Evaluation of the intracellular lipid-lowering effect of polyphenols extract from highland barley in HepG2 cells

Active ingredients from highland barley have received considerable attention as natural products for developing treatments and dietary supplements against obesity.In practical application,the research of food combinations is more significant than a specific food component.This study investigated the lipid-lowering effect of highland barley polyphenols via lipase assay in vitro and HepG2 cells induced by oleic acid(OA).Five indexes,triglyceride(TG),total cholesterol(T-CHO),low density lipoprotein-cholesterol(LDL-C),aspartate aminotransferase(AST),and alanine aminotransferase(ALT),were used to evaluate the lipidlowering effect of highland barley extract.We also preliminary studied the lipid-lowering mechanism by Realtime fluorescent quantitative polymerase chain reaction(q PCR).The results indicated that highland barley extract contains many components with lipid-lowering effects,such as hyperoside and scoparone.In vitro,the lipase assay showed an 18.4%lipase inhibition rate when the additive contents of highland barley extract were 100μg/m L.The intracellular lipid-lowering effect of highland barley extract was examined using 0.25 mmol/L OA-induced HepG2 cells.The results showed that intracellular TG,LDL-C,and T-CHO content decreased by 34.4%,51.2%,and 18.4%,respectively.ALT and AST decreased by 51.6%and 20.7%compared with the untreated hyperlipidemic HepG2 cells.q PCR results showed that highland barley polyphenols could up-regulation the expression of lipid metabolism-related genes such as PPARγand Fabp4.

Reduction of the oxidative damage to H_(2)O_(2)-induced HepG2 cells via the Nrf2 signalling pathway by plant flavonoids Quercetin and Hyperoside

Hyperoside and quercetin are similar in molecular structures.In this study,the antioxidant regulatory targets of hyperoside and quercetin are mainly in the nuclear factor(erythroid-2-derived)-related factor 2(Nrf2)pathway predicted by network pharmacology.And the antioxidant effect and mechanism of hyperoside and quercetin were measured and compared in H_(2)O_(2)-induced Hep G2 cells and Caenorhabditis elegans.The findings indicated that quercetin was more effective than hyperoside in reducing oxidative damage,which was proved by improved cell viability,decreased reactive oxygen species(ROS)production,decreased cellular apoptosis,and alleviated mitochondrial damage.In addition,quercetin was more efficient than hyperoside in enhancing the expression of Nrf2-associated m RNAs,increasing the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT),and reducing the cellular malondialdehyde(MDA)content.Quercetin was superior to hyperoside in prolonging the lifespan of worms,decreasing the accumulation of lipofuscin,inhibiting ROS production,and increasing the proportion of skn-1 in the nucleus.With the Nrf2 inhibitor ML385,we verified that quercetin and hyperoside primarily protected the cells against oxidative damage via the Nrf2 signalling pathway.Furthermore,molecular docking and dynamics simulations demonstrated that the quercetin-Kelch-like ECH-associated protein 1(Keap1)complex was more stable than the hyperoside-Keap1 complex.The stable structure of the complex might hinder the binding of Nrf2 and Keap1 to release Nrf2 and facilitate its entry into the nucleus to play an antioxidant role.Overall,quercetin had a better antioxidant than hyperoside.

Naringin ameliorates H_(2)O_(2)-induced oxidative damage in cells and prolongs the lifespan of female Drosophila melanogaster via the insulin signaling pathway

Naringin exists in a wide range of Chinese herbal medicine and has proven to possess several pharmacological properties.In this study,PC12,HepG2 cells,and female Drosophila melanogaster were used to investigate the antioxidative and anti-aging effects of naringin and explore the underlying mechanisms.The results showed that naringin inhibited H_(2)O_(2)-induced decline in cell viability and decreased,the content of reactive oxygen species in cells.Meanwhile,naringin prolonged the lifespan of flies,enhanced the abilities of climbing and the resistance to stress,improved the activities of antioxidant enzymes,and decreased malondialdehyde content.Naringin also improved intestinal barrier dysfunction and reduced abnormal proliferation of intestinal stem cells.Moreover,naringin down-regulated the mRNA expressions of inr,chico,pi 3k,and akt-1,and up-regulated the mRNA expressions of dilp2,dilp3,dilp5,and foxo,thereby activating autophagy-related genes and increasing the number of lysosomes.Furthermore,the mutant stocks assays and computer molecular simulation results further indicated that naringin delayed aging by inhibiting the insulin signaling(IIS)pathway and activating the autophagy pathway,which was consistent with the result of network pharmacological predictions.

