【作者】 房良华；

【导师】 刘沈林；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的：探讨透脓散提取液抗肿瘤作用及其可能的机制。方法：应用液相色谱-质谱联用技术测定透脓散总提物成分；MTT法检测不同浓度透脓散提物对不同肿瘤细胞(人淋巴瘤Raji细胞,人肠癌LOVO细胞)的增殖抑制率；流式细胞术检测透脓散提液对Raji细胞和LOVO细胞凋亡率及细胞周期的影响；RT-PCR检测透脓散提取液对Raji细胞和LOVO细胞PI3K/AKT信号通路相关分子(PI3K,Akt,m-TOR, p70S6K1,Bad,NF-KB,caspase9,caspase3)的mRNA的表达；Westernblot法检测透脓散提取液对Raji细胞和LOVO细胞PI3K/AKT信号通路相关分子(PI3K,p-Akt,p-mTOR,p-p70S6K1, p-Bad,NF-KB,caspase9,caspase3)蛋白表达的变化；急性毒性实验检测透脓散用药的安全性；根据荷肠癌LOVO细胞裸鼠移植瘤的体积和移植瘤重量计算透脓散提取液的抑瘤率；HE染色光镜观察肿瘤组织的形态结构。免疫组织化学法检测透脓散提取液对肿瘤组织P13K/AKT信号通路相关分子蛋白的表达：ELISA检测裸鼠血清Th1/Th2型细胞因子的含量。结果：1透脓散提取液主要的化学成分为：毛蕊异黄酮葡萄糖苷,芒柄花苷洋,川芎内酯Ⅰ,毛蕊异黄酮,芒柄花黄素,黄芪甲苷,藁本内酯,且醇提液的成分明显高于水提液。2透脓散提取液可抑制Raji细胞和LOVO细胞的增殖活性并具有明显剂量和时间依赖性。对Raji细胞的72小时半数抑制率为(Half maximal inhibitory concentration,IC50)817.142μg/mL,对LOVO细胞的48小时IC50为：347.603μg/mL (258.526μg/mL to471.545μg/mL)。3透脓散提取液可诱导Raji细胞,LOVO细胞凋亡并阻滞细胞周期在G1期。125μg/mL,250μg/mL和500μg/mL透脓散提取液干预Raji细胞,48h后细胞的凋亡率分别为：12.23±1.98%(P<0.05),20.97+3.96%(P<0.01)和30.4±4.87%(P<0.01),与空白对照组6.02±1.01%相比具有显著的统计学意义。G1期的比例逐渐增高,分别为：44.80%±2.36%(P<0.05),55.48%+2.71%(P<0.01),66.76%±2.25%(P<0.01)与对照组39.66%±2.01%相比具有统计学意义。应用62.5μg/mL,125μg/mL,250μg/mL透脓散醇提液干预LOVO细胞48h后,LOVO细胞的凋亡率分别为：12.8%±1.58%(P<0.05),19.9%±2.16%,(P<0.01),29.57±2.76%(P<0.01)与对照组(7.3%±1.06%)相比具有显著的统计学意义。G1期细胞的比例逐渐增多,分别为：45.45%±2.54%(P<0.05),56.13%±2.54%(P<0.01),66.05%+2.54%(P<0.01)与对照组40.02%＋0.69%相比具有统计学意义。250μg/mL透脓散提取物干预Raji细胞48小时后明显降低Raji细胞NF-κB mRNA及蛋白的表达,增加Bad, caspase-9, caspase-3mRNA及蛋白的表达且具有明显的剂量依赖性。125μg/mL透脓散醇提液干预LOVO细胞48小时后明显降低PI3K, AKT, mTOR, p70s6kl mRNA表达,而Caspase-9,Caspase-3mRNA表达明显增加,PI3K, p-AKT, p-mTOR, p-p70s6kl蛋白表达降低,而Caspase-9, Caspase-3蛋白表达增加,同样具有明显的剂量依赖性。透脓散提取液最大给药量40000mg/kg时对裸鼠未见明显毒性。高、中、低剂量组的透脓散提取液抑瘤率分别为：67.11%、48.68%、36.84%。免疫组化显示透脓散组P13K,p-Akt, p-mTOR, p-P70S6K1, VEGF, CD31蛋白表达降低,caspase-9, caspase-3蛋白表达增加,且随着剂量的增高,作用逐渐增强。此外,透脓散能调节移植瘤裸鼠的免疫功能,上调Thl型细胞因子(IL-2,IL-12),下调Th2型细胞因子(IL-6,IL-10),从而纠正移植瘤裸鼠机体Thl/Th2漂移,且此效应具有明显的剂量依赖性。结论：1透脓散提取液可从基因转录和蛋白表达两个层面调控PI3K/AKT信号转导通路中多个分子的表达,从而发挥其抑制Raji细胞、LOVO细胞的增殖,诱导细胞凋亡,阻滞细胞停滞在G1期的作用。2透脓散提取液可能通过调控PI3K/AKT信号转导通路中多个分子的表达、纠正移植瘤裸鼠的Th1/Th2漂移从而发挥抑制LOVO裸鼠移植瘤的生长的作用。3透脓散可作为一种有潜力治疗恶性肿瘤的经典方剂。更多还原

【Abstract】 AIM:To investigate the anti-cancer effects of Tou Nong San extracts (TNSEs) and its possible mechanism.METHODS:LC-MS Assay for TNS extract. The cytotoxic effect of TNSE on Raji cells and LOVO cells was measured by3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle and apoptosis were determined by flow cytometry(FCM).The molecular mechanisms of TNSE-mediated apoptosis were further investigated by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the mRNA expression of nuclear factor-KB (NF-κB), Bcl-xL and Bcl-2-associated death promoter (Bad), caspase-9and caspase-3,PI3K, Akt, mTOR, p70s6k1, and by Western blotting to detect the proteins expression of NF-κB, Bad,PI3K,p-Akt, p-mTOR, p-p70s6k1, caspase-9, caspase-3.The safty of TNSEs was detected by acute toxicity testing.The inhibition ratio was gained on the base of the weight and volume of transplanted tumor, morphosis of tumor was detected by hematoxylin and eosin (HE)stain, the proteins expression of PI3K,p-Akt,p-mTOR,p-p70s6k1, caspase-9, caspase-3,VEGF,CD31were detect by immuno-histochemical(IHC) method and the content of cytokine of Thl/Th2was tested by Enzyme-Linked Immunosorbent Assay(ELISA).RESULTS:The contents of7major components in TNSEs were Calycosin-7-O-b-D-glucoside, Formononetin-7-O-b-D-glucoside senkyunolide