Cytoprotective effects of quercetin and its sugar-containing natural congeners in cultured HEK293 cells injured by anoxia/hypoglycemia and the structure-effect relationship thereto

Objective To comparatively study anti-free radical and cytoprotective effects of quercetin(Q)and its monoglucoside isoquercetin(I),diglucoside rutin(R),which differs only in glycosyl-substitution at C-3 position of the molecules,using anoxia/hypoglycemia-induced cell injury model and thereby to explore the structure-effect relationship thereto.Methods The cell injury model was established by HEK293 cells cultured in vitro with Na2S2O3 plus sugar-free Earle's fluid as incubation medium;Cell survival rate(CSR),total antioxidant capacity(TAC),SOD and LDH levels were determined;The effect intensity of the 3 flavonoids were compared by means of IC50,the concentration required to achieve 50% inhibition of the changes in the above indices in injured cells.Results Q,I and R all concentration-dependently elevated CSR,TAC and SOD,and reduced LDH level;the all of IC50s for the above indices were ranked in order of IC50,QI>R.Conclusions The 3 structurally similar flavoloids all have significant and concentration-dependent anti-free radical and cyto-protective effects with the intensity being in order of aglycone>monoglucoside>diglucoside;the substitution of-OH by sugar group at C-3 position of flavoloids and increase in the sugar-substituent number are associated with the effect intensity reduced;namely,the intensity of these effects of flavonoids is negatively related the substutution by sugar group at C-3 position.     

高效液相色谱法同时测定葡萄籽中芦丁、槲皮素与异槲皮素的含量

目的 建立高效液相色谱法测定葡萄籽中的芦丁、槲皮素与异槲皮素的含量.方法 葡萄籽经提取处理得到黄酮苷元,通过高效液相色谱法测定芦丁、槲皮素、异槲皮素的含量来计算总黄酮的含量.在Agilent 1200系统中,室温条件下使用Symmetry C18柱(4.6 mm×250 mm,5.0 μm),以乙腈-0.4％磷酸溶液(40∶60)为流动相;流速1.0mL· min-1;检测波长501 nm.结果 芦丁、槲皮素、异槲皮素在0.2～1.6 μg线性关系均良好,相关系数r分别为0.999 1、0.999 4、0.999 0,平均回收率分别为101.0％、98.2％、99.7％,RSD分别为1.3％、2.7％、1.6％(n=6).结论 该法灵敏度高、专属性强,可以通过测定葡萄籽中的芦丁、槲皮素、异槲皮素来推算其总黄酮含量,对葡萄籽的药用成分价值作出评估.     

槲皮素及其水溶性衍生物对HL-60细胞生长影响的比较

目的　研究并比较槲皮素及其硫酸酯钠盐衍生物 (槲皮素单硫酸酯钠、槲皮素二硫酸酯钠 )对HL 6 0细胞生长的影响。方法　细胞存活力以台盼兰染色法测定 ;蛋白激酶CK 2活性以连接到CK 2底物上的 [γ 3 2 P]GTP的3 2 P放射活度来检测 ;细胞周期变化以流式细胞仪检测。结果　槲皮素、槲皮素单硫酸酯钠和槲皮素二硫酸酯钠均可抑制HL 6 0细胞的生长 ,槲皮素和槲皮素单硫酸酯钠对HL 6 0细胞生长的抑制作用差异无显著性 (P >0 0 5 ) ,槲皮素二硫酸酯钠的作用则比槲皮素弱得多 (P <0 0 1) ;槲皮素可抑制HL 6 0细胞中蛋白激酶CK 2的活性 ,而槲皮素单硫酸酯钠和槲皮1999 0 5 14收稿 ,1993 0 7 15修回 广东省自然科学基金资助课题 (No 940 5 86)和广东省高教厅重点学科资助课题 (No 93 0 4)作者简介 :翁　云 ,女 ,2 7岁 ,硕士 ;梁念慈 ,男 ,5 8岁 ,教授 ,博士生导师 ,广东医学院院长。研究方向 :生化药理学素二硫酸酯钠对HL 6 0细胞内CK 2活性无明显影响 ;槲皮素阻断HL 6 0细胞于G2 /M期 ,而槲皮素单硫酸酯钠和槲皮素二硫酸酯钠均阻断细胞于G0 /G1期。结论　槲皮素、槲皮素单硫酸酯钠和槲皮素二硫酸酯钠均为潜在的肿瘤细胞生长抑制剂 ,但它们的抑制机制却不相同 ,槲皮素 7 OH对维持槲皮素的功能有重要作用。     

