皂荚提取物对肝癌细胞小鼠TGF-β/Smads信号系统调控的影响
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摘要
目的：全世界平均每年约有100万人死于肝癌。近年来,研究肿瘤细胞中信号转导通路的作用成为阐明肿瘤发生发展机制和寻找有效生物学治疗手段的热点。研究表明TGF-β/Smads信号异常的传导是肿瘤浸润和转移的重要因素。中医认为肿瘤的形成病机很复杂,认为痰浊内阻是肿瘤形成的病机关键。著名医学家朱丹溪在他的主要著作都列有痰门,探讨其理法方药,尤其在肿瘤的发病因素的认识上提出与痰有关。皂荚是具有“祛痰化湿、散结消肿”功效的抗肿瘤的常用中药。我们希望通过研究皂荚提取物(乙酸乙酯提取)对小鼠肝癌细胞TGF-β/Smads信号系统调控的影响,为皂荚提取物(乙酸乙酯提取)是否具有抗肝癌的作用提供参考。
方法：
第一部分：理论探讨
1从中医理论上阐明痰和肿瘤的关系。强调痰、毒和瘀相互影响、相互转化、相互胶着是肿瘤发生和发展的关键。
2提出从“痰”论治肝癌的可行性。并阐明化痰药“皂荚”具有抗癌作用的前期研究依据。
第二部分：皂荚的文献研究
从皂荚的一般特性研究；皂荚的历史研究探讨；含有皂荚的方剂研究；皂荚的药理学研究和皂荚的临床应用等方面阐明皂荚的特点和研究价值。
第三部分：实验研究
我们采用清洁级的昆明种小鼠60只。通过随机方法(随机数字表法)将实验动物分为6组,实验分为阴性对照组(吐温80溶剂)；皂荚提取物(乙酸乙酯提取)3个浓度组(26.0mg/d、13.0mg/d、7.8mg/d);阳性对照组(金龙胶囊组)；空白对照组(不造模组)。每组10只小鼠。造模采用小鼠右腋皮下接种H22肝癌细胞悬液的方法。造模后第2天到第16天,按实验分组和要求(每只灌胃量为0.4ml,已经配好浓度)进行灌胃。造模后第17天处死小鼠,收集标本进行各项检查。实验一：观察各组小鼠大体情况；各组小鼠死亡率；各组小鼠剥瘤称重,计算各组肿瘤抑制率和病理学观察。实验二：检测各组小鼠血清中的γ-谷氨酰转移酶(GGT),碱性磷酸酶(ALP),总蛋白(TP),白蛋白(ALB)和甲胎蛋白(AFP)的浓度。实验三：实时荧光定量的方法检测各组小鼠肝脏组织中的Smad4mRNA和Smad7mRNA的表达。实验四：免疫组化的方法检测Smad4和Smad7蛋白质的表达。
结果：
实验一：皂荚提取物对接种H22肝癌细胞荷瘤小鼠一般生物学特性和病理学的观察
1鼠大体观察：空白对照组小鼠活动正常,精神状态很好,毛发正常,并且有光泽,饮食正常,大小便正常。皂荚提取物(高剂量组),皂荚提取物(中剂量组),皂荚提取物(低剂量组)和阳性对照组(金龙胶囊组),各组小鼠毛发脱落、有稀疏感、光泽度欠佳,精神不振,活动明显减少,饮食较空白对照组明显减少,饮水少较空白对照组明显减少不明显,大小便情况较空白对照组少,对外界反应慢。阴性对照组(溶剂土温80)的情况类似。右侧前肢接种H22肝癌细胞的小鼠,接种部位可见大小不等的肿块。有部分小鼠腹腔部位出现腹部胀大,用指尖触诊,有明显波动感,考虑为形成的腹水。
2昆明种小鼠的死亡率达21.67%。
3各组小鼠肿瘤重量比较：皂荚提取物(高)平均肿瘤重量2.87±0.34,皂荚提取物(中)平均肿瘤重量3.68±0.34,皂荚提取物(低)平均肿瘤重量3.64±0.36,阳性对照组平均肿瘤重量2.60±0.36与阴性对照组平均肿瘤重量4.73±0.40比较明显降低,P<0.01；阴性对照组,皂英提取物(中),皂荚提取物(低)与阳性对照组比较明显增高,P<0.01；皂荚提取物(高)与阳性对照组比较肿瘤重量无统计学差异,P>0.05。
各组小鼠肿瘤抑制率：阳性对照组为45%；阴性对照组为0；皂荚提取物高剂量组39%；皂荚提取物中剂量组22%；皂荚提取物低剂量组为23%。
4病理学观察：空白对照组小鼠肝脏的肉眼和显微镜下观察都表现正常,模型组各组小鼠总体上较空白对照组(非模型组)有更多的不正常的表现。
阴性对照组,皂荚提取物中剂量组和皂荚提取物低剂量组病理学观察：肉眼大体观察：小鼠的肝脏体积较空白对照组小鼠明显增大；质地较空白对照组硬度增强；肝脏表面可见多个散在的灰白结节。阴性对照组最多,皂荚提取物中剂量组和皂荚提取物低剂量组比阴性对照组略少。光学显微镜下：肝小叶结构不清晰,肝细胞索排列不整齐。细胞核形态明显异常,肝细胞有部分细胞核增大,或核质比例增大。
阳性对照组,皂荚提取物高剂量组病理学观察：肉眼大体观察：小鼠的肝脏体积较空白对照组小鼠略有增大；质地较空白对照组硬度略有增强,但不及阴性对照组；肝脏表面可以偶见散在的灰白结节。阳性对照组和皂荚提取物高剂量组没有明显差别。光学显微镜下：肝小叶结构欠清晰,肝细胞索排列不整齐。细胞核形态异常不明显。推测皂荚提取物高剂量组能通过对肝癌癌瘤有明显抑制癌细胞生长的作用,从而延缓肝癌细胞造成肝脏组织的进一步损伤,从而起到保护肝脏的目的。
实验二：皂荚提取物对接种H22肝癌细胞荷瘤小鼠肝功能相关指标的影响
小鼠血清GGT水平：阳性对照组平均值23±2 U／L、皂荚提取物组(高)平均值23±2 U／L与阴性对照组平均值38±2U／L比较明显降低,P<0.01；皂荚提取物组(中)平均值27±2U／L,皂荚提取物组(低)平均值37±2U／L与阴性对照组平均值比较降低P<0.05。皂荚提取物组(中)平均值,皂荚提取物组(低)平均值,阴性对照组平均值与阳性对照组平均值比较明显降低,P<0.01。
小鼠血清ALP水平：阳性对照组平均值25±5 IU／L、皂荚提取物组(高)平均值34±6 IU／L、皂荚提取物组(中)平均值56±5 IU／L与阴性对照组平均值90±8 IU／L比较明显降低,P<0.01；皂荚提取物组(低)平均值75±7水平于阴性对照组比较降低P<0.05。皂荚提取物组(高)皂荚提取物组(中),皂荚提取物组(低),阴性对照组ALP水平于阳性对照组比较明显降低,P<0.01。
