干预第10天时,与NC组比较,荷瘤小鼠的力竭游泳时间显著减少(P<0.01)；与TC组比较,NC组小鼠的力竭游泳时间显著延长(P=0.000), PTX组小鼠的力竭游泳时间显著减少(P=0.014)、而WKKL组(P=0.381)、WKKL+PTX组(P=0.717)的游泳时间与之相比无显著差异。与PTX组比较,其余4组小鼠的力竭游泳时间显著延长(P<0.05～0.01)。干预20天时,与NC组比较,荷瘤组小鼠的力竭游泳时间显著减少(P<0.01)。与TC组比较,NC组小鼠的力竭游泳时间显著延长(P=0.000),而PTX组、WKKL组、WKKL+PTX组的游泳时间无显著差异(P>0.05)。与PTX组比较,NC组、WKKL组、WKKL+PTX组小鼠的力竭游泳时间显著延长(P<0.05～0.01)。②不同时间点的同组小鼠力竭游泳时间比较：NC组小鼠与干预前比较,干预第10天(P=0.000)、干预第20天(P=0.000)的游泳时间显著延长：与干预第10天比较,干预第20天时小鼠的游泳时间显著延长(P=0.004)。TC组小鼠与干预前比较,干预第10天(P=0.015),干预第20天的游泳时间均显著下降(P=0.000)；与干预第10天比较,干预第20天时小鼠的游泳能力显著下降(P=0.000)。PTX组小鼠与干预前比较,干预第10天(P=0.000)、干预第20天(P=0.000)的游泳时间均显著下降；与干预第10天比较,干预第20天时小鼠的游泳能力显著下降(P=0.000)。WKKL组小鼠与干预前比较,干预第10天小鼠的游泳能力均呈现下降的趋势(P=0.241),但无显著变化,干预第20天时小鼠的游泳能力显著下降(P=0.007)；与干预第10天比较,干预第20天时小鼠的游泳能力无显著变化(P=0.638)。WKKL+PTX组小鼠与干预前比较,干预第10天(P=0.051)时小鼠的游泳能力无显著下降,干预第20天时小鼠的游泳能力显著下降(P=0.039)；与干预第10天比较,干预第20天时小鼠的游泳能力无显著变化(P=0.464)。
(5)维康颗粒对荷瘤小鼠肿瘤生长的抑制作用：实验结束时,与TC组比较,WKKL+PTX组(P=0.007)\PTX组(P=0.037)的瘤重均显著减轻。与TC组比较,WKKL+PTX组、WKKL组、PTX组的肿瘤体积显著变小(P<0.05～0.01)。WKKL+PTX组、WKKL组和PTX组3组小鼠的抑瘤率分别为22.24%、15.36%和16.89%。
(6)维康颗粒对荷瘤小鼠脏器指数的影响：①脾指数：与NC组比较,其余4组的脾指数均显著升高(P=0.01)。与TC组比较,PTX组、WKKL组、WKKL+PTX组的脾指数无显著差异(P>0.05)。与PTX组比较,WKKL组、WKKL+PTX组的脾指数无显著差异(P>0.05)。②胸腺指数：与NC组比较,其余4组的胸腺指数显著下降(P<0.01)。与TC组比较,PTX组的胸腺指数显著下降(P=0.001),WKKL组(P=0.460)、WKKL+PTX组(P=0.588)小鼠的胸腺指数无显著差异。与PTX组比较,WKKL组(P=0.010)、WKKL+PTX组(P=0.006)小鼠的胸腺指数显著升高。③肾上腺指数：与NC组比较,TC组、PTX组的肾上腺指数显著升高(P<0.01)。与TC组比较,WKKL+PTX组小鼠的肾上腺指数显著降低(P=0.037)。与PTX组比较,WKKL组、WKKL+PTX组小鼠的肾上腺指数无显著差异(P>0.05)。
(7)维康颗粒对乳腺癌小鼠CRF血浆代谢组学的研究：①五组小鼠之间血浆NMR的分析：用PLS-DA模型,最终计算出5组小鼠血浆样本共有2个主成分,5组之间基本分离,且各组相关集中,组间可基本区分。②NC组与TC组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和NC组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到16个有统计学意义的变量(P<0.05～0.01)。与NC组小鼠比较,TC组小鼠血浆中的乳酸、甘油磷酸酯、精胺、亮氨酸、丙氨酸、葡萄糖-6-磷酸、3-羟基丁酸水平升高,肌醇、磷酸胆碱、辅酶A、辛酰甘氨酸、3-羟基异戊酸、神经酸、甲基丙二酸、天冬氨酸水平下降。③TC组与PTX组小鼠组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和PTX组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,两组小鼠两组样本之间显著区分,但PTX组样本之间较TC组分散。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到13个有统计学意义的变量(P<0.05～0.01)。与TC组小鼠比较,PTX组小鼠血浆中的瓜氨酸水平升高,肌醇、磷酸胆碱、异柠檬酸、甘油磷酸酯、亚油酸、谷氨酰胺、辅酶A、天冬氨酸、丝氨酸、高半胱氨酸、赤藓糖水平下降。④TC组与WKKL组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和WKKL组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到1个有统计学意义的变量(P<0.05～0.01)。与TC组小鼠比较,WKKL组小鼠血浆中的甘油磷酸酯水平升高。⑤TC组与WKKL+PTX组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和WKKL+PTX组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到17个有统计学意义的变量(P<0.05～0.01)。与TC组小鼠比较,WKKL+PTX组小鼠血浆中的的肌醇、二羟基丙酮、磷酸胆碱、亚精胺、3-羟基丁酸、丙二酸、天冬氨酸、丝氨酸、L-缬氨酸、丙酮酸、琥珀酸半醛、氨基己酸、β-丙氨酸水平下降,丙酰甘氨酸、瓜氨酸、异亮氨酸、辛酰甘氨酸、2-羟基丁酸水平升高。⑥PTX组与WKKL+PTX组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,PTX组和WKKL+PTX组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到5个有统计学意义的变量(P<0.05-0.01)。与PTX组小鼠比较,WKKL+PTX组小鼠血浆中的神经酸、辅酶A、磷酸烯醇丙酮酸、肌醇、赤藓糖水平升高。⑦WKKL组与WKKL+PTX组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,WKKL组和WKKL+PTX组两组分离趋势增强,但仍存在一定的重叠,两组尚未能完全区分开来。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到12个有统计学意义的变量(P<0.05～0.01)。与WKKL组小鼠比较,WKKL+PTX组小鼠血浆中的丙酰甘氨酸、瓜氨酸、甘油磷酸酯、2-羟基丁酸、辛酰甘氨酸、异亮氨酸、皮质酮、异阿魏酸、四氢叶酸、苯丙氨酸、丙氨酸水平升高,磷酸胆碱水平下降。
2.维康颗粒缓解化疗引起的相关性疲劳的基因组学机制：
(1)维康颗粒对化疗小鼠的一般情况的影响：给药干预前,3组小鼠之间的精神状态、活动情况、饮食以及皮毛外观等一般情况未见有显著差别。各组小鼠均活泼好动,皮毛光洁整齐,眼睛有神,对食物敏感,逃避反应快。整个实验过程中,NC组小鼠的上述情况未有显著变化。与NC组的小鼠比较,其余2组化疗小鼠均逐渐出现毛发稀疏、聚集、不活泼、行动迟缓和进食减少等现象,但WKKL+PTX组小鼠的一般情况较PTX组要好。干预前,各组小鼠的体重无显著差别(F=0.066,P=0.936)。NC组小鼠的体重逐渐增加,干预7天时,NC组小鼠的体重较干预前显著增加(P=0.002), PTX组小鼠体重较干预前减少、WKKL+PTX组小鼠体重较干预前增加,但是无显著差异。与PTX组小鼠体重相较,NC组(P=0.001), WKKL+PTX组(P=0.002)均有显著性差异。干预14天时,NC组及WKKL+PTX组小鼠的体重较干预前显著增加(P=0.05～0.01),但较干预1周时无显著增加。PTX组小鼠的体重较干预前(P=0.009)、干预7天(P=0.008)时均显著减轻。与PTX组小鼠体重比较,NC组(P=0.000)、WKKL+PTX组(P=0.000)的小鼠体重均分别有显著差异。
(2)维康颗粒对化疗小鼠的抗疲劳作用：①同一时间点各组小鼠力竭游泳时间的比较：干预前,3组小鼠的力竭游泳时间无显著差异(P=0.992)。干预第7天时,与NC组比较,PTX组(P=0.000)、WKKL+PTX组(P=0.001)小鼠的力竭游泳时间显著减少；但PTX组与WKKL+PTX组小鼠的力竭游泳时间无显著差异。干预14天时,与NC组比较,PTX组(P=0.000)、WKKL+PTX组(P=0.002)小鼠的力竭游泳时间显著减少。与PTX组比较,NC组(P=0.000)、WKKL+PTX组(P=0.007)组小鼠的力竭游泳时间显著延长(P=0.01)。②不同时间点的同组小鼠力竭游泳时间结果：随着干预时间的增加,NC组小鼠的游泳能力呈现逐步增加的趋势,PTX组小鼠的游泳能力呈现逐渐下降的趋势,WKKL+PTX组小鼠的游泳能力呈现波动性的增加趋势。对于NC组小鼠而言,与干预前比较,干预第7天(P=0.026)、干预第14天(P=0.011)的游泳时间均显著延长；与干预第10天比较,干预第20天时小鼠的游泳时间显著延长(P=0.022)。对于PTX组小鼠而言,与干预前比较,干预第7天(P=0.029)、干预第14天(P=0.002)的游泳时间均显著下降；与干预第7天比较,干预第14天时小鼠的游泳能力无显著差异(P=0.053)。对于WKKL+PTX组小鼠而言,与干预前比较,干预第7天时(P=0.336)、干预第14天时(P=0.284)小鼠的游泳能力先下降后上升,但无显著差异；与干预第7天比较,干预第14天时小鼠的游泳能力无显著变化(P=0.134)。
(3)维康颗粒对化疗小鼠骨骼肌基因组学的研究：对PTX组、WKKL+PTX组和正常对照组芯片中每张芯片上44171条基因的比值Ratio,根据前面所设条件进行分析,得出PTX组vs正常对照组与PTX组vs WKKL+PTX组的交集存在差异表达基因共47条。其中,PTX组表达水平上调(Ratio值大于2)的有39条,表达水平下调(Ratio值小于0.5)的有8条。①参与线粒体能量代谢的基因有Atp4a、Pla2glb、Pikfyve、Lpin2、Pld4、Ppm1h、Acot10等。其中PTX组基因Atp4a表达下调,WKKL+PTX组基因Atp4a表达上调。PTX组基因Pla2glb表达上调,而WKKL+PTX组基因Pla2glb表达下调。②参与炎症反应、体液免疫反应、细胞粘附、防御应答等与免疫功能相关的基因如S100a9、Igh-6、Ghrl等。PTX组S100a9、Igh-6呈上调趋势,Ghrl表达下调,WKKL+PTX组S100a9、Igh-6呈下调趋势,Ghrl表达上调。③两组的差异表达基因涉及到多个信号转导通路,包括VEGF signaling pathway(血管内皮细胞因子信号通路)、Fc epsilon RI signaling pathway (Fc epsilon RI信号通路)、MAPK signaling pathway(丝裂原活化蛋白激酶信号通路)、GnRH signaling pathway(促性腺激素释放激素信号通路)、Phosphatidylinositol signaling system(磷脂酰肌醇信号系统)等。
结论
1、本实验中的乳腺癌小鼠模型存在CRF,且CRF的程度随肿瘤的生长而加重。乳腺癌小鼠CRF的发生机制可能与肿瘤本身的增殖所导致的三羧酸循环、脂肪酸代谢、β-丙氨酸代谢、苹果酸-天冬氨酸穿梭、线粒体电子传递链、氨基酸代谢、葡萄糖-丙氨酸循环、糖异生等多个代谢通路紊乱有关。
2、应用PTX化疗可加重乳腺癌小鼠的CRF,且CRF的程度随化疗的时间延长而加重。PTX加重乳腺癌小鼠的CRF的发生机制可能与PTX引起小鼠丙酮酸代谢、三羧酸循环、氨基酸代谢、亚精胺和精胺代谢、脂肪酸代谢、半乳糖代谢、肌醇代谢、磷酸肌醇代谢、甘氨酸和丝氨酸代谢、同型半胱氨酸降解、甘油脂质代谢、甘油磷酸穿梭等多个代谢通路紊乱有关。
3、单独应用WKKL能显著改善荷瘤小鼠的一般情况、延长小鼠生存时间,实验过程中有缓解乳腺癌小鼠CRF的作用的趋势,但无统计学差异。WKKL对乳腺癌小鼠CRF的影响可能是通过调节脂质代谢、甘油磷酸穿梭、磷脂的合成、线粒体电子传递链等多个代谢通路有关。
4、WKKL和PTX联合应用能显著改善由肿瘤本身及肿瘤化疗所引起的CRF,且效果比单独应用WKKL好。WKKL和PTX联合应用缓解乳腺癌小鼠的CRF作用机制可能是通过调节三羧酸循环、脂肪酸代谢、丙酸代谢、丙酮酸代谢、亚精胺和精胺代谢、氨基酸代谢、葡萄糖-丙氨酸循环、糖异生、丙酮酸代谢、肌醇代谢、甘油磷酸穿梭、线粒体电子传递链、苹果酸-天冬氨酸穿梭、磷脂的合成等多个代谢通路有关。
5、WKKL能显著改善由化疗所引起的疲劳,WKKL缓解化疗所引起的疲劳的作用机制可能是通过对能量代谢、免疫功能、信号转导通路(MAPK信号传导通路、GnRH信号传导通路等)及细胞因子等方面相关的基因表达的影响有关。
Objective
To evaluate effect of WKKL alleviates CRF which caused by the tumor and the chemotherapy of paclitaxel (PTX) in breast cancer mice CRF model. Reveal the mechanism of WKKL alleviates CRF and chemotherapy related fatigue.
Methods
1. The metabolism mechanism of WKKL alleviates CRF.
Before the experiment, all female BALB/c mice were trained in the swimming tank (25℃±1℃) for2days (5min/day). Then, exclude the mice which the swimming time is too long or too short and too excited or too quiet. Randomly selected10mice as normal controls (NS, po,20ml/kg, qd) mice, and the rest mice were prepared for breast cancer cell inoculation.4TI breast cancer cells were inoculated in subcutaneous of the fourth pair breast fat pad region of mouse right side of the abdominal wall to prepare the mouse model of CRF. Each mouse was injected0.1ml4T1breast cancer cells (1×106cells/ml). When the tumor grows to50mm3and100mm3(about day7after inoculation), the mice were divided in to four groups (10mice/group) according to the tumor volume. The four groups are tumor control (NS, po,20ml/kg, qd) group, PTX (PTX, ip,10mg/kg, q2d) group, WKKL (WKKL, po,1.5g/kg, qd) group and WKKL+PTX (WKKL, po,1.5g/kg qd; PTX, ip,10mg/kg, q2d) group. Then the following aspects of the experiment were carried out on the5groups of mice (NC group, TC group, PTX group, WKKL group and WKKL+ PTX):
