Neferine from Nelumbo nucifera induces autophagy through the inhibition of PI3K/Akt/mTOR pathway and ROS hyper generation in A549 cells

Previously we have reported that neferine from the medicinal plant Nelumbo nucifera, inhibited cancer cell proliferation by inducing apoptosis. The present study was focused on the action mechanism of neferine in inducing autophagy in lung cancer cells. Neferine markedly inhibited A549 cell proliferation in a dose dependent manner. Acidic vesicular accumulation was observed in neferine treated cells as an indication of autophagy. Neferine could induce the conversion of LC3B-I to LC3B-II without affecting the expression levels of PI3KCIII and Beclin1. It has been observed that neferine mediated autophagy is dependent on inhibition of PI3K/Akt/mTOR signaling by neferine. Neferine treatment could also lead to the ROS hypergeneration and depletion of cellular antioxidant, GSH. The results demonstrate that neferine-induced autophagy is mediated through ROS hypergeneration and mTOR inhibition. Taken together, the present study unveils a novel mechanism of action of neferine on lung cancer cells in the induction of autophagy.     

3种黄酮对脂肪肝细胞氧化应激的影响

目的：评价表没食子儿茶素没食子酸酯(EGCG)、表儿茶素、柚皮素3种食源性黄酮对降低脂肪肝细胞模型中氧化应激水平的作用。方法：采用油酸诱导的HepG2细胞建立脂肪肝模型,根据细胞存活率、乳酸脱氢酶(LDH)释放量和活性氧产生量反映细胞损伤程度及氧化应激状态,通过黄酮物质处理后的脂肪肝细胞模型活性氧减少量评价其缓解脂肪肝氧化应激状态的功效。结果：EGCG的抗氧化能力优于表儿茶素,两种物质的抗氧化能力随浓度增加而增强,呈量效关系。柚皮素的抗氧化作用最弱,100μmol/L柚皮素具有促氧化作用。     

Coumestrol induces senescence through protein kinase CKII inhibition-mediated reactive oxygen species production in human breast cancer and colon cancer cells

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC₅₀ of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent K_i value of 7.67 μM. Coumestrol at 50 μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21^Cip1/WAF1 pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22^phox siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.     

Antioxidant behaviour of carotenoids highly accumulated in HepG2 cells

The antioxidant behaviour of major dietary carotenoids accumulated at high concentrations in human hepatoma HepG2 cells was evaluated, in comparison with α-tocopherol. The cells that accumulated carotenoids and α-tocopherol at levels higher than the values reported in the human liver were exposed to mild oxidative stress with tert-butylhydroperoxide. β-Carotene (>2.6 nmol/mg protein) and astaxanthin (>1.8 nmol/mg protein) significantly suppressed lipid peroxidation, while β-cryptoxanthin and lutein did not. α-Tocopherol remarkably suppressed lipid peroxidation with an IC₅₀ value of 0.16 nmol/mg protein. Neither α-tocopherol nor any of the carotenoids except for lycopene showed pro-oxidant action even at high cellular concentrations. The antioxidant behaviours of carotenoids in a cellular milieu were quite different from those previously found in liposomes and homogeneous solutions. Further studies are required to assess the implications of the antioxidant behaviours found in the cultured cells on human health.     

Investigation of the cytotoxicity of food-grade nanoemulsions in Caco-2 cell monolayers and HepG2 cells

Nanoemulsions represent one of the emerging formulations for nutraceutical delivery. However, the possible toxicity associated with the small droplet size (diameter <200 nm) is still unknown. In this study, three nanoemulsions emulsified by modified starch, Tween 20 and whey protein isolate, respectively, were prepared and their cytotoxicity was examined by comparing with the corresponding micron-sized emulsions. Caco-2 cell monolayers were used to mimic the small intestine epithelium. Integrity of the cell membrane and tight junctions was tested by measuring the lactate dehydrogenase leakage and transepithelial electrical resistance, respectively. All three nanoemulsions did not reveal significant difference from their micron-sized counterparts, suggesting no apparent toxicity of the nanoemulsions on the small intestine. Meanwhile, the possible hepatic toxicity was investigated using MTT assay on HepG2 cells. It was found that nanoemulsions made with modified starch and whey protein isolate, but not Tween 20, affected the cell viability/proliferation more than did the micron-sized emulsions. Further in vivo investigation is required to examine the possible hepatic toxicity of nanoemulsions.     

