α7 烟碱型乙酰胆碱受体在电针减轻肢体缺血再灌注兔肺损伤中的作用

目的:观察α7烟碱型乙酰胆碱受体(α7nAChR)在电针减轻肢体缺血再灌注诱发兔肺损伤中的作用。方法:40只健康雄性新西兰大白兔,采用随机数字表法分为假手术组、模型组、电针组和α-银环蛇毒素(α-BGT)组(n=10)。采用夹闭股动脉3 h、再灌注4 h的方法制备兔肢体缺血再灌注模型;电针组、α-BGT组于模型制备前1～4 d(30 min/次,1次/d)及模型制备过程中电针足三里穴和肺俞穴(电流l～2 mA,频率2/15 Hz,疏密波);α-BGT组于模型制备前30min腹腔注射α7nAChR拮抗剂α-BGT(1μg/kg)。再灌注4 h时采集颈动脉血样,随后处死兔取肺组织,观察病理学结果并行肺损伤评分,计算肺湿/干重(W/D)比值。采用ELISA法检测肺组织肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)含量,Western blot法检测α7nAChR蛋白表达水平。结果:模型组、电针组和α-BGT组再灌注4 h时肺组织W/D比值分别为6.89±1.07、4.56±0.75、8.68±1.49,均明显高于假手术组(2.83±0.43,P0.05);肺损伤评分分别为7.52±1.05、4.16±0.68、9.23±1.57,均明显高于假手术组(1.19±0.22,P0.05);TNF-α分别为(13.3±2.3)pg/mg、(11.0±1.6)pg/mg、(14.1±3.0)pg/mg,均明显高于假手术组[(3.2±0.3) pg/mg,P0.05];IL-1β分别为(9.2±2.0)pg/mg、(7.4±1.7)pg/mg、(10.5±1.9)pg/mg,均明显高于假手术组[(2.1±0.5) pg/mg,P0.05];IL-6分别为(14.2±2.3)pg/mg、(11.7±2.0)pg/mg、(14.8±1.8)pg/mg,均明显高于假手术组[(4.2±0.7)pg/mg, P0.05];α7nAChR表达水平分别为(0.55±0.09)、(0.87±0.14)、(0.33±0.08),均明显高于假手术组(0.23±0.04,P0.05)。与模型组比较,电针组再灌注4h时肺组织W/D比值、肺损伤评分、肺组织TNF-α、IL-1β、IL-6含量均显著降低(P0.05),电针组α7nAChR表达均显著上调(P0.05)。与电针组比较,α-BGT组再灌注4h时肺组织W/D比值、肺损伤评分、肺组织TNF-α、IL-1β、IL-6含量升高(P0.05),α7nAChR表达下调(P0.05)。结论:α7nAChR表达上调,可能参与了电针减轻肢体缺血再灌注诱发兔肺损伤的过程。     

电针对肢体缺血再灌注诱发兔肺损伤的影响

目的:观察电针肺俞穴和足三里穴对肢体缺血再灌注诱发兔肺损伤的影响。方法:40只健康雄性新西兰大白兔,采用随机数字表法分为假手术组、模型组、电针组和非穴位电针组(每组n=10)。采用夹闭股动脉3 h、再灌注4 h的方法制备兔肢体缺血再灌注损伤模型。电针组于模型制备前1～4 d(30 min/次,1次/d)及模型制备过程中电针肺俞穴和足三里穴(疏密波,刺激电流l～2 mA,频率2/15 Hz,波宽0.2～0.6 ms,以兔出现轻微肌颤为宜);非穴位电针组以相同的参数电针刺激肺俞穴和足三里穴旁开0.5 cm非经非穴处。再灌注4 h时处死兔,取肺组织行肺损伤评分,计算肺湿/干重(W/D)比值;测定肺组织髓过氧化物酶(MPO)活性、Western blotting法检测肺组织核因子-κBp65 (NF-κBp65)及细胞间黏附分子-1(ICAM-1)蛋白表达水平。结果:模型组、电针组和非穴位电针组再灌注4 h时肺组织W/D比值分别为6.75±0.36、4.90±0.23、6.68±0.39,均明显高于假手术组(3.09±0.15, P0.05);上述三组肺损伤评分分别为8.12±1.05、4.38±0.64、8.23±0.98,均明显高于假手术组(0.87±0.29, P0.05);上述三组MPO活性分别为(5.18±0.45)U/g、(3.76±0.33) U/g、(5.24±0.39) U/g,均明显高于假手术组[(1.59±0.25) U/g,P0.05];与假手术组相比,模型组、电针组和非穴位电针组ICAM-1及NF-κBp65表达水平升高;与模型组比较,电针组再灌注4 h时肺组织W/D比值、肺损伤评分、MPO活性、ICAM-1及NF-κBp65表达水平降低(P0.05)。结论:电针肺俞穴和足三里穴可减轻肢体缺血再灌注诱发的兔肺损伤,其机制可能与抑制NF-κB激活,从而减少ICAM-1介导的中性粒细胞聚集有关。     