I, Calycosin,formononetin, Astragaloside IV, Z-Ligustilide.TNSEs inhibited Raji cells and LOVO cells proliferation in dose-and time-dependent manners. The Half maximal inhibitory concentration(IC50) of Raji cells was817.142μg/mL at72h,and that of LOVO cells was347.603μg/mL at48h. The apoptosis and Glaresst of these two cell lines were found after treated with TNSEs. After48h treatment with various concentrations of TNSEs (125,250and500μg/mL), the apoptosis rates of Raji cells were12.23±1.98%(P<0.05),20.97±3.96%(P<0.01) and30.4±4.87%(P<0.01), respectively, compared with that of the control (6.02±1.01%).and the G1arrest was also found that was The proportion of G1cells was44.80%±2.36%(P<0.05),55.48%±2.71%(P<0.01),66.76%±2.25%(P<0.01) respectively, compared with that of the control (39.66%±2.01%).After48h treatment with various concentrations of TNSE (62.5μg/mL,125μg/mL,and250μg/mL),the apoptosis rates of LOVO cell were12.8%±1.58%(P<0.05),19.9%±2.16%,(P<0.01)and29.57%±2.76%(P<0.01), respectively, compared with that of the control(7.3%±1.06%).The proportion of G1cells was45.45%±2.54%(P<0.05),56.13%±2.54%(P<0.01),66.05%±2.54%(P<0.01) respectively, compared with that of the control (40.02%±0.69%).RT-PCR demonstrated that NF-κB mRNA expression was significantly downregulated in Raji cells treated with250μg/mL TNSEs for48h (P<0.05), while Bad, caspase-9and caspase-3mRNA levels were upregulated (P<0.05). Moreover, TNSEs treatment resulted in down-regulation of NF-κB protein expression and strikingly up-regulated proteins expression of Bad, cleaved caspase-9, cleaved caspase-3in a dose-dependent manner, as determined by western blot.Furthermore,the similar results were found in LOVO cells treated with125μg/mL TNSEs for48h. The mRNA expression of PI3K, AKT, mTOR, P70s6k1was significantly downregulated (P<0.05), while caspase-9and caspase-3mRNA levels were upregulated (P<0.05,), which was accompanied by significantly down-regulation of the proteins of PI3K,p-AKT,p-mTOR, p-P70s6k1and strikingly up-regulation of the proteins of cleaved caspase-9, cleaved caspase-3in a dose-dependent fashion, as determined by western blot.There is no any toxicity at the maximum administration40000mg/kg for the mice, so40000mg/kg is safe dose and used for the next experiments. The inhibition rate of the implanted tumor in these three groups(high dosage group,middle dosage group,low dosage group)was67.11%,48.68%,36.84%respectively.The proteins expression of PI3K, p-Akt, p-mTOR,p-P70S6K1, VEGF,CD31were decreased and that of caspase-9,caspase-3were increased in implanted tumor of nude mouse treated with different concentration TNSEs as detected with IHC. Further-moer,TNSEs could regulate the nude mouses’immunity through upragulating Th1cytockines including IL-2, IL-12and down-regulating Th2cytockines, including IL-6IL-10, rectifying the drift of Thl/Th2,which was also in a dose dependant manner.Conclusion:1TNSEs could reduce proliferation, cause apoptosis and cell-cycle arrest in Raji cells and LOVO cells, which might be associated with the regulation of PI3K/AKT signaling pathway from two levels of gene transduction and protein expression.2TNSEs inhibited the growth of implanted tumor of nude mouse which was increasingly associated with regulating multiple molecules in the P3K/AKT pathway and with rectifying the drift of Thl/Th2.3TNS is a hopeful classic Chinese Medical Formula for tumor therapy.更多还原