α-硫辛酸对高糖诱导的血管内皮细胞凋亡的保护作用

目的 观察α-硫辛酸对高糖诱导的血管内皮细胞凋亡是否有保护作用.方法 以不同浓度α-硫辛酸干预高糖作用下的血管内皮细胞(ECV304),共同孵育72h,以Annexin-FITC/PI双染流式细胞术和TUNEL法检测细胞凋亡.结果 高糖明显增加内皮细胞凋亡(P＜0.01);以不同浓度α-硫辛酸干预,内皮细胞凋亡明显减少(P＜0.01),这种作用具有浓度依赖性.结论 α-硫辛酸对高糖诱导的血管内皮细胞凋亡有保护作用.     

TNF-α诱导脑胶质瘤细胞凋亡过程中NF-κB、IκB活性的研究

目的探讨核转录因子-κB(NF-κB)及其抑制因子(IκB)在肿瘤坏死因子-α(TNF-α)诱导人脑胶质瘤细胞株U 251细胞凋亡过程中生物活性的变化。方法应用流式细胞仪检测TNF-α对U-251细胞生长的抑制和诱导凋亡作用。用免疫组化方法检测肿瘤细胞p65的表达,用W estern b lot检测IκB蛋白的变化。结果TNF-α可诱导U 251细胞凋亡,激活细胞中p65并诱导IκB降解;抗氧化剂二硫碳吡咯烷醇(PDTC)可抑制TNF-α诱导的IκB的降解及NF-κB的激活,增强TNF-α诱导U 251细胞的凋亡。结论TNF-α在诱导U 251细胞凋亡的过程中可激活NF-κB。抑制NF-κB活性,可增强TNF-α诱导细胞凋亡的作用。     

N-乙酰半胱氨酸通过抑制JAK2/STAT3信号通路对高糖诱导的内皮细胞损伤的保护作用

目的 探讨N-乙酰半胱氨酸(NAC)能否通过抑制JAK2/STAT3信号通路对高糖诱导的内皮细胞损伤起保护作用。方法 体外分别应用不同浓度NAC对人脐静脉内皮细胞(HUVEC)进行预处理,筛选出NAC减轻高糖诱导的HUVEC细胞毒性的最佳浓度。使用最佳浓度的NAC预处理高糖诱导的HUVEC,通过CCK-8检测细胞存活率,Hoechst33258核染色荧光显微镜照相法检测内皮细胞凋亡的形态学改变,Western blot检测JAK2/STAT3信号通路的蛋白表达水平,DCFH-DA检测细胞内活性氧水平,ELISA检测相关炎症因子细胞间黏附分子1(ICAM-1)、核因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)及白细胞介素1β(IL-1β)、IL-6和IL-8的含量;荧光探针JC-1检测线粒体膜电位的变化。结果 应用7 mmol/L NAC预处理HUVEC 30 min可明显减轻高糖诱导的HUVEC损伤,使细胞存活率升高,细胞凋亡数量减少,细胞凋亡蛋白cleaved Caspase-3表达减少,胞内活性氧堆积及线粒体膜电位丢失减少(P＜0.01)。NAC能抑制高糖对p-JAK2、p-STAT3表达的上调作用(P＜0.01),同时可抑制高糖诱导的炎症反应,使炎症因子ICAM-1、NF-κB、TNF-α及白细胞介素1β(IL-1β)、IL-6和IL-8的水平下降(P＜0.01)。结论NAC能够通过抑制JAK2/STAT3信号通路对高糖诱导的HUVEC损伤起到保护作用。     

