【作者】 张翠丽；

【导师】 谢克勤；

【作者基本信息】 山东大学 ， 卫生毒理学， 2012， 博士

【摘要】 1.研究目的肝癌是最常见的肝脏恶性肿瘤,在肿瘤发病率中位居第六,在肿瘤死因中排名第三,但至今仍无有效的治疗方法,因此迫切需要一些有效低毒的抗癌剂预防或抑制肝癌的进展。二乙基亚硝胺(N-nitrosodiethylamine, NDEA)是最重要的环境致癌物之一,是所有动物和人类的潜在致癌物,广泛存在于奶酪、大豆、处理的肉制品、含酒精的饮料、烟草制品、化妆品和农用化学品中,因此NDEA诱发的肝癌已经成为重大的公共卫生问题。大蒜油(garlic oil, GO)是用水蒸气蒸馏法从大蒜中提取的挥发性有机硫化合物的总称,包括30多种硫化物,其中二烯丙基硫、二烯丙基二硫、二烯丙基三硫是其主要的生物活性成分。因为NDEA的代谢活化和活性氧的大量产生是其诱发肝癌的关键步骤,而GO对多种代谢酶有调节作用,同时可清除活性氧,推测GO可能对NDEA诱发的肝癌有预防作用,因此本研究的主要目的是评价GO对NDEA诱发的大鼠肝癌的预防效果并探讨其作用机制。2.研究方法2.1.雄性SPF级Wistar大鼠60只,随机分为4组：正常对照组、NDEA模型组、GO低剂量处理组和GO高剂量处理组。GO处理组提前灌胃20和40mg/kg.bw (2ml/kg,5次/周)的GO1周后,除正常对照组外,其余大鼠均灌胃给予1Omg/kg.bw的NDEA (2ml/kg,5次/周),连续给予20周,灌胃NDEA前4小时GO处理组继续灌胃不同浓度的GO, NDEA模型组灌胃等体积(2ml/kg)的玉米油。20周后处死动物,分别从结节发生率、结节数目、血清学生化指标、组织病理学改变、肝细胞增殖能力等方面评价GO对NDEA诱发的大鼠肝癌的预防效果。2.2.用苯胺作为底物检测肝组织中细胞色素P4502E1(CYP2E1)的活性；使用荧光分光光度计分别测量CYP1A1和CYP1A2催化乙氧基试卤灵或甲基试卤灵脱乙氧基或脱甲基生成试卤灵时荧光强度的变化,计算出肝组织中CYP1A1和CYP1A2的活性；分别使用过氧化氢异丙苯、2,4-二氯-1-硝基苯和利尿酸作为底物检测胞浆中谷胱甘肽毓基转移酶(GSTalpha. GST mu和GST pi)的活性；用对硝基酚作为底物检测尿苷二磷酸-葡萄糖醛酸转移酶(UGT)的活性。2.3.用Trizol试剂提取肝脏总RNA,反转录获得cDNA,用实时定量PCR仪检测肝组织中代谢酶(Cyp, Gst, Ugt)的mRNA表达水平。2.4.将肝脏按1：4(质量/体积比)比例加入匀浆缓冲液(50mM Tris,6.4mM氯化镁,0.2M蔗糖,pH=7.5),冰浴匀浆,12000g,高速离心20分钟(4℃),将上清液用RIPA强裂解液变性溶解后,105,000g离心60分钟,离心后上清为胞浆部分,用Western blotting测定GST alpha、GSTmu、GST pi的蛋白含量,沉淀为微粒体部分,用Western blotting测定CYP2E1、CYP1A1、CYP1A2和UGT1A6的蛋白含量。2.5.肝组织按1：9(质量／体积比)加入匀浆液(0.0M Tris-HCl,0.0001MEDTA-2Na,0.01M葡萄糖、0.8%氯化钠)匀浆,1000×g离心20分钟(4℃),收集上清。通过测量肝匀浆上清中硫代巴比妥酸的蓄积量反应机体的脂质过氧化(Lipid peroxidation, LPO)水平,结果用丙二醛(malondialdehyde,MDA)含量表示。MDA、谷胱甘肽(reduced glutathione, GSH)含量测定和超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、谷胱甘肽还原酶(glutathione reductase, GR)、谷胱甘肽过氧化物酶(glutathione peroxidase, GPx)和谷胱甘肽巯基转移酶(glutathione S-transferase, GST)活性测定参照试剂盒说明书进行。2.6.将肝脏组织按质量/体积比1：5加入预冷的强RIPA裂解液[50mM Tris-HCl,pH7.4,50mM NaCl,1%曲拉通X-100,1%脱氧胆酸钠,0.1%SDS,50mMβ-巯基乙醇、1mM钒酸钠、5mM氟化钠、1%Cocktail蛋白酶抑制剂,用前数分钟内加入苯甲基磺酰氟(phenylmethanesulfonyl fluoride, PMSF),使PMSF的最终浓度为1mM],3000转/分钟匀浆,冰浴放置30分钟后14,000×g离心15分钟,用Western blotting检测肝匀浆中磷酸肌醇3激酶(phosphoinositide3-kinase, PI3K)、蛋白激酶B(也称AKT)、核因子κB(nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB)及凋亡相关蛋白(Bcl-2、Bcl-xl、β-arrestin-2、Bax、Caspase-3)的变化。2.7.用Trizol试剂提取肝脏中总RNA,反转录获得cDNA,用实时定量PCR仪检测肝组织中凋亡相关基因(Bcl-2、Bcl-xl、β-arrestin-2、Bax、Caspase-3)的mRNA表达水平。3.研究结果3.1.GO对NDEA诱发的肝癌的预防效果3.1.1.GO和NDEA对肝脏外观的影响大鼠长期(20周)低剂量接触NDEA (10mg/kg.bw)后,所有大鼠肝脏表面均散在大小不等的灰白色结节,即结节发生率为100%,肝脏明显肿大,有4例形成明显的巨瘤块,直径大于3cm,而且有明显的坏死灶；GO处理组肝脏表面结节明显减少,体积减小,GO高、低剂量处理组结节发生率分别为73%和87%。3.1.2.GO和NDEA对大鼠体重和相对肝重的影响NDEA模型组大鼠体重增长缓慢,随着NDEA染毒时间的延长,大鼠平均体重增重逐渐减少,动物处理14周后,NDEA模型组大鼠体重呈现负增长。肝重增大,所以相对肝重明显增加；与NDEA模型组相比,GO处理组动物体重增重明显增加(P<0.05或P<0.01)；实验结束时,与NDEA模型组相比,GO低、高剂量组大鼠体重明显增加,肝重减小,因而肝脏系数明显下降。3.1.3.GO和NDEA对血清生化指标的影响与正常对照组比较,NDEA模型组大鼠血清中ALT、AST、ALP、γ-GT的活性和AFP的含量均明显升高(P<0.01),表明NDEA处理使大鼠肝脏发生明显的肝损伤和癌前病变；GO低、高剂量处理组各项指标较NDEA模型组明显降低(P<0.05或P<0.01)；两个GO处理组间比较无统计学差异(P>0.05)。3.1.4.GO和NDEA对肝脏病理组织学的影响正常对照组大鼠肝组织结构正常,无自发性肝癌,肝小叶结构清晰,肝细胞结构完整,无明显的纤维化和胶原聚集。与正常对照组相比,NDEA模型组正常肝小叶结构被破坏,癌细胞分化程度低：细胞异型性明显如多核、核固缩、核内空泡等,细胞核增大,核仁增大增多,染色质粗糙,分裂相多见,胞浆稀少,可见瘤巨细胞,癌巢常推挤或浸润周围组织。GO处理组镜下可见肝小叶结构相对较好,癌细胞分化程度高。天狼猩红染色结果表明,正常对照组几乎未见天狼猩红阳性着色,NDEA模型组天狼猩红染色阳性区域明显增加；与NDEA模型组相比,GO处理组天狼猩红染色阳性区域明显减少,肝组织纤维化程度明显减轻。3.1.5.GO和NDEA对肝脏中PCNA表达的影响免疫组织化学结果显示,正常对照组大鼠肝脏病理切片偶见PCNA阳性染色,但NDEA模型组可见大量的PCNA阳性染色(P<0.01)；与NDEA模型组相比,GO预处理明显减少了PCNA的表达,并存在剂量-效应关系,表明GO明显阻断了NDEA诱导的细胞增殖。3.2.GO对肝脏Ⅰ相和Ⅱ相代谢酶的影响3.2.1.GO和NDEA对Ⅰ相代谢酶活性、mRNA水平和蛋白含量的影响3.2.1.1.GO和NDEA对Ⅰ相代谢酶活性的影响与正常对照组相比,NDEA处理20周后,大鼠肝脏中CYP2E1和CYP1A2的活性分别降低了72%和89%,而CYP1A1的活性升高了66%；与NDEA模型组相比,GO显著抑制了NDEA诱导的CYP2E1、CYP1A2的活性降低和CYP1A1的活性升高(P<0.05或P<0.01)3.2.1.2.GO和NDEA对Ⅰ相代谢酶mRNA水平的影响与正常对照组相比,NDEA模型组大鼠Cyplal基因表达水平升高了约1倍,Cyp2el和Cyp1a2的基因表达水平分别降低了26%和51%(P<0.01)；与NDEA模型组相比,长期处理高剂量的GO部分逆转了NDEA诱导的Cyp基因的变化(P<0.01或P<0.05)。3.2.1.3.GO和NDEA对Ⅰ相代谢酶蛋白水平的影响与正常对照组相比,NDEA模型组大鼠CYP2E1和CYP1A2的蛋白含量分别降低了97%和96%(P<0.01),预处理GO有效的抑制了NDEA诱导的CYP2E1和CYP1A2的蛋白含量的降低(P<0.01)。