银杏叶提取物对AFB1致大鼠肝癌抑制作用的研究
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摘要
原发性肝癌(Primary hepatic carcinoma, PHC)是人类比较常见的一种恶性肿瘤,在全球范围内的发病率排第五,死亡率排第三。我国是原发性肝癌高发的国家,每年因原发性肝癌死亡的人数有23万之多,占全部恶性肿瘤死亡率近20%,仅次于胃癌位居第二位,在江苏启东及广西扶绥地区原发性肝癌的发病率尤其突出。原发性肝癌具有诊治难、进展快、预后差的特点,严重危害着人们的健康,必须进一步加强原发性肝癌的防治研究。
流行病学及动物学实验研究表明,在我国和一些非洲国家,慢性乙型肝炎病毒(HBV)感染和饮食中黄曲霉毒素(Aflatoxins, AFT)污染是原发性肝癌高发人群的2个主要危险因素。而且,这两个因素之间能够相互协同,共同促进肝癌的发生发展。AFT是由黄曲霉菌和寄生曲霉菌所产生的一种代谢产物,化学上属二呋喃香豆素的衍生物。根据其分子结构的不同,天然产生的AFT分为B1、B2、G1及G2四种异构体。在我国,许多的食物、粮油、药品原料、农副产品及其制成品均易被AFT所污染,其中又以黄曲霉素B1(Aflatoxin B1, AFB1)的污染最为广泛,毒理作用也最强。AFB1是目前所知的最强致癌物质之一。研究表明,食物中黄曲霉毒素的含量多少与肝癌的发生呈正相关,这已被许多流行病学实验病理学研究所证实。
研究表明,AFB1在没有经过代谢活化之前的母体化合物是无致癌性的,它进入机体后必须通过体内的Ⅰ相药物代谢酶细胞色素P450系统作用,生物代谢活化后才能表现出毒性作用,被吸收的AFB1进入机体后,经Ⅰ相药物代谢酶代谢转化,形成黄曲霉毒素M1(Aflatoxin M1, AFM1)、黄曲霉毒素P1(Aflatoxin P1, AFP1)、黄曲霉毒素Q1(Aflatoxin Q1, AFQ1)及黄曲霉毒素醇,后三者被认为无活性而通过尿液或粪便排泄出体外。而AFM1和AFB1可被细胞色素P450lA2(Microsomal cytochrome P4501A2,CYP1A2)、细胞色素P4503A4(Microsomal cytochrome P4503A4, CYP3A4)代谢活化为能与细胞大分子物质(DNA或蛋白)结合形成活性中间体AFM1-8,9环氧化物(AFM1-exo-8,9-epoxide)及AFB1-8,9环氧化物(AFB1-exo-8,9-epoxide, AFBO)方才具有基因毒性及致癌性。作为催化这一代谢反应的关键酶——细胞色素P450,主要存在于肝细胞内,其中又以CYP3A4的含量最多及活性最高。AFBO能自发和核酸及蛋白质等生物大分子相结合而形成相应加合物。AFB1和DNA共价结合形成AFB1-DNA加合物是引起基因突变的基础,也是导致原发性肝癌发生的第一步。AFB1-DNA加合物主要存在于肝细胞内,除部分被体内Ⅱ相代谢酶如谷胱苷肽转移酶(GST)等作用下转为无毒物排出体外,由于分子内电子场的改变,也可自发形成其它DNA加合物,导致DNA损伤。
AFT严重危害着人类的健康,为了防止或者减少其造成的各种危害,除了避免食物被污染及去除毒素等措施外,用各种化学的方法抑制、延迟或者逆转癌变过程,并防止癌症发生的途径也日益被人们所重视。研究发现,一些植物性化学物如叶绿素、茶多酚、黄酮类化合物等在肿瘤化学预防这一邻域有着独特的防癌作用。通过在AFT高污染地区补充植物化学物制剂或其含量较高的食物来预防AFT致肝癌效应的作用越来越受到众多学者的重视。食物及其成分作为化学预防剂具有安全、易接受的优点,是目前开展AFB1相关恶性肿瘤化学预防剂的重点研究领域之一。
银杏(Ginkogo biloba L)为银杏科植物,其叶和果均具有重要的药用价值。标准银杏叶提取物(Ginkgo Biloba Extract)被国际通称为EGb761,一般含有24%的黄酮甙类及5-7%的银杏特有的萜烯类。近来研究发现EGb761及其组分可以通过抗氧化、抗血管生成及其基因调节而具有抗癌的作用。因此,目前对银杏叶的药用价值研究已从最初在心、脑血管系统疾病的应用扩展至抗肿瘤领域。前期实验研究证实EGb761对AFB1诱导的大鼠肝癌有抑制作用,但其具体作用机制尚未完全明确。研究发现在实验大鼠肝癌表达的众多基因中,IGF-Ⅱ、P16Ink4a、NADE、GST-Pi及UGT等肿瘤相关基因的表达明显大于正常肝脏,p75NTR、P53、AKR7A及COX-2等基因与大鼠肝癌的关系尤为突出。本研究通过动态观察EGb761在AFB1诱发大鼠肝癌实验过程中对肝脏相关代谢酶及上述肿瘤相关基因表达的影响,进一步探讨EGb761化学阻断大鼠肝癌发生发展中的分子生物学机制。为肝癌防治药物的研制提供更多的实验依据,将有很大的经济及社会效益。
方法
建立AFB1致PHC大鼠模型：70只雄性4周龄Wistar大鼠适应环境后,按体重随机将其分为3组：AFB1组,25只,喂饲正常饲料,于第4周开始腹腔注AFB1至第52周；EGb761+AFB1组,25只,每天喂饲含2g/kgEGb761饲料至实验结束,腹腔注AFB1剂量及时间同AFB1组；空白对照组,20只,喂饲正常饲料,不给AFB1处理。分别于实验第13周、23周、33周、43周、53周及63周对全部动物进行剖腹肝活检,第73周结束实验,处死全部实验动物。所取肝组织标本分成3块,一块立即制备肝匀浆,分离出肝微粒体进行肝代谢酶CYP3A4及GSH的检测；一块冷冻保存于-80℃,用于检测肿瘤相关基因的表达情况；一块放入10%中性福尔马林溶液内固定后常规石蜡包埋,用于HE染色。采用Real time-PCR及Western Blotting技术相对定量分析IGF-Ⅱ、p16Ink4a、NADE及p75NTR等肿瘤相关基因的表达情况,并对各蛋白表达的相关性进行统计分析。
结果
1.大鼠肿瘤发生情况：
第73周实验结束时,各组有效动物数及肿瘤发生情况如下：AFB1组17只,发生恶性肿瘤13例(76.5%),其中10例为肝细胞肝癌(Hepatocellular carcinoma, HCC),其余为胆管细胞癌2例、纤维肉瘤1例,原发性肝癌发生率为70.6%(12/17)；EGb761+AFB1组17只,发生恶性肿瘤6例(35.3%),其中HCC为5例,胆管细胞癌1例,原发性肝癌发生率为35.3%(6/17)。对照组16只,无肿瘤发生；EGb761+AFB1组肝癌发生率显著低于AFB1组(P<0.01)。
2.大鼠肝脏组织形态学变化情况
实验过程中,由AFB1诱发的肝脏恶性肿瘤最早出现在第52周,为纤维肉瘤,第一例肝癌的出现在第55周,最早出现肿瘤及第一例肝癌的大鼠均为AFB1组大鼠。镜下动态观察肝活检组织病理切片显示AFB1诱发大鼠肝癌的癌变过程大致可以分为三个阶段,第一阶段(13周～33周),出现不同程度的肝细胞变性；第二阶段(34周～53周)出现异常肝细胞增生灶及增生结节,主要为嗜碱性结节；第三阶段(54周～73周)肝病变进一步加重,逐渐可见癌结节。EGb761+AFB1组大鼠在诱癌各阶段的肝脏损害均较同时段AFB1组要轻,发生增生性病变比AFB1组亦较迟,对照组无上述改变。
3.EGb761对肝药酶的影响
3.1大鼠肝组织代谢酶CYP3A4活性的变化情况
在整个实验过程中,各组大鼠肝组织代谢酶CYP3A4活性在第23周及53周时呈现双波峰变化；CYP3A4酶活性在AFB1组、EGb761+AFB1组,除第13周外,其他时段均高于对照组；AFB1组在第53周及63周时CYP3A4的活性显著于高于EGb761+AFB1组,差异有统计学意义(P<0.05)。
3.2大鼠肝组织GSH含量的变化情况
在整个实验过程中,AFB1组和EGb761+AFB1组肝组织GSH含量在第13至33周时较正常对照组要低,随着实验的进展,呈现逐渐下降趋势,但AFB1组下降过程明显要快于EGb761+AFB1组,在第43周及53周时差异最明显(P<0.05)。而正常对照组在整个实验过程中各时段的变化并不明显。
4.EGb761对大鼠肝癌发生过程中肿瘤相关基因表达的影响
4.1EGb761对大鼠肝组织p16Ink4a mRNA及相应蛋白表达的影响
