Pinolenic Acid Downregulates Lipid Anabolic Pathway in HepG2 Cells

Pine nut oil (PNO) was reported to reduce lipid accumulation in the liver. However, the specific effect of pinolenic acid (18:3, all‐cis‐Δ5,9,12), a unique component of PNO, on lipid metabolism has not been studied. We hypothesized that pinolenic acid downregulates the lipid anabolic pathway in HepG2 cells. HepG2 cells were incubated in serum‐free medium supplemented with 50 μM bovine serum albumin (BSA), palmitic acid, oleic acid, γ‐linolenic acid, pinolenic acid, eicosapentaenoic acid (EPA), or α‐linolenic acid for 24 h. Lipid accumulation was determined by Oil Red O (ORO) staining. The mRNA levels of genes related to fatty acid biosynthesis (SREBP1c, FAS, SCD1, and ACC1), fatty acid oxidation (ACC2, PPARα, CPT1A, and ACADL), cholesterol synthesis (SREBP2 and HMGCR), and lipoprotein uptake (LDLr) and of genes that may be involved in the downregulation of the lipogenic pathway (ACSL3, ACSL4, and ACSL5) were determined by qPCR. LDLR protein levels were measured by Western blot analysis. The mRNA levels of SREBP1c, FAS, and SCD1 were significantly downregulated by pinolenic acid treatment compared to BSA control (53, 54, and 38 % lower, respectively). In addition, the mRNA levels of HMGCR, ACSL3, and LDLr were significantly lower (30, 30, and 43 % lower, respectively), and ACSL4 tended to be lower in the pinolenic acid group (20 % lower, P = 0.082) relative to the control group. In conclusion, pinolenic acid downregulated the lipid anabolic pathway in HepG2 cells by reducing expression of genes related to lipid synthesis, lipoprotein uptake, and the regulation of the lipogenic pathway.     

Indomethacin and Diclofenac Hybrids with Oleanolic Acid Oximes Modulate Key Signaling Pathways in Pancreatic Cancer Cells

Our earlier studies showed that coupling nonsteroidal anti-inflammatory drugs (NSAIDs) with oleanolic acid derivatives increased their anti-inflammatory activity in human hepatoma cells. The aim of this study was to evaluate their effect on the signaling pathways involved in inflammation processes in human pancreatic cancer (PC) cells. Cultured PSN-1 cells were exposed for 24 h (30 µM) to OA oxime (OAO) derivatives substituted with benzyl or morpholide groups and their conjugates with indomethacin (IND) or diclofenac (DCL). The activation of NF-κB and Nrf2 was assessed by the evaluation of the translocation of their active forms into the nucleus and their binding to specific DNA sequences via the ELISA assay. The expression of NF-κB and Nrf2 target genes was evaluated by R-T PCR and Western blot analysis. The conjugation of IND or DCL with OAO derivatives increased cytotoxicity and their effect on the tested signaling pathways. The most effective compound was the DCL hybrid with OAO morpholide (4d). This compound significantly reduced the activation and expression of NF-κB and enhanced the activation and expression of Nrf2. Increased expression of Nrf2 target genes led to reduced ROS production. Moreover, MAPKs and the related pathways were also affected. Therefore, conjugate 4d deserves more comprehensive studies as a potential PC therapeutic agent.     

Streptozotocin-Induced Cytotoxicity, Oxidative Stress and Mitochondrial Dysfunction in Human Hepatoma HepG2 Cells