川芎嗪调控PKCβ_1减轻波动高糖诱导人脐静脉内皮细胞损伤的实验研究

目的观察川芎嗪对波动高糖诱导人脐静脉内皮细胞损伤及蛋白激酶Cβ_1(PKCβ_1)表达的影响。方法分离、鉴定并培养人脐静脉内皮细胞,实验共分为7组:正常低糖(N)组、稳定高糖(W)组、波动高糖(B)组、稳定高糖+LY333531(WL)组、波动高糖+LY333531(BL)组、波动高糖+川芎嗪(TMP)组和波动高糖+二甲双胍(MH)组。8d后检测各组细胞凋亡率、肿瘤坏死因子α(TNF-α)、可溶性细胞间黏附分子1(sICAM-1)、晚期氧化蛋白产物(AOPP)和总抗氧化能力(T-AOC)水平及细胞PKCβ_1蛋白表达情况。结果与N组相比,W组和B组HUVECs总凋亡率、TNF-α、sICAM-1、AOPP含量、PKCβ_1蛋白表达均显著升高(P0.01),T-AOC显著降低(P0.01),且W组和B组间差异有统计学意义(P0.05或P0.01)。应用TMP和MH干预具有与LY333531阻断相似的效应,可显著降低细胞的总凋亡率、TNF-α、sICAM-1、AOPP含量及PKCβ_1蛋白表达(P0.01),同时可显著升高T-AOC水平(P0.01)。结论波动性高糖状态具有显著加重人脐静脉内皮细胞损伤的效应,且该效应与PKCβ_1表达水平显著升高密切相关;川芎嗪对波动性高血糖状态下的血管内皮功能具有明显的保护作用,其作用机制与其显著抑制PKCβ_1的过表达进而减轻氧化应激和炎症反应密切相关。     

依达拉奉对Aβ25-35诱导分化PC12细胞NF-κB p65基因表达的影响

目的 探讨依达拉奉(MCI-186)对淀粉样β蛋白25-35(Aβ25-35)诱导的PC12细胞NF-κB p65基因表达的影响.方法 实验对象分为3组:MCI-186保护组(MCI-186 20 μmol/L,Aβ25-35 30 μmol/L)、Aβ25-35干预组(Aβ25-35 30 μmol/L)和正常对照组.采用MTT法测定细胞生存率;RT-PCR检测NF-κB p65 mRNA表达变化,Western印迹法检测NF-κB p65蛋白表达的变化.结果 Aβ25-35能增加NF-κB p65 mRNA及其蛋白的表达,促进细胞凋亡;MCI-186能抑制NF-κB p65 mRNA及其蛋白的表达,抑制细胞凋亡.结论 MCI-186具有神经细胞保护作用,可能的机制与其抑制NF-κB p65 mRNA及其蛋白表达有关.     

血管内皮生长因子拮抗内皮细胞凋亡的实验研究

目的:研究血管内皮生长因子(VEGF)对血管内皮细胞凋亡及凋亡相关基因Bcl-2与apo-1/Fas mRNA表达的影响。方法:将体外培养的人脐静脉内皮细胞(HUVEC)分成对照组、肿瘤坏死因子-α(TNF-α)处理组、TNF-α加VEGF处理组;采用原位末端标记法、流式细胞术观察各组细胞的凋亡发生情况,通过RT-PCR法观察各组细胞中凋亡相关基因Bcl-2与apo-1/Fas mRNA表达的变化。结果:TNF-α处理组凋亡细胞和apo-1/Fas mRNA的表达明显高于对照组和TNF-α加VEGF处理组,而Bcl-2 mRNA表达情况相反。结论:VEGF能拮抗TNF-α诱导的内皮细胞凋亡,其抗凋亡的机制可能与其上调Bcl-2 mRNA表达与下调apo-1/Fas mRNA表达有关。     

罗汉果皂苷提取物通过调控miR-199a-3p对高糖诱导的肾小管上皮细胞损伤的影响

目的 探究罗汉果皂苷提取物对高糖诱导的肾小管上皮细胞损伤的影响及潜在分子机制。方法 体外培养人肾小管上皮细胞HK-2,采用高糖和罗汉果皂苷提取物干预HK-2细胞，细胞计数试剂盒(CCK-8)检测细胞活力，流式细胞术分析细胞凋亡率，Western印迹分析B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达，实时荧光定量聚合酶链式反应(RT-qPCR)检测miR-199a-3p的表达，酶联免疫吸附试验(ELISA)检测肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-8的表达。结果 高糖能够抑制HK-2细胞活力，促进细胞凋亡和Bax的表达，抑制Bcl-2的表达，提高TNF-α、IL-6和IL-8的含量；罗汉果皂苷提取物能够抑制高糖诱导的HK-2细胞凋亡，提高细胞活力，促进miR-199a-3p的表达，降低TNF-α、IL-6和IL-8的含量；过表达miR-199a-3p能够抑制高糖诱导的HK-2细胞损伤，下调miR-199a-3p能够逆转罗汉果皂苷提取物对高糖诱导的HK-2细胞损伤保护作用。结论 罗汉果皂苷提取物能够通过上调miR-199a-3p的表达对高...     

