摘要
肝细胞癌是严重危害人类健康的主要恶性肿瘤之一,尤其在我国及东南亚和非洲地区更为明显,我国肝癌死亡率位居恶性肿瘤第二位,而广西则是我国肝癌高发地区,肝癌死亡率位居恶性肿瘤的首位,并且呈显著上升态势。肝癌发病急骤,发展迅速,多数患者就诊时已属中、晚期,预后极差。即使肝细胞癌早期采取有效的综合治疗措施,5年生存率也处于50%左右,因此,开展肝癌危险因素及发病机制究、开展三级预防工作尤为重
要。至今公认的HBV感染、黄曲霉毒素B1(AFB1)摄入及居民饮用水源污染被认为是导致肝癌的三大危险因素。AFB1在肝癌高发地区污染相当严重,尽管开展了“防霉去毒”等防癌宣传教育工作,由于种种原因,居民粮食中难免受AFB1不同程度污染,人类在日常生活中很难避免暴露于AFB1之中。因此,探讨具有抗AFB1致癌作用的天然物具有十分重要意义。AFB1致癌机理不是十分清楚,但有研究报道,AFB1在机体内通过肝脏氧化代谢酶系的作用,才能形成具有致癌活性的中间体或形成复合物,使AFB1降低或失去生物活性。这类酶系的代表主要有CYPs和GSTs,而它们与AFB1致肝癌密切相关的主要有CYP3A4和GST-Pi酶。金花茶含有多种氨基酸、茶多酚等活性物质,具有抗氧化、抗血管生成及调节基因表达的作用,其能否作为化学干预剂阻断AFB1致大鼠肝癌发生以及其抗癌作用的机制,是本文研究的主要内容及目标。目前国内外尚未见同类研究报道。
方法
选取Wistar大鼠70只,按体重随机将其分为A、B、C 3组。A组:AFB1组,25只,按照200μg/kg·BW腹腔注AFB1至52W。B组:金花茶组,25只,每天以灌胃的方法饲以30%金花茶浓缩液,腹腔注AFB1剂量同A组。C组:20只,为空白对照组,不给AFB1和金花茶处理。每周称各组动物体重并按照体重计算注射AFB1的量。每10周进行抽血及肝活检,第73周结束实验,处死各组动物,取血、肝及肿瘤组织。选取不同时期肝组织应用荧光分光光度法检测肝组织CYP3A4酶活性及免疫组化法检测GST-Pi、STAT3蛋白的表达情况。
结果
1.大鼠肿瘤发生情况:
AFB1诱发大鼠肝恶性肿瘤发生的最早时间在第52W,第一例肝癌的发生在第55W,均为AFB1组大鼠。在诱癌不同阶段肝组织的损伤程度有明显差异,且三组间肝脏损伤程度亦不一致。金花茶干预组各阶段大鼠肝脏损害均较AFB1组轻,增生性病变发生比AFB1组亦较晚,大鼠平均生存时间及肝癌发生率亦显著低于AFB1组。对照组无上述改变。
第73W实验结束时,恶性肿瘤发生率AFB1组和金花茶组分别为76.5%(13/17)和25.0%(4/16),肝细胞癌发生率AFB1组和金花茶组分别为58.8%(10/17)和18.8%(3/16),与AFB1组相比,恶性肿瘤发生率和HCC发生率显著高于金花茶组(P=0.0053和P=0.0324)。对照组无肿瘤发生。
2.大鼠肝组织CYP3A4酶活性的变化情况
在整个实验过程中,动态观察各组肝组织CYP3A4酶活性,结果显示各组CYP3A4酶活性在23W和53W呈现双波峰变化;AFB1组和金花茶组CYP3A4酶活性在23W升高到顶峰,AFB1组在53W和63W,CYP3A4酶活性高于金花茶组和对照组,差别有统计学意义(P <0.05)。对照组CYP3A4酶活性在整个实验过程中,除33W和63W出现明显降低外,其他各阶段变化不大。
3.大鼠肝组织GST-Pi蛋白的表达情况
GST-Pi阳性染色定位于细胞质中,阳性细胞呈灶状或片状分布。在诱癌过程中,AFB1组和金花茶组GST-Pi蛋白表达随着诱癌时间的延长而逐渐增强,尤其在AFB1组更明显,在33W、53W、73W时GST-Pi的表达率:AFB1组为73%、86.7%、93.3%,金花茶组为66.7%、80.0%、80.0%,前者均相应高于后者。对照组在73W时表达率为10%,其他时段均为阴性表达。同一时段不同组间比较,在第33W至73W,AFB1组及金花茶组显著高于对照组,差别有统计学意义(P<0.05),在第33W至63W,AFB1组高于金花茶组,但无显著性差异,在第73W,AFB1组显著高于金花茶组和对照组,差别有统计学意义(P=0.000)。其余各组之间或同组不同时段之间大鼠GST-Pi蛋白的表达均无显著差异(P>0.05)。
4.大鼠肝组织STAT3蛋白表达情况
STAT3阳性染色定位于细胞质中,阳性细胞呈散在或灶性分布。AFB1组和金花茶组,在诱癌过程中STAT3蛋白表达随诱癌时间的延长而逐渐增强,在33W、53W和73W时STAT3的表达率:AFB1组为20.0%、53.3%、86.7%,金花茶组为13.3%、40.0%、60.0%。同一时段不同组间比较,在上述时段AFB1组及金花茶组高于对照组,差别有统计学意义(P<0.05),在第53W至73W,AFB1组高于金花茶组,但无显著性差异(P > 0.05)。
结论
1.金花茶浓缩液具有抑制AFB1诱发大鼠肝癌的发生率,具有抗AFB1致肝癌作用,可作为潜在的人类肝癌化学预防剂。
2.在AFB1诱癌过程中,金花茶浓缩液能抑制CYP3A4酶的活力,从而减少前致癌物的代谢,降低其致癌性及其化学性肝损伤达到保护肝脏的作用。
3.在AFB1诱癌过程中,金花茶浓缩液能抑制GST-Pi和STAT3蛋白表达,阻止细胞分裂和恶性增殖,延缓肿瘤的发生,可能与启动子CpG岛甲基化和启动STAT3下游基因的活化,并阻断了某些重要抑癌基因的活性有关。
Hepatocellular carcinoma (HCC) is the one of major health care problem of cancer mortality worldwide. Especially, HCC is more obvious in China and southeast Asian and Africa. And it is the second leading cause of cancer death in China, and the mortality of HCC is the first most common cancer in Guangxi province. And that have a significant rise. The patients of hepatocellular carcinoma have the characteristics with the difficult of diagnosis and treatment, illness progress and development are rapid and poor prognosis. But all of Middle-late stage patients with the prognosis were poor. Even the early, the effective treatments methods of HCC were taken, the rates of 5-years survival also have 50%. So that, the works of HCC risk factors and pathogenesis research, and to develop the tertiary prevention had were particularly important.