Streptozotocin (STZ) is an antibiotic often used in the treatment of different types of cancers. It is also highly cytotoxic to the pancreatic beta-cells and therefore is commonly used to induce experimental type 1 diabetes in rodents. Resistance towards STZ-induced cytotoxicity in cancer cells has also been reported. Our previous studies have reported organ-specific toxicity and metabolic alterations in STZ-induced diabetic rats. STZ induces oxidative stress and metabolic complications. The precise molecular mechanism of STZ-induced toxicity in different tissues and carcinomas is, however, unclear. We have, therefore, investigated the mechanism of cytotoxicity of STZ in HepG2 hepatoma cells in culture. Cells were treated with different doses of STZ for various time intervals and the cytotoxicity was studied by observing the alterations in oxidative stress, mitochondrial redox and metabolic functions. STZ induced ROS and RNS formation and oxidative stress as measured by an increase in the lipid peroxidation as well as alterations in the GSH-dependent antioxidant metabolism. The mitochondria appear to be a highly sensitive target for STZ toxicity. The mitochondrial membrane potential and enzyme activities were altered in STZ treated cells resulting in the inhibition of ATP synthesis. ROS-sensitive mitochondrial aconitase activity was markedly inhibited suggesting increased oxidative stress in STZ-induced mitochondrial toxicity. These results suggest that STZ-induced cytotoxicity in HepG2 cells is mediated, at least in part, by the increase in ROS/RNS production, oxidative stress and mitochondrial dysfunction. Our study may be significant for better understanding the mechanisms of STZ action in chemotherapy and drug induced toxicity.     

微波萃取、高效液相色谱法测定番石榴叶中的齐墩果酸和熊果酸

建立了微波萃取、高效液相色谱测定番石榴叶中齐墩果酸和熊果酸的方法。采用分式析因设计实验考察了微波萃取参数的影响,番石榴叶粒度和萃取时间是影响齐墩果酸和熊果酸的微波萃取产率的最重要因素。优化的微波萃取条件如下:60~80 mesh番石榴叶、10 mL乙醇、80℃和10 min。微波萃取与索氏萃取得到的齐墩果酸和熊果酸产率一致,然而,前者仅需10 min,后者则需要4 h。     

Structure of Sphingolipids From Sea Cucumber Cucumaria frondosa and Structure-Specific Cytotoxicity Against Human HepG2 Cells

To investigate the relationship between structure and activity, three glucocerebroside series (CFC‐1, CFC‐2 and CFC‐3), ceramides (CF‐Cer) and long‐chain bases (CF‐LCB) of sea cucumber Cucumaria frondosa (C. frondosa) were isolated and evaluated in HepG2 cells. The molecular species of CFC‐1, CFC‐2 and CFC‐3 and CF‐Cer were identified using reversed‐phase liquid chromatography with heated electrospray ionization coupled to high‐resolution mass spectrometry (RPLC‐HESI‐HRMS), and determined on the basis of chemical and spectroscopic evidence: For the three glucocerebroside series, fatty acids (FA) were mainly saturated (18:0 and 22:0), monounsaturated (22:1, 23:1 and 24:1) and 2‐hydroxyl FA (2‐HFA) (23:1 h and 24:1 h), the structure of long‐chain bases (LCB) were dihydroxy (d17:1, d18:1 and d18:2) and trihydroxy (t16:0 and t17:0), and the glycosylation was glucose; For CF‐Cer, FA were primarily saturated (17:0) and monounsaturated (16:1 and 19:1), the structure of LCB were dihydroxy (d17:1 and d18:1), and trihydroxy (t16:0). The results of cell experiment indicated that all of three glucocerebroside series, CF‐Cer and CF‐LCB exhibited an inhibitory effects on cell proliferation. Moreover, CFC‐3 was most effective in three glucocerebrosides to HepG‐2 cell viability. The inhibition effect of CF‐LCB was the strongest, and the inhibition effect of CF‐Cer was much stronger than glucocerebrosides.     

Inhibition of Human Group IIA-Secreted Phospholipase A2 and THP-1 Monocyte Recruitment by Maslinic Acid

