PI3K/AKT相关信号通路在猪牙皂提取物抑制人肝癌细胞SMMC-7721中的表达
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摘要
一、研究目的
本课题通过理论与实验研究，探讨猪牙皂提取物正丁醇部位、乙醇部位抑制人肝癌细胞SMMC-7721的效果，并从分子水平、蛋白水平研究该药物对PI3K/AKT相关细胞信号通路的影响，阐述猪牙皂抗肝癌的可能机理。
二、研究方法
第一部分理论研究
1.中医防治原发性肝癌具有减少癌变、减轻临床症状、减少术后症状及复发、提高免疫力、提高生存质量和远期疗效的优势。
2.中医治疗原发性肝癌可以从痰瘀出发，化痰药猪牙皂具有抗肝癌的研究价值。
第二部分实验研究
1.根据人临床用量（猪牙皂生药量1.5g.70kg.d-1）、人与大鼠的等效剂量倍数，倍增至7倍为实验低剂量，按1：2：4比例，设为低、中、高剂量组，将40只Wistar雄性大鼠随机分为4组，即：对照（空白血清）组、正丁醇部位低剂量(0.15g.kg.d-1)组、中剂量(0.3g.kg.d-1)组和高剂量(0.6g.kg.d-1)组，每组10只大鼠。连续灌胃三天，心脏采血，同组血清合并。利用噻唑蓝还原法(MTT法)检测猪牙皂提取物正丁醇部位含药血清对人肝癌细胞SMMC-7721的抑制率。
2.根据人临床用量（猪牙皂生药量1.5g.70kg.d-1）、人与大鼠的等效剂量倍数，倍增至7倍为实验低剂量，按1：2：4比例，设为低、中、高剂量组，将220只Wistar雄性大鼠随机分为22组，即：对照（空白血清）组1组，乙醇部位含药血清按30%、60%、95%、30%+60%、60%+95%、30%+95%、30%+60%+95%分为7个浓度，每个浓度再分别按低、中、高剂量各设1个实验组，乙醇部位含药血清总共分为21个组，每组10只大鼠。连续灌胃三天，心脏采血，同组的血清合并。利用MTT法检测猪牙皂提取物乙醇部位含药血清对人肝癌细胞SMMC-7721的抑制率。
3．实验分为3组:空白组（人肝癌细胞SMMC-7721,未加任何药物），猪牙皂组（将实验二的猪牙皂提取物正丁醇部位中剂量组按10%浓度加入人肝癌细胞SMMC-7721），索拉非尼组（将4μmol/l的索拉非尼加入人肝癌细胞SMMC-7721）。采用蛋白印迹法、实时荧光定量PCR分别检测各组0h、0.5h、3h、8h、25h、48h作用时间点的VEGF、PTEN、PI3K、AKT、mTOR、Caspase9。
4.统计学分析
4.1猪牙皂提取物正丁醇部位各剂量组对SMMC-721肝癌细胞的抑制率应用SPSS18.0进行分析处理，各组检测数据用均数±标准差(x±s)表示，P<0.05为差异有统计学意义。
4.2猪牙皂提取物乙醇部位各浓度、各剂量组对SMMC-721肝癌细胞的抑制率应用SPSS18.0进行分析处理。观察指标采用x士s表示指标平均水平及变异性，析因设计方差分析检验不同浓度乙醇、不同剂量猪牙皂的主效应和交互作用，当两者存在交互作用时，进一步做单独效应分析，固定乙醇浓度在某个水平对猪牙皂不同剂量之间进行比较，同样固定猪牙皂在某个水平对乙醇不同浓度之间进行比较。P<0.05为差异有统计学意义。
4.3所有数据录入计算机建立数据库，应用SAS9.0进行分析处理。观察指标采用x士s表示指标平均水平及变异性，利用重复测量方差分析比较索拉非尼和猪牙皂在同一作用时间对肝癌细胞AKT、Caspase9、mTOR、PI3K、PTEN、VEGF六个基因表达的差异，比较索拉非尼在不同作用时间对肝癌细胞六个基因表达的差异，比较猪牙皂在不同作用时间对肝癌细胞六个基因表达的差异。P<0.05为差异有统计学意义。
三、研究结果
1.猪牙皂提取物正丁醇部位含药血清低剂量组与对照组比较差异（P<0.05）有统计学意义，与中剂量组比较（P>0.05）无统计学意义。中剂量组与对照组比较（P<0.05）有统计学意义，与高剂量比较（P<0.05）有统计学意义。高剂量与对照组比较（P>0.05）无统计学意义。
2.猪牙皂提取物乙醇部位含药血清，60%浓度乙醇低剂量组、30%+60%浓度乙醇高剂量、30%+60%+95%浓度乙醇中剂量分别与空白组比较，无统计学差异（P＞0.05）；其它各浓度组、各剂量组分别与空白组比较均有统计学差异（P＜0.05）。猪牙皂提取物乙醇部位不同浓度之间有统计学差异（P＜0.05），不同剂量之间有统计学差异（P＜0.05），不同浓度与不同剂量之间有统计学差异（P<0.05）。
3.对比猪牙皂组和索拉菲尼组在0h/0.5h/3h/8h/25h/48h对六个基因的影响，结果显示：
①P TEN的表达：Western blot检测中将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，PTEN表达无差异（P＞0.05），索拉非尼组中，0.5h/3h/8h/25h与空白组比较PTEN表达增加（P＜0.01），48h比较表达无明显变化（P＞0.05）；猪牙皂组中，0.5h/3h/8h与空白组比较PTEN表达明显增加（P＜0.01），25h与空白组比较PTEN表达明显增加（P＜0.05），48h比较表达无明显变化（P＞0.05）。与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h对PTEN的上调表达无明显差异（P＞0.05）。而在25h，猪牙皂对PTEN的上调表达要低于索拉非尼组（P＜0.01）；在48h，两组对PTEN的表达均无改变（P＞0.05）。PCR检测中将两组在0h/0.5h/3h/8h/25h/48h的PTEN扩增倍数进行比较，通过统计学分析，25h、48h比较都有统计学差异（P＜0.05），0h/0.5h/3h/8h均无统计学差异（P＞0.05）；将索拉菲尼组中，0.5h/3h/8h/25h/48h的PTEN扩增倍数分别与0h比较，除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。猪牙皂组中，0.5h/3h/8h/25h/48h的PTEN扩增倍数分别与0h比较，均有显著性差异（P＜0.01）。另外，索拉菲尼组中各时间点PTEN扩增倍数进行两两比较，在0.5h与25h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P<0.01）；将猪牙皂组中，0.5h/3h/8h/25h/48h的PTEN扩增倍数进行两两比较也均有显著性差异（P <0.01）。
②Caspase9的表达：western blot检测中空白组中，5个时间点与0h的Caspase9表达无变化（P＞0.05）；将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，Caspase9表达无差异（P＞0.05），索拉非尼组中，3h/8h与空白组比较Caspase9表达显著增加（P＜0.01），0.5h/25h与空白组比较Caspase9表达显著增加（P＜0.05），48h比较表达无明显变化（P＞0.05）；猪牙皂组中，0.5h/3h/8h/25h与空白组比较Caspase9表达明显增加（P＜0.01）,48h比较表达无明显变化（P＞0.05）。与索拉非尼组相比，猪牙皂组在0h/0.5h/25h/48h对Caspase9的上调表达无明显差异（P＞0.05）。而在3h/8h，猪牙皂对Caspase9的上调表达要高于索拉非尼组（P＜0.01）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的Caspase9扩增倍数进行比较，通过统计学分析，3h、8h比较有显著性差异（P＜0.01），0h/0.5h/25h/48h均无统计学差异（P＞0.05）；将索拉菲尼组和猪牙皂中，0.5h/3h/8h/25h/48h的Caspase9扩增倍数分别与0h比较，两组均有除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。另外，分别将两组内各时间点Caspase9扩增倍数进行两两比较，索拉菲尼组在0.5h与25h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）；猪牙皂组在0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
③mTOR的表达：western blot检测中将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，mTOR表达无差异（P＞0.05），索拉非尼组中，0.5h/48h与空白组比较mTOR表达减少（P＜0.05），3h/8h/25h与空白组比较mTOR表达显著减少（P＜0.01）；猪牙皂组中，3h/8h与空白组比较mTOR表达明显减少（P＜0.01），0.5h/25h/48h与空白组比较mTOR表达减少（P＜0.05）。与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h/25h对mTOR的下调表达无明显差异（P＞0.05）。而在48h，猪牙皂对mTOR的下调表达要弱于索拉非尼组（P＜0.01）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的mTOR扩增倍数进行比较，通过统计学分析，48h比较有显著性差异（P＜0.01），0h/0.5h/3h/8h/25h均无统计学差异（P＞0.05），将索拉菲尼组和猪牙皂，在0.5h/3h/8h/25h/48h的mTOR扩增倍数分别在组内与0h比较，均有显著性差异（P＜0.01）。另外，将各时间点mTOR扩增倍数进行两两比较，两组也均有在0.5h与25h、3h与48h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
