气／质联用法检测反式花生四烯酸对糖尿病大鼠视网膜微血管的硝化应激损伤评价

背景反式花生四烯酸（TAAs）是NO2介导的花生四烯酸异构化的主要产物,是硝化应激的特异性脂质标志物。NO2介导的损伤是缺血性视网膜病变可能的发病机制之一,先前的研究证实高糖可以引发硝化应激。目的应用气／质联用（GC／MS）法检测TAAs水平以评价高糖引发的硝化应激对视网膜微血管的损害程度。方法2周龄雄性SD大鼠100只随机分为模型组和正常对照组,应用链脲佐菌素（STZ）60mg／kg一次性腹腔注射诱导糖尿病动物模型。应用改良合成法,花生四烯酸（AA）经环氧化及去氧化制得TAAs的标准品14E—AA。GC／MS检测造模后2、4、8、12、16周时血清中TAAs及AA的含量,选择离子79,计算峰面积之比。结果50只大鼠成功建立糖尿病模型,其高血糖状态持续在实验期。14E—AA纯度达到97．3％,可作为标准品使用。糖尿病大鼠造模成功50只,每组10只,造模后2周、4周模型组大鼠血清中TAAs／AA（反式与）顺式之比与正常对照组相比差异无统计学意义（t=-0．376、t=-0．642,P〉0．05）;造模后8、12、16周糖尿病大鼠血清中TAAs／AA值为0．1042、0．1568和0．1895,明显高于对照组的0．0832、0．0910和0．1100,差异均有统计学意义（t=-36．409、t=-166．714、t=-148．212,P〈0．05）。结论糖尿病大鼠血清中TAAs／AA水平随病程的延长而增加,提示硝化应激对视网膜微血管的损伤程度与病程的长短有关。建立GC／MS检测TAAs体系可为缺血性微血管病变的研究提供新的评价指标。     

番荔枝内酯类聚醚类似物AA005抗白血病细胞体外增殖效应及其可能的机制

本研究探讨番荔枝内酯类聚醚类似物AA005广泛的体外抗白血病细胞增殖效应及对急性早幼粒白血病细胞系NB4的可能作用机制。采用CCK-8试剂盒检测AA005对多种白血病细胞系增殖的影响;台盼蓝染色法检测细胞活率的改变;瑞氏染色后显微镜观察NB4细胞形态学结构的变化;流式细胞术分析细胞死亡形式;WesternBlot检测caspase-3的活化情况及其底物PARP-1的剪切情况;流式细胞术检测低浓度AA005(<200 nmol/L)对细胞周期的影响。结果表明,高浓度AA005(>200 nmol/L)能够抑制多种白血病细胞的增殖,呈浓度依赖性;不同浓度的AA005作用于NB4、U937、K562白血病细胞48 h后,绝大多数细胞死亡;以NB4细胞为例,AA005作用后出现类似凋亡形态特征;AA005几乎可同时诱导细胞发生早期凋亡及晚期凋亡;AA005诱导细胞死亡过程中仅有微弱的caspase-3活化及其底物PARP-1的剪切,caspase广谱抑制剂Z-VAD-fmk不能抑制AA005所致的细胞死亡;非毒性浓度的AA005(<200 nmol/L)引起细胞G2/M期阻滞。结论:番荔枝内酯类聚醚类似物AA005具有广泛的抗白血病细胞增殖效应,其机制可能与AA005诱导白血病细胞死亡及引起细胞G2/M期阻滞有关。     

血小板反应蛋白-1上调介导反式花生四烯酸对糖尿病大鼠的硝化应激损伤

Background  Trans-arachidonic acids (TAAs), newly discovered markers of nitrative stress and the major products of nitrogen dioxide (NO₂˙)-mediated isomerization of arachidonic acid (AA), represent a new mechanism of NO₂˙-induced toxicity. It has been reported that TAAs were generated in oxygen-induced microvascular degeneration model and TAAs were also generated in a diabetic retinopathy (DR) model. In this study, we examined high glucose-induced nitrative stress damage and TAAs levels and explored the possible mechanisms for DR caused by reactive nitrogen species.

Methods  Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. Bovine retinal capillary endothelial cells (BRECs) were selectively cultured and incubated with normal or high glucose. The serum TAAs and AA in diabetic rats were measured by the gas chromatography and mass spectrometry (GC/MS) method. The ratio of peak area of TAAs to AA with selected ion of 79 was estimated by a group t-test. Thrombospondin-1 (TSP-1) in the rat retinas and BRECs extracts were examined by Western blotting. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) protein was examined by Western blotting in BRECs incubated with high glucose. 

Results  The TAAs to AA ratio (TAAs/AA) was significantly increased in the serum at 8, 12 and 16 weeks after STZ injection (P <0.05), with no noticeable change found at 2 or 4 weeks (P >0.05). Expression of TSP-1 in the retina of diabetic rats was progressively elevated according to the duration of diabetes. TSP-1 expression was increased in BRECs incubated with high glucose at 48 hours. Moreover, high glucose also increased ERK1/2 expression, which peaked at 30 minutes and then decreased in the following 48 hours.

Conclusion  An elevation of TAAs/AA is associated with high glucose-induced nitrative stress, which probably involves upregulation of TSP-1 through activating ERK1/2.