热休克蛋白27磷酸化在高糖诱导的人脐静脉内皮细胞凋亡中的作用

目的研究热休克蛋白27磷酸化在高糖诱导的人脐静脉内皮细胞凋亡中的作用。方法取健康剖腹产孕妇的脐静脉内皮细胞(HUVEC)进行培养,选择2～3代细胞进行实验。①检测高糖对内皮细胞中热休克蛋白27(HSP-27)蛋白活性的影响:实验分正常细胞组与高糖组。正常细胞组不做处理直接提取细胞总蛋白,高糖组为30.5mmol/L高糖分别干预细胞24、48、72h后提取总蛋白。②检测槲皮素对高糖诱导的内皮细胞中HSP-27活性的影响:实验分为正常细胞组,高糖组和高糖+槲皮素组。正常细胞组不做处理,高糖组为30.5mmol/L高糖干预细胞;高糖+槲皮素组为槲皮素(10μmol/L)先与细胞孵育1h后再加入刺激物高糖(30.5mmol/L),各细胞组培养48h后提取总蛋白。①与②中细胞总蛋白均采用特异性Phospho-HSP27抗体的蛋白免疫印迹法检测HSP-27的活性;采用Annexin-Ⅴ-PI染色流式细胞技术检测人脐静脉内皮细胞凋亡。分组及干预情况同②,比较各组间细胞的凋亡率。结果高糖呈时间依赖性诱导人脐静脉内皮细胞中HSP-27磷酸化,30.5mmol/L高糖作用24、48、72h,HSP-27磷酸化水平较正常对照组分别提高了100%(P<0.05),182.5%(P<0.01),117%(P<0.05)。而HSP-27选择性阻断剂槲皮素(10μmol/L)作用48h可以抑制高糖诱导的HSP-27磷酸化,与高糖组相比槲皮素组HSP-27磷酸化水平降低了52.6%(P<0.01),与此同时,可使凋亡增加,内皮细胞凋亡率较高糖组增高了37.6%(P<0.01)。结论 HSP-27磷酸化可能在高糖诱导的人脐静脉内皮细胞凋亡中起保护作用。     

Protective effect of osteoprotegerin in vascular endothelial cells cultured with high glucose and its possible mechanism

目的 探讨护骨素对高糖诱导的血管内皮细胞保护作用及机制.方法 人脐静脉内皮细胞分别在正糖(5 mmol/L葡萄糖)、高糖(25 mmol/L葡萄糖)、高糖+护骨素(25 mmol/L葡萄糖+2 μg/ml护骨素)、高渗(5mmol/L葡萄糖+20 mmol/L甘露醇)环境下培养72小时.以流式细胞术测定各组细胞的凋亡;Western blot法分析各组细胞磷酸化核转录因子(NF-κB)抑制蛋白激酶β (p-IκKβ)、NF-κB抑制蛋白激酶β(IκKβ)、NF-κB抑制蛋白α(IκBα)、bcl-2及bax表达水平.结果 (1)与正糖组相比,高糖组内皮细胞凋亡率显著增加(P＜0.01);高糖+护骨素组细胞凋亡明显低于高糖组,而又高于正糖组(P＜0.05);(2)与正糖组相比,高糖组p-IκKβ表达显著增高(P＜0.01),IκBo和bcl-2表达显著降低(P＜0.01);高糖+护骨素组p-IκKβ明显低于高糖组,而又高于正糖组(P＜0.05),IκBα和Bcl-2表达水平明显高于高糖组,而又低于正糖组(P＜0.05).结论 高糖通过激活IκKβ/NF-κB信号通路引起血管内皮细胞凋亡;护骨素具有抗高糖诱导的IκKβ/NF-κB活性作用,从而降低内皮细胞凋亡,保护内皮细胞.     