黄蜀葵花总黄酮介导AKT1信号通路拮抗HepG-2细胞脂质沉积研究

目的:探讨黄蜀葵花总黄酮(TFA)对棕榈酸(PA)诱导HepG-2细胞脂质沉积模型的干预效应及机制。方法:体外培养人肝源HepG-2细胞,应用PA处理以构建细胞脂质沉积模型,然后从黄蜀葵花中提取TFA,以TFA或蛋白激酶B(AKT)抑制剂作为干预剂,对PA诱导的细胞脂质沉积模型进行预处理,应用油红O染色、酶联免疫吸附实验和免疫印迹实验(Western blotting)检测细胞脂质代谢、AKT1和脂联素(APN)信号通路蛋白表达水平。结果:与PA模型组比较,TFA或AKT抑制剂干预后,细胞脂质沉积程度(%)、游离脂肪酸(FFAs)和甘油三酯(TG)水平降低,而APN水平升高(P<0.05)。Western blotting实验显示PA模型组AKT1和固醇调节元件结合蛋白1(SREBP-1)表达水平上调而APN和糖原合成酶激酶3β(GSK3β)水平下调(P<0.05)。与PA模型组比较,TFA或AKT抑制剂干预显示AKT1下调而APN水平上调,其下游信号GSK3β表达上调而SREBP-1表达下调(P<0.05)。结论:TFA拮抗PA诱导的HepG-2细胞脂质沉积可能与其介导AKT1抑制及APN信号通路激活有关。

Protective effect of brain and muscle arnt-like protein-1 against ethanol-induced ferroptosis by activating Nrf2 in mice liver and HepG2 cells

Alcohol abuse has recently become a serious health concern worldwide,and the incidence of alcoholic liver disease(ALD)is rapidly increasing with high morbidity and mortality.Ferroptosis is a newly recognized form of regulated cell death caused by the iron-dependent accumulation of lipid peroxidation.Here we showed that the circadian clock protein brain and muscle arnt-like protein-1(BMAL1)in hepatocytes is both necessary and sufficient to protect against ALD by mitigating ferroptosis.U pon exposure to alcohol(5%Lieber-DeCarli liquid alcohol diet for 10 days before binged alcohol with 5 g/kg body weight in vivo,300 mmol/L for 12 h in vitro,respectively),the content of iron,reactive oxygen species(ROS)and malondialdehyde(MDA)was boosted signifi cantly while glutathione(GSH)was decreased that mainly based on the downregulated protein expression of ferritin heavy chain(FTH),ferroportin(FPN),heme oxygenase1(HO-1)and anti-cystine/glutamate antiporter(SLC7A11),while these changes could be abolished by ferroptosis inhibitor Ferrostatin-1[Fer-1(5 mg/kg body weight for 10 days in vivo,10μmol/L for 2 h in vitro,respectively)].Further study indicated that the alcohol could activate the protein expression of BMAL1 which exerts a protective effect against ferroptosis through promoting nuclear factor erythroid 2-related factor 2(Nrf2)translocation into nuclear and subsequently stimulating its downstream proteins FTH,FPN,glutathione peroxidase 4 activity(GPX4),HO-1,SLC7A11,while knockdown of BMAL1 and Nrf2 by RNA interference further downregulated the expression of these protein and thus promoting ferroptosis in response to alcohol.Collectively,our results unveiled that the protective action of BMAL1 during alcohol challenge depends on its ability to activate Nrf2-ARE antiferroptosis pathway and targeting hepatic BMAL1 to dampen hepatic ferroptosis signaling may have therapeutic potential for ALD.