(1) The effect of WKKL on the general situations of the tumor-bearing mice:The Weight, activity, mental state, fur appearance changes, and diet of mice in each group were observed during the experiment. The general situations of the mice was evaluated for each week.
(2) The effect of WKKL on the behavior of the tumor-bearing mice:All mice were conducted open field test at the12th day after administration. The total movement distance, the distance of peripheral lattice, the distance of central lattice, frequency of grooming, climbing frequency and stool amount of mice in the experimental tank were observed.30min after the last administration, observe the immobility time of all mice by the TS-200tail suspension test instrument.
(3) The effect of WKKL on the survival time of tumor-bearing mice:Observe the survival time of mice of the behavior experiment without any interventions. When all mice were died, calculate the mean survival time, median survival time and the life extension rate of mice in each group.
(4) The effect of WKKL on the anti-fatigue effects of the tumor-bearing mice: Take another of the5groups of mice. Then observe the exhaustive swimming time of all mice in the weight-loaded swimming test. A tin wire (7%of body weight) was loaded on the tail root of each mouse. The mice were tested three times (0d,10d and20d after administration).
(5) The effect of WKKL on the tumor growth inhibition of the tumor-bearing mice:Take another of the5groups of mice. Two-dimensional measurements were taken with calipers every three days and calculate the tumor volume. After the last administration, all mice were sacrificed, collected the blood and separate the plasma, stored at-80℃for metabolomics research. Then peel the tumor tissue and weighted. And the tumor inhibitory rate was calculated.
(6) The effect of WKKL on the organs index of the tumor-bearing mice: weighted the spleen, thymus and adrenal gland take from the previous experiment, and calculate the organs index.
(7) The effect of WKKL on the plasma metabolomics of CRF mice model: Metabolomic finger printing was acquired by1H-NMR. Multivariate data statistic (PCA,PLS-DA,OPLS-DA) was used to pattern recognition. Then we got the5groups of mice plasma metabolic profiles. Then we compare the data group between group. Then chosen the VIP and normalized integral value filter out the differences metabolites (potential molecular markers) in plasma between groups. The endogenous metabolites were assayed by HMBD data base and other document. Then, analyze the changed metabolic pathways. At last, reveal the mechanism of CRF and how WKKL alleviates the CRF.
2. The gene mechanism of WKKL alleviates chemotherapy related fatigue:
Before the experiment,25female BALB/c mice were trained in the swimming tank (25℃±1℃) for2days (5min/day). Then, exclude the mice which the swimming time is too long or too short and too excited or too quiet. The mice were divided in to three groups as normal controls (NS, po,20ml/kg, qd) group, PTX (PTX, ip,10mg/kg, q2d) group, and WKKL+PTX (WKKL, po,1.5g/kg qd; PTX, ip,10mg/kg, q2d) group. Then the following aspects of the experiment were carried out on the3groups of mice:
(1) The effect of WKKL on the general situation of the chemotherapy-bearing mice:The Weight, activity, mental state, diet, and fur appearance changes of mice in each group were observed during the experiment. The general situation of the mice was evaluated for each week.
(2) The effect of WKKL on the anti-fatigue effects of the chemotherapy-bearing mice:Observe the exhaustive swimming time of all mice in the weight-loaded swimming test. A tin wire (7%of body weight) was loaded on the tail root of each mouse. The mice were tested three times (0d,7d and14d after administration).
(3) The effect of WKKL on the skeletal muscle genomics of chemotherapy related fatigue mice model:Gene chip technology was acquired to obtain the original genetic information in the skeletal muscles of mice in the experiments, Affmetrix GCOSvl.4(gene chip operating software versionl.4) was used to of obtain the fluorescence signal intensity and ratio calculation and statistical analysis to get single analysis results of each chip to chip quality control process. Compare the difference of gene expression between the three groups. Analyze the related pathways, reveal the correlation of fatigue mechanism of chemotherapy, and the mechanism of WKKL alleviate chemotherapy related fatigue.
Results
1. The metabolism mechanism of WKKL alleviates CRF.
(1) The effect of WKKL on the general situation of the tumor-bearing mice: Before the administration, the general mental state of mice between the5groups had no significant difference. Mice in each group were lively. All mice were sensitive to food and escape quickly. The situation of mice in the NC group keeps the same during the experiment. Compared with NC group mice, the other4groups mice's hair were gradually sparse, and they gradually inactive, move slowly and eating less. At the end of the experiment, the general situation of mice in this4groups from the poor to relatively good order were TC group, PTX group, WKKL+PTX group and WKKL group. Before administration the body weight of mice in each group had no significant difference (F=0.100,P=0.982). However, after1week administration, body weight in NC group were significantly increased compared to the previous (P<0.01), and significantly heavier than PTX(P=0.000). After2weeks administration, body weight in NC group were significantly increased compared to the beginning (P<0.01), but there had no significant increase compared with intervention1st week (P>0.05). The body weight of mice in NC group were significantly heavier than other4groups (P<0.01). The body weight of mice in TC group, WKKL group and WKKL+PTX group had no significant changes to the beginning (P>0.05), but significant changes to the1st week (P<0.05). The body weight of mice in PTX group had significant changes to the beginning and1st week (P<0.01). Compared with TC group mice, the body weight of mice in PTX group had significantly changes (P<0.01). Compared with PTX group mice, the body weight of mice in WKKL group and WKKL+PTX group had significant changes (P<0.01). After3weeks administration, the body weight of mice in each group maintain at the level of the2week.
