七叶皂苷钠通过抑制4EBP1的磷酸化进而减缓CCl4诱导的大鼠肝纤维化

目的研究七叶皂苷钠(Sodium Aescinate,SA)对四氯化碳诱导的大鼠肝纤维化的抑制作用。方法41只健康♂SD大鼠随机分为正常对照组(n=5)、模型组(n=18)以及七叶皂苷钠治疗组(n=18)。在8周实验结束后取大鼠肝脏组织,HE染色观察肝脏组织的形态结构,Masson染色观察胶原纤维增生,免疫组化检测相关蛋白的表达;MTT法测定大鼠肝星状细胞HSC-T6细胞增殖,流式细胞仪检测细胞凋亡,Western blot法检测细胞蛋白的表达。结果七叶皂苷钠可以抑制四氯化碳诱导的大鼠肝纤维化形成,抑制肝星状细胞的增殖,并能促进其凋亡,且能抑制p-4EBP1、I型胶原及Ⅲ型胶原的表达。结论七叶皂苷钠通过抑制4EBP1磷酸化进而抑制肝脏纤维化的进程。     

苦参碱和氧化苦参碱对二乙基亚硝胺诱发大鼠肝癌作用的影响

目的　研究苦参碱和氧化苦参碱对二乙基亚硝胺诱发大鼠肝癌作用的影响。方法　采用二乙基亚硝胺法诱发大鼠肝癌 ,观察腹腔注射苦参碱 2 5mg·kg-1和氧化苦参碱10 5mg·kg-130d后 ,大鼠肝表面癌结节数、肝 /体重比和血清中丙氨酸氨基转移酶 (ALT)、γ 谷氨酰转肽酶 (γ GT)、碱性磷酸酶 (ALP)的变化。结果　氧化苦参碱组大鼠肝表面癌结节数、肝 /体重比和血清ALT、γ GT明显低于模型组 (P<0 0 5 ) ;苦参碱组大鼠肝表面癌结节数和血清γ GT明显低于模型组 (P <0 0 5 )。结论　苦参碱和氧化苦参碱 ,尤其是氧化苦参碱 ,不仅能保护肝细胞免受损伤 ,而且能抑制肿瘤细胞增长     

白藜芦醇对胆道梗阻再通大鼠肝功能的保护作用

目的 研究白藜芦醇(Res)对胆道梗阻再通大鼠肝损害的保护作用及机制。方法 雄性Wistar大鼠60只,随机分为4组,即A组:假手术组,B组:梗阻性黄疸模型组,C组:梗阻性黄疸模型＋胆道再通组,D组:梗阻性黄疸＋胆道再通 +Res干预组。连续给药7 d,实验结束后检测各组大鼠血清中总胆红素(TBIL)、直接胆红素(DBIL)、丙氨酸氨基转氨酶(ALT)水平;RT-PCR检测肝组织沉默信息调控因子1(SIRT1)mRNA的表达,Western blot检测肝组织SIRT1蛋白和核因子-κB(NF-κB)蛋白表达,免疫组化检测过氧化物酶体增殖物激活受体α(PPARα)蛋白在肝脏中的表达,原位末端标记(TUNEL)法检测肝细胞凋亡。结果 与假手术组比较,模型组血清ALT水平升高,SIRT1 mRNA及蛋白、PPARα蛋白表达降低,NF-κB蛋白表达升高,细胞凋亡率升高(P<0.05);而造模后胆道再通组与模型组比较,血清ALT水平降低,SIRT1 mRNA及蛋白、PPARα蛋白表达增加,NF-κBp蛋白表达降低,细胞凋亡率降低(P<0.05);白藜芦醇组与C组比较:血清ALT水平降低,SIRT1 mRNA及蛋白、PPARα蛋白表达增加,NF-κBp蛋白表达降低,细胞凋亡率降低(P<0.05)。结论 Res可能通过激活SIRT1抑制NF-κB,发挥抗炎、抗凋亡作用,通过促进PPARα的表达发挥抗氧化作用,从而减轻胆道梗阻再通大鼠的肝损害,促进肝功能恢复。     

