The expression of efflux and uptake transporters are regulated by statins in Caco-2 and HepG2 cells

Aim： Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and efflux/uptake transporters. There is little information on the regulation of these proteins in human cells after statin therapy. In this study, the effects of atorvastatin and simvastatin on mRNA expression of efflux （ABCB1, ABCG2 and ABCC2） and uptake （SLCO1B1, SLCO2B1 and SLC22A1） drug transporters in Caco-2 and HepG2 cells were investigated.
Methods： Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins. Results： Differences in mRNA basal levels of the transporters were as follows： ABCC2〉ABCG2〉ABCB1〉SLCO1B1〉〉〉SLC22A1〉SLCO2B1 for HepG2 ceils, and SLCO2B1〉〉ABCC2〉ABCB1〉ABCG2〉〉〉SLC22A1 for Caco-2 cells. While for HepG2 cells, ABCC2, ABOG2 and SLCO2B1 mRNA levels were significantly up-regulated at 1, 10 and 20 pmol/L after 12 or 24 h treatment, in Caco-2 cells, only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin. Interestingly, whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells, in Caco-2 cells the statin significantly down-regulated ABCB1, ABCC2, SLC22A1, and SLCO2B1 mRNA levels after 12 or 24 h treatment.
Conclusion： These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters, and this effect depends on the cell type. Furthermore, alterations in the expression levels of drug transporters in the liver and/or intestine may con- tribute to the variability in oral disposition of statins.     

Comparative cytotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells in culture

The use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics has increased significantly owing to their antibacterial and antifungal properties. As a consequence, the need for validated rapid screening methods to assess their toxicity is necessary to ensure consumer safety. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential cytotoxicity of food‐ and cosmetic‐related nanoparticles. The two cell culture models were utilized to compare the potential cytotoxicity of 20‐nm silver. The average size of the silver nanoparticle determined by our transmission electron microscopy (TEM) analysis was 20.4 nm. The dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The concentration of the 20‐nm silver solution determined by our inductively coupled plasma–mass spectrometry (ICP‐MS) analysis was 0.962 mg ml^–1. Our ICP‐MS and TEM analysis demonstrated the uptake of 20‐nm silver by both HepG2 and Caco2 cells. Cytotoxicity, determined by the Alamar Blue reduction assay, was evaluated in the nanosilver concentration range of 0.1 to 20 µg ml^–1. Significant concentration‐dependent cytotoxicity of the nanosilver in HepG2 cells was observed in the concentration range of 1 to 20 µg ml^–1 and at a higher concentration range of 10 to 20 µg ml^–1 in Caco2 cells compared with the vehicle control. A concentration‐dependent decrease in dsDNA content was observed in both cell types exposed to nanosilver but not controls, suggesting an increase in DNA damage. The DNA damage was observed in the concentration range of 1 to 20 µg ml^–1. Nanosilver‐exposed HepG2 and Caco2 cells showed no cellular oxidative stress, determined by the dichlorofluorescein assay, compared with the vehicle control in the concentration range used in this study. A concentration‐dependent decrease in mitochondria membrane potential in both nanosilver exposed cell types suggested increased mitochondria injury compared with the vehicle control. The mitochondrial injury in HepG2 cells was significant in the concentration range of 1 to 20 µg ml^–1, but in Caco2 cells it was significant at a higher concentration range of 10 to 20 µg ml^–1. These results indicated that HepG2 cells were more sensitive to nanosilver exposure than Caco2 cells. It is generally believed that cellular oxidative stress induces cytotoxicity of nanoparticles. However, in this study we did not detect any nanosilver‐induced oxidative stress in either cell type at the concentration range used in this study. Our results suggest that cellular oxidative stress did not play a major role in the observed cytotoxicity of nanosilver in HepG2 and Caco2 cells and that a different mechanism of nanosilver‐induced mitochondrial injury leads to the cytotoxicity. The HepG2 and Caco2 cells used this study appear to be targets for silver nanoparticles. The results of this study suggest that the differences in the mechanisms of toxicity induced by nanosilver may be largely as a consequence of the type of cells used. This differential rather than universal response of different cell types exposed to nanoparticles may play an important role in the mechanism of their toxicity. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, are excellent systems for screening cytotoxicity of silver nanoparticles. These long established cell culture models and simple assays used in this study can provide useful toxicity and mechanistic information that can help to better inform safety assessments of food‐ and cosmetic‐related silver nanoparticles. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