(2) The effect of WKKL on the behavior of the tumor-bearing mice:(a) open-field experimental results:The farthest total movement distances were the mice in NC group, and PTX group were the least (PTC=0.014、PPTX=0.003). Compared with mice in NC group, the total movement distance of mice in TC group and PTX group were significantly differences (PTC=0.000)、PPTX=0.000). The farthest distance of peripheral lattice was mice in NC group, and TC group (P=0.006) were the least. Compared with mice in NC group, the distance of peripheral lattice of TC group, PTX group and WKKL group of mice were significantly reduced, and had no significant difference in WKKL+PTX group(P>0.05). Compared with mice in TC group, the stool amount in NC group, PTX group and WKKL+PTX group were significantly decreased (P<0.05～0.01). Compared with mice in PTX group, the stool amount in NC group, TC group, WKKL Group and WKKL+PTX group were significantly increased (P<0.05～0.01). The frequencies of climbing of mice in NC group were the most, and least in WKKL group. Compared with mice in NC group, the frequencies of climbing of mice in WKKL group and WKKL+PTX group were significantly decreased (P<0.05～0.01). The mice in PTX group and WKKL+PTX group had higher frequencies of grooming than other groups, the mice in TC group had the least. Compared with mice in NC group, the frequency of grooming of mice in TC group and WKKL group were significantly decreased (P<0.05～0.01). Compared with mice in TC group, the frequency of grooming of mice in NC group, PTX group and WKKL+PTX group were significantly increased (P<0.01). Compared with mice in PTX group, the frequency of grooming of mice in TC group and WKKL group were significantly decreased (P<0.05～0.01).(b) Tail suspension test results: The immobility time of mice in TC group were longer than NC group, but there was no significant difference between the two groups (P=0.308). Compared with mice in TC group, the immobility time of mice in WKKL group (P=0.004) and WKKL+PTX group (P=0.001) were significantly shorter
(3) The effect of WKKL on the survival time of tumor-bearing mice:Compared with mice in TC group, the median survival time and the average survival time in other3groups were significantly prolonged (P<0.05-0.01). Compared with mice in PTX group, the median survival time and the average survival time in WKKL+PTX group were significantly prolonged (P=0.039), and significantly decreased in TC group. Compared with mice in TC group, the survival time of WKKL+PTX group, WKKL group and PTX group were prolonged21.61%,13.07%and8.54%, respectively.
(4) The effect of WKKL on the anti-fatigue effects of the tumor-bearing mice:(a) Swimming time between the groups at same time points:There were no significant differences between the5groups (P=0.992) at the beginning. At10th day after administration, the swimming ability of mice in NC group were significantly longer than TC group, PTX group and WKKL+PTX group (P=0.01). Compared with mice in TC group, the swimming time in PTX group, WKKL group and WKKL+PTX group had no significant differences (P=0.05). Compared with mice in PTX group, the swimming ability of mice in NC group and WKKL group were significantly prolonged (P=0.05～0.01). At20th day after administration, the swimming ability of mice in NC group were significantly longer than other4groups (P=0.000) Compared with mice in TC group, the swimming time of mice in PTX group, WKKL group and WKKL+PTX group had no significant differences (P>0.05). Compared with mice in PTX group, the swimming ability of mice in WKKL+PTX group and WKKL group were significantly prolonged (P<0.01).(b) Swimming time at different time points of each group mice:With the intervention time increased, the swimming ability of mice in NC group showed a gradual increase trend, and a gradual decrease trend in TC group, PTX group and WKKL+PTX group. Compared with the mice in NC group at0day, the swimming ability was significantly increased at10th day and20lh day (P=0.05～0.01). Compared with the mice in TC group at0day, the swimming ability had no significantly changes at10th day (P>0.05), but was significantly decreased at20th day (P<0.01). Compared with the mice in PTX group at0day, the swimming ability was significantly decreased at10th day and20th day (P<0.01). Compared with the mice in WKKL group at0day, the swimming ability had no significantly changes at10th day (P>0.05), but was significantly decreased at20th day (P<0.05). Compared with the mice in WKKL+PTX group at0day, the swimming ability was significantly decreased at10th day and20th day (P=0.05～0.01).
(5) The effect of WKKL on the tumor growth inhibition of the tumor-bearing mice:Compared with mice in TC group, the tumor weight in WKKL+PTX group (P=0.007) and PTX group (P=0.037) were significantly lighter. Compared with mice in TC group, the tumor volume in WKKL+PTX group were significantly smaller (P<0.05). The tumor inhibition rates of WKKL+PTX group, WKKL group and PTX group were22.24%,15.36%and16.89%, respectively.
(6) The effect of WKKL on the organs index of the tumor-bearing mice:(a) Spleen index:Compared with mice in NC group, the spleen index in tumor bearing mice (TC group, PTX group, WKKL group and WKKL+PTX group) were significantly increased (P<0.01). Compared with mice in TC group, the spleen index in PTX group, WKKL group and WKKL+PTX group have no difference (P>0.05). Compared with mice in PTX group, the spleen index in WKKL group and WKKL+PTX group have no difference (P>0.05).(b) Thymus index:The thymus index of mice in NC group was higher than other groups, and the least was in PTX group. Compared with mice in NC group, the thymus index of mice in tumor bearing mice (TC group, PTX group, WKKL group and WKKL+PTX group) were significantly decreased (P<0.01). Compared with mice in TC group, the thymus index of mice of PTX group were significantly decreased (P=0.001). Compared with mice in PTX group, the thymus index of mice in WKKL group (P=0.010) and WKKL+PTX group (P=0.006) were significantly increased,(c) Adrenal index: Compared with mice in NC group, the adrenal index of mice in TC group and PTX group were significantly increased (P<0.01). Compared with mice in TC group, the adrenal index of mice in WKKL+PTX group were significantly decreased (P=0.037). Compared with mice in PTX group, the adrenal index of mice in WKKL group and WKKL+PTX group had no difference (P>0.05).
(7) The effect of WKKL on the plasma metabolomics of CRF mice model:(a) The5groups1H-NMR-based plasma analysis of CRF mice:The samples of5groups were divided by the PLS-DA model, and calculated2principal components,(b) Compare between NC group and TC group:The PCA and PLS-DA model can divided the samples of NC group and TC group. And the OPLS-DA model was divided better. Then, found16significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in NC group, the levels of Lactic acid, glycerol phosphate, spermine, leucine, alanine, glucose-6-phosphoric acid,3-hydroxy butyric acid were increased in TC group, and the levels of Choline, inositol phosphate, coenzyme A, octanoyl glycine,3-hydroxy isovaleric acid, acid, methyl malonic acid, aspartic acid were decreased in TC group,(c) Compare between TC group and PTX group:The PCA and PLS-DA model were divided the samples of TC group and PTX group. And the OPLS-DA model was divided better. Then, found13significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the levels of citrulline were increased in PTX group, and the levels of Choline, inositol phosphate, citric acid, glycerol phosphate, linoleic acid, glutamine, coenzyme A, aspartic acid, serine, cysteine, erythrose were decreased in PTX group,(d) Compare between TC group and WKKL group:The PCA and PLS-DA model can divided the samples of TC group and WKKL group. And the OPLS-DA model was divided better. Then, found1significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the level of glycerol3-phosphate was increased in WKKL group,(e) Compare between TC group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of TC group and WKKL+PTX group. And the OPLS-DA model was divided better. Then, found17significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the levels of Propionyl glycine, citrulline, isoleucine, octanoyl glycine,2-hydroxy butyric acid were increased in WKKL+PTX group, and the levels of Inositol, dihydroxy acetone, phosphoric acid, choline, spermine,3-hydroxy butyric acid and malonic acid, aspartic acid, serine, L-valine, pyruvic acid, succinic acid and aldehyde, amino caproic acid, beta alanine were decreased in WKKL+PTX group.(f) Compare between PTX group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of PTX group and WKKL+PTX group. And the OPLS-DA model was divided better. Then, found5significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in PTX group, the levels of Nervonic acid, coenzyme A, phosphoenolpyruvate, inositol and erythrose were increased in WKKL+PTX group,(g) Compare between WKKL group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of PTX group and WKKL+PTX group, but here are still some overlap between the two groups. And the OPLS-DA model was divided better. Then, found12significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in WKKL group, the levels of Propionyl glycine, citrulline, glycerophosphate,2-hydroxy butyric acid, octanoyl glycine, isoleucine, corticosterone, folic acid, ferulic acid, hydrogen, phenylalanine, alanine were increased the levels of Choline phosphatein was decreased in WKKL+PTX group.
2. The gene mechanism of WKKL alleviates chemotherapy related fatigue:
(1) The effect of WKKL on the general situation of the chemotherapy-bearing mice:Before the administration, the general mental state, activity, diet, and fur appearance of mice between the3groups had no significant difference. Mice in each group were lively. Their fur was bright, clean and tidy. Their eyes were bright. All mice were sensitive to food and escape quickly. The situation of mice in the NC group keeps the same during the experiment. Compared with NC group mice, the other2groups mice's hair were gradually sparse, and they gradually inactive, move slowly and eating less. At the end of the experiment, the general situation of mice in this2groups from the poor to relatively good order were PTX group, WKKL+PTX group. Before administration the body weight of mice in each group had no significant difference (F=0.066, P=0.936). However, after1week administration, body weight in NC group were significantly increased compared to the previous (P=0.002) After2weeks administration, body weight in NC (P=0.001) and WKKL+PTX group (P=0.002) were significantly increased compared to the beginning, but there had no significant increase compared with intervention1st week (P>0.05). Compared with PTX group mice, the body weight of mice in NC group (P=0.000) and WKKL+PTX group (P=0.000) had significant changes.
