大鼠肢体缺血/再灌注后肝脏iNOS表达的变化及其意义

目的:探讨肢体缺血/再灌注(I/R)致肝损伤时肝组织iNOS表达的变化及其意义.方法:夹闭大鼠双侧股动脉根部4 h、开放2～24 h,制备肢体I/R模型.RT-PCR检测肝组织iNOSmRNA表达的改变,免疫组化染色法观察iNOS蛋白及过氧亚硝基阴离子(ONOO-)的生成与分布,比色法测定肝组织MDA含量及SOD活性;对肢体I-R大鼠用氨基胍抑制iNOS活性后,观察其肝组织的病理学变化.结果:肢体I-R后,肝组织iNOS mRNA的表达水平较对照组显著上调(P＜0.05),肝组织内出现大量iNOS及ONOO-阳性肝细胞,肝组织MDA含量升高及SOD活性降低均与对照组有显著性差异(P＜0.01).应用氨基胍抑制iNOS活性,使肢体I-R所致肝组织病变减轻.结论:肢体I/R后,肝组织iNOSmRNA及蛋白表达显著上调,所诱生的高浓度NO参与介导了肢体I/R引发的肝脏损伤.     

血红素氧合酶-1 mRNA在离体大鼠心肌缺血预处理中的变化

目的:观察血红素氧合酶-1(HO-1)mRNA在缺血预适应中的变化.方法:实验动物随机分为对照组(CN)、缺血/再灌损伤组(I/R)、缺血预适应 缺血/再灌损伤组(PC).结果:PC组的心功能恢复率高于I/R组(P<0.05),MDA含量低于I/R组(P<0.05),HO-1 mRNA又高于I/R组(P<0.05).结论:HO-1mRNA表达上调与缺血预适应保护缺血/再灌注损伤心肌有关.     

血红素氧合酶-1在离体大鼠心肌缺血预适应中的作用

目的:分别观察给予HO-1诱导剂和抑制剂对心肌相对缺血再灌注损伤和缺血预适应的影响,探讨HO-1在缺血预适应中的作用.方法:实验动物随机分为对照组(CN)、缺血/再灌损伤组(I/R)、缺血预适应 缺血/再灌损伤组(PC)、HO-1诱导剂 缺血/再灌损伤组(HM)、HO-1抑制剂 缺血预适应组(ZP).心肌缺血/再灌损伤采用相对缺血/再灌损伤模型,缺血预适应则为相对缺血5min恢复5min,反复2次.测定心功能、MDA及HO-1活性变化.结果:HM组HO-1活性升高,心功能恢复率均显著高于IR组(P＜0.01),MDA含量显著低于IR组(P＜0.05).ZP组活性降低,心功能恢复率显著低于PC组(P＜0.05),MDA含量显著高于PC组(P＜0 05).结论:HO-1是缺血预适应释放的内源性活性物质之一.     

血红素氧合酶-1参与心肌细胞延迟预适应

目的:探讨血红素氧合酶-1(HO-1)在心肌细胞缺血预适应(IPC)中的作用。方法:实验分5组:正常对照组(CN组)、缺血/再灌注组(I/R组)、缺血预适应+缺血/再灌注组(PC组)、ZnPP(HO-1抑制剂)+缺血预适应+缺血/再灌注组(ZP组)和Hemin(HO-1诱导剂)+缺血/再灌注组(HE组)。测定HO-1mRNA表达、心肌细胞存活率、胞内[Ca2+]i和细胞培养液中MDA含量等。结果:PC组和HE组HO-1mRNA表达量和细胞存活率显著高于I/R组,而MDA含量和胞内[Ca2+]i则皆显著低于I/R组,PC组各指标与ZP组间有显著性差异,与HE组66较未见显著性差异。结论:IPC可以诱导HO-1mRNA表达,对心肌细胞缺血/再灌注损伤产生延迟保护作用。     

