Polaprezinc (N-(3-aminopropionyl)-L-histidinato zinc) ameliorates dextran sulfate sodium-induced colitis in mice

OBJECTIVE: Polaprezinc (N-(3-Aminopropionyl)-L-histidinato zinc), an anti-ulcer drug, has been reported to have an anti-inflammatory action in several inflammatory diseases. The aim of this study was to investigate the effect of polaprezinc on dextran sulfate (DSS)-induced colitis in mice. MATERIAL AND METHODS: Mice with colitis induced by DSS were intrarectally treated with polaprezinc (15 mg/kg) or zinc sulfate (7.5 mg/kg) every day after the administration of DSS for 7 days. Disease activity index (DAI) and histological tissue damage were assessed. Levels of myeloperoxidase (MPO) activity, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in the colon were measured. Expression of heat shock protein (HSP) 25 and HSP70 in the colon was analyzed by Western blot analysis. RESULTS: DAI and histological scores were remarkably reduced in polaprezinc-treated mice with DSS-induced colitis. Polaprezinc suppressed the increase of MPO activity and the production of TNF-alpha and IFN-gamma in the colon tissues of mice with DSS-induced colitis. Expression of HSP25 and HSP70 was remarkably up-regulated in the colon tissues of polaprezinc-treated mice during DSS treatment. CONCLUSIONS: Polaprezinc suppresses DSS-induced colitis in mice, partly through inhibition of production of pro-inflammatory cytokine, suppression of neutrophils accumulation and cytoprotection by overexpression of HSPs. Polaprezinc could be useful in the treatment of inflammatory bowel diseases.     

PEP-1-CAT融合蛋白预处理对过氧化氢诱导的内皮细胞氧化应激损伤的保护作用

目的采用体外培养的人脐静脉内皮细胞株(CRL-1730),研究 PEP-1肽介导人过氧化氢酶(CAT)穿透细胞的能力,并探讨 PEP-1-CAT 对过氧化氢(H_2O_2)诱导的内皮细胞氧化应激损伤是否有保护作用。方法构建原核表达质粒 pET15b-PEP-1-CAT,将其在基因工程菌中表达出 N 端带有6个组氨酸“标签”(His-tag)的重组蛋白,并通过金属镍螯合亲和层析对其进行纯化,将纯化得到的PEP-1-CAT 融合蛋白加入体外培养的人脐静脉内皮细胞,通过 Western blot 来分析其转导能力,同时对转导人细胞内的蛋白进行酶活性检测。然后以0.5 mmol/L H_2O_2处理细胞建立内皮细胞氧化应激损伤的模型,检测不同浓度的 PEP-1-CAT 蛋白对氧化应激下细胞存活率、乳酸脱氢酶(LDH)活性及丙二醛(MDA)含量的影响。结果 PEP-1-CAT 融合蛋白能以时间、剂量依赖的方式高效转导进入细胞内。正常内皮细胞 LDH 活性和细胞存活率分别为(540.6±65.7)U/L和100%;H_2O_2处理组 LDH活性显著高[(849.3±95.1)U/L,P<0.01],细胞存活率明显低[(37.23±5.68)%,P<0.01],MDA含量亦较正常组显著高[(8.23±1.58)nmol/L 比(2.46±1.42)nmol/L,P<0.01]。用不同浓度的PEP-1-CAT 对细胞进行预保护,均可显著提高细胞存活率、降低 LDH 释放量,并提高细胞的抗氧化能力,表现为 MDA 含量较 H_2O_2处理组低(P<0.05或<0.01)。结论 PEP-1-CAT 融合蛋白能以天然活性形式高效转导入人脐静脉内皮细胞,且转导的蛋白能有效对抗氧化应激损伤。这种蛋白转导方式,为用 CAT 防治各种与氧化应激损伤有关的疾病提供了一个新的治疗途径。     

Vascular endothelial growth factor protects hepatoma cells against oxidative stress-induced cell death