人参皂甙Rb1在核因子NF-E2相关因子2/血红素氧合酶-1通路减轻肠缺血再灌注致急性肺损伤中的分子机制

目的 探讨人参皂甙Rb1对肠缺血/再灌注致急性肺损伤的保护效应及核因子NF-E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)通路参与该效应的分子机制.方法 成年雄性C57BL/6J小鼠随机分为5组:假手术组(S组);肠缺血/再灌注组(I/R组);再灌注+Rb1组(I/R +Rb1组);全反式维甲酸(ATRA)+再灌注组(ATRA+ I/R组);ATRA+再灌注+Rb1组(ATRA+ I/R+Rb1组).采用肠缺血/再灌注模型,Western blot检测肺组织Nrf2、HO-1表达变化;酶联免疫吸附试验(ELISA)法检测肺组织肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、IL-10水平;检测超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;检测肺湿/干比及肺组织病理损伤评分.结果 与S组比较,其他4组Nrf2、HO-1蛋白表达,TNF-α、IL-6、MDA含量,肺组织湿/干重比及肺组织病理评分增高(P＜0.05);与1/R组比较,1/R+ Rb1组Nrf2,HO-1蛋白表达,TNF-α,IL-6,MDA含量,肺组织湿/干重比及肺组织病理评分降低(P＜0.05);与I/R+ Rb1组比较,ATRA+ I/R组,ATRA+ I/R+ Rb1组Nrf2、HO-1蛋白表达,NF-α、IL-6、MDA含量,肺组织湿/干重比及肺组织病理评分增高(P＜0.05).SOD活性、IL-10水平与上述变化相反.结论 肠缺血/再灌注可引起急性肺损伤,人参皂甙Rb1后处理能通过激活Nrf2/HO-1通路减轻肠缺血/再灌注所致肺损伤.     