过氧化氢诱导人肝癌细胞产生氧化应激最佳作用浓度的研究

不同浓度过氧化氢作用人肝癌细胞(HepG2)4 h后,用单细胞凝胶电泳技术测定DNA损伤状况,分光光度法测定MDA含量、SOD及GSH-Px活性,研究过氧化氢的最佳作用浓度,构建体外氧化应激细胞模型。实验结果表明,过氧化氢的最佳作用浓度为100μmol/L,作用细胞的时间选择4 h,可以成功构建以过氧化氢为氧化应激诱导剂的HepG2细胞体外氧化应激模型。     

Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway

Ovarian cancer is a significant malignancy for women in the western world, and its death rate has remained unchanged over the past 50 years, leaving room for proper chemoprevention. Kaempferol is a natural flavonoid widely distributed in fruits and vegetables, and epidemiological studies have found a negative correlation between kaempferol consumption and ovarian cancer risk. To understand the mechanism behind this negative correlation, we investigated kaempferol's ability to induce apoptosis in A2780/CP70, A2780/wt, and OVCAR-3 ovarian cancer cell lines. Kaempferol inhibited cell proliferation but did not cause necrosis in all 3 cell lines. For the apoptosis, caspase 3/7 levels were induced in a concentration-dependent manner by kaempferol treatment, with A2780/wt cells being the most responsive. This induction can be diminished by pre-treatment with a caspase-9 inhibitor, indicating an intrinsic apoptosis pathway. Western blot analysis revealed that protein levels of Bcl-x(L) were decreased in ovarian cancer cells, while p53, Bad, and Bax proteins were up-regulated by kaempferol treatment. Our data indicate that kaempferol induces apoptosis in ovarian cancer cells through regulating pro-apoptotic and anti-apoptotic protein expressions in the intrinsic apoptosis pathways, and is a good candidate for the chemoprevention of ovarian cancers in humans. Further studies in animal models and clinical trials are therefore warranted.     

小麦低聚肽对HepG2细胞胆固醇代谢的影响

目的:在对小麦低聚肽的基础理化成分和分子量分布进行分析的基础上,观察不同浓度的小麦低聚肽对人肝癌细胞株HepG2胆固醇代谢的影响。方法:将培养的HepG2细胞随机分为5组:对照组、2、4、6、8g/L小麦低聚肽实验组。经小麦低聚肽作用24h后,进行反转录-聚合酶链反应（RT-PCR）,定量分析低密度脂蛋白受体（LDLR）和3-羟基-3-甲基戊二酸单酰辅酶A还原酶（HMGCR）mRNA的表达。结果:与对照组相比,经小麦低聚肽作用后,各实验组HepG2细胞LDLR mRNA表达明显增加（p<0.05或p<0.01）,其中6g/L小麦低聚肽组的LDLR mRNA表达增加最多;HMGCR mRNA表达均降低,2、4、6g/L小麦低聚肽组的HMGCR mRNA表达极显著降低（均为p<0.01）,其中2g/L小麦低聚肽组的HMGCR mRNA表达降低最多。结论:不同浓度的小麦低聚肽均能升高HepG2细胞内LDLR mRNA水平和降低HMGCR mRNA水平,从而起到降低胆固醇的作用,为其开发利用提供了一定的实验依据和理论基础。      

中国沙棘乙醇提取物的体外及对HepG2细胞的抗氧化活性

本文从生化角度全面分析了中国沙棘乙醇提取物的体外抗氧化活性,并在评价细胞毒性的基础上,以Hep G2细胞为模型,利用流式细胞仪评估了提取物的细胞抗氧化能力。结果表明,中国沙棘乙醇提取物对ABTS自由基具有良好的清除效果,当浓度为5 mg/m L时,其清除能力与阳性对照BHT相当;中国沙棘乙醇提取物对NO自由基也有良好的清除效果,但其整体清除能力不及BHT;当浓度为5 mg/m L时,中国沙棘乙醇提取物对亚油酸脂质过氧化表现抑制,但其抑制效果低于BHT;中国沙棘乙醇提取物的总抗氧化能力随浓度的增大而增强,当浓度为5 mg/m L时,其总抗氧化能力与同浓度下的BHT接近。在浓度为0.05~5 mg/m L范围内,中国沙棘乙醇提取物对Hep G2细胞不显示毒性;当处理浓度为0.5、1、5 mg/m L时,中国沙棘乙醇提取物的阳性细胞率与阳性对照相比分别降低了75.22%、72.36%和78.2%。中国沙棘乙醇提取物不仅在体外条件下具有良好的抗氧化活性,而且在Hep G2细胞内表现出良好的抗氧化能力。      

二氢杨梅素和杨梅苷抑制HepG2细胞的协同作用

目的:探讨藤茶中的主要活性成分二氢杨梅素(DMY)和杨梅苷(MYT)抑制HepG2细胞增殖的协同作用.方法:通过对体外培养的细胞分别给予DMY、MYT和DMY:MYT为1∶4、1∶2、1∶1、2∶1、4∶1、8∶1的混合物,采用MTT法和荧光染色法测定其对HepG2细胞活力的影响,并用合用指数(CI)和等效曲线法分析二...     