(2) The effect of WKKL on the anti-fatigue effects of the chemotherapy-bearing mice:(a) Swimming time between the groups at same time points:There were no significant differences between the3groups (P=0.992) at the beginning. At7th day after administration, the swimming ability of mice in NC group were significantly longer than PTX group(P=0.000) and WKKL+PTX group(P=0.001). Compared with mice in PTX group, the swimming ability of mice in NC group (P=0.000) and WKKL+PTX group (P=0.007) were significantly prolonged. At14th day after administration, the swimming ability of mice in NC group were significantly longer than other2groups (P=0.01). Compared with mice in PTX group, the swimming ability of mice in WKKL+PTX group and NC group were significantly prolonged (P <0.01).(b) Swimming time at different time points of each group mice:With the intervention time increased, the swimming ability of mice in NC group showed a gradual increase trend, and a gradual decrease trend in PTX group and a volatility trend in WKKL+PTX group. Compared with the mice in NC group at0day, the swimming ability was significantly increased at14th day (P=0.026). Compared with the mice in PTX group at0day, the swimming ability was significantly decreased at14th day (P=0.002)
(3)The effect of WKKL on gene mechanism:There were47different genes between PTX and WKKL+PTX group.39were up-regulated, and8were down-regulated. Atp4a, Pla2g1b, Pikfyve, Lpin2, Pld4, Ppm1h, Acot10, etc. participated in the mitochondrial energy metabolism. S100a9, Igh-6, Ghrl, etc. participated in in the inflammatory response, humoral immune response, cell adhesion, defense response. There were involved in multiple signal transduction pathways, including VEGF signaling pathway, Fc epsilon RI signaling pathwaysignaling pathway and MAPK signaling pathway GnRH signaling pathway and Phosphatidylinositol signaling system, etc.
Conclusion
(1) The mouse inoculation with4TI breast cancer cells at the breast fat pad region of the right side of the abdominal wall subcutaneous exist CRF. The degrees of CRF were related to the tumor growth. The occurrence of CRF might related to the disturbance of the metabolic pathways of tricarboxylic acid cycle, fatty acid metabolism, β-alanyl acid metabolism, amino acid metabolism, gluconeogenesis, pyruvate metabolism,glucose alanine cycle, mitochondrial electron transport chain and malate-aspartate shuttle.
(2) PTX can aggravated CRF, and the degrees of CRF were aggravated according to the chemotherapy period prolonged. The mechanism of PTX aggravated CRF might related to the disturbance of the metabolic pathways of Pyruvate metabolism, tricarboxylic acid cycle, metabolism of amino acid metabolism, and spermine and spermine, metabolism of fatty acid metabolism, Lipid metabolism, glycerol phosphate shuttle, synthesis of phospholipids, mitochondrial electron transport chain.
(3) WKKL can improve the general situation of the tumor-bearing mice, prolong the survival time. WKKL can't alleviate CRF. Thoes effect of WKKL might related to the function of regulated the metabolic pathways of inositol metabolism, glycerol lipid metabolism, glycerol phosphate shuttle and mitochondrial electron transport chain.
(4) The combination therapy of WKKL and PTX can significantly alleviate CRF which caused by the breast cancer and the chemotherapy of PTX. The effect of alleviate CRF of the combination therapy of WKKL and PTX was better than only use WKKL. The mechanism of combination therapy of WKKL and PTX alleviate CRF might related to the function of regulated the metabolic pathways of The Krebs cycle, metabolism of fatty acid metabolism, propionic acid, pyruvate metabolism, spermine and spermine metabolism, amino acid metabolism, sugar, glucose and alanine cycle dysplasia, pyruvate metabolism, muscle metabolism of alcohol, glycerol phosphate shuttle, mitochondrial electron transport chain, malic acid-aspartate shuttle, the synthesis of phospholipids.
(5) WKKL can improve the fatigue of the chemotherapy-bearing mice, The mechanism of WKKL alleviate chemotherapy related fatigue might related to the genes expression of energy metabolism, immune function, signal transduction pathways.
近年来,随着乳腺癌的诊疗技术的进步以及人们自我保健意识的加强,乳腺癌患者的死亡率正逐渐下降,生活中经历过或正在经历乳腺癌的人们越来越多。肿瘤相关性疲劳(cancer related fatigue, CRF)的产生与癌症或癌症的治疗相关,并且干扰患者正常生活,极大地影响了癌症患者的生活质量。因此,如何提高这部分日益增多的人群的生活质量就变得十分重要。
目前,CRF越来越引起国内外学者的关注,但CRF发生的病理生理学机制仍然不是很清楚。现代西医学临床上仍缺乏有效的干预手段,这为中医药干预CRF提供了展示的机遇。CRF的治疗要立足于肿瘤疾病本身,中医认为正气不足是肿瘤发生的内在根本原因,气血阴阳失衡、脏腑功能紊乱是它的病理基础,其基本病理变化除正气亏虚外,更有气滞、血瘀、痰结、湿聚、热毒等相互纠结,日久积滞而成有形之肿块。CRF是在肿瘤的基础上形成的。因此,可认为CRF是一种病因明确,治疗多变,以虚损为主,夹有实邪的病证,病理属性总属本虚标实；正气不足,毒瘀内存为其基本病机。因此中医药干预CRF在尽可能消除疲劳相关原因之外,还应强调中医的扶正与祛邪相辅相成,辨证论治,重视整体调节,立足于补养脾肾,行气祛瘀。中医理论指出：“脾为后天之本,气血生化之源,主肌肉四肢”、“肝藏血,肝主筋,为罢极之本”、“肾为先天之本,生命之根,主骨生髓,为作强之官,伎巧出焉”。故认为疲劳与中医肺、脾、肝、肾功能失调密切相关。在此理论基础上,本课题组研制出防治疲劳的中药复方——维康颗粒,从肺、脾、肝、肾着手,具有滋补肝肾、健脾润肺、益气养阴之功效,从而通过多种途径改善疲劳,提高人的生活质量。