曲西立滨对大鼠实验性早期肝癌的干预效果评价

目的探讨曲西立滨(triciribine, TCBN)对二乙基亚硝胺(diethylnitrosamine, DEN)诱发的大鼠早期肝癌的干预效果。方法 48只Wistar雄性大鼠,随机分为4组:正常对照组、TCBN对照组、DEN模型组和TCBN干预组(TCBN+DEN),每组12只。TCBN对照组和干预组大鼠腹腔注射TCBN(0.5 mg/kg·bw, 5次/周)16周。给予TCBN 1周后,DEN模型组和TCBN干预组开始灌胃DEN(10 mg/kg·bw, 5次/周),连续14周。TCBN给药结束后,处死大鼠。评价各组大鼠体重、肝重、肝脏系数、肝表面癌结节数目的变化,并对肝进行病理组织学检查;检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)和γ-谷氨酰转肽酶(γ-GT)的活性。免疫组织化学法检测甲胎蛋白(alpha-fetoprotein, AFP)的表达。结果与正常对照组相比,TCBN对照组动物无明显异常;DEN模型组大鼠体重明显降低,肝重和肝脏系数明显增加,血清ALT、AST、ALP和γ-GT的活性升高,AST/ALT比值下降,差异均有统计学意义(P0.05);与DEN模型组相比,TCBN干预组大鼠各项指标均明显改善,差异均有统计学意义(P0.05)。病理学及免疫组织化学结果表明,TCBN干预后肝脏表面癌结节数目明显减少,切片中癌结节面积明显缩小,无明显的AFP阳性染色。结论 TCNB的干预明显抑制了DEN诱发的大鼠早期肝癌的进展。     

鬼针草总黄酮对肝纤维化大鼠治疗作用及机制探讨

目的观察鬼针草总黄酮(total flavones of bidens bip-innata L,TFB)对肝纤维化大鼠的治疗作用及机制。方法采用CCl4诱导大鼠肝纤维化模型,观察TFB对肝纤维化大鼠血清中HA、PCⅢ、CⅣ,肝组织中Hyp含量和肝脏病理组织学及肝组织中胶原增生程度的影响。同时采用α-SMA和TUNEL双重染色观察TFB对肝纤维化大鼠肝星状细胞(he-patic stellate cells,HSC)凋亡的影响。另体外分离培养HSC,MTT法观察TFB对HSC增殖的影响,电镜和流式细胞术法观察TFB对HSC凋亡的影响。结果TFB能降低肝纤维化大鼠血清中HA、PCⅢ、CIV和肝组织中Hyp含量,改善其肝脏病理损伤程度,减少肝纤维化大鼠肝组织中胶原的增生,抑制HSC的活化、增殖,促进活化的HSC凋亡。结论TFB对肝纤维化大鼠有很好的治疗作用,其抑制HSC活化、增殖,促进活化的HSC凋亡可能是TFB治疗肝纤维化的重要机制之一。     

超声引导康莱特（中药制剂）瘤内注射治疗大鼠移植性肝肿瘤的疗效观察

目的 探讨康莱特局部注射治疗大鼠肝肿瘤的疗效及作用机制.方法 SD肝内移植瘤大鼠模型30只,随机分为超声引导下经皮肝瘤内注射生理盐水组、无水乙醇组、康莱特组各10只.全部大鼠在接种6天后每隔5天向瘤内注入治疗药物共3次,至造模后21天处死.处死前比较各组大鼠体重、肿瘤大小、血细胞各项参数、肝肾功能.处死大鼠取移植瘤组织行病理组织学等检查.结果 三组大鼠处死前体重、肿瘤生长指数、Ki-67指数两两比较差异有统计学意义.处死前各组大鼠血细胞各项参数与造模前比较无统计学意义.乙醇组处死前肝肾功能与造模前比较差异有统计学意义.三组肿瘤细胞凋亡指数比较,康莱特组明显高于盐水组及乙醇组.结论 康莱特瘤内注射治疗大鼠肝肿瘤有一定的疗效,其抑制肿瘤的作用机制为促进肿瘤细胞凋亡,抑制肿瘤细胞增殖.     