槲皮素对高同型半胱氨酸血症家兔血管内皮细胞的保护作用

目的　评估槲皮素对高同型半胱氨酸血症家兔血管内皮细胞的影响。方法　制造家兔高同型半胱氨酸损伤血管内皮细胞模型 ,同时灌胃给予槲皮素 5 0、10 0和 2 0 0mg·kg-1,30d后采血进行循环内皮细胞计数 ,测定血清MDA、SOD、NO和血浆ET ,取胸主动脉作血管反应性测定 ,并进行组织学检查。结果　与损伤模型组相比 ,槲皮素给药组循环内皮细胞降低 ,MDA、ET水平降低 ,而NO和SOD水平升高 ,对ACh的内皮依赖性舒张反应趋近正常 ,对NTG的内皮非依赖性舒张反应无影响 ,组织学检查也证实槲皮素有效地对抗高同型半胱氨酸造成的血管内皮细胞损伤。结论　槲皮素对高同型半胱氨酸损伤家兔血管内皮细胞有保护作用。     

RP-HPLC法测定新疆哈萨克族常用药材包尔胡特果实中芦丁和槲皮素的含量

目的建立测定新疆包尔胡特(Rhamnus cathartica L.)果实中芦丁和槲皮素含量的方法。方法色谱柱:依利特Hypersil ODS2C18(260mm×4.6mm,5μm)配预柱;芦丁的流动相:甲醇-10mL·L~(-1)乙酸溶液(70∶30),槲皮素的流动相:甲醇-4mL·L~(-1)磷酸溶液(35∶65);流速均为1.0mL·min~(-1);柱温:30℃;芦丁检测波长:257nm,槲皮素检测波长:360nm;进样量均为10μL。结果芦丁质量浓度在43.411 0~165.600 0μg·mL~(-1)范围内线性关系良好(r=0.999 7),平均加样回收率为99.84%,RSD值为1.45%;槲皮素质量浓度在5.454 0~87.264 0μg·mL~(-1)范围内线性关系良好(r=0.999 9),平均加样回收率为99.83%,RSD值为1.49%。结论该方法简便、准确、重复性好,可用于包尔胡特果实原料中芦丁和槲皮素的含量测定。     

Kinetic studies of the effects of Temodal and quercetin on astrocytoma cells

The aim of the present study was to investigate the kinetics of the effects exerted by Temodal and quercetin on the survival of the human astrocytoma MOGGCCM cell line. Our results indicate that quercetin was toxic and induced necrosis, whereas Temodal induced autophagy-mediated cell death most effectively. The amount of cell death directly correlated with drug concentration and length of exposure. During combined administration of both drugs, Temodal attenuated the cytotoxic effects of quercetin. Combinations of both drugs were effective in inducing programmed cell death, but the type of cell death was concentration-dependent. Co-administration of Temodal (100 μM) with a low quercetin concentration (5 μM) resulted in a very significant induction of autophagy; however, after treatment with quercetin at a higher concentration (30 μM), apoptosis became the primary mechanism of cell death. The sequence of drug administration was also important. The highest number of dead cells was observed after simultaneous administration of both drugs or after pre-incubation with Temodal followed by treatment with quercetin. Apoptosis was identified through activation of the mitochondrial pathway including cleavage of caspase-3 and release of cytochrome c. Autophagy was identified through increased levels of LC3II. Our results indicate that Temodal and quercetin are synergistic inducers of programmed cell death, better together than applied separately. This drug combination appears to be a potent and promising therapeutic relevant to the treatment of gliomas.     

