A possible mechanism of action for azelaic acid in the human epidermis

Summary Azelaic acid, and other saturated dicarboxylic acids (C₉-C₁₂), are shown to be competitive inhibitors of tyrosinase (K _I azelaic acid = 2.73×10^–3 M) and of membrane-associated thioredoxin reductase (K _I azelaic acid = 1.25×10^–5 M). The monomethyl ester of azelaic acid does not inhibit thioredoxin reductase, but it does inhibit tyrosinase, although double the concentration is necessary compared with azelaic acid (K _I azelaic acid monomethyl ester = 5.24×10^–3 M). Neither azelaic acid nor its monomethyl ester inhibit tyrosinase when catechol is used as a substrate instead of l-tyrosine. Therefore, the weak inhibitory action of azelaic acid on tyrosinase appears to be due to the competition of a single carboxylate group on this inhibitor for the -carboxylate binding site of the l-tyrosine substrate on the enzyme active site. Based on the inhibitor constant on tyrosinase, at least cytotoxic levels of azelaic acid would be required for the direct inhibition of melanin biosynthesis in melanosomes if this mechanism is responsible for depigmentation in the hyperpigmentation disorders lentigo maligna and melasma. Alternatively only 10^–5 M azelaic acid is required to inhibit thioredoxin reductase. This enzyme is shown to regulate tyrosinase through a feedback mechanism involving electron transfer to intra-cellular thioredoxin, followed by a specific interaction between reduced thioredoxin and tyrosinase. Furthermore, the thioredoxin reductase/thioredoxin system is shown to be a principal electron donor for the ribonucleotide reductases which regulates DNA synthesis. Inhibition of thioredoxin reductase by azelaic acid provides a rationale for both its depigmenting property and the reversible inhibition of DNA synthesis observed in cultured epidermal cells and also in some of the bacteria associated with acne vulgaris.     

Growth and survival of B-chronic lymphocytic leukaemia cells

Although chronic lymphocytic leukaemia of B-cell type (B-CLL) is the most common form of leukaemia in the Western world, several questions about the biology of B-CLL remain to be clarified. To obtain a conceptual model for B-CLL, defined as a relentless accumulation of resting B-CLL cells, it is particularly relevant to ask which cell type is the normal counterpart of B-CLL; what is the site of proliferation; which signals are involved in the recruitment and induction of proliferation and which signals contribute to the survival of the B-CLL cells? The significance of the studies on B-CLL cellsin vitro for the interpretation of thein vivo situation may be questioned since they oversimplify the multiple and complex cellular interactions that occurin vivo. However, thein vitro studies have been instrumental in elucidating signals that may regulate growth, differentiation and survival of B-CLL cells. This knowledge, herein reviewed, can be used to put forward a hypothesis on B-CLL cell regulationin vivo.     

硫氧还蛋白硝基化在多柔比星诱导的乳鼠心肌细胞凋亡中的作用

 目的：探讨硫氧还蛋白(thioredoxin,Trx)硝基化在多柔比星诱导的乳鼠心肌细胞凋亡过程中的作用。方法：体外分离培养新生SD大鼠心肌细胞,直接给予多柔比星刺激和预孵育过氧亚硝基阴离子(ONOO-)清除剂锰(III)四(1-甲基-4-吡啶基)卟啉(MnTMPyP)后再给予多柔比星刺激。MTT检测细胞存活率,细胞凋亡荧光Hoechst 33258试剂盒检测细胞凋亡情况,分光光度法检测caspase-3的活性,Western blotting法检测聚腺苷酸二磷酸核糖聚合酶1剪切片段［cleaved poly(ADP-ribose) polymerase-1,cleaved PARP-1］、凋亡信号调节激酶1 (apoptosis signal-regulating kinase 1, ASK1)、磷酸化ASK1（p-ASK1）、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase, p38 MAPK）和磷酸化p38 MAPK（p-p38 MAPK）的表达,免疫沉淀法检测Trx-NT和Trx-硝基酪氨酸的形成。结果：给予多柔比星后,心肌细胞Hoechst荧光染色观察到明显凋亡,MnTMPyP预保护后,凋亡情况减轻。多柔比星组与正常组相比,Trx硝基化水平升高,caspase-3活性、cleaved PARP-1和p-p38 MAPK表达增加(P<005),Trx-ASK1和p-ASK1表达减少(P<005)。MnTMPyP预保护组与多柔比星组相比,Trx硝基化水平降低(P<005),caspase-3活性、cleaved PARP-1和p-p38 MAPK表达降低(P<005),Trx-ASK1和p-ASK1表达增加(P<005)。结论: 多柔比星刺激心肌细胞后,Trx硝基化水平和细胞凋亡明显增加;给予ONOO-清除剂MnTMPyP后,Trx硝基化程度和凋亡情况得到明显改善。Trx硝基化可能是多柔比星诱导心肌细胞凋亡的机制之一。     