小鼠血清TP：阳性对照组平均值70±2、皂荚提取物组(高)平均值65±6、皂荚提取物组(中)平均值59±3和皂荚提取物组(低)平均值58±3的TP水平于阴性对照组平均值47±4比较明显增高,P<0.01；。皂荚提取物组(中),皂荚提取物组(低)和阴性对照组的TP水平于阳性对照组比较明显降低,P<0.01。
小鼠血清ALB：阳性对照组平均值30±2、皂荚提取物组(高)平均值29±1的小鼠ALB水平于阴性对照组平均值22±2比较明显增高,P<0.01。皂荚提取物组(中)平均值25±3,皂荚提取物组(低)平均值23±2和阴性对照组的小鼠ALB水平于阳性对照组比较明显降低,P<0.01。
小鼠血清AFP：结果各组小鼠AFP的数值均没有测出。实验三：皂荚提取物对接种H22肝癌细胞荷瘤小鼠TGF-β/Smads信号转导通路smad4 mRNA和smad7 mRNA表达的影响
Smad4mRNA表达：扩增倍数阳性对照组平均值0.566±0.092和皂荚提取物高剂量组平均值0.471±0.069与阴性对照组平均值0.320±0.099比较明显增高,P<0.01；皂荚提取物中剂量组平均值0.363±0.058和皂荚提取物低剂量组平均值0.280±0.067与阳性对照组比较比较明显降低,P<0.01。
Smad7mRNA表达：扩增倍数阳性对照组平均值2.007±0.499、皂荚提取物组(高剂量组)平均值2.230±0.491的小鼠肝脏组织中Smad7mRNA表达水平于阴性对照组平均值3.537±0.401比较明显降低,P<0.01。
皂荚提取物组(中剂量组)平均值3.106±0.410,皂荚提取物组(低剂量组)平均值3.613±0.416和阴性对照组小鼠肝组织中Smad7mRNA表达水平于阳性对照组比较明显增高,P<0.01。
实验四：皂荚提取物对接种H22肝癌细胞荷瘤小鼠TGF-β/Smads信号转导通路smad4和smad7蛋白表达的影响
Smad4蛋白表达：阳性对照组的小鼠肝脏Smad4蛋白表达水平平均值2973.190±248.540与阴性对照组平均值1196.399±837比较明显增高,P<0.01。皂荚提取物组(高剂量组)平均值2295.187±820.119与阴性对照组比较增高,P<0.05。
阴性对照组的小鼠肝脏Smad4蛋白表达水平与阳性对照组比较明显降低,P<0.01。皂荚提取物组(中剂量组)平均值1558.835±302.801和皂荚提取物组(低剂量组)平均值988.614±338.995与阳性对照组比较降低,P<0.05。
Smad7蛋白表达：阳性对照组平均值1454.381±302.405和皂荚提取物组(高剂量组)平均值1494.202±399.553的小鼠肝脏Smad7蛋白表达水平与阴性对照组平均值2239.589±272.892比较明显降低,P<0.01。皂荚提取物组(中剂量组)平均值1496.981±505.121与阴性对照组比较降低,P<0.05。
阴性对照组和皂荚提取物组(低剂量组)平均值2330.056±177.105的小鼠肝脏Smad7蛋白表达水平与阳性对照组比较明显增高,P<0.01。
结论：皂荚提取物(乙酸乙酯提取)的抗肝癌的作用可能有下列机理实现：①改善荷瘤小鼠的生存质量而抑制肿瘤对机体的损伤。②通过抑制接种部位肿瘤体的大小而起到抗癌的作用。③通过保护荷瘤小鼠的肝脏功能而起到保护肝脏的作用④皂荚提取物高剂量组(26.0mg／d)能通过增强Smad4mRNA的表达和下调Smad7mRNA的表达。⑤皂荚提取物高剂量组(26.0mg／d)能通过增强Smad4蛋白的表达和下调Smad7蛋白的表达。综合起到抗癌作用。
Objective:About 100 million people worldwide each year die from liver cancer. In recent years, studies of tumor cell signal transduction pathway into the development of mechanisms and clarify the tumor biology to find an effective treatment for hot spots. The results show that abnormal signal transduction TGF-β/Smads tumor invasion and metastasis is an important factor. TCM pathogenesis of tumor formation is complex and that the phlegm resistance is the key to the pathogenesis of tumor formation. Zhu Dan-famous medical scientist in his major works are listed with sputum door of their hair cut herbs, especially in tumors raised awareness of risk factors related with sputum. Acacia is a "phlegm dampness, Sanjiexiaozhong" anti-tumor efficacy of drugs commonly used. We hope that by studying the acacia extract (ethyl acetate extract) on liver cancer cell signaling system in the regulation of TGF-β/Smads for acacia extract (ethyl acetate extract) it has a reference against liver cancer.