BACKGROUND: The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk-1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease. METHODS: Cell viability was determined by test method with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by western blot analysis. RESULTS: The c-Met tyrosine phosphorylation in PLC/PRF/5 hepatoma cells was increased by treatment with 20 ng/mL hepatocyte growth factor (HGF), and extracellular signal-regulated kinase (ERK) was also activated. Although Flk-1 was phosphorylated in response to VEGF (>50 ng/mL), phosphorylated ERK was not detected at these concentrations. A total of 5.0 and 10 micromol/L hydrogen peroxide (H(2)O(2)) caused cell death in a dose-dependent manner after 24 h. On western blot analysis at 1 h with H(2)O(2), rapid phosphorylation of both ERK1/2 and c-Jun NH(2)-terminal kinase (JNK) was observed. In the first 6 h, H(2)O(2) induced cell death for 58.4 +/- 6.8%, whereas the presence of 100 ng/mL VEGF improved the survival rate to 77.2 +/- 4.2%. The VEGF significantly decreased H(2)O(2)-induced cell death after 12 h, whereas HGF (20 ng/mL) did not have a similar effect. When cells were incubated with 5 micromol/L H(2)O(2), expression of VEGF protein was detected. Furthermore, H(2)O(2)-induced phosphorylation of ERK and JNK was also reduced by VEGF (100 ng/mL). In contrast, HGF did not induce phosphorylation of ERK and JNK. CONCLUSION: Hepatoma cells might be able to survive under continuous oxidative stress through expression of VEGF.     

Na+/Ca2+ exchanger: target for oxidative stress in salt-sensitive hypertension

The Na+/Ca2+ exchanger regulates intracellular calcium ([Ca2+]i), and attenuation of Na+/Ca2+ exchange by oxidative stress might lead to dysregulation of [Ca2+]i. We have shown that the Na+/Ca2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na+/Ca2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45Ca2+ uptake (reverse mode) and [Ca2+]i elevation (forward mode) in the presence and absence of H2O2 and peroxynitrite. Our results showed that 45Ca2+ uptake in SNCX cells was attenuated at 500 and 750 micromol/L H2O2 (63+/-12% and 25+/-7%, respectively; n=16) and at 50 and 100 micromol/L peroxynitrite (47+/-9% and 22+/-9%, respectively; n=16). In RNCX cells, 45Ca2+ uptake was attenuated at only 750 and 100 micromol/L H2O2 and peroxynitrite (61+/-9% and 63+/-6%, respectively; n=16). In addition, the elevation in [Ca2+]i was greater in SNCX cells than in RNCX cells in response to 750 micromol/L H2O2 (58+/-5.5 vs 17+/-4.1 nmol/L; n=13) and 100 micromol/L peroxynitrite (33+/-5 vs 11+/-6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca2+]i that is found in this model of salt-sensitive hypertension.     

Mitochondrial sources of H2O2 generation play a key role in flow-mediated dilation in human coronary resistance arteries

We previously showed that hydrogen peroxide (H2O2) contributes to flow-induced dilation in human coronary resistance arteries (HCRAs); however, the source of this H2O2 is not known. We hypothesized that the H2O2 is derived from superoxide (O2*-) generated by mitochondrial respiration. HCRAs were dissected from right atrial appendages obtained from patients during cardiac surgery and cannulated with micropipettes. H2O2-derived radicals and O2*- were detected by electron spin resonance (ESR) using BMPO as the spin trap and by histofluorescence using hydroethidine (HE, 5 micromol/L) and dichlorodihydrofluorescein (DCFH, 5 micromol/L). Diameter changes to increases in pressure gradients (20 and 100 cm H2O) were examined in the absence and the presence of rotenone (1 micromol/L), myxothiazol (100 nmol/L), cyanide (1 micromol/L), mitochondrial complex I, III, and IV inhibitors, respectively, and apocynin (3 mmol/L), a NADPH oxidase inhibitor. At a pressure gradient of 100 cm H2O, ubisemiquinone and hydroxyl radicals were detected from effluents of vessels. Including superoxide dismutase and catalase in the perfusate reduced the ESR signals. Relative ethidium and DCFH fluorescence intensities in HCRAs exposed to flow were enhanced (1.45+/-0.15 and 1.57+/-0.12, respectively compared with no-flow) and were inhibited by rotenone (0.87+/-0.17 and 0.95+/-0.07). Videomicroscopic studies showed that rotenone and myxothiazol blocked flow-induced dilation (% max. dilation at 100 cm H2O: rotenone, 74+/-3% versus 3+/-13%; myxothiazol, 67+/-3% versus 28+/-4%; P<0.05). Neither cyanide nor apocynin altered flow-induced dilation. These results suggest that shear stress induced H2O2 formation, and flow-induced dilation is derived from O2*- originating from mitochondrial respiration.     

