Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine‐induced hepatocellular carcinoma

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long‐term diseases including hepatocellular carcinoma (HCC ). Melatonin has been reported to alleviate promotion and progression of HCC , but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN ) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg^?1d^?1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK 1ε, Rev‐erbα, and Rev‐erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT 1 receptor and reduced levels of the hypoxia‐inducible factors Hif‐1α and Hif‐2α. An increased expression of p21, p53, and PARP 1/2, a higher Bax/Bcl‐2 ratio, and a lower expression of Cyclin D1, CDK 6, HSP 70, HSP 90, and GRP 78 proteins were also observed in melatonin‐treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV ‐ERB agonist SR 9009 in human Hep3B cells, and BMAL 1 knocking down attenuated the pro‐apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.     

Chemoprevention of liver cancer by plant polyphenols

Primary liver cancer or hepatocellular carcinoma (HCC) is one of the most frequent tumors representing the fifth commonest malignancy worldwide and the third cause of mortality from cancer. Currently, the treatments for HCC are not so effective and new strategies are needed for its fight. Chemoprevention, the use of natural or synthetic chemical agents to reverse, suppress or prevent carcinogenesis is considered an important way for confronting HCC. Many of the chemopreventive agents are phytochemicals, namely non-nutritive plant chemicals with protective or disease preventive properties. In this review, we focus on plant polyphenols, one of the most important classes of phytochemicals, their chemopreventive properties against HCC and discuss the molecular mechanisms accounting for this activity.     

二乙基亚硝胺诱导大鼠肝癌模型的建立及评价

目的：探讨二乙基亚硝胺诱发大鼠肝癌模型的方法。方法：选取体重150～200 g的雄性Wistar大鼠120只,随机分为建模组和对照组。建模组实验大鼠以25 mg/kg的剂量进行二乙基亚硝胺腹腔注射,每周3次,并喂饲含30%酪蛋白和0.05%苯巴比妥钠的饲料。对照组大鼠以建模组的剂量和注射频率用生理盐水进行腹腔注射。在实验第10～18周,每隔2周对实验大鼠肝脏的肿瘤发病情况进行影像检查,将检查结果为阳性的大鼠处死做解剖分析,检查结果为阴性的大鼠继续饲养。结果：在18周时诱癌成功率为76.67%,病理学结果显示,建模组实验大鼠肝脏的病变包括了肝细胞的损伤坏死期、增生硬化期和癌变期。结论：通过长时间多次小剂量二乙基亚硝胺腹腔注射的方法,成功建立了二乙基亚硝胺的大鼠肝癌模型,此模型是研究人体肝癌成因及发展的理想可靠的活体动物模型。     

复方半枝莲防治二乙基亚硝胺诱发大鼠肝癌的研究

目的 :研究复方半枝莲 (SBC)对二乙基亚硝胺 (DEN)诱发大鼠肝癌的防治作用。方法 :利用免疫组化、流式细胞仪、血清和组织生化等检测方法 ,分别在第 14周和第 2 4周观察 SBC对 DEN诱发的肝癌形成过程的影响。结果 :中药组 14周时大鼠肝脏异型性增生灶明显较模型组少 ,2 4周时形成的肝癌结节小而少 ,模型组、中药组肝癌发生率分别为 75 .0 %、5 0 .0 % ;免疫组化显示中药组大鼠肝组织谷胱甘肽 - S-转移酶胎盘型阳性灶面积明显低于模型组 ;肝组织匀浆上清液谷胱甘肽 - S-转移酶含量以及血清 λ-谷氨酰转移酶、碱性磷酸酶、谷丙转氨酶含量也明显低于模型组 ;流式细胞仪检测结果显示 ,中药组大鼠肝细胞 G0 - G1 期比例下降 ,G2 - M期比例升高。结论 :SBC能抑制癌前病变 ,延缓肝癌的形成 ,降低肝癌发病率 ,其作用机制之一可能为阻滞 G2 - M期细胞进展 ,从而抑制DEN引起的肝细胞的去分化和恶性增殖     

Echographic detection of diethylnitrosamine-induced liver tumors in rats and the effect of the intratumoral injection of an inhibitor of c-Jun N-terminal kinase