④P I3K的表达：western blot检测中空白组中，5个时间点与0h的PI3K表达无变化（P＞0.05）；将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，PI3K表达无差异（P＞0.05），索拉非尼组中，3h/8h与空白组比较PI3K表达显著减少（P＜0.01），0.5h与空白组比较PI3K表达减少（P＜0.05），25h/48h与空白组比较表达无明显变化（P＞0.05）；猪牙皂组中，3h/8h/25h/48h与空白组比较PI3K表达显著减少（P＜0.01），0.5h比较表达无明显变化（P＞0.05）。将猪牙皂组与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h对PI3K表达调控较为相似，且在3h/8h对PI3K的下调表达无明显差异（P＞0.05）。而在25h/48h，猪牙皂对PI3K的下调表达要弱于索拉非尼组（P＜0.05）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的PI3K扩增倍数进行比较，通过统计学分析，25h、48h比较都有显著性差异（P＜0.01），0h/0.5h/3h/8h均无统计学差异（P＞0.05）；将猪牙皂组和索拉菲尼组，在0.5h/3h/8h/25h/48h的PI3K扩增倍数组内分别与0h比较，两组均有显著性差异（P＜0.01）。另外，将各时间点PI3K扩增倍数进行两两比较，索拉菲尼组中0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P<0.01）；猪牙皂组中，3h与8h比较无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
⑤AKT的表达：western blot检测中将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，AKT表达无差异（P＞0.05），索拉非尼组中，0.5h/3h/8h/25h/48h与空白组比较AKT表达显著减少（P＜0.01）；猪牙皂组中，0.5h/3h/8h/25h/48h与空白组比较AKT表达同样明显减少（P＜0.01）。与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h/25h/48h对AKT表达调控均无明显差异（P＞0.05）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的AKT扩增倍数进行比较，通过统计学分析均无统计学差异（P＞0.05），索拉菲尼组中，0.5h/3h/8h/25h/48h的AKT扩增倍数分别与0h比较，除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。而猪牙皂组中，0.5h/3h/8h/25h/48h的AKT扩增倍数分别与0h比较，均有显著性差异（P＜0.01）。另外，将各时间点AKT扩增倍数进行两两比较，两组都有0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P<0.01）。
⑥VEGF的表达：western blot检测中空白组中，5个时间点与0h均VEGF的表达无变化（P＞0.05）；将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，VEGF表达无差异（P＞0.05），说明三组在0h的VEGF表达无明显差异，索拉非尼组中，0.5h/3h/8h/25h/48h与0h比较VEGF表达显著减少（P＜0.01）；猪牙皂组中，0.5h/3h/8h/25h/48h与0h比较VEGF表达同样明显减少（P＜0.01）。并且将猪牙皂组与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h/25h/48h对VEGF表达调控均无明显差异（P＞0.05）。 PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的VEGF扩增倍数进行比较，通过统计学分析，两组在6个时间点比较均无统计学差异（P＞0.05），将猪牙皂组和索拉菲尼组中，0.5h/3h/8h/25h/48h的VEGF扩增倍数分别与0h比较，两组均有除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。另外，将各时间点VEGF扩增倍数进行两两比较，索拉菲尼组在3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有统计学差异（P <0.05），而猪牙皂组0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
四、结论
1.猪牙皂提取物正丁醇部位含药血清低剂量组、中剂量组对人肝癌细胞SMMC-7721均具有抑制效果，高剂量组无抑制作用。
2.猪牙皂提取物乙醇部位除60%浓度乙醇低剂量组、30%+60%浓度乙醇高剂量、30%+60%+95%浓度乙醇中剂量组外，其它不同浓度组及其高、中、低剂量组均能抑制人肝癌细胞SMMC-7721；各浓度组之间、及其高、中、低剂量组之间也存在差异。
3.通过对比空白组、索拉菲尼组和猪牙皂组对肝癌细胞中六个基因的表达影响，我们可以看到：
①猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现PTEN基因表达上调，说明皂荚提取物与索拉非尼均能通过调控PTEN基因的上调，从而抑制肿瘤血管生成、抑制肝癌细胞增殖。
②猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现Caspase9基因表达上调，说明皂荚提取物与索拉非尼均能通过调控Caspase9基因的上调，从而诱导肝癌细胞的凋亡。
③猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现mTOR基因、AKT基因、PI3K基因表达下调，说明皂荚提取物与索拉非尼均能通过调控mTOR基因、AKT基因、PI3K基因的下调，阻断PI3k／Akt／mTOR信号通路，从而抑制肝癌细胞的生长。
④猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现VEGF基因表达下调，说明皂荚提取物与索拉非尼均能通过调控VEGF基因的下调，从而抑制肿瘤血管生成。
⑤对于6个时间点猪牙皂提取物正丁醇部位和索拉非尼作用于肝癌细胞调控6个基因的表达，两组均在3h和8h时对AKT基因、mTOR基因、PI3K基因、VEGF基因的下调作用最为显著；两组均在8h时对PTEN基因上调作用最为显著；索拉非组在8h对Caspase9基因上调作用最为显著，皂荚提取物在3h、8h对Caspase9基因上调作用均较为显著。
⑥皂荚提取物与索拉非尼对肝癌细胞中6个基因的调控较为相似，比较皂荚提取物组与索拉非尼组，除在25h，索拉非尼对PTEN上调要强于猪牙皂；在3h/8h/25h，猪牙皂对Caspase9上调要明显强于索拉非尼；在25h/48h，索拉非尼对mTOR下调要强于猪牙皂；在48h，猪牙皂对PI3K下调要强于索拉非尼。4个基因其余时间段和VEGF、AKT的调控均较为相同。
1.Objectives
This issue through theoretical and experimental study to clarify n-butanolposition and ethanol position of Gleditsiae extract can inhibit human hepatomaSMMC-7721cells results, while the expression of PI3K/AKT signalingpathways from molecular level, protein level of is one of the mechanism ofanti-hepatoma.
2.Methods
The first part is theoretical study
(1)TCM can prevent and treat the primary liver cance, it also relieveclinical symptoms, decrease postoperative symptoms and recurrence, enhanceimmunity, improve the quality of survival and the forward curative effect.
(2)The treatment of primary liver cancer through Chinese medicine canstarting from can start from the phlegm and blood stasis, phlegm medicineGleditsiae has anti-hepatoma research value.
The second part is the experimental study
(1)According to the human clinical dose (Gleditsiae crude drug1.5g.70kg.d-1), human and rat equivalent dose multiples doubled to seventimes as the experimental low-dose, according to1:2:4,the40Male Wistar ratswere randomly divided into four groups, namely: control (blank serum) group,n-butanol low dose (0.15g.kg.d-1) group, middle dose (0.3g.kg.d-1) Groupsand high-dose (0.6g.kg.d-1) groups,each group is10rats. Intragastrically three days, extract blood from heart,then combine with serum from the samegroups. Using the MTT reduction method (MTT assay) Gleditsiae n-butanolextract containing serum on human hepatoma SMMC-7721cells inhibitionrate.
(2)According to the human clinical dosage (quantity of crude drugsGleditsiae is1.5g.70kg.d-1), human and rat multiple dose equivalent, doubledto7times as the low dose experiments. according to1:2:4, the220MaleWistar rats were randomly divided into22groups: control (blank serum)Group1, containing serum on ethanol site at30%,60%,95%,30%+60%,60%+95%,30%+95%,30%+60%+95%is divided into seven levels, theneach concentration was divided into low, medium and high doses, so totalgroup of experimental is21groups. each group has10rats. Intragastricallythree days, extract blood from heart, combined with serum from the samegroup. Using the MTT assay Gleditsiae parts ethanol extract containing serumon human hepatoma SMMC-7721cells inhibition rate.
(3)The experiment was divided into three groups: control group (humanhepatoma cells SMMC-7721, did not add any medication), Gleditsiae group(the second experiment Gleditsiae n-butanol extract dose group added at aconcentration of10%human hepatoma cell line SMMC-7721), sorafenibgroup (the4μmol/l of sorafenib adding human hepatoma cell SMMC-7721).Western blot analysis using real-time quantitative PCR were used to detecteach group0h,0.5h,3h,8h,25h,48h reaction time points VEGF, PTEN, PI3K,AKT, mTOR, Caspase9.
3. Statistical analysis
3.1Inhibition rate in each dose group of SMMC-721hepatocellularcarcinoma cells can be expressed by mean±standard deviation (±SD);singlefactor analysis of variance was used for comparison of inhibition rate groups;If the difference was statistically significant, the comparisons of each groups difference was useing Bonferrni method；so the data using SPSS18.0statisticalanalysis software for processing; take P <0.05as significance level in singlefactor analysis of variance; pairwise comparison test level when0.05/compare times.
3.2Inhibition rate in each dose group of SMMC-721hepatocellularcarcinoma cells using SPSS18.0analysis. Observation indexes using x士s toexpress index average level and variability; Factorial design analysis ofvariance is to test the main effects and interaction effects of differentconcentrations of ethanol and different doses of Gleditsia sinensis; whenthere was an interaction between the two, it is wise to carry out a furtherseparate effects analysis. fixed ethanol concentration at a certain level,thencompare the different dose of Fructus Gleditsiae.At the same time, fixedFructus Gleditsiae at a certain level,then compare the different concentrationsof ethano. P <0.05was considered statistically significant.
3.3All data were input into the computer to establish a database, usingSAS9.0to analysis.using士s to express index average level and variability.compared Sola Fini and Fructus Gleditsiae at the same time on hepatoma cellsAKT, Caspase9, mTOR, PI3K, PTEN, VEGF six gene expression usingrepeated measures analysis of variance; compared Sola Fini and FructusGleditsiae in the different time on six gene expression in hepatoma cells. P<0.05was considered statistically significant.
4.Results
(1) Between the low-dose group and the control group, the difference (P<0.05) was statistically significant. compared with the low dose group, themiddle dose group (P>0.05) was not statistically significance. compared withthe middle dose group,the control group and high Dose group had astatistically significant.At the same time,there was no difference between thecontrol group and High-dose group.
(2) Gleditsiae parts ethanol extract serum containing60%ethanolconcentration of low-dose group,30%+60%concentration of high doses ofethanol,30%+60%+95%concentration ethanol doses were compared withthe control group, there were no statistical Learn the difference; comparedwith the control group,other various concentrations and each dose group werestatistically significant. Gleditsiae parts of different concentrations of ethanolextract were significantly different respective, there are significant differencesamoung different doses, different concentrations were significantly differentbetween different doses.
(3) By comparing the blank group, the sola fini and Gleditsiae group sixgene expression in liver cells, we can see:
①PTEN expression: Western blot analysis in the sorafenib group andthe control group, the control group and the group were in the Gleditsiae0hcomparison, no significant difference (P>0.05) PTEN expression sorafenibgroup,0.5h/3h/8h/25h comparison with the control group PTEN expressionincreased (P <0.01),48h comparison showed no significant change (P>0.05);Gleditsiae group,0.5h/3h/8h PTEN expression in comparison with the controlgroup was significantly increased (P<0.01),25h PTEN expression incomparison with the control group was significantly increased (P <0.05),48hcomparison showed no significant change (P>0.05). Group compared withsorafenib, Gleditsiae group showed no significant difference (P>0.05) onPTEN in0h/0.5h/3h/8h upregulation. In25h, Gleditsiae upregulate theexpression of PTEN is lower than the sorafenib group (P <0.01); at48h, theexpression of PTEN in both groups had no change (P>0.05). PCR detected inthe two groups of PTEN in0h/0.5h/3h/8h/25h/48h of amplification werecompared by statistical analysis,25h,48h comparison has significantdifference (P <0.05),0h/0.5h/3h/8h no significant difference (P>0.05); thesorafenib group, PTEN amplification of multiple0.5h/3h/8h/25h/48h compared with0h respectively, in addition to the differences in0h and48h nostatistically significant (P>0.05) outside,0.5h/3h/8h/25h of amplification and0h were significant differences (P<0.01). Gleditsiae group, PTENamplification multiple0.5h/3h/8h/25h/48h respectively compared with0h,there were significant differences (P <0.01). In addition, sorafenib group ateach time point of amplification of PTEN pairwise comparison,0.5h and25hshowed no significant difference (P>0.05), pairwise comparisons weresignificant differences in the other time points (P <0.01); Gleditsiae group,PTEN amplification of multiple0.5h/3h/8h/25h/48h pairwise comparison alsoshowed significant differences (P <0.01).
②Caspase9of expression: western blot detection of the control group,the five time points and0h 's expression did not change Caspase9(P>0.05);the sorafenib group and the control group, the control group and the groupwere in the Gleditsiae compare0h, Caspase9showed no difference (P>0.05),sorafenib group,3h/8h Caspase9comparison with the control group wassignificantly increased (P<0.01),0.5h/25h Caspase9comparison with thecontrol group was significantly increased (P <0.05),48h comparison showedno significant change (P>0.05); Gleditsiae group was significantly increased0.5h/3h/8h/25h Caspase9comparison with the control group (P <0.01),48hcomparison showed no significant change (P>0.05). Group compared withsorafenib, Gleditsiae set in0h/0.5h/25h/48h upregulated on Caspase9's nosignificant difference (P>0.05). In3h/8h, Gleditsiae upregulate the expressionof Caspase9higher than the sorafenib group (P <0.01). PCR detection in thesorafenib group, Gleditsiae group were amplified in0h/0.5h/3h/8h/25h/48hmultiples of Caspase9compare by statistical analysis,3h,8h There was asignificant difference (P <0.01),0h/0.5h/25h/48h no significant difference (P>0.05); the sorafenib group and Gleditsiae,0.5h/3h/8h/25h/48h the Caspase9multiples were amplified compared with0h, with both groups except in48h 0h no significant difference (P>0.05) outside,0.5h/3h/8h/25h of amplificationand0h were significant differences (P <0.01). Additionally, respectively, in thetwo groups at each time point Caspase9amplification of multiple pairwisecomparison, sorafenib group0.5h and25h showed no significant difference(P>0.05), pairwise comparisons were other time points significant differences(P<0.01); Gleditsiae set in0.5h and25h,3h and8h showed no significantdifference (P>0.05), pairwise comparisons of other time points weresignificant differences (P <0.01).
③mTOR expression of: western blot detection in the sorafenib groupand the control group, the control group and the group were in the Gleditsiae0h comparison, no significant difference (P>0.05) mTOR expression sorafenibgroup,0.5h/48h comparison with the control group mTOR expressiondecreased (P<0.05),3h/8h/25h mTOR expression comparison with the controlgroup significantly reduced (P<0.01); Gleditsiae group,3h/8h mTORcomparison with the control group was significantly reduced (P<0.01),0.5h/25h/48h comparison with the control group mTOR expression wasdecreased (P<0.05). Group compared with sorafenib, Gleditsiae set in0h/0.5h/3h/8h/25h on down the expression of mTOR was no significantdifference (P>0.05). In48h, Gleditsiae to downregulate the expression ofmTOR is weaker than the sorafenib group (P <0.01). PCR detection in thesorafenib group, Gleditsiae group were amplified in0h/0.5h/3h/8h/25h/48h ofmTOR multiple comparison, through statistical analysis,48h there was asignificant difference (P <0.01),0h/0.5h/3h/8h/25h no significant difference(P>0.05), the sorafenib group and Gleditsiae, in0.5h/3h/8h/25h/48h multiplesof mTOR were amplified in the group with0h, there were significantdifferences (P <0.01). In addition, at each time point mTOR amplify multiplepairwise comparison, the two groups were also in0.5h and25h,3h and48hshowed no significant difference (P>0.05), the other time points were pairwise comparison significant differences (P <0.01).
④PI3K expression of: western blot detection of the control group, thefive time points and0h of PI3K expression did not change (P>0.05); thesorafenib group and the control group, the control group and the group werein the Gleditsiae compare0h, PI3K expression was no difference (P>0.05),sorafenib group,3h/8h PI3K expression comparison with the control groupsignificantly decreased (P <0.01),0.5h comparison with the control groupdecreased expression of PI3K (P <0.05),25h/48h comparison with the controlgroup showed no significant change (P>0.05); Gleditsiae group,3h/8h/25h/48h PI3K expression comparison with the control groupsignificantly decreased (P <0.01),0.5h comparison showed no significantchange (P>0.05). The Gleditsiae group compared with sorafenib, Gleditsiaeset in0h/0.5h/3h/8h for regulating the expression of PI3K is similar, and nosignificant difference in3h/8h expression (P>0.05) on the reduction ofPI3K. In25h/48h, Gleditsiae to downregulate the expression of PI3K isweaker than the sorafenib group (P<0.05). PCR detection in the sorafenibgroup, Gleditsiae groups were0h/0.5h/3h/8h/25h/48h of PI3K amplificationmultiples compared through statistical analysis,25h,48h comparison hassignificant difference (P<0.01),0h/0.5h/3h/8h no significant difference (P>0.05); Gleditsiae sorafenib group and the group of PI3K amplification in0.5h/3h/8h/25h/48h compared with0h within multiple groups were twogroups were significantly different (P <0.01). In addition, at each time pointPI3K amplification of multiple pairwise comparison, sorafenib group0.5hand25h,3h and8h showed no significant difference (P>0.05), pairwisecomparisons were other time points significant differences (P<0.01);Gleditsiae group,3h was no significant difference (P>0.05), pairwisecomparisons of other time points were significant differences (P <0.01).
⑤expression of AKT: western blot detection in the sorafenib group and the control group, the control group and the group were in the Gleditsiae0hcomparison, no significant difference (P>0.05) AKT expression sorafenibgroup,0.5h/significantly reduce3h/8h/25h/48h comparison with the controlgroup AKT expression (P <0.01); Gleditsiae group,0.5h/3h/8h/25h/48h AKTexpression comparison with the control group also decreased significantly (P<0.01). Group compared with sorafenib, Gleditsiae set for regulating theexpression of AKT was no significant difference (P>0.05) in0h/0.5h/3h/8h/25h/48h. PCR detection in the sorafenib group, Gleditsiaegroup were amplified in0h/0.5h/3h/8h/25h/48h of AKT compare multipleanalyzes were not statistically different (P>0.05) by statistics, sorafenib group,AKT amplification of multiple0.5h/3h/8h/25h/48h compared with0hrespectively, except in0h and48h was no significant difference (P>0.05)outside,0.5h/3h/8h/25h and0h amplification multiples are significantdifferences (P<0.01). The Gleditsiae group,0.5h/3h/8h/25h/48h ofamplification of AKT respectively compared with0h, there were significantdifferences (P <0.01). In addition, at each time point AKT amplification ofmultiple pairwise comparison, both groups0.5h and25h,3h and8h showedno significant difference (P>0.05), pairwise comparison of the other timepoints were significantly difference (P <0.01).
⑥VEGF expression is: western blot detection of the control group, theexpression of five time points0h both VEGF and no change (P>0.05); thesorafenib group and the control group, the control group with Gleditsiaegroup were conducted at0h comparison, VEGF expression was no difference(P>0.05), described three groups0h no significant difference in the expressionof VEGF sorafenib group,0.5h/3h/8h/25h/48h VEGF expression wassignificantly reduced when compared with0h (P <0.01); Gleditsiae group,0.5h/3h/8h/25h/48h0h comparison with the same expression of VEGF wassignificantly reduced (P <0.01). And the Gleditsiae sorafenib group compared with Gleditsiae set in0h/0.5h/3h/8h/25h/48h on VEGF expression andregulation were not significantly different (P>0.05). PCR detection in thesorafenib group, Gleditsiae group were amplified in0h/0.5h/3h/8h/25h/48hmultiples of VEGF were compared by statistical analysis, the two groups atsix time points showed no statistically significant difference (P>0.05),Gleditsiae sorafenib group and the group,0.5h/3h/8h/25h/48h ofamplification of VEGF compared with0h respectively, with both groupsexcept in0h48h the difference was not statistically significant (P>0.05)outside,0.5h/3h/8h/25h of amplification and0h were significant differences(P <0.01). In addition, at each time point of amplification VEGF pairwisecomparison, sorafenib group and8h3h showed no significant difference (P>0.05), statistically significant pairwise comparison are other time points (P <0.05), while the pig teeth soap0.5h and25h,3h and8h showed no significantdifference (P>0.05), pairwise comparisons were significant differences (P<0.01) the other time points.
Conclusions
(1) Gleditsiae n-butanol extract containing serum low-dose group, middledose group on human hepatoma SMMC-7721cells were inhibitory effect ofhigh-dose group had no inhibitory effect.
(2) Gleditsiae parts in addition to the concentration of60%ethanolextract ethanol low dose group, high dose of30%+60%concentrationethanol,30%+60%+95%concentration ethanol dose group, other groups ofdifferent concentrations and high, medium and low dose group can inhibithuman liver cancer cell SMMC-7721; The concentration between group, andits high, there is also a difference between middle and low dose group.
(3) Comparing to the control group, sorafenib group and fructusgleditsiae extract soap HCC cells affect the expression of genes for the sixgroups, we can see that
①gleditsiae extractparts of n-butanol and Sorafenib HCC cells werefound in the PTEN gene upregulation, indicating acacia extract with sorafenibcould increase by regulating PTEN gene, thereby inhibiting tumorangiogenesis, inhibit hepatoma cell proliferation.
②gleditsiae extractparts of n-butanol and the role of sorafenib in HCCcells were found Caspase9upregulated genes, indicating acacia extract withsorafenib can increase Caspase9by regulating genes, thus inducing apoptosisof hepatoma cells.
③gleditsiae extractparts of n-butanol and Sorafenib HCC cells werefound in the mTOR gene, AKT gene, PI3K genes downregulated, indicatingacacia extract with sorafenib can by regulating mTOR gene, AKT gene, genedown-regulated PI3K, blocking PI3k/Akt/mTOR signaling pathway, therebyinhibiting the growth of hepatoma cells.
④gleditsiae extractparts of n-butanol and the role of sorafenib in HCCcells were downregulated VEGF gene appears, indicating acacia extract withsorafenib could by down regulation of VEGF gene, thereby inhibiting tumorangiogenesis.
⑤For the six time points Gleditsiae and parts of n-butanol extract ofsorafenib in HCC cells regulate the expression of the role of six genes in bothgroups during3h and8h of AKT gene, mTOR gene, PI3K gene, VEGF genedownward effect the most significant; groups were8h when PTEN geneupregulation of the most significant; sorafenib group8h Caspase9geneupregulation most significant acacia extract3h,8h Caspase9geneupregulation were more significant.
⑥acacia extract with Sorafenib HCC cells regulate six groups of genesis similar, relatively acacia extract group and sorafenib, except in25h,sorafenib PTEN raised better than fructus gleditsiae extract soap; in3h/8h/25h, fructus gleditsiae extract soap on Caspase9raised significantly stronger than sorafenib; in25h/48h, sorafenib reduced mTOR better thanfructus gleditsiae extract soap; at48h, fructus gleditsiae extract soap on PI3Kdown better than sorafenib Nigeria.4groups of genes rest period and VEGFgenes, regulatory genes are more AKT same.
本课题通过理论与实验研究，探讨猪牙皂提取物正丁醇部位、乙醇部位抑制人肝癌细胞SMMC-7721的效果，并从分子水平、蛋白水平研究该药物对PI3K/AKT相关细胞信号通路的影响，阐述猪牙皂抗肝癌的可能机理。
二、研究方法
第一部分理论研究
1.中医防治原发性肝癌具有减少癌变、减轻临床症状、减少术后症状及复发、提高免疫力、提高生存质量和远期疗效的优势。
2.中医治疗原发性肝癌可以从痰瘀出发，化痰药猪牙皂具有抗肝癌的研究价值。
第二部分实验研究
1.根据人临床用量（猪牙皂生药量1.5g.70kg.d-1）、人与大鼠的等效剂量倍数，倍增至7倍为实验低剂量，按1：2：4比例，设为低、中、高剂量组，将40只Wistar雄性大鼠随机分为4组，即：对照（空白血清）组、正丁醇部位低剂量(0.15g.kg.d-1)组、中剂量(0.3g.kg.d-1)组和高剂量(0.6g.kg.d-1)组，每组10只大鼠。连续灌胃三天，心脏采血，同组血清合并。利用噻唑蓝还原法(MTT法)检测猪牙皂提取物正丁醇部位含药血清对人肝癌细胞SMMC-7721的抑制率。
2.根据人临床用量（猪牙皂生药量1.5g.70kg.d-1）、人与大鼠的等效剂量倍数，倍增至7倍为实验低剂量，按1：2：4比例，设为低、中、高剂量组，将220只Wistar雄性大鼠随机分为22组，即：对照（空白血清）组1组，乙醇部位含药血清按30%、60%、95%、30%+60%、60%+95%、30%+95%、30%+60%+95%分为7个浓度，每个浓度再分别按低、中、高剂量各设1个实验组，乙醇部位含药血清总共分为21个组，每组10只大鼠。连续灌胃三天，心脏采血，同组的血清合并。利用MTT法检测猪牙皂提取物乙醇部位含药血清对人肝癌细胞SMMC-7721的抑制率。
3．实验分为3组:空白组（人肝癌细胞SMMC-7721,未加任何药物），猪牙皂组（将实验二的猪牙皂提取物正丁醇部位中剂量组按10%浓度加入人肝癌细胞SMMC-7721），索拉非尼组（将4μmol/l的索拉非尼加入人肝癌细胞SMMC-7721）。采用蛋白印迹法、实时荧光定量PCR分别检测各组0h、0.5h、3h、8h、25h、48h作用时间点的VEGF、PTEN、PI3K、AKT、mTOR、Caspase9。
4.统计学分析
4.1猪牙皂提取物正丁醇部位各剂量组对SMMC-721肝癌细胞的抑制率应用SPSS18.0进行分析处理，各组检测数据用均数±标准差(x±s)表示，P<0.05为差异有统计学意义。
4.2猪牙皂提取物乙醇部位各浓度、各剂量组对SMMC-721肝癌细胞的抑制率应用SPSS18.0进行分析处理。观察指标采用x士s表示指标平均水平及变异性，析因设计方差分析检验不同浓度乙醇、不同剂量猪牙皂的主效应和交互作用，当两者存在交互作用时，进一步做单独效应分析，固定乙醇浓度在某个水平对猪牙皂不同剂量之间进行比较，同样固定猪牙皂在某个水平对乙醇不同浓度之间进行比较。P<0.05为差异有统计学意义。
4.3所有数据录入计算机建立数据库，应用SAS9.0进行分析处理。观察指标采用x士s表示指标平均水平及变异性，利用重复测量方差分析比较索拉非尼和猪牙皂在同一作用时间对肝癌细胞AKT、Caspase9、mTOR、PI3K、PTEN、VEGF六个基因表达的差异，比较索拉非尼在不同作用时间对肝癌细胞六个基因表达的差异，比较猪牙皂在不同作用时间对肝癌细胞六个基因表达的差异。P<0.05为差异有统计学意义。
三、研究结果
1.猪牙皂提取物正丁醇部位含药血清低剂量组与对照组比较差异（P<0.05）有统计学意义，与中剂量组比较（P>0.05）无统计学意义。中剂量组与对照组比较（P<0.05）有统计学意义，与高剂量比较（P<0.05）有统计学意义。高剂量与对照组比较（P>0.05）无统计学意义。
2.猪牙皂提取物乙醇部位含药血清，60%浓度乙醇低剂量组、30%+60%浓度乙醇高剂量、30%+60%+95%浓度乙醇中剂量分别与空白组比较，无统计学差异（P＞0.05）；其它各浓度组、各剂量组分别与空白组比较均有统计学差异（P＜0.05）。猪牙皂提取物乙醇部位不同浓度之间有统计学差异（P＜0.05），不同剂量之间有统计学差异（P＜0.05），不同浓度与不同剂量之间有统计学差异（P<0.05）。
3.对比猪牙皂组和索拉菲尼组在0h/0.5h/3h/8h/25h/48h对六个基因的影响，结果显示：
①P TEN的表达：Western blot检测中将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，PTEN表达无差异（P＞0.05），索拉非尼组中，0.5h/3h/8h/25h与空白组比较PTEN表达增加（P＜0.01），48h比较表达无明显变化（P＞0.05）；猪牙皂组中，0.5h/3h/8h与空白组比较PTEN表达明显增加（P＜0.01），25h与空白组比较PTEN表达明显增加（P＜0.05），48h比较表达无明显变化（P＞0.05）。与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h对PTEN的上调表达无明显差异（P＞0.05）。而在25h，猪牙皂对PTEN的上调表达要低于索拉非尼组（P＜0.01）；在48h，两组对PTEN的表达均无改变（P＞0.05）。PCR检测中将两组在0h/0.5h/3h/8h/25h/48h的PTEN扩增倍数进行比较，通过统计学分析，25h、48h比较都有统计学差异（P＜0.05），0h/0.5h/3h/8h均无统计学差异（P＞0.05）；将索拉菲尼组中，0.5h/3h/8h/25h/48h的PTEN扩增倍数分别与0h比较，除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。猪牙皂组中，0.5h/3h/8h/25h/48h的PTEN扩增倍数分别与0h比较，均有显著性差异（P＜0.01）。另外，索拉菲尼组中各时间点PTEN扩增倍数进行两两比较，在0.5h与25h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P<0.01）；将猪牙皂组中，0.5h/3h/8h/25h/48h的PTEN扩增倍数进行两两比较也均有显著性差异（P <0.01）。
②Caspase9的表达：western blot检测中空白组中，5个时间点与0h的Caspase9表达无变化（P＞0.05）；将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，Caspase9表达无差异（P＞0.05），索拉非尼组中，3h/8h与空白组比较Caspase9表达显著增加（P＜0.01），0.5h/25h与空白组比较Caspase9表达显著增加（P＜0.05），48h比较表达无明显变化（P＞0.05）；猪牙皂组中，0.5h/3h/8h/25h与空白组比较Caspase9表达明显增加（P＜0.01）,48h比较表达无明显变化（P＞0.05）。与索拉非尼组相比，猪牙皂组在0h/0.5h/25h/48h对Caspase9的上调表达无明显差异（P＞0.05）。而在3h/8h，猪牙皂对Caspase9的上调表达要高于索拉非尼组（P＜0.01）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的Caspase9扩增倍数进行比较，通过统计学分析，3h、8h比较有显著性差异（P＜0.01），0h/0.5h/25h/48h均无统计学差异（P＞0.05）；将索拉菲尼组和猪牙皂中，0.5h/3h/8h/25h/48h的Caspase9扩增倍数分别与0h比较，两组均有除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。另外，分别将两组内各时间点Caspase9扩增倍数进行两两比较，索拉菲尼组在0.5h与25h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）；猪牙皂组在0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
③mTOR的表达：western blot检测中将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，mTOR表达无差异（P＞0.05），索拉非尼组中，0.5h/48h与空白组比较mTOR表达减少（P＜0.05），3h/8h/25h与空白组比较mTOR表达显著减少（P＜0.01）；猪牙皂组中，3h/8h与空白组比较mTOR表达明显减少（P＜0.01），0.5h/25h/48h与空白组比较mTOR表达减少（P＜0.05）。与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h/25h对mTOR的下调表达无明显差异（P＞0.05）。而在48h，猪牙皂对mTOR的下调表达要弱于索拉非尼组（P＜0.01）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的mTOR扩增倍数进行比较，通过统计学分析，48h比较有显著性差异（P＜0.01），0h/0.5h/3h/8h/25h均无统计学差异（P＞0.05），将索拉菲尼组和猪牙皂，在0.5h/3h/8h/25h/48h的mTOR扩增倍数分别在组内与0h比较，均有显著性差异（P＜0.01）。另外，将各时间点mTOR扩增倍数进行两两比较，两组也均有在0.5h与25h、3h与48h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
④P I3K的表达：western blot检测中空白组中，5个时间点与0h的PI3K表达无变化（P＞0.05）；将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，PI3K表达无差异（P＞0.05），索拉非尼组中，3h/8h与空白组比较PI3K表达显著减少（P＜0.01），0.5h与空白组比较PI3K表达减少（P＜0.05），25h/48h与空白组比较表达无明显变化（P＞0.05）；猪牙皂组中，3h/8h/25h/48h与空白组比较PI3K表达显著减少（P＜0.01），0.5h比较表达无明显变化（P＞0.05）。将猪牙皂组与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h对PI3K表达调控较为相似，且在3h/8h对PI3K的下调表达无明显差异（P＞0.05）。而在25h/48h，猪牙皂对PI3K的下调表达要弱于索拉非尼组（P＜0.05）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的PI3K扩增倍数进行比较，通过统计学分析，25h、48h比较都有显著性差异（P＜0.01），0h/0.5h/3h/8h均无统计学差异（P＞0.05）；将猪牙皂组和索拉菲尼组，在0.5h/3h/8h/25h/48h的PI3K扩增倍数组内分别与0h比较，两组均有显著性差异（P＜0.01）。另外，将各时间点PI3K扩增倍数进行两两比较，索拉菲尼组中0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P<0.01）；猪牙皂组中，3h与8h比较无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
⑤AKT的表达：western blot检测中将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，AKT表达无差异（P＞0.05），索拉非尼组中，0.5h/3h/8h/25h/48h与空白组比较AKT表达显著减少（P＜0.01）；猪牙皂组中，0.5h/3h/8h/25h/48h与空白组比较AKT表达同样明显减少（P＜0.01）。与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h/25h/48h对AKT表达调控均无明显差异（P＞0.05）。PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的AKT扩增倍数进行比较，通过统计学分析均无统计学差异（P＞0.05），索拉菲尼组中，0.5h/3h/8h/25h/48h的AKT扩增倍数分别与0h比较，除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。而猪牙皂组中，0.5h/3h/8h/25h/48h的AKT扩增倍数分别与0h比较，均有显著性差异（P＜0.01）。另外，将各时间点AKT扩增倍数进行两两比较，两组都有0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P<0.01）。
⑥VEGF的表达：western blot检测中空白组中，5个时间点与0h均VEGF的表达无变化（P＞0.05）；将空白组与索拉菲尼组、空白组与猪牙皂组分别在0h进行比较，VEGF表达无差异（P＞0.05），说明三组在0h的VEGF表达无明显差异，索拉非尼组中，0.5h/3h/8h/25h/48h与0h比较VEGF表达显著减少（P＜0.01）；猪牙皂组中，0.5h/3h/8h/25h/48h与0h比较VEGF表达同样明显减少（P＜0.01）。并且将猪牙皂组与索拉非尼组相比，猪牙皂组在0h/0.5h/3h/8h/25h/48h对VEGF表达调控均无明显差异（P＞0.05）。 PCR检测中将索拉非尼组、猪牙皂组分别在0h/0.5h/3h/8h/25h/48h的VEGF扩增倍数进行比较，通过统计学分析，两组在6个时间点比较均无统计学差异（P＞0.05），将猪牙皂组和索拉菲尼组中，0.5h/3h/8h/25h/48h的VEGF扩增倍数分别与0h比较，两组均有除在0h与48h差异无统计学意义（P＞0.05）外，0.5h/3h/8h/25h与0h扩增倍数均有显著性差异（P＜0.01）。另外，将各时间点VEGF扩增倍数进行两两比较，索拉菲尼组在3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有统计学差异（P <0.05），而猪牙皂组0.5h与25h、3h与8h比较均无统计学差异（P>0.05），其它时间点的两两比较均有显著性差异（P <0.01）。
四、结论
1.猪牙皂提取物正丁醇部位含药血清低剂量组、中剂量组对人肝癌细胞SMMC-7721均具有抑制效果，高剂量组无抑制作用。
2.猪牙皂提取物乙醇部位除60%浓度乙醇低剂量组、30%+60%浓度乙醇高剂量、30%+60%+95%浓度乙醇中剂量组外，其它不同浓度组及其高、中、低剂量组均能抑制人肝癌细胞SMMC-7721；各浓度组之间、及其高、中、低剂量组之间也存在差异。
3.通过对比空白组、索拉菲尼组和猪牙皂组对肝癌细胞中六个基因的表达影响，我们可以看到：
①猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现PTEN基因表达上调，说明皂荚提取物与索拉非尼均能通过调控PTEN基因的上调，从而抑制肿瘤血管生成、抑制肝癌细胞增殖。
②猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现Caspase9基因表达上调，说明皂荚提取物与索拉非尼均能通过调控Caspase9基因的上调，从而诱导肝癌细胞的凋亡。
③猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现mTOR基因、AKT基因、PI3K基因表达下调，说明皂荚提取物与索拉非尼均能通过调控mTOR基因、AKT基因、PI3K基因的下调，阻断PI3k／Akt／mTOR信号通路，从而抑制肝癌细胞的生长。
④猪牙皂提取物正丁醇部位与索拉非尼作用于肝癌细胞均出现VEGF基因表达下调，说明皂荚提取物与索拉非尼均能通过调控VEGF基因的下调，从而抑制肿瘤血管生成。
⑤对于6个时间点猪牙皂提取物正丁醇部位和索拉非尼作用于肝癌细胞调控6个基因的表达，两组均在3h和8h时对AKT基因、mTOR基因、PI3K基因、VEGF基因的下调作用最为显著；两组均在8h时对PTEN基因上调作用最为显著；索拉非组在8h对Caspase9基因上调作用最为显著，皂荚提取物在3h、8h对Caspase9基因上调作用均较为显著。
⑥皂荚提取物与索拉非尼对肝癌细胞中6个基因的调控较为相似，比较皂荚提取物组与索拉非尼组，除在25h，索拉非尼对PTEN上调要强于猪牙皂；在3h/8h/25h，猪牙皂对Caspase9上调要明显强于索拉非尼；在25h/48h，索拉非尼对mTOR下调要强于猪牙皂；在48h，猪牙皂对PI3K下调要强于索拉非尼。4个基因其余时间段和VEGF、AKT的调控均较为相同。
1.Objectives
This issue through theoretical and experimental study to clarify n-butanolposition and ethanol position of Gleditsiae extract can inhibit human hepatomaSMMC-7721cells results, while the expression of PI3K/AKT signalingpathways from molecular level, protein level of is one of the mechanism ofanti-hepatoma.
2.Methods
The first part is theoretical study
(1)TCM can prevent and treat the primary liver cance, it also relieveclinical symptoms, decrease postoperative symptoms and recurrence, enhanceimmunity, improve the quality of survival and the forward curative effect.
(2)The treatment of primary liver cancer through Chinese medicine canstarting from can start from the phlegm and blood stasis, phlegm medicineGleditsiae has anti-hepatoma research value.
The second part is the experimental study
(1)According to the human clinical dose (Gleditsiae crude drug1.5g.70kg.d-1), human and rat equivalent dose multiples doubled to seventimes as the experimental low-dose, according to1:2:4,the40Male Wistar ratswere randomly divided into four groups, namely: control (blank serum) group,n-butanol low dose (0.15g.kg.d-1) group, middle dose (0.3g.kg.d-1) Groupsand high-dose (0.6g.kg.d-1) groups,each group is10rats. Intragastrically three days, extract blood from heart,then combine with serum from the samegroups. Using the MTT reduction method (MTT assay) Gleditsiae n-butanolextract containing serum on human hepatoma SMMC-7721cells inhibitionrate.
(2)According to the human clinical dosage (quantity of crude drugsGleditsiae is1.5g.70kg.d-1), human and rat multiple dose equivalent, doubledto7times as the low dose experiments. according to1:2:4, the220MaleWistar rats were randomly divided into22groups: control (blank serum)Group1, containing serum on ethanol site at30%,60%,95%,30%+60%,60%+95%,30%+95%,30%+60%+95%is divided into seven levels, theneach concentration was divided into low, medium and high doses, so totalgroup of experimental is21groups. each group has10rats. Intragastricallythree days, extract blood from heart, combined with serum from the samegroup. Using the MTT assay Gleditsiae parts ethanol extract containing serumon human hepatoma SMMC-7721cells inhibition rate.
(3)The experiment was divided into three groups: control group (humanhepatoma cells SMMC-7721, did not add any medication), Gleditsiae group(the second experiment Gleditsiae n-butanol extract dose group added at aconcentration of10%human hepatoma cell line SMMC-7721), sorafenibgroup (the4μmol/l of sorafenib adding human hepatoma cell SMMC-7721).Western blot analysis using real-time quantitative PCR were used to detecteach group0h,0.5h,3h,8h,25h,48h reaction time points VEGF, PTEN, PI3K,AKT, mTOR, Caspase9.
3. Statistical analysis
3.1Inhibition rate in each dose group of SMMC-721hepatocellularcarcinoma cells can be expressed by mean±standard deviation (±SD);singlefactor analysis of variance was used for comparison of inhibition rate groups;If the difference was statistically significant, the comparisons of each groups difference was useing Bonferrni method；so the data using SPSS18.0statisticalanalysis software for processing; take P <0.05as significance level in singlefactor analysis of variance; pairwise comparison test level when0.05/compare times.
3.2Inhibition rate in each dose group of SMMC-721hepatocellularcarcinoma cells using SPSS18.0analysis. Observation indexes using x士s toexpress index average level and variability; Factorial design analysis ofvariance is to test the main effects and interaction effects of differentconcentrations of ethanol and different doses of Gleditsia sinensis; whenthere was an interaction between the two, it is wise to carry out a furtherseparate effects analysis. fixed ethanol concentration at a certain level,thencompare the different dose of Fructus Gleditsiae.At the same time, fixedFructus Gleditsiae at a certain level,then compare the different concentrationsof ethano. P <0.05was considered statistically significant.
3.3All data were input into the computer to establish a database, usingSAS9.0to analysis.using士s to express index average level and variability.compared Sola Fini and Fructus Gleditsiae at the same time on hepatoma cellsAKT, Caspase9, mTOR, PI3K, PTEN, VEGF six gene expression usingrepeated measures analysis of variance; compared Sola Fini and FructusGleditsiae in the different time on six gene expression in hepatoma cells. P<0.05was considered statistically significant.
4.Results
(1) Between the low-dose group and the control group, the difference (P<0.05) was statistically significant. compared with the low dose group, themiddle dose group (P>0.05) was not statistically significance. compared withthe middle dose group,the control group and high Dose group had astatistically significant.At the same time,there was no difference between thecontrol group and High-dose group.
(2) Gleditsiae parts ethanol extract serum containing60%ethanolconcentration of low-dose group,30%+60%concentration of high doses ofethanol,30%+60%+95%concentration ethanol doses were compared withthe control group, there were no statistical Learn the difference; comparedwith the control group,other various concentrations and each dose group werestatistically significant. Gleditsiae parts of different concentrations of ethanolextract were significantly different respective, there are significant differencesamoung different doses, different concentrations were significantly differentbetween different doses.
(3) By comparing the blank group, the sola fini and Gleditsiae group sixgene expression in liver cells, we can see:
①PTEN expression: Western blot analysis in the sorafenib group andthe control group, the control group and the group were in the Gleditsiae0hcomparison, no significant difference (P>0.05) PTEN expression sorafenibgroup,0.5h/3h/8h/25h comparison with the control group PTEN expressionincreased (P <0.01),48h comparison showed no significant change (P>0.05);Gleditsiae group,0.5h/3h/8h PTEN expression in comparison with the controlgroup was significantly increased (P<0.01),25h PTEN expression incomparison with the control group was significantly increased (P <0.05),48hcomparison showed no significant change (P>0.05). Group compared withsorafenib, Gleditsiae group showed no significant difference (P>0.05) onPTEN in0h/0.5h/3h/8h upregulation. In25h, Gleditsiae upregulate theexpression of PTEN is lower than the sorafenib group (P <0.01); at48h, theexpression of PTEN in both groups had no change (P>0.05). PCR detected inthe two groups of PTEN in0h/0.5h/3h/8h/25h/48h of amplification werecompared by statistical analysis,25h,48h comparison has significantdifference (P <0.05),0h/0.5h/3h/8h no significant difference (P>0.05); thesorafenib group, PTEN amplification of multiple0.5h/3h/8h/25h/48h compared with0h respectively, in addition to the differences in0h and48h nostatistically significant (P>0.05) outside,0.5h/3h/8h/25h of amplification and0h were significant differences (P<0.01). Gleditsiae group, PTENamplification multiple0.5h/3h/8h/25h/48h respectively compared with0h,there were significant differences (P <0.01). In addition, sorafenib group ateach time point of amplification of PTEN pairwise comparison,0.5h and25hshowed no significant difference (P>0.05), pairwise comparisons weresignificant differences in the other time points (P <0.01); Gleditsiae group,PTEN amplification of multiple0.5h/3h/8h/25h/48h pairwise comparison alsoshowed significant differences (P <0.01).
②Caspase9of expression: western blot detection of the control group,the five time points and0h 's expression did not change Caspase9(P>0.05);the sorafenib group and the control group, the control group and the groupwere in the Gleditsiae compare0h, Caspase9showed no difference (P>0.05),sorafenib group,3h/8h Caspase9comparison with the control group wassignificantly increased (P<0.01),0.5h/25h Caspase9comparison with thecontrol group was significantly increased (P <0.05),48h comparison showedno significant change (P>0.05); Gleditsiae group was significantly increased0.5h/3h/8h/25h Caspase9comparison with the control group (P <0.01),48hcomparison showed no significant change (P>0.05). Group compared withsorafenib, Gleditsiae set in0h/0.5h/25h/48h upregulated on Caspase9's nosignificant difference (P>0.05). In3h/8h, Gleditsiae upregulate the expressionof Caspase9higher than the sorafenib group (P <0.01). PCR detection in thesorafenib group, Gleditsiae group were amplified in0h/0.5h/3h/8h/25h/48hmultiples of Caspase9compare by statistical analysis,3h,8h There was asignificant difference (P <0.01),0h/0.5h/25h/48h no significant difference (P>0.05); the sorafenib group and Gleditsiae,0.5h/3h/8h/25h/48h the Caspase9multiples were amplified compared with0h, with both groups except in48h 0h no significant difference (P>0.05) outside,0.5h/3h/8h/25h of amplificationand0h were significant differences (P <0.01). Additionally, respectively, in thetwo groups at each time point Caspase9amplification of multiple pairwisecomparison, sorafenib group0.5h and25h showed no significant difference(P>0.05), pairwise comparisons were other time points significant differences(P<0.01); Gleditsiae set in0.5h and25h,3h and8h showed no significantdifference (P>0.05), pairwise comparisons of other time points weresignificant differences (P <0.01).
③mTOR expression of: western blot detection in the sorafenib groupand the control group, the control group and the group were in the Gleditsiae0h comparison, no significant difference (P>0.05) mTOR expression sorafenibgroup,0.5h/48h comparison with the control group mTOR expressiondecreased (P<0.05),3h/8h/25h mTOR expression comparison with the controlgroup significantly reduced (P<0.01); Gleditsiae group,3h/8h mTORcomparison with the control group was significantly reduced (P<0.01),0.5h/25h/48h comparison with the control group mTOR expression wasdecreased (P<0.05). Group compared with sorafenib, Gleditsiae set in0h/0.5h/3h/8h/25h on down the expression of mTOR was no significantdifference (P>0.05). In48h, Gleditsiae to downregulate the expression ofmTOR is weaker than the sorafenib group (P <0.01). PCR detection in thesorafenib group, Gleditsiae group were amplified in0h/0.5h/3h/8h/25h/48h ofmTOR multiple comparison, through statistical analysis,48h there was asignificant difference (P <0.01),0h/0.5h/3h/8h/25h no significant difference(P>0.05), the sorafenib group and Gleditsiae, in0.5h/3h/8h/25h/48h multiplesof mTOR were amplified in the group with0h, there were significantdifferences (P <0.01). In addition, at each time point mTOR amplify multiplepairwise comparison, the two groups were also in0.5h and25h,3h and48hshowed no significant difference (P>0.05), the other time points were pairwise comparison significant differences (P <0.01).
④PI3K expression of: western blot detection of the control group, thefive time points and0h of PI3K expression did not change (P>0.05); thesorafenib group and the control group, the control group and the group werein the Gleditsiae compare0h, PI3K expression was no difference (P>0.05),sorafenib group,3h/8h PI3K expression comparison with the control groupsignificantly decreased (P <0.01),0.5h comparison with the control groupdecreased expression of PI3K (P <0.05),25h/48h comparison with the controlgroup showed no significant change (P>0.05); Gleditsiae group,3h/8h/25h/48h PI3K expression comparison with the control groupsignificantly decreased (P <0.01),0.5h comparison showed no significantchange (P>0.05). The Gleditsiae group compared with sorafenib, Gleditsiaeset in0h/0.5h/3h/8h for regulating the expression of PI3K is similar, and nosignificant difference in3h/8h expression (P>0.05) on the reduction ofPI3K. In25h/48h, Gleditsiae to downregulate the expression of PI3K isweaker than the sorafenib group (P<0.05). PCR detection in the sorafenibgroup, Gleditsiae groups were0h/0.5h/3h/8h/25h/48h of PI3K amplificationmultiples compared through statistical analysis,25h,48h comparison hassignificant difference (P<0.01),0h/0.5h/3h/8h no significant difference (P>0.05); Gleditsiae sorafenib group and the group of PI3K amplification in0.5h/3h/8h/25h/48h compared with0h within multiple groups were twogroups were significantly different (P <0.01). In addition, at each time pointPI3K amplification of multiple pairwise comparison, sorafenib group0.5hand25h,3h and8h showed no significant difference (P>0.05), pairwisecomparisons were other time points significant differences (P<0.01);Gleditsiae group,3h was no significant difference (P>0.05), pairwisecomparisons of other time points were significant differences (P <0.01).
⑤expression of AKT: western blot detection in the sorafenib group and the control group, the control group and the group were in the Gleditsiae0hcomparison, no significant difference (P>0.05) AKT expression sorafenibgroup,0.5h/significantly reduce3h/8h/25h/48h comparison with the controlgroup AKT expression (P <0.01); Gleditsiae group,0.5h/3h/8h/25h/48h AKTexpression comparison with the control group also decreased significantly (P<0.01). Group compared with sorafenib, Gleditsiae set for regulating theexpression of AKT was no significant difference (P>0.05) in0h/0.5h/3h/8h/25h/48h. PCR detection in the sorafenib group, Gleditsiaegroup were amplified in0h/0.5h/3h/8h/25h/48h of AKT compare multipleanalyzes were not statistically different (P>0.05) by statistics, sorafenib group,AKT amplification of multiple0.5h/3h/8h/25h/48h compared with0hrespectively, except in0h and48h was no significant difference (P>0.05)outside,0.5h/3h/8h/25h and0h amplification multiples are significantdifferences (P<0.01). The Gleditsiae group,0.5h/3h/8h/25h/48h ofamplification of AKT respectively compared with0h, there were significantdifferences (P <0.01). In addition, at each time point AKT amplification ofmultiple pairwise comparison, both groups0.5h and25h,3h and8h showedno significant difference (P>0.05), pairwise comparison of the other timepoints were significantly difference (P <0.01).
⑥VEGF expression is: western blot detection of the control group, theexpression of five time points0h both VEGF and no change (P>0.05); thesorafenib group and the control group, the control group with Gleditsiaegroup were conducted at0h comparison, VEGF expression was no difference(P>0.05), described three groups0h no significant difference in the expressionof VEGF sorafenib group,0.5h/3h/8h/25h/48h VEGF expression wassignificantly reduced when compared with0h (P <0.01); Gleditsiae group,0.5h/3h/8h/25h/48h0h comparison with the same expression of VEGF wassignificantly reduced (P <0.01). And the Gleditsiae sorafenib group compared with Gleditsiae set in0h/0.5h/3h/8h/25h/48h on VEGF expression andregulation were not significantly different (P>0.05). PCR detection in thesorafenib group, Gleditsiae group were amplified in0h/0.5h/3h/8h/25h/48hmultiples of VEGF were compared by statistical analysis, the two groups atsix time points showed no statistically significant difference (P>0.05),Gleditsiae sorafenib group and the group,0.5h/3h/8h/25h/48h ofamplification of VEGF compared with0h respectively, with both groupsexcept in0h48h the difference was not statistically significant (P>0.05)outside,0.5h/3h/8h/25h of amplification and0h were significant differences(P <0.01). In addition, at each time point of amplification VEGF pairwisecomparison, sorafenib group and8h3h showed no significant difference (P>0.05), statistically significant pairwise comparison are other time points (P <0.05), while the pig teeth soap0.5h and25h,3h and8h showed no significantdifference (P>0.05), pairwise comparisons were significant differences (P<0.01) the other time points.
Conclusions
(1) Gleditsiae n-butanol extract containing serum low-dose group, middledose group on human hepatoma SMMC-7721cells were inhibitory effect ofhigh-dose group had no inhibitory effect.
(2) Gleditsiae parts in addition to the concentration of60%ethanolextract ethanol low dose group, high dose of30%+60%concentrationethanol,30%+60%+95%concentration ethanol dose group, other groups ofdifferent concentrations and high, medium and low dose group can inhibithuman liver cancer cell SMMC-7721; The concentration between group, andits high, there is also a difference between middle and low dose group.
(3) Comparing to the control group, sorafenib group and fructusgleditsiae extract soap HCC cells affect the expression of genes for the sixgroups, we can see that
①gleditsiae extractparts of n-butanol and Sorafenib HCC cells werefound in the PTEN gene upregulation, indicating acacia extract with sorafenibcould increase by regulating PTEN gene, thereby inhibiting tumorangiogenesis, inhibit hepatoma cell proliferation.
②gleditsiae extractparts of n-butanol and the role of sorafenib in HCCcells were found Caspase9upregulated genes, indicating acacia extract withsorafenib can increase Caspase9by regulating genes, thus inducing apoptosisof hepatoma cells.
③gleditsiae extractparts of n-butanol and Sorafenib HCC cells werefound in the mTOR gene, AKT gene, PI3K genes downregulated, indicatingacacia extract with sorafenib can by regulating mTOR gene, AKT gene, genedown-regulated PI3K, blocking PI3k/Akt/mTOR signaling pathway, therebyinhibiting the growth of hepatoma cells.
④gleditsiae extractparts of n-butanol and the role of sorafenib in HCCcells were downregulated VEGF gene appears, indicating acacia extract withsorafenib could by down regulation of VEGF gene, thereby inhibiting tumorangiogenesis.
⑤For the six time points Gleditsiae and parts of n-butanol extract ofsorafenib in HCC cells regulate the expression of the role of six genes in bothgroups during3h and8h of AKT gene, mTOR gene, PI3K gene, VEGF genedownward effect the most significant; groups were8h when PTEN geneupregulation of the most significant; sorafenib group8h Caspase9geneupregulation most significant acacia extract3h,8h Caspase9geneupregulation were more significant.
⑥acacia extract with Sorafenib HCC cells regulate six groups of genesis similar, relatively acacia extract group and sorafenib, except in25h,sorafenib PTEN raised better than fructus gleditsiae extract soap; in3h/8h/25h, fructus gleditsiae extract soap on Caspase9raised significantly stronger than sorafenib; in25h/48h, sorafenib reduced mTOR better thanfructus gleditsiae extract soap; at48h, fructus gleditsiae extract soap on PI3Kdown better than sorafenib Nigeria.4groups of genes rest period and VEGFgenes, regulatory genes are more AKT same.
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