PPARγ基因靶向shRNA真核表达载体的构建及表达

目的研究短发夹RNA(shorthairpin RNA,shRNA)真核表达载体在人脐静脉内皮细胞(human umbilical veinendo-thelial cell,HUVECs)中对人过氧化物酶体增殖物激活受体γ(PPARγ)基因表达的抑制作用。方法设计3条靶向PPARγ的shRNA,经退火成互补双链,克隆到质粒pGPU6/GFP/Neo中构建重组载体,经酶切鉴定和测序确认后,将3个重组表达载体转染到HUVECs,利用Western blot检测并筛选出抑制效果最好的重组表达载体。结果通过酶切鉴定和测序分析,靶向PPARγ的3个pGPU6/GFP/Neo-shR-NA重组载体构建成功,Western blot结果显示pGPU6/GFP/NeoshRNAPPARγ3可有效抑制高糖诱导HUVECs中PPARγ基因的表达,抑制率为63.2%。结论 PPARγ基因靶向shRNA真核表达载体构建成功,且能有效抑制HUVECs中PPARγ基因的表达。     

Up-regulation of PPARgamma in myocardial infarction

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are key regulators for cardiac energy metabolism after myocardial injury. We hypothesized, that PPARs are regulated in myocardial infarction (MI) and their activity is modulated by angiotensin receptor blockers (ARBs). METHODS: Following induction of MI, male rats were treated with placebo or the ARB irbesartan for three weeks. PPARalpha, beta/delta and gamma protein expression and gene expression of PPAR target genes and glucose transporters were measured. PPARgamma-protein expression was analyzed by immunofluorescence. RESULTS: MI decreased LVP and dp/dtmax and increased LVEDP, this effect was counteracted by irbesartan. PPARalpha and PPARbeta/delta protein expression was not altered in MI and was not affected by irbesartan. PPARgamma protein content was increased in the infarcted area and localized to cardiac myocytes and fibroblasts. In parallel, expression of CTGF was increased 10-fold in the infarcted zone. PPAR target genes (CD36, MCAD, ACO and GLUT4) were significantly decreased in infarcted tissue, and this was unaffected by irbesartan. However, CD36 and ACO in the non-infarcted areas were up-regulated by irbesartan. CONCLUSION: Endogenous up-regulation of PPARgamma in MI is insufficient to counteract the decrease in metabolic genes, but parallels an increase in the profibrotic mediator CTGF. Irbesartan increases fatty acid oxidating enzymes after MI independent of PPARgamma regulation.     

格列酮类降糖作用研究进展

格列酮类化学上属于噻唑烷二酮类化合物。格列酮类增加靶组织对胰岛素的敏感性,降低胰岛素抵抗,是一类新型口服降糖药,被称之为胰岛素增敏剂。格列酮类对正常动物和Ⅰ型糖尿病模型无降糖作用,对遗传胰岛素抵抗模型和人工胰岛素抵抗模型疗效佳。胰岛素抵抗的研究需要正糖夹技术进行检测,正糖夹的评价指标主要是平衡状态下的葡萄糖输注速率。格列酮类降糖作用机制目前认为与过氧化物酶体增殖激活受体有关,格列酮类作用于PPARγ,间接参与胰岛素的信号传导,导致增强胰岛素的效应,但确切机制尚未清楚。     

PPARs和NFкB在高脂血症大鼠内皮细胞中表达的研究

目的:探讨核转录因子PPARα、PPARγ和NFκB在正常及高脂血症大鼠主动脉内皮细胞中表达的变化以及fibrate干预后炎性因子IL-6的变化.方法:取正常及高脂血症大鼠模型主动脉冰冻切片行免疫组化分析PPARα、PPARγ、NFκB和IL-6的表达,PPARs的人工合成配体Gemfibrate和Bzafibrate干预后的变化及其关系.结果:正常大鼠主动脉上有PPARα、PPARγ、NFκB和IL-6的弱表达.高脂血症时,主动脉内皮细胞层PPARα、PPARγ、NFκB、IL-6的表达增强,其OD值较正常大鼠显著增加(P＜0.05),NFκB的活化明显.Fibrates药物干预后,血脂下降(除HDL),PPARα、PPARγ进一步活化,IL-6的表达减弱,对NFκB的表达无明显影响.结论:核转录因子PPARs和NFκB在高脂血症人鼠内皮细胞中均显著增加.Fi-brates类药物可通过活化相应受体PPARs部分抑制炎性因子的分泌.     