The anti-cancerous mechanism of licochalcone A on human hepatoma cell HepG2 based on the miRNA omics

To explore the function of licochalcone A as an anticancer phytochemical on HepG2 cells and investigate its potential mechanisms,we analyzed the microRNAs(miRNAs)expression profile of HepG2 cells in response to licochalcone A(70μmol/L)in vitro.102 dysregulated miRNAs were detected,and SP1 was expected as the transcription factor that regulates the functions of most screened miRNAs.A sum of 431 targets,the overlap of predicted mRNAs from TargetScan,miRDB,and miRtarbase were detected as the targets for these dysregulated miRNAs.FoxO signaling pathway was the hub pathway for the targets.A protein-protein interaction network was structured on the STRING platform to discover the hub genes.Among them,PIK3R1,CDC42,ESR1,SMAD4,SUMO1,KRAS,AGO1,etc.were screened out.Afterwards,the miRNA-target networks were established to screen key dysregulated miRNAs.Two key miRNAs(hsa-miR-133b and hsa-miR-145-5p)were filtered.Finally,the miRNA-target-transcription factor networks were constructed for these key miRNAs.The networks for these key miRNAs included three and two transcription factors,respectively.These identified miRNAs,transcription factors,targets,and regulatory networks may offer hints to understand the molecular mechanism of licochalcone A as a natural anticarcinogen.

mRNA transcriptome profiling of human hepatocellular carcinoma cells HepG2 treated with Catharanthus roseus-silver nanoparticles

BACKGROUND The demand for the development of cancer nanomedicine has increased due to its great therapeutic value that can overcome the limitations of conventional cancer therapy.However,the presence of various bioactive compounds in crude plant extracts used for the synthesis of silver nanoparticles(AgNPs)makes its precise mechanisms of action unclear.AIM To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G.Don(C.roseus)-AgNPs.METHODS The proliferative activity of hepatocellular carcinoma(HepG2)and normal human liver(THLE3)cells treated with C.roseusAgNPs were measured using MTT assay.The RNA samples were extracted and sequenced using BGIseq500 platform.This is followed by data filtering,mapping,gene expression analysis,differentially expression genes analysis,Gene Ontology analysis,and pathway analysis.RESULTS The mean IC 50 values of C.roseusAgNPs on HepG2 was 4.38±1.59μg/mL while on THLE3 cells was 800±1.55μg/mL.Transcriptome profiling revealed an alteration of 296 genes.C.roseusAgNPs induced the expression of stress-associated genes such as MT,HSP and HMOX-1.Cellular signalling pathways were potentially activated through MAPK,TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest.The alteration of ARF6,EHD2,FGFR3,RhoA,EEA1,VPS28,VPS25,and TSG101 indicated the uptake of C.roseus-AgNPs via both clathrin-dependent and clathrinindependent endocytosis.CONCLUSION This study provides new insights into gene expression study of biosynthesised AgNPs on cancer cells.The cytotoxicity effect is mediated by the aberrant gene alteration,and more interestingly the unique selective antiproliferative properties indicate the C.roseusAgNPs as an ideal anticancer candidate.

Anti-diabetic potential of apigenin,luteolin,and baicalein via partially activating PI3K/Akt/GLUT-4 signaling pathways in insulin-resistant HepG2 cells

Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities.In this study,apigenin,luteolin,and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant(IR)HepG2 cells.All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4(GLUT4)and phosphor-glycogen synthase kinase(GSK-3β).These fl avonoids signifi cantly inhibited the production of reactive oxygen species(ROS)and advanced glycation end-products(AGEs),which were closely related to the suppression of the phosphorylation form of NF-κB and P65.The expression levels of insulin receptor substrate-1(IRS-1),insulin receptor substrate-2(IRS-2)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway in IR-HepG2 cells were all partially activated by the fl avonoids,with variable effects.Furthermore,the intracellular metabolic conditions of the fl avonoids were also evaluated.