CYP1Al在正常对照组表达水平很低,长期低剂量处理NDEA显著增加了CYP1A1的蛋白表达水平(约为正常对照组的500倍)(P<0.01)；与NDEA模型组相比,GO低、高剂量处理组CYP1A1的蛋白表达水平分别降低了40%和81%(P<0.01)。3.2.2.GO和NDEA对Ⅱ相代谢酶GST alpha、GST mu和GST pi活性、mRNA水平和蛋白含量的影响3.2.2.1.GO和NDEA对Ⅱ相代谢酶GSTalpha、GST mu和GST pi活性的影响与正常对照组相比,NDEA处理20周后,大鼠肝脏中GST alpha和GSTmu的活性分别降低了37%和26%,而肿瘤标志物GST pi的活性升高了2.1倍；与NDEA模型组相比,GO显著抑制了NDEA诱导的GST alpha、GST mu的活性降低和GST pi的活性升高(P<0.01)。3.2.2.2.GO和NDEA对Ⅱ相代谢酶Gst alpha、Gst mu和Gst pi mRNA水平的影响与正常对照组相比,NDEA模型组大鼠Gst pi基因表达水平升高了67%,Gst alpha和Gst mu的基因表达水平分别降低了37%和24%(P<0.01)；与NDEA模型组相比,低、高剂量GO处理组均明显逆转了NDEA诱导的Gst基因的变化(P<0.01或P<0.05),而且有剂量-效应关系。3.2.2.3.GO和NDEA对Ⅱ相代谢酶GST alpha、GST mu和GST pi蛋白含量的影响GST pi在正常对照组未检测到,而NDEA模型组GST pi的蛋白表达水平很高。与NDEA模型组相比,高剂量GO处理组GST pi的蛋白表达水平明显下降(P<0.01)；而GO低、高剂量处理组GST alpha和GST mu的蛋白表达水平明显回升(P<0.01),并有剂量-效应关系。3.2.3.GO和NDEA对Ⅱ相代谢酶UGT活性、mRNA水平和蛋白含量的影响3.2.3.1.GO和NDEA对Ⅱ相代谢酶UGT活性的影响与正常对照组相比,NDEA处理20周后,大鼠肝脏中UGT的总活性降低了47%(P<0.01)；预处理GO可明显抑制NDEA诱导的UGT总活性的降低(P<0.01)。3.2.3.2.GO和NDEA对Ⅱ相代谢酶Ugt1a1和Ugt1a6mRNA表达水平的影响与正常对照组相比,NDEA模型组大鼠Ugt1a6基因的表达水平降低了35%(P<0.01)；与NDEA模型组相比,低、高剂量GO处理组均明显抑制了NDEA诱导的Ugt1a6基因的降低(P<0.05或P<0.01)。然而不同组Ugt1al的mRNA表达水平无明显改变。3.2.3.3.GO和NDEA对Ⅱ相代谢酶UGTlA6蛋白含量的影响与正常对照组相比,NDEA模型组大鼠UGT1A6的蛋白表达水平降低了约50%(P<0.01)；与NDEA模型组相比,低、高剂量GO处理组均明显抑制了NDEA诱导的UGT1A6蛋白含量的的降低(P<0.05或P<0.01),并且有剂量-效应关系。3.3.GO和NDEA对脂质过氧化和抗氧化系统的影响3.3.1.GO和NDEA对大鼠肝脏中脂质过氧化产物MDA含量的影响NDEA长期低剂量摄入引起大鼠肝脏中脂质过氧化产物MDA含量的明显增加,约为正常对照组的1.6倍(P<0.01)。与NDEA模型组相比,GO预处理显著降低了肝脏中MDA的含量,其中GO低、高剂量处理组MDA含量分别降低了34%和44%(P<0.01),而GO低、高剂量处理组MDA含量与正常对照组相比无统计学差异(P>0.05)。3.3.2.GO和NDEA对大鼠肝脏中抗氧化物质GSH含量的影响与正常对照组相比,NDEA长期低剂量摄入导致大鼠肝脏中抗氧化物质GSH的耗竭,NDEA模型组的GSH含量仅为正常对照组的6%(P<0.01)。与NDEA模型组相比,GO预处理明显增加了肝脏中抗氧化物质GSH的含量,其中GO低、高剂量处理组GSH的含量分别是NDEA模型组的2.4倍和7.4倍(P<0.01))。3.3.3.GO和NDEA对大鼠肝脏中抗氧化酶活性的影响与正常对照组相比,NDEA长期低剂量摄入导致大鼠肝脏中不同抗氧化酶活性的明显降低(P<0.01)；GO预处理明显逆转了NDEA诱导的抗氧化酶活性的降低,与NDEA模型组相比,GO低、高剂量处理组SOD、CAT、GPx、GR和GST的活性分别升高了29%、27%、75%、47%、122%(P<0.05或P<0.01)和24%、34%、94%、98%、187%(P<0.01)。3.3.4.GO抑制了NDEA诱导的8-OHdG的形成大鼠持续暴露于NDEA20周后,肝组织DNA中8-OHdG的含量是正常对照组的2.4倍(P<0.01)。提前给予GO处理后,抑制了NDEA诱导的8-OHdG的升高；与NDEA模型组相比,GO低、高剂量组8-OHdG的含量分别降低了29%(P<0.05)和44%(P<0.01)。3.4. PI3K-AKT-NFκB及凋亡通路在GO预防NDEA肝癌中的作用3.4.1.GO和NDEA对PI3K的影响PI3K的两个亚基p85和p110的蛋白含量在NDEA慢性染毒大鼠肝组织中均明显增加,与正常对照组相比,p85和p110的蛋白含量分别为正常对照组的1.8倍(P<0.01)和2.5倍(P<0.01)；提前给予GO,可显著抑制NDEA诱导的p85和p110的蛋白含量的增加,与NDEA模型组相比,GO低、高剂量组p85的蛋白含量分别降低了41%(P<0.01)和44%(P<0.01),p110的蛋白含量分别降低了24%(P<0.05)和61%(P<0.01)。3.4.2.GO和NDEA对AKT和磷酸化AKT的影响总AKT在NDEA慢性染毒大鼠肝组织中蛋白表达明显增加,是正常对照组的1.7倍(P<0.01)。大鼠提前灌胃GO后再给予NDEA,显著抑制了大鼠肝脏中总AKT的蛋白表达；与NDEA模型组相比,GO低、高剂量组总AKT的蛋白含量分别降低了41%和47%(P<0.01)。磷酸化AKT是AKT的活性形式,NDEA慢性染毒大鼠后,p-AKT(Thr308)和p-AKT (Ser473)的蛋白含量均明显增加,与正常对照组相比, p-AKT(Thr308)和p-AKT (Ser473)的蛋白含量分别为正常对照组的3.0倍和12.6倍(P<0.01)；NDEA染毒前大鼠灌胃不同剂量的GO,可显著抑制NDEA诱导的p-AKT(Thr308)和p-AKT (Ser473)的增加,其中,与NDEA模型组相比,GO低、高剂量组p-AKT (Thr308)的蛋白含量分别降低了13%和35%(P<0.05),p-AKT(Ser473)的蛋白含量分别降低了36%(P<0.05)和64%(P<0.01)。3.4.3.GO和NDEA对NF-κB和其抑制蛋白IκB的影响大鼠慢性染毒NDEA后,肝组织中P-IκB的含量明显减少,与正常对照组相比,P-IκB的蛋白含量减少了77%(P<0.01)；大鼠预处理GO可显著增加IκB的磷酸化,与NDEA模型组相比,GO低、高剂量组P-IκB的蛋白含量分别为NDEA模型组的1.5倍和2.7倍(P<0.05)。与P-IκB的变化情况相反,大鼠慢性染毒NDEA后,肝组织中NF-kBp65的含量显著增加,约为正常对照组的1.9倍(P<0.01)；大鼠提前给予GO后,对NDEA诱导的p65蛋白含量的增加有明显的抑制作用,其中,与NDEA模型组相比,GO低、高剂量处理组p65的蛋白含量分别降低了33%和39%(P<0.01)。3.4.4.GO和NDEA对抗凋亡蛋白Bcl-2和Bcl-xl的影响3.4.4.1.GO和NDEA对Bcl-2和Bd-xl mRNA表达水平的影响与正常对照组相比,NDEA模型组Bcl-2和Bcl-xl的mRNA表达水平明显增加,分别是正常对照组mRNA表达水平的3.8倍和1.5倍(P<0.01)；GO预处理显著抑制了NDEA诱导的Bcl-2和Bcl-xl的mRNA表达水平的增加(P<0.01),与NDEA模型组相比,GO低、高剂量处理组Bcl-2的mRNA表达水平分别降低了69%和80%(P<0.01)；Bcl-xl的mRNA表达水平分别降低了51%和41%(P<0.01)。而且GO低、高剂量处理组均可将Bcl-2和Bcl-xl的mRNA表达水平调节至正常水平,与正常对照组相比无统计学差异(P>0.05)。3.4.4.2.GO和NDEA对Bcl-2和Bcl-xl蛋白表达水平的影响NDEA长期低剂量暴露后,导致NDEA模型组Bcl-2和Bcl-xl的蛋白含量明显增加,分别是正常对照组的1.6倍和4.0倍(P<0.01)；GO预处理也显著抑制了NDEA诱导的Bcl-2和Bcl-xl的蛋白含量的增加,与NDEA模型组相比,Bcl-2的蛋白含量在GO低、高剂量处理组分别降低了41%(P<0.01)和60%(P<0.01)；Bcl-xl的蛋白含量在GO低、高剂量处理组分别降低了33%(P<0.01)和80%(P<0.01)。3.4.5.GO和NDEA对促凋亡蛋白Bax的影响3.4.5.1.GO和NDEA对Bax的mRNA表达水平的影响与正常对照组相比,NDEA模型组Bax的mRNA表达水平明显减少,约为正常对照组的53%(P<0.01)；GO预处理显著抑制了NDEA诱导的Bax的mRNA表达水平的减少,与NDEA模型组相比,GO低、高剂量组Bax的mRNA表达水平分别为NDEA模型组的1.7倍和2.7倍(P<0.01),而且GO高剂量处理组Bax的mRNA表达水平高于正常对照组(P<0.05),显著促进了NDEA诱导的肝癌细胞的凋亡。