2016-2018年天津儿童医院含β2受体激动剂药品使用情况分析

目的了解天津市儿童医院含β2受体激动剂药品的使用情况,为临床药品管理和合理用药提供参考。方法收集天津市儿童医院2016-2018年门诊药房使用含β2受体激动剂药品的相关数据信息,统计药品相关指标,并分析其变化趋势。结果 2016-2018年含β2受体激动剂药品的用药金额及使用率呈现逐年递增趋势,尤其在2018年,口服用含β2受体激动剂药品的用药金额大幅增长,使用率明显增高。应用含β2受体激动剂药品的用药频度(DDDs)排序前3位的药品依次是盐酸丙卡特罗片、盐酸丙卡特罗口服液、布地奈德福莫特罗粉吸入剂(80μg∶4.5μg);日均费用(DDC)最高的是硫酸特布他林雾化溶液,口服药品中DDC最高的是氨溴特罗口服液。结论天津市儿童医院含β2受体激动剂药品的用药金额和使用率与儿童呼吸系统疾病季节性发病特点密切相关,临床应用基本合理,同时应及时了解临床用药现状,规范管理,合理用药。     

Effect of pH, mucin and bovine serum on rifampicin permeability through Caco-2 cells

Rifampicin, a poorly soluble drug, has great importance in therapeutics as it is the main drug used to treat tuberculosis. The characterization of its permeability and the factors that influence it represent an important tool for predicting its bioavailability. Caco‐2 cell monolayers were used as models of the intestinal mucosa to assess the uptake and transport of rifampicin and the effects of various experimental conditions were investigated, in order to establish the influence of these variables on rifampicin permeability. Different pHs (5.8, 6.8 and 7.4) in the apical medium, the presence or absence of mucin (3.0% w/v) in the donor site and the presence or absence of bovine serum albumin (4.0% v/v) in the receptor chamber were the evaluated conditions. The quantification of rifampicin in the apical or basolateral chambers was performed by a validated HPLC‐UV method. The change in the donor chamber pH showed that permeability values were greater at pH 6.8, although this increase does not result in an alteration of the qualitative classification of rifampicin, which has high permeability. Mucin and bovine serum showed no effects on the permeability of rifampicin at the concentration tested. Overall, the current study suggests that pH, artificial mucin and bovine serum proteins have no influence on rifampicin permeability. Copyright © 2012 John Wiley & Sons, Ltd.     

澳洲茄边碱对肝癌 HepG2细胞增殖及凋亡的影响

目的：探讨澳洲茄边碱（SM ）对 HepG2细胞增殖、凋亡的影响及其可能作用机制。方法用不同浓度SM 5、10、15和20μg／ml分别处理 HepG2细胞3、6、12和24 h ,并设不加药的对照组。采用MTT法检测 HepG2细胞增殖,DAPI染色法观察细胞核形态的变化,流式细胞术检测细胞凋亡和周期,Western blot法检测B细胞淋巴瘤‐白血病2（Bcl‐2）、Bcl相关X蛋白（Bax）、半胱天冬氨酸蛋白酶3（Caspase‐3）及Ki67的蛋白表达。结果与对照组相比,SM 呈剂量依赖性地抑制HepG2细胞增殖,促进HepG2细胞凋亡,将细胞周期阻滞于G2／M期,并且上调Bax和Caspase‐3蛋白表达,下调Bcl‐2和Ki67蛋白表达（P＜0．05或P＜0．01）。结论 SM 能有效抑制 HepG2细胞的增殖,促进细胞凋亡的发生;SM上调Bax和Caspase‐3表达,下调Bcl‐2和Ki67表达,细胞周期阻滞于G2／M期可能是其发挥上述作用的机制。     

Resveratrol is efficiently glucuronidated by UDP-glucuronosyltransferases in the human gastrointestinal tract and in Caco-2 cells