Yet, we were recognized at now, the three risk factors of HBV infection, aflatoxin (AFB1) intake and people drinking water pollution were considered to be causes HCC. The regions high rates of HCC occurrence were pretty serious by AFB1 pollution. Although launched "mouldproof to poison" education work such as cancer propaganda. For a variety of reasons, the foods were contaminated by high-risk pollution with AFB1 inordinately. The lifes of human in daily were exposured difficult to avoid in the areas of high-risk pollution with AFB1. Therefore, to investigate the significance have been found for prophylaxis and treatment HCC that to find the formation against natural objects or its extract could fight AFB1 induced HCC in these areas. However, the mechanisms of carcinogenic by AFB1-induced were not very clear. But studies have reported, AFB1 within the body have role of oxidative metabolism by the liver enzyme could become biotransformations form active intermediates with carcinogenicity. Because of this epoxy cures with high activity and low stability in the body, and the adduct make AFB1 reduce or lose biological activity. The CYPs and GSTs of drug metabolizing enzymes mainly have CYP3A4 and GST-Pi as well as several subtribes which close-related with drug metabolism that mediated AFB1 metabolic activation and detoxification. The Camellia chrysantha(Hu)Tuyama (CCT) contains many amino acids, tea polyphenol and many kinds of composition. CCT have the role of block cell proliferation through anti-oxidation, anti-angiogenic and regulate gene expression. But so far, the reports have not yet been research about CCT to restrain AFB1causing to hepatocellular carcinoma. This paper is to make it clear that AFB1 cause liver cancer of extracting effective components and analysis by CCT working on AFB1 induced cancer animal models, By immunohistochemical techniques, it
observes the effects with dynamically the expression level of related gene with process of AFB1 causing liver cancer, and to investigate the molecular biology mechanism of the blocking hepatocellular carcinoma occurrence and development. Similar research report has not been seen in domestic and foreign.
Methods
Establish the model wistar Rats in Aflatoxin B1-induced hepatic carcinoma, 70 Wistar rats were divided randomly into A, B and C groups. Groups A was AFB1 group with 25 rats. The specific injection to 53w of experiment and dosage about AFB1:200ug/kg weight. Group B was CCT group with 25 rats, from the experimental in the first week began feeding containing 30% concentrate CCT of the stomach every irrigation experimental end of experiment, AFB1 was injected with dosage and intra peritoneal time same to group A; Group C was control group with 20 rats, in the first week, from the experiment began to end feeding normal feed, not giving AFB1 processing end. Animals were sacrificed at the 73rd-week and liver tissues were collected. The each animal are be weight every week once, and according to the amount of weight calculation AFB1 injections. The each animal of liver biopsies were had performed every ten weeks. Animals were sacrificed at the 73rd-week and serums, hepatic tissues and hepatocarcinoma tissues were collected. Using the hepatic microsomal mixed-function oxidase enzyme system dynamically examined the CYP3A4 activity by quantitative fluorescence spectrophotometry and the expressions of GST-Pi, STAT3 protein were examined by immunohistochemical staining with hepatic tissues different poit-times in hepatocarcinogenesis induced by AFB1 in rats.