巨噬细胞亚型转变在大鼠肾脏缺血/再灌注损伤中的作用

目的：探讨巨噬细胞在大鼠肾脏缺血/再灌注损伤过程中的亚型转变及意义。方法：将30只雄性SD大鼠随机分成假手术组（Sham,n=6）和缺血/再灌组（IRI,夹闭肾动脉45 min,n=24）。IRI组分别于术后0、6、24和72 h取肾组织,每个时相组6只大鼠。用HE染色观察肾组织损伤程度;免疫组化染色检测细胞增殖核抗原（PCNA）的表达;实时定量RT-PCR检测巨噬细胞移动抑制因子（MIF） mRNA的表达;免疫组织荧光染色检测MIF、单核巨噬细胞趋化蛋白-1（MCP-1）以及活化巨噬细胞标志物CD68的表达,流式细胞分析检测巨噬细胞M1和M2亚型的分布特征。结果：病理结果显示大鼠肾局部损伤情况和炎症细胞浸润程度在24 h时最为严重,之后逐渐恢复。PCNA在再灌后表达明显增加,6 h达峰值,72 h表达下降。相比于正常组,再灌组大鼠肾组织中MIF的mRNA和蛋白表达明显升高;MCP-1表达则在6 h达峰值,随后下降;而CD68阳性的巨噬细胞数量明显增加,24 h达峰值,72 h表达下降。更进一步研究发现缺血/再灌注6 h时,M1亚型分布达最高值;之后随着缺血/再灌注时间延长,M1亚群相对含量开始下调,M2随之升高。结论：在肾脏缺血/再灌注早期,M1巨噬细胞介导的组织损伤发挥主要作用,随后M2型表达逐渐上调,并通过促进细胞增殖修复肾组织损伤。     

N-乙酰半胱氨酸对大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达的影响

目的:探讨大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达情况及NAC的保护作用机制.方法:45只雄性SD大鼠随机分成三组:假手术组(Sham组,n=5);缺血再灌注损伤组(I/R 组,n=20)缺血60min后分别再灌注1、3、6、12h;N-乙酰半胱氨酸组(NAC组,n=20):先自阴茎背静脉给大鼠注射溶于生理盐水的NAC,20min后再按I/R组处理.在各规定的再灌注时间点,分别采用western-blot和免疫组化方法测定肝组织中NF-κB和ICAM-1的表达.结果:I/R组和NAC组再灌注1、3、6、12h后,NF-k B的表达均明显高于Sham组(p＜0.01),于再灌注3h达到高峰;ICAM-1的表达均明显高于Sham组(p＜0.01),于再灌注6h达到高峰.NAC组再灌注1、3、6h与VR组相同时间点比较:NF-k B和ICAM-1的表达均低于I/R组(p＜0.05).NAC组再灌注12h与I/R组相同时间点比较:NF-K B和ICAM一1的表达虽然在数值上有所减少,但统计学上无差异(p＞0.05).结论:大鼠肝脏缺血再灌注后NF-κB和ICAM-1表达增加,NAC可抑制NF-k B激活,减少ICAM-1表达减轻大鼠肝脏缺血再灌注损伤.     

吸入一氧化碳防治肢体缺血／再灌注所致肝脏损伤的实验研究

目的：观察吸入外源性一氧化碳（CO）对肢体缺血／再灌注（I／R）所致肝脏损伤的防治作用。方法：健康SD大鼠100只,随机分为假手术（S）、假手术吸入CO（SC）、I／R、I／R吸入CO（RC）组。通过夹闭股动脉4h、再开放6—72h、10d复制肢体L／R致肝脏损伤模型。S、I／R组吸入普通医用空气,SC、RC组吸入含CO（体积分数为0．05％）的医用空气。光镜观察肝组织病理学变化,全自动生化分析仪检测血谷丙转氨酶（GPT）,流式细胞仪检测肝细胞凋亡百分比及bax、bcl-2的表达水平。结果：S组与SC组比较,各项观察指标无显著差别;与SC组比较,I/R及RC组肝组织呈病理改变,血清GPT及肝细胞凋亡百分比明显升高;I／R组肝细胞bax蛋白的表达水平明显升高。和L／R组相比。RC组肝组织损伤程度减轻,血清GPT、肝细胞凋亡百分比及bax蛋白的表达水平明显降低,而肝细胞bcl-2蛋白的表达水平显著升高。结论：吸入适量外源性CO对肢体I／R所致肝脏损伤有防治效应。     

