硫化氢对MDA及GSH在大鼠肝星状细胞氧应激中表达的影响

目的:探讨硫化氢对MDA与GSH在大鼠肝星状细胞氧应激中表达的影响.方法:将HSCT6分为8组:空白组(B组,HSC-T6)、对照组(C组,B组+500 μmol/L Fe-NTA)、NaHS组(N1:C组+20 μmol/L NaHS;N2:C组+100 μmol/L NaHS;N3:C组+200μmol/L NaHS)、格列苯脲组(G1:C组+20μmol/LGLBN;G2:C组+200 μmol/L GLBN;G3:C组+700 μmol/L GLBN).用MDA试剂盒测试血清MDA含量;用GSH试剂盒测定上清中GSH含量.结果:24 h后MDA含量对照组和空白组有明显差异(P<0.05),而GSH含量12与24 h均有明显差异(均P<0.05).给予NaHS后,MDA含量24 h N2和N3组与对照组均有明显差异(4.48±0.07 nmol/mg prot,3.58±0.02 nmol/mg protvs 5.05±0.07 nmol/mg prot,均P<0.05),12与24 h GSH含量N2和N3组与对照组有明显差异(35.57±2.02 mg/g prot,36.92±2.30 mg/g protvs 33.64±2.95 mg/g prot;36.49±2.08 mg/gprot,37.59±2.03 mg/g prot vs 31.06±3.08 mg/g prot,均P<0.05).给予GLBN处理后,各项指标结果与给予NaHS处理后结果相对应,各组与相对应组比较均有明显差异(均P<0.05).结论:氧应激下硫化氢对HSC细胞具有保护作用,可抑制肝纤维化的发生发展.     

染氟小鼠成骨细胞系MC3T3-E1细胞中还原型谷胱甘肽和氧化型谷胱甘肽观察

目的 观察染氟小鼠成骨细胞系MC3T3-E1细胞中还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)的水平.方法 采用噻唑蓝(MTT)法检测不同染氟条件下[F-剂量分别为0(对照)、0.5、1.0、2.0、4.0、8.0、12.0、20.0 mg/L]MC3T3-E1细胞1、2、4、10 d的细胞活性.另外,按染氟剂量,将MC3T3-E1细胞分为0(对照)、2、8、20 mg/L组,分别染氟2、4、10 d,采用高效液相色谱—串联四极杆质谱技术检测细胞内GSH、GSSG和谷氨酰胺(Gln)水平.结果 染氟1d时2.0 mg/L组细胞活性(0.57±0.05)明显高于对照组(0.49±0.03,P＜0.01);染氟4d时8.0、12.0 mg/L组细胞活性(0.49±0.07、0.47±0.09)明显低于对照组(0.63±0.06,P＜ 0.05或＜ 0.01);染氟10d时8.0 mg/L组细胞活性(1.52±0.29)明显高于对照组(0.86±0.23),而20.0 mg/L组细胞活性(0,54±0.07)明显低于对照组(P均＜0.01).染氟2、10d时20 mg/L组细胞中GSH水平[(13.92±4.63)、(0.53±0.30) μmol/L]明显低于相应的对照组[(26.42±3.67)、(24.85±5.68)μmol/L,P均＜0.01].染氟2d时2 mg/L组、染氟4d时8 mg/L组、染氟10 d时8 mg/L组细胞内GSSG水平[(1.12±0.62)、(2.13±0.62)、(2.97±1.30)μmol/L]明显高于相应的对照组[(0.55±0.22)、(1.46±0.46)、(1.35±0.50)μmol/L,P＜ 0.05或＜ 0.01].染氟4d时2 mg/L组和染氟10d时8、20 mg/L组细胞内Gln水平[(62.80±17.41)、(122.26±19.51)、(19.38±8.11) μmol/L]明显低于相应的对照组[(83.28±14.32)、(147.15±16.95) rmol/L,P均＜0.05或＜ 0.01].结论 染氟能明显改变成骨细胞内的GSH、GSSG和Gln水平,从而影响细胞内氧化还原平衡态.     