在第13周、33周时,各组p16Ink4a mRNA的表达量差异不明显(P>0.05)。至第53、73周时,AFB1组、EGb761+AFB1组p16Ink4a mRNA的表达量均较对照组下调(P<0.05),AFB1组下调最明显,与EGb761+AFB1组比较差异有统计学意义(P<0.05)。
在第13周、33周时,p16Ink4a蛋白在各组的表达水平无明显差异(P>0.05)。至第53周、73周时,AFBl组和EGb761+AFB1组中表达量较对照组均有所下调(P<0.05)。p16Ink4a蛋白在EGb761+AFB1组表达水平较同时段AFB1组要高,差异有统计学意义(P<0.05)。
4.2EGb761对大鼠肝组织P53mRNA表达的影响
P53mRNA的表达量于第13周时在各组大鼠肝组织中均有低度表达,差异不明显；至第33、53及73周时,AFB1组、EGb761+AFB1组的表达量均较对照组上调(P<0.01)；但P53mRNA在EGb761+AFB1组的表达量较同时段AFB1组要高,差异有统计学意义(P<0.01)。
4.3EGb761对大鼠肝组织IGF-ⅡmRNA及蛋白表达的影响
第13周、33周时,IGF-ⅡmRNA在各组肝组织中均有表达,但无明显差异(P>0.05)；至第53周、73周时,与对照组比较,AFB1组和EGb761+AFB1组中IGF-ⅡmRNA的表达均明显上调(P<0.01)；EGb761+AFB1组中IGF-ⅡmRNA表达水平较同时段AFB1组明显要低,差异有统计学意义(P<0.01)。
第13周、33周时,IGF-Ⅱ蛋白在各组肝组织中均为低表达或几乎不表达,无明显差异；至第53周、73周时,IGF-Ⅱ蛋白在AFB1组和EGb761+AFB1组均为高表达,明显高于对照组；且随肝癌的发生发展有逐渐增高趋势；第53周、73周时,EGb761+AFB1组IGF-Ⅱ蛋白表达水平始终低于同期AFBl组,差异有统计学意义(P<0.01)。
4.4EGb761对大鼠肝组织NADE mRNA及蛋白表达的影响
自第33周开始,NADE mRNA的表达量在AFB1组和EGb761+AFB1组均较同时段正常对照组要高,差异有统计学意义(P<0.05),两组间差异不明显(P>0.05)。
第33、53及73周时,AFB1、EGb761+AFB1两组中NADE蛋白的表达量均较正常对照组明显要高,差异有统计学意义(P<0.01)；两组间比较,EGb761+AFB1组表达较AFB1组要高,但差异无统计学意义。
4.5EGb761对大鼠肝组织p75NTR mRNA及蛋白表达的影响
在实验开始的第13至第33周,各组的p75NTR mRNA表达量差异不明显。到第53周及第73周时,在AFB1组及EGb761+AFB1组中的p75NTRmRNA表达量较正常对照组明显下调(P<0.05)；其中AFB1组下调更为明显,与EGb761+AFB1组比较,差异有统计学意义(P<0.01)。
实验至第53周时开始,p75NTR蛋白在AFB1组及EGb761+AFB1组中的表达量较正常对照组明显下降(P<0.01)；AFB1组p75NTR蛋白表达量较EGb761+AFB1组要低,差异有统计学意义(P<0.05)。
4.6EGb761对大鼠肝组织COX-2mRNA表达的影响
在实验开始的第13及33周时,COX-2mRNA在AFB1组、EGb761+AFB1组均呈低表达状态,差异无统计学意义；至第53周及73周时COX-2mRNA在AFB1组及EGb761+AFB1组均呈高表达量状态,与正常对照组比较差异明显(P<0.01)；而两组间比较差异无统计学意义(P>0.05)；空白对照组的COX-2mRNA在整个实验过程中,都呈低表达甚至无表达状态。
4.7EGb761对大鼠肝组织UGT mRNA表达的影响
在实验过程中的第13、33、53及73周时,UGT mRNA在AFB1组及EGb761+AFB1组中表达量均较正常对照组明显上调,差异有统计学意义(P<0.01)；其中UGT在EGb761+AFB1组中的表达量在各时段较AFBl组显著要高,差异有统计学意义(P<0.01)。
4.8EGb761对大鼠肝组织AKR7A mRNA表达的影响
在实验开始的第13周,AKR7A mRNA的表达量在AFB1组及EGb761+AFB1组中较正常对照组均要明显增高,差异有统计学意义(P<0.01)；第33、53周及73周,EGb761+AFB1组中的表达量要高于AFB1组(P<0.01)。
4.9EGb761对大鼠肝组织GST-Pi mRNA表达的影响
在整个实验过程中,GST-Pi mRNA的表达量在AFB1组及EGb761+AFB1组中较正常对照组均要明显增高,差异有统计学意义(P<0.01)；至第33、53周时,AFB1组的表达量要高于EGb761+AFB1组,但无显著性差异(P>0.05)；至第73周,AFB1组的表达量要显著高于EGb761+AFB1组,差异有统计学意义(P<0.01)。
5.0p16Ink4a、IGF-ⅡNADE及P75NTR蛋白表达的相互关系
在实验第73周、53周及33周时,AFB1组及EGb761+AFB1组中p16Ink4a蛋白的表达与p75NTR蛋白的表达呈显著的正相关关系(P<0.05)。
结论
1.银杏叶提取物能有效降低AFB1诱发肝癌的发生率,具有防癌作用,可成为人类与AFB1相关性肝癌的化学预防剂。
2.在AFB1诱发大鼠肝癌过程中,银杏叶提取物能抑制肝脏Ⅰ相酶CYP3A4的活性,从而减少具有基因毒性及致癌性的AFBO的形成,降低其致癌性。
3.银杏叶提取物能提高大鼠肝脏GSH的含量水平,从而提高机体的抗氧化能力,减轻AFB1诱癌过程中自由基所致的脂质过氧化损伤,亦可能是其发挥抗癌作用的机制之一。
4.银杏叶提取物在AFB1诱发大鼠肝癌过程中,能够上调抑癌基因p16Ink4a、P53及p75NTR的表达水平,下调肝癌病变相关增殖基因IGF-Ⅱ的表达量,可能是其抗癌作用的分子生物学基础之一。
5.银杏叶提取物在AFB1诱发的大鼠肝癌病变过程中,可以显著提高肝组织相关解毒代谢酶UGT及AKR7A的表达水平,以增强机体自身对真菌毒素的解毒能力。
6.银杏叶提取物在AFB1诱发的大鼠肝癌病变过程中,能够下调GST-Pi的表达水平,可能与GST-Pi启动子区的甲基化现象有关。
7.银杏叶提取物在AFB1诱发的大鼠肝癌病变过程中,p16Ink4a蛋白与p75NTR蛋白的表达呈正相关关系,提示银杏叶提取物可通过同时调节不同的基因即阻滞了细胞周期,又诱导了相应肿瘤细胞的凋亡,这可能是其抗癌机制之一。
Background and Objective
Primary hepatic carcinoma (PHC) is more common malignant tumor in human. The incidence of PHC is the fifth most common cancer and the mortality rate is the third leading cause of cancer in worldwide. PHC is the second cause leading cancer death in China. In any given year20%of total fatalities caused by malignant tumor are PHC in China. The incidence and fatalities of PHC is especially striking in Qidong of Jiangsu and Fusui Country of Guangxi province where PHC is the first most common cancer. The patients of PHC have the characteristics with the difficult of diagnosis and treatment, illness progress fast and poor prognosis. PHC is a terrible disease and this situation is very harmful to people's health.