MiR-21靶向调控Mfn2对缺血再灌注损伤肾小管上皮NRK-52E细胞自噬及凋亡的影响

摘要 目的：探讨miR-21对缺血再灌注损伤肾小管上皮细胞自噬及凋亡的影响及其与线粒体融合素2（mitochondria fusion protein mitofusin2,Mfn2）的靶向关系。方法：将大鼠近端肾小管上皮细胞株NRK-52E细胞按处理方式不同分组：I/R+control mimics组（转染control mimics后缺氧3 h/复氧3 h）,I/R+miR-21mimics组（转染miR-21mimics后缺氧3 h/复氧3 h）,I/R组（缺氧3 h/复氧3 h）及对照组（正常培养）。选取30只Sprague-Dawley(SD)大鼠,随机分为假手术组、缺血再灌注模型组(I/R组)。取大鼠肾组织进行HE染色,自动生化分析仪检测大鼠血清尿素氮（BUN）、肌酐（Cr）,四甲基偶氮唑盐比色法（MTT）检测细胞增殖能力,TUNEL法检测细胞凋亡,实时荧光定量PCR检测细胞自噬和凋亡相关基因LC3-Ⅱ、LC3-Ⅰ、Beclin1、Bcl-2、Bax及Mfn2 mRNA表达,Western blot法检测细胞自噬和凋亡相关蛋白的表达,荧光素酶实验验证miR-21与Mfn2的靶向关系。结果：Sham组大鼠血清BUN、Cr水平,大鼠肾组织细胞凋亡率高于I/R组（P<0.05）。I/R组大鼠肾组织肾小管结构紊乱,大量炎症细胞浸润。Sham组大鼠肾组织miR-21水平高于I/R组（P<0.05）。48 和 72 h 时,I/R+miR-21 mimics组细胞活力明显低于I/R+control mimics组,I/R组及对照组（P<0.05）,I/R组细胞活力低于对照组（P<0.05）。I/R+miR-21mimics组凋亡率显著高于I/R+control mimics组,I/R组及对照组（P<0.05）,I/R组凋亡率显著高于对照组（P<0.05）。与对照组比较,I/R组细胞Beclin1、LC3-Ⅱ/LC3-Ⅰ、Bax蛋白及基因mRNA表达量升高,Bcl-2蛋白及基因mRNA表达量降低（P<0.05）;与I/R组比较,I/R+miR-21mimics组细胞Beclin1、LC3-Ⅱ/LC3-Ⅰ、Bax蛋白及基因mRNA表达量升高,Bcl-2蛋白及基因mRNA表达量降低（P<0.05）。miR-21与Mfn2具有靶向关系。结论：miR-21可靶向Mfn2促进肾缺血再灌注损伤引起的凋亡及自噬。     

芍药苷减轻四氯化碳诱导的急性肝损伤

为研究芍药苷对四氯化碳(CCl_4)诱导的急性肝损伤的保护作用及相关机制。将36只健康雄性C57BL/J小鼠随机分成6组:空白组,芍药苷对照组,CCl_4模型组(0.1%,20 mL/kg),高、中、低剂量芍药苷+CCl_4组(10 mg/kg,30 mg/kg,100 mg/kg),每组6只。24 h后眼球取血收集血清,测定丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST)活性。用苏木精-伊红(HE)染色,观察肝脏的组织学改变;试剂盒测定肝组织中SOD、GSH-PX、CAT的活性及MDA和GSH含量;ELISA法检测血清中TNF-α、IL-6含量;试剂盒检测小鼠肝组织中Caspase-3的活性;q RT-PCR检测肝组织中HO-1 mRNA的表达。实验发现PAE降低小鼠血清ALT、AST水平,改善肝脏的病理形态;芍药苷抑制CCl_4诱导氧化应激,升高肝组织HO-1 mRNA水平;降低TNF-α、IL-6含量;芍药苷减低肝组织中Caspase-3活性,减少肝细胞凋亡。由此可知,芍药苷可保护CCl_4诱导的急性肝细胞损伤,该保护作用可能与抑制脂质过氧化,减少促炎细胞因子产生,减少肝细胞凋亡,促进抗氧化蛋白表达有关。     

bFGF对脑缺血再灌注大鼠海马及顶叶皮质神经元的保护作用

目的本实验应用大脑中动脉栓塞（MCAO）模型,观察bFGF对脑缺血再灌注损伤后海马及顶叶皮质中Wnt通路抑制因子Dickkopf-1（DKK-1）和Wnt通路中pCatenin的表达作用的影响,以探讨Wnt通路对缺血性脑损伤的作用机制,为临床治疗缺血性脑血管病提供实验依据。方法应用线栓法制作大鼠局灶性脑缺血再灌注模型,大脑中动脉阻塞1h再灌注损伤24h,采用免疫组织化学SABC法及RT-PCR法检测海马及顶叶皮质CA1区神经元β-Catenin和DKK-1mRNA的表达。结果正常sham组,大鼠海马组织DKK-1 mRNA表达较少,β-Catenin阳性产物在细胞质内有所表达;I／R组,DKK-1 mRNA表达明显增多,β-Catenin在胞质内表达明显减少;bFGF组,大鼠海马组织DKK-1 mRNA表达较I／R组明显减少,而海马细胞质内β-Catenin表达较I／R组明显增加。结论bFGF抑制缺血神经元凋亡,参与DKK-1 mRNA和β-Catenin的调节,对缺血神经元有保护作用。     