丹酚酸A对四氯化碳诱导的大鼠肝纤维化的防治作用及机制研究

目的探讨丹参水溶性成分丹酚酸A对四氯化碳(CCl4)诱导的大鼠肝纤维化的防治作用及机制研究。方法将36只SD大鼠随机分为正常对照组(对照组)、肝纤维化组(模型组)和丹酚酸A给药组(给药组)。通过50%CCl4花生油溶液(1 m L·kg-1)灌胃诱导建立肝纤维化模型。给药组同时每日1次腹腔注射丹酚酸A 10 mg·kg-1。建模与给药期间观察大鼠基本生活状况,6周后处死大鼠,观察大鼠肝脏大体形态;用HE染色法进行肝脏病理学观察;全自动生化分析仪检测血清谷丙转氨酶(ALT)及谷草转氨酶(AST)的水平;放射免疫法测定血清透明质酸(HA)、Ⅳ型胶原(CⅣ)、层黏蛋白(LN)和Ⅲ型前胶原肽(PⅢP)的含量;Western blot检测肝脏组织中Bcl-2和Bax蛋白的表达。结果与模型组相比,丹酚酸A给药组能够显著降低大鼠血清ALT和AST的水平,明显降低大鼠血清肝纤维化指标,并改善大鼠肝脏病理组织学结构(P<0.05)。与模型组相比,丹酚酸A给药组能够促进肝脏组织中Bcl-2蛋白的表达,抑制Bax蛋白表达(P<0.05)。结论丹酚酸A可能是通过调节大鼠肝脏Bcl-2和Bax蛋白的表达来发挥其抗肝纤维化的作用。     

Several new targets of antitumor agents

甲胎蛋白作为肝癌生长的促进因子，是抗肝癌药的新靶点．用药物抑制或封闭癌基因表达为治疗肿瘤开辟了新途径．诱导肿瘤细胞向正常细胞分化已成为肿瘤药物治疗的又一崭新策略．诱导肿瘤细胞凋亡是许多抗肿瘤药的共同作用方式；用自杀基因靶向治疗肿瘤优化了目前的化学治疗，具有广阔的应用前景．     

白藜芦醇对SGC-7901肿瘤细胞增殖的影响

目的研究白藜芦醇(Res)对SGC-7901肿瘤细胞增殖的影响。方法MTT法测定Res对SGC-7901细胞抑制率;流式细胞仪检测Res对肿瘤细胞凋亡和肿瘤细胞周期的影响;荧光显微镜从形态学鉴定肿瘤细胞凋亡。结果Res明显抑制肿瘤细胞的增殖,且呈一定剂量依赖关系,其IC50为164.69μmol·L-1,当Res剂量为44、88、176μmol·L-1时,肿瘤细胞的抑制率为:29.693%、33.986%、85.634%;此外,肿瘤细胞周期G1期细胞的比例减少,S期细胞的比例增加,G2/M期减小;通过形态学观察发现,随着Res剂量的增加,镜下凋亡细胞比例逐渐增加,肿瘤细胞的核质比减小,细胞核高度固缩,染色程度加深,凋亡小体的数量不断增加。结论Res明显抑制SGC-7901细胞的增殖且诱导其凋亡。     

重组人干扰素α-2b联合卡介苗治疗膀胱肿瘤的实验研究

目的：观察联合应用重组人干扰素α-2b（rhIFNα-2b）与卡介苗治疗大鼠膀胱肿瘤的疗效并探讨其机制。方法：膀胱灌注N-甲基亚硝基脲（MNU）诱导建立Wistar大鼠膀胱肿瘤模型,于灌注MNU后第10周开始治疗,第13周处死所有大鼠．免疫组织化学检测肿瘤组织CD3^＋、CD4^＋．CD8^＋细胞的数量,TUNEL（末端脱氧核苷酰基转移酶介导性dUTP切口末端标记）法检测肿瘤细胞的凋亡。结果：免疫组化证实联合用药组免疫细胞的浸润明显。TUNEL凋亡检测显示联合用药组凋亡显著。结论：联合干扰素和卡介苗治疗大鼠膀胱肿瘤能明显抑制大鼠膀胱肿瘤的生长,其机制可能是通过增加免疫细胞浸润和诱导肿瘤细胞凋亡增加而产生作用的。     