三萜皂苷Saxifragifolin D抑制人肝癌耐药细胞HepG2/ADM生长并诱导细胞凋亡作用研究

目的研究Saxifragifolin D(SD)对人肝癌耐药细胞HepG2/ADM的生长抑制及诱导凋亡作用。方法采用MTT法观察SD对HepG2/ADM细胞的增殖抑制作用,应用流式细胞仪分析SD对细胞周期的影响,AnnexinⅤ-FITC/PI双染检测凋亡细胞比率,JC-1染色观察SD对细胞内线粒体膜电位的影响,Western blot检测凋亡相关蛋白caspase-9,caspase-3和PARP的激活及c-Raf,MEK和ERK蛋白的表达和磷酸化水平。结果 SD可以明显抑制人肝癌耐药细胞HepG2/ADM的增殖。细胞周期检测发现SD诱导细胞产生亚二倍体凋亡峰,同时细胞凋亡率也由对照组的5.3%增加到34.8%和47.8%。线粒体膜电位检测结果显示SD导致细胞内线粒体膜电位的明显降低。Western blot检测结果表明caspase-9,caspase-3被激活,PARP被剪切活化,cytochrome C由线粒体释放至胞质,c-Raf、MEK和ERK蛋白的磷酸化水平降低。结论 SD可以抑制人肝癌耐药细胞HepG2/ADM增殖并诱导其凋亡,作用机制可能与线粒体功能障碍及抑制c-Raf/MEK/ERK通路的活化有关。

姜黄素对肝癌耐药细胞HepG2/ADM的增殖和阿霉素耐药性的影响

目的分析姜黄素对肝癌耐药细胞HepG2/ADM的增殖和阿霉素耐药性的影响。方法 (1)将HepG2/ADM细胞分为不同浓度药物组(加入5μg/mL、10μg/mL、20μg/mL、40μg/mL、60μg/mL的姜黄素)、阴性对照组(接种细胞但不加药物)及空白对照组(不接种细胞,只加培养基),培养24、48及72 h后检测细胞增殖情况。(2)将HepG2细胞、HepG2/ADM细胞分为药物组、阴性对照组(接种细胞但不加药物)及空白对照组(不接种细胞,只加培养基)。HepG2细胞药物组加入不同浓度阿霉素(0.1μg/mL、0.2μg/mL、0.4μg/mL、0.8μg/mL、1.6μg/mL);HepG2/ADM细胞药物组分两个亚组,一组加入不同浓度阿霉素(1.5μg/mL、3μg/mL、6μg/mL、12μg/mL、24μg/mL),另一组加入5μg/mL姜黄素和不同浓度阿霉素(1.5μg/mL、3μg/mL、6μg/mL、12μg/mL、24μg/mL)。培养48 h后检测细胞增殖情况,计算姜黄素的逆转倍数以及HepG2/ADM细胞耐药指数。(3)将HepG2/ADM细胞分为对照组(只加培养基)、阿霉素组(14μg/mL阿霉素)、姜黄素组(5μg/mL姜黄素)、阿霉素与姜黄素联合组(14μg/mL阿霉素+5μg/mL姜黄素)。作用48 h后,检测各组磷酸化p38丝裂原活化蛋白激酶(p38MAPK)蛋白的表达。结果 (1)不同浓度的姜黄素均可抑制肝癌耐药细胞HepG2/ADM的增殖。姜黄素分别作用48、72 h后,HepG2/ADM细胞的增殖抑制率随着药物浓度的增加而增加(均P<0.05);在5、10、20、40μg/mL姜黄素作用下HepG2/ADM细胞的增殖抑制率随着作用时间的延长而增加(均P<0.05)。(2)HepG2/ADM细胞对阿霉素的耐药指数为6.81,对阿霉素呈中度耐药;5μg/mL姜黄素对HepG2/ADM细胞阿霉素耐药性的逆转倍数为1.49。(3)对照组磷酸化p38MAPK蛋白水平高于其他组(均P<0.05);姜黄素组、阿霉素组磷酸化p38MAPK蛋白水平均高于阿霉素与姜黄素联合组(均P<0.05),但姜黄素组与阿霉素组之间差异无统计学意义(P>0.05)。结论姜黄素不仅可体外抑制肝癌耐药细胞HepG2/ADM的增殖,还可逆转其对阿霉素的耐药性,而这可能与姜黄素抑制p38MAPK的磷酸化有关。