Resveratrol (3, 5, 4'-trihydroxy-trans-stilbene), a natural polyphenol present in grapes and peanuts, has been reported to exert a variety of potentially therapeutic effects. The aim of this study was to determine the contribution of the gastrointestinal (GI) tract to the glucuronidation of this compound and its cis-isomer, which also occurs naturally. For this purpose, glucuronidation of the two resveratrol isomers was investigated in human microsomes prepared from: stomach, duodenum, four segments of the remaining small intestine (S-1 to S-4) and colon, and from the human intestinal cell lines Caco-2 and PD-7. cis- and trans-Resveratrol were efficiently glucuronidated in the GI tract with the formation of both 3-O- and 4'-O-glucuronides, however, the two stereoisomers were glucuronidated at different rates depending on the donor and the segment considered. Microsomes prepared from Caco-2 and PD-7 cells also efficiently glucuronidated cis-resveratrol and, to a lesser extent, the trans-isomer, however, only the 3-O-glucuronide was formed. Among the UDP-glucuronosyltransferases (UGT) that are known to be expressed in the GI tract, the isoforms UGT1A1, 1A6, 1A8, 1A9 and 1A10 were active in glucuronidating trans- and/or cis-resveratrol. The results demonstrate that the GI tract may contribute significantly to the first pass metabolism of these naturally occurring polyphenols.     

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells

Exposure to nanosilver found in food‐ and cosmetics‐related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN‐forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food‐ and cosmetics‐related chemicals including nanosilver. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by a flow cytometric in vitro micronucleus assay

Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food‐related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric‐based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma–mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma–mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration‐dependent micronucleus formation sevenfold at 10 µg ml^–1 concentration in attached cell conditions and 1.3‐fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2‐fold at a concentration of 10 µg ml^–1 both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver‐induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

白鲜碱对HepG2细胞的毒性作用及其机制

目的研究白鲜碱的肝细胞毒性作用及其毒性机制。方法白鲜碱2.5～800μmol·L-1与HepG2细胞作用24 h,用MTT法检测细胞存活率并计算IC50值,用乳酸脱氢酶(LDH)释放实验检测细胞膜损伤。白鲜碱25～100μmol·L-1与HepG2细胞作用4,24或48 h,用试剂盒方法分别检测细胞培养液中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、谷胱甘肽S-转移酶(GST)和谷氨酰转肽酶(GGT)活性。用倒置显微镜观察细胞形态变化;激光共聚焦扫描显微镜检测细胞线粒体膜电位的变化。结果白鲜碱25～800μmol· L-1与HepG2细胞作用24 h对HepG2细胞存活的抑制作用随着浓度的增加而降低(r=0.965,P<0.05),IC50值为(283±27)μmol·L-1。与溶剂对照组相比,白鲜碱12.5～50μmol·L-1作用24 h,HepG2细胞LDH释放率显著升高(P<0.01)。白鲜碱100和200μmol·L-1可引起HepG2细胞形态发生明显变化,细胞皱缩脱落,细胞数目减少,并使HepG2细胞线粒体膜电位下降(P<0.05,P<0.01)。白鲜碱100和200μmol·L-1与HepG2细胞作用24 h可使细胞培养液中ALT和AST活性显著升高,并呈浓度依赖性(r=0.995,P<0.05和r=0.996,P<0.05),线粒体膜电位亦明显下降(r=0.978,P<0.05)。与溶剂对照组相比,白鲜碱100和200μmol·L-1与HepG2细胞作用4 h,细胞培养液中GST活性明显升高(P<0.05);作用24 h,GST活性升高呈浓度依赖性(r=0.987,P<0.05)。白鲜碱200μmol· L-1作用48 h导致HepG2细胞培养液中GGT活性升高(P<0.05)。结论较高浓度的白鲜碱(≥100μmol· L-1)具有潜在的肝毒性,细胞膜损伤和线粒体损伤可能是其肝毒性作用机制之一。     