PI3K/Akt/Nrf2信号通路在电针刺减轻兔内毒素休克诱发急性肺损伤中的作用

目的:评价磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/核因子E2相关因子2(Nrf2)信号通路在电针刺减轻兔内毒素休克诱发急性肺损伤中的作用。方法:健康清洁级雄性新西兰大白兔80只,随机分成8组(n=10):对照组(C组)、内毒素休克诱发急性肺损伤模型组(L组)、渥曼青霉素+模型组(WL组)、渥曼青霉素组(W组)、二甲基亚砜组(D组)、模型+电针刺组(EL)、模型+电针刺激非穴位组(SEL)、模型+电针刺+渥曼青霉素组(ELW)。EL组、ELW组于模型制备前1~4 d及制备过程中电针刺激兔双侧足三里和肺俞穴,SEL组用同样方法电针刺激足三里和肺俞穴旁开0.5 cm处。L组、WL组、EL组、SEL组和ELW组经耳缘静脉注射脂多糖(LPS)5 mg/kg,C组、W组和D组给予等容量生理盐水。WL组、W组和ELW组在模型制备前30 min静注渥曼青霉素0.6 mg/kg,D组静注0.08 mL/kg DMSO,C组、L组静注生理盐水0.08 mL/kg。注射LPS或生理盐水后6 h,采集动脉血,检测TNF-α和IL-10浓度,处死动物后取肺组织,进行病理学损伤评分,测定SOD活性及MDA含量及检测p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白的表达。结果:L组、WL组、EL组、SEL组和ELW组肺损伤评分分别为(6.8±0.9)、(8.3±1.1)、(4.5±0.6)、(6.5±0.7)、(5.9±0.9),血清TNF-α浓度分别为(3.14±0.23)ng/mL、(4.62±0.32)ng/mL、(2.03±0.18)ng/mL、(3.18±0.25)ng/mL、(3.96±0.28)ng/mL,以及肺组织MDA含量分别为(4.34±0.39)nmol/mg、(6.29±0.54)nmol/mg、(3.09±0.26)nmol/mg、(4.31±0.35)nmol/mg、(5.74±0.47)nmol/mg,均较C组升高,IL-10浓度分别为(5.37±0.47)pg/mL、(3.14±0.25)pg/mL、(6.98±0.54)pg/mL、(5.34±0.43)pg/mL、(5.19±0.39)pg/mL,以及SOD含量分别为(67.6±3.8)U/mg、(42.3±2.6)U/mg、(87.5±5.6)U/mg、(62.3±4.1)U/mg、(57.4±3.5)U/mg,较C组降低,肺组织p-Akt蛋白分别为(1.58±0.29)、(1.23±0.21)、(1.93±0.32)、(1.52±0.28)、(1.46±0.24),HO-1蛋白分别为(0.67±0.09)、(0.48±0.07)、(1.05±0.12)、(0.69±0.08)、(0.74±0.09),Nrf2核蛋白分别为(0.84±0.11)、(0.61±0.09)、(1.37±0.18)、(0.89±0.12)、(0.94±0.13),以及总蛋白分别为(2.14±0.43)、(1.82±0.32)、(3.25±0.58)、(2.18±0.45)、(2.46±0.49),表达水平较C组上调(P0.05),W组、D组上述各指标差异无统计学意义(P0.05);与L组比较,EL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量降低,而IL-10浓度及SOD活性升高,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平上调,WL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P0.05),而SEL组上述各指标差异无统计学意(P0.05);与EL组比较,ELW组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P0.05);与ELW组比较,WL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P0.05)。结论:PI3K/Akt/Nrf2信号通路介导电针刺激足三里和肺俞穴减轻兔内毒素休克诱发急性肺损伤的作用,机制可能与上调HO-1表达有关。     

电针刺对兔肢体缺血再灌注诱发肺损伤内质网应激的影响

目的:评价电针对肢体缺血再灌注诱发兔肺损伤时肺组织内质网应激的影响。方法:40 只健康雄性新西兰大白兔,采用随机数字表法分为4 组:假手术组、模型组、电针组和非穴位电针组,每组10 只。采用夹闭股动脉3 h、再灌注4 h的方法制备兔肢体缺血再灌注损伤模型。电针组于模型制备前4 d( 每天早8 点,30 min/ 次,1 次/d) 及模型制备过程中电针肺俞穴和足三里穴( 疏密波,刺激电流l~2 mA,频率2/15 Hz,波宽0.2~0.6 ms),以兔出现轻微肌颤为宜;非穴位电针组以相同的参数电针刺激肺俞穴和足三里穴旁开0.5 cm 非经非穴处。再灌注4 h 时处死兔,取肺组织行HE 染色和肺损伤评分,计算肺湿重/ 干重(W/D) 比值;采用TUNEL 法确定肺泡上皮细胞凋亡指数(AI) ;采用Western blotting 法检测肺组织葡萄糖调节蛋白78(GRP78)、CCAAT/ 增强子结合蛋白同源蛋白(CHOP) 及caspase-12 表达水平。结果:与假手术组比较,其余3 组的肺损伤评分、W/D 比值及肺泡上皮细胞AI 均升高,肺组织GRP78、CHOP 及caspase-12 表达均上调,差异有统计学意义(P <0.05) ;与模型组比较,电针组肺损伤评分、W/D 比值及肺泡上皮细胞AI 降低,肺组织GRP78、CHOP 及caspase-12 表达下调,差异有统计学意义(P <0.05)。结论:电针刺激肺俞穴和足三里穴减轻兔肢体缺血再灌注诱发肺损伤的机制可能与抑制内质网应激诱导、减轻肺泡上皮细胞凋亡有关。     