Protective effects of hydrogen peroxide against ischemia/reperfusion injury in perfused rat hearts.

Among the several mechanisms proposed for ischemic preconditioning (IPC), generation of reactive oxygen species (ROS) is reported to be involved in the cardioprotective effects of IPC. The present study was designed to investigate whether repetitive exposure to hydrogen peroxide (H(2)O(2)) can protect the myocardium against subsequent ischemia/reperfusion injury, and whether the H(2)O(2)-induced cardioprotection is related to the preservation of energy metabolism. Langendorff-perfused rat hearts were exposed to two, 5 min episodes of IPC or to various concentrations of H(2)O(2) twice and then to 35 min global ischemia and 40 min reperfusion. Using (31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy, cardiac phosphocreatine (PCr) and ATP and intracellular pH (pH(i)) were monitored. IPC and the treatment with 2 micromol/L H(2)O(2) significantly improved the post-ischemic recovery of left ventricular developed pressure (LVDP) and the PCr and ATP compared with those of the control ischemia/reperfusion (LVDP: 36.9 +/-7.4% of baseline in control hearts, 84.0+/-3.5% in IPC, 65.4+/-3.8% in H(2)O(2); PCr: 51.1+/-5.3% in control hearts, 81.4+/-5.5% in IPC, 81.7+/-5.2% in H(2)O(2); ATP: 12.3+/-1.6% in control hearts; 30.0+/-2.8% in IPC, 28.6+/-2.3% in H(2)O(2), mean +/- SE, p<0.05). However, lower (0.5 micromol/L) or higher (10 micromol/L) concentration of H(2)O (2) had no effect. There were significant linear correlations between mean LVDP and high-energy metabolites after 40 min reperfusion in H(2)O(2)-treated hearts. In IPC-treated hearts, the mean LVDP was greater than that in the 2 micromol/L H(2)O(2)-treated hearts under similar levels of high-energy metabolites. IPC also ameliorated intracellular acidification (6.38+/-0.03 in control hearts, 6.65+/-0.04 in IPC, p<0.05), but treatment with H(2)O(2) did not affect pH(i) during ischemia (6.40+/-0.05 in H(2)O(2)). In conclusion, H(2)O(2) had protective effects against ischemia/reperfusion injury and the effects were related to the preservation of energy metabolism. IPC could have additional protective mechanisms that are associated with the amelioration of intracellular acidosis during ischemia.     