Thymoquinone inhibits cell proliferation through regulation of G1/S phase cell cycle transition in N-nitrosodiethylamine-induced experimental rat hepatocellular carcinoma

Dysregulated cell proliferation and tumorigenesis is frequently encountered in several cancers including hepatocellular carcinogenesis (HCC). Thus, agents that inhibit cell proliferation and restrain hepatic tumorigenesis through cell cycle regulation have a beneficial effect in the treatment of hepatocellular carcinogenesis. The present study was aimed to investigate the efficacy of thymoquinone (TQ), an active compound derived from the medicinal plant Nigella sativa, on N-nitrosodiethylamine (NDEA) [0.01% in drinking water for 16 weeks]-induced hepatocarcinogenesis in experimental rats. After experimental period, the hepatic nodules, liver injury markers and tumor markers levels were substantially increased in NDEA induced liver tumors in rats. However, TQ (20 mg/kg body weight) treatment greatly reduced liver injury markers and decreased tumor markers and prevented hepatic nodule formation and reduced tumor multiplicity in NDEA induced hepatic cancer bearing rats and this was evident from argyrophilic nucleolar organizer region (AgNORs) staining. Moreover, the uncontrolled cell proliferation was assessed by specific cell proliferative markers [proliferating cell nuclear antigen (PCNA) and Ki67] by immunofluorescence, immunoblot and analysis of mRNA expression. Simultaneously, we assessed the activity of TQ on G1/S phase cell cycle regulation with specific cell cycle proteins (p21^WAF1/CIP1, CDK4, Cyclin D1 and Cyclin E) by immunoprecipitation in experimental rats. Treatment with TQ significantly reduced the detrimental alterations by abrogating cell proliferation, which strongly induced G1/S arrest in cell cycle transition. In conclusion, our results suggest that TQ has a potent anti proliferative activity by regulating the G1/S phase cell cycle transition and exhibit a beneficial role in the treatment of HCC.     

Immunomodulatory activity of resveratrol: discrepant in vitro and in vivo immunological effects

trans-Resveratrol is a dietary polyphenolic compound present in grapes, which has been shown to exhibit strong anti-inflammatory, antioxidant, and chemopreventive activities. In this study we have compared the in vitro and in vivo effects of resveratrol on the development of various cell-mediated immune responses, including mitogen/antigen-induced T cell proliferation, induction of cytotoxic T lymphocytes (CTLs), interleukin-2 (IL-2) induced lymphokine activated killer cells, and cytokine production. We found significant suppression (>90%) of the mitogen/antigen-induced T cell proliferation and development of allo-antigen specific CTLs in vitro with resveratrol at a concentration of 25 microM. Intragastric administration of resveratrol (2 mg daily) to mice for 4 weeks showed no effect on age-related gain in body weight, peripheral blood cell counts (WBC, RBC, or platelets), or the cellularity of bone marrow or spleen. The CD4(+) and CD8(+) T cells in spleen or colony-forming units-total in the marrow also remained unaffected by treatment with resveratrol. Spleen cells, which were stimulated in vitro after being removed from mice which had been administered resveratrol for 2 or 4 weeks, showed no significant change in IL-2 or concanavalin A induced proliferation of T cells or production of IL-2 induced lymphokine activated killer cells. Further, the production of in interferon-gamma and IL-12 was not affected by administration of resveratrol, but production of tumor necrosis factor-alpha was reduced. Even when conducted entirely in vivo, treatment with resveratrol was found to only marginally reduce allo-antigen induced T cell proliferation and the generation of CTLs in the draining lymph nodes. Thus, even though resveratrol strongly inhibits T cell proliferation and production of cytolytic cells in vitro, oral administration of resveratrol for 4 weeks does not induce hematologic or hematopoietic toxicity, and only marginally reduces the T cell-mediated immune responses.     