川芎嗪对家兔肺缺血/再灌注损伤时Fas/FasL基因表达的影响

目的:探讨川芎嗪对肺缺血/再灌注损伤(PI/RI)时Fas/FasL基因表达的影响。方法:采用在体兔单肺原位缺血-再灌注模型。实验兔90只,随机均分为假手术对照组(Sham)、肺缺血/再灌注组(I/R)和肺缺血/再灌注加川芎嗪治疗组(LGT)。每组又分为再灌注1h3、h、5 h三个亚组,每个亚组10只,分别于再灌注1 h3、h、5 h三个时点取左肺组织,观察Fas/Fas配体(Fas/FasL)mRNA定位表达、凋亡指数(AI)、肺组织湿、干重比(W/D)、肺损伤组织学定量评价指标(IQA)及光镜、电镜下的组织形态学改变。结果:肺再灌注1 h、3 h、5 h,LGT组Fas/FasLmRNA在肺小动脉内(外)膜、肺小静脉内膜、肺泡上皮及肺支气管上皮弱阳性表达,与I/R组同一时点比较阳性表达明显减弱;AI、W/D和IQA值显著低于I/R组(P<0.01和P<0.05);肺组织形态学异常改变不同程度减轻。结论:川芎嗪可下调肺组织Fas/FasL mRNA的表达而减轻细胞凋亡,对PI/RI发挥积极的防护作用。     

Induction of heme oxygenase-1 protects mouse liver from apoptotic ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is the main cause of primary graft dysfunction of liver allografts. Cobalt-protoporphyrin (CoPP)–dependent induction of heme oxygenase (HO)-1 has been shown to protect the liver from I/R injury. This study analyzes the apoptotic mechanisms of HO-1-mediated cytoprotection in mouse liver exposed to I/R injury. HO-1 induction was achieved by the administration of CoPP (1.5 mg/kg body weight i.p.). Mice were studied in in vivo model of hepatic segmental (70 %) ischemia for 60 min and reperfusion injury. Mice were randomly allocated to four main experimental groups (n = 10 each): (1) A control group undergoing sham operation. (2) Similar to group 1 but with the administration of CoPP 72 h before the operation. (3) Mice undergoing in vivo hepatic I/R. (4) Similar to group 3 but with the administration of CoPP 72 h before ischemia induction. When compared with the I/R mice group, in the I/R+CoPP mice group, the increased hepatic expression of HO-1 was associated with a significant reduction in liver enzyme levels, fewer apoptotic hepatocytes cells were identified by morphological criteria and by immunohistochemistry for caspase-3, there was a decreased mean number of proliferating cells (positively stained for Ki67), and a reduced hepatic expression of: C/EBP homologous protein (an index of endoplasmic reticulum stress), the NF-κB’s regulated genes (CIAP2, MCP-1 and IL-6), and increased hepatic expression of IκBa (the inhibitory protein of NF-κB). HO-1 over-expression plays a pivotal role in reducing the hepatic apoptotic IR injury. HO-1 may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation.     

内源性一氧化碳减轻大鼠双侧后肢缺血再灌注所致的肺损伤

通过观察血红素氧化酶（HO）阻断剂－－锌原卟啉（ZnPP)对肺组织、肺泡间质多形核白细胞数目肺组织丙二醛含量和湿重干重之比的影响,并对肺组织HO活性和血内碳氧血红蛋白水平（COHb）进行检测,以探讨内源性HO／一氧化碳（CO）在肢体缺血再灌注（I／R）所致肺损伤中的作用。结果发现,大鼠双侧后肢I／R可导致急性肺损伤,同时使肺组织中HO活性和血内COHb水平显著升高;应用ZnPP预处理可使HO活性和COHb水平显著降低,但肺损伤却进一步加重。上述实验结果表明,肢体I／R致肺损伤时,肺组织中HO活性和内源性CO生成增多或减轻大鼠肢体I／R所致的肺损伤。     