Epidemiological research and experimental study of zoology evidenced that chronic hepatitis B virus (HBV) infection and diet aflatoxins (AFT) pollution are thought to be two largely responsible for the high incidence of PHC in southern Africa and southeast China. However, The two factors can be synergies between, jointly promote the development of PHC. AFT is a metabolic product of aspergillus flavus and Parasitic aspergillus and the chemical is belong to two furan coumarin derivatives. According to the different of its molecular structure, the natural AFT divided into B1, B2, G1and G2. In our country, a lot of food, grain and oil, drugs raw materials, agricultural and sideline products and finished goods are contaminated by AFT, which Aflatoxin B1(Aflatoxin B1, AFB1) of the pollution is the most extensive and toxicological effect is the most strongest. At present, AFB1is one of the strongest carcinogenic substance. Research shows that the positive correlation between the amount of AFB1in food and the occurrence of hepatocellular carcinoma. It has been confirmed by many epidemic research institutions in Asia and Africa.
Research shows that, the parent compound of AFB1is without carcinogenicity before metabolic activation. After entering the body, this chemical is biotransformed by cytochrome P450system to the reactive intermediante AFB1-exo-8,9-epoxide (AFBO)exert its gene toxicity and carcinogenicity. Cytochrome P450is the key enzyme when it catalytics the metabolic reactions. cytochrome P450is mainly exist in the liver cells, which the content of cytochrome P4503A4is the most and activity is the highest.
AFBO can combined with biological macromolecules such as nucleic acids and proteins to form corresponding the adducts. AFB1and DNA covalent bonding form AFB1-DNA adducts is formed the basis of genetic mutations, also which led to the first step of Primary hepatic carcinoma. AFB1-DNA adducts exist mainly in liver cells. Part of AFB1-DNA adducts are converted into non-toxic substance and eliminated from the body under the action of the body Ⅱ phase metabolic enzymes such as Glutathione S-transferase (GST). Due to the change of the electronic field in molecules, AFB1can also add other DNA spontaneously formed and lead to the DNA damage.
AFT is very harmful to human health. In order to prevent or reduce the harm caused by AFT, all kinds of methods were used such as avoid the food pollution and remove toxins measures and so on. Chemoprevention is a pharmacological approach to suppress, delay or reverse cancerous process, and to prevent cancer ways have also increasingly concerned by people. Research shows that some plant chemicals such as chlorophyll, tea polyphenol, flavonoids compounds has a unique anti-cancer effects in the neighborhood of cancer prevention. Through add chemical preparation plants or its content of food is higher in high pollution area to prevent the function of AFT lead to PHC attracted more and more researchers'attention. Food and its composition as chemical prevention agent has the advantages such as safe, easy to accept. It became one of the important fields in conducting chemical preventive research.
Gingko (Ginkogo biloba L) for ginkgo secco plant, its leaves and fruit has important medicinal value. Ginkgo biloba leaf extract, Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, typically contains about24%flavonoid glycosides and about5-7%terpene trilactones that are unique to Ginkgo. Recent research shows that EGb761and its components has the function of antitumor through anti-oxidation, antiangiogenic and gene regulation. So, the study of EGb761on the application of cardiovascular system disease and cerebrovascular disorder has been expanded to antitumor territory.
The experimental study was confirmed that EGb761has Inhibition on Aflatoxin B1-induced Hepatocarcinogenesis in wistar Rats. However, the specific mechanism was not completely clear. In recent years, the studies found that the expression of related genes in hepatocellular carcinoma of experimental rat such as IGF-II, NADE, P16INK4a, GST-Pi, UGT etc were significantly greater than normal liver, and the relationship of P53, AKR7A, P75NTR, COX-2with the tumor are very outstanding. Therefore, we investigated dynamic the influence of EGb761on liver related metabolic enzymes and tumor gene expression on Aflatoxin B1-induced Hepatocarcinogenesis in wistar Rats. We investigated the molecular biology mechanism of the blocking hepatocellular carcinoma occurrence and development. For the drug of prevention liver cancer, it can provide more experimental basis. There will be a great economic and social benefits.
Methods
The model of aflatoxin B1-induced Primary hepatic carcinoma was established in wistar Rats. Among the totale of70, four-weeks old male wistar Rats were divided randomly into three groups:AFB1, EGb761+AFB1and normal control groups. AFB1group:25rats were exposed to AFB1by intraperitoneally injection from the fourth week experimental and feeding normal feed in the first week until the end of experiment. AFB1+EGb761group:25rats were exposed to AFB1done as AFB1group and were fed feedstuff containing2g/kg EGb761from the experiment in the first week until the end of experiment. Control groups,20rats were not exposed to AFB1and were fed normal feed in the first week from the beganing until the end of experiment. During hepatocarcinogenesis, liver biopsies were performed on all of animals at13thW,23thW,33thW,43thW,53thW and63thW of the experiment. All survivals animals were sacrificed by cervical dislocation at73rd-week and liver tissues and tumor were collected. The Liver tissue samples were separated into three pieces. Microsomal and cytosolic fractions were prepared from fresh liver tissue and applied to detect the hepatic drug-metabolizing enzymes CYP3A4activity and GSH. One section of liver tissue samples snap-frozen in liquid N2and stored at-80℃for investigated related gene expression of the tumor. Some liver tissues were immerged in10%formalin fixation and paraffin embedded for HE dyeing. The relative quantitative analysis technology Real time-PCR and Western Blotting were used to investigate the genes expression situation in the tumors such as IGF-II, p16Ink4a, P53, NADE and p75NTR etc. The correlation of the protein expression were analysed by statistics.
Results
1. The data of rats tumor occurrence:
The effective numbers of animals and tumorigenesis effective at the end of experiment (73W)as follows:the effective numbers of animals in AFB1group was17, occurred malignant tumor13cases (76.5%), including hepatocellular carcinoma (HCC)10cases, bile duct carcinoma two cases, fibrosarcoma one case, PHC incidence was70.6%(12/17); EGb761+AFB1group was17, occurred malignant tumor6cases (35.3%), including hepatocellular carcinoma5cases, bile duct carcinoma one case, PHC incidence was35.3%(6/17); The control group was16, no tumor was occurrenced. The incidence of PHC in EGb761+AFB1group was significantly lower than that in AFB1group (P<0.01).
2. The morphology change of rat liver tissue:
The earliest time AFB1-induced rat liver malignant tumors(including fibrosarcoma) formation was at52thW. The first case of Primary hepatic carcinoma was occured in AFB1group rats at the55thW.