3.4.5.2.GO和NDEA对Bax蛋白表达水平的影响与正常对照组相比,NDEA模型组Bax蛋白含量明显下降,约为正常对照组含量的58%(P<0.01)；GO预处理显著抑制了NDEA诱导的Bax蛋白含量的降低,与NDEA模型组相比,GO低、高剂量组Bax的蛋白含量分别为NDEA模型组的2.5倍和4.4倍(P<0.05),而且GO高剂量处理组Bax的蛋白表达水平高于正常对照组(P<0.01)。3.4.6.GO和NDEA对Bcl-2/Bax比值的影响因为与正常对照组相比,NDEA模型组Bcl-2的mRNA表达水平和蛋白含量均明显增加,而Bax的mRNA表达水平和蛋白含量显著减少,所以NDEA模型组大鼠肝脏中Bcl-2/Bax比值明显增加,其中mRNA表达水平的Bcl-2/Bax比值约为正常对照组的7.1倍(P<0.01),蛋白含量的Bcl-2/Bax比值约为正常对照组的2.7倍(P<0.01),因此与正常对照组相比NDEA模型组的细胞凋亡受到明显抑制；GO预处理显著抑制了NDEA诱导的Bcl-2/Bax比值的升高,与NDEA模型组相比,Bcl-2/Bax比值明显减小,其中mRNA表达水平的Bcl-2/Bax比值在GO低、高剂量处理组分别降低了82%(和93%(P<0.01),蛋白含量的Bcl-2/Bax比值在GO低、高剂量处理组分别降低了76%和91%(P<0.01),其中GO高剂量组的Bcl-2/Bax比值在mRNA水平和蛋白水平均低于正常对照组。3.4.7.GO和NDEA对促凋亡执行蛋白酶Caspase-3的影响3.4.7.1.GO和NDEA对Caspase-3的mRNA表达水平的影响与正常对照组相比,NDEA模型组Caspase-3的mRNA表达水平明显减少,约为正常对照组的77%(P<0.05)；GO预处理显著抑制了NDEA诱导的Caspase-3的mRNA表达水平的减少,与NDEA模型组相比,GO低、高剂量组Caspase-3的mRNA表达水平分别为NDEA模型组的1.5倍和2.3倍(P<0.01),而且GO高剂量处理组的Caspase-3的mRNA表达水平显著高于正常对照组(P<0.01)。3.4.7.2.GO和NDEA对Caspase-3的蛋白表达水平的影响与正常对照组相比,NDEA模型组Caspase-3的蛋白含量显著降低,约为正常对照组的62%(P<0.01)；提前给予GO对NDEA引起的Caspase-3的蛋白含量的减少有明显的抑制作用,与NDEA模型组相比,GO低、高剂量组Caspase-3的蛋白含量均约为NDEA模型组的3.0倍(P<0.01),而且也显著高于正常对照组的蛋白表达水平(P<0.01)。3.4.8.GO和NDEA对β-arrestin-2的影响NDEA长期低剂量处理大鼠诱导了β-arrestin-2的mRNA表达和蛋白表达,与正常对照组相比,NDEA模型组的β-arrestin-2的mRNA表达水平和蛋白含量分别为正常对照组的1.9倍和3.4倍(P<0.01)；大鼠提前给予不同剂量的GO显著抑制了NDEA诱导的β-arrestin-2的mRNA和蛋白水平的表达增加,与NDEA模型组相比,β-arrestin-2的mRNA表达水平在GO低、高剂量组分别降低了36%和43%(P<0.01),(3-arrestin-2的蛋白含量在GO低、高剂量组分别降低了52%和69%(P<0.01)。4.结论4.1.分别从结节发生率、结节数目、血清生化指标、病理组织学改变、细胞的增殖能力等方面证实了GO对NDEA诱发的大鼠肝癌进展有明显的预防作用；4.2.GO预防NDEA诱发的肝癌的机制可能与其调节肝脏Ⅰ相代谢酶酶(包括CYP2E1、CYP1A2、CYP1A1)的活性和表达水平、上调Ⅱ相代谢酶(GSTalpha, GSTmu、GST pi、UGT1A1、UGT1A6)的活性和表达水平有关；4.3.大鼠慢性染毒NDEA (10mg/kg.bw,5次／周,20周),可显著增加机体的脂质过氧化水平；提前预处理GO可明显缓解NDEA引起的脂质过氧化；4.4.GO显著抑制了NDEA诱导的GSH含量的减少和SOD、CAT、GPx、GR、GST活性的降低,并明显减轻了NDEA诱导的DNA的氧化损伤,这可能是GO预防NDEA肝癌进展的重要机制之一；4.5.大鼠慢性染毒NDEA后,活化了PI3K-AKT信号通路,并激活下游的NF-κB信号分子,从而抑制凋亡；提前预处理GO可显著抑制NDEA对PI3K-AKT-NF-κB-抗凋亡这一信号通路的激活,这可能是GO预防NDEA肝癌进展的重要机制之一。更多还原

【Abstract】 1. ObjectiveHepatocarcinoma is the most common malignant liver tumors. Hepatocarcinoma ranked sixth in tumor incidence and third in tumor death cause sequence. The conventional therapy of hepatocarcinoma gives little hope for restoration of health because of poor diagnosis and serious side effects. Therefore, developing more effective and less toxic anticancer agents, including natural products, is necessary to prevent or retard the process of hepatocarcinogenesis. N-nitrosodiethylamine (NDEA), which exists widespreadly in nature such as in cheese, soybean, processed meats, alcoholic beverages, tobacco products, cosmetics and agricultural chemicals, is one of the most important environmental carcinogens. So the hepatocarcinoma induced by NDEA has become a major public health problem.The steam distillation method is widely used to extract and condense volatile OSCs from garlic and the final oily product is called garlic oil (GO), which contains more than30OSCs. It has been reported that diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are the three major components involved in GO. Accumulating evidence has demonstrated that the metabolic activation of NDEA and overproduction of reactive oxygen species (ROS) play key roles in the etiology of hepatocarcinoma. Because GO may regulate many metabolic enzymes and can scavenge ROS, we speculate GO could prevent NDEA-induced hepatocarcinogenesis. Therefore, the present study was designed to evaluate the effects of GO on NDEA-induced hepatocarcinogenesis and to explore the mechanisms.2. Methods2.1.60Male Wistar rats were randomly divided into4groups, i.e. normal control group, NDEA group, and two different doses of GO groups. The rats in GO group were treated with GO (20or40mg/kg body weight) by gavage for21weeks (5time/week), while other animals received equal