Potential effects of L-NAME on alcohol-induced oxidative stress

AIM: Nitric oxide (NO) is a highly reactive oxidant synthesized from L-arginine by nitric oxide synthase (NOS). NO may cause injury through the generation of potent radicals. Nw-nitro-L-arginine methyl ester (L-NAME) is a non-selective inhibitor of NOS. We aimed to evaluate whether L-NAME treatment had protective effects against oxidative stress in rats intragastrically fed with ethanol during a 4 wk-period. METHODS: Thirty-six male Wistar rats were divided into 3 equal groups: group 1 (control group-isocaloric dextrose was given), group 2 (6 g/kg·d ethanol-induced group) and group 3 (both ethanol 6 g/kg·d and L-NAME 500 mg/L in drinking water-given group). Animals were sacrificed at the end of 4 wk-experimental period, and intracardiac blood and liver tissues were obtained. Biochemical measurements were performed both in plasma and in homogenized liver tissues. Alanine amino transferase (ALT), aspartate amino transferase (AST), malondialdehyde (MDA), NO, superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels were measured by spectrophotometry. RESULTS: ALT and AST in group 2 (62 U/L and 128 U/L, respectively) were higher than those in group 1 (24 U/L and 38 U/L) and group 3 (37 U/L and 81 U/L) (P<0.001 for both). Plasma and tissue levels of MDA in group 2 (4.66 μmol/L and 0.55 μmol/mg protein) were higher than in group 1 (2.65 μmol/L and 0.34 nmol/mg protein) and group 3 (3.43 μmol/L and 0.36 nmol/mg protein) (P<0.001 for both). Plasma and liver tissue levels of NO in group 2 (54.67 μmol/L and 586.50 nmol/mg protein) were higher than in group 1 (34.67 μmol/L and 435.33 nmol/mg protein) and group 3 (27.50 μmool/L and 412.75 nmol/mg protein ) (P<0.001 for both). Plasma and liver tissue SOD activities in group 2 (15.25 U/mL and 5.38 U/ mg protein, respectively) were lower than in group 1 (20.00 U/mL and 8.13 U/ mg protein) and group 3 (19.00 U/mL and 6.93 U/ mg protein) (P<0.001 for both). Plasma and liver tissue CAT activities in group 2 (145 U/mL and 37 U/ mg protein, respectively) were lower than in group 1 (176 U/mL and 73 U/mg protein) and group 3 (167 U/mL and 61 U/mg protein) (P<0.001 for both). Meanwhile, erythrocytes and liver tissue levels of GSH in group 2 (4.12 mg/g Hb and 5.38 nmol/mg protein, respectively) were lower than in group 1 (5.52 mg/g Hb and 4.49 nmol/mg protein) and group 3 (5.64 mg/g Hb and 4.18 nmol/mg protein) (P<0.001 for both). CONCLUSION: Our findings show that L-NAME may produce a restorative effect on ethanol-induced liver damage via decreasing oxidative stress and increasing antioxidant status.     

老年糖尿病患者自由基与微血管并发症关系的探讨

目的　探讨老年糖尿病患者自由基和抗氧化能力的变化及其与微血管并发症的关系。　方法　测定 6 5例老年糖尿病患者和 6 5例健康老年对照者血浆或红细胞中脂质过氧化物 (LPO)、超氧化物岐化酶 (SOD)、过氧化氢酶 (CAT)、谷胱甘肽过氧化物酶 (GSH PX)、维生素C(VC)、维生素E(VE)、β 胡萝卜素 (β CAR)、谷胱甘肽 (GSH) ,同时测定患者的空腹血糖、餐后 2h血糖、糖化血红蛋白(HbA1c)、空腹和餐后 2hC肽、血脂、尿微量白蛋白排泄率、肌电图。　结果　老年糖尿病患者LPO(42 97± 6 99)nmol/g高于健康老年组 (31 5 9± 7 4 4 )nmol/g ,SOD(1712 4 4± 15 7 0 4 )U/L、CAT(2 17 0 1± 2 9 36 ) μg/g、GSH PX(2 1 0 1± 3 38)× 10 -10 U/RBC、VC(40 98± 10 5 1) μmol/L、VE(16 4 4± 2 4 5 ) μmol/L、β CAR(1 19± 0 2 3) μmol/L、GSH(0 98± 0 16 )nmol/L低于健康老年组〔分别为 (192 8 38± 14 3 4 4 )U/L、(2 6 4 4 0± 6 3 5 5 ) μg/g、(2 5 16± 6 4 1)× 10 -10 U/RBC、(5 2 2 3± 10 5 1) μmol/L、(2 3 0 4± 5 38) μmol/L、(1 6 3± 0 4 0 ) μmol/L、(1 2 5± 0 2 0 )nmol/L〕 ,合并糖尿病微血管并发症者变化更加明显 ,LPO和年龄呈正相关 (r=0 310 ,P <0 0 5 ) ,SOD、C     