大鼠肺缺血期肢体缺血处理对肺缺血/再灌注损伤的保护作用

目的 观察大鼠肺缺血期双后肢缺血处理对肺缺血/再灌注损伤的影响.方法 将24只SD大鼠随机分为3组(n=8):缺血再灌注组(I/R组)、肺缺血期肢体缺血处理组(LIPER组)和假手术组(S组).I/R组开胸后左侧肺门阻断45 min,再灌注120 min.LIPER组左侧肺门阻断45 min,再灌注120 min,左侧肺门阻断5 min时阻断双后肢血供5 min,再灌注5 min,反复4次处理.S组开胸后旷置观察165 min.再灌注120 min时采动脉血样作血气分析;再灌注120 min后处死大鼠,取左肺组织,测定超氧化物歧化酶(SOD)、髓过氧化物酶(MPO)活性及丙二醛(MDA)含量;计算湿干比(W/D);肺组织行病理切片,苏木素-伊红(HE)染色,观察病理形态改变,并进行肺损伤评分.结果 I/R组和LIPER组血氧分压(PaO2)值分别为(58.5±3.1)和(71.0±3.5)mmHg(1 mmHg =0.133 kPa,P＜0.05)、W/D值分别为6.20±0.32和5.39 ±0.29(P ＜0.05)、SOD活性分别为(14.21±1.14)和(33.78 ±2.06) U/mg(P＜0.05)、MDA含量分别为(1.32±0.09)和(0.81±0.05) nmol/mg(P＜0.05)、MPO活性分别为(4.30 ±0.22)和(2.01 ±0.17) U/g(P＜0.05)、急性肺损伤评分值分别为(12.13±1.13)和(6.75±0.71)分(P＜0.05).结论 肺缺血期双后肢缺血处理具有减轻肺缺血/再灌注损伤的作用.     

二氮嗪对幼龄大鼠深低温脑缺血再灌注后氧自由基及细胞凋亡的影响

目的 探讨二氮嗪对幼龄大鼠深低温脑缺血再灌注后氧自由基和细胞凋亡的影响及相关的脑保护作用机制.方法 将54只3周龄健康sD大鼠随机分为假手术组、模型组和二氮嗪组,每组18只,建立深低温脑缺血再灌注模型.于术后24 h处死大鼠,检测脑组织超氧化物歧化酶(SOD)、丙二醛的含量,Western Blot法检测脑组织细胞质细胞色素C含量,免疫组化法检测脑组织细胞质Caspase-3含量.结果 模型组脑组织中SOD含量为(198±41)U/mg,低于假手术组的(321±36)U/rag(P<0.01);丙二醛含量为(212±21)nmol/mg,高于假手术组的(100±23)nmol/mg(P<0.01);细胞色素C蛋白表达(0.72±0.09)和Caspase-3蛋白表达(83±10)均高于假手术组(0.17±0.02和115±9)(P<0.01).二氮嗪组脑组织SOD含量为(264±34)U/rag,高于模型组(P<0.05);而丙二醛含量(174±19)nmol/mg、细胞色素C蛋白表达(0.41±0.05)和Caspase-3蛋白表达(99±11)均低于模型组(P<0.05).结论 二氮嗪对幼龄大鼠深低温脑缺血再灌注损伤具有脑保护作用,其机制与抑制氧自由基产生和细胞凋亡有关.     