金松酸对HepG2细胞中甘油三酯蓄积的影响

为探讨香榧金松酸(SA)对甘油三酯(TG)蓄积的影响及其作用机制,本研究以HepG2为模型细胞,首先采用MTT法确定油酸(OA)与SA的临界安全浓度,然后通过油红染色法观察细胞内脂质积累情况并定量测定了细胞内TG含量,最后利用RT-qPCR分析SA对细胞脂质代谢关键基因的影响。结果显示：OA和SA总添加量为500 mol/L,随着SA替换量从0%上升到100%,细胞内TG蓄积明显降低,从0.033 mmol/g减低到0.017 mmol/g;脂肪酸合成关键基因FAS、ACC1、SCD1、SREBP1的表达量分别降低了40.92%、62.01%、25.56%、43.79%,脂肪酸氧化关键基因SCD1、PPARα的表达量分别提高了143.94%、445.11%,TG分解基因ATGL的表达量增加了123.71%。因此,SA显著抑制了细胞TG蓄积,其可能机制是SA通过上调TG分解代谢关键酶同时下调TG合成代谢关键酶。作为天然食物成分,SA在减肥食品开发方面具有良好的应用前景。     

基于微流控技术的橙黄决明素对HepG2细胞的体外安全性评估

目的 本研究将微流控技术运用到HepG2细胞模型构建及对橙黄决明素的潜在体外安全性评估中。方法 以鼠尾胶原Ⅰ型（1.3 mg/mL）+明胶（7.5%）构建仿真人体微环境,对乙酰氨基酚（APAP）作为阳性对照组,通过不同浓度橙黄决明素对HepG2细胞的增殖活性、活/死细胞染色和功能性生化指标进行体外安全性评估。结果 该平台实现了HepG2细胞的稳定培养与应用,经72 h培养,细胞成团增殖。橙黄决明素连续处理48 h后计算细胞存活率,结果表明随着橙黄决明素浓度的增加,0、50、100和200μmol/L细胞存活率分别为100.0%、95.3%、90.3%和81.6%,与空白对照组比较,尤以200μmol/L差异最为显著（P<0.05）;红色标记的死细胞数量随橙黄决明素浓度增加逐渐增多,绿色标记的活细胞数量则逐渐减少;尿素氮（BUN）含量随着浓度的增加而显著降低,与空白对照组（0μmol/L）比较,50～200μmol/L组差异具有显著性（P<0.05）,且200μmol/L与APAP组BUN含量接近;谷丙转氨酶、谷草转氨酶和乳酸脱氢酶活性之间随着浓度的变化均无显著性差异。结论 ...     

水飞蓟素对丙烯酰胺诱导人肝癌细胞氧化损伤的保护作用

研究水飞蓟素对人肝癌细胞系（human hepatocellular liver carcinoma cell line,HepG2）增殖的影响以及 对食品加工过程中产生的丙烯酰胺所致肝细胞毒性的预防效果,同时对其保护机制进行探讨。通过噻唑蓝法检测 水飞蓟素对HepG2细胞增殖的影响和对丙烯酰胺诱发HepG2细胞毒性的保护效果;利用荧光探针法对HepG2细胞 内活性氧的变化进行检测,并运用比色法分别检测细胞内脂质、蛋白质及DNA的氧化损伤标志物（丙二醛、蛋 白羰基和8-羟基脱氧鸟苷）含量;利用酶活力试剂盒检测细胞内过氧化氢酶（catalase,CAT）、超氧化物歧化酶 （superoxide dismutase,SOD）和谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活力的变化。结果显示, 12～96 μg/mL的水飞蓟素对HepG2细胞无毒性;水飞蓟素可以抑制丙烯酰胺引起的细胞存活率降低和氧自由基水平 升高,减少丙烯酰胺对脂质、蛋白质和DNA的氧化损伤,并提高细胞内抗氧化物酶CAT、SOD和GSH-Px活力。研 究证明,水飞蓟素能够缓解丙烯酰胺对肝细胞造成的氧化损伤。     

Low abundance of mitophagy markers is associated with reactive oxygen species overproduction in cows with fatty liver and causes reactive oxygen species overproduction and lipid accumulation in calf hepatocytes