Dual effects of quercetin in doxorubicin‐induced nephrotoxicity in rats and its modulation of the cytotoxic activity of doxorubicin on human carcinoma cells

Quercetin (QUR) has been shown to induce anti‐, as well as, pro‐oxidant effects depending on the dose and on the redox state of the cell. This study investigated the effects of different doses of QUR on doxorubicin (DOX)‐induced nephrotoxicity in rats with emphasis on the suggested mechanisms and its modulation of the cytotoxic activity of DOX on different human carcinoma cell lines. Three doses of QUR (10, 50, and 100 mg kg^?1) were administered orally to adult male albino rats for 14 days, in the presence or absence of nephrotoxicity induced by a single intraperitoneal injection of DOX (15 mg kg^?1) at day 7 of the experiment. Moreover, the effect of QUR in the presence of DOX on the cell viability of four different human cancer cell lines; PC3, HELA, MCF7, and HEPG2 was studied. Results showed that the lowest dose of QUR was more effective in preserving renal function (kidney index, blood urea nitrogen, serum creatinine, renal malondialdehyde, nitric oxide, reduced glutathione, catalase activity, renal expressions of TNF‐α, IL‐1B, iNOS, and caspase‐3, and renal histopathology) than higher doses. Alternatively, the pro‐oxidant and pro‐inflammatory mechanisms have been reflected at highest QUR dose. In conclusion, QUR protected against DOX‐induced nephrotoxicity with a provision to dosage adjustment. Furthermore, QUR did not interfere but rather enhanced the cytotoxic effects of DOX on different human cancer cell lines. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 624–636, 2016.     

槲皮素对HL-60细胞中p53、bcl-2基因表达的影响

目的探讨槲皮素对人早幼粒白血病细胞株（ＨＬ ６０）细胞中ｐ５３、ｂｃｌ ２基因表达的影响。方法采用台盼蓝排除法确定活细胞数和免疫细胞化学ＳＰ染色法分别检测细胞中ｐ５３、ｂｃｌ ２基因表达。结果槲皮素对ＨＬ ６０细胞的增殖有明显的抑制作用,当其作用２４ｈ后,其ＩＣ５０为４６６μｍｏｌ·Ｌ－１,槲皮素对ＨＬ ６０细胞中ｐ５３、ｂｃｌ ２基因表达均有一定的抑制作用,且呈明显的剂量依赖关系。结论槲皮素抑制ｐ５３、ｂｃｌ ２的表达可能是其抑制ＨＬ ６０细胞增殖和生长的重要机制之一。     

PGE_1对培养乳鼠心肌细胞缺氧/复氧损伤的保护作用

目的　研究PGE1对纯化培养心肌细胞缺血再灌注损伤的保护作用及机制。方法　采用纯化培养的心肌细胞建立缺氧 /复氧损伤模型 ,实验分 5组 :正常细胞培养组、缺氧 /复氧损伤模型组、缺氧 /复氧损伤 +PGE1(5、15、4 5 μg·L-1)组。分别用黄嘌呤氧化酶法测定SOD活力 ,硫代巴比妥酸显色法测定MDA含量 ,MTT染色法测定线粒体脱氢酶活性改变并测定LDH含量变化。结果　PGE1可明显提高SOD、LDH活性 ,降低MDA含量并可提高线粒体脱氢酶活性。结论　PGE1具有明显的抗缺氧复氧损伤、保护心肌作用     

Acute effects of diesel exhaust particles and cisplatin on oxidative stress in cultured human kidney (HEK 293) cells,and the influence of curcumin thereon

Particulate air pollution with particle diameters less than 2.5 μm contribute to respiratory and extra-respiratory morbidity and mortality. We have recently reported the first in vivo experimental evidence that Diesel exhaust particles (DEP) in the lung aggravated the renal, pulmonary, and systemic effects of cisplatin (CP)-induced acute renal failure in rats. This in vitro study sought to determine whether and to what extent does DEP exposure exacerbate the effects of CP-induced oxidative stress in human embryonic kidney (HEK-293) cells, and to examine if these effects could be mitigated/prevented with curcumin (the yellow pigment isolated from turmeric). Cells viability, cysteine uptake and oxidative stress indices [glutathione (GSH), total antioxidant capacity (TAC), and the activities of antioxidant enzymes (catalase; glutathione peroxidase; superoxide dismutase)] were evaluated in all study groups. DEP aggravated the CP- induced HEK-293 cells toxicity, as evidenced by decreasing cell viability and by inducing oxidative stress (GSH depletion, TAC impairment, and antioxidant enzymes inhibition). DEP, but not CP, significantly reduced cysteine uptake. Curcumin prevented the observed DEP and CP-induced cellular insults. These findings suggest that DEP augmented the CP-induced toxicity in HEK-293 cells. Curcumin exhibited a strong potential for protection against DEP and CP-induced cytotoxicity.     