儿茶素对Caco-2细胞胆固醇摄取的影响及机制研究

目的： 研究儿茶素（Catechin，Cat）对Caco-2细胞胆固醇摄取的影响，以及可能的作用机制。方法： 利用胆固醇：甲基β环糊精复合物（Chol：MβCD）建立Caco-2细胞脂质蓄积模型。不同浓度的儿茶素（20、40、60 μmol·L^-1）处理细胞24 h，结合油红O染色法观察细胞内的脂质蓄积，酶法测定细胞内胆固醇含量及分布，qRT-PCR及Westernblot检测胆固醇代谢相关基因NPC1L1和SREBP-2的表达。结果： 与空白组相比，Chol：MβCD处理组细胞内红色脂滴颗粒以及胆固醇含量明显增加。20~60 μmol·L^-1儿茶素不仅可以不同程度地减少细胞内红染脂滴的形成，而且可以显著降低细胞内总胆固醇和游离胆固醇的含量，以及胆固醇酯在总胆固醇中的比例。此外，儿茶素可以剂量依赖性地降低胆固醇代谢相关基因NPC1L1及SREBP-2的mRNA和蛋白表达。其中以60 μmol·L^-1 Cat处理组的作用最为显著（P<0.01）。结论： 儿茶素可能通过下调胆固醇代谢相关基因NPC1L1和SREBP-2的表达，进而减少Caco-2细胞摄取胆固醇及蓄积。     

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by fluorescent microscopy of cytochalasin B‐blocked micronucleus formation

As a consequence of the increased use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics to prevent fungal and bacterial growth, there is a need for validated rapid screening methods to assess the safety of nanoparticle exposure. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential genotoxicity of 20‐nm nanosilver. The average silver nanoparticle size as determined by transmission electron microscopy (TEM) was 20.4 nm. Dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The silver concentration in a 20‐nm nanosilver solution determined by the inductively coupled plasma–mass spectrometry (ICP‐MS) analysis was 0.962 mg ml^?1. Analysis by ICP‐MS and TEM demonstrated the uptake of 20‐nm silver by both HepG2 and Caco2 cells. Genotoxicity was determined by the cytochalasin B‐blocked micronucleus assay with acridine orange staining and fluorescence microscopy. Concentration‐ and time‐dependent increases in the frequency of binucleated cells with micronuclei induced by the nanosilver was observed in the concentration range of 0.5 to 15 µg ml^?1 in both HepG2 and Caco2 cells compared with the control. Our results indicated that HepG2 cells were more sensitive than Caco2 cells in terms of micronuclei formation induced by nanosilver exposure. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, represent potential screening models for prediction of genotoxicity of silver nanoparticles by in vitro micronucleus assay. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

The roles of alpha- and beta-adrenoceptor stimulation in myocardial ischaemia

beta-Adrenoceptor (AR) ligands have been the mainstay of cardiovascular therapy for decades, with beta-AR antagonist being used for hypertension, angina and myocardial infarction and adrenaline in use for cardiopulmonary resuscitation for nearly 100 years. Ischaemia of the heart through coronary artery occlusion causes cell injury and death through necrosis and apoptosis. Reperfusion of the ischaemic myocardium results in cardiac dysfunction and infarction. Stimulation of alpha- and beta-ARs in the ischaemic heart have variable and inconsistent effects depending on when the agonist is applied. This review describes the different effects of stimulation of the three established beta-AR subtypes (beta(1)-, beta(2)- and beta(3)-ARs) either before ischaemia (preconditioning) or during ischaemia and reperfusion of the heart (postconditioning). Brief periods of ischaemia preceding a major ischaemic episode can have a protective effect against post-ischaemia-reperfusion damage, known as ischaemic preconditioning. This review considers the role of endogenous catecholamines released during preconditioning and the nature of the adrenoceptor subtypes that mediate these effects. The clinical significance of this to the use of beta-AR antagonists is considered. The transduction pathways and effects on apoptosis of the cardioprotective and deleterious effects of AR activation are considered. This commentary reviews the literature and attempts to bring together a unified synopsis of the effects of adrenoceptor stimulation in myocardial ischaemia and the potential clinical relevance.     

Glucose regulation of dipeptidyl peptidase IV gene expression is mediated by hepatocyte nuclear factor-1alpha in epithelial intestinal cells