熊果酸对肝星状细胞增殖与凋亡的影响

目的 体外观察熊果酸对肝星状细胞增殖与凋亡的影响,探讨熊果酸诱导肝星状细胞凋亡的可能作用机制. 方法 将不同浓度熊果酸作用于肝星状细胞HSC-T6及肝细胞L02,分别在药物作用24、48、72 h后用四甲基偶氮唑盐法检测熊果酸对HSC-T6及L02细胞增殖的影响;流式细胞仪检测熊果酸对HSC-T6凋亡的影响;光学显微镜观察熊果酸作用后细胞形态学变化情况;免疫细胞化学法检测HSC-T6中Bcl-2、Bax和Caspase-3蛋白的表达情况. 结果 各种浓度的熊果酸均可抑制HSC-T6细胞的增殖,且呈剂量-时间依赖性;当熊果酸浓度为25、50、75μmol/L时可促进L02细胞增殖,浓度＞75μmol/L则表现为抑制L02细胞增殖.在病理形态学方面,熊果酸作用HSC-T6细胞48 h后,光学显微镜下可见细胞缩小变圆、核浓缩等.25、50、75 μmol/L熊果酸作用HSC-T6细胞48 h后,流式细胞仪检测显示细胞凋亡率分别为10.30%±3.85%、21.87%±4.46%、31.33%±6.18%,比对照组(2.93%±1.60%)明显升高(P＜0.01).免疫细胞化学显示Bax及Caspase-3蛋白表达较对照组升高(P＜0.05),且呈剂量依赖性,而Bcl-2蛋白表达水平与对照组无明显差异(P＞0.05).结论 在体外熊果酸可较明显地抑制HSC-T6细胞增殖,诱导其凋亡;对L02细胞的生长具有双向调节作用.熊果酸诱导HSC-T6细胞凋亡可能与降低Bcl-2/Bax比值、激活Caspase-3蛋白有关.     

阻塞性睡眠呼吸暂停低通气综合征患者呼出气凝集液中过氧化氢浓度

目的测定阻塞性睡眠呼吸暂停低通气综合征(OSAHS)患者呼出气凝集液(EBC)中的过氧化氢(H2O2)浓度,观察其改变及与病情程度的相关性。方法收集EBC 42份,其中OSAHS患者32份(吸烟者18份,非吸烟者14份),非OSAHS对照组10份。荧光法测定睡前和晨起EBC中的H2O2浓度。结果OSAHS组患者睡前EBC中H2O2浓度与对照组比较差异无统计学意义(F= 0．01,P=0．99)。晨起EBC中H2O2浓度OSAHS不吸烟组和吸烟组分别为(1．82±1．12)、(1．65±0．89)μmol／L,较对照组(0．71±0．36)μmol／L明显增高(F=5．11,P=0．01)。其浓度亦分别较睡前[(0．87±0．45)、(0．88±0．36)μmol／L]升高(t分别为2．95、3．43,P均<0．01)。对照组睡前[(0．86±0．46)μmol／L]和晨起[(0．71±0．36)μmol／L]差异无统计学意义(t=0．81,P=0．43)。晨起EBC中H2O2浓度与夜间最低血氧饱和度(SpO2min)呈负相关(β=-0．36,P=0．02),晨起与睡前H2O2浓度变化的差值亦与SpO2min呈负相关(β=-0．38,P=0．01)。结论OSAHS患者由于夜间反复出现缺氧-再氧和,呼吸道局部氧应激反应在晨起增加。     

过表达HSP20的慢病毒载体对H2O2诱导的大鼠心肌细胞凋亡的影响

目的构建过表达大鼠热休克蛋白20(HSP20)基因慢病毒载体,探讨其对H2O2诱导的大鼠H9C2心肌细胞凋亡的影响。方法构建大鼠HSP20基因pHIV-HSP20过表达质粒慢病毒,与包装质粒psPAX2、pMD2G共转染293FT细胞,检测其转染效率;包装慢病毒并转染H9C2心肌细胞;72 h后观察其转染效率,RT-PCR法检测细胞HSP20 mRNA表达,CCK-8、Hochest33258染色法检测H2O2诱导后H9C2心肌细胞凋亡情况。结果成功构建了HSP20慢病毒过表达载体,经293FT细胞包装后,其对H9C2细胞72 h的转染效率为95%;与慢性病毒组比较,H9C2细胞转染72 h后HSP20 mRNA水平显著升高,细胞活力下降,心肌细胞凋亡率明显降低(P均<0.01)。结论过表达HSP20能显著抑制H2O2诱导的H9C2心肌细胞凋亡。     