槲皮素对HL-60细胞中p53、bcl-2基因表达的影响

目的探讨槲皮素对人早幼粒白血病细胞株（ＨＬ６０）细胞中ｐ５３、ｂｃｌ２基因表达的影响。方法采用台盼蓝排除法确定活细胞数和免疫细胞化学ＳＰ染色法分别检测细胞中ｐ５３、ｂｃｌ２基因表达。结果槲皮素对ＨＬ６０细胞的增殖有明显的抑制作用,当其作用２４ｈ后,其ＩＣ５０为４６６μｍｏｌ·Ｌ－１,槲皮素对ＨＬ６０细胞中ｐ５３、ｂｃｌ２基因表达均有一定的抑制作用,且呈明显的剂量依赖关系。结论槲皮素抑制ｐ５３、ｂｃｌ２的表达可能是其抑制ＨＬ６０细胞增殖和生长的重要机制之一。     

趋化因子受体CCR6的克隆及其在HEK293细胞中的表达

目的：构建人趋化因子受体6（CCR6）cDNA序列的真核表达载体，并了解其在HEK293细胞中的表达。方法：提取人淋巴结总RNA，通过逆转录PCR扩增出CCR6基因片段，并构建真核表达载体pcDNA3，1（＋）-CCR6；重组载体通过脂质体转染HEK293细胞，免疫荧光法鉴定CCR6的表达。结果：酶切鉴定和序列分析证实重组质粒含有CCR6编码序列．转染实验表明重组质粒能在HEK293细胞中表达出具有活性的CCR6片段。结论：CCR6真核表达载体构建及表达成功，为下一步CCR6拈抗剂的筛选奠定了基础。     

高效液相色谱法评价贵州金钱草代谢产物槲皮素和山奈素的多样性

目的： 建立金钱草中槲皮素和山奈素的HPLC测定方法,用以测定贵州省不同来源地的21份金钱草中槲皮素和山奈素的含量,并对其多样性进行分析。方法： 采用Alltima C₁₈柱（5 μm,250 mm×4.6 mm）,以甲醇-0.4%磷酸（50∶50,V/V）为流动相,柱温：25℃,流速：1.0 mL·min^-1,检测波长为360 nm;用SPSS 19.0软件对不同来源地金钱草中槲皮素和山奈素进行聚类分析。结果： 槲皮素和山奈素分别在0.028 6~0.196 5 mg （r=0.999 7）和0.047 7~0.381 4 mg （r=0.999 9）的定量范围内,色谱峰面积与进样量呈良好的线性关系,相关系数均大于0.999。槲皮素和山奈素在精密度实验、重复性实验和稳定性试验中的RSD分别为0.24%和0.58%、0.95%和1.32%、1.02%和1.08%,RSD均小于1.40％,加样平均回收率为99.42%和98.36%。不同来源地的21份金钱草中槲皮素和山奈素含量差异较大。结论： 用HPLC法测定金钱草中槲皮素和山奈素含量简单、精确、重复性高,贵州金钱草中槲皮素和山奈素含量差异较大,具有丰富的多样性及一定的地域性。     

The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.     