1. Dipeptidyl peptidase IV (DPP-IV) is a new drug target in the treatment of Type 2 diabetes. Dipeptidyl peptidase IV enzyme activity is significantly altered in Type 2 diabetic patients with hyperglycaemia, but the underlying molecular mechanisms remain unclear. 2. The first aim of the present study was to clarify whether glucose regulates DPP-IV enzyme activity. To address this, DPP-IV gene expression and enzyme activity were measured in Caco2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) concentrations of glucose. We observed that high glucose inhibited DPP-IV gene expression and enzyme activity. 3. The second aim of the present study was to investigate whether hepatocyte nuclear factor (HNF)-1alpha contributes to glucose regulation of DPP-IV gene expression. To explore this question, associations between the gene expression of DPP-IV and HNF-1alpha were examined in Caco-2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) glucose. We found that the pattern of glucose-regulated DPP-IV gene expression is similar to that of HNF-1alpha. Moreover, to elucidate whether glucose regulation of DPP-IV gene expression is affected when HNF-1alpha is inhibited, we produced two stable cell lines in which a dominant-negative mutant HNF-1alphaR271G or basic vectors were stably expressed. We found that glucose regulation of DPP-IV gene expression was compromised in HNF-1alphaR271G cells, but was well maintained in basic vector cells. 4. These results suggest that glucose regulation of DPP-IV gene expression is mediated by HNF-1alpha.     

Curcumin-induced genotoxicity and antigenotoxicity in HepG2 cells

Curcumin, a polyphenolic yellow pigment found in turmeric, is commonly used as a coloring agent in foods, drugs, and cosmetics. In our previous study, we found that low levels of curcumin did not increase the reactive oxygen species (ROS) formation and caused no damage to DNA in human hepatoma G2 (HepG2) cells, but at high doses, curcumin imposed oxidative stress and damaged DNA. In the present study, we are determined to investigate the genotoxic and antigenotoxic effects of curcumin using HepG2 cell line, a relevant in vitro model to detect the cytoprotective, antigenotoxic, and cogenotoxic agents. The results of micronucleus (MN) assays showed that, on one hand, curcumin at the high tested concentrations (8 and 16 μg/ml) displayed a small but significant increase in the frequency of MN, and on the other hand, it was observed that the low tested concentration (2 μg/ml) significantly reduced the MN formation induced by the chemotherapeutic agent cyclophosphamide. The present results indicate that curcumin shows both genotoxicity and antigenotoxicity depending on its concentration.     

山奈酚对HepG2细胞增殖的影响及诱导其凋亡的机制研究

目的研究山奈酚对人肝癌细胞(HepG2细胞)增殖与凋亡的影响及其机制。方法将HepG2细胞分为空白组和实验组,实验组以CCK-8法检测10个不同浓度的(10,20,30,40,50,60,70,80,90,100μmol·L^-1)山奈酚处理HepG2细胞24 h后的存活率。最终以3个浓度(20,40,50μmol·L^-1)山奈酚作为低、中、高3个浓度实验组。以免疫印迹法检测核苷酸结合寡聚化域样受体蛋白3(NLRP3)和死骨片-1(P62)蛋白表达水平(灰度值)。结果山奈酚处理HepG2细胞24 h后,空白组与低、中、高3个浓度实验组HepG2细胞的存活率分别为(100.00±0.00)%,(87.92±3.13)%,(77.92±4.40)%和(70.53±4.19)%;上述这4组的NLRP3表达水平分别为0.27±0.05,0.50±0.03,0.71±0.08和0.93±0.10;上述这4组的P62表达水平分别为0.54±0.06,0.76±0.05, 0.87±0.04和1.09±0.10。上述指标:3个浓度实验组与空白组比较,差异均有统计学意义(P<0.05,P<0.01,P<0.001)。结论山奈酚可有效抑制HepG2细胞增殖、抑制其自噬并诱导其凋亡,其机制可能为有效促进P62的沉积,同时能诱导凋亡经典途径中NLRP3的合成,从而抑制HepG2细胞自噬并促进其凋亡。     

吡非尼酮对肝癌HepG2细胞增殖和凋亡的影响(“豪森杯”优秀论文三等奖)

目的：研究吡非尼酮（pirfenidone,PF）对人肝癌细胞系HepG2增殖和凋亡的影响。方法：CCK-8法测定不同浓度PF对HepG2细胞增殖活性的影响;Hoechst 33258荧光染色法观察PF处理后HepG2细胞形态的变化;流式细胞仪检测细胞凋亡率。结果：PF对HepG2细胞具有显著增殖抑制作用,并呈浓度和时间依赖性;Hoechst 33258染色可见PF处理后细胞出现典型的凋亡形态学变化;流式细胞仪检测结果显示,与空白组比较,PF处理后的HepG2细胞凋亡率显著增加（P﹤0.01）。结论：PF对人肝癌细胞系HepG2细胞增殖具有抑制作用,且与诱导HepG2细胞凋亡有关。     