因此,本论文从主补肝脾肾,兼以理气祛瘀着手,选择维康颗粒,用其在乳腺癌小鼠模型上进行干预CRF的相关实验研究,以期为临床运用维康颗粒干预CRF提供实验依据,并揭示其作用机制。
目的
通过建立乳腺癌小鼠CRF模型,评价自制中药复方维康颗粒对由肿瘤本身及肿瘤化疗所引起的乳腺癌小鼠CRF的作用,并明确中药复方维康颗粒缓解CRF的代谢组学机制及缓解化疗引起的相关性疲劳的基因组学机制。
方法
1.维康颗粒缓解CRF的代谢组学机制：
实验前,将所有雌性BALB/c小鼠置于游泳箱适应性游泳训练2天,5min/天,水温25℃±1℃。剔除游泳时间过长和过短的小鼠以及过于兴奋和过于安静的小鼠。然后,随机抽取10只小鼠作为正常对照(NS, po,20ml/kg, qd)组,剩余小鼠为待制备乳腺癌小鼠CRF模型组。将1×105cells/ml的4T1乳腺癌细胞接种于小鼠右侧腹壁第4对乳房脂肪垫区域的皮下,制备乳腺癌小鼠CRF模型。当瘤块长到50mm3～100mm3(接种后第7天)的时候,按瘤体积随机分为4组,每组10只,分别为肿瘤对照(NS, po,20ml/kg, qd)组、紫杉醇(PTX, ip,10mg/kg, q2d)组、维康颗粒(WKKL, po,6g/kg, qd)组、维康颗粒+紫杉醇(WKKL, po,6g/kg, qd; PTX, ip,10mg/kg, q2d)组。上述5组小鼠分别进行以下几个方面的实验：
(1)维康颗粒对荷瘤小鼠的一般情况的影响：观察实验过程中各组小鼠的体重、精神状态、活动情况、饮食以及皮毛外观变化。每周对上述小鼠的一般情况进行一次评价。
(2)维康颗粒对荷瘤小鼠的行为学影响：给药的第12天对所有小鼠进行旷场实验。主要观察小鼠在实验箱内的总运动距离、外周格运动距离、中央格运动距离、理毛次数、大便粒数、爬壁次数。于末次给药后30min,观察小鼠6min内的悬尾不动时间。
(3)维康颗粒的对荷瘤小鼠的生存时间的影响：末次药后,不再给予任何干预措施,动态观察荷瘤小鼠生存时间,并计算各组小鼠的中位生存时间、平均生存时间及生命延长率。
(4)维康颗粒对荷瘤小鼠的抗疲劳作用：于开始给药前、给药后的第10天、第20天,用7%体重的铅坠系于小鼠尾部,将小鼠放入水温25℃±1℃的游泳箱中进行力竭游泳测试,并记录力竭游泳时间。
(5)维康颗粒对荷瘤小鼠肿瘤生长的抑制作用：另取上述4组荷瘤小鼠,各组小鼠自接受治疗开始,每3天测量肿瘤体积。在末次给药1小时后,摘眼球采血,分离血浆,置-80℃保存,用于代谢组学研究。并剥离瘤块并称重,计算抑瘤率。
(6)维康颗粒对荷瘤小鼠脏器指数的影响：取前一实验中分离并称重的脾脏、胸腺、肾上腺等器官,按对应的每只小鼠的体重计算脏器指数。
(7)维康颗粒对乳腺癌小鼠CRF血浆代谢组学的研究：应用核磁共振氢谱(1H-NMR)技术获取前面实验中小鼠血浆中的原始代谢指纹图谱信息,通过数据采集处理获得各样本数据信息,采用多元数据统计方法(PCA、PLS-DA、 OPLS-DA)模式识别等方法进行数据处理,得到5组小鼠血浆代谢概况,并进行组间两两比较。最后结合模型VIP值、归一化积分值筛选出两两组间小鼠血浆有差异的代谢物(潜在分子标志物),结合数据库和文献进行代谢物鉴定,并分析相关代谢途径变化,揭示乳腺癌小鼠CRF的发生机制、WKKL缓解乳腺癌小鼠CRF的作用机制。
2.维康颗粒缓解化疗引起的相关性疲劳的基因组学机制：
另取25只雌性BALB/c小鼠置于小鼠于游泳箱(高30cm,直径25cm)适应性游泳训练2天,5min/天,水温25℃±1℃。剔除游泳时间过长和过短的小鼠以及过于兴奋和过于安静的小鼠。按游泳时间随机分为3组,每组6只,共18只。分别为正常对照(NS, po,20ml/kg, qd)组、紫杉醇(PTX, ip,10mg/kg, q2d)组、维康颗粒+紫杉醇(WKKL, po,6g/kg, qd; PTX, ip,10mg/kg, q2d)组。上述3组小鼠分别进行以下几个方面的实验：
(1)维康颗粒对化疗小鼠的一般情况的影响：观察实验过程中各组小鼠的体重、精神状态、活动情况、饮食以及皮毛外观变化。每周对上述小鼠的一般情况进行一次评价。
(2)维康颗粒对化疗小鼠的抗疲劳作用：于开始给药前、给药后的第7天、第14天,用7%体重的铅坠系于小鼠尾部,将小鼠放入水温25℃±1℃的游泳箱中进行力竭游泳测试,并记录力竭游泳时间。
(3)维康颗粒对化疗小鼠骨骼肌基因组学的研究：应用基因芯片技术获取前面实验中小鼠骨骼肌中的原始基因信息,应用Affmetrix的GCOSvl.4(genechip operating software versionl.4)对荧光信号的强度和比值进行计算和统计分析,得到各芯片的单张分析结果,对芯片进行质量控制处理。用SAM (significant analysis of microarray)分析法,总体比较PTX组与正常对照组,PTX组与WKKL+PTX组间的基因表达有无显著性差异。结合数据库和文献进行基因成分、功能、进程鉴定,并分析相关通路变化,揭示化疗相关性疲劳的发生机制、WKKL缓解化疗相关性疲劳的作用机制。
结果
1.维康颗粒缓解CRF的代谢组学机制：
(1)维康颗粒对荷瘤小鼠的一般情况的影响：给药干预前,5组小鼠之间的精神状态、活动情况、饮食以及皮毛外观等一般情况未见有显著差别。各组小鼠均活泼好动,皮毛光洁整齐,眼睛有神,对食物敏感,逃避反应快。整个实验期间,NC组小鼠均保持活泼好动、皮毛光洁整齐、对食物敏感、逃避快。与NC组的小鼠比较,其余4组荷瘤小鼠均逐渐出现毛发稀疏、不活泼、行动迟缓和进食减少等现象。实验结束时,4组荷瘤小鼠的一般情况从差到相对较好依次为PTX组、TC组、WKKL+PTX组、WKKL组。干预前,各组小鼠的体重无显著差别(F=0.100,P=0.982)。NC组小鼠的体重逐渐增加。而随着肿瘤的生长,在干预2周和第3周时,TC组小鼠的体重均较NC组显著减轻(P=0.000)、PTX组小鼠的体重均较TC组显著减轻(P<0.05～0.01)、WKKL+PTX组小鼠的体重较PTX组显著增加(P<0.05～0.01)。
(2)维康颗粒对荷瘤小鼠的行为学影响：①旷场实验结果：TC组、PTX组小鼠的5min内的总运动距离(PTC组=0.014、PPTX组=0.003)、外周格运动距离(PTC组=0.000、PPTX组=0.000)较NC组小鼠均显著减少。而WKKL+PTX组小鼠的外周格运动距离较TC组(P=0.006)、PTX组(P=0.044)小鼠显著增加。TC组小鼠较NC组小鼠的爬壁次数显著减少(P=0.039)、理毛次数显著减少(P=0.006)。PTX组小鼠较TC组小鼠的大便粒数显著减少(P=0.000)、爬壁次数显著减少(P=0.003)。WKKL+PTX组小鼠较TC组小鼠的大便粒数显著减少(P=0.000); WKKL+PTX组小鼠的大便粒数较PTX组增加,但是无显著意义。②悬尾实验结果：与NC组小鼠相比,TC组小鼠的不动时间有所延长,但无显著差异(P=0.308)。与TC组小鼠比较,WKKL组(P=0.004)、WKKL+PTX组(P=0.001)小鼠的不动时间显著缩短。
(3)维康颗粒的对荷瘤小鼠的生存时间的影响：与TC组比较,PTX组(P=0.043)、WKKL组(P=0.007)、WKKL+PTX组(P=0.000)小鼠的中位生存时间及平均生存时间均显著延长。与PTX组比较,WKKL+PTX组小鼠的中位生存时间及平均生存时间均显著延长(P=0.039)。与TC组小鼠相比,WKKL+PTX组、WKKL组和PTX组3组小鼠的生命延长率分别为21.61%、13.07%和8.54%。
(4)维康颗粒对荷瘤小鼠的抗疲劳作用：①同一时间点各组小鼠力竭游泳时间的比较：干预前,5组小鼠的力竭游泳时间无显著差异(P=0.992)。干预第10天时,与NC组比较,荷瘤小鼠的力竭游泳时间显著减少(P<0.01)；与TC组比较,NC组小鼠的力竭游泳时间显著延长(P=0.000), PTX组小鼠的力竭游泳时间显著减少(P=0.014)、而WKKL组(P=0.381)、WKKL+PTX组(P=0.717)的游泳时间与之相比无显著差异。与PTX组比较,其余4组小鼠的力竭游泳时间显著延长(P<0.05～0.01)。干预20天时,与NC组比较,荷瘤组小鼠的力竭游泳时间显著减少(P<0.01)。与TC组比较,NC组小鼠的力竭游泳时间显著延长(P=0.000),而PTX组、WKKL组、WKKL+PTX组的游泳时间无显著差异(P>0.05)。与PTX组比较,NC组、WKKL组、WKKL+PTX组小鼠的力竭游泳时间显著延长(P<0.05～0.01)。②不同时间点的同组小鼠力竭游泳时间比较：NC组小鼠与干预前比较,干预第10天(P=0.000)、干预第20天(P=0.000)的游泳时间显著延长：与干预第10天比较,干预第20天时小鼠的游泳时间显著延长(P=0.004)。TC组小鼠与干预前比较,干预第10天(P=0.015),干预第20天的游泳时间均显著下降(P=0.000)；与干预第10天比较,干预第20天时小鼠的游泳能力显著下降(P=0.000)。PTX组小鼠与干预前比较,干预第10天(P=0.000)、干预第20天(P=0.000)的游泳时间均显著下降；与干预第10天比较,干预第20天时小鼠的游泳能力显著下降(P=0.000)。WKKL组小鼠与干预前比较,干预第10天小鼠的游泳能力均呈现下降的趋势(P=0.241),但无显著变化,干预第20天时小鼠的游泳能力显著下降(P=0.007)；与干预第10天比较,干预第20天时小鼠的游泳能力无显著变化(P=0.638)。WKKL+PTX组小鼠与干预前比较,干预第10天(P=0.051)时小鼠的游泳能力无显著下降,干预第20天时小鼠的游泳能力显著下降(P=0.039)；与干预第10天比较,干预第20天时小鼠的游泳能力无显著变化(P=0.464)。
(5)维康颗粒对荷瘤小鼠肿瘤生长的抑制作用：实验结束时,与TC组比较,WKKL+PTX组(P=0.007)\PTX组(P=0.037)的瘤重均显著减轻。与TC组比较,WKKL+PTX组、WKKL组、PTX组的肿瘤体积显著变小(P<0.05～0.01)。WKKL+PTX组、WKKL组和PTX组3组小鼠的抑瘤率分别为22.24%、15.36%和16.89%。
(6)维康颗粒对荷瘤小鼠脏器指数的影响：①脾指数：与NC组比较,其余4组的脾指数均显著升高(P=0.01)。与TC组比较,PTX组、WKKL组、WKKL+PTX组的脾指数无显著差异(P>0.05)。与PTX组比较,WKKL组、WKKL+PTX组的脾指数无显著差异(P>0.05)。②胸腺指数：与NC组比较,其余4组的胸腺指数显著下降(P<0.01)。与TC组比较,PTX组的胸腺指数显著下降(P=0.001),WKKL组(P=0.460)、WKKL+PTX组(P=0.588)小鼠的胸腺指数无显著差异。与PTX组比较,WKKL组(P=0.010)、WKKL+PTX组(P=0.006)小鼠的胸腺指数显著升高。③肾上腺指数：与NC组比较,TC组、PTX组的肾上腺指数显著升高(P<0.01)。与TC组比较,WKKL+PTX组小鼠的肾上腺指数显著降低(P=0.037)。与PTX组比较,WKKL组、WKKL+PTX组小鼠的肾上腺指数无显著差异(P>0.05)。