Microscopically dynamic observation of liver biopsy histopathology shows that AFB1-induced rats liver cancerous process can be divided into three stages: The first stage (13thW-33thW), there were some different degrees of liver cell denaturation appear in rats liver. The second stage (34thW-53thW), the abnormal liver cell hyperplasia and focal hyperplasia nodules, mainly for basophilic nodules were developed. The third stage (54thW-73thW), liver disease become accentuate further, gradually visible carcinoma nodules. In the date using EGb761intervention the liver damages in different stage were lighter than AFB1group at the same stage. The proliferative lesion was also later than AFB1group and the control group was not changes alone.
3. Effect of EGb761on hepatic tissues metabolic enzymes
3.1The changes of CYP3A4activity in rat hepatic tissues
During the whole experimental process, the activity of CYP3A4in AFB1group and EGb761+AFB1group had different levels of changes. The activity of CYP3A4were formed double peaks at23thW and53thW. The activity of CYP3A4in AFB1group and EGb761+AFB1group were higher than that in control group except13thW. The activity of CYP3A4increased significantly during53thW and63thW in AFB1group compared to EGb761+AFB1group (P＜O.05). The activity of CYP3A4in control group was decreased at the33thW and63thW. There are no change significantly in other stage.
3.2The changes of GSH conten in rat hepatic tissues
The content of GSH in rat hepatic tissues is lower in AFB1group and EGb761+AFB1group compared to control group. Along with the progress of the experiment, the content of GSH decreased gradually in AFB1groups and EGb761+AFB1group. The content of GSH decreased significantly during43W and53W in AFB1group compared to EGb761+AFB1group (P＜0.05). The change of GSH in normal control group was not obvious during each period of the whole process of experiment.
4. Effect of EGb761on the expression of related cncer genes
4.1Effect of EGb761on the expression of p16Ink4a mRNA and corresponding protein in rat hepatic tissues
The expression of p16Ink4a mRNA is not significantly higher or lower in each group at13thW and33thW(P>0.05), however, in AFB1group and EGb761+AFB1group were significantly lower than that in the control groups during53thW and73thW(P<0.05). The expression of p16Ink4a mRNA in AFB1group was significantly lower than that in the EGb761+AFB1group from53thW to73thW (P＜0.05).
The p16Ink4a protein expressions were not significant difference in each group during13thW and33thW(P>0.05), however, in AFB1group and EGb761+AFB1group were significantly lower than that in the control groups during53thW and73thW(P<0.05). The p16Ink4a protein expression in EGb761+AFB1group was significantly higher than that in the AFB1groups (P＜0.05).
4.2Effect of EGb761on the expression of P53mRNA in rat hepatic tissues
The expression of P53mRNA was low expression and no significant difference in each group during13thW. The expression of P53mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during33thW,53thW and73thW(P<0.01). The expression of P53mRNA in EGb761+AFB1group was significantly higher than that in the AFB1group (P＜0.01).
4.3Effect of EGb761on the expression of IGF-II mRNA and corresponding protein in rat hepatic tissues
The expression of IGF-II mRNA was not significant difference in each group during13thW and33thWCP>0.05). The expression of IGF-II mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control groups during53thW and73thW. The expression of IGF-II mRNA in EGb761+AFB1group was significantly lower than that in the AFB1group (P＜0.01).
The IGF-II protein expressions were low expression or not expression in each group during13thW and33thW. There was not significant difference in each group during13thW. Along with the progress of the experiment, the IGF-II protein expressions increased gradually. The IGF-II protein expressions in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during53thW and73thW. The IGF-II protein expressions in EGb761+AFB1group was significantly lower than that in AFB1group during53thW and73thW (P<0.05).
4.4Effect of EGb761on the expression of NADE mRNA and corresponding protein in rat hepatic tissues
The expression of NADE mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from33thW to73thW and the difference have statistical significance (P<0.01). The expression of NADE mRNA in EGb761+AFB1group was higher than that in the AFB1group. However, there was not statistical significance on the differences between AFB1andEGb761+AFB1group(P>0.05).
The NADE protein expression in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from33thW to73thW (P<0.05). The NADE protein expression in EGb761+AFB1group was slight higher than that in the AFB1group, however, there was not statistical significance on the difference among them (P>0.05).
4.5Effect of EGb761on the expression of p75NTR mRNA and corresponding protein in rat hepatic tissues
The expression of p75NTR mRNA was not significantly difference in each group during13thW and33thW(P>0.05). The expression of p75NTR mRNA in AFB1group and EGb761+AFB1group were significantly lower than that in the control group during53thWand73thW (P＜0.05). The expression of p75NTR mRNA in EGb761+AFB1group was higher than that in the AFB1group (P＜0.01).
The p75NTR protein expression in AFB1group and EGb761+AFB1group were significantly lower than that in the control group from55thW to73thW(P<0.01). The p75NTR protein expression in AFB1group was significantly lower than that in the EGb761+AFBl group from55thW to73thW (P＜0.05).
4.6Effect of EGb761on the expression of COX-2mRNA in rat hepatic tissues
The expression of COX-2mRNA were low expression in AFB1group and EGb761+AFB1group during13thW and33thW. The expression of COX-2mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during53thW and73thW (P<0.01). There was not significantly difference between AFB1group and EGb761+AFB1group(P>0.05). The expression of COX-2mRNA was low or no expression in control group from13thW to73thW.
4.7Effect of EGb761on the expression of UGT mRNA in rat hepatic tissues
The expression of UGT mRNA in AFB1group and EGb761+AFB1group are significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P<0.01). The expression of UGT mRNA in EGb761+AFB1group are significantly higher than that in the AFB1group during whole of the experimental process and the difference have statistical significance (P<0.01).
4.8Effect of EGb761on the expression of AKR7A mRNA in rat hepatic tissues
The expression of AKR7A mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P＜0.01). The expression of AKR7A mRNA in EGb761+AFB1group are significantly higher than that in the AFB1group(P<0.01).
4.9Effect of EGb761on the expression of GST-Pi mRNA in rat hepatic tissues
The expression of GST-Pi mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P<0.01). The AFB1group are not significantly higher than that in AFB1+EGb761group during33thW and53thW. The expression of GST-Pi mRNA in AFB1group was not only significantly higher than that in the control group, but also higher than those in the EGb761+AFB1group during73thW(P<0.01).
5.0Effect of EGb761on the mutual relations of p16Ink4a, IGF-II, NADE and p75NTR protein expression
The protein expression between p16Ink4a and p75NTR in in AFB1group and EGb761+AFB1group was positive correlation during73thW,55thW and33thW(P <0.01).
Conclusions
1. EGb761can reduce the incidence of PHC induced by AFB1. It indicated that EGb761has the effects of anti-cancer development and may be used at the chemoprevention for human PHC related with AFB1.
2. EGb761can restrain the liver enzymes I phase CYP3A4activity, reduced the AFBO formation, and weaken the carcinogenicity of AFB1.
3. EGb761can improve the content of rat liver GSH level, so as to improve the body's anti-oxidation ability, reduced lipid peroxidation damage caused by free radicals in the carcinogenesis induced by AFBl, which is one of the mechanism of EGb761anti-cancer effects.
4. In the carcinogenesis AFB1-induced, EGb761can upregulate the expressive level of tumor-suppressor genes such as P16Ink4a, p53and P75NTR, downregulate the expressive level of IGF-II related proliferation which is one of the foundation of molecular biology of anti-cancer effects.
5. In order to enhance the detoxify ability against the mycotoxin's for organism itself, EGb761can greatly improve the expressive level of metabolic enzymes related detoxification in the liver tissue such as UGT and AKR7A, during hepatocarcinogenesis aflatoxin B1-induced in Wistar Rats.
6. EGb761can cut the expression level of GST-Pi during hepatocarcinogenesis aflatoxin Bl-induced in Wistar Rats.This may be due to the methylation about promotor of CpG-island.