人参皂苷Rh2对人肝癌细胞HepG2/ADM耐药逆转作用及其机制研究

目的人参皂苷Rh2能抑制多种恶性肿瘤细胞增殖,但缺少Rh2对多药耐药的肝癌细胞的敏感性研究。文中主要探讨人参皂苷Rh2对人肝癌细胞株HepG2/ADM多药耐药性[盐酸多柔比星(ADM)、5-氟尿嘧啶(5-Fu)、顺铂(DDP)、长春新碱(VCR)]的逆转作用及机制。方法 MTT法检测(0~250μg/m L)Rh2对HepG2/ADM细胞活力的影响;MTT法筛选最佳逆转耐药的Rh2浓度。细胞分为空白对照组、ADM组和ADM+40μg/m LRh2组。空白对照组:不加药物处理;ADM组:给予ADM处理48 h;ADM+40μg/m LRh2组:给予40μg/m L的Rh2预处理30 min后,给予ADM处理48 h。流式细胞术检测Rh2对细胞内Rh-123荧光强度的影响;RT-PCR法检测MDR1基因的表达;Western blot检测P-gp、Bax、Bcl-2、cleved caspase-3蛋白水平。结果与HepG2细胞相比,HepG2/ADM对ADM、DDP、5-FU、VCR 4种化疗药物的耐药指数分别为32.95、4.63、4.20、4.81。经过40μg/m L的Rh2作用HepG2/ADM细胞48 h后,耐药细胞对4种化疗药的敏感性增强且IC50明显下降,其耐药性的逆转倍数分别为3.70、3.53、2.64、2.55倍。耐药细胞内储留的Rh-123的荧光强度通过流式细胞仪检测结果显示,与ADM组比较,加入40μg/m L Rh2后,细胞内Rh-123的荧光强度明显增高(65.83±1.78 vs 78.21±1.26,P<0.01)。RT-PCR结果显示,ADM+40μg/m L Rh2组MDR1表达较ADM组显著降低(0.48±0.02 vs 0.86±0.05,P<0.05)。Western blot结果显示,ADM+40μg/m L Rh2组P-gp蛋白水平亦较ADM组明显降低(0.97±0.04 vs 1.91±0.03,P<0.01);ADM+40μg/m L Rh2组Bax和cleaved caspase-3表达较ADM组明显增加(1.76±0.04 vs 1.25±0.02,38.26±5.45 vs 0.42±0.04,P<0.05),同时发现Bcl-2表达明显减少(1.25±0.05 vs 1.86±0.03,P<0.05)。结论人参皂苷Rh2能有效逆转HepG2/ADM细胞的多药耐药性,其作用机制可能与降低MDR1、P-gp表达、增加细胞内药物积累以及介导Bax/Bcl-2信号通路有关。

HIFU逆转人肝癌细胞HepG2/Adm多药耐药的实验研究

背景与目的:超声波能够改变细胞膜通透性,增强化疗药物对肿瘤细胞的杀伤作用。本研究评价高强度聚焦超声及其联合阿霉素(Adriamycin,ADM)对人肝癌多药耐药细胞株HepG2/Adm的效应,并探讨其作用机制。方法:取对数生长期的HepG2、HepG2/Adm细胞进行实验,分为HepG2、HepG2(超声波照射5s)、HepG2/Adm、HepG2/Adm(超声波照射5s)4组。用MTT法检测处理前后耐药细胞对抗癌药物的敏感性;用流式细胞仪检测肿瘤细胞表面mdr1基因表达产物P170的表达及细胞内阿霉素浓度;利用SP免疫组化法观察细胞膜及核膜上P-糖蛋白(P-glycoprotein,P-gp)的表达。结果:频率0.8MHz、焦域声强460W/cm2连续照射5s,能够部分逆转HepG2/Adm细胞的耐药性,HepG2/Adm细胞P-gp的表达活性下降83.1%,耐药细胞内ADM浓度增加88%,对ADM、cDDP、MMC、5-FU和MTX相对逆转效率分别为66.4%、63.4%、89.4%、72.4%和75.0%。结论:高强度聚焦超声可提高人肝癌细胞HepG2/Adm内药物浓度,降低细胞膜及核膜表面P-gp表达,从而部分逆转肿瘤细胞多药耐药。