Role of 5-lipoxygenase in resveratrol mediated suppression of 7,12-dimethylbenz(α)anthracene-induced mammary carcinogenesis in rats

Substantial evidences suggest that lipoxygenase-catalyzed products have a strong influence on the development and progression of human cancers. The dietary phytochemical resveratrol has become a focus of intense research owing to its roles in cancer prevention. A single tail vein injection of 7,12-dimethylbenz(a)anthracene (DMBA) was given at a dose of 0.5mg/0.2 ml oil emulsion/100g body weight at 50 days of age of female Sprague-Dawley rats. Rats were treated with resveratrol from 2 weeks before DMBA injection (5 weeks of animal age) and continued to 24 weeks of the experimentation at a dose of 100 μg/rat in the diet. We observed that resveratrol acts as a potent 5-lipoxygenase (5-LOX) inhibitor obtained from natural sources. Our result indicated that resveratrol is a strong antioxidant in reducing lipid peroxidation and preventing DNA damage. It significantly decreased the extent of DNA strand break, inhibited abnormal cell proliferation as evidenced by BrdU labeling index and also induced apoptosis in carcinogen-challenged rat mammary tissue. Increased TGF-β1 expression in resveratrol treated rats is thought to be one of the factors inducing apoptosis to suppress DMBA-induced mammary carcinogenesis.     

Regulation of PKB/Akt-pathway in the chemopreventive effect of lactoferrin against diethylnitrosamine-induced hepatocarcinogenesis in rats

BackgroundAbnormal activation of protein kinase B (PKB) is associated with many cancers. This makes inhibition of PKB signaling pathway a promising strategy for cancer therapy. Lactoferrin (Lf) has been reported for its inhibition of tumor growth and metastasis, however, the mechanism is not completely understood. Its anti-hepatocarcinogenic activity has not taken the deserved recognition despite the additional advantages of Lf as an antiviral against hepatitis C virus, the main cause of hepatocellular carcinoma (HCC), and as a targeting ligand for delivering chemotherapeutics to hepatoma cells.MethodsThis study evaluated the anti-hepatocarcinogenic effect of Lf, and the role of PKB in this effect using diethylnitrosamine (DENA)-induced HCC rat model, and a primary cell culture prepared from the induced hepatic lesions (DENA?HCC cell culture).ResultsUp-regulation of activated PKB in the hepatocytes of rats with DENA-induced HCC was observed, as measured biochemically in the liver homogenate, and localized immunohistochemically. This was accompanied by increment of hepatocytes proliferation, and expression of vascular endothelial growth factor and endothelial nitric oxide synthase. Involvement of PKB in DENA-induced HCC was confirmed by the observed decrease in cell proliferation in DENA?HCC cell culture that was treated with PKB inhibitor. In Lf-treated rats, a dose-dependent chemopreventive effect was observed, with decreased expression and activation of PKB, amelioration of the other DENA-induced alterations, and stimulation of apoptosis. In vitro, Lf blocked PKB activator-induced cell proliferation.ConclusionThese findings support the chemopreventive activity of Lf against HCC, and suggest regulation of PKB-pathway as a potential mechanism underlying this effect.     

The protective effect of resveratrol on dimethylnitrosamine-induced liver fibrosis in rats

Oxidative stress in liver injury is a major pathogenetic factor in progress of liver fibrosis. Resveratrol, a representative antioxidant derived from grapes, has been reported to show widespread pharmacological properties. In this study, we investigated the protective effects of resveratrol on dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Rats were treated with resveratrol daily by oral gavage for seven days after a single intraperitoneal injection of DMN (40 mg/kg). Resveratrol remarkably recovered body and liver weight loss due to DMNinduced liver fibrosis. Liver histology showed that resveratrol alleviated the infiltration of inflammatory cells and fibrosis of liver tissue. Resveratrol decreased the level of malondialdehyde and increased the levels of glutathione peroxidase and superoxide dismutase. Also, resveratrol significantly inhibited the mRNA expression of inflammatory mediators including inducible nitric oxide, tumor necrosis factor-alpha and interleukin-1beta. In addition, resveratrol showed not only reduced mRNA expression of fibrosis-related genes such as transforming growth factor beta 1, collagen type I, and alpha-smooth muscle actin, but also a significant decrease of hydroxyproline in rats with DMN-induced liver fibrosis. Our results suggest that resveratrol could be used to treat liver injury and fibrosis and be useful in preventing the development of liver fibrosis and cirrhosis.     