7. There was a positive correlation between the protein expression of p16Ink4a and p75NTR during hepatocarcinogenesis aflatoxin B1-induced in Wistar Rats. This phenomenon was indicated that EGb761can adjust different genes that block the cell cycle, and induced apoptosis of corresponding of tumor cells through the meanwhile, this could be one of its anti-cancer mechanism.
流行病学及动物学实验研究表明,在我国和一些非洲国家,慢性乙型肝炎病毒(HBV)感染和饮食中黄曲霉毒素(Aflatoxins, AFT)污染是原发性肝癌高发人群的2个主要危险因素。而且,这两个因素之间能够相互协同,共同促进肝癌的发生发展。AFT是由黄曲霉菌和寄生曲霉菌所产生的一种代谢产物,化学上属二呋喃香豆素的衍生物。根据其分子结构的不同,天然产生的AFT分为B1、B2、G1及G2四种异构体。在我国,许多的食物、粮油、药品原料、农副产品及其制成品均易被AFT所污染,其中又以黄曲霉素B1(Aflatoxin B1, AFB1)的污染最为广泛,毒理作用也最强。AFB1是目前所知的最强致癌物质之一。研究表明,食物中黄曲霉毒素的含量多少与肝癌的发生呈正相关,这已被许多流行病学实验病理学研究所证实。
研究表明,AFB1在没有经过代谢活化之前的母体化合物是无致癌性的,它进入机体后必须通过体内的Ⅰ相药物代谢酶细胞色素P450系统作用,生物代谢活化后才能表现出毒性作用,被吸收的AFB1进入机体后,经Ⅰ相药物代谢酶代谢转化,形成黄曲霉毒素M1(Aflatoxin M1, AFM1)、黄曲霉毒素P1(Aflatoxin P1, AFP1)、黄曲霉毒素Q1(Aflatoxin Q1, AFQ1)及黄曲霉毒素醇,后三者被认为无活性而通过尿液或粪便排泄出体外。而AFM1和AFB1可被细胞色素P450lA2(Microsomal cytochrome P4501A2,CYP1A2)、细胞色素P4503A4(Microsomal cytochrome P4503A4, CYP3A4)代谢活化为能与细胞大分子物质(DNA或蛋白)结合形成活性中间体AFM1-8,9环氧化物(AFM1-exo-8,9-epoxide)及AFB1-8,9环氧化物(AFB1-exo-8,9-epoxide, AFBO)方才具有基因毒性及致癌性。作为催化这一代谢反应的关键酶——细胞色素P450,主要存在于肝细胞内,其中又以CYP3A4的含量最多及活性最高。AFBO能自发和核酸及蛋白质等生物大分子相结合而形成相应加合物。AFB1和DNA共价结合形成AFB1-DNA加合物是引起基因突变的基础,也是导致原发性肝癌发生的第一步。AFB1-DNA加合物主要存在于肝细胞内,除部分被体内Ⅱ相代谢酶如谷胱苷肽转移酶(GST)等作用下转为无毒物排出体外,由于分子内电子场的改变,也可自发形成其它DNA加合物,导致DNA损伤。
AFT严重危害着人类的健康,为了防止或者减少其造成的各种危害,除了避免食物被污染及去除毒素等措施外,用各种化学的方法抑制、延迟或者逆转癌变过程,并防止癌症发生的途径也日益被人们所重视。研究发现,一些植物性化学物如叶绿素、茶多酚、黄酮类化合物等在肿瘤化学预防这一邻域有着独特的防癌作用。通过在AFT高污染地区补充植物化学物制剂或其含量较高的食物来预防AFT致肝癌效应的作用越来越受到众多学者的重视。食物及其成分作为化学预防剂具有安全、易接受的优点,是目前开展AFB1相关恶性肿瘤化学预防剂的重点研究领域之一。
银杏(Ginkogo biloba L)为银杏科植物,其叶和果均具有重要的药用价值。标准银杏叶提取物(Ginkgo Biloba Extract)被国际通称为EGb761,一般含有24%的黄酮甙类及5-7%的银杏特有的萜烯类。近来研究发现EGb761及其组分可以通过抗氧化、抗血管生成及其基因调节而具有抗癌的作用。因此,目前对银杏叶的药用价值研究已从最初在心、脑血管系统疾病的应用扩展至抗肿瘤领域。前期实验研究证实EGb761对AFB1诱导的大鼠肝癌有抑制作用,但其具体作用机制尚未完全明确。研究发现在实验大鼠肝癌表达的众多基因中,IGF-Ⅱ、P16Ink4a、NADE、GST-Pi及UGT等肿瘤相关基因的表达明显大于正常肝脏,p75NTR、P53、AKR7A及COX-2等基因与大鼠肝癌的关系尤为突出。本研究通过动态观察EGb761在AFB1诱发大鼠肝癌实验过程中对肝脏相关代谢酶及上述肿瘤相关基因表达的影响,进一步探讨EGb761化学阻断大鼠肝癌发生发展中的分子生物学机制。为肝癌防治药物的研制提供更多的实验依据,将有很大的经济及社会效益。
方法
建立AFB1致PHC大鼠模型：70只雄性4周龄Wistar大鼠适应环境后,按体重随机将其分为3组：AFB1组,25只,喂饲正常饲料,于第4周开始腹腔注AFB1至第52周；EGb761+AFB1组,25只,每天喂饲含2g/kgEGb761饲料至实验结束,腹腔注AFB1剂量及时间同AFB1组；空白对照组,20只,喂饲正常饲料,不给AFB1处理。分别于实验第13周、23周、33周、43周、53周及63周对全部动物进行剖腹肝活检,第73周结束实验,处死全部实验动物。所取肝组织标本分成3块,一块立即制备肝匀浆,分离出肝微粒体进行肝代谢酶CYP3A4及GSH的检测；一块冷冻保存于-80℃,用于检测肿瘤相关基因的表达情况；一块放入10%中性福尔马林溶液内固定后常规石蜡包埋,用于HE染色。采用Real time-PCR及Western Blotting技术相对定量分析IGF-Ⅱ、p16Ink4a、NADE及p75NTR等肿瘤相关基因的表达情况,并对各蛋白表达的相关性进行统计分析。
结果
1.大鼠肿瘤发生情况：
第73周实验结束时,各组有效动物数及肿瘤发生情况如下：AFB1组17只,发生恶性肿瘤13例(76.5%),其中10例为肝细胞肝癌(Hepatocellular carcinoma, HCC),其余为胆管细胞癌2例、纤维肉瘤1例,原发性肝癌发生率为70.6%(12/17)；EGb761+AFB1组17只,发生恶性肿瘤6例(35.3%),其中HCC为5例,胆管细胞癌1例,原发性肝癌发生率为35.3%(6/17)。对照组16只,无肿瘤发生；EGb761+AFB1组肝癌发生率显著低于AFB1组(P<0.01)。
2.大鼠肝脏组织形态学变化情况
实验过程中,由AFB1诱发的肝脏恶性肿瘤最早出现在第52周,为纤维肉瘤,第一例肝癌的出现在第55周,最早出现肿瘤及第一例肝癌的大鼠均为AFB1组大鼠。镜下动态观察肝活检组织病理切片显示AFB1诱发大鼠肝癌的癌变过程大致可以分为三个阶段,第一阶段(13周～33周),出现不同程度的肝细胞变性；第二阶段(34周～53周)出现异常肝细胞增生灶及增生结节,主要为嗜碱性结节；第三阶段(54周～73周)肝病变进一步加重,逐渐可见癌结节。EGb761+AFB1组大鼠在诱癌各阶段的肝脏损害均较同时段AFB1组要轻,发生增生性病变比AFB1组亦较迟,对照组无上述改变。
3.EGb761对肝药酶的影响
3.1大鼠肝组织代谢酶CYP3A4活性的变化情况
在整个实验过程中,各组大鼠肝组织代谢酶CYP3A4活性在第23周及53周时呈现双波峰变化；CYP3A4酶活性在AFB1组、EGb761+AFB1组,除第13周外,其他时段均高于对照组；AFB1组在第53周及63周时CYP3A4的活性显著于高于EGb761+AFB1组,差异有统计学意义(P<0.05)。
3.2大鼠肝组织GSH含量的变化情况
在整个实验过程中,AFB1组和EGb761+AFB1组肝组织GSH含量在第13至33周时较正常对照组要低,随着实验的进展,呈现逐渐下降趋势,但AFB1组下降过程明显要快于EGb761+AFB1组,在第43周及53周时差异最明显(P<0.05)。而正常对照组在整个实验过程中各时段的变化并不明显。
4.EGb761对大鼠肝癌发生过程中肿瘤相关基因表达的影响
4.1EGb761对大鼠肝组织p16Ink4a mRNA及相应蛋白表达的影响