volume of corn oil. From the second week, the rats in GO and NDEA groups were orally received NDEA (10mg/kg body weight,5time/week), while the animals in normal control group were administered volume of saline. At the end of week21, all animals were sacrificed by decapitation and blood was collected. The preventive effects of GO on NDEA induced hepatocarcinoma were evaluated by nodule incidence, number of nodules, serum biochemical indices, histopathological changes and cell proliferation capacity.2.2. The activity of CYP2E1was measured with aniline as the substrate. The activities of CYP1A1and CYP1A2were detected by measurement of the dealkylation of ethoxyresorufin and methoxyresorufin using Hitachi fluorescence spectrophotometer. The activities of cytosolic GST alpha, GST mu and GST pi were determined using cumene hydroperoxide (CuOOH),2,4-dichloro-l-nitrobenzene (DCNB) and ethacrynic acid as substrates. UDP-glucuronosyl-transferase (UGT) activity was determined with p-nitrophenol (PNP) as a substrate.2.3. Total RNA was isolated from the rat livers using Trizol reagent according to the manufacture’s instructions. Complementary DNA was synthesized using the RevertAidTM First Strand cDNA Synthesis Kit. The levels of gene expression of metabolic enzymes in liver tissues were quantified by qRT-PCR.2.4. The liver tissues were homogenized in lysis buffer (50mM Tris,6.4mM magnesium chloride,0.2M sucrose, pH=7.5), then were centrifuged at12OOOg for20min. The supernatant was dissolved in strongly RIPA lysis buffer, and then was centrifuged at105,000g for60min. The supernatant and pellet were the cytosolic and microsomal samples, respectively. The protein contents of GST alpha, GST mu, GST pi in the supernatant and CYP2E1, CYP1A1, CYP1A2, UGT1A6in the pellet were measured by Western blotting.2.5. The liver tissues were homogenized in9volumes of ice-cold buffer (pH7.4) containing0.01M Tris-HCl,0.0001M EDTA-2Na,0.01M saccharose sucrose, and 0.8%saline. The homogenates were centrifuged at1000g for20min at4℃. The supernatant was collected. Lipid peroxidation (LPO) was determined by measuring the accumulation of thiobarbituric acid reactive substance and expressed as MDA content. The levels of MDA, GSH and the activities of antioxidant enzymes including SOD, CAT, GPx, GR, and GST were assayed using commercial assay kits according to the manufacturer’s instructions.2.6. The liver tissues were homogenized in lysis buffer (50mM Tris-HCl,150mM NaCl,1%Triton X-100,1%sodium deoxychlolate,0.1%sodium dodecylsulphate,50mM beta-glycerophosphate,1mM Na3VO4,5mM NaF,1%cocktail protein inhibitors and1mM phenylmethylsulfphonylfloride), then were centrifuged at14OOOg for15min. Western blotting was used to detect the protein contents of phosphoinositide3-kinase (PI3K), AKT, NF-κB, Bcl-2, Bcl-xl, β-arrestin-2, Bax and Caspase-3.2.7. Total RNA was isolated from the rat livers using Trizol reagent. Complementary DNA was synthesized using cDNA Synthesis Kit. The levels of gene expression of apoptotic related genes (Bcl-2, Bcl-xl, β-arrestin-2, Bax and Caspase-3) in liver tissues were quantified by qRT-PCR.3. Results3.1. The preventive effect of GO on NDEA-induced hepatocarcinoma in rats3.1.1. Morphological changes of the liverThe appearance of livers in normal control group was normal and no macroscopically detectable nodules. The rats in NDEA group revealed enlarged liver. The nodule incidence of NDEA group was100%and the maximun diameter of nodules was about10mm. At the same time, there were clear necrosis regions in livers of NDEA-treated rats. Interestingly, a significant reduction in liver enlargement, nodule incidence and average nodule numbers per nodule-bearing liver was observed in GO-treated rats compared to that of NDEA group.3.1.2. Changes of body weight and relative liver weightIn the first week following NDEA treatment, the