Maslinic acid is a natural pentacyclic triterpenoid which has anti‐inflammatory properties. A recent study showed that secretory phospholipase A₂ (sPLA₂) may be a potential binding target of maslinic acid. The human group IIA (hGIIA)‐sPLA₂ is found in human sera and their levels are correlated with severity of inflammation. This study aims to determine whether maslinic acid interacts with hGIIA‐sPLA₂ and inhibits inflammatory response induced by this enzyme. It is shown that maslinic acid enhanced intrinsic fluorescence of hGIIA‐sPLA₂ and inhibited its enzyme activity in a concentration‐dependent manner. Molecular docking revealed that maslinic acid binds to calcium binding and interfacial phospholipid binding site, suggesting that it inhibit access of catalytic calcium ion for enzymatic reaction and block binding of the enzyme to membrane phospholipid. The hGIIA‐sPLA₂ enzyme is also responsible in mediating monocyte recruitment and differentiation. Results showed that maslinic acid inhibit hGIIA‐sPLA₂‐induced THP‐1 cell differentiation and migration, and the effect observed is specific to hGIIA‐sPLA₂ as cells treated with maslinic acid alone did not significantly affect the number of adherent and migrated cells. Considering that hGIIA‐sPLA₂ enzyme is known to hydrolyze glyceroacylphospholipids present in lipoproteins and cell membranes, maslinic acid may bind and inhibit hGIIA‐sPLA₂ enzymatic activity, thereby reduces the release of fatty acids and lysophospholipids which stimulates monocyte migration and differentiation. This study is the first to report on the molecular interaction between maslinic acid and inflammatory target hGIIA‐sPLA₂ as well as its effect towards hGIIA‐sPLA₂‐induced THP‐1 monocyte adhesive and migratory capabilities, an important immune‐inflammation process in atherosclerosis.     

Cyclopropaneoctanoic Acid 2‐Hexyl Upregulates the Expression of Genes Responsible for Lipid Synthesis and Release in Human Hepatic HepG2 Cells

Recently we have found cyclopropaneoctanoic acid 2‐hexyl (CPOA2H) in humans and demonstrated its elevated levels in patients with metabolic diseases associated with hypertriglyceridemia. However, it is still unclear whether CPOA2H may influence lipid metabolism in lipogenic tissues. To verify this, HepG2 hepatocytes and 3T3‐L1 adipocytes were cultured with various concentrations of CPOA2H, and then the expressions of genes associated with lipid metabolism were determined. Incubation with CPOA2H at concentrations found in patients with metabolic diseases enhanced the expression of hepatocyte genes associated with lipid synthesis and release, in particular, the fatty acid synthase gene (nearly 20‐fold increase in the mRNA level). In contrast, incubation with CPOA2H caused the downregulation of most adipocyte genes associated with lipid synthesis, whereas the level of leptin mRNA was increased. These findings suggest that CPOA2H may contribute to hypertriglyceridemia in patients with metabolic diseases, upregulating the expression of hepatocyte genes responsible for lipid synthesis and release.     

人NIRF基因真核表达质粒的构建及其在HepG2.2.15细胞中的表达

目的构建人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达NIRF蛋白。方法从HeLa细胞中提取总RNA,设计特异性引物,通过RT-PCR法扩增NIRF基因编码区全长序列,插入到pIRES2-EGFP真核表达载体中,构建重组真核表达质粒pIRES2-EGFP-NIRF,转染HepG2.2.15细胞,检测细胞中NIRF基因mRNA的转录水平及蛋白的表达水平。结果重组真核表达质粒pIRES2-EGFP-NIRF经双酶切及测序鉴定证明构建正确,转染HepG2.2.15细胞后,可检测到细胞中NIRF基因mRNA的转录及蛋白的表达。结论已成功构建了人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达了NIRF蛋白,为下一步研究其在肿瘤组织中的功能奠定了基础。     

Inhibitory Effects of Ursolic Acid on the Stemness and Progression of Human Breast Cancer Cells by Modulating Argonaute-2