酰基化ghrelin抑制高糖诱导的人血管内皮细胞凋亡

目的近年来研究证明高糖引起的血管内皮细胞凋亡可能加重糖尿病动脉粥样硬化,本研究旨在探讨酰基化ghrelin能否抑制高糖诱导的人血管内皮细胞凋亡。方法应用吖啶橙形态学染色及TUNEL、流式细胞分析仪、分光光度计研究高糖及ghrelin预处理后内皮细胞凋亡及caspase-3活性情况。结果内皮细胞暴露于高浓度葡萄糖(33.3mmol/L)72h与正常水平葡萄糖(5.5mmol/L)相比凋亡细胞数量显著增加,酰基化ghrelin(10-7mol/L)预处理24h后显著降低高糖诱导的凋亡。同时高糖环境下凋亡蛋白caspase-3活性增高,酰基化ghrelin预处理后明显降低caspase-3活性。结论酰基化ghrelin可以抑制高糖诱导的血管内皮细胞凋亡及凋亡蛋白caspase-3的表达,可能在防治糖尿病动脉粥样硬化的过程中起到一定作用。     

Effect of erythropoietin on endothelial cell apoptosis induced by high glucose

Erythropoietin (Epo) has been reported to inhibit apoptosis of neuron and erythroid cells. In this study, we examined an effect of high glucose on apoptosis of endothelial cells and investigated an anti-apoptotic effect of Epo. Human aortic endothelial cells were incubated with normal or high glucose for 72 h, and apoptotic cells were detected by TUNEL assay. Simultaneously, Epo (100 U/ml) was added to the high glucose medium to examine an inhibitory effect on the apoptosis induced by high glucose. Activity of caspase-3 was also measured using a specific substrate. To investigate a possible mechanism of Epo's action on apoptosis, phosphorylation of Akt was examined by applying Epo. Incubation with high glucose increased apoptosis of endothelial cells, whereas this effect was prevented by co-incubation with Epo. Caspase-3 activity was also increased (1.4-fold) by incubation with high glucose, and the activation of caspase-3 was normalized to the control level by co-incubation with Epo. Furthermore, Epo-induced phosphorylation of Akt in dose-dependent manner. In conclusion, we demonstrated that incubation with high glucose activated caspase-3 and induced apoptosis of endothelial cells. Epo was shown to phosphorylate Akt, leading to the inhibition of caspase-3 activation and apoptosis induced by high glucose. These results suggest that reduced production of Epo in patients with end-stage of nephropathy may accelerate diabetic angiopathy and that replacing therapy with Epo might inhibit endothelial cell apoptosis and diabetic angiopathy.     

Biphasic effects of free radical scavengers against bleomycin-induced pulmonary fibrosis

Oxidative stress plays a critical role in the development of pulmonary fibrosis. However, the effects of treatment with anti-oxidant agents against pulmonary fibrosis have not yet been thoroughly investigated. In this study, the effect of MCI-186, a novel free radical scavenger, on bleomycin-induced pulmonary fibrosis was investigated. Bleomycin (0.05units/mouse) was administered intratracheally into C57Bl/6 mice. MCI-186 was given to bleomycin-treated mice intraperitoneally from (i) day -3 to day 7, or from (ii) day 10 to day 28 after bleomycin administration in successive days. At 28 days after bleomycin administration, pulmonary fibrosis was then assessed by lung histology and hydroxyproline. MCI-186 inhibited H(2)O(2)-induced DNA damage in bronchial epithelium in vitro. MCI-186 decreased the lipid peroxide content, a marker for DNA damage, in the lung and reduced 8-OHdG positive cells in the lung in vivo. During the early period (day -3 to day 7) administration, MCI-186 partially attenuated bleomycin-induced pulmonary fibrosis. However, during the late period (day 10 to day 28) MCI-186 exacerbated pulmonary fibrosis, based on the histology and hydroxyproline content. In this condition, MCI-186 in the late period decreased the number of apoptosis cells induced by bleomycin, and therefore it might contribute to the deterioration of pulmonary fibrosis. These data indicate that MCI-186, radical scavenger, has a biphasic effect on bleomycin-induced pulmonary fibrosis in mice. Careful attention should be paid before clinical application of new remedies for pulmonary fibrosis.     