rats began to show a slow growth. Along with the NDEA exposure, the mean weight gain decreased gradually. After14weeks of exposure, the body weight in NDEA group showed negative growth. NDEA treatment increased the relative liver weight when compared with the normal control group (P<0.01). Administration of20and40mg/kg.bw GO significantly reduced the relative liver weight when compared with the NDEA group (P<0.01).3.1.3. Serum biochemical assays.NDEA administration led to the elevation of serum ALT, AST, ALP, γ-GT and AFP levels compared with those in normal control group (P<0.01), indicating liver damage and preneoplastic lesion. All these above adverse effects of NDEA were suppressed by GO.3.1.4The histological changes of the liverThe histological examinations were performed to evaluate the effects of GO and NDEA on the morphological changes and the collagen accumulation. The liver tissues of rats in normal control group revealed regular hepatic lobules without obvious fibrotic area, which were illustrated by H&E staining and sirius red staining, respectively. In contrast, the liver sections from the rats in the NDEA group showed collapse of hepatic lobules and the obvious pleomorphism such as multiple nucleoli, pyknotic nuclei, and intranuclear vacuoles. Furthermore, the sirius red positive areas were markedly increased in the NDEA-treated rats. Interestingly, the liver sections of rats in GO pretreatment groups showed fewer degenerative changes, and less sirius red positive areas.3.1.5. The changes of PCNA expression by immunohistochemistryThe positive staining of PCNA occasionally appeared in the liver sections of the normal control group, which was dramatically increased in the hepatic sections of NDEA group. However, GO pretreatment significantly decreased the number of PCNA positive nuclei when compared with the NDEA group.3.2. The changes of phase Ⅰ and phase Ⅱ metabolic enzymes3.2.1. Effects of GO and NDEA on the activities, mRNA and protein levels of phase Ⅰ enzymes 3.2.1.1. Effects of GO and NDEA on the activities of phase I enzymesCompared with the normal control group, the activities of CYP2E1and CYP1A2were decreased by72%and89%(P<0.01), respectively, while the activity of CYP1A1was increased by66%in rats of NDEA group (P<0.01). The changes induced by NDEA were inhibited by GO pretreatment.3.2.1.2. Effects of GO and NDEA on the mRNA levels of phase I enzymesCompared with the normal control group, the mRNA levels of Cyp2e1and Cyp1a1were decreased by26%and51%(P<0.01), respectively, while the mRNA level of Cyp1a1was about2-fold in rats of NDEA-groups (P<0.01). GO significantly inhibited these changes induced by NDEA.3.2.1.3. Effects of GO and NDEA on the protein levels of phase I enzymesCompared with the normal control group, the protein levels of CYP2E1and. CYP1A2were decreased by97%and96%(P<0.01). GO pretreatment can inhibit these decreases induced by NDEA. The protein level of CYP1A1in rats of NDEA group was almost50-fold greater than the corresponding control value (P<0.01). Compared with the NDEA group, the protein content of CYP1A1was decreased by40%and81%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively.3.2.2. Effects of GO and NDEA on the activities, mRNA and protein levels of GST alpha, GST mu and GST pi3.2.2.1. Effects of GO and NDEA on the activities of GST alpha, GST mu and GST piCompared with the control value, the activities of GST alpha and GST mu were slightly but significantly decreased, however, the activity of GST pi was sharply increased (about3.1fold) in NDEA group (P<0.01). Compared with the NDEA group, GO pretreatment significantly inhibited the activation of GST pi, and the inactivation of GST alpha and GST mu (P<0.01). Interestingly, all these above changes induced by NDEA were simultaneously ameliorated by GO pretreatment.3.2.2.2. Effects of GO and NDEA on