Mitochondria are the main site of fatty acid oxidation and reactive oxygen species (ROS) formation. Damaged or dysfunctional mitochondria induce oxidative stress and increase the risk of lipid accumulation. During the process of mitophagy, PTEN induced kinase 1 (PINK1) accumulates on damaged mitochondria and recruits cytoplasmic Parkin to mitochondria. As an autophagy receptor protein, sequestosome-1 (p62) binds Parkin-ubiquitinated outer mitochondrial membrane proteins and microtubule-associated protein 1 light chain 3 (LC3) to facilitate degradation of damaged mitochondria. In nonruminants, clearance of dysfunctional mitochondria through the PINK1/Parkin-mediated mitophagy pathway contributes to reducing ROS production and maintaining metabolic homeostasis. Whether PINK1/Parkin-mediated mitophagy plays a similar role in dairy cow liver is not well known. Thus, the objective of this study was to investigate mitophagy status in dairy cows with fatty liver and its role in free fatty acid (FFA)-induced oxidative stress and lipid accumulation. Liver and blood samples were collected from healthy dairy cows (n = 10) and cows with fatty liver (n = 10) that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Calf hepatocytes were isolated from 5 healthy newborn female Holstein calves (1 d of age, 30–40 kg). Hepatocytes were transfected with small interfering RNA targeted against PRKN for 48 h or transfected with PRKN overexpression plasmid for 36 h, followed by treatment with FFA (0.3 or 1.2 mM) for 12 h. Mitochondria were isolated from fresh liver tissue or calf hepatocytes. Serum concentrations of β-hydroxybutyrate were higher in dairy cows with fatty liver. Hepatic malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were greater in cows with fatty liver. The lower protein abundance of PINK1, Parkin, p62, and LC3-II in hepatic mitochondrial fraction of dairy cows with fatty liver indicated the mitophagy was impaired. In hepatocytes, knockdown of PRKN decreased protein abundance of p62 and LC3-II in the mitochondrial fraction, and increased contents of triacylglycerol (TG), MDA, and H₂O₂. In addition, protein abundances of PINK1, Parkin, p62, and LC3-II were lower in the mitochondrial fraction from hepatocytes treated with 1.2 mM FFA than the hepatocytes treated with 0.3 mM FFA, whereas the content of TG, MDA, and H₂O₂ increased. In 1.2 mM FFA-treated hepatocytes, PRKN overexpression increased protein abundance of p62 and LC3-II in the mitochondrial fraction and decreased contents of TG, MDA, and H₂O₂. Together, our data demonstrate that low abundance of mitophagy markers is associated with ROS overproduction in dairy cows with fatty liver and impaired mitophagy induced by a high concentration of FFA promotes ROS production and lipid accumulation in female calf hepatocytes.     

The role of reactive oxygen species in the induction of Ty1 retrotransposition in Saccharomyces cerevisiae

Here we provide evidence for a dependence between the increased production of reactive oxygen species and the activation of Ty1 retrotransposition. We have found that the strong activator of Ty1 mobility, methylmethane sulphonate, can not induce Ty1 retrotransposition in cells with compromised mitochondrial oxidative phosphorylation (rho^?; sco1Δ), which is the major source for production of reactive oxygen species (ROS) in Saccharomyces cerevisiae. The quantitative estimation of superoxide anions in living cells showed that rho⁺ cells exposed to methylmethane sulphonate increase Ty1 retrotransposition and superoxide levels. The increase of superoxide anions by the superoxide generator menadione is accompanied by induction of Ty1 mobility without any treatment with a DNA‐damaging agent. Higher frequencies of retrotransposition were found in rho⁺ and rho^? cells treated with exogenously added hydrogen peroxide or in cells with disrupted YAP1 gene characterized by increased intracellular levels of hydrogen peroxide. These data indicate that increased levels of ROS may have an independent and key role in the induction of Ty1 retrotransposition. Copyright © 2010 John Wiley & Sons, Ltd.     

薰衣草精油对HepG2细胞的抗增殖作用研究

研究了薰衣草精油对HepG2细胞的生长抑制作用。用溴化二苯偶氮盐（MTT）法观察薰衣草精油和20%的薰衣草精油含药血清对HepG2的生长抑制作用;HE染色法观察薰衣草精油对HepG2细胞形态学的影响;流式细胞术检测薰衣草精油对HepG2细胞凋亡的影响。结果:薰衣草精油明显抑制HepG2细胞的增殖,并呈浓度依赖性;20%的薰衣草精油血清对HepG2细胞的抑制率与5-氟尿嘧啶血清相比无显著性差异;薰衣草精油处理HepG2后细胞数及体积减少,胞浆和细胞核浓缩;不同质量浓度的薰衣草精油作用HepG2后出现明显的凋亡细胞群及坏死细胞。结果表明,薰衣草精油可以通过诱导细胞凋亡或坏死的方式抑制HepG2细胞的生长。