氧自由基诱导鸭乙型肝炎病毒DNA整合及其细胞传代研究

目的探讨氧自由基对鸭乙型肝炎病毒（DHBV）DNA在受感染细胞DNA上整合发生的影响以及整合后DHBVDNA的细胞传代性。方法选择鸡肝癌细胞株—LMHD21-6,在其由单个细胞培养至3×107～5×107个细胞（共23代）的过程中,用低浓度H2O2（10.0μmol/L）作用于细胞;TUNEL法检测细胞DNA链断裂情况;Southernblot技术观察细胞DNA上新的整合型DHBVDNA的发生情况。结果(1)低浓度H2O2（≤10.0μmol/L）导致的细胞死亡率仅为32.0%,但可诱导细胞DNA链断裂致细胞调亡,而H2O2浓度＞10.0μmol/L时其细胞死亡率＞50.0%;(2)低浓度H2O2作用的细胞DNA上新的DHBVDNA整合发生率为50.0%（6/12）,而无H2O2作用的发生率仅8.33%（1/12）,两者比较差异有显著意义（P＜0.05＝;(3)含新的整合型DHBADNA的细胞在无H2O2作用下,再由单个细胞培养至第23代,其子代细胞DNA上可见与其母代细胞DNA上新的整合型DHBVDNA碱基大小完全一致的DHBVDNA带。结论氧自由基为DHBVDNA整合发生的重要诱因之一,新的整合型DHBVDNA可有较稳定的细胞传代。     

Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries

BACKGROUND: Increased vascular superoxide anion (.O(2)(-)) production contributes to endothelial dysfunction and hypertension in animal models of cardiovascular disease. Observations in experimental animals suggest that angiotensin II (Ang II) increases.O(2)(-) production by activation of vascular NAD(P)H oxidase. We studied the sources of.O(2)(-) production in human blood vessels and investigated whether, and by what mechanism, Ang II might alter vascular.O(2)(-) production. METHODS AND RESULTS: Internal mammary arteries (IMAs) and saphenous veins (SVs) were collected at the time of cardiac surgery. Vessels were incubated in Krebs buffer at 37 degrees C.O(2)(-) was measured by lucigenin chemiluminescence. Basal. O(2)(-) concentrations were greater in IMAs than SVs. Inhibitors of NAD(P)H oxidase (10 micromol/L to 200 micromol/L diphenyleneiodonium) and xanthine oxidase (1 mmol/L allopurinol) caused reductions in.O(2)(-) concentrations in both IMAs and SVs. Western blotting of superoxide dismutase proteins demonstrated similar expression in IMAs and SVs. Vessels were also incubated in the presence or absence of Ang II (1 pmol/L to 1 micromol/L). Ang II increased.O(2)(-) production in IMAs at 4 hours of incubation (control, 978+/-117 pmol. min(-1). mg(-1); 1 micromol/L of Ang II, 1690+/-213 pmol. min(-1). mg(-1); n=27, P=0.0001, 95% CI 336, 925) but not in SVs. This effect was completely inhibited by coincubation of IMAs with DPI (100 micromol/L), a nonspecific Ang II antagonist ([sar(1), thre(8)]-Ang II, 1 micromol/L) and a specific Ang II type 1 (AT(1)) receptor antagonist (losartan, 1 micromol/L). Conclusions-. O(2)(-) production is greater in human IMAs than in SVs. NAD(P)H oxidase and xanthine oxidase are sources of.O(2)(-) production in these vessels. The vasoactive peptide Ang II increases.O(2)(-) production in human arteries by an AT(1) receptor-dependent mechanism.     