the mRNA levels of Gst alpha, Gst mu and Gst piThe mRNA levels of Gst alpha and Gst mu in rats of NDEA group were decreased by37%and24%(P<0.01) compared with those in normal control group, respectively, while the mRNA levels of Gst pi was increased by67%(P<0.01). The changes of the mRNA levels induced by NDEA were also significantly attenuated by GO pretreatment.3.2.2.3. Effects of GO and NDEA on the protein levels of GST alpha, GST mu and GST piGST pi was not detected in the normal control group. NDEA significantly induced GST pi protein expression, but reduced the protein levels of GST alpha and GST mu. Again, GO obviously suppressed the decreases of the GST alpha and GST mu protein levels and the increase of the GST pi protein level.3.2.3. Effects of GO and NDEA on the activities, mRNA and protein levels of UGTs3.2.3.1. Effect of GO and NDEA on the activity of UGTsThe activity of UGTs in rats of NDEA group was decreased by47%compared with the control value (P<0.01), which was significantly inhibited by GO pretreatment.3.2.3.2. Effects of GO and NDEA on the mRNA levels of UgtsCompared with the normal control group, the mRNA level of Ugt1a6was significantly decreased by35%(P<0.01) in NDEA group. GO suppressed NDEA-induced decrease of the mRNA level of Ugt1a6. However, no significant alteration of the mRNA level of Ugt1a1was observed in all groups.3.2.3.3. Effect of GO and NDEA on the protein level of UGT1A6The protein level of UGT1A6in NDEA group was significantly decreased by50%(P<0.01), when compared to normal control group. GO pretreatment significantly suppressed NDEA-induced decrease of the protein level of UGT1A6.3.3. Effects of GO and NDEA on LPO and antioxidant systems in NDEA-induced hepatocarcinoma3.3.1. Effect of GO and NDEA on MDA level in rat liverNDEA significantly increased the MDA level (about1.6-fold), when compared to the normal control group. Compared with that of NDEA group, the MDA level was decreased by34%and44%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively. No significant difference of MDA level was observed between GO treatment groups and normal control group (P>0.05).3.3.2. Effect of GO and NDEA on GSH level in rat liverCompared with the normal control group, the GSH level was significantly decreased by94%in NDEA group. Compared with the NDEA group, GO pretreatment significantly increased the GSH level. The GSH level was about2.4-fold and7.4-fold in20and40mg/kg.bw GO treatment groups(P<0.01), respectively, when compared to the NDEA group.3.3.3. Effects of GO and NDEA on the activities of antioxidant enzymes in rat liverNDEA caused significant decreases in the activities of SOD, CAT, GPx, GR, and GST (P<0.01). Compared with NDEA group, GO pretreatment significantly increased the activities of SOD, CAT, GPx, GR and GST by29%,27%,75%,47%,122%(P<0.05or P<0.01) and24%,34%,94%,98%,187%(P<0.01) in20and40mg/kg.bw GO treatment groups, respectively.3.3.4. GO inhibited the formation of8-OHdG in liver DNA induced by NDEAAfter20weeks of NDEA treatment, the content of8-OHdG was augmented to2.4-fold (P<0.01) compared with that in normal control group. However, this elevation of8-OHdG induced by NDEA was significantly reduced in GO treatment groups and decreased by29%(P<0.05) and44%(P<0.01) in20and40mg/kg.bw treatment groups, respectively.3.4. Effects of GO and NDEA on PI3K-AKT-NFκB and apoptosis3.4.1. Eeffect of GO and NDEA on PI3KNDEA caused significant increases in the protein contents of p85and p110. Compared with the normal control group, the protein contents of p85and p110were about1.8-fold (P<0.01) and2.5-fold (P<0.01) in NDEA group. GO pretreatment can significantly inhibit the increases induced by NDEA. Compared with those of NDEA group, the protein content of p85was decreased by41%and44%(P<0.01) in20and40mg/kg.bw GO pretreatment groups; while the protein content of p110was decreased by24%and61%in20and40mg/kg.bw GO pretreatment groups (P<0.01), respectively. 