在第13周、33周时,各组p16Ink4a mRNA的表达量差异不明显(P>0.05)。至第53、73周时,AFB1组、EGb761+AFB1组p16Ink4a mRNA的表达量均较对照组下调(P<0.05),AFB1组下调最明显,与EGb761+AFB1组比较差异有统计学意义(P<0.05)。
在第13周、33周时,p16Ink4a蛋白在各组的表达水平无明显差异(P>0.05)。至第53周、73周时,AFBl组和EGb761+AFB1组中表达量较对照组均有所下调(P<0.05)。p16Ink4a蛋白在EGb761+AFB1组表达水平较同时段AFB1组要高,差异有统计学意义(P<0.05)。
4.2EGb761对大鼠肝组织P53mRNA表达的影响
P53mRNA的表达量于第13周时在各组大鼠肝组织中均有低度表达,差异不明显；至第33、53及73周时,AFB1组、EGb761+AFB1组的表达量均较对照组上调(P<0.01)；但P53mRNA在EGb761+AFB1组的表达量较同时段AFB1组要高,差异有统计学意义(P<0.01)。
4.3EGb761对大鼠肝组织IGF-ⅡmRNA及蛋白表达的影响
第13周、33周时,IGF-ⅡmRNA在各组肝组织中均有表达,但无明显差异(P>0.05)；至第53周、73周时,与对照组比较,AFB1组和EGb761+AFB1组中IGF-ⅡmRNA的表达均明显上调(P<0.01)；EGb761+AFB1组中IGF-ⅡmRNA表达水平较同时段AFB1组明显要低,差异有统计学意义(P<0.01)。
第13周、33周时,IGF-Ⅱ蛋白在各组肝组织中均为低表达或几乎不表达,无明显差异；至第53周、73周时,IGF-Ⅱ蛋白在AFB1组和EGb761+AFB1组均为高表达,明显高于对照组；且随肝癌的发生发展有逐渐增高趋势；第53周、73周时,EGb761+AFB1组IGF-Ⅱ蛋白表达水平始终低于同期AFBl组,差异有统计学意义(P<0.01)。
4.4EGb761对大鼠肝组织NADE mRNA及蛋白表达的影响
自第33周开始,NADE mRNA的表达量在AFB1组和EGb761+AFB1组均较同时段正常对照组要高,差异有统计学意义(P<0.05),两组间差异不明显(P>0.05)。
第33、53及73周时,AFB1、EGb761+AFB1两组中NADE蛋白的表达量均较正常对照组明显要高,差异有统计学意义(P<0.01)；两组间比较,EGb761+AFB1组表达较AFB1组要高,但差异无统计学意义。
4.5EGb761对大鼠肝组织p75NTR mRNA及蛋白表达的影响
在实验开始的第13至第33周,各组的p75NTR mRNA表达量差异不明显。到第53周及第73周时,在AFB1组及EGb761+AFB1组中的p75NTRmRNA表达量较正常对照组明显下调(P<0.05)；其中AFB1组下调更为明显,与EGb761+AFB1组比较,差异有统计学意义(P<0.01)。
实验至第53周时开始,p75NTR蛋白在AFB1组及EGb761+AFB1组中的表达量较正常对照组明显下降(P<0.01)；AFB1组p75NTR蛋白表达量较EGb761+AFB1组要低,差异有统计学意义(P<0.05)。
4.6EGb761对大鼠肝组织COX-2mRNA表达的影响
在实验开始的第13及33周时,COX-2mRNA在AFB1组、EGb761+AFB1组均呈低表达状态,差异无统计学意义；至第53周及73周时COX-2mRNA在AFB1组及EGb761+AFB1组均呈高表达量状态,与正常对照组比较差异明显(P<0.01)；而两组间比较差异无统计学意义(P>0.05)；空白对照组的COX-2mRNA在整个实验过程中,都呈低表达甚至无表达状态。
4.7EGb761对大鼠肝组织UGT mRNA表达的影响
在实验过程中的第13、33、53及73周时,UGT mRNA在AFB1组及EGb761+AFB1组中表达量均较正常对照组明显上调,差异有统计学意义(P<0.01)；其中UGT在EGb761+AFB1组中的表达量在各时段较AFBl组显著要高,差异有统计学意义(P<0.01)。
4.8EGb761对大鼠肝组织AKR7A mRNA表达的影响
在实验开始的第13周,AKR7A mRNA的表达量在AFB1组及EGb761+AFB1组中较正常对照组均要明显增高,差异有统计学意义(P<0.01)；第33、53周及73周,EGb761+AFB1组中的表达量要高于AFB1组(P<0.01)。
4.9EGb761对大鼠肝组织GST-Pi mRNA表达的影响
在整个实验过程中,GST-Pi mRNA的表达量在AFB1组及EGb761+AFB1组中较正常对照组均要明显增高,差异有统计学意义(P<0.01)；至第33、53周时,AFB1组的表达量要高于EGb761+AFB1组,但无显著性差异(P>0.05)；至第73周,AFB1组的表达量要显著高于EGb761+AFB1组,差异有统计学意义(P<0.01)。
5.0p16Ink4a、IGF-ⅡNADE及P75NTR蛋白表达的相互关系
在实验第73周、53周及33周时,AFB1组及EGb761+AFB1组中p16Ink4a蛋白的表达与p75NTR蛋白的表达呈显著的正相关关系(P<0.05)。
结论
1.银杏叶提取物能有效降低AFB1诱发肝癌的发生率,具有防癌作用,可成为人类与AFB1相关性肝癌的化学预防剂。
2.在AFB1诱发大鼠肝癌过程中,银杏叶提取物能抑制肝脏Ⅰ相酶CYP3A4的活性,从而减少具有基因毒性及致癌性的AFBO的形成,降低其致癌性。
3.银杏叶提取物能提高大鼠肝脏GSH的含量水平,从而提高机体的抗氧化能力,减轻AFB1诱癌过程中自由基所致的脂质过氧化损伤,亦可能是其发挥抗癌作用的机制之一。
4.银杏叶提取物在AFB1诱发大鼠肝癌过程中,能够上调抑癌基因p16Ink4a、P53及p75NTR的表达水平,下调肝癌病变相关增殖基因IGF-Ⅱ的表达量,可能是其抗癌作用的分子生物学基础之一。
5.银杏叶提取物在AFB1诱发的大鼠肝癌病变过程中,可以显著提高肝组织相关解毒代谢酶UGT及AKR7A的表达水平,以增强机体自身对真菌毒素的解毒能力。
6.银杏叶提取物在AFB1诱发的大鼠肝癌病变过程中,能够下调GST-Pi的表达水平,可能与GST-Pi启动子区的甲基化现象有关。
7.银杏叶提取物在AFB1诱发的大鼠肝癌病变过程中,p16Ink4a蛋白与p75NTR蛋白的表达呈正相关关系,提示银杏叶提取物可通过同时调节不同的基因即阻滞了细胞周期,又诱导了相应肿瘤细胞的凋亡,这可能是其抗癌机制之一。
Background and Objective
Primary hepatic carcinoma (PHC) is more common malignant tumor in human. The incidence of PHC is the fifth most common cancer and the mortality rate is the third leading cause of cancer in worldwide. PHC is the second cause leading cancer death in China. In any given year20%of total fatalities caused by malignant tumor are PHC in China. The incidence and fatalities of PHC is especially striking in Qidong of Jiangsu and Fusui Country of Guangxi province where PHC is the first most common cancer. The patients of PHC have the characteristics with the difficult of diagnosis and treatment, illness progress fast and poor prognosis. PHC is a terrible disease and this situation is very harmful to people's health.