Methods:
PartⅠ:Theory
1 From the Chinese medicine theory to clarify the relationship between sputum and tumor. Stressed sputum, drugs and stasis interaction, mutual transformation, tumorigenesis and mutual stalemate is key to development. 2 raised from the "phlegm" On the feasibility of liver cancer treatment. And set out phlegm medicine "acacia" anti-cancer based on a preliminary study.
PartⅡ:the literature of acacia
From the general properties of acacia; acacia historical study; with the prescription of acacia; acacia pharmacological research and clinical applications such as acacia acacia clarify the characteristics and research value.
PartⅢ:Experimental Study
We use the Kunming mice of clean grade 60. Through the random method (random number table) to experimental animals were divided into 6 groups, were divided into negative control group (Tween 80 solvent); honey locust extract (ethyl acetate extraction) 3 concentrations (26.0mg/d,13.0mg/d,7.8mg/d); positive control group (Jinlong Capsule group); blank control group (not in the model).10 mice in each group. Modeling using the right mouse inoculated subcutaneously with H22 liver cell suspension method. 2 days after modeling, to 16 days, the two experimental groups and requirements (amount of each administered 0.4ml, already with a good concentration) were administered.17 days after modeling, the mice were killed to collect samples of the inspection. Experiment 1: the general situation of mice; mortality of mice in each group; stripping of mice tumor weight, tumor inhibition rate of each group and Pathology. Experiment 2:detection of serum levels ofγ-glutamyl transferase (GGT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB) and alpha-fetoprotein (AFP) concentration. Experiment 3:Real-time fluorescence quantitative method to detect the liver tissue of mice and Smad7mRNA Smad4mRNA expression. Experiment 4:Immunohistochemistry was used to detect the expression of Smad4 and Smad7 protein.
Results:
Experiment 1:acacia extract inoculated mice bearing H22 hepatoma cell biological characteristics of normal and pathological observations
1 observation:control activities in the normal mice, in good spirits, normal hair, and shiny, eating normal, urine normal. Acacia extract (high dose group), acacia extract (middle dose group), acacia extract (low dose) and the positive control group (dragon capsule), hair loss in mice in each group, there are sparse feel, gloss poor, listlessness, decreased activity, diet significantly reduced compared with the control group, less drinking water than the control group decreased obvious, toilet cases less than the control group, the outside world is slow to respond. Negative control (solvent soil temperature of 80) will be similar. Right flank of the mice inoculated with H22 hepatoma cells, the inoculation site lumps of varying sizes. Some parts of mouse peritoneal swollen abdomen, palpation with the fingertips, there is obviously a sense of volatility, consider the formation of ascites.
2 Kunming 21.67% mortality in mice.
3 Comparison of tumor weight of mice in each group:acacia extract (high) the average tumor weight of 2.87±0.34, acacia extract (in) the average tumor weight of 3.68±0.34, acacia extract (lower) the average tumor weight of 3.64±0.36, positive control group The average tumor weight of 2.60±0.36 and the average tumor weight of negative control group decreased significantly compared to 4.73±0.40, P<0.01; the negative control group, acacia extract (middle), acacia extract (low) and the positive control group was significantly higher, P<0.01; acacia extract (high) and the positive control group no significant difference in tumor weight, P>0.05.
The mice tumor inhibitory rate:45% positive control; negative control group was 0; acacia extract high-dose group 39%; acacia extracts dose group 22%; acacia extract low-dose group was 23%.
4 Pathological observation:the control group's eye and liver under the microscope all appear normal, model group mice in each group overall than the control group (non-model group) have more abnormal performance.
Negative control group, acacia extracts dose group and low dose of acacia extract pathological observation:visual general observation:the liver volume than the control group mice increased significantly; texture than the control group increased hardness; liver surface shows multiple nodules scattered in the gray. The most negative group, acacia extracts dose group and low dose group of acacia extract the control group slightly less than the negative. Optical microscope:hepatic lobules was not clear, liver cells arranged in irregular claims. Obviously abnormal nuclear shape, some nuclei of liver cells increases, or increases the proportion of nuclear transfer.
Positive control group, high dose of acacia extract pathological observation:general observation of the naked eye:the liver volume than the control group mice increased slightly; texture and hardness than the control group slightly increased, but less than the negative control group; liver surface can be scattered in the gray nodules occasionally. Positive control group and the acacia extract high-dose group was not significantly different. Optical microscope:hepatic lobules unclear, the liver cells arranged in irregular claims. Abnormal nuclear morphology was not obvious. Speculated that the high dose group of acacia extract through the inhibition of liver cancer tumors are significant growth of cancer cells, liver cells and thus delay further liver damage caused to serve the purpose of protecting the liver.
Experiment 2:inoculation of acacia extract on mice bearing H22 liver cancer cells related to indicators of liver function
levels of serum GGT:the positive control group average of 23±2 U/L, acacia extract group (high) mean 23±2 U/L and the negative control group averaged 38±2U/L compared significantly lower, P<0.01; acacia extract group (middle) mean 27±2U/L, acacia extract group (low) average of 37±2U/L compared with the negative control group average lower P<0.05. Acacia extract group (middle), on average, acacia extract group (low), on average, the average negative control group compared with the positive control group mean decreased, P<0.01.
Serum ALP level:the positive control group mean 25±5 IU/ L, acacia extract group (high) mean 34±6 IU/L, acacia extract group (middle), average 56±5 IU/L and negative control group, mean 90±8 IU/L were significantly decreased, P<0.01; acacia extract group (low) average of 75+7 levels lower in the negative control group P<0.05. Acacia extract group (high), acacia extract group (middle), acacia extract group (low), ALP level in negative control group compared with the control group decreased significantly, P<0.01.
Serum TP:positive control group average of 70±2, acacia extract group (high) mean 65±6, acacia extract group (middle), mean 59±3 and acacia extract group (low) average of 58±3 of the TP level in the negative control group average of 47±4 were significantly increased, P<0.01;. Acacia extract group (middle), acacia extract group (low) and the negative control group TP level in the positive control group decreased significantly, P<0.01.
Serum ALB:positive control group mean 30±2, acacia extract groups (high) mean 29±1 in the mouse ALB level in the negative control group average of 22±2 were significantly increased, P <0.01. Acacia extract group (middle), mean 25±3, acacia extract group (low) average of 23±2 and the negative control group of mice ALB level in the positive control group decreased significantly, P<0.01.
Serum AFP:Results mice AFP values were not measured.
Experiment 3:acacia extract inoculated mice bearing H22 hepatoma cells TGF-β/Smads signal transduction pathway in smad4 mRNA and smad7 mRNA expression
Smad4mRNA expression:positive amplification control group mean 0.566±0.092 and acacia extract high-dose group averaged 0.471±0.069 and the average negative control group was significantly higher compared 0.320±0.099, P<0.01; acacia extracts dose group was Value of 0.363±0.058 and acacia extract low-dose group averaged 0.280±0.067 compared with positive control group decreased significantly, P<0.01.
Smad7mRNA expression:positive amplification control group mean 2.007±0.499, acacia extract group (high dose) mean 2.230±0.491 in the mouse liver tissue Smad7mRNA expression level in the negative control group average of 3.537±0.401 significantly lower compared, P<0.01.
Acacia extract group (middle dose group) mean 3.106±0.410, acacia extract group (low dose group) average of 3.613±0.416 and the negative control group of mice the expression of liver tissue Smad7mRNA compared with the control group significantly increased, P<0.01.
Experiment 4:acacia extract inoculated mice bearing H22 liver cancer cell signal transduction pathway TGF-β/Smads smad4 and smad7 protein expression
Smad4 protein expression:positive control group of mice liver protein levels of Smad4 average 2973.190±248.540 and the average of the negative control group was significantly higher compared 1196.399±837, P<0.01. Acacia extract group (high dose) average 2295.187±820.119 increased compared with the negative control group, P<0.05.
Negative control group of mice liver Smad4 protein levels compared with the positive control group decreased significantly, P<0.01. Acacia extract group (middle dose group) average 1558.835±302.801 and acacia extract group (low dose) average 988.614±338.995 decreased compared with the positive control group, P <0.05.
Smad7 protein expression:positive control group average of 1454.381±302.405 and acacia extract group (high dose group) average 1494.202±399.553 Smad7 protein expression in mouse liver, the level of the average negative control group was decreased significantly compared 2239.589±272.892, P< 0.01. Acacia extract group (middle dose group) average 1496.981±505.121 decreased compared with the negative control group, P<0.05. Negative control group and acacia extract group (low dose) average 2330.056±177.105 in the mouse liver Smad7 protein levels compared with the positive control group was significantly higher, P<0.01.
Conclusion:The acacia extract (ethyl acetate extract) of anti-liver cancer cells may have mechanisms to achieve the following:①to improve the quality of life of mice and inhibit the tumor on the body damage.②inoculation site by inhibiting the size of the tumor play a role in cancer.③tumor-bearing mice by protecting liver function and play a role in protecting the liver④acacia extract the high dose group (26.0mg/d) through enhanced expression and reduced Smad7mRNA Smad4mRNA expression.⑤acacia extract high-dose group (26.0mg/d) through increased Smad4 protein expression and reduced Smad7 protein. Play a role in comprehensive cancer.
方法：
第一部分：理论探讨
1从中医理论上阐明痰和肿瘤的关系。强调痰、毒和瘀相互影响、相互转化、相互胶着是肿瘤发生和发展的关键。
2提出从“痰”论治肝癌的可行性。并阐明化痰药“皂荚”具有抗癌作用的前期研究依据。
第二部分：皂荚的文献研究
从皂荚的一般特性研究；皂荚的历史研究探讨；含有皂荚的方剂研究；皂荚的药理学研究和皂荚的临床应用等方面阐明皂荚的特点和研究价值。
第三部分：实验研究
我们采用清洁级的昆明种小鼠60只。通过随机方法(随机数字表法)将实验动物分为6组,实验分为阴性对照组(吐温80溶剂)；皂荚提取物(乙酸乙酯提取)3个浓度组(26.0mg/d、13.0mg/d、7.8mg/d);阳性对照组(金龙胶囊组)；空白对照组(不造模组)。每组10只小鼠。造模采用小鼠右腋皮下接种H22肝癌细胞悬液的方法。造模后第2天到第16天,按实验分组和要求(每只灌胃量为0.4ml,已经配好浓度)进行灌胃。造模后第17天处死小鼠,收集标本进行各项检查。实验一：观察各组小鼠大体情况；各组小鼠死亡率；各组小鼠剥瘤称重,计算各组肿瘤抑制率和病理学观察。实验二：检测各组小鼠血清中的γ-谷氨酰转移酶(GGT),碱性磷酸酶(ALP),总蛋白(TP),白蛋白(ALB)和甲胎蛋白(AFP)的浓度。实验三：实时荧光定量的方法检测各组小鼠肝脏组织中的Smad4mRNA和Smad7mRNA的表达。实验四：免疫组化的方法检测Smad4和Smad7蛋白质的表达。
结果：
实验一：皂荚提取物对接种H22肝癌细胞荷瘤小鼠一般生物学特性和病理学的观察
1鼠大体观察：空白对照组小鼠活动正常,精神状态很好,毛发正常,并且有光泽,饮食正常,大小便正常。皂荚提取物(高剂量组),皂荚提取物(中剂量组),皂荚提取物(低剂量组)和阳性对照组(金龙胶囊组),各组小鼠毛发脱落、有稀疏感、光泽度欠佳,精神不振,活动明显减少,饮食较空白对照组明显减少,饮水少较空白对照组明显减少不明显,大小便情况较空白对照组少,对外界反应慢。阴性对照组(溶剂土温80)的情况类似。右侧前肢接种H22肝癌细胞的小鼠,接种部位可见大小不等的肿块。有部分小鼠腹腔部位出现腹部胀大,用指尖触诊,有明显波动感,考虑为形成的腹水。
2昆明种小鼠的死亡率达21.67%。
3各组小鼠肿瘤重量比较：皂荚提取物(高)平均肿瘤重量2.87±0.34,皂荚提取物(中)平均肿瘤重量3.68±0.34,皂荚提取物(低)平均肿瘤重量3.64±0.36,阳性对照组平均肿瘤重量2.60±0.36与阴性对照组平均肿瘤重量4.73±0.40比较明显降低,P<0.01；阴性对照组,皂英提取物(中),皂荚提取物(低)与阳性对照组比较明显增高,P<0.01；皂荚提取物(高)与阳性对照组比较肿瘤重量无统计学差异,P>0.05。
各组小鼠肿瘤抑制率：阳性对照组为45%；阴性对照组为0；皂荚提取物高剂量组39%；皂荚提取物中剂量组22%；皂荚提取物低剂量组为23%。
4病理学观察：空白对照组小鼠肝脏的肉眼和显微镜下观察都表现正常,模型组各组小鼠总体上较空白对照组(非模型组)有更多的不正常的表现。
阴性对照组,皂荚提取物中剂量组和皂荚提取物低剂量组病理学观察：肉眼大体观察：小鼠的肝脏体积较空白对照组小鼠明显增大；质地较空白对照组硬度增强；肝脏表面可见多个散在的灰白结节。阴性对照组最多,皂荚提取物中剂量组和皂荚提取物低剂量组比阴性对照组略少。光学显微镜下：肝小叶结构不清晰,肝细胞索排列不整齐。细胞核形态明显异常,肝细胞有部分细胞核增大,或核质比例增大。
阳性对照组,皂荚提取物高剂量组病理学观察：肉眼大体观察：小鼠的肝脏体积较空白对照组小鼠略有增大；质地较空白对照组硬度略有增强,但不及阴性对照组；肝脏表面可以偶见散在的灰白结节。阳性对照组和皂荚提取物高剂量组没有明显差别。光学显微镜下：肝小叶结构欠清晰,肝细胞索排列不整齐。细胞核形态异常不明显。推测皂荚提取物高剂量组能通过对肝癌癌瘤有明显抑制癌细胞生长的作用,从而延缓肝癌细胞造成肝脏组织的进一步损伤,从而起到保护肝脏的目的。
实验二：皂荚提取物对接种H22肝癌细胞荷瘤小鼠肝功能相关指标的影响
小鼠血清GGT水平：阳性对照组平均值23±2 U／L、皂荚提取物组(高)平均值23±2 U／L与阴性对照组平均值38±2U／L比较明显降低,P<0.01；皂荚提取物组(中)平均值27±2U／L,皂荚提取物组(低)平均值37±2U／L与阴性对照组平均值比较降低P<0.05。皂荚提取物组(中)平均值,皂荚提取物组(低)平均值,阴性对照组平均值与阳性对照组平均值比较明显降低,P<0.01。
小鼠血清ALP水平：阳性对照组平均值25±5 IU／L、皂荚提取物组(高)平均值34±6 IU／L、皂荚提取物组(中)平均值56±5 IU／L与阴性对照组平均值90±8 IU／L比较明显降低,P<0.01；皂荚提取物组(低)平均值75±7水平于阴性对照组比较降低P<0.05。皂荚提取物组(高)皂荚提取物组(中),皂荚提取物组(低),阴性对照组ALP水平于阳性对照组比较明显降低,P<0.01。
小鼠血清TP：阳性对照组平均值70±2、皂荚提取物组(高)平均值65±6、皂荚提取物组(中)平均值59±3和皂荚提取物组(低)平均值58±3的TP水平于阴性对照组平均值47±4比较明显增高,P<0.01；。皂荚提取物组(中),皂荚提取物组(低)和阴性对照组的TP水平于阳性对照组比较明显降低,P<0.01。
小鼠血清ALB：阳性对照组平均值30±2、皂荚提取物组(高)平均值29±1的小鼠ALB水平于阴性对照组平均值22±2比较明显增高,P<0.01。皂荚提取物组(中)平均值25±3,皂荚提取物组(低)平均值23±2和阴性对照组的小鼠ALB水平于阳性对照组比较明显降低,P<0.01。
小鼠血清AFP：结果各组小鼠AFP的数值均没有测出。实验三：皂荚提取物对接种H22肝癌细胞荷瘤小鼠TGF-β/Smads信号转导通路smad4 mRNA和smad7 mRNA表达的影响
Smad4mRNA表达：扩增倍数阳性对照组平均值0.566±0.092和皂荚提取物高剂量组平均值0.471±0.069与阴性对照组平均值0.320±0.099比较明显增高,P<0.01；皂荚提取物中剂量组平均值0.363±0.058和皂荚提取物低剂量组平均值0.280±0.067与阳性对照组比较比较明显降低,P<0.01。
Smad7mRNA表达：扩增倍数阳性对照组平均值2.007±0.499、皂荚提取物组(高剂量组)平均值2.230±0.491的小鼠肝脏组织中Smad7mRNA表达水平于阴性对照组平均值3.537±0.401比较明显降低,P<0.01。
皂荚提取物组(中剂量组)平均值3.106±0.410,皂荚提取物组(低剂量组)平均值3.613±0.416和阴性对照组小鼠肝组织中Smad7mRNA表达水平于阳性对照组比较明显增高,P<0.01。
实验四：皂荚提取物对接种H22肝癌细胞荷瘤小鼠TGF-β/Smads信号转导通路smad4和smad7蛋白表达的影响
Smad4蛋白表达：阳性对照组的小鼠肝脏Smad4蛋白表达水平平均值2973.190±248.540与阴性对照组平均值1196.399±837比较明显增高,P<0.01。皂荚提取物组(高剂量组)平均值2295.187±820.119与阴性对照组比较增高,P<0.05。
阴性对照组的小鼠肝脏Smad4蛋白表达水平与阳性对照组比较明显降低,P<0.01。皂荚提取物组(中剂量组)平均值1558.835±302.801和皂荚提取物组(低剂量组)平均值988.614±338.995与阳性对照组比较降低,P<0.05。
Smad7蛋白表达：阳性对照组平均值1454.381±302.405和皂荚提取物组(高剂量组)平均值1494.202±399.553的小鼠肝脏Smad7蛋白表达水平与阴性对照组平均值2239.589±272.892比较明显降低,P<0.01。皂荚提取物组(中剂量组)平均值1496.981±505.121与阴性对照组比较降低,P<0.05。
阴性对照组和皂荚提取物组(低剂量组)平均值2330.056±177.105的小鼠肝脏Smad7蛋白表达水平与阳性对照组比较明显增高,P<0.01。
结论：皂荚提取物(乙酸乙酯提取)的抗肝癌的作用可能有下列机理实现：①改善荷瘤小鼠的生存质量而抑制肿瘤对机体的损伤。②通过抑制接种部位肿瘤体的大小而起到抗癌的作用。③通过保护荷瘤小鼠的肝脏功能而起到保护肝脏的作用④皂荚提取物高剂量组(26.0mg／d)能通过增强Smad4mRNA的表达和下调Smad7mRNA的表达。⑤皂荚提取物高剂量组(26.0mg／d)能通过增强Smad4蛋白的表达和下调Smad7蛋白的表达。综合起到抗癌作用。
Objective:About 100 million people worldwide each year die from liver cancer. In recent years, studies of tumor cell signal transduction pathway into the development of mechanisms and clarify the tumor biology to find an effective treatment for hot spots. The results show that abnormal signal transduction TGF-β/Smads tumor invasion and metastasis is an important factor. TCM pathogenesis of tumor formation is complex and that the phlegm resistance is the key to the pathogenesis of tumor formation. Zhu Dan-famous medical scientist in his major works are listed with sputum door of their hair cut herbs, especially in tumors raised awareness of risk factors related with sputum. Acacia is a "phlegm dampness, Sanjiexiaozhong" anti-tumor efficacy of drugs commonly used. We hope that by studying the acacia extract (ethyl acetate extract) on liver cancer cell signaling system in the regulation of TGF-β/Smads for acacia extract (ethyl acetate extract) it has a reference against liver cancer.