The role of radix hedysari polysaccharide on the human umbilical vein endothelial cells (HUVECs) induced by high glucose

BackgroundDiabetes mellitus can cause a wide variety of vascular complications and it is one of the major risk factors for cardiovascular diseases (CVD). High glucose can induce vascular endothelial cell apoptosis. In this study, we investigated the effect of radix hedysari polysaccharide (HPS) on the depression of apoptosis of human umbilical vein endothelial cells (HUVECs) induced by high glucose.MethodsHUVECs were treated with media containing 30 mM glucose in the presence or absence of vitamin C or HPS. The level of intracellular reactive oxygen species (ROS) and apoptosis of HUVECs was measured with flow cytometry. Expression of c-Jun NH₂-terminal kinase (JNK) and caspase-3 were testified by real-time quantitative RT-PCR and immunofluorescence.ResultsHigh glucose was capable of eliciting the overexpression of JNK during the treatment procedure. Moreover, we found that the caspase-3 became overexpressed in apoptosis induced by high glucose; HPS could inhibit apoptosis under high glucose and suppress the generation of ROS and the overexpression of JNK and caspase-3. The effect of HPS on ROS quenching, inhibition of JNK and caspase-3 overexpression at the concentration of 100 μg/ml was similar to that of vitamin C at the concentration of 100 μM.ConclusionThe findings of the present study may suggest that HPS play a protection role on HUVECs against apoptosis induced by high glucose.     

Inhibition of neointimal hyperplasia development by MCI-186 is correlated with downregulation of nuclear factor-kappaB pathway.

BACKGROUND: Atherosclerosis is a progressing inflammatory response mediated by various signaling molecules among which nuclear factor kappaB (NF-kappaB) is thought to have a pivotal role. This study demonstrated the efficacy of antioxidant MCI-186 in preventing the progression of atherosclerosis by inhibiting signaling molecules such as NF-kappaB. METHODS AND RESULTS: Balloon injury of intima was performed in the right common carotid artery of Japanese male white rabbits, which were then fed a 1% high cholesterol diet for 4 weeks, after assigning them to either the control (n=7) or MCI-186 (0.5 mg .kg(-1) . day(-1), n=7) group. Histological analysis revealed a reduction in neointimal thickness and lipid deposition in the subendothelial area of the MCI-186 group. Immunohistochemical analysis revealed attenuation of E-selectin expression, macrophage migration and proliferation of smooth muscle cells in the MCI-186 treated group. In in vitro studies, rabbit aorta smooth muscle cells were incubated with rIL-1betain either the presence or absence of MCI-186. MCI-186 significantly inhibited rIL-1beta-induced proliferation of smooth muscle cells from rabbit aorta, as well as the activation of NF-kappaB. Moreover, western blot analysis showed the inhibitory action of MCI-186 on the nuclear translocation of NF-kappaB in human umbilical vein endothelial cells under rIL-1betastimulation. CONCLUSIONS: MCI-186 could provide a novel therapeutic strategy for atherosclerosis by inhibiting the NF-kappaB pathway.     

Fas (CD95)- and tumor necrosis factor-mediated apoptosis in liver endothelial cells: role of caspase-3 and the p38 MAPK.