Epidemiological research and experimental study of zoology evidenced that chronic hepatitis B virus (HBV) infection and diet aflatoxins (AFT) pollution are thought to be two largely responsible for the high incidence of PHC in southern Africa and southeast China. However, The two factors can be synergies between, jointly promote the development of PHC. AFT is a metabolic product of aspergillus flavus and Parasitic aspergillus and the chemical is belong to two furan coumarin derivatives. According to the different of its molecular structure, the natural AFT divided into B1, B2, G1and G2. In our country, a lot of food, grain and oil, drugs raw materials, agricultural and sideline products and finished goods are contaminated by AFT, which Aflatoxin B1(Aflatoxin B1, AFB1) of the pollution is the most extensive and toxicological effect is the most strongest. At present, AFB1is one of the strongest carcinogenic substance. Research shows that the positive correlation between the amount of AFB1in food and the occurrence of hepatocellular carcinoma. It has been confirmed by many epidemic research institutions in Asia and Africa.
Research shows that, the parent compound of AFB1is without carcinogenicity before metabolic activation. After entering the body, this chemical is biotransformed by cytochrome P450system to the reactive intermediante AFB1-exo-8,9-epoxide (AFBO)exert its gene toxicity and carcinogenicity. Cytochrome P450is the key enzyme when it catalytics the metabolic reactions. cytochrome P450is mainly exist in the liver cells, which the content of cytochrome P4503A4is the most and activity is the highest.
AFBO can combined with biological macromolecules such as nucleic acids and proteins to form corresponding the adducts. AFB1and DNA covalent bonding form AFB1-DNA adducts is formed the basis of genetic mutations, also which led to the first step of Primary hepatic carcinoma. AFB1-DNA adducts exist mainly in liver cells. Part of AFB1-DNA adducts are converted into non-toxic substance and eliminated from the body under the action of the body Ⅱ phase metabolic enzymes such as Glutathione S-transferase (GST). Due to the change of the electronic field in molecules, AFB1can also add other DNA spontaneously formed and lead to the DNA damage.
AFT is very harmful to human health. In order to prevent or reduce the harm caused by AFT, all kinds of methods were used such as avoid the food pollution and remove toxins measures and so on. Chemoprevention is a pharmacological approach to suppress, delay or reverse cancerous process, and to prevent cancer ways have also increasingly concerned by people. Research shows that some plant chemicals such as chlorophyll, tea polyphenol, flavonoids compounds has a unique anti-cancer effects in the neighborhood of cancer prevention. Through add chemical preparation plants or its content of food is higher in high pollution area to prevent the function of AFT lead to PHC attracted more and more researchers'attention. Food and its composition as chemical prevention agent has the advantages such as safe, easy to accept. It became one of the important fields in conducting chemical preventive research.
Gingko (Ginkogo biloba L) for ginkgo secco plant, its leaves and fruit has important medicinal value. Ginkgo biloba leaf extract, Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, typically contains about24%flavonoid glycosides and about5-7%terpene trilactones that are unique to Ginkgo. Recent research shows that EGb761and its components has the function of antitumor through anti-oxidation, antiangiogenic and gene regulation. So, the study of EGb761on the application of cardiovascular system disease and cerebrovascular disorder has been expanded to antitumor territory.
The experimental study was confirmed that EGb761has Inhibition on Aflatoxin B1-induced Hepatocarcinogenesis in wistar Rats. However, the specific mechanism was not completely clear. In recent years, the studies found that the expression of related genes in hepatocellular carcinoma of experimental rat such as IGF-II, NADE, P16INK4a, GST-Pi, UGT etc were significantly greater than normal liver, and the relationship of P53, AKR7A, P75NTR, COX-2with the tumor are very outstanding. Therefore, we investigated dynamic the influence of EGb761on liver related metabolic enzymes and tumor gene expression on Aflatoxin B1-induced Hepatocarcinogenesis in wistar Rats. We investigated the molecular biology mechanism of the blocking hepatocellular carcinoma occurrence and development. For the drug of prevention liver cancer, it can provide more experimental basis. There will be a great economic and social benefits.
Methods
The model of aflatoxin B1-induced Primary hepatic carcinoma was established in wistar Rats. Among the totale of70, four-weeks old male wistar Rats were divided randomly into three groups:AFB1, EGb761+AFB1and normal control groups. AFB1group:25rats were exposed to AFB1by intraperitoneally injection from the fourth week experimental and feeding normal feed in the first week until the end of experiment. AFB1+EGb761group:25rats were exposed to AFB1done as AFB1group and were fed feedstuff containing2g/kg EGb761from the experiment in the first week until the end of experiment. Control groups,20rats were not exposed to AFB1and were fed normal feed in the first week from the beganing until the end of experiment. During hepatocarcinogenesis, liver biopsies were performed on all of animals at13thW,23thW,33thW,43thW,53thW and63thW of the experiment. All survivals animals were sacrificed by cervical dislocation at73rd-week and liver tissues and tumor were collected. The Liver tissue samples were separated into three pieces. Microsomal and cytosolic fractions were prepared from fresh liver tissue and applied to detect the hepatic drug-metabolizing enzymes CYP3A4activity and GSH. One section of liver tissue samples snap-frozen in liquid N2and stored at-80℃for investigated related gene expression of the tumor. Some liver tissues were immerged in10%formalin fixation and paraffin embedded for HE dyeing. The relative quantitative analysis technology Real time-PCR and Western Blotting were used to investigate the genes expression situation in the tumors such as IGF-II, p16Ink4a, P53, NADE and p75NTR etc. The correlation of the protein expression were analysed by statistics.
Results
1. The data of rats tumor occurrence:
The effective numbers of animals and tumorigenesis effective at the end of experiment (73W)as follows:the effective numbers of animals in AFB1group was17, occurred malignant tumor13cases (76.5%), including hepatocellular carcinoma (HCC)10cases, bile duct carcinoma two cases, fibrosarcoma one case, PHC incidence was70.6%(12/17); EGb761+AFB1group was17, occurred malignant tumor6cases (35.3%), including hepatocellular carcinoma5cases, bile duct carcinoma one case, PHC incidence was35.3%(6/17); The control group was16, no tumor was occurrenced. The incidence of PHC in EGb761+AFB1group was significantly lower than that in AFB1group (P<0.01).
2. The morphology change of rat liver tissue:
The earliest time AFB1-induced rat liver malignant tumors(including fibrosarcoma) formation was at52thW. The first case of Primary hepatic carcinoma was occured in AFB1group rats at the55thW.
Microscopically dynamic observation of liver biopsy histopathology shows that AFB1-induced rats liver cancerous process can be divided into three stages: The first stage (13thW-33thW), there were some different degrees of liver cell denaturation appear in rats liver. The second stage (34thW-53thW), the abnormal liver cell hyperplasia and focal hyperplasia nodules, mainly for basophilic nodules were developed. The third stage (54thW-73thW), liver disease become accentuate further, gradually visible carcinoma nodules. In the date using EGb761intervention the liver damages in different stage were lighter than AFB1group at the same stage. The proliferative lesion was also later than AFB1group and the control group was not changes alone.