The stemness and metastasis of cancer cells are crucial features in determining cancer progression. Argonaute-2 (AGO2) overexpression was reported to be associated with microRNA (miRNA) biogenesis, supporting the self-renewal and differentiation characteristics of cancer stem cells (CSCs). Ursolic acid (UA), a triterpene compound, has multiple biological functions, including anticancer activity. In this study, we find that UA inhibits the proliferation of MDA-MB-231 and MCF-7 breast cancer cell lines using the CCK-8 assay. UA induced a significant decrease in the fraction of CSC in which it was examined by changes in the expression of stemness biomarkers, including the Nanog and Oct4 genes. UA altered invasion and migration capacities by significant decreases in the levels of epithelial-to-mesenchymal transition (EMT) proteins of slug and vimentin. Furthermore, the co-reduction in oncogenic miRNA levels (miR-9 and miR-221) was a result of the down-modulation in AGO2 in breast cancer cells in vitro. Mechanically, UA increases PTEN expression to inactivate the FAK/PI3K/Akt/mTOR signaling pathway and the decreased level of c-Myc in quantitative RT-PCR and Western blot imaging analyses. Our current understanding of the anticancer potential of UA in interrupting between EMT programming and the state of CSC suggests that UA can contribute to improvements in the clinical practice of breast cancer.     

Effect of Quercetin and Fingolimod,Alone or in Combination,on the Sphingolipid Metabolism in HepG2 Cells

Combinations of anti-cancer drugs can overcome resistance to therapy and provide new more effective treatments. In this work we have analyzed the effect of the polyphenol quercetin and the anti-cancer sphingosine analog fingolimod on the sphingolipid metabolism in HepG2 cells, since sphingolipids are recognized as mediators of cell proliferation and apoptosis in cancer cells. Treatment of hepatocellular carcinoma HepG2 cells with quercetin and fingolimod, alone or in combination, induced different degrees of sphingomyelin (SM) reduction and a corresponding activation of neutral sphingomyelinase (nSMase). Western blot analysis showed that only treatments containing quercetin induced up-regulation of nSMase expression. The same treatment caused elevation of ceramide (CER) levels, whereas the observed alterations in sphingosine (SPH) content were not statistically significant. The two tested drugs induced a reduction of the pro-proliferative sphingolipid, sphingosine 1 phosphate (S1P), in the following order: quercetin, fingolimod, quercetin + fingolimod. The activity of the enzyme responsible for CER hydrolysis, alkaline ceramidase (ALCER) was down-regulated only in the incubations involving quercetin and fingolimod did not affect this activity. The enzyme, maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was down-regulated by incubations in the following order: quercetin, fingolimod, quercetin + fingolimod. Western blot analysis showed down-regulation in SK1 expression upon quercetin but not upon fingolimod treatment. Studies on the effect of quercetin and fingolimod on the two proteins associated with apoptotic events, AKT and Bcl-2, showed that only quercetin, alone or in combination, down-regulated the activity of the two proteins. The reported observations provide information which can be useful in the search of novel anti-tumor approaches, aiming at optimization of the therapeutic effect and maximal preservation of healthy tissues.     

2-羟基-3-萘甲酸生产工艺条件的优化研究

通过对中和及酸析过程的分析,采用改进后的参数指标进行中和及酸析得到的2-羟基-3-萘甲酸,产品为大颗粒、亮黄色,含量98.78%,收率80.13%(以消耗的2-萘酚计),并实现了在大生产中的应用。     

Identification of Cyclopropaneoctanoic Acid 2-Hexyl in Human Adipose Tissue and Serum

Fatty acids containing a cyclopropane ring in their structure (cyclopropane FA) have been found in a wide variety of bacteria, a number of protozoa, and Myriapoda. Little is known about cyclopropane FA in mammal, especially in human tissues. The present study deals with the identification of cyclopropane FA in adipose tissue and serum of humans and rats. Fatty acids extracted from the adipose tissue and serum obtained from obese women during bariatric surgery were methylated and analyzed on GC–MS. We have identified: cyclopropaneoctanoic acid 2-hexyl, cyclopropaneoctanoic acid 2-octyl, cyclopropanenonanoic acid, and 2-[[2-[(2-ethylcyclopropyl)methyl]cyclopropyl]methyl] acid in human adipose tissue. We confirmed the presence of cyclopropaneoctanoic acid 2-hexyl by derivatization of FA extracted from human adipose tissue to picolinyl esters. Cyclopropaneoctanoic acid 2-hexyl was the main cyclopropane FA (approximately 0.4 % of total fatty acids in human adipose tissue, and about 0.2 % of total fatty acids in the serum). In adipose tissue cyclopropaneoctanoic acid 2-hexyl was found mainly in triacylglycerols, whereas in serum in phospholipids and triacylglycerols. The cyclopropaneoctanoic acid 2-hexyl has also been found in serum, and adipose tissue of rats in amounts comparable to humans. The content of cyclopropaneoctanoic acid 2-hexyl decreased in adipose tissue of rats maintained on a restricted diet for 1 month. In conclusion, we demonstrated that cyclopropaneoctanoic acid 2-hexyl is present in human adipose tissue and serum. Adipose tissue cyclopropaneoctanoic acid 2-hexyl is stored mainly in triacylglycerols and the storage of this cyclopropane FA is affected by food restriction.     