3.4.2. Effects of GO and NDEA on AKT and AKT phosphorylationNDEA significantly increased the protein content of total AKT (about1.7-fold), when compared with the normal control group. Compared with that of NDEA group, the protein content of total AKT was decreased by41%and47%in20and40mg/kg.bw GO pretreatment groups (P<0.01), respectively.Phosphorylated AKT is the active form of AKT. NDEA administration led to the elevation of the protein contents of p-AKT (Thr308) and p-AKT (Ser473). The protein contents of p-AKT (Thr308) and p-AKT (Ser473) in NDEA group was about3.0-fold and12.6-fold (P<0.01), when compared to the normal control group.Compared with the NDEA group, the protein content of p-AKT (Thr308) was decreased by13%and35%in20and40mg/kg.bw GO pretreatment groups (P<0.05), while the protein content of p-AKT (Ser473) was decreased by36%(P<0.05) and64%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively.3.4.3. Effects of GO and NDEA on IκB and NF-κBCompared with the normal control group, the protein level of p-IκB was decreased by77%(P<0.01) in NDEA group. GO pretreatment can enhance the phosphorylation of IκB. Compared with the NDEA group, the protein content of p-IκB was about1.5-fold and2.7-fold (P<0.05) in20and40mg/kg.bw GO pretreatment groups, respectively. NDEA significantly increased the protein content of NF-κB p65(about1.9-fold), when compared with the normal control group. Compared with that of NDEA group, the protein content of NF-kB p65was decreased by33%and39%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively.3.4.4. Effects of GO and NDEA on Bcl-2and Bcl-xl3.4.4.1. Effects of GO and NDEA on the mRNA levels of Bcl-2and Bcl-xlQuantitative real-time PCR results showed significant increases in mRNA expression levels of anti-apoptotic Bcl-2and Bcl-xl in NDEA group. Compared with the normal control group, the mRNA expression levels of Bcl-2and Bcl-xl in NDEA group were increased to3.8-fold and1.5-fold (P<0.01), respectively. Compared with the NDEA group, the mRNA level of Bcl-2in20and40mg/kg.bw GO pretreatment groups was decreased by69%and80%(P<0.01), respectively, while the mRNA expression level of Bcl-xl was decreased by51%and41%(P<0.01), respectively.3.4.4.2. Effects of GO and NDEA on the protein levels of Bcl-2and Bcl-xlCompared with the normal control group, the protein contents of Bcl-2and Bcl-xl in NDEA group were increased to1.6-fold and4.0-fold (P<0.01), respectively. GO pretreatment inhibited the increases of Bcl-2and Bcl-xl induced by NDEA treatment. Compared with the NDEA group, the protein content of Bcl-2in20and40mg/kg.bw GO pretreatment groups was decreased by41%and60%(P<0.01), respectively, while the protein content of Bcl-xl was decreased by33%and80%(P<0.01), respectively.3.4.5. Effect of GO and NDEA on Bax3.4.5.1. Effect of GO and NDEA on the mRNA level of BaxCompared with the normal control group, the mRNA expression level of Bax in NDEA group was decreased by47%(P<0.01). Compared with the NDEA group, the mRNA level of Bax in20and40mg/kg.bw GO pretreatment groups was increased to1.7-fold and2.7-fold (P<0.01), respectively. Furthermore, the mRNA level of Bax in40mg/kg.bw GO pretreatment group was higher than that of the normal control group (P<0.05).3.4.5.2. Effect of GO and NDEA on the protein level of BaxCompared with the normal control group, the protein content of Bax in NDEA group was decreased by42%(P<0.01). Compared with the NDEA group, the protein content of Bax in20and40mg/kg.bw GO pretreatment groups was increased to2.5-fold and4.4-fold (P<0.01), respectively. Furthermore, the protein content of Bax in40mg/kg.bw GO pretreatment group was higher than that in the normal control group (P<0.01).3.4.6. Effect of GO and NDEA on Bcl-2/Bax ratioCompared with the normal control