白藜芦醇烟酸酯抑制人肝癌细胞HepG2增殖并诱导细胞的凋亡

目的：观察白藜芦醇的修饰物白藜芦醇烟酸酯（ResT）对人肝癌细胞HepG2生长增殖的影响及诱导凋亡的作用。方法：用不同浓度的ResT处理HepG2细胞，MTT法检测ResT对HepG2细胞生长增殖的抑制作用；应用Hochest荧光染色法观察凋亡细胞的发生；流式细胞术（FCM）检测分析细胞周期和细胞凋亡率；比色法测定Caspase-3酶活性。结果：ResT抑制HepG2细胞的增殖并呈现一定的量效和时效关系，HepG2细胞与ResT作用后出现典型的凋亡细胞形态改变，FCM分析显示大部分细胞阻滞于G1期，S期细胞比例降低。且药物作用组出现凋亡峰。药物作用12、24、48h后，细胞的凋亡率分别为8．7％、21．1％、和32.7％。显示ResT诱导的细胞凋亡作用随时间的延长而增加，同时Caspase-3酶活性显著增强。结论：ResT可抑制人肝癌细胞HepG2的生长增殖，其作用机制之一可能与阻滞细胞于G1期及诱导细胞凋亡有关。     

Comparison of ceftibuten transport across Caco-2 cells and rat jejunum mounted on modified Ussing chambers

Ceftibuten uptake into Caco-2 cells and intestinal brush border membrane vesicles is mediated by the dipeptide transport system (PEPT1). The apical to basolateral transport characteristics of ceftibuten across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was examined. Mannitol was used as a paracellular marker along with trans-epithelial electrical resistance (TEER) for monitoring tight junction permeability. Transport across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was linear across the concentration range 0.25-10 mM. The net flux of mannitol and ceftibuten was higher across rat jejunum compared with Caco-2 cells. At a donor concentration of 0.25 mM, ceftibuten transport across Caco-2 cells was found to be pH dependent. Glycyl proline, a dipeptide, and 2,4- dinitrophenol, an energy poison, caused a reduction in the permeability of 0.25 mM ceftibuten across Caco-2 cells. Benzoic acid and adipic acid also inhibited transcellular transport of ceftibuten. At a donor concentration of 0.25 mM, passive paracellular transport accounts for about 60% and the active carrier mediated mechanism accounts for about 40% of ceftibuten transport across Caco-2 cells. None of the inhibitors however, had a significant effect on ceftibuten transport across rat jejunum mounted on a modified Ussing chamber at a donor concentration of 0.25 mM. In the concentration range 0.25-10 mM, ceftibuten is predominantly transported by paracellular mechanisms across rat jejunum and a mixture of active and passive transport across Caco-2 cells.     

Dichlorodiphenyldichloroethylene exposure reduces r‐GCS via suppressed Nrf2 in HepG2 cells

p,p′‐dichlorodiphenyldichloroethylene (p,p′‐DDE), the major isomer of persistent 1,1‐Bis(4‐chlorophenyl)?2,2,2‐trichloroethane metabolite, is highly associated with the risk of liver cancer. γ‐glutamyl‐cysteine synthetase (γ‐GCS), which is the rate‐limiting enzyme of glutathione (GSH) biosynthesis and an important scavenger of reactive oxygen species (ROS), is considered as a potential therapeutic target for many cancers. However, the association between the body burden of p,p′‐DDE and γ‐GCS has not been fully established. Here, we indicated that low doses of p,p′‐DDE exposure promoted the proliferation and decreased γ‐GCS activity of HepG2 cells in a dose‐ and time‐dependent manner. In addition, p,p′‐DDE elevated ROS content and attenuated glutathione peroxidase activity. This was accompanied with inhibitions of NF‐E2‐related factor 2 (Nrf2) at the mRNA and protein levels. ROS inhibitor supplement could significantly reverse these effects. Moreover, the addition of the proteasome inhibitor, MG132, strongly reversed the p,p′‐DDE‐reduced Nrf2 expression and γ‐GCS activity. Consistently, GSH content was in line with the alteration of γ‐GCS. Collectively, the results indicate that p,p′‐DDE treatment downregulates γ‐GCS activity in HepG2 cells by inducing ROS‐mediated Nrf2 loss. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 350–359, 2016.