去氢表雄酮通过Akt信号通路诱导肝癌细胞凋亡和细胞周期阻滞

目的 研究去氢表雄酮（DHEA）及其代谢物去氢表雄酮硫酸酯（DHEAs）对HepG2和HT-29细胞抑制增殖、促进凋亡及诱导细胞周期阻滞的作用。方法 应用MTT比色法检测不同浓度（1、10、50、100、200μmol／L）的DHEA或DHEAs与HepG2和HT-29细胞孵育8、24、48、72 h对两种细胞系的生长抑制作用;应用流式细胞术检测不同浓度的DHEA或DHEAs对细胞凋亡及细胞周期的变化;采用Western blot检测细胞内磷酸化Akt（Ser473,Thr308）蛋白的水平。结果 （1）不同浓度DHEA作用细胞24h时,HepG2的存活率24h时分别为92．7％±0．9%、84．7％±1．2％、62．4％±0．8％、49．5％±0．8％和50．7％±0．3％,HT-29细胞的存活率分别为92．5％±0.4％、89．5％±0．7％、80．5％±1．1％、67.5％±1.5％和70．6％±0．6％,与对照组相比,DHEA明显抑制HepG2和HT-29两种细胞的生长。在浓度为100μmol／L作用24 h时作用明显,而DHEAs对HepG2及HT-29细胞的增殖无明显影响。（2）100μmol／L的DHEA显著抑制两种细胞周期进程, HepG2细胞G0／G1期细胞比例显著升高,可以达到68．4％±2．0％,而对照组为48．6％±1．2％。HT-29细胞在100μmol／L时的G0／G1期的比率为90．3％±2．7％,而对照组仅为59．0％±1．2％,S及G2／M期细胞明显减少。（3）100μmol／L DHEA作用24 h时能显著诱导HepG2细胞凋亡（凋亡率为18．6％±2．2％）,而DHEAs却无此作用。（4）HepG2细胞在100μmol／L和200μmol／L DHEA作用24 h后,磷酸化Akt（Thr^308）、磷酸化Akt（Set^473）蛋白表达显著降低,这种作用在应用PI3K抑制剂和PI3K激活剂后分别被增强和消除。结论 DHEA对HepG2和HT-29两种肿瘤细胞系均具有较强的抗增殖作用。而对于不同的肿瘤细胞系,DHEA可能通过调节Akt的信号通路来诱导细胞凋亡,还可能通过阻滞细胞周期,使其阻滞在G0／G1期。DHEAs对细胞的生长没有明显的作用。     

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC—7721 cells

AIM:Cyclooxygenase-2(COX-2)has been suggested to be associated with carcinogenesis.We sought to investigate the effect of the selective COX-2 inhibitor,Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS:This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line Various concentrations of Nimesulide(0,200μmol/L,300μmol/L,400μmol/L)were added and incubated.Cell proliferation was detected with MTT colorimetric assay,cell proliferation was detected with MTT colorimetric assay,cell apoptosis by electron microscopy,flow cytometry and TUNEL.RESULTS:Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the controla group.The duration lowerst inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%,the highest inhibition rate was 58.49%,After incubation with Nimesulide for 72h,the most highest apotosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%&#177;1.62%,vs2.24%&#177;0.26%and 21.23&#177;1.78vs2.01&#177;0.23(P&lt;0.05).CONCLUSION:The selective COX-2 inhibitor,Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells,The apoptosis rate and the apoptosis index are dose-dependent.Under electron microscope SMMC-7721 cells incubated with 300 μmol and 400μmol Nimesulide show apoptotic characteristics With the clarification of the mechanism of selective COX-2 inhibitors,Thtese COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.     

多发性骨髓瘤患者p16基因甲基化及砷剂诱导去甲基化的研究

目的探讨p16基因启动子区CpG岛甲基化在多发性骨髓瘤(MM)患者发病中的作用以及三氧化二砷(As2O3)诱导p16基因的去甲基化作用。方法采用巢式甲基特异性PCR法检测MM患者以及利用As2O3作用前后人MM细胞株U266的p16基因的甲基化状态,RTPCR检测U266细胞用药前后p16基因mRNA的表达变化,生长曲线、MTT法检测As2O3对细胞的生长和增殖抑制。利用流式细胞仪检测DNA含量分析法探讨As2O3对骨髓瘤细胞系U266周期的影响。结果MM患者p16基因的甲基化比例为54.8%,U266细胞存在p16基因甲基化,p16基因不表达,As2O3作用后p16基因甲基化程度明显减弱至消失,与未处理组相比,不同剂量作用72h后p16基因表达阳性条带灰度值与βactin比值分别为0.22±0.10、0.59±0.11、0.68±0.09,阳性对照灰度比值为0.77±0.13,其差异有统计学意义(P<0.01)。结论p16基因甲基化在MM患者中较为常见,这可能为MM的诊断和治疗提供借鉴;As2O3可诱导p16基因去甲基化,使p16基因表达上调,恢复其活性,为去甲基化治疗提供新思路。     

Inhibitory effect of polaprezinc on the inflammatory response to Helicobacter pylori.