生长抑素及质子泵抑制剂对大鼠胰腺腺泡细胞慢活化电压依赖性钾离子通道的影响

目的 通过检测慢活化电压依赖性外向钾离子通道电流(Iks)的变化,探讨生长抑素及质子泵抑制剂(PPI)对胰腺腺泡细胞慢活化电压依赖性钾离子通道及胰腺外分泌的影响.方法 分离SD大鼠胰腺组织,取得胰腺腺泡细胞悬液,用EPC10膜片钳放大器记录电压钳制下的不同组别胰腺腺泡细胞Iks的电流值并比较其间差异.分组如下:冲洗液处理为对照组,5μmol/L和20μmol/L 293B组,5 nmol/L促胰液素组,5 nmol/L促胰液素+100 nmol/L或10 nmol/L或1 nmol/L生长抑素组,0.3 mmol/L 8溴—环单磷酸腺苷(8Br-cAMP)组,0.3 mmol/L 8Br-cAMP+100 nmol/L生长抑素组,1 μmol/L乙酰胆碱(Ach)组,1μmol/L Ach+100 nmol/L生长抑素组,5 nmol/L促胰液素+10-5mol/L或10-6mol/L或10-7mol/L奥美拉唑组,1μmol/L Ach+10-5 mol/L或10-6 mol/L或10-7 mol/L奥美拉唑组.组间比较采用单因素方差分析,P＜0.05为差异有统计学意义.结果 大鼠胰腺腺泡细胞静息膜电位为(-40±0.8)mV,当去极化至+10 mV时,对照组的Iks为(420.0±3.2) pA.经5 μmol/L和20 μmol/L 293B作用后,大鼠胰腺腺泡细胞Iks减少为对照组的(60.4±4.2)％和(30.2±3.1)％(F=6.87,P＜0.05).5 nmol/L促胰液素刺激后,Iks增加为(823.0±2.2)pA,分别加入100 nmol/L、10 nmol/L和1 nmol/L生长抑素,Iks降低至(510.0±3.2)pA,(584.0±2.8)pA和(789.0±6.9)pA(F=5.67,P＜0.05);分别加入10-5 mol/L、10-6 mol/L和10-7mol/L奥美拉唑后,Iks峰值未见明显变化,分别为(806.5±3.6)pA,(814.8±3.2) pA和(816.3±2.9) pA (P＞0.05).1 μmol/L Ach刺激后,Iks的峰值为(966.0±3.2) pA;加入100 nmol/L生长抑素后,Iks峰值为(942.0±6.3)pA;分别加入10-5 mol/L,10-6 mol/L和10-7 mol/L奥美拉唑后,其Iks峰值未见明显变化,分别为(956.3±10.3) pA,(957.5±8.6)pA和(960.0±8.4)pA (P＞0.05).结论 生长抑素能抑制胰腺腺泡细胞慢活化电压依赖性钾离子通道的开放,PPI对该通道没有明显抑制作用.     

吴茱萸次碱对大鼠动脉增龄性改变的干预影响

目的观察吴茱萸次碱对大鼠动脉增龄性变化的影响,并探讨其作用机制。方法选择Wistar大鼠32只,分为3月龄组、12月龄组、18月龄组和干预组,每组8只。各组检测超氧化物歧化酶(SOD)、丙二醛以及谷胱甘肽(GSH)含量;实时PCR法检测内皮型一氧化氮合酶(eNOS)mRNA表达;Western blot法测eNOS蛋白和磷酸化蛋白表达。结果与12月龄组比较,18月龄组丙二醛水平明显升高,SOD、GSH、eNOS mRNA、磷酸化eNOS和eNOS蛋白表达明显降低(P<0.05);与18月龄组比较,干预组丙二醛[(4.96±0.22)nmol/mg vs(7.26±0.35)nmol/mg]水平明显降低,SOD[(232.07±11.16)U/mg vs(149.88±13.77)U/mg]、GSH[(12.27±1.42)μg/mg vs(5.40±0.79)μg/mg]、eNOS mRNA[(1.39±0.15)vs(0.74±0.18)]、磷酸化eNOS[(204.67±3.07)vs(121.45±2.54)]和eNOS蛋白[(591.44±2.98)vs(469.57±1.79)]表达明显升高(P<0.05);3月龄组与12月龄组上述指标比较,差异无统计学意义(P>0.05)。结论吴茱萸次碱可以提高自然增龄大鼠动脉血管的抗氧化能力,增加eNOS的表达,从而发挥延缓大鼠血管衰老的作用。     