使用腺病毒过表达TXNIP对INS-1胰岛细胞 损伤和凋亡的影响

【摘要】 目的 使用腺病毒转染技术,通过在胰岛细胞过表达TXNIP蛋白及C247S点突变的TXNIP,观察TXNIP过表达是否可以引起正常培养条件下的胰岛细胞发生损伤和凋亡,并通过与C247S位点突变导致丧失与Trx结合能力的突变TXNIP过表达组的比较,分析TXNIP引起细胞损伤和凋亡的下游途径,以进一步明确TXNIP在糖尿病时引起各器官细胞损伤的机制。方法 将用正常糖脂浓度培养的胰岛细胞分为4组：正常培养组、正常+Ad-eGFP空病毒组、正常 Ad-TXNIP过表达组、正常 Ad-TXNIPC247S点突变过表达组。结果 ①与Ad-eGFP组相比,过表达组TXNIP的mRNA表达、TXNIP蛋白水平、Trx与TXNIP的结合量、LDH活性、caspase-3活性、p38激酶活性均显著增高,而 Trx活性显著降低;C247S点突变过表达组TXNIP的mRNA表达、TXNIP蛋白水平、LDH活性、caspase-3活性均显著增高、Trx与TXNIP的结合量、Trx活性、p38激酶活性均无明显变化。②与过表达组相比,点突变过表达组Trx活性明显升高、LDH活性、caspase-3活性较低,而Trx与TXNIP的结合量和p38激酶活性明显降低 。结论 单纯过表达TXNIP可以引起正常糖脂浓度培养条件下的胰岛细胞发生损伤和凋亡,TXNIP介导细胞损伤和凋亡的机制一方面与其抑制Trx活性,增加自由基损伤、增加ASK1-p38激酶依赖的凋亡途径有关,同时也有不依赖结合抑制Trx的途径存在。TXNIP的表达上调是糖尿病引起胰岛细胞损伤和凋亡的重要原因。     

外源性硫氧还蛋白对高糖、高脂刺激引起的H9c2细胞损伤和凋亡的影响

目的 观察给予外源性硫氧还蛋白（Trx）后对高糖、高脂刺激引起的心肌细胞损伤和凋亡的影响,并分析其可能机制.方法 将处于对数生长期的H9c2心肌细胞随机分为三组：正常对照组（普通DMEM培养基葡萄糖5 mmol/L加入20 mmol/L甘露醇）、高糖高脂组（DMEM高糖25 mmol/L培养基加入高脂600 μmol/L软脂酸）和Trx干预组（在高糖高脂组的培养基中加入3 ＊9滋mol/L Trx）.培养48 h后,分别收集培养基上清与细胞,进行指标检测.结果 与正常对照组比较,高糖高脂组乳酸脱氢酶及caspase-3活性均显著升高（P＜0.01）.给予Trx干预后,与高糖高脂组比较,Trx干预组乳酸脱氢酶及caspase-3活性均显著下降（P＜0.01）.与正常组比较,高糖高脂组Trx的表达量未见明显改变（P＞0.05）,但是Trx活性显著下降（P＜0.01）.结论 高糖、高脂刺激可以引起H9c2心肌细胞损伤和凋亡,其机制与内源性Trx活性的降低有关.外源性补充Trx可明显提高Trx的活性,减轻心肌细胞的损伤和凋亡.     