Recently, we showed that TNF enhances the susceptibility of endothelial cells from murine liver sinusoids (LEC) to Fas-mediated apoptosis, suggesting that signals transduced by Fas and TNF receptors may synergistically increase intracellular death signals in these cells. In this work we evaluated whether caspase-3 and p38 are involved in LEC apoptosis induced by Fas and TNF. Here we show that LEC treated with Fas agonist (Jo2 mAb at 0.1 microg/ml) and TNF had a greater caspase-3 activity (twofold increase) than cells treated with each factor alone. There was a strong correlation between caspase-3 activity and cell killing induced by Jo2/TNF, indicating that this caspase plays a critical role in this process. Likewise, there was a significant increase in caspase-8 activity in LEC treated with Jo2 and TNF, compared with untreated cells or cells treated with each factor alone. Apoptosis of LEC induced by Jo2/TNF was partially reversed by SB203580, a p38 inhibitor, suggesting that p38 is involved in apoptosis of these cells. To our knowledge, this is the first report that apoptosis induced by Fas/TNF in LEC is associated with coactivation of both caspase-3 and p38. Potentially, both caspase-3 and p38 may be of great importance in endothelial cell pathology as molecular targets for preventing vascular damage due to endothelial cell apoptosis.     

Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells

Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insulin inhibits tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Because insulin has been shown to stimulate the protein kinase Akt, we investigated whether activation of Akt contributes to the apoptosis-suppressive effect of insulin and characterized the downstream signaling pathway. Incubation with insulin dose-dependently prevented apoptosis induced by TNF-alpha (50 ng/mL). The extent of apoptosis suppression by insulin was similar to the effect of vascular endothelial growth factor. Pharmacological inhibition of Akt activation or overexpression of a dominant-negative Akt mutant prevented the antiapoptotic effect of insulin. Furthermore, we investigated the effect of TNF-alpha on Akt phosphorylation by Western blot analysis with the use of a phosphospecific Akt antibody. Incubation of HUVECs with TNF-alpha induced a marked dephosphorylation of Akt. Insulin counteracted this TNF-alpha-induced dephosphorylation of Akt. Furthermore, we investigated the downstream signaling events. Akt has been shown to mediate its apoptosis-suppressive effects via phosphorylation of Bad or caspase-9. However, incubation with insulin did not lead to enhanced phosphorylation of Bad at Ser 136 or Ser 112. In contrast, insulin inhibited caspase-9 activity and prevented caspase-9-induced apoptosis. Mutation of the Akt site within caspase-9 significantly reduced the apoptosis-suppressive effect of insulin. The present study demonstrates an important role for insulin-mediated Akt activation in the prevention of endothelial cell apoptosis, which may importantly contribute to cell homeostasis and the integrity of the endothelium. In endothelial cells, Akt seems to mediate its antiapoptotic effect, at least in part, via phosphorylation of caspase-9 rather than Bad.     

肝细胞生长因子对糖基化终产物诱导内皮细胞凋亡的影响

目的探讨糖基化终产物对内皮细胞的凋亡作用,以及肝细胞生长因子对内皮细胞凋亡的影响。方法体外培养人脐静脉内皮细胞,予不同浓度糖基化终产物及肝细胞生长因子干预,分为实验对照组及100 mg/L、200mg/L4、00 mg/L糖基化终产物组和400 mg/L糖基化终产物 100μg/L肝细胞生长因子组,采用四甲基偶氮唑蓝比色法测定各组内皮细胞生长抑制率,通过吖啶橙荧光染色观察细胞形态学变化,流式细胞术测定Annexin V-FITC/PI双染标记的细胞凋亡率,检测肝细胞生长因子对糖基化终产物诱导内皮细胞凋亡的影响;蛋白免疫印迹法分析各组凋亡基因Bax、Bcl-2蛋白的表达及酶联反应法测定细胞凋亡蛋白酶3的活性。结果肝细胞生长因子能明显降低糖基化终产物对内皮细胞生长的抑制作用(P<0.01);糖基化终产物诱导培养的内皮细胞出现明显的凋亡形态学改变,在一定浓度范围内,内皮细胞凋亡率与糖基化终产物的浓度和作用时间呈依赖关系,肝细胞生长因子干预后可显著降低不同时间的内皮细胞凋亡率(P<0.05);肝细胞生长因子作用内皮细胞抗凋亡基因Bcl-2表达明显升高(P<0.01),而促凋亡基因Bax表达无明显变化(P>0.05);细胞凋亡蛋白酶3活性显著降低(P<0.05)。结论糖基化终产物诱导人内皮细胞凋亡,而肝细胞生长因子抑制糖基化终产物诱导的内皮细胞凋亡,其作用机制可能是上调抗凋亡基因Bcl-2水平、抑制细胞凋亡蛋白酶3的激活。     