Helicobacter pylori-infected gastrointestinal mucosa is frequently infiltrated by polymorphonuclear leukocytes (PMN) and monocytes, and these invading cells have been implicated in gastrointestinal mucosal inflammation. To clarify the efficacy of polaprezinc, a chelate compound consisting of zinc and L-carnosine, against H pylori-induced inflammation including PMN infiltration, the in vitro effects of this drug on interleukin (IL)-8 production by an established gastric cancer cell line (MKN 45 cells) and on PMN-endothelial cell adhesive interactions was investigated. Polaprezinc and zinc sulphate inhibited IL-8 production by MKN 45 cells in response to stimulation with H pylori water extract (HPE) in a dose-dependent manner from 10(-7) M to 10(-5) M. In addition, the expression of CD11b and CD18 on PMN and PMN-dependent adhesion to endothelial cells elicited by HPE was inhibited by polaprezinc and zinc sulphate in a concentration-dependent manner. L-carnosine did not have any effects on IL-8 production or PMN-endothelial cell interactions. These results suggest that polaprezinc, mainly the zinc component, may inhibit H pylori-induced PMN-mediated gastric inflammation by attenuating CD11b/CD18 expression on PMN and IL-8 production from gastric epithelial cells.     

内源性硫化氢在慢性阻塞性肺疾病患者中的变化及意义

目的研究内源性硫化氢(H2S)在慢性阻塞性肺疾病(COPD)发病中的作用。方法COPD急性加重组(AECOPD组)27例、稳定期COPD组37例和健康对照组13名,在入选时测定血清H2S和一氧化氮(NO)水平、肺功能、诱导痰细胞分类计数,对AECOPD患者行超声心动图和血气分析。结果(1)血清H2S水平稳定期COPD组[(50·8±2·5)μmol/L]比健康对照组[(39·8±1·6)μmol/L]、AECOPD组[(33·5±2·2)μmol/L]均显著增加(P均<0·01)。(2)AECOPD组吸烟者血清H2S[(28·1±1·3)μmol/L]比非吸烟者[(39·4±3·9)μmol/L,P<0·05]和健康非吸烟者显著降低[(39·8±1·6)μmol/L,P<0·01]。(3)稳定期COPD组不同程度气流阻塞患者血清H2S水平呈线性下降趋势(P<0·05),COPD全球创议(GOLD)Ⅲ期[(45·1±4·1)μmol/L]较Ⅰ期患者[(70·2±6·2)μmol/L]血清H2S水平显著下降(P<0·05)。(4)AECOPD组伴有肺动脉高压患者血清H2S水平显著降低[(26·3±2·2)、(36·2±2·5)μmol/L,P<0·05]。(5)血清H2S与NO、第一秒用力呼气容积占预计值百分比(FEV1占预计值%)、诱导痰淋巴细胞计数、诱导痰巨噬细胞计数均呈正相关(r=0·278~0·533,P均<0·05或0·01),与肺动脉收缩压(PASP)、诱导痰中性粒细胞计数均呈负相关(r=-0·561、-0·422,P=0·011、0·001)。结论内源性H2S可能参与COPD气流阻塞的发病,作为一种无创指标监测疾病严重程度和活动度具有一定意义。     

Induction of apoptosis of human gastric carcinoma SGC-7901 cell line by 5, 7-dihydroxy-8-nitrochrysin in vitro