脂多糖诱导大鼠主动脉血红素氧合酶-1表达及其对血管反应性的影响

目的：探讨血红素－HO－1－CO－cGMP道路对内毒素血症大鼠主动脉血管张力的影响及其分子机制。方法：用离体血管环张力测定技术,观察静脉注射脂多糖（LPS）6h,大鼠胸主动脉环（TARs)对苯肾上腺素（PE）累积收缩反应。分别用一氧化碳（CO）供体正缺血红素（He),血红素氧合酶－1（HO－1）抑制剂锌原卟啉（ZnPP-IX),鸟苷酸环化酶（sGC)抑制剂亚甲兰（MB）预卵育后,测定TARs对PE收缩反应的变化。分别测定主动脉中CO含量,HO－1活性,Western blot测定HO－1蛋白含量,RT－PCR检测HO－1 mRNA表达的改变。结果：LPS组TARs对PE累积收缩反应明显降低,ZnPP-IX可部分逆转低收缩反应,MB可完全逆转低收缩反应,而用He可加重低收缩反应状态;LPS组动脉组织中CO的含量上升,HO－1活性、蛋白表达量和mRNA表达均明显增加。结论：LPS可使主动脉HO－1基因表达上调,蛋白含量及酶活性明显增加,表明启动血红素－HO－1－CO－cGMP通路,是介导ES大鼠主动脉低收缩反应重要机制之一。     

Higenamine reduces apoptotic cell death by induction of heme oxygenase-1 in rat myocardial ischemia-reperfusion injury

Pharmacological modulation of heme oxygenase (HO) gene expression may have significant therapeutic potential in oxidant-induced disorders, such as ischemia reperfusion (I/R) injury. Higenamine is known to reduce ischemic damages by unknown mechanism(s). The protective effect of higenamine on myocardial I/R-induced injury was investigated. Ligation of rat left anterior descending coronary artery for 30 min under anesthesia was done and followed by 24 h reperfusion before sacrifice. I/R-induced myocardial damages were associated with mitochondria-dependent apoptosis as evidenced by the increase of cytochrome c release and caspase-3 activity. Administration of higenamine (bolus, i.p) 1 h prior to I/R-injury significantly decreased the release of cytochrome c, caspase-3 activity, and Bax expression but up-regulated the expression of Bcl-2, HO-1, and HO enzyme activity in the left ventricles, which were inhibited by ZnPP IX, an enzyme inhibitor of HO-1. In addition, DNA-strand break-, immunohistochemical-analysis, and TUNEL staining also supported the anti-apoptotic effect of higenamine in I/R-injury. Most importantly, administration of ZnPP IX inhibited the beneficial effect of higenamine. Taken together, it is concluded that HO-1 plays a core role for the protective action of higenamine in I/R-induced myocardial injury.     

ADSCs-exo attenuates hepatic ischemia–reperfusion injury after hepatectomy by inhibiting endoplasmic reticulum stress and inflammation

Hepatic ischemia–reperfusion (I/R) injury commonly occurs during liver surgery. Exosomes from adipose-derived stem cells (ADSCs-exo) induce a hepatoprotective effect during hepatic I/R injury. This study aimed to investigate the possible mechanism by which ADSCs-exo attenuates hepatic I/R injury in rats. Rats were randomly divided into four groups: Sham, I30R + PH, ADSCs, and ADSCs-exo groups. Liver tissues were collected immediately after 24 h of reperfusion for further analyses. The content of inflammatory factors in liver tissue was detected using enzyme-linked immunosorbent assay. The pathological changes in liver tissue were analyzed using HE staining. Transmission electron microscopy was used to visualize the ultrastructural changes of hepatocytes. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to detect the expression of endoplasmic reticulum stress (ERS)-related genes and proteins. Liver histomorphology and hepatocyte ultrastructure changes improved after ADSCs-exo treatment. Moreover, ADSCs-exo treatment significantly downregulated tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 levels while upregulating IL-10 levels. Western blot analysis suggested that the protein expressions of GRP78, p-PERK, p-eIF2α, p-IRE1α, XBP1s, ATF-6, ATF-4, CHOP, p-JNK, cleaved-Caspase-3, cleaved Caspase-9, and cleaved Caspase-12 significantly decreased after ADSCs-exo treatment. RT-qPCR results demonstrated that mRNA expression of GRP78, IRE1α, XBP1, ATF-6, ATF-4, CHOP, JNK, Caspase-3, Caspase-9, and Caspase-12 markedly reduced after ADSCs-exo treatment. In conclusion, ADSCs-exo protects against hepatic I/R injury after hepatectomy by inhibiting ERS and inflammation. Therefore, ADSCs-exo can be considered as a viable option for the treatment of hepatic I/R injury.