3. Effect of EGb761on hepatic tissues metabolic enzymes
3.1The changes of CYP3A4activity in rat hepatic tissues
During the whole experimental process, the activity of CYP3A4in AFB1group and EGb761+AFB1group had different levels of changes. The activity of CYP3A4were formed double peaks at23thW and53thW. The activity of CYP3A4in AFB1group and EGb761+AFB1group were higher than that in control group except13thW. The activity of CYP3A4increased significantly during53thW and63thW in AFB1group compared to EGb761+AFB1group (P＜O.05). The activity of CYP3A4in control group was decreased at the33thW and63thW. There are no change significantly in other stage.
3.2The changes of GSH conten in rat hepatic tissues
The content of GSH in rat hepatic tissues is lower in AFB1group and EGb761+AFB1group compared to control group. Along with the progress of the experiment, the content of GSH decreased gradually in AFB1groups and EGb761+AFB1group. The content of GSH decreased significantly during43W and53W in AFB1group compared to EGb761+AFB1group (P＜0.05). The change of GSH in normal control group was not obvious during each period of the whole process of experiment.
4. Effect of EGb761on the expression of related cncer genes
4.1Effect of EGb761on the expression of p16Ink4a mRNA and corresponding protein in rat hepatic tissues
The expression of p16Ink4a mRNA is not significantly higher or lower in each group at13thW and33thW(P>0.05), however, in AFB1group and EGb761+AFB1group were significantly lower than that in the control groups during53thW and73thW(P<0.05). The expression of p16Ink4a mRNA in AFB1group was significantly lower than that in the EGb761+AFB1group from53thW to73thW (P＜0.05).
The p16Ink4a protein expressions were not significant difference in each group during13thW and33thW(P>0.05), however, in AFB1group and EGb761+AFB1group were significantly lower than that in the control groups during53thW and73thW(P<0.05). The p16Ink4a protein expression in EGb761+AFB1group was significantly higher than that in the AFB1groups (P＜0.05).
4.2Effect of EGb761on the expression of P53mRNA in rat hepatic tissues
The expression of P53mRNA was low expression and no significant difference in each group during13thW. The expression of P53mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during33thW,53thW and73thW(P<0.01). The expression of P53mRNA in EGb761+AFB1group was significantly higher than that in the AFB1group (P＜0.01).
4.3Effect of EGb761on the expression of IGF-II mRNA and corresponding protein in rat hepatic tissues
The expression of IGF-II mRNA was not significant difference in each group during13thW and33thWCP>0.05). The expression of IGF-II mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control groups during53thW and73thW. The expression of IGF-II mRNA in EGb761+AFB1group was significantly lower than that in the AFB1group (P＜0.01).
The IGF-II protein expressions were low expression or not expression in each group during13thW and33thW. There was not significant difference in each group during13thW. Along with the progress of the experiment, the IGF-II protein expressions increased gradually. The IGF-II protein expressions in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during53thW and73thW. The IGF-II protein expressions in EGb761+AFB1group was significantly lower than that in AFB1group during53thW and73thW (P<0.05).
4.4Effect of EGb761on the expression of NADE mRNA and corresponding protein in rat hepatic tissues
The expression of NADE mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from33thW to73thW and the difference have statistical significance (P<0.01). The expression of NADE mRNA in EGb761+AFB1group was higher than that in the AFB1group. However, there was not statistical significance on the differences between AFB1andEGb761+AFB1group(P>0.05).
The NADE protein expression in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from33thW to73thW (P<0.05). The NADE protein expression in EGb761+AFB1group was slight higher than that in the AFB1group, however, there was not statistical significance on the difference among them (P>0.05).
4.5Effect of EGb761on the expression of p75NTR mRNA and corresponding protein in rat hepatic tissues
The expression of p75NTR mRNA was not significantly difference in each group during13thW and33thW(P>0.05). The expression of p75NTR mRNA in AFB1group and EGb761+AFB1group were significantly lower than that in the control group during53thWand73thW (P＜0.05). The expression of p75NTR mRNA in EGb761+AFB1group was higher than that in the AFB1group (P＜0.01).
The p75NTR protein expression in AFB1group and EGb761+AFB1group were significantly lower than that in the control group from55thW to73thW(P<0.01). The p75NTR protein expression in AFB1group was significantly lower than that in the EGb761+AFBl group from55thW to73thW (P＜0.05).
4.6Effect of EGb761on the expression of COX-2mRNA in rat hepatic tissues
The expression of COX-2mRNA were low expression in AFB1group and EGb761+AFB1group during13thW and33thW. The expression of COX-2mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group during53thW and73thW (P<0.01). There was not significantly difference between AFB1group and EGb761+AFB1group(P>0.05). The expression of COX-2mRNA was low or no expression in control group from13thW to73thW.
4.7Effect of EGb761on the expression of UGT mRNA in rat hepatic tissues
The expression of UGT mRNA in AFB1group and EGb761+AFB1group are significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P<0.01). The expression of UGT mRNA in EGb761+AFB1group are significantly higher than that in the AFB1group during whole of the experimental process and the difference have statistical significance (P<0.01).
4.8Effect of EGb761on the expression of AKR7A mRNA in rat hepatic tissues
The expression of AKR7A mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P＜0.01). The expression of AKR7A mRNA in EGb761+AFB1group are significantly higher than that in the AFB1group(P<0.01).
4.9Effect of EGb761on the expression of GST-Pi mRNA in rat hepatic tissues
The expression of GST-Pi mRNA in AFB1group and EGb761+AFB1group were significantly higher than that in the control group from13thW to73thW and the difference have statistical significance (P<0.01). The AFB1group are not significantly higher than that in AFB1+EGb761group during33thW and53thW. The expression of GST-Pi mRNA in AFB1group was not only significantly higher than that in the control group, but also higher than those in the EGb761+AFB1group during73thW(P<0.01).
5.0Effect of EGb761on the mutual relations of p16Ink4a, IGF-II, NADE and p75NTR protein expression
The protein expression between p16Ink4a and p75NTR in in AFB1group and EGb761+AFB1group was positive correlation during73thW,55thW and33thW(P <0.01).
Conclusions
1. EGb761can reduce the incidence of PHC induced by AFB1. It indicated that EGb761has the effects of anti-cancer development and may be used at the chemoprevention for human PHC related with AFB1.
2. EGb761can restrain the liver enzymes I phase CYP3A4activity, reduced the AFBO formation, and weaken the carcinogenicity of AFB1.
3. EGb761can improve the content of rat liver GSH level, so as to improve the body's anti-oxidation ability, reduced lipid peroxidation damage caused by free radicals in the carcinogenesis induced by AFBl, which is one of the mechanism of EGb761anti-cancer effects.
4. In the carcinogenesis AFB1-induced, EGb761can upregulate the expressive level of tumor-suppressor genes such as P16Ink4a, p53and P75NTR, downregulate the expressive level of IGF-II related proliferation which is one of the foundation of molecular biology of anti-cancer effects.
5. In order to enhance the detoxify ability against the mycotoxin's for organism itself, EGb761can greatly improve the expressive level of metabolic enzymes related detoxification in the liver tissue such as UGT and AKR7A, during hepatocarcinogenesis aflatoxin B1-induced in Wistar Rats.
6. EGb761can cut the expression level of GST-Pi during hepatocarcinogenesis aflatoxin Bl-induced in Wistar Rats.This may be due to the methylation about promotor of CpG-island.
7. There was a positive correlation between the protein expression of p16Ink4a and p75NTR during hepatocarcinogenesis aflatoxin B1-induced in Wistar Rats. This phenomenon was indicated that EGb761can adjust different genes that block the cell cycle, and induced apoptosis of corresponding of tumor cells through the meanwhile, this could be one of its anti-cancer mechanism.
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