β-榄香烯对血管紧张素Ⅱ诱导的HSC T6细胞增殖和迁移及RhoA的影响

目的 探讨β榄香烯对血管紧张素(ANG)Ⅱ诱导的肝星状细胞(HSC)增殖迁移及RhoA信号的影响. 方法体外培养HSC,应用ANGⅡ诱导HSC增殖迁移,采用四甲基偶氮唑盐法测定细胞增殖,Transwell小室检测细胞迁移,RT-PCR检测RhoA mRNA,Western blot检测RhoA蛋白.结果 1、2、4、8、10 μmol/L ANGⅡ组A490分别为0.129±0.004、0.143±0.016、0.166±0.012、0.183±0.004、0.283+0.014,与空白对照组(0.110+0.002)比较,ANGⅡ可显著促进HSC增殖,F=112.640,P<0.01;10、8、4 μmol/L的ANG Ⅱ可显著诱导HSC迁移,细胞迁移数分别为每高倍视野(17.40±2.07)、(12.60±1.14)、(9.00±1.58)个,与空白对照组[(1.60±0.55)个]比较,F=117.496,P<0.01.10、5、2.5mg/L的β榄香烯组A490分别为0.076±0.005、0.086±0.003、0.105±0.038,显著低于4μmol/L ANG Ⅱ组,F=95.706,P<0.01;10、5、2.5 mg/L的β-榄香烯组细胞迁移数分别为每高倍视野(1.33±0.58),(2.67±0.58)、(1.67±0.58)个,与4μmol/L ANGⅡ组比较,F=55.600,P<0.01,差异有统计学意义.4 μmol/L ANGⅡ组可显著诱导RhoA蛋白(相对表达量0.975±0.001)及mRNA(相对表达量0.951±0.045)表达,经10、5、2.5 mg/L的β-榄香烯作用后RhoA蛋白相对表达量分别为0.077±0.032、0.538 ± 0.026、0.701±0.078,RhoA mRNA相对表达量分别为0.734±0.038、0.845±0.036、0.881±0.027,较4 μ mol/L ANG Ⅱ组显著下降,F值分别为217.119,18.010,P值均<0.01.结论 ANG Ⅱ可显著诱导HSC增殖、辽移,随剂量的增加诱导作用加强,β-榄香烯可有效抑制ANG Ⅱ诱导的HSC增殖迁移,并能抑制HSC表达RhoA.     

前列腺素E1对梗阻性黄疸大鼠肝脏缺血再灌注损伤时白介素-1β表达的影响

目的:探讨前列腺素(PG)E1对梗阻性黄疸肝脏缺血再灌注损伤的保护作用以及对IL-1β表达的影响.方法:将♂Wistar大鼠随机分为PGE1处理组(PG组,n=18)和生理盐水对照组(NS组,n=18).参照Yoshidome法结扎并切断胆总管建立梗阻性黄疸模型,1 wk后Pringle法阻断肝门15 min,再灌注后建立胆道再通.于缺血前15 min至再灌注60 min,PG组经门静脉持续泵入PGE1 0.5 μg/(kg·min),NS组给予等量生理盐水.于再灌注1、6和24 h 3个时点取材,检测血清ALT,AST,总胆红素(total bilirubin,TBIL),直接胆红素(direct bilirubin,DBIL)水平,测定肝组织GSH,MDA含量.ELISA法测定肝组织IL-1β表达,并观察肝脏病理组织学改变.结果:再灌注各时点PG组血清ALT水平(1 h:1939±1427 nkat/L vs 5596±2975 nkat/L;6 h:3409±1708 nkat/L vs 9279±4404 nkat/L;24 h:1434±274 nkat/L vs 2264±630 nkat/L)和AST(1 h:21746±12083 nkat/L vs 37552±12382 nkat/L;6 h:55039±35471 nkat/L vs 98811±11126 nkat/L;24 h:9394±1662nkat/L vs 27664±15856 nkat/L)、肝组织MDA含量(1 h:0.89±0.18 μmol/g vs 1.21±0.24 μmol/g;6 h:1.08±0.23 μmol/g vs 1.45±0.13 μmol/g;24 h:1.03±0.08 μmol/g vs 1.45±0.26 μmol/g)以及肝组织IL-1β表达水平(1 h:304.1±67.9 ng/L vs 362.8±137.1 ng/L;6 h:376.8±74.6 ng/L vs 618.8±217.8 ng/L;24 h:273.0±69.0 ng/L vs 373.0±71.7 ng/L)均显著低于NS组(P＜0.05),而肝组织GSH含量显著高于NS组(1 h:945.1±121.2 mg/g vs 720.0±80.1 mg/g;6 h:753.5±118.6 mg/g vs 553.6±140.0 mg/g;24 h:768.0±135.9 mg/g vs 596.3±36.4 mg/g,P＜0.05),但两者胆红素水平无明显差异(P＞0.05).PG组肝脏病理组织学损伤程度也较NS组明显减轻.结论:PGE1下调肝组织IL-1β表达,对梗阻性黄疸肝脏缺血再灌注损伤具有保护作用.     