远隔肢体缺血预处理对兔单肺缺血-再灌注损伤细胞凋亡的影响

目的 从细胞凋亡的角度探讨远隔肢体缺血预处理影响兔肺缺血-再灌注(I-R)损伤的可能机制.方法 18只日本大耳白兔随机均分为三组:缺血-再灌注组(I-R组)、肢体缺血预处理组(R组)、假手术组(S组).建立兔在体左肺缺血-再灌注(I-R)损伤模型.通过采用脱氧核苷酸末端转移酶介导的DNA原位末端缺口标记技术(TUNEL)检测再灌注3 h时凋亡指数(AI)的变化,予Westernblotting检测肺组织中Bcl-2、Bax蛋白的表达情况.结果 与S组比较,I-R组肺组织细胞凋亡指数和Bax蛋白显著增高(P＜0.01),而Bcl-2蛋白含量显著降低(P＜0.01),Bcl-2/Bax比值降低(P＜0.05).R组细胞凋亡指数和Bcl-2蛋白表达明显高于I-R组(P＜0.01),而Bax蛋白的含量明显低于I-R组(P＜0.05),Bcl-2/Bax比值增高(P＜0.05).结论 远隔肢体缺血预处理可上调肺组织Bcl-2蛋白,下调Bax蛋白表达,增加Bcl-2/Bax比值,抑制肺组织细胞凋亡,从而对肺缺血-再灌注损伤起到保护作用.     

缺血后处理对小鼠肠缺血再灌注致肾损伤时Nrf2蛋白表达的影响

目的 评价缺血后处理对小鼠肠缺血再灌注致肾损伤时核因子E2相关因子2(Nrf2)蛋白表达的影响.方法 健康雄性C57BL/6J小鼠36只,9～12周,采用随机数字表法,将其随机分为3组(n=12):假手术组(S组)、缺血再灌注组(I/R组)、缺血后处理+缺血再灌注组(IPO组).采用夹闭肠系膜上动脉根部45 min恢复灌注的方法制备小鼠肠缺血再灌注损伤模型,IPO组于缺血45 min时再灌注30s,缺血30s,重复3次后恢复灌注.于再灌注2h时采集颈动脉血样,然后处死小鼠,取肾组织,测定血清BUN、Cr和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)水平,检测肾组织Nrf2和HO-1蛋白表达、MDA含量、SOD活性、TNF-α、IL-6和IL-10的含量.显微镜下观察肾组织病理学结果,并行病理学损伤评分.结果 与S组比较,I/R组血清BUN、Cr和NAGL浓度升高,肾脏组织Nrf2及HO-1蛋白表达上调,MDA含量升高,SOD活性降低,肾脏组织病理学损伤评分升高(P＜0.05);与I/R组比较,IPO组血清BUN、Cr和NAGL浓度降低,肾脏组织Nrf2及HO-1蛋白表达上调,MDA含量降低,SOD活性升高,肾脏组织病理学损伤评分降低(P＜0.05).各组肾脏组织TNF-α、IL-6和IL-10含量比较差异无统计学意义(P＞0.05).结论 缺血后处理可减轻小鼠肠缺血再灌注致肾损伤,其机制可能与促进Nrf2蛋白表达,从而上调HO-1蛋白表达有关.     

大鼠肺缺血再灌注时Nrf2与铁死亡的关系

目的:评价大鼠肺缺血再灌注时核因子E2相关因子2(Nrf2)与铁死亡的关系。方法:健康雄性SD大鼠54只,体重220~250 g,采用随机数字表法分为3组( n＝18)：假手术组(Sham组)、肺缺血再灌注组(IR组)和肺缺血再灌注+Nrf2激动剂萝卜硫素组(IR+SFP组)。采用夹闭左肺门60 min再灌...     