Allopregnanolone与GABA在大鼠大脑的皮层细胞中对kainate诱导乳酸脱氢酶释放的不同作用

目的:观察allopregnanolone与γ氨基丁酸(GABA)对兴奋性毒性的作用.方法:对原代培养大鼠大脑的皮层细胞,采用kainate(KA)处理,诱发兴奋性毒性.通过测定释放至细胞培养基中的乳酸盐脱氢酶(LDH)活性。观察allopregna-nolone与GABA对兴奋性毒性的作用.结果:短期 或长期KA处理,均增加 LDH活性的释放,其作用呈剂量依赖性,EC_(50)值分别为(0.16±0.03)mol/L和(0.257±0.15)mmol/L.短期(3 h)allopreg-nanolone (10-1000 nmol/L)处理对0.2 mmol/L KA诱导的 LDH活性的释放无明显影响.而长期(24 h)allopregnanolone(10-1000 nmol/L)处理抑制KA诱导的LDH活性的释放,其作用呈剂量依赖性,EC_(50)值为(436±19)nmol/L.短期GABA(0.1-100μmo 1/L)处理,加剧KA(0.2 mmol/L)诱导的 LDH活性的释放,其作用呈剂量依赖性,EC_(50)值为(2.7±1.0)μmol/L.长期GABA处理,对KA诱导的LDH活性的释放无明显影响.结论;allopregnanolone和 GABA对KA诱导的兴奋性毒性有不同作用.     

Comparison of the effects of vitamin E and/or quercetin in attenuating chronic cyclosporine A-induced nephrotoxicity in male rats

1. The aim of the present study was to investigate the effects of vitamin E and/or quercetin (Q) on renal function, oxygen radical concentrations in the kidney and some anti-oxidant enzyme activities in rats treated with cyclosporine A (CsA). 2. Groups of rats (270 +/- 15 g), on standard rat chow and water, received all their treatments by gavage for either 4 or 8 weeks. Control groups received either olive oil (0.5 mL) or 25% ethanol (0.5 mL) + olive oil (0.5 mL) per day as vehicle. All experimental groups received 25 mg CsA/kg per day in 0.5 mL olive oil. The vitamin E group received 100 mg vitamin E/kg per day in olive oil in addition to CsA treatment. The quercetin group received 15 mg of Q/kg per day in 0.5 mL of 25% ethanol in addition to CsA treatment. The vitamin E + quercetin group received the two anti-oxidants at the concentrations given in addition to CsA treatment. 3. Quercetin, at a concentration less than one-quarter of vitamin E, was more efficient in lowering blood urea nitrogen, serum creatinine and kidney malondialdehyde in CsA-treated rats. However, neither of the two anti-oxidants was able to normalize these analytes to control values after either 4 or 8 weeks treatment. 4. Quercetin (50 micromol/kg per day) elevated all renal anti-oxidant enzyme activities to values observed in the negative controls. However, vitamin E (232 micromol/kg per day) only normalized glutathione peroxidase activity at the end of either 4 or 8 weeks treatment. Combination treatment with the two anti-oxidants abolished all the ill-effects of CsA. 5. Combination treatment with the two anti-oxidants of renal transplant patients receiving CsA may be beneficial in ameliorating the chronic nephrotoxic effects of the important immunosuppressive drug CsA.     

骨碎补对体外培养人牙髓细胞增殖及超微结构的影响

目的:探讨骨碎补对体外培养的人牙髓细胞增殖及超微结构的影响。方法:组织块法体外培养人牙髓细胞。水提醇沉法提取骨碎补有效成分,以不同浓度骨碎提取液作用于体外培养的人牙髓细胞。MTT法检测骨碎补各浓度组对人牙髓细胞增殖的作用。扫描电镜和透射电镜下观察骨碎补对人牙髓细胞超微结构的影响。结果:原代培养的人牙髓细胞呈梭形或多角形。各浓度骨碎补均对体外培养的人牙髓细胞有促增殖作用,其中以100mg.L-1质量浓度组促增殖作用最明显。电镜下实验组人牙髓细胞表面枝状嵴丰富,细胞周围可见细胞外基质;细胞浆内有丰富粗面内质网和游离的核糖体,核内常染色质均匀分散,异染色质少。结论:骨碎补对体外培养的人牙髓细胞有明显的促增殖作用。     