(7)维康颗粒对乳腺癌小鼠CRF血浆代谢组学的研究：①五组小鼠之间血浆NMR的分析：用PLS-DA模型,最终计算出5组小鼠血浆样本共有2个主成分,5组之间基本分离,且各组相关集中,组间可基本区分。②NC组与TC组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和NC组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到16个有统计学意义的变量(P<0.05～0.01)。与NC组小鼠比较,TC组小鼠血浆中的乳酸、甘油磷酸酯、精胺、亮氨酸、丙氨酸、葡萄糖-6-磷酸、3-羟基丁酸水平升高,肌醇、磷酸胆碱、辅酶A、辛酰甘氨酸、3-羟基异戊酸、神经酸、甲基丙二酸、天冬氨酸水平下降。③TC组与PTX组小鼠组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和PTX组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,两组小鼠两组样本之间显著区分,但PTX组样本之间较TC组分散。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到13个有统计学意义的变量(P<0.05～0.01)。与TC组小鼠比较,PTX组小鼠血浆中的瓜氨酸水平升高,肌醇、磷酸胆碱、异柠檬酸、甘油磷酸酯、亚油酸、谷氨酰胺、辅酶A、天冬氨酸、丝氨酸、高半胱氨酸、赤藓糖水平下降。④TC组与WKKL组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和WKKL组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到1个有统计学意义的变量(P<0.05～0.01)。与TC组小鼠比较,WKKL组小鼠血浆中的甘油磷酸酯水平升高。⑤TC组与WKKL+PTX组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,TC组和WKKL+PTX组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到17个有统计学意义的变量(P<0.05～0.01)。与TC组小鼠比较,WKKL+PTX组小鼠血浆中的的肌醇、二羟基丙酮、磷酸胆碱、亚精胺、3-羟基丁酸、丙二酸、天冬氨酸、丝氨酸、L-缬氨酸、丙酮酸、琥珀酸半醛、氨基己酸、β-丙氨酸水平下降,丙酰甘氨酸、瓜氨酸、异亮氨酸、辛酰甘氨酸、2-羟基丁酸水平升高。⑥PTX组与WKKL+PTX组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,PTX组和WKKL+PTX组各自集中,两组可完全区分。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到5个有统计学意义的变量(P<0.05-0.01)。与PTX组小鼠比较,WKKL+PTX组小鼠血浆中的神经酸、辅酶A、磷酸烯醇丙酮酸、肌醇、赤藓糖水平升高。⑦WKKL组与WKKL+PTX组组间两两比较：采用PCA、PLS-DA方法进行数据处理和模式识别后,WKKL组和WKKL+PTX组两组分离趋势增强,但仍存在一定的重叠,两组尚未能完全区分开来。进一步用OPLS-DA模型处理数据后,两组样本集中趋势更显著,分类效果更好。综合载荷矩阵图、S图、VIP值、归一化积分值共筛选得到12个有统计学意义的变量(P<0.05～0.01)。与WKKL组小鼠比较,WKKL+PTX组小鼠血浆中的丙酰甘氨酸、瓜氨酸、甘油磷酸酯、2-羟基丁酸、辛酰甘氨酸、异亮氨酸、皮质酮、异阿魏酸、四氢叶酸、苯丙氨酸、丙氨酸水平升高,磷酸胆碱水平下降。
2.维康颗粒缓解化疗引起的相关性疲劳的基因组学机制：
(1)维康颗粒对化疗小鼠的一般情况的影响：给药干预前,3组小鼠之间的精神状态、活动情况、饮食以及皮毛外观等一般情况未见有显著差别。各组小鼠均活泼好动,皮毛光洁整齐,眼睛有神,对食物敏感,逃避反应快。整个实验过程中,NC组小鼠的上述情况未有显著变化。与NC组的小鼠比较,其余2组化疗小鼠均逐渐出现毛发稀疏、聚集、不活泼、行动迟缓和进食减少等现象,但WKKL+PTX组小鼠的一般情况较PTX组要好。干预前,各组小鼠的体重无显著差别(F=0.066,P=0.936)。NC组小鼠的体重逐渐增加,干预7天时,NC组小鼠的体重较干预前显著增加(P=0.002), PTX组小鼠体重较干预前减少、WKKL+PTX组小鼠体重较干预前增加,但是无显著差异。与PTX组小鼠体重相较,NC组(P=0.001), WKKL+PTX组(P=0.002)均有显著性差异。干预14天时,NC组及WKKL+PTX组小鼠的体重较干预前显著增加(P=0.05～0.01),但较干预1周时无显著增加。PTX组小鼠的体重较干预前(P=0.009)、干预7天(P=0.008)时均显著减轻。与PTX组小鼠体重比较,NC组(P=0.000)、WKKL+PTX组(P=0.000)的小鼠体重均分别有显著差异。
(2)维康颗粒对化疗小鼠的抗疲劳作用：①同一时间点各组小鼠力竭游泳时间的比较：干预前,3组小鼠的力竭游泳时间无显著差异(P=0.992)。干预第7天时,与NC组比较,PTX组(P=0.000)、WKKL+PTX组(P=0.001)小鼠的力竭游泳时间显著减少；但PTX组与WKKL+PTX组小鼠的力竭游泳时间无显著差异。干预14天时,与NC组比较,PTX组(P=0.000)、WKKL+PTX组(P=0.002)小鼠的力竭游泳时间显著减少。与PTX组比较,NC组(P=0.000)、WKKL+PTX组(P=0.007)组小鼠的力竭游泳时间显著延长(P=0.01)。②不同时间点的同组小鼠力竭游泳时间结果：随着干预时间的增加,NC组小鼠的游泳能力呈现逐步增加的趋势,PTX组小鼠的游泳能力呈现逐渐下降的趋势,WKKL+PTX组小鼠的游泳能力呈现波动性的增加趋势。对于NC组小鼠而言,与干预前比较,干预第7天(P=0.026)、干预第14天(P=0.011)的游泳时间均显著延长；与干预第10天比较,干预第20天时小鼠的游泳时间显著延长(P=0.022)。对于PTX组小鼠而言,与干预前比较,干预第7天(P=0.029)、干预第14天(P=0.002)的游泳时间均显著下降；与干预第7天比较,干预第14天时小鼠的游泳能力无显著差异(P=0.053)。对于WKKL+PTX组小鼠而言,与干预前比较,干预第7天时(P=0.336)、干预第14天时(P=0.284)小鼠的游泳能力先下降后上升,但无显著差异；与干预第7天比较,干预第14天时小鼠的游泳能力无显著变化(P=0.134)。
(3)维康颗粒对化疗小鼠骨骼肌基因组学的研究：对PTX组、WKKL+PTX组和正常对照组芯片中每张芯片上44171条基因的比值Ratio,根据前面所设条件进行分析,得出PTX组vs正常对照组与PTX组vs WKKL+PTX组的交集存在差异表达基因共47条。其中,PTX组表达水平上调(Ratio值大于2)的有39条,表达水平下调(Ratio值小于0.5)的有8条。①参与线粒体能量代谢的基因有Atp4a、Pla2glb、Pikfyve、Lpin2、Pld4、Ppm1h、Acot10等。其中PTX组基因Atp4a表达下调,WKKL+PTX组基因Atp4a表达上调。PTX组基因Pla2glb表达上调,而WKKL+PTX组基因Pla2glb表达下调。②参与炎症反应、体液免疫反应、细胞粘附、防御应答等与免疫功能相关的基因如S100a9、Igh-6、Ghrl等。PTX组S100a9、Igh-6呈上调趋势,Ghrl表达下调,WKKL+PTX组S100a9、Igh-6呈下调趋势,Ghrl表达上调。③两组的差异表达基因涉及到多个信号转导通路,包括VEGF signaling pathway(血管内皮细胞因子信号通路)、Fc epsilon RI signaling pathway (Fc epsilon RI信号通路)、MAPK signaling pathway(丝裂原活化蛋白激酶信号通路)、GnRH signaling pathway(促性腺激素释放激素信号通路)、Phosphatidylinositol signaling system(磷脂酰肌醇信号系统)等。
结论
1、本实验中的乳腺癌小鼠模型存在CRF,且CRF的程度随肿瘤的生长而加重。乳腺癌小鼠CRF的发生机制可能与肿瘤本身的增殖所导致的三羧酸循环、脂肪酸代谢、β-丙氨酸代谢、苹果酸-天冬氨酸穿梭、线粒体电子传递链、氨基酸代谢、葡萄糖-丙氨酸循环、糖异生等多个代谢通路紊乱有关。
2、应用PTX化疗可加重乳腺癌小鼠的CRF,且CRF的程度随化疗的时间延长而加重。PTX加重乳腺癌小鼠的CRF的发生机制可能与PTX引起小鼠丙酮酸代谢、三羧酸循环、氨基酸代谢、亚精胺和精胺代谢、脂肪酸代谢、半乳糖代谢、肌醇代谢、磷酸肌醇代谢、甘氨酸和丝氨酸代谢、同型半胱氨酸降解、甘油脂质代谢、甘油磷酸穿梭等多个代谢通路紊乱有关。
3、单独应用WKKL能显著改善荷瘤小鼠的一般情况、延长小鼠生存时间,实验过程中有缓解乳腺癌小鼠CRF的作用的趋势,但无统计学差异。WKKL对乳腺癌小鼠CRF的影响可能是通过调节脂质代谢、甘油磷酸穿梭、磷脂的合成、线粒体电子传递链等多个代谢通路有关。
4、WKKL和PTX联合应用能显著改善由肿瘤本身及肿瘤化疗所引起的CRF,且效果比单独应用WKKL好。WKKL和PTX联合应用缓解乳腺癌小鼠的CRF作用机制可能是通过调节三羧酸循环、脂肪酸代谢、丙酸代谢、丙酮酸代谢、亚精胺和精胺代谢、氨基酸代谢、葡萄糖-丙氨酸循环、糖异生、丙酮酸代谢、肌醇代谢、甘油磷酸穿梭、线粒体电子传递链、苹果酸-天冬氨酸穿梭、磷脂的合成等多个代谢通路有关。
5、WKKL能显著改善由化疗所引起的疲劳,WKKL缓解化疗所引起的疲劳的作用机制可能是通过对能量代谢、免疫功能、信号转导通路(MAPK信号传导通路、GnRH信号传导通路等)及细胞因子等方面相关的基因表达的影响有关。
Objective
To evaluate effect of WKKL alleviates CRF which caused by the tumor and the chemotherapy of paclitaxel (PTX) in breast cancer mice CRF model. Reveal the mechanism of WKKL alleviates CRF and chemotherapy related fatigue.