核因子E2相关因子/血红素加氧合酶1通路在硫化氢减轻心搏骤停大鼠血脑屏障损伤中的作用

目的 探讨核因子E2相关因子(nuclear factor E2 related factor,Nrf2)/血红素加氧合酶1(heme oxygenase-1,HO-1)通路是否参与硫化氢(hydrogen sulfide,H2S)减轻心搏骤停大鼠血脑屏障(blood-brain barrier,BBB)损伤及参与该效应的可能机制. 方法 采用经食管电刺激法建立大鼠心搏骤停心肺复苏(cardiopulmonary resuscitation,CPR)模型,缺血时间4 min.140只成年雄性SD大鼠按随机数字表法分为5组:假手术组(Sham组,20只)、心搏骤停模型组(CPR组,30只)、硫氢化钠(sodium hydrogen sulfide,NaHS)组(30只)、NaHS+溶剂[二甲基亚砜(dimethyl sulfoxide,DMSO)]对照组(NaHS+DMSO组,30只)、NaHS+Nrf2抑制剂维甲酸(retinoic acid,RA)组(NaHS+RA组,30只).Sham组仅进行麻醉,经口气管插管,经股动静脉置管操作,其他4组均行电刺激诱发心搏骤停;NaHS组与NaHS+RA组于自主循环恢复即刻经股静脉注射NaHS(0.5 mg/kg),此后持续泵入NaHS(1.5 mg·kg-1,h-1)3 h;NaHS+RA组于实验前1周每天及复苏即刻腹腔注射RA 10 mg/kg;NaHS+DMSO组于实验前1周每天及复苏即刻腹腔给予等容量溶剂DMSO,CPR组于复苏即刻静脉给予等量生理盐水.复苏成功后连续观察7d,记录大鼠生存情况;于复苏后1、3、7d评价神经功能;于复苏后24 h检测脑含水量、BBB通透性,Western blot法检测Nrf2、HO-1、BBB紧密连接蛋白occludin表达变化,免疫组织化学染色检测基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达变化.结果 大鼠的生存率、神经功能评分、Nrf2和HO-1表达及occludin表达量比较,NaHS组均高于CPR组(P＜0.05),而NaHS+RA组低于NaHS组(P＜0.05);脑含水量和伊文思蓝(evans blue,EB)含量及MMP-9阳性细胞数比较,NaHS组显著低于CPR组(P＜0.05),而NaHS+RA组较NaHS组增高(P＜0.05).NaHS组与NaHS+DMSO组各项结果比较,差异无统计学意义(P＞0.05). 结论 H2S可减轻心搏骤停大鼠BBB损伤,可能与H2S激活Nrf2/HO-1通路、抑制MMP-9表达、减少Occludin蛋白丢失有关.     

左卡尼汀对肾缺血再灌注损伤的抗氧化作用及其机制

目的研究左卡尼汀对大鼠肾缺血再灌注损伤的抗氧化作用并探讨其机制。方法将大鼠随机分为3组：对照组（C组）,缺血再灌注组（IR组）,左卡尼汀组（LC组）。C组不予缺血再灌注处理,IR组及LC组建立肾脏IR模型。再灌注6h后检测各组血清肌酐（Cr）和尿素氮（BUN）水平;测定肾组织超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量;RT-PCR检测肾组织核因子E2相关因子2（Nrf2）、血红素氧化酶-1（HO-1）mRNA含量;Western-blot检测各组肾组织Nrf2及HO-1蛋白表达水平。结果 LC组血清Cr、BUN水平低于IR组[（74.17±12.80）μmol/L、（24.28±2.58）mmol/L vs.（112.83±17.45）μmol/L、（35.13±6.01）mmol/L],差异具有统计学意义（P〈0.01）。LC组肾组织SOD活性高于IR组[（39.55±6.61）kU/g vs.（28.05±4.37）kU/g],差异具有统计学意义（P〈0.01）;MDA显著降低于IR组[（4.15±0.69）μmol/g vs.（6.12±1.08）μmol/g],差异具有统计学意义（P〈0.01）。IR组Nrf2、HO-1mRNA及蛋白表达水平高于C组（P〈0.01）,低于LC组（P〈0.01）。结论左卡尼汀对肾脏缺血再灌注损伤具有明显保护作用,其机制可能为激活Keapl-Nrf2-ARE通路进而诱导HO-1的表达。     