Methods:
PartⅠ:Theory
1 From the Chinese medicine theory to clarify the relationship between sputum and tumor. Stressed sputum, drugs and stasis interaction, mutual transformation, tumorigenesis and mutual stalemate is key to development. 2 raised from the "phlegm" On the feasibility of liver cancer treatment. And set out phlegm medicine "acacia" anti-cancer based on a preliminary study.
PartⅡ:the literature of acacia
From the general properties of acacia; acacia historical study; with the prescription of acacia; acacia pharmacological research and clinical applications such as acacia acacia clarify the characteristics and research value.
PartⅢ:Experimental Study
We use the Kunming mice of clean grade 60. Through the random method (random number table) to experimental animals were divided into 6 groups, were divided into negative control group (Tween 80 solvent); honey locust extract (ethyl acetate extraction) 3 concentrations (26.0mg/d,13.0mg/d,7.8mg/d); positive control group (Jinlong Capsule group); blank control group (not in the model).10 mice in each group. Modeling using the right mouse inoculated subcutaneously with H22 liver cell suspension method. 2 days after modeling, to 16 days, the two experimental groups and requirements (amount of each administered 0.4ml, already with a good concentration) were administered.17 days after modeling, the mice were killed to collect samples of the inspection. Experiment 1: the general situation of mice; mortality of mice in each group; stripping of mice tumor weight, tumor inhibition rate of each group and Pathology. Experiment 2:detection of serum levels ofγ-glutamyl transferase (GGT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB) and alpha-fetoprotein (AFP) concentration. Experiment 3:Real-time fluorescence quantitative method to detect the liver tissue of mice and Smad7mRNA Smad4mRNA expression. Experiment 4:Immunohistochemistry was used to detect the expression of Smad4 and Smad7 protein.
Results:
Experiment 1:acacia extract inoculated mice bearing H22 hepatoma cell biological characteristics of normal and pathological observations
1 observation:control activities in the normal mice, in good spirits, normal hair, and shiny, eating normal, urine normal. Acacia extract (high dose group), acacia extract (middle dose group), acacia extract (low dose) and the positive control group (dragon capsule), hair loss in mice in each group, there are sparse feel, gloss poor, listlessness, decreased activity, diet significantly reduced compared with the control group, less drinking water than the control group decreased obvious, toilet cases less than the control group, the outside world is slow to respond. Negative control (solvent soil temperature of 80) will be similar. Right flank of the mice inoculated with H22 hepatoma cells, the inoculation site lumps of varying sizes. Some parts of mouse peritoneal swollen abdomen, palpation with the fingertips, there is obviously a sense of volatility, consider the formation of ascites.
2 Kunming 21.67% mortality in mice.
3 Comparison of tumor weight of mice in each group:acacia extract (high) the average tumor weight of 2.87±0.34, acacia extract (in) the average tumor weight of 3.68±0.34, acacia extract (lower) the average tumor weight of 3.64±0.36, positive control group The average tumor weight of 2.60±0.36 and the average tumor weight of negative control group decreased significantly compared to 4.73±0.40, P<0.01; the negative control group, acacia extract (middle), acacia extract (low) and the positive control group was significantly higher, P<0.01; acacia extract (high) and the positive control group no significant difference in tumor weight, P>0.05.
The mice tumor inhibitory rate:45% positive control; negative control group was 0; acacia extract high-dose group 39%; acacia extracts dose group 22%; acacia extract low-dose group was 23%.
4 Pathological observation:the control group's eye and liver under the microscope all appear normal, model group mice in each group overall than the control group (non-model group) have more abnormal performance.
Negative control group, acacia extracts dose group and low dose of acacia extract pathological observation:visual general observation:the liver volume than the control group mice increased significantly; texture than the control group increased hardness; liver surface shows multiple nodules scattered in the gray. The most negative group, acacia extracts dose group and low dose group of acacia extract the control group slightly less than the negative. Optical microscope:hepatic lobules was not clear, liver cells arranged in irregular claims. Obviously abnormal nuclear shape, some nuclei of liver cells increases, or increases the proportion of nuclear transfer.