Methods
1. The metabolism mechanism of WKKL alleviates CRF.
Before the experiment, all female BALB/c mice were trained in the swimming tank (25℃±1℃) for2days (5min/day). Then, exclude the mice which the swimming time is too long or too short and too excited or too quiet. Randomly selected10mice as normal controls (NS, po,20ml/kg, qd) mice, and the rest mice were prepared for breast cancer cell inoculation.4TI breast cancer cells were inoculated in subcutaneous of the fourth pair breast fat pad region of mouse right side of the abdominal wall to prepare the mouse model of CRF. Each mouse was injected0.1ml4T1breast cancer cells (1×106cells/ml). When the tumor grows to50mm3and100mm3(about day7after inoculation), the mice were divided in to four groups (10mice/group) according to the tumor volume. The four groups are tumor control (NS, po,20ml/kg, qd) group, PTX (PTX, ip,10mg/kg, q2d) group, WKKL (WKKL, po,1.5g/kg, qd) group and WKKL+PTX (WKKL, po,1.5g/kg qd; PTX, ip,10mg/kg, q2d) group. Then the following aspects of the experiment were carried out on the5groups of mice (NC group, TC group, PTX group, WKKL group and WKKL+ PTX):
(1) The effect of WKKL on the general situations of the tumor-bearing mice:The Weight, activity, mental state, fur appearance changes, and diet of mice in each group were observed during the experiment. The general situations of the mice was evaluated for each week.
(2) The effect of WKKL on the behavior of the tumor-bearing mice:All mice were conducted open field test at the12th day after administration. The total movement distance, the distance of peripheral lattice, the distance of central lattice, frequency of grooming, climbing frequency and stool amount of mice in the experimental tank were observed.30min after the last administration, observe the immobility time of all mice by the TS-200tail suspension test instrument.
(3) The effect of WKKL on the survival time of tumor-bearing mice:Observe the survival time of mice of the behavior experiment without any interventions. When all mice were died, calculate the mean survival time, median survival time and the life extension rate of mice in each group.
(4) The effect of WKKL on the anti-fatigue effects of the tumor-bearing mice: Take another of the5groups of mice. Then observe the exhaustive swimming time of all mice in the weight-loaded swimming test. A tin wire (7%of body weight) was loaded on the tail root of each mouse. The mice were tested three times (0d,10d and20d after administration).
(5) The effect of WKKL on the tumor growth inhibition of the tumor-bearing mice:Take another of the5groups of mice. Two-dimensional measurements were taken with calipers every three days and calculate the tumor volume. After the last administration, all mice were sacrificed, collected the blood and separate the plasma, stored at-80℃for metabolomics research. Then peel the tumor tissue and weighted. And the tumor inhibitory rate was calculated.
(6) The effect of WKKL on the organs index of the tumor-bearing mice: weighted the spleen, thymus and adrenal gland take from the previous experiment, and calculate the organs index.
(7) The effect of WKKL on the plasma metabolomics of CRF mice model: Metabolomic finger printing was acquired by1H-NMR. Multivariate data statistic (PCA,PLS-DA,OPLS-DA) was used to pattern recognition. Then we got the5groups of mice plasma metabolic profiles. Then we compare the data group between group. Then chosen the VIP and normalized integral value filter out the differences metabolites (potential molecular markers) in plasma between groups. The endogenous metabolites were assayed by HMBD data base and other document. Then, analyze the changed metabolic pathways. At last, reveal the mechanism of CRF and how WKKL alleviates the CRF.
2. The gene mechanism of WKKL alleviates chemotherapy related fatigue:
Before the experiment,25female BALB/c mice were trained in the swimming tank (25℃±1℃) for2days (5min/day). Then, exclude the mice which the swimming time is too long or too short and too excited or too quiet. The mice were divided in to three groups as normal controls (NS, po,20ml/kg, qd) group, PTX (PTX, ip,10mg/kg, q2d) group, and WKKL+PTX (WKKL, po,1.5g/kg qd; PTX, ip,10mg/kg, q2d) group. Then the following aspects of the experiment were carried out on the3groups of mice:
(1) The effect of WKKL on the general situation of the chemotherapy-bearing mice:The Weight, activity, mental state, diet, and fur appearance changes of mice in each group were observed during the experiment. The general situation of the mice was evaluated for each week.
(2) The effect of WKKL on the anti-fatigue effects of the chemotherapy-bearing mice:Observe the exhaustive swimming time of all mice in the weight-loaded swimming test. A tin wire (7%of body weight) was loaded on the tail root of each mouse. The mice were tested three times (0d,7d and14d after administration).
(3) The effect of WKKL on the skeletal muscle genomics of chemotherapy related fatigue mice model:Gene chip technology was acquired to obtain the original genetic information in the skeletal muscles of mice in the experiments, Affmetrix GCOSvl.4(gene chip operating software versionl.4) was used to of obtain the fluorescence signal intensity and ratio calculation and statistical analysis to get single analysis results of each chip to chip quality control process. Compare the difference of gene expression between the three groups. Analyze the related pathways, reveal the correlation of fatigue mechanism of chemotherapy, and the mechanism of WKKL alleviate chemotherapy related fatigue.
Results
1. The metabolism mechanism of WKKL alleviates CRF.
(1) The effect of WKKL on the general situation of the tumor-bearing mice: Before the administration, the general mental state of mice between the5groups had no significant difference. Mice in each group were lively. All mice were sensitive to food and escape quickly. The situation of mice in the NC group keeps the same during the experiment. Compared with NC group mice, the other4groups mice's hair were gradually sparse, and they gradually inactive, move slowly and eating less. At the end of the experiment, the general situation of mice in this4groups from the poor to relatively good order were TC group, PTX group, WKKL+PTX group and WKKL group. Before administration the body weight of mice in each group had no significant difference (F=0.100,P=0.982). However, after1week administration, body weight in NC group were significantly increased compared to the previous (P<0.01), and significantly heavier than PTX(P=0.000). After2weeks administration, body weight in NC group were significantly increased compared to the beginning (P<0.01), but there had no significant increase compared with intervention1st week (P>0.05). The body weight of mice in NC group were significantly heavier than other4groups (P<0.01). The body weight of mice in TC group, WKKL group and WKKL+PTX group had no significant changes to the beginning (P>0.05), but significant changes to the1st week (P<0.05). The body weight of mice in PTX group had significant changes to the beginning and1st week (P<0.01). Compared with TC group mice, the body weight of mice in PTX group had significantly changes (P<0.01). Compared with PTX group mice, the body weight of mice in WKKL group and WKKL+PTX group had significant changes (P<0.01). After3weeks administration, the body weight of mice in each group maintain at the level of the2week.
(2) The effect of WKKL on the behavior of the tumor-bearing mice:(a) open-field experimental results:The farthest total movement distances were the mice in NC group, and PTX group were the least (PTC=0.014、PPTX=0.003). Compared with mice in NC group, the total movement distance of mice in TC group and PTX group were significantly differences (PTC=0.000)、PPTX=0.000). The farthest distance of peripheral lattice was mice in NC group, and TC group (P=0.006) were the least. Compared with mice in NC group, the distance of peripheral lattice of TC group, PTX group and WKKL group of mice were significantly reduced, and had no significant difference in WKKL+PTX group(P>0.05). Compared with mice in TC group, the stool amount in NC group, PTX group and WKKL+PTX group were significantly decreased (P<0.05～0.01). Compared with mice in PTX group, the stool amount in NC group, TC group, WKKL Group and WKKL+PTX group were significantly increased (P<0.05～0.01). The frequencies of climbing of mice in NC group were the most, and least in WKKL group. Compared with mice in NC group, the frequencies of climbing of mice in WKKL group and WKKL+PTX group were significantly decreased (P<0.05～0.01). The mice in PTX group and WKKL+PTX group had higher frequencies of grooming than other groups, the mice in TC group had the least. Compared with mice in NC group, the frequency of grooming of mice in TC group and WKKL group were significantly decreased (P<0.05～0.01). Compared with mice in TC group, the frequency of grooming of mice in NC group, PTX group and WKKL+PTX group were significantly increased (P<0.01). Compared with mice in PTX group, the frequency of grooming of mice in TC group and WKKL group were significantly decreased (P<0.05～0.01).(b) Tail suspension test results: The immobility time of mice in TC group were longer than NC group, but there was no significant difference between the two groups (P=0.308). Compared with mice in TC group, the immobility time of mice in WKKL group (P=0.004) and WKKL+PTX group (P=0.001) were significantly shorter
(3) The effect of WKKL on the survival time of tumor-bearing mice:Compared with mice in TC group, the median survival time and the average survival time in other3groups were significantly prolonged (P<0.05-0.01). Compared with mice in PTX group, the median survival time and the average survival time in WKKL+PTX group were significantly prolonged (P=0.039), and significantly decreased in TC group. Compared with mice in TC group, the survival time of WKKL+PTX group, WKKL group and PTX group were prolonged21.61%,13.07%and8.54%, respectively.
(4) The effect of WKKL on the anti-fatigue effects of the tumor-bearing mice:(a) Swimming time between the groups at same time points:There were no significant differences between the5groups (P=0.992) at the beginning. At10th day after administration, the swimming ability of mice in NC group were significantly longer than TC group, PTX group and WKKL+PTX group (P=0.01). Compared with mice in TC group, the swimming time in PTX group, WKKL group and WKKL+PTX group had no significant differences (P=0.05). Compared with mice in PTX group, the swimming ability of mice in NC group and WKKL group were significantly prolonged (P=0.05～0.01). At20th day after administration, the swimming ability of mice in NC group were significantly longer than other4groups (P=0.000) Compared with mice in TC group, the swimming time of mice in PTX group, WKKL group and WKKL+PTX group had no significant differences (P>0.05). Compared with mice in PTX group, the swimming ability of mice in WKKL+PTX group and WKKL group were significantly prolonged (P<0.01).(b) Swimming time at different time points of each group mice:With the intervention time increased, the swimming ability of mice in NC group showed a gradual increase trend, and a gradual decrease trend in TC group, PTX group and WKKL+PTX group. Compared with the mice in NC group at0day, the swimming ability was significantly increased at10th day and20lh day (P=0.05～0.01). Compared with the mice in TC group at0day, the swimming ability had no significantly changes at10th day (P>0.05), but was significantly decreased at20th day (P<0.01). Compared with the mice in PTX group at0day, the swimming ability was significantly decreased at10th day and20th day (P<0.01). Compared with the mice in WKKL group at0day, the swimming ability had no significantly changes at10th day (P>0.05), but was significantly decreased at20th day (P<0.05). Compared with the mice in WKKL+PTX group at0day, the swimming ability was significantly decreased at10th day and20th day (P=0.05～0.01).