Many putative endocrine disruptors inhibit prostaglandin synthesis

Background

Prostaglandins (PGs) play key roles in development and maintenance of homeostasis of the adult body. Despite these important roles, it remains unclear whether the PG pathway is a target for endocrine disruption. However, several known endocrine-disrupting compounds (EDCs) share a high degree of structural similarity with mild analgesics.

Objectives and Methods

Using cell-based transfection and transduction experiments, mass spectrometry, and organotypic assays together with molecular modeling, we investigated whether inhibition of the PG pathway by known EDCs could be a novel point of endocrine disruption.

Results

We found that many known EDCs inhibit the PG pathway in a mouse Sertoli cell line and in human primary mast cells. The EDCs also reduced PG synthesis in ex vivo rat testis, and this reduction was correlated with a reduced testosterone production. The inhibition of PG synthesis occurred without involvement of canonical PG receptors or the peroxisome proliferator–activated receptors (PPARs), which have previously been described as targets of EDCs. Instead, our results suggest that the compounds may bind directly into the active site of the cyclooxygenase (COX) enzymes, thereby obstructing the conversion of arachidonic acid to PG precursors without interfering with the expression of the COX enzymes. A common feature of the PG inhibitory EDCs is the presence of aromatic groups that may stabilize binding in the hydrophobic active site of the COX enzymes.

Conclusion

Our findings suggest a hitherto unknown mode of action by EDCs through inhibition of the PG pathway and suggest new avenues to investigate effects of EDCs on reproductive and immunological disorders that have become increasingly common in recent decades.     

中药来源过氧化物酶体增殖物激活受体激动剂在治疗糖尿病方面的研究进展

过氧化物酶体增殖物激活受体(peroxisome proliferator activated receptors,PPARs)是配体激活的转录因子核受体超家族成员之一,其激动剂可以用于治疗糖尿病及其并发症。一些中药提取物具有PPARs激动剂活性,已被证实可以治疗实验性糖尿病及其并发症。综述了在中药中发现的已被阐明结构的PPARs激动剂,为其研究开发及糖尿病治疗药物的设计提供参考。     

Conjugated linoleic acid prevents cell growth and cytokine production induced by TPA in human keratinocytes NCTC 2544

Conjugated linoleic acid (CLA) is reported to have anti-cancer activity, based on animal and in vitro studies. Since it has been suggested that CLA anti-carcinogenic effect stems from its anti-inflammatory properties, this study investigated whether CLA can prevent cell proliferation induced by TPA in human keratinocytes NCTC 2544 contemporary to inhibition of inflammation.     

Multitargeted bioactive ligands for PPARs discovered in the last decade

Type 2 diabetes took insulin resistance as the main clinical manifestation. PPARs have been reported to be the therapeutic targets of metabolic disorders, such as obesity, hypertension, diabetes, and cardiovascular disease. Previously, PPARγ agonist rosiglitazone was restricted in clinic due to cardiomyocytes infarction, weight gain, and other serious side‐effects, which were mainly due to the single and selective PPARγ agonism. In recent years, multitarget‐directed PPAR agonists with synergistic reaction as well as fewer side‐effect have been the hot topic in designing promising agents. In this review, we updated and generalized the development of PPARγ partial agonists, PPARγ antagonists, PPARα/γ dual agonists, PPARδ partial agonists, PPARδ antagonists, PPARα/δ dual agonists, PPARγ/δ dual agonists, and PPARα/γ/δ pan‐agonists published in recent decade. Most of these molecules were modified from known structures or came from high‐throughput screening. Among these molecules, some were expected to be promising drugs against metabolic disorders, while others seemed to provide new insight for designing novel PPAR agents.