2-Chlorohexadecanal and 2-Chlorohexadecanoic Acid Induce COX-2 Expression in Human Coronary Artery Endothelial Cells

2-Chlorohexadecanal (2-ClHDA), a 16-carbon chain chlorinated fatty aldehyde that is produced by reactive chlorinating species attack of plasmalogens, is elevated in atherosclerotic plaques, infarcted myocardium, and activated leukocytes. We tested the hypothesis that 2-ClHDA and its metabolites, 2-chlorohexadecanoic acid (2-ClHA) and 2-chlorohexadecanol (2-ClHOH), induce COX-2 expression in human coronary artery endothelial cells (HCAEC). COX-2 protein expression increased in response to 2-ClHDA treatments at 8 and 20 h. 2-ClHA also increased COX-2 expression following an 8 h treatment. Quantitative PCR showed that 2-ClHDA treatment increased COX-2 mRNA over 8 h, while 2-ClHA treatment led to a modest increase by 1 h and those levels remained constant over 8 h. 2-ClHDA led to a significant increase in 6-keto-PGF(1alpha) release (a measure of PGI(2) release) by HCAEC. These data suggest that 2-ClHDA and its metabolite 2-ClHA, which are produced during leukocyte activation, may alter vascular endothelial cell function by upregulation of COX-2 expression.     

QC法测定福辛普利钠中异辛酸和新戊酸的残留量

建立了气相色谱法测定福辛普利钠中异辛酸和新戊酸的残留量。采用DB-wax(50 m×0.32 mm,3μm)色谱柱,FID检测,以氮气为载气,起始温度50℃,保持5 min后,以40℃/min的速度升至170℃,保持10 min,以甲醇为稀释液,正戊酸为内标物,直接进样。结果显示,福辛普利钠中异辛酸和新戊酸完全分开,浓度在考察范围内与峰面积具有良好的线性关系,相关系数r为0.9994和0.9991,精密度RSD均小于10%,平均回收率分别为98.9%、97.8%均在90%~110%之间。本法快速、灵敏、准确,可用于福辛普利钠中异辛酸和新戊酸的测定。     

CLA Reduces Inflammatory Mediators from A427 Human Lung Cancer Cells and A427 Conditioned Medium Promotes Differentiation of C2C12 Murine Muscle Cells

Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor “in vivo”, excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1β and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.     

一种用腐殖酸助滤生产硝磷酸液的新方法

在以硝酸和磷矿为起始原料生产硝基肥的工艺中：在硝酸萃取磷矿过程中加入腐殖酸,过滤工段利用腐殖酸助滤,实现了固液顺利分离;滤饼简单洗涤并中和后去造有机肥,滤液经后续工序去造硝基肥,这样不外排滤渣,养分也不损失,做到了资源的循环利用。     

古龙酸纳滤浓缩过程的膜污染及膜清洗

研究了古龙酸纳滤浓缩过程中膜污染的原因,在此基础上,对纳滤膜清洗剂进行了筛选,并考察了不同化学清洗剂对膜通量恢复率的影响,并使用电镜观测的方法（SEM）对膜污染及清洗后的效果进行了定性分析。结果表明,选用含酶的复合清洗剂S-001,膜通量恢复率达到97．8％。