(5) The effect of WKKL on the tumor growth inhibition of the tumor-bearing mice:Compared with mice in TC group, the tumor weight in WKKL+PTX group (P=0.007) and PTX group (P=0.037) were significantly lighter. Compared with mice in TC group, the tumor volume in WKKL+PTX group were significantly smaller (P<0.05). The tumor inhibition rates of WKKL+PTX group, WKKL group and PTX group were22.24%,15.36%and16.89%, respectively.
(6) The effect of WKKL on the organs index of the tumor-bearing mice:(a) Spleen index:Compared with mice in NC group, the spleen index in tumor bearing mice (TC group, PTX group, WKKL group and WKKL+PTX group) were significantly increased (P<0.01). Compared with mice in TC group, the spleen index in PTX group, WKKL group and WKKL+PTX group have no difference (P>0.05). Compared with mice in PTX group, the spleen index in WKKL group and WKKL+PTX group have no difference (P>0.05).(b) Thymus index:The thymus index of mice in NC group was higher than other groups, and the least was in PTX group. Compared with mice in NC group, the thymus index of mice in tumor bearing mice (TC group, PTX group, WKKL group and WKKL+PTX group) were significantly decreased (P<0.01). Compared with mice in TC group, the thymus index of mice of PTX group were significantly decreased (P=0.001). Compared with mice in PTX group, the thymus index of mice in WKKL group (P=0.010) and WKKL+PTX group (P=0.006) were significantly increased,(c) Adrenal index: Compared with mice in NC group, the adrenal index of mice in TC group and PTX group were significantly increased (P<0.01). Compared with mice in TC group, the adrenal index of mice in WKKL+PTX group were significantly decreased (P=0.037). Compared with mice in PTX group, the adrenal index of mice in WKKL group and WKKL+PTX group had no difference (P>0.05).
(7) The effect of WKKL on the plasma metabolomics of CRF mice model:(a) The5groups1H-NMR-based plasma analysis of CRF mice:The samples of5groups were divided by the PLS-DA model, and calculated2principal components,(b) Compare between NC group and TC group:The PCA and PLS-DA model can divided the samples of NC group and TC group. And the OPLS-DA model was divided better. Then, found16significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in NC group, the levels of Lactic acid, glycerol phosphate, spermine, leucine, alanine, glucose-6-phosphoric acid,3-hydroxy butyric acid were increased in TC group, and the levels of Choline, inositol phosphate, coenzyme A, octanoyl glycine,3-hydroxy isovaleric acid, acid, methyl malonic acid, aspartic acid were decreased in TC group,(c) Compare between TC group and PTX group:The PCA and PLS-DA model were divided the samples of TC group and PTX group. And the OPLS-DA model was divided better. Then, found13significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the levels of citrulline were increased in PTX group, and the levels of Choline, inositol phosphate, citric acid, glycerol phosphate, linoleic acid, glutamine, coenzyme A, aspartic acid, serine, cysteine, erythrose were decreased in PTX group,(d) Compare between TC group and WKKL group:The PCA and PLS-DA model can divided the samples of TC group and WKKL group. And the OPLS-DA model was divided better. Then, found1significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the level of glycerol3-phosphate was increased in WKKL group,(e) Compare between TC group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of TC group and WKKL+PTX group. And the OPLS-DA model was divided better. Then, found17significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the levels of Propionyl glycine, citrulline, isoleucine, octanoyl glycine,2-hydroxy butyric acid were increased in WKKL+PTX group, and the levels of Inositol, dihydroxy acetone, phosphoric acid, choline, spermine,3-hydroxy butyric acid and malonic acid, aspartic acid, serine, L-valine, pyruvic acid, succinic acid and aldehyde, amino caproic acid, beta alanine were decreased in WKKL+PTX group.(f) Compare between PTX group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of PTX group and WKKL+PTX group. And the OPLS-DA model was divided better. Then, found5significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in PTX group, the levels of Nervonic acid, coenzyme A, phosphoenolpyruvate, inositol and erythrose were increased in WKKL+PTX group,(g) Compare between WKKL group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of PTX group and WKKL+PTX group, but here are still some overlap between the two groups. And the OPLS-DA model was divided better. Then, found12significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in WKKL group, the levels of Propionyl glycine, citrulline, glycerophosphate,2-hydroxy butyric acid, octanoyl glycine, isoleucine, corticosterone, folic acid, ferulic acid, hydrogen, phenylalanine, alanine were increased the levels of Choline phosphatein was decreased in WKKL+PTX group.
2. The gene mechanism of WKKL alleviates chemotherapy related fatigue:
(1) The effect of WKKL on the general situation of the chemotherapy-bearing mice:Before the administration, the general mental state, activity, diet, and fur appearance of mice between the3groups had no significant difference. Mice in each group were lively. Their fur was bright, clean and tidy. Their eyes were bright. All mice were sensitive to food and escape quickly. The situation of mice in the NC group keeps the same during the experiment. Compared with NC group mice, the other2groups mice's hair were gradually sparse, and they gradually inactive, move slowly and eating less. At the end of the experiment, the general situation of mice in this2groups from the poor to relatively good order were PTX group, WKKL+PTX group. Before administration the body weight of mice in each group had no significant difference (F=0.066, P=0.936). However, after1week administration, body weight in NC group were significantly increased compared to the previous (P=0.002) After2weeks administration, body weight in NC (P=0.001) and WKKL+PTX group (P=0.002) were significantly increased compared to the beginning, but there had no significant increase compared with intervention1st week (P>0.05). Compared with PTX group mice, the body weight of mice in NC group (P=0.000) and WKKL+PTX group (P=0.000) had significant changes.
(2) The effect of WKKL on the anti-fatigue effects of the chemotherapy-bearing mice:(a) Swimming time between the groups at same time points:There were no significant differences between the3groups (P=0.992) at the beginning. At7th day after administration, the swimming ability of mice in NC group were significantly longer than PTX group(P=0.000) and WKKL+PTX group(P=0.001). Compared with mice in PTX group, the swimming ability of mice in NC group (P=0.000) and WKKL+PTX group (P=0.007) were significantly prolonged. At14th day after administration, the swimming ability of mice in NC group were significantly longer than other2groups (P=0.01). Compared with mice in PTX group, the swimming ability of mice in WKKL+PTX group and NC group were significantly prolonged (P <0.01).(b) Swimming time at different time points of each group mice:With the intervention time increased, the swimming ability of mice in NC group showed a gradual increase trend, and a gradual decrease trend in PTX group and a volatility trend in WKKL+PTX group. Compared with the mice in NC group at0day, the swimming ability was significantly increased at14th day (P=0.026). Compared with the mice in PTX group at0day, the swimming ability was significantly decreased at14th day (P=0.002)
(3)The effect of WKKL on gene mechanism:There were47different genes between PTX and WKKL+PTX group.39were up-regulated, and8were down-regulated. Atp4a, Pla2g1b, Pikfyve, Lpin2, Pld4, Ppm1h, Acot10, etc. participated in the mitochondrial energy metabolism. S100a9, Igh-6, Ghrl, etc. participated in in the inflammatory response, humoral immune response, cell adhesion, defense response. There were involved in multiple signal transduction pathways, including VEGF signaling pathway, Fc epsilon RI signaling pathwaysignaling pathway and MAPK signaling pathway GnRH signaling pathway and Phosphatidylinositol signaling system, etc.
Conclusion
(1) The mouse inoculation with4TI breast cancer cells at the breast fat pad region of the right side of the abdominal wall subcutaneous exist CRF. The degrees of CRF were related to the tumor growth. The occurrence of CRF might related to the disturbance of the metabolic pathways of tricarboxylic acid cycle, fatty acid metabolism, β-alanyl acid metabolism, amino acid metabolism, gluconeogenesis, pyruvate metabolism,glucose alanine cycle, mitochondrial electron transport chain and malate-aspartate shuttle.
(2) PTX can aggravated CRF, and the degrees of CRF were aggravated according to the chemotherapy period prolonged. The mechanism of PTX aggravated CRF might related to the disturbance of the metabolic pathways of Pyruvate metabolism, tricarboxylic acid cycle, metabolism of amino acid metabolism, and spermine and spermine, metabolism of fatty acid metabolism, Lipid metabolism, glycerol phosphate shuttle, synthesis of phospholipids, mitochondrial electron transport chain.
(3) WKKL can improve the general situation of the tumor-bearing mice, prolong the survival time. WKKL can't alleviate CRF. Thoes effect of WKKL might related to the function of regulated the metabolic pathways of inositol metabolism, glycerol lipid metabolism, glycerol phosphate shuttle and mitochondrial electron transport chain.
(4) The combination therapy of WKKL and PTX can significantly alleviate CRF which caused by the breast cancer and the chemotherapy of PTX. The effect of alleviate CRF of the combination therapy of WKKL and PTX was better than only use WKKL. The mechanism of combination therapy of WKKL and PTX alleviate CRF might related to the function of regulated the metabolic pathways of The Krebs cycle, metabolism of fatty acid metabolism, propionic acid, pyruvate metabolism, spermine and spermine metabolism, amino acid metabolism, sugar, glucose and alanine cycle dysplasia, pyruvate metabolism, muscle metabolism of alcohol, glycerol phosphate shuttle, mitochondrial electron transport chain, malic acid-aspartate shuttle, the synthesis of phospholipids.
(5) WKKL can improve the fatigue of the chemotherapy-bearing mice, The mechanism of WKKL alleviate chemotherapy related fatigue might related to the genes expression of energy metabolism, immune function, signal transduction pathways.
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