丹皮酚对肝癌HepG2/ADM细胞株多药耐药性的逆转作用及其机制

目的探讨丹皮酚体外给药对肝癌耐药细胞株Hep G2/阿霉素(ADM)多药耐药性的逆转作用及其可能分子机制。方法采用人的肝癌细胞株HepG2,体外ADM浓度递增法诱导建立耐药肝癌细胞HepG2/ADM耐药模型。分别用0、5、10、25、50、100μmol/L的丹皮酚培养液培养HepG2/ADM、Hep G2细胞48 h,选定25、50μmol/L丹皮酚进行下一步实验。分别采用ADM(0.01、0.2、0.5、0.75、1.5、3.0μmol/L)和氟尿嘧啶(0、1、2、4、10、20、40μmol/L)单独刺激,及25、50μmol/L丹皮酚联合上述各浓度的氟尿嘧啶及ADM联合刺激2种细胞株。采用MTT法检测HepG2/ADM、HepG2细胞的增殖水平,流式细胞仪检测Hep G2/ADM、HepG2细胞内细胞毒药物含量,蛋白免疫印迹法检测肿瘤细胞P-糖蛋白表达水平。结果 ADM抑制Hep G2/ADM、HepG2细胞株的IC50依次为1.48、124.14μmol/L,耐药指数为83.9;丹皮酚(25、50μmol/L)能够明显增强ADM、氟尿嘧啶对HepG2/ADM细胞株的细胞毒作用,并降低IC50值(P均<0.01),但对HepG2细胞的细胞毒作用无明显影响;丹皮酚(25、50μmol/L)能够明显增加HepG2/ADM细胞株对ADM的摄取能力(P均<0.01),但对HepG2细胞株无明显影响;丹皮酚能显著降低HepG2/ADM细胞株P-糖蛋白表达水平(P均<0.01),但对HepG2细胞株P-糖蛋白水平无明显影响。结论丹皮酚对HepG2/ADM细胞株多药耐药性具有明显的逆转作用,可能与其抑制HepG2/ADM细胞株P-糖蛋白表达、增加细胞毒药物细胞内摄取有关。

负载抗原的DC与CIK共培养对多药耐药肝癌细胞HepG2/ADM的杀伤作用及其机制研究

目的观察负载抗原的树突状细胞(DC)与细胞因子诱导的杀伤细胞(CIK)对高表达P-糖蛋白(Pgp)的多药耐药(MDR)的肝癌细胞株HepG2/ADM细胞的杀伤作用和机制研究。方法用常规方法诱导健康志愿者外周血中单个核细胞产生DC和CIK细胞,制备HepG2/ADM细胞冻融抗原后冲击DC,并与CIK细胞分别共培养24、48、72、96h,并将未负载抗原的DC和CIK细胞共培养作为对照。采用流式细胞术鉴定DC、CIK细胞的表型,采用CCK-8试剂检测HepG2/ADM细胞的增殖活力。用RT-PCR检测各组细胞内mdr-1mRNA的水平变化,Western blot检测细胞内P-gp蛋白水平的变化。结果与未负载抗原的DC-CIK相比,负载抗原的DC-CIK表面分子表达明显增高(P<0.05),对HepG2/ADM细胞增殖活力抑制作用更加明显(P<0.05)。RT-PCR和Western blot结果分别显示,随着作用时间的延长,未负载抗原的DC-CIK和负载抗原DC-CIK的HepG2/ADM细胞内的mdr-1mRNA和P-gp蛋白水平分别都有明显的降低(P<0.05),而且后者的抑制作用更明显(P<0.05)。结论经抗原冲击的DC和CIK共培养可以明显提高对多药耐药HepG2/ADM细胞株的杀伤活性,其机制可能与抑制与MDR密切相关的mdr-1基因和及其编码的P-gp蛋白水平有关。

粗毛纤孔菌三萜成分分析及其对HepG 2细胞模型的脂质调节作用

为探索粗毛纤孔菌中的总三萜活性成分及其体外降脂能力,采用超高液相色谱-飞行时间质谱联用技术(UPLC-TOF-MS/MS)对粗毛纤孔菌中的总三萜进行定性分析,并通过油酸诱导建立HepG_(2)细胞高脂模型,以HepG_(2)细胞高脂模型的存活率、脂质积累量和血脂四项为指标,考察粗毛纤孔菌纯化后总三萜的体外降脂能力。根据UPLC-TOF-MS/MS得到的洗脱顺序、保留时间、分子量、质谱信息,结合文献,共鉴定出粗毛纤孔菌中三萜成分8种。体外实验表明,400μmol/L油酸孵育HepG_(2)细胞24 h时,HepG_(2)细胞内的脂质积累量最高。相较于其他浓度组,总三萜质量浓度为250μg/mL时,高脂HepG_(2)细胞内的脂质含量下降最为显著,总胆固醇、甘油三酯、低密度脂蛋白胆固醇的清除率分别达到41.86%、44.44%、44.02%,高密度脂蛋白胆固醇的增长率达到57.33%。研究结果证实了粗毛纤孔菌三萜的体外降脂功能,为开发新型降血脂功能食品提供了实验依据。

辐射诱导多药耐药肝癌细胞HepG2/ADM凋亡的实验研究

目的研究联合应用放、化疗对多药耐药肝癌细胞HepG2/ADM凋亡的影响并探讨诱导凋的机制.方法比较放化疗、单纯放疗、单纯化疗的诱导凋亡作用,将HepG2/ADM分为6组,比较各组凋亡率;应用RT-PCR法检测各组HepG2/ADM细胞p53、bcl-2和bax的表达.结果经处理因素作用后,组1～6HepG2/ADM细胞凋亡率分别为2.8±0.3%,5.2±0.5%,10.9±2.3%,31.7±4.1%,11.7±2.8%,35.2±4.5%.放化疗与单纯化疗相比诱导HepG2/ADM凋亡率明显增高(P＜0.05).RT-PCR检测组1～6 p53表达分别为:0.22±0.04、0.23±0.02、0.31±0.04、0.55±0.02、0.35±0.04、0.57±0.05;bcl-2表达分别为:0.34±0.03、0.3l±0.04、0.25±0.02、0.14±0.02、0.26±0.03、0.10±0.03;bax表达分别为:0.21±0.02、0.25±0.02、0.29±0.03、0.38±0.03、0.29±0.03、0.41±0.06.结论放化疗联合应用能够显著增加耐药细胞HepG2/ADM凋亡率,这种作用与p53、bax、bcl-2表达调节有关.

慢病毒介导BC047440基因沉默逆转HepG2/ADM细胞化疗耐药性机制的探讨

目的利用siRNA方法沉默HepG2/ADM细胞系中BC047440基因,观察其受抑制后对细胞多药耐药性的影响及其分子机制。方法建立BC047440蛋白在HepG2/ADM细胞系siRNA干扰模型,采用Western blot检测BC047440蛋白的表达;实验分3组:BC047440-shRNA组、control-shRNA组和HepG2/ADM组。CCK-8法分析阿霉素对细胞生长抑制率的影响,流式细胞仪检测细胞凋亡及细胞周期分布;Western blot检测BC047440沉默后NF-κB蛋白表达的变化;Real-time PCR检测Survivin、CCNL1基因mRNA水平的变化。结果成功建立BC047440蛋白siRNA干扰模型;NF-κB蛋白表达BC047440-shRNA组约为HepG2/ADM组67.69%,约为control-shRNA组67.39%;细胞毒性实验中24、48 h阿霉素对BC047440-shRNA组细胞毒性作用明显增强;流式细胞仪检测结果显示BC047440-shRNA组细胞凋亡明显增多,细胞周期分布多停留于G1/G0期,S期细胞明显减少。Real-time PCR检测BC047440-shRNA组Survivin基因mRNA表达约为con-trol-shRNA组37%,约为HepG2/ADM组42%;检测BC047440-shRNA组CCNL1基因mRNA表达约为control-shRNA组3.5倍,约为HepG2/ADM组2.4倍。结论沉默BC047440基因可通过NF-κB信号通路及其下游Survivin、CCNL1信号逆转HepG2/ADM细胞的多药耐药性,提示BC047440基因可能是形成肝癌多药耐药的重要分子之一。