AIM： To investigate the effect of 5, 7-dihydroxy-8- nitrochrysin （NOChR） on apoptosis of human gastric carcinoma SGC-7901 cell line.
METHODS： SGC-7901 cells were cultured in vitro and the inhibitory effect of NOChR on proliferation of SGC-7901 cells was measured by using an Ml-r assay. NOChR-induced apoptosis rate of SGC-7901 cells was detected using flow cytometry （FCM） with PI staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of NOChR on the proxisome proliferator-activated receptor-γ （PPARγ）, Bcl-2 and Bax protein expression of SGC-7901 cells was analyzed by Western blot.
RESULTS： MIF assay showed that NOChR markedly inhibited proliferation of SGC-7901 cells in a dose- dependent manner, and when ICso was 4.14 μmol/L, the potency of NOChR was 10 times than that of lead compound, chrysin （ChR, IC50 was 40.56 μmol/L）, and was similar to 5-fluorouracil （5-FU, IC50 was 4.51 μmol/L）. FCM with propidium iodide （PI） staining demonstrated that the apoptosis rates of SGC-7901 cells treated with 1.25, 5.00 and 20.00 μmol/L NOChR for 48 h were 9.8% 4- 0.2%, 36.8% 4- 1.9% and 45.5% 4- 3.5%, respectively, and were significantly higher when treated with 5.00 and 20.00 μmol/L NOChR than that with 20.00 μmol/L ChR （12.9% 4- 1.5%）. DNA agarose gel electrophoresis showed that treatment of SGC-7901 cells with 20.00 μmol/L NOChR for 48 h resulted in typical DNA ladder bands of DNA of SGC-7901 cells, which could be eliminated by treating with 10.00 μmol/L GW9662, a blocker of PPARy. Western blot analysis revealed that after 24 h of treatment with 20.00 μmol/L NOChR, PPARgamma and Bax protein expression of SGC-7901 cells increased but Bcl-2 expression decreased; however, pre-incubation with 10.00 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 20.00 μmol/L NOChR on Bax and Bcl-2 protein expression of SGC-7901 cells.
CONCLUSION： NOChR induces apoptosis of SGO7901 cell lines by activating PPARy and decreasing ra     

Enhanced H(2)O(2)-induced cytotoxicity in "epithelioid" smooth muscle cells: implications for neointimal regression

Vascular smooth muscle cells (SMCs) are phenotypically diverse. Although most medial SMCs can be classified as "fusiform," others are of the "epithelioid" phenotype. Proliferation and apoptosis of epithelioid SMCs may contribute importantly to neointimal formation and regression, respectively. Because reactive oxygen species (ROS) are increased in vascular injury and can induce apoptosis of SMCs, we compared the effects of ROS on epithelioid and fusiform SMCs. Epithelioid and fusiform SMC lines were clonally isolated from rat aortic media and studied under similar conditions and passage numbers. H(2)O(2) produced dose- and time-dependent cytotoxicity that was enhanced in epithelioid compared with fusiform cells. After 24-hour exposure to 50 micromol/L H(2)O(2), epithelioid cell numbers were reduced by 34+/-5% versus a 3+/-5% (P<0.05) reduction in fusiform cell numbers. Similar results were obtained whether H(2)O(2) was administered to growth-arrested or growing cells or when epithelioid and fusiform cells were exposed to extracellular O(2)(.-). To investigate whether apoptosis contributed to enhanced ROS-induced cytotoxicity in epithelioid SMCs, terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining was performed. The incidence of TUNEL positivity was 5-fold increased in epithelioid versus fusiform SMCs after treatment with 50 micromol/L H(2)O(2) (19+/-1% epithelioid versus 5+/-1% fusiform, P<0.05). Enhanced H(2)O(2)-induced apoptosis in epithelioid SMCs was confirmed by DNA laddering. Furthermore, when balloon-injured aortas were exposed to H(2)O(2) ex vivo, enhanced apoptosis was observed in neointimal compared with medial SMCs. These results suggest that epithelioid SMCs exhibit enhanced sensitivity to ROS-induced apoptosis, which may play an important role in neointimal regression.