Intravenous administration of glutathione protects parenchymal and non-parenchymal liver cells against reperfusion injury following rat liver transplantation

AIM:To investigate the effects of intravenous administrationof the antioxidant glutathione (GSH) on reperfusion injuryfollowing liver transplantation.METHODS:Livers of male Lewis rats were transplantedafter 24 h of hypothermic preservation in University ofWisconsin solution in a syngeneic setting.During a 2-hreperfusion period either saline (controls,n=8) or GSH(50 or 100 μmol/(h·kg),n=5 each) was continuouslyadministered via the jugular vein.RESULTS:Two hours after starting reperfusion plasmaALT increased to 1 457±281 U/L (mean±SE) in controlsbut to only 908±187 U/L (P<0.05) in animals treated with100 μmol GSH/(h·kg).No protection was conveyed by50μmol GSH/(h·kg).Cytoprotection was confirmed bymorphological findings on electron microscopy:GSHtreatment prevented detachment of sinusoidal endothelialcells (SECs) as well as loss of microvilli and mitochondrialswelling of hepatocytes.Accordingly,postischemic bile flowincreased 2-fold.Intravital fluorescence microscopy revealeda nearly complete restoration of sinusoidal blood flow and asignificant reduction of leukocyte adherence to sinusoidsand postsinusoidal venules.Following infusion of 50μmol and100 μmol GSH/(h·kg),plasma GSH increased to 65±7 mol/Land 97±18 mol/L,but to only 20±3 mol/L in untreated recipients.Furthermore,plasma glutathione disulfide (GSSG) increasedto 7.5±1.0 mol/L in animals treated with 100μmol/(h·kg) GSHbut infusion of 50μmol GSH/(h·kg) did not raise levels ofuntreated controls (1.8±0.5 mol/L vs 2.2±0.2 mol/L).CONCLUSION:Plasma GSH levels above a critical level mayact as a “sink” for ROS produced in the hepatic vasculature during reperfusion of liver grafts.Therefore,GSH can beconsidered a candidate antioxidant for the Drevention ofreperfusion injury after liver transplantation,in particularsince it has a low toxicity in humans.     

谷胱甘肽转移酶μ基因缺失和氧化损伤与系统性红斑狼疮相关性研究

目的　检测系统性红斑狼疮 (SLE)患者谷胱甘肽转移酶 μ(GSTμ)基因缺失及血中一氧化氮 (NO)、脂质过氧化物 (LPO)、超氧化物歧化酶 (SOD)、谷胱甘肽过氧化物酶 (GSH Px)和谷胱甘肽 (GSH)含量 ,分析其与SLE发病之间的关系。方法　用PCR法检测 87例SLE患者和 40名健康对照组的GSTμ基因 ,用化学分析法测上述 5项指标。 结果　SLE患者GSTμ基因缺失率达 6 9 0 % ,明显高于对照组的 47 5 %。SLE活动期NO(79± 18) μmol/L、LPO (10 4± 2 0 ) μmol/L明显高于稳定期和对照组的水平。SLE活动期SOD (12 86± 2 5 2 ) μU/L、GSH Px (78± 14)U/mg、GSH (0 37±0 0 5 )mg/ g明显低于稳定期及对照组水平。在SLE稳定期GSH (1 0 0± 0 14)mg/ g ,仍明显低于对照组水平。血清NO水平与LPO呈显著直线正相关 ,与SOD、GSH Px、GSH呈显著直线负相关。抗dsDNA与NO、LPO呈显著直线正相关。在SLE活动期 ,GSTμ基因缺失者LPO (11 4± 2 2 ) μmol/L明显高于GSTμ基因携带者的水平 ,SOD (1111± 2 18) μU/L、GSH Px (6 7± 14)U/mg、GSH (0 2 4±0 0 4)mg/g明显低于GSTμ基因携带者的水平。在SLE稳定期GSTμ基因缺失者的SOD和GSH水平仍低于GSTμ基因携带者。 结论　GSTμ基因缺失可能是SLE发病的遗传因素之一 ,SLE