Background and Aim:  There have so far been few reports describing echographic studies of chemically-induced carcinogenesis in rodent livers. Using echography, we observed diethylnitrosamine-induced liver tumors in rats and examined the effect of an intratumoral injection of an inhibitor of c-Jun N-terminal kinase.
Methods:  Male Wistar rats were given 100 ppm of diethylnitrosamine for 6 weeks and their liver nodules were examined by echography weekly. The size of the nodules was measured and they were examined histologically. The effect of SP600125, an inhibitor of c-Jun N-terminal kinase, on the growth of rat hepatoma cell line McA-RH7777 was tested in vitro . Thereafter, SP600125 was injected into the liver nodules under echographic guidance in vivo and the changes in the proliferating cell nuclear antigen expression and size of the nodules were examined.
Results:  The four distinct lobes of rat livers were clearly observed by transabdominal echography. The nodules in the livers were first detected 6 weeks after the treatment began, when they were as small as 1.6 mm in diameter. The nodules thereafter became more malignant histologically as they grew larger than 4 mm. SP600125 decreased the expression of proliferating cell nuclear antigen and the growth of McA-RH7777 cells. After SP600125 was injected in vivo , the proliferating cell nuclear antigen level and the growth rate of the rat liver nodules all significantly decreased.
Conclusions:  Our results indicate that echography is quite useful for follow-up studies of liver carcinogenesis in rats, and c-Jun N-terminal kinase might be another therapeutic target in liver neoplasms.     

二乙基亚硝胺联合N-亚硝基吗啉诱导大鼠肝癌模型的实验病理研究

目的建立二乙基亚硝胺（diethylnitrosamine，DEN）联合Ⅳ-亚硝基吗啉（N-nitrosomorpholine，NMOR）诱导SD大鼠的肝癌模型并初步动态观察肝脏病理形态变化及肝癌肺转移。方法按100mS／kg体重腹腔注射一次DEN，第二天开始给予含100ppmNMOR饮水供鼠自由饮用的方法诱导SD大鼠肝癌模型，病理常规HE染色动态观察。结果病理学检查证实DEN＋NMOR成功诱导出具有转移能力的肝癌模型，20周时肝癌发生率为87．5％（21／24），肺转移发生率为47．6％（10／21）。病理过程大致经过肝细胞损伤期、肝细胞增生硬化期、肝细胞癌变和肺转移发生等时期。结论该肝癌模型可以作为研究人类肝癌发生、发展和转移的一种理想的动物模型。     

THE IMMUNOHISTOCHEMISTRY AND IN SITU cDNA-mRNA HYBRIDIZATION OF CARBAMYL PHOSPHATE SYNTHETASE I IN ENZYME-ALTERED LIVER CELLS DURING CARCINOGENESIS

The changes of carbamyl phosphate synthetase I(CPS 1)in diethylnitrosamine-(DEN)-inducedenzyme-altered liver cells were studied by means of immunohistochemical(PAP)and in situcDNA-mRNA hybridization methods.The experimental rats were treated with DEN,2-acetylaminofluorene(2-AAF)and 2/3 hepatectomy according to Solt-Farber's protocol andwere further promoted by oral daily administration of 0.05% phenobarbital in drinking water.The results showed that the average number of lesions showing abnormal expression of CPS1 was relatively constant over the course of the experiment(8 months),while the numberof normally expressing lesions gradually decreased.The former lesions were also largerin volume than the latter ones.We conclude that in DEN-initiated lesions the abnormallyexpressed CPS 1 lesions may grow continuously,thus leading to the formation of largernodules.We also suspect that some of these lesions have increased tendencies to developinto tumors.     

茅台酒与乙醇影响二乙基亚硝胺引发小鼠肝细胞癌的比较

目的：观察茅台酒与乙醇在协同二乙基亚硝胺（DEN）引发小鼠肝细胞癌（HCC）的差异。方法：74只雄性C57BL／6J小鼠随机分为正常对照组、茅台酒组、乙醇组、DEN组、茅台酒干预组及乙醇干预组,实验35周末检测各组小鼠血清谷氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）与肝组织匀浆中丙二醛（MDA）水平,观察肝组织病理学变化和HCC相关磷脂酰肌醇蛋白聚糖3（GPC3）表达。结果：肝功能检测发现,茅台酒干预组与正常对照组比较,ALT和MDA差异无统计学意义（P〉0．05）,而AST高于正常对照组,差异有统计学意义（P〈0．05）;乙醇干预组ALT、AST与ADM高于正常对照组、茅台酒组、乙醇组、DEN组、茅台酒干预组,均P〈0．05;病理组织学检查发现乙醇干预组小鼠肝纤维化程度和GPC3表达明显高于其他各组,P〈0．05。茅台干预组肝脏细胞学检查未见癌前病变及HCC,仅在汇管区有少量PGC3表达。结论：乙醇可以协同DEN诱发小鼠HCC,茅台酒没有这种协同作用。