相思子蛋白P2抗肝癌作用及对端粒酶活性影响

目的研究相思子蛋白P2对肝癌细胞生长的抑制作用及机制。方法体外实验:MTT法检测相思子蛋白P2对人肝癌HepG2细胞增殖的抑制作用。流式细胞仪检测对HepG2细胞周期和细胞凋亡的影响。TRAP-SYBR-Green染色法检测相思子蛋白P2作用前后细胞端粒酶活性的改变。体内实验:相思子蛋白P2 50、75、100μg.kg-1连续灌胃给药10 d,观察对小鼠肝癌H22移植性肿瘤的生长抑制作用。小鼠口服给药急性毒性观察。结果相思子蛋白P2可明显抑制HepG2细胞增殖,IC50值为5.172×10-3 mg.L-1。在5×10-5～1×10-3 mg.L-1剂量作用下,可引起HepG2细胞凋亡。相思子蛋白P2主要将细胞周期滞留在S期,从而抑制了肿瘤细胞增殖。同时可使细胞端粒酶活性明显降低,其下调端粒酶活性的作用随药物浓度的增加而明显增强。相思子蛋白P2对小鼠H22肝癌细胞生长有明显抑制作用,100μg.kg-1抑瘤率达62.47%,且对胸腺和脾脏指数的影响较环磷酰胺小。小鼠口服给药LD50值为6.77 mg.kg-1。结论相思子蛋白P2体内外均可明显抑制肝癌细胞的生长,其抗肿瘤作用可能与下调细胞端粒酶活性、改变细胞周期分布,诱导细胞凋亡有关。     

miR-331—3p在肝细胞癌中表达的研究

目的运用实时荧光定量PCR（qRT—PCR）技术检测miR-331—3p在肝癌细胞株和肝癌组织中的表达,探讨其在肝癌中表达的临床意义及潜在临床价值。方法采用基于2^-△△Cr的qRT—PCR检测miR-331—3p在正常肝细胞株（HL-7702）、不同侵袭转移能力的肝癌细胞株（HepG2、MHCC97-H、HCCLM3）的表达,同时收集5例正常肝组织、30例肝癌组织及癌旁组织,进行定量分析,并分析miR-331—3p与肝癌患者临床病理特征的关系。结果与正常肝细胞株相比,miR-331—3p在肝癌细胞株中表达下调,且随着肝癌细胞株侵袭转移程度增加,其表达下调越明显（P〈0．05）;与正常肝组织相比。肝癌组织中miR-331—3p有不同程度的表达下调（P〈0．05）,且与肝癌患者肿瘤多发结节（P=0．036）、低分化程度（P=0．035）以及伴有静脉浸润（P=0．016）等临床病理特征相关。结论miR-331-3p在肝癌的发生、发展过程可能发挥重要作用,miR-331—3p有望成为肝癌新的生物标志物或预后因子。     

Resveratrol inhibits dimethylnitrosamine-induced hepatic fibrosis in rats

Resveratrol, a phytoalexin found in grapes and red wines, has been reported to exhibit a wide range of pharmacological properties. In this study, we investigated the protective effect of resveratrol on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Oral administration of resveratrol (20 mg/kg daily for 4 weeks) remarkably prevented the DMN-induced loss in body and liver weight, and inhibited the elevation of serum alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin levels. Resveratrol also increased serum albumin and hepatic glutathione levels and reduced the hepatic level of malondialdehyde due to its antioxidant effect. Furthermore, DMN-induced elevation of hydroxyproline content was reduced in the resveratrol treated rats, the result of which was consistent with the reduction in type I collagen mRNA expression and the histological analysis of liver tissue stained with Sirius red. The reduction in hepatic stellate cell activation, as assessed by α-smooth muscle actin staining, and the reduction in transforming growth factor-β1 mRNA expression were associated with resveratrol treatment. In conclusion, resveratrol exhibited in vivo hepatoprotective and antifibrogenic effects against DMN-induced liver injury, suggesting that resveratrol may be useful in the prevention of the development of hepatic fibrosis.