A comparative study of the role of crocin and sitagliptin in attenuation of STZ-induced diabetes mellitus and the associated inflammatory and apoptotic changes in pancreatic β-islets

Type 1 diabetes mellitus (T1DM) describes a complex group of metabolic disorders associated with elevated blood glucose levels and increased risks of complications development. Exploring new drug therapies would reduce the increased diabetes-associated morbidity and mortality and will reduce the excessive health care costs. Crocin is the major active ingredient of saffron. In the current study, DM was induced by single intraperitoneal injection of streptozocin (50 mg/kg).DM progression was associated with a significant increase in blood glucose level with reduced insulin and increased glucagon secretion. Pancreatic malondialdehyde (MDA) content significantly escalated, while superoxide dismutase (SOD) activity, reduced glutathione (GSH) concentration, catalase activity, thioredoxin level and serum total antioxidant capacity significantly declined. This was associated with a significant increase in pancreatic caspase-3 contents and pancreatic infiltration with inflammatory cells in β-islets. Both sitagliptin and crocin significantly reduced blood glucose levels, enhanced pancreatic insulin expression and secretion and suppressed glucagon secretion with enhancement of anti-oxidant defenses and reduction of oxidative burden, with evident anti-inflammatory impacts. Interestingly, the effect of crocin on DM indices, inflammatory and apoptotic changes was comparable to that of sitagliptin; the standard oral hypoglycemic agent. Nevertheless, crocin had a superior effect compared to sitagliptin on blood sugar level, β-islets diameter and insulin immune-reactivity. In conclusion, crocin reduced blood glucose level mainly via reduction of oxidative burden, modulation of apoptotic pathway and attenuation of pancreatic inflammation.     

介导人硫氧还蛋白基因转移的重组腺病毒构建

目的应用基因重组方法构建携带人硫氧还蛋白(hTRX)基因的复制缺陷型重组腺病毒。方法用内切酶、RT-PCR、测序方法获得目的基因片段,然后将其cDNA克隆至表达载体pShuttle构建hTRX的重组真核细胞表达载体pShuttle-hTRX,在HeLa细胞中进行瞬时表达检测。从真核表达载体pShuttle-hTRX切下目的基因,并插入至腺病毒载体构建hTRX的重组腺病毒载体Adeno-hTRX,经PCR方法亦证明了成功构建腺病毒重组体。重组腺病毒在293细胞中扩增、纯化。结果成功构建了携带人硫氧还蛋白基因的复制缺陷型重组腺病毒,并以多种方法证实了构建载体的正确性。结论正确构建的人硫氧还蛋白重组腺病毒载体,可为基因治疗心血管系统疾病打下良好基础。     

Insulin counteracts glucotoxic effects by suppressing thioredoxin-interacting protein production in INS-1E beta cells and in Psammomys obesus pancreatic islets

Aims/hypothesis  In type 2 diabetes, glucose toxicity leads to beta cell apoptosis with decreased beta cell mass as a consequence. Thioredoxin-interacting protein (TXNIP) is a critical mediator of glucose-induced beta cell apoptosis. Since hyperglycaemia leads to elevated serum insulin, we hypothesised that insulin is involved in the regulation of TXNIP protein levels in beta cells. Methods  We studied the production of TXNIP in INS-1E beta cells and in islets of Psammomys obesus, an animal model of type 2 diabetes, in response to glucose and different modulators of insulin secretion. Results  TXNIP production was markedly augmented in islets from diabetic P. obesus and in beta cells exposed to high glucose concentration. In contrast, adding insulin to the culture medium or stimulating insulin secretion with different secretagogues suppressed TXNIP. Inhibition of glucose and fatty acid-stimulated insulin secretion with diazoxide increased TXNIP production in beta cells. Nitric oxide (NO), a repressor of TXNIP, enhanced insulin signal transduction, whereas inhibition of NO synthase abolished its activation, suggesting that TXNIP inhibition by NO is mediated by stimulation of insulin signalling. Treatment of beta cells chronically exposed to high glucose with insulin reduced beta cell apoptosis. Txnip knockdown mimicking the effect of insulin prevented glucose-induced beta cell apoptosis. Conclusions/interpretation  Insulin is a potent repressor of TXNIP, operating a negative feedback loop that restrains the stimulation of TXNIP by chronic hyperglycaemia. Repression of TXNIP by insulin is probably an important compensatory mechanism protecting beta cells from oxidative damage and apoptosis in type 2 diabetes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.