group, the mRNA and protein expression levels of Bcl-2were increased in NDEA group, while the mRNA and protein expression levels of Bax were decreased. So the ratio of Bcl-2/Bax in NDEA group was significantly increased. Compared with the normal control group, the Bcl-2/Bax ratio of mRNA level was increased to7.1-fold (P<0.01), while the Bcl-2/Bax ratio of protein content was increased to2.7-fold (P<0.01) in NDEA group. GO pretreatment inhibited the increases of Bcl-2/Bax ratio induced by NDEA treatment. Compared with the NDEA group, the Bcl-2/Bax ratio of mRNA level in20and40mg/kg.bw GO pretreatment groups was decreased by82%and93%(P<0.01), respectively, while the Bcl-2/Bax ratio of protein content was decreased by76%and91%(P<0.01), respectively. Furthermore, the Bcl-2/Bax ratio in40mg/kg.bw GO pretreatment group was lower than that in the normal control group.3.4.7. Effect of GO and NDEA on Caspase-33.4.7.1. Effect of GO and NDEA on the mRNA level of Caspase-3Compared with the normal control group, the mRNA expression level of Caspase-3in NDEA group was decreased by23%(P<0.05). Compared with the NDEA group, the mRNA level of Bax in20and40mg/kg.bw GOpretreatment groups was increased to1.5-fold and2.3-fold (P<0.01), respectively. Furthermore, the mRNA level of Caspase-3in40mg/kg.bw GOpretreatment group was higher than that in the normal control group (P<0.01).3.4.7.2. Effect of GO and NDEA on the protein level of Caspase-3Compared with the normal control group, the protein content of Caspase-3in NDEA group was decreased by38%(P<0.01). Compared with the NDEA group, the protein content of Bax in20and40mg/kg.bw GO pretreatment groups was increased to about3.0-fold (P<0.01). Furthermore, the protein content of Caspase-3in20and40mg/kg.bw GO pretreatment groups was higher than that in the normal control group (P<0.01).3.4.8. Effect of GO and NDEA on β-arrestin-2Recently, β-arrestin-2was shown to mediate anti-apoptotic signaling, so we detected the expression levels of β-arrestin-2in liver tissues. Compared with the normal control group, the mRNA and protein expression levels of β-arrestin-2in NDEA group were increased to1.9-fold and3.4-fold (P<0.01), respectively. Pretreatment with GO caused marked decreases at both the mRNA and protein levels. Compared with the NDEA group, the mRNA level of β-arrestin-2in20and40 mg/kg.bw GO pretreatment groups was decreased by36%and43%(P<0.01), respectively, while the protein expression level of β-arrestin-2was decreased by52%and69%(P<0.01), respectively.4. Conclusion4.1. GO obviously prevented NDEA-induced hepatocarcinogenesis in terms of nodule incidence, number of nodules, serum biochemical parameters, histopathological changes and cell proliferation capacity.4.2. GO regulated the activities and expression levels of phase I metabolizing enzyme (CYP2E1, CYP1A2, CYP1A1) and phase Ⅱ metabolizing enzymes (GST alpha, GST mu, GST pi, UGT1A1, UGT1A6), which maybe contribute to its prevention against NDEA-induced hepatocarcinogenesis.4.3. Chronic exposure of rats to NDEA (lOmg/kg.bw,5times/week,20weeks) significantly increased lipid peroxidation; GO pretreatment alleviated lipid peroxidation induced by NDEA.4.4. GO significantly inhibited NDEA-induced reduction of GSH content and decrease of SOD, CAT, GPx, GR, GST activities, and notably relieved DNA oxidative damage induced by NDEA, which might be associated with its prevention against NDEA-induced hepatocarcinogenesis.4.5. Chronic exposure of rats to NDEA activated the PI3K-AKT signaling pathway and downstream NF-κB signaling molecule, thus inhibiting apoptosis. GO pretreatment significantly inhibited the activation of PI3K-AKT-NF-KB-anti-apoptosis signaling pathway, which suggested the downregulation of PI3K-AKT-NF-κB-anti-apoptosis by GO pretreatment was involved in its prevention against NDEA-induced hepatocarcinogenesis.更多还原