Effects of MCI-186 (edaravone), a novel free radical scavenger, upon experimental atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Recent evidence suggests that oxidative stress may play a role in the development of atherosclerosis. MCI-186 (3-methyl-1-phenyl-1-phyrazolin-5-one, edaravone) is a novel free radical scavenger, but it remains unclear whether free radical scavengers would be effective for the prevention of the disease. METHODS AND RESULTS: Experimental atherosclerosis was induced in apolipoprotein E-deficient mice fed a high-fat diet containing 0.3% cholesterol. Mice were treated with an intraperitoneal injection of either MCI-186 1 mg/kg per day or MCI-186 10 mg/kg per day on alternate days over 4 weeks. Fatty streak lesion was suppressed by MCI-186 10 mg/kg per day administration, but not by mg/kg per day. Immunohistochemical analysis showed that macrophage and CD4+ T-cell accumulation and oxidative stress overload in the fatty streak lesion were suppressed in mice that received MCI-186 treatment. CONCLUSIONS: MCI-186 administration suppressed the development of atherosclerosis, associated with reduced expression of both immune-activated cells and oxidative stress in fatty streak plaques.     

高糖高脂对培养成年大鼠心肌细胞损伤观察及机制初探

目的建立成年大鼠心肌细胞培养模型,观察给予高糖、高脂刺激后是否发生心肌细胞损伤和凋亡,并分析其可能机制。方法将分离所得成年大鼠心肌细胞接种入培养皿后随机分为2组进行培养。正常对照组：以M199培养基（Media199）加入5mmol／L葡萄糖与20mmol／L甘露醇作为正常对照培养基培养细胞;高糖高脂组：以M199加入高糖（25mmol／L葡萄糖）高脂（600μmol／L软脂酸）作为培养基培养模拟糖尿病。培养48h后,分别收集培养基上清与细胞,进行心肌损伤和凋亡指标测定。结果高糖高脂组乳酸脱氢酶（LDH）,细胞凋亡蛋白caspase-3、caspase-8、caspase-9活性显著升高,与正常对照组比较,差异均具有统计学意义（P均〈0．01）。结论在培养的成年大鼠心肌细胞,高糖、高脂可引起心肌细胞损伤和凋亡,这种凋亡与easpase-8和caspase-9依赖的凋亡途径有关。     

Thiamine and benfotiamine prevent increased apoptosis in endothelial cells and pericytes cultured in high glucose

BACKGROUND: High glucose induces pathological alterations in small and large vessels, possibly through increased formation of AGE, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. We showed previously that thiamine and benfotiamine correct delayed replication and increase lactate production in endothelial cells subjected to high glucose. We now aim at verifying the effects of thiamine and benfotiamine on cell cycle, apoptosis, and expression of adhesion molecules in endothelial cells and pericytes, under high ambient glucose. METHODS: Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/L) or high (28 mmol/L) glucose, with or without thiamine or benfotiamine, 50 or 100 micro mol/L. Apoptosis was determined by two separate ELISA methods, measuring DNA fragmentation and caspase-3 activity, respectively. Cell cycle and integrin subunits alpha3, alpha5, and beta1 concentration were measured by flow cytometry. RESULTS: Apoptosis was increased in high glucose after 3 days of culture, both in endothelium and pericytes. Thiamine and benfotiamine reversed such effects. Neither cell cycle traversal nor integrin concentrations were modified in these experimental conditions. CONCLUSIONS: Thiamine and benfotiamine correct increased apoptosis due to high glucose in cultured vascular cells. Further elucidations of the mechanisms through which they work could help set the basis for clinical use of this vitamin in the prevention and/or treatment of diabetic microangiopathy.