多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用

目的观察多沙唑嗪(racemic-doxazosin,rac-DOX)及其对映体(S-DOX、R-DOX)对大鼠血管平滑肌细胞(vascularsmooth muscle cells,VSMCs)增殖的影响。方法采用培养的大鼠主动脉血管平滑肌细胞,应用MTT比色法,测定VSMCs的增殖活力,Giemsa’s染色观察VSMCs的形态学改变。结果在3~30μmol.L-1浓度范围,S-DOX、R-DOX和rac-DOX作用于VSMCs 96 h,均抑制大鼠胸主动脉VSMCs增殖活动;但是,S-DOX的抑制作用强于同浓度R-DOX(P<0.05),与同浓度rac-DOX的抑制作用无差别。以30μmol.L-1浓度的药物处理VSMCs 48 h、72 h和96 h;S-DOX对主动脉VSMCs增殖的抑制率依次为28.67%、34.51%和56.89%,R-DOX为22.59%、26.66%和45.79%,rac-DOX为21.88%、32.84%和52.04%。以不同浓度的药物处理大鼠胸主动脉VSMCs 96 h,rac-DOX、S-DOX和R-DOX抑制VSMCs增殖达40%的浓度(IC40)分别为(12.1±2.6)、(10.2±1.3)和(20.9±2.2)μmol.L-1;R-DOX的IC40值大于rac-DOX和S-DOX(P<0.01)。S-DOX处理后,VSMCs出现细胞体积变小,核固缩,核边集等形态学改变。结论S-DOX和R-DOX对大鼠胸主动脉VSMCs的抗增殖作用具有化学结构的立体选择性,S-DOX的抗VSMCs增殖作用明显强于R-DOX。     

Inhibitory effects of amiloride on the current mediated by native GABAA receptors in cultured neurons of rat inferior colliculus

1. The diuretic amiloride is known to modulate the activity of several types of ion channels and membrane receptors in addition to its inhibitory effects on many ion transport systems. However, the effects of amiloride on some important ion channels and receptors, such as GABA_A receptors, in the central nervous system have not been characterized. 2. In the present study, we investigated the functional action of amiloride on native GABA_A receptors in cultured neurons of rat inferior colliculus using whole‐cell patch‐clamp recordings. 3. Amiloride reversibly inhibited the amplitude of the GABA‐induced current (I_GABA) in a concentration‐dependent manner (IC₅₀ 454 ± 24 μmol/L) under conditions of voltage‐clamp with a holding potential at ?60 mV. The inhibition depended on drug application mode and was independent of membrane potential. Amiloride did not change the reversal potential of I_GABA. Moreover, amiloride induced a parallel right‐ward shift in the concentration–response curve for I_GABA without altering the maximal value and Hill coefficient. 4. The present study shows that amiloride competitively inhibits the current mediated by native GABA_A receptors in the brain region, probably via a direct action on GABA‐binding sites on the receptor. The findings suggest that the functional actions of amiloride on GABA_A receptors may result in possible side‐effects on the central nervous system in the case of direct application of this drug into the cerebrospinal fluid for treatment of diseases such as brain tumours.     

Inhibitory effects of crocetin on high glucose-induced apoptosis in cultured human umbilical vein endothelial cells and its mechanism

Dysfunction of endothelial cell is considered as a major cause of vascular complications in diabetes. Crocetin has been shown to have strong antioxidant activities. In present study, we tested whether crocetin inhibited high glucose-induced apoptosis in cultured human umbilical vein endothelial cells (HUVEC_s) and to explore its possible mechanism. Exposure to high glucose (33 mM) for 72h induced a pronounced increase in apoptosis compared with normal glucose (5.5 mM), as evaluated by cell chromatin staining with Hoechst 33,258 and cell death detection ELISA. High glucose attenuated activation of Akt and endothelial nitric oxide synthase (eNOS). Crocetin (0.1 μM, 1.0 μM) prevented high glucose-induced apoptosis, which correlates with the increase of activation of p-Akt, following the up-regulation of eNOS and NO production. Pretreatment with phosphatidylinositol 3′ kinase (Pl3K) inhibitor LY294002 or eNOS inhibitor N^G-nitro-arginine methyl ester (LN or L-NAME) inhibited crocetin’ effect on p-Akt or eNOS, respectively. For the first time, results of our study suggest that crocetin inhibits high glucose-induced apoptosis, at least partly, via Pl3K/Akt/eNOS pathway in HUVEC_s and crocetin may exert a beneficial effect in preventing diabetes-associated cardiovascular complications.