美洲大蠊提取物逆转人肝癌细胞HepG2/ADM多药耐药的机制研究

目的:研究美洲大蠊提取物脱脂膏及CⅡ-3(分别简称"脱脂膏""CⅡ-3")逆转耐阿霉素(ADM)人肝癌细胞HepG2/ADM多药耐药的作用机制。方法:采用MTT法考察不同质量浓度索拉非尼(阳性对照)、脱脂膏和CⅡ-3对HepG2/ADM细胞的毒性作用,并计算20%抑制浓度(IC20)。试验设置敏感组、耐药组、索拉非尼组、脱脂膏组和CⅡ-3组,敏感组使用HepG2细胞,后4组均使用HepG2/ADM细胞。敏感组和耐药组细胞给予常规培养基,其余3组细胞给予相应药物(浓度均为IC20)。采用激光扫描共聚焦显微技术测定细胞中ADM含量;采用Western blotting法测定细胞中凋亡相关蛋白[B细胞淋巴瘤2(Bcl-2)、剪切型胱天蛋白酶9 p37(Cleaved-Caspase-9 p37)]的表达水平;分别采用实时荧光定量-聚合酶链式反应法和免疫细胞化学染色法检测细胞中多药耐药相关基因mRNA及蛋白[P-糖蛋白(P-gp)(MDR1基因表达产物)、肺耐药蛋白(LRP)、乳腺癌耐药相关蛋白(BCRP)]和酶介导多药耐药途径中相关基因mRNA及蛋白[谷胱甘肽转移酶(GST-π)、DNA拓扑异构酶Ⅱ(TopoⅡ)]的表达水平。结果:索拉非尼、脱脂膏、CⅡ-3对HepG2/ADM细胞的IC20分别为(2.40±0.16)、(200.44±27.52)、(18.00±1.82)μg/mL。与敏感组比较,耐药组细胞中Bcl-2、P-gp、LRP、BCRP、TopoⅡ蛋白表达水平以及MDR1、LRP、BCRP、GST-πm RNA表达水平均显著升高(P<0.05或P<0.01)。与耐药组比较,脱脂膏组和CⅡ-3组细胞中ADM含量显著增加(P<0.05或P<0.01),MDR1 m RNA表达水平和LRP、BCRP、GST-πm RNA及其蛋白表达水平均显著降低(P<0.05或P<0.01);CⅡ-3组细胞中Bcl-2蛋白表达水平、TopoⅡmRNA表达水平均显著降低(P<0.01),Cleaved-Caspase-9 p37蛋白表达水平显著升高(P<0.05)。结论:脱脂膏、CⅡ-3可通过减少药物外排、促进细胞凋亡、减少多药耐药相关基因和酶介导多药耐药途径中相关基因的mRNA及其蛋白的表达等方式,逆转HepG2/ADM细胞的多药耐药性,且CⅡ-3的效果优于脱脂膏。

Hepatocellular carcinoma HepG2 cell apoptosis and caspase-8 and Bcl-2 expression induced by injectable seed extract of Coix lacryma-jobi

BACKGROUND:Many Chinese herbs,especially herbal injections,have been shown to have anti-tumor effects in recent years.However,since most reports focus on the clinical effectiveness of these herbs,their mechanisms of action are not well understood.In this study,we assessed apoptosis in the hepatocellular carcinoma (HCC) cell line HepG2 induced by an injectable extract from the seed of Coix lacryma-jobi (Semen coicis,SC),and monitored the expression of Bcl-2 and caspase-8.METHODS:Injectable SC was applied to HepG2 cells at different concentrations and the cells were collected 12,24 and 48 hours later.5-fluorouracil was used as a positive control group,and fluorescence-activated cell-sorting cytometry was used to measure the apoptosis rate of HepG2 cells and the expression of Bcl-2 and caspase-8 proteins.RESULTS:SC induced apoptosis in HepG2 cells in a concentration and time-dependent manner,and the expression of caspase-8 was elevated and prolonged.However,it did not significantly influence the expression of Bcl-2.CONCLUSION:Injectable SC may induce apoptosis in HCC cells by regulating the expression of caspase-8.

Partial Beclin 1 silencing aggravates doxorubicin-and Fasinduced apoptosis in HepG2 cells

AIM： To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment. METHODS： Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry. Levels of Beclin 1, BCI-XL and cytochrome c, and the cleavage of poly （ADP-ribose） polymerase （PARP） were assayed by using Western blots. RESULTS： Beclin 1 expression decreased by 75% 72 h after Beclin 1 siRNA transfection. Partial Beclin 1 silencing significantly increased the percentage of subG1 cells 24 and 40 h after treatment with doxorubicin or anti-Fas antibody, respectively, and this potentiation was abrogated by treatment with a pan-caspase inhibitor. Partial Beclin 1 silencing also increased PARP cleavage, mitochondrial membrane depolarization and cytosolic cytochrome c. The pro-apoptotic consequences of partial Beclin 1 silencing were not associated with a decline in Bcl-XL expression.CONCLUSION： Partial Beclin 1 silencing aggravates mitochondrial permeabilization and apoptosis in HepG2 cells treated with an anti-Fas antibody or with doxorubicin.