参附注射液对大鼠肢体缺血再灌注后肝脏损伤的保护作用

目的观察参附注射液对大鼠肢体缺血再灌注损伤后肝功能、血红素加氧酶-1（HO-1）表达的影响,并对其保护机制做一初步探讨。方法 64只清洁级SD雄性大鼠,用随机数字表法随机分为4组,每组16只,分别为假手术组、缺血再灌注组、参附干预组、参附＋锌原卟啉Ⅸ（Znpp）干预组。假手术组：大鼠麻醉后仅分离不夹闭股动脉,分离血管前10 min以7.5 mL/kg腹腔注射生理盐水;缺血再灌注组：夹闭股动脉前10 min以7.5 mL/kg腹腔注射生理盐水,夹闭股动脉缺血3 h,再灌注4 h;参附干预组：夹闭股动脉前10 min以7.5 mL/kg腹腔注射参附注射液,夹闭股动脉缺血3 h,再灌注4 h;参附＋Znpp干预组：术前30 min腹腔注射Znpp 5 mg/kg,余同参附干预组。再灌注完毕后取材,取外周静脉血测血清谷丙转氨酶（GPT）、谷草转氨酶（GOT）含量;取肝组织测定肝组织中丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性;采用免疫组织化学法测定肝脏组织中HO-1蛋白表达;光镜下观察肝脏病理学改变。结果 1与假手术组比较,各肢体缺血再灌注造模组MDA含量均明显升高（P〈0.05）,SOD活性明显降低（除参附干预组外,P〈0.05）,GPT、GOT含量均明显升高（P〈0.05）,HO-1蛋白表达明显升高（P〈0.05）。2与缺血再灌注组比较,参附干预组MDA含量明显降低（P〈0.05）,SOD活性明显升高（P〈0.05）,血清GPT、GOT含量明显降低（P〈0.05）,肝组织中HO-1蛋白表达升高（P〈0.05）。3与参附干预组比较,参附＋Znpp干预组MDA含量明显升高（P〈0.05）,SOD活性明显降低（P〈0.05）,血清GPT、GOT含量明显升高（P〈0.05）,而肝组织HO-1蛋白表达差异无统计学意义（P〉0.05）。结论肢体缺血再灌注可造成肝脏功能损伤,给予参附注射液预处理可以减轻肝脏损害程度,这种保护作用可能与参附注射液预处理上调HO-1蛋白在肝组织中的表达、抑制氧自由基生成?     

Pulmonary expression of inducible heme-oxygenase after ischemia/reperfusion of the lower extremities in rats

BACKGROUND: Expression of inducible heme-oxygenase (HO-1) has been shown to be increased in various inflammatory disorders, which may confer a protective role. The aim of our study was to assess pulmonary expression of HO-1 after ischemia/reperfusion (I/R) of the lower limbs in rats. MATERIALS AND METHODS: We compared three groups of rats (n = 5/group): one Sham group, and two I/R groups (aorta cross-clamped for 2 h followed by 2 h of reperfusion), one of which pre-treated with Zn-protoporphyrin (Zn-PP), a competitive inhibitor of HO (50 micromol/kg, i.p.). At the end of experiment, lungs were harvested for determination of HO activity and HO-1 expression by Western blot and immunohistochemistry. Lung injury was assessed by bronchoalveolar lavage, histological study, and determination of the lung Evans Blue dye content, an index of microvascular permeability. RESULTS: I/R of the lower limbs was responsible for acute lung injury (ALI), characterized by neutrophilic infiltration (87 +/- 20 x 10(3) neutrophils/mm(3), Sham group versus 191 +/- 38 x 10(3) neutrophils/mm(3), I/R group; P < 0.002) and an increase in lung Evans blue dye content: (74 +/- 6 microg/g, Sham group versus 122 +/- 48 microg/g, I/R group; P < 0.05). Pre-treatment with Zn-PP further increases the Evans Blue content (122 +/- 48 microg/g, I/R group versus 179 +/- 23 microg/g Zn-PP group P < 0.05) and the neutrophilic infiltration. Pulmonary heme-oxygenase activity, and HO-1 content were increased after I/R. (10.5 +/- 12 pmol bilirubin/mg protein/h, Sham group versus 101.2 +/- 66 pmol bilirubin/mg protein/h, I/R group; P < 0.02). Immunohistochemistry revealed that the expression of HO-1 was mainly localized to inflammatory cells. CONCLUSIONS: ALI following I/R of the lower limbs in rats is associated with an increase of pulmonary expression of HO-1, inhibition of this expression increase the severity of ALI.     

Protective effect of ischemