丹参水提物对乳鼠心肌细胞缺氧-复氧损伤的保护作用

本文研究丹参水提物对乳鼠心肌细胞缺氧-复氧损伤的保护作用。以乳鼠心室肌进行心肌细胞培养并建立缺氧-复氧损伤模型,检测不同浓度丹参水提物预处理后细胞的存活率,乳酸脱氢酶(LDH)、肌酸激酶(CK)、天冬氨酸转移酶(AST)的泄漏量及Akt的磷酸化水平。丹参水提物对缺氧-复氧损伤的心肌细胞具有明显的保护作用,这可能与它激活PI3K/Akt信号通路有关。     

缺氧-复氧对培养的大鼠心肌细胞损伤与细胞凋亡的影响

目的:探讨体外培养的大鼠心肌细胞在缺氧(缺血)和缺氧-复氧(再灌注)状态下心肌细胞损伤和细胞凋亡情况。方法:常规方式培养SD大鼠心肌细胞。给予模拟缺血(缺氧)溶液、模拟复氧(再灌注)液以及终浓度为200U/mL的超氧化无歧化酶(SOD)干预处理,制备缺氧-复氧损伤组(H/R组)、SOD+缺氧-复氧损伤组(SOD+H/R组)和缺氧预处理组(HP组)模型,未经处理的为正常对照组(control组)。结果:屹H/R组的细胞存活率较对照组明显降低(P<0.01);H/R+SOD组和HP组的细胞存活率虽然较正常对照组明显降低(P<0.05),但比H/R组为高(P<0.05);亿H/R组培养液中培养液中心肌酶活性显著高于HP组和H/R+SOD组(P<0.01)。役H/R组心肌细胞内的脂质过氧化产物丙二醛(MDA)含量也明显增高,与HP组和H/R+SOD组的心肌细胞内MDA含量比较有显著性差异(P<0.05);而HP组、H/R组与对照组间比较,无明显差异(P>0.05);均提示了缺氧-复氧过程中心肌细胞损伤严重,也说明了缺氧(血)预处理或SOD均有细胞保护作用。臆H/R组心肌细胞内游离钙含量增加,显著高于HP组和H/R+SOD组(P<0.01);逸流式细胞仪测定出H/R组心肌细胞凋亡率也明显高于HP组和H/R+SOD组(P<0.01)。心肌细胞凋亡的数量与细胞内钙的增加呈正相关(P<0.01)。肄电镜下所见H/R组的许多细胞表现为胞浆减少,核深染、固缩状态,异染色质不均匀,部分线粒体呈空泡变性,或是肌浆网囊泡变等凋亡期或凋亡前期样改变,少数呈凋亡向坏死方面改变;而其他组则少见此些变化。结论:体外培养的心肌细胞在缺氧-复氧情况下同样可加重心肌细胞损伤,可能是通过降低细胞内钙离子浓度而起作用的。     

Does maternal nicotine exposure during gestation increase the injury severity of small intestine in the newborn rats subjected to experimental necrotizing enterocolitis

Background/Purpose

The aim of this study was to evaluate the effects of maternal nicotine exposure during gestation on injury severity of small intestine in the newborn rats subjected to hypoxia-reoxygenation and cold stress.

Methods

A total of 21 Sprague-Dawley pregnant rats were divided into 3 equal groups. The groups were labeled as group 1, control group; group 2, hypoxia-reoxygenation group; and group 3, nicotine-hypoxia-reoxygenation group. The rats of group 3 were exposed to nicotine via subcuticular injection for the last week of gestation (2 mg/kg/d). Newborn rats were collected immediately after birth to prevent suckling of maternal milk (40 rat pups in group 1, 43 rat pups in group 2, and 41 rat pups in group 3). Litters in groups 2 and 3 were stressed twice daily with asphyxia followed by cold (4°C for 10 minutes) stress to induce hypoxic intestinal injury which is relevant to human necrotizing enterocolitis. Breathing 100% CO₂ for 10 minutes in a chamber followed by 10-minute 100% O₂ breathing was the asphyxia model repeated twice daily. After hypoxia-reoxygenation and cold stress, newborn rats were returned to their mother's cages. This protocol was repeated for the following 2 days, and the rat pups were decapitated on the third day. Using this protocol of asphyxia and cold stress, all of neonatal rats developed clinical and pathological signs of hypoxia-induced intestinal injury. The entire gastrointestinal tract was removed and examined macroscopically. A 2-cm section of distal ileum from each animal was taken for histopathological and biochemical examinations. Histological changes in ileal architecture were scored and graded from 1 to 5. The remaining intestinal tissues of the animals were used for lipid peroxidation analysis.

Results

Typical signs of hypoxia-induced intestinal injury were observed in the 2 experimental groups (groups 2 and 3) macroscopically. There were more grades 3 and 4 injuries in group 3 (P < .05). The malondialdehyde levels were elevated in groups 2 and 3 (P < .001). The malondialdehyde levels of the group 3 were also significantly higher than group 2 (P < .01).

Conclusions

Maternal nicotine exposure during gestation results in higher grade histological injury in newborn rats subjected to hypoxia-reoxygenation and cold stress.     

丙泊酚降低高糖环境下H9C2心肌细胞缺氧后的损伤

目的探讨小窝蛋白3(Cav-3)在丙泊酚降低高糖环境下H9C2心肌细胞缺氧后损伤中的作用。方法培养大鼠原代H9C2心肌细胞,构建细胞缺氧-复氧模型。将心肌细胞分为六组:正常培养组(NC组)、高糖培养组(HG组)、高糖缺氧-复氧组(HR组)、丙泊酚处理组(P组)、Cav-3抑制剂组(PI组)和DMSO溶剂组(D组)。比较六组心肌细胞损伤程度、氧化应激反应、线粒体功能、Cav-3蛋白含量、蛋白激酶B(AKT)及信号传导及转录激活因子3(STAT3)活化情况。结果与HR组比较,P组细胞活力、总SOD(T-SOD)活性、线粒体活力、ATP含量、Cav-3蛋白含量、AKT及STAT3活化明显增加,LDH、CK-MB活性、cTnI含量、Bcl-2相关X蛋白(Bax)与B淋巴细胞瘤-2(Bcl-2)比值(Bax/Bcl-2)明显下降,含半胱氨酸的天冬氨酸蛋白水解酶-3(caspase 3)及含半胱氨酸的天冬氨酸蛋白水解酶-9(caspase-9)蛋白含量、MDA浓度、JC-1染色的荧光绿红比明显降低,DCF-DA荧光阳性细胞数、MPTP开放明显减少(P0.05);与P组比较,PI组和D100组细胞活力、T-SOD活性、线粒体活力、ATP含量、Cav-3蛋白含量明显降低,AKT及STAT3活化明显减少(P0.05),LDH、CK-MB活性、cTnI含量、Bax/Bcl-2值、caspase-3及caspase-9蛋白含量、MDA浓度、JC-1染色的荧光绿红比明显升高,DCF-DA荧光阳性细胞数、MPTP开放明显增加(P0.05)。结论丙泊酚通过上调Cav-3减少高糖环境下H9C2心肌细胞的缺氧后线粒体的损伤及细胞的死亡。     

NADPH氧化酶亚单位nox-1在心肌细胞急性缺氧复氧损伤时的变化及心肌营养素-1的作用

目的： 探讨心肌细胞急性缺氧复氧损伤时NADPH氧化酶亚单位nox-1的变化及心肌营养素-1的作用。方法: 用改良的方法培养出生1-３ d的乳鼠心肌细胞,分为6组：(1)对照组;(2)缺氧复氧组;(3)缺氧复氧+CT-1组;(4)缺氧复氧+CT-1+LY294002组 (PIK3/Akt 阻断剂);（5）缺氧复氧+CT-1+PD98059组（ERK 阻断剂）;（6）缺氧复氧+CT-1+助溶剂DMSO组。 CT-1 的浓度为10 μg/L。MTS法测定心肌细胞的存活率,四氯四乙基苯丙咪唑基羰化青碘化物（JC1）检测心肌细胞线粒体膜电位(Δψm),二氯荧光黄双乙酸盐（DCFH-CA）检测细胞活性氧（ROS）,流式细胞仪检测心肌细胞凋亡率。Nox-1蛋白采用Western blotting检测。结果: 缺氧复氧培养后心肌细胞凋亡率及细胞内ROS较对照组明显增加,分别是(19.7%±1.4% vs 2.1%±0.5%, 14.07%±1.25% vs 3.54%±0.86%, P<0.05),而心肌细胞存活率显著降低,线粒体膜电位（Δψm）下降;nox-1表达明显升高。CT-1处理的心肌细胞,较缺氧复氧组心肌细胞存活率明显上升 (87.0%±7.3%),而心肌细胞凋亡率及细胞内ROS 显著减少,Δψm水平增加,nox-1蛋白表达下调。而CT-1的这些作用能被PIK3/Akt和ERK阻断剂抑制。结论: 心肌细胞急性缺氧复氧损伤时NADPH氧化酶亚单位nox-1表达上调,而心肌营养素-1能通过下调nox-1表达,发挥对心肌细胞保护作用。     

红花黄色素对幼鼠缺氧-复氧损伤的保护作用

目的：观察幼鼠缺氧-复氧后脑水肿和脂质过氧化作用的变化及红花黄色素对其影响和意义。方法：采用SD幼鼠缺氧-复氧模型，于缺氧前30min腹腔注射红花黄色素(7g生药／kg体重)。幼鼠窒息缺氧40min后，再复氧24h、48h，用干湿质量法测定脑组织含水量。以硫代巴比妥酸法测定丙二醛(MDA)含量，用黄瞟呤氧化酶法测超氧化物歧化酶(SOD)活性。观察红花黄色素对缺氧-复氧24h、48h时脑组织含水量、MDA及SOD活性的影响。结果：缺氧-复氧48h时脑组织含水量、MDA含量明显增加，而SOD活性下降，红花黄色素能减轻脑组织水肿程度，抑制MDA生成，增强SOD活性。结论：红花黄色素通过减少脂质过氧化作用对缺氧-复氧脑损伤有保护作用。     

PI3K/Akt信号途径在二氮嗪抑制缺氧-复氧大鼠心肌微血管内皮细胞凋亡中的意义

目的 研究磷脂酰肌醇-3-激酶/Akt (PI3K/Akt)信号途径在二氮嗪抑制缺氧-复氧(H-R)大鼠心肌微血管内皮细胞(MMECs)凋亡中的意义.方法 将培养的SD大鼠MMECs随机分为正常对照组(C组)、缺氧-复氧组(H-R组,缺氧2h复氧2 h)、二氮嗪+缺氧-复氧组(DZ组,二氮嗪100 μmol/L预处理2 h+缺氧2h复氧2 h)、PI3K特异性阻滞剂LY294002+二氮嗪+缺氧-复氧组(LY294002组,LY294002 100 μmol/L预处理2 h+二氮嗪100μmol/L预处理2 h+缺氧2h复氧2 h).Hoechst染色观察凋亡细胞结构,Annexin V-FITC/PI双染法测定细胞凋亡率,RT-PCR和Western blot分别检测Akt的mRNA和蛋白表达水平.结果 与C组比较,H-R组细胞凋亡率升高,Akt mRNA和蛋白表达增加(P＜0.05或P＜0.01);与H-R组比较,DZ组细胞凋亡率降低,Akt mRNA和蛋白表达增加(P＜0.05或P＜0.01);与DZ组比较,LY294002组细胞凋亡率升高,Akt mRNA和蛋白表达减少(P＜0.01).结论 H-R损伤引起MMECs凋亡.线粒体三磷酸腺苷敏感性钾通道(mito-KATP)开放后通过PI3K/Akt信号途径能部分抑制该作用.     

镁对血管内皮细胞缺氧再复氧损伤的保护作用

目的探讨镁离子对人脐静脉血管内皮细胞缺氧再复氧损伤的保护作用及其可能机制。方法取人脐静脉分离内皮细胞培养传代 ,至第三代分为对照组、缺氧复氧组和硫酸镁组。用缺氧再复氧诱导血管内皮细胞损伤。镁离子组浓度分别为 (2、4、8、11、16mmol/L)。对照组常规培养 ,硫酸镁组加入不同浓度的镁离子预处理后缺氧再复氧。分别测定细胞培养液中一氧化氮 (NO) ,丙二醛 (MDA)及内皮素(ET-1)的含量。结果缺氧复氧组NO含量较对照组明显降低 (x缺氧 ±svsx对照 ±s,P<0.01) ,MDA与ET -1含量显著上升 (x缺氧 ±svsx对照 ±s,P<0.01) ,硫酸镁组NO含量较缺氧复氧组明显升高(x硫酸镁±svsx缺氧 ±s,P<0.05) ,MDA和ET -1含量显著降低 (x硫酸镁±svsx缺氧±s,P<0.05) ,且一定范围内呈剂量依赖关系。结论缺氧再复氧能造成VECs损伤 ,镁离子能保护缺氧再复氧造成的VECs损伤 ,其机制可能与抗脂质过氧化、稳定细胞膜及促进自由基清除有关。     

Effects of aging and every-other-day feeding on the levels of oxygen radicals in rat brain slices

Caloric and food restriction attenuate oxidative stress. The effect of aging and every-other-day (EOD) feeding on oxygen radical-dependent chemiluminescent intensity was examined in ex vivo brain slices from Fischer rats during oxygenation and hypoxia-reoxygenation with lucigenin, a chemilumigenic probe used for detecting superoxide anion radicals. The chemiluminescent intensity increased during reoxygenation after hypoxic treatment, and the chemiluminescence in the brain slices at the baseline and during reoxygenation increased with age. However, no difference was observed in the superoxide-dependent chemiluminescence between brain slices prepared from the aged rats fed EOD and those fed ad libitum. Our results indicated that age-dependent increases in superoxide production might be associated with enhanced oxidative stress in aged Fischer rat brains. However, the present study newly indicated that decreased superoxide production might not be a major causal factor in caloric and food restriction attenuated oxidative stress.     

右美托咪定联合Tol样受体4抑制剂对缺氧复氧心肌细胞凋亡和炎症反应的影响及其机制

目的探讨右美托咪定(DEX)联合Toll样受体4(TLR4)抑制剂TAK-242对缺氧复氧(H/R)心肌细胞凋亡和炎症反应的影响及其机制。方法心肌细胞H9C2分为对照(Con)组、H/R组(H/R损伤)、DEX组(1.0μmol/L DEX,再行H/R损伤)、TAK-242组(30μmol/L TAK-242,再行H/R损伤)和DEX+TAK-242组(30μmol/L TAK-242及1.0μmol/L DEX,再行H/R损伤处理)。各组细胞复氧培养6 h后,采用MTT法、流式细胞仪、试剂盒、酶联免疫吸附法检测细胞增殖、凋亡、乳酸脱氢酶(LDH)释放率、白介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)含量,Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解的caspase-3(cleaved caspase-3)、TLR4和核因子B p65(NF-κB p65)蛋白表达。结果五组细胞凋亡率、LDH释放率、Bax、Bcl-2、cleaved caspase-3、TLR4、NF-κB p65蛋白表达水平、IL-1β、TNF-α含量比较差异均有统计学意义(F=316.938、330.004、839.933、169.750、378.365、476.535、298.527、99.219、293.498,P<0.05)。与Con组相比,H/R组细胞的凋亡率、LDH释放率、Bax、cleaved caspase-3、TLR4和NF-κB p65蛋白表达水平、细胞上清液中IL-1β和TNF-α含量均明显升高(均P<0.05),Bcl-2蛋白表达水平明显降低(均P<0.05)。与H/R组相比,DEX组、TAK-242组和DEX+TAK-242组的细胞凋亡率、LDH释放率、Bax蛋白、cleaved caspase-3、TLR4和NF-κB p65蛋白、IL-1β、TNF-α的表达水平均明显降低,Bcl-2蛋白表达水平明显升高(均P<0.05)。与DEX组、TAK-242组相比,DEX+TAK-242组的细胞凋亡率、LDH释放率、Bax、cleaved caspase-3、TLR4和NF-κB p65蛋白表达、IL-1β、TNF-α表达水平更低,Bcl-2蛋白的表达更高(均P<0.05)。结论DEX和TAK-242联合可协同抑制H/R引起的心肌细胞凋亡和炎症反应,其作用机制可能与协同抑制TLR4/NF-κB通路有关。