Positive control group, high dose of acacia extract pathological observation:general observation of the naked eye:the liver volume than the control group mice increased slightly; texture and hardness than the control group slightly increased, but less than the negative control group; liver surface can be scattered in the gray nodules occasionally. Positive control group and the acacia extract high-dose group was not significantly different. Optical microscope:hepatic lobules unclear, the liver cells arranged in irregular claims. Abnormal nuclear morphology was not obvious. Speculated that the high dose group of acacia extract through the inhibition of liver cancer tumors are significant growth of cancer cells, liver cells and thus delay further liver damage caused to serve the purpose of protecting the liver.
Experiment 2:inoculation of acacia extract on mice bearing H22 liver cancer cells related to indicators of liver function
levels of serum GGT:the positive control group average of 23±2 U/L, acacia extract group (high) mean 23±2 U/L and the negative control group averaged 38±2U/L compared significantly lower, P<0.01; acacia extract group (middle) mean 27±2U/L, acacia extract group (low) average of 37±2U/L compared with the negative control group average lower P<0.05. Acacia extract group (middle), on average, acacia extract group (low), on average, the average negative control group compared with the positive control group mean decreased, P<0.01.
Serum ALP level:the positive control group mean 25±5 IU/ L, acacia extract group (high) mean 34±6 IU/L, acacia extract group (middle), average 56±5 IU/L and negative control group, mean 90±8 IU/L were significantly decreased, P<0.01; acacia extract group (low) average of 75+7 levels lower in the negative control group P<0.05. Acacia extract group (high), acacia extract group (middle), acacia extract group (low), ALP level in negative control group compared with the control group decreased significantly, P<0.01.
Serum TP:positive control group average of 70±2, acacia extract group (high) mean 65±6, acacia extract group (middle), mean 59±3 and acacia extract group (low) average of 58±3 of the TP level in the negative control group average of 47±4 were significantly increased, P<0.01;. Acacia extract group (middle), acacia extract group (low) and the negative control group TP level in the positive control group decreased significantly, P<0.01.
Serum ALB:positive control group mean 30±2, acacia extract groups (high) mean 29±1 in the mouse ALB level in the negative control group average of 22±2 were significantly increased, P <0.01. Acacia extract group (middle), mean 25±3, acacia extract group (low) average of 23±2 and the negative control group of mice ALB level in the positive control group decreased significantly, P<0.01.
Serum AFP:Results mice AFP values were not measured.
Experiment 3:acacia extract inoculated mice bearing H22 hepatoma cells TGF-β/Smads signal transduction pathway in smad4 mRNA and smad7 mRNA expression
Smad4mRNA expression:positive amplification control group mean 0.566±0.092 and acacia extract high-dose group averaged 0.471±0.069 and the average negative control group was significantly higher compared 0.320±0.099, P<0.01; acacia extracts dose group was Value of 0.363±0.058 and acacia extract low-dose group averaged 0.280±0.067 compared with positive control group decreased significantly, P<0.01.
Smad7mRNA expression:positive amplification control group mean 2.007±0.499, acacia extract group (high dose) mean 2.230±0.491 in the mouse liver tissue Smad7mRNA expression level in the negative control group average of 3.537±0.401 significantly lower compared, P<0.01.
Acacia extract group (middle dose group) mean 3.106±0.410, acacia extract group (low dose group) average of 3.613±0.416 and the negative control group of mice the expression of liver tissue Smad7mRNA compared with the control group significantly increased, P<0.01.
Experiment 4:acacia extract inoculated mice bearing H22 liver cancer cell signal transduction pathway TGF-β/Smads smad4 and smad7 protein expression
Smad4 protein expression:positive control group of mice liver protein levels of Smad4 average 2973.190±248.540 and the average of the negative control group was significantly higher compared 1196.399±837, P<0.01. Acacia extract group (high dose) average 2295.187±820.119 increased compared with the negative control group, P<0.05.
Negative control group of mice liver Smad4 protein levels compared with the positive control group decreased significantly, P<0.01. Acacia extract group (middle dose group) average 1558.835±302.801 and acacia extract group (low dose) average 988.614±338.995 decreased compared with the positive control group, P <0.05.
Smad7 protein expression:positive control group average of 1454.381±302.405 and acacia extract group (high dose group) average 1494.202±399.553 Smad7 protein expression in mouse liver, the level of the average negative control group was decreased significantly compared 2239.589±272.892, P< 0.01. Acacia extract group (middle dose group) average 1496.981±505.121 decreased compared with the negative control group, P<0.05. Negative control group and acacia extract group (low dose) average 2330.056±177.105 in the mouse liver Smad7 protein levels compared with the positive control group was significantly higher, P<0.01.
Conclusion:The acacia extract (ethyl acetate extract) of anti-liver cancer cells may have mechanisms to achieve the following:①to improve the quality of life of mice and inhibit the tumor on the body damage.②inoculation site by inhibiting the size of the tumor play a role in cancer.③tumor-bearing mice by protecting liver function and play a role in protecting the liver④acacia extract the high dose group (26.0mg/d) through enhanced expression and reduced Smad7mRNA Smad4mRNA expression.⑤acacia extract high-dose group (26.0mg/d) through increased Smad4 protein expression and reduced Smad7 protein. Play a role in comprehensive cancer.
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