Results
1. The datas of rats tumor occurrence,
The earliest time that AFB1 evoked rat liver malignant tumor (fibrosarcoma) formation is at 52W, and the first case occurrence of hepatocellular carcinoma is AFB1 group rats in the 55W.They are all AFB1 group rats. Microscopically dynamic observation biopsy liver histopathology shows the rat liver AFB1 induced cancers process can appear different degrees of liver cell degeneration,and all so it have Significant differences and different damage degrees each other about three groups. Using CCT intervention rats induced cancer in various stages of liver damage is lighter than AFB1, and happened proliferative lesion also later than AFB1 group, average survival time, rats and final hepatocellular carcinoma incidence significantly lower than AFB1 group. The control group was without the changes.
The end of experiment at 73W, the rats of occurred malignant tumor in group AFB1 and CCT were 76.5 percent (13/17) and 25.0 percent (4/16), the rats of hepatocellular carcinoma in group AFB1 and CCT were 58.8 percent (10/17) and 18.8 percent (3/16). In group AFB1, the rats of occurred malignant tumor and hepatocellular carcinoma are significantly higher than the CCT groups (P = 0.0053 and P = 0.0324). The control groups have no rat tumor occurrence.
2. The changes of CYP3A4 activity in rat hepatic tissues metabolic enzymes
In the whole experiment process, The CYP3A4 metabolic enzymes activity show increased gradually with peak value at the 23rd-week and then decreased gradually, in each groups with dynamic observation. And the CYP3A4 activity increased again at the 53rd-week, forming double peaks. CYP3A4 activities decreased significantly during53rd- and 63rd-week in AFB1 and CCT group compared to control group (P<0.05). The level of CYP3A4 activity in control group was decreased at the 33rd-week and 63rd-week, and no change significantly in other stage.
3. The expression of GST -Pi in rat hepatic tissues
GST-Pi positive dyeing located in cell plasma, positive cells is multifocal dyeing or flake distribution. In the whole experiment process, the expression of protein were gradually rise heighten with the time extend induced cancer, especially obvious in AFB1 group. The rates of GST-Pi in AFB1 group were 73%, 86.7%, 93.3% and in CCT group were 66.7%, 80.0%, 80.0% at 33W, 53W and 73W. The rates of GST-Pi in AFB1 group are corresponding higher than in CCT group. And that the control group was 10% at the 73W, and the expression was negative in other time. The same time is compared among different groups, the expression of GST-Pi in AFB1 groups and CCT groups are significantly higher than the control groups (P < 0.05) from 33W to 73W, the AFB1 groups are no significantly higher than and CCT groups from 33W to 63W. AFB1 group is not only significantly higher than CCT group, but also higher than those in the control groups (P = 0.000) in the 73W. The GST-Pi protein expressions were no significant difference (P > 0.05) at different time-points between groups or between group rats.
4. The expression of STAT3 in rat hepatic tissues
STAT3 positive dyeing located in cell hepatic tissues plasma, positive cells in scattered upon or dyeing focal distribution. Induced cancer process, In group AFB1 and group CCT , the rates expressions of STAT3 protein were gradually rise heighten with the time extend induced cancer, The rates of STAT3 in AFB1 group were 20.0%, 53.3%, 86.7% and in CCT group was 13.3%, 40.0%, 60.0% at 33W, 53W and 73W. And that the control group was 10% at the 53W and 73W, and the expression was negative in other time. The same time is compared among different groups, the expression of STAT3 in CCT groups are significantly higher than the control groups (P < 0.05) from 53W to 73W, the AFB1 groups are no significantly higher than and CCT groups from 53W to 73W (P > 0.05).
Conclusions
1. CCT concentrate had inhibited the rates of hepatocellular carcinoma incidence in AFB1 induced. The effects of hepatocellular carcinoma have prevented, that likely to become human hepatocellular carcinoma with AFB1 correlation of potential chemoprevention agent.
2. The CYP3A4 activity was inhibited during hepatocarcinogenesis in CCT groups. This may be due to decrease of carcinogens metabolic and that reduce carcinogenicity and hepatic injury of chemical to attain the role of protect liver.
3. In the carcinogenic process, the expression of GST - Pi and STAT3 were inhibited during hepatocarcinogenesis in CCT groups, and so it could prevent cell division and malign hyperplasia and delay the development of tumor. This may be due to the methylation about promotor of CpG-island and activation of STAT3 downstream gene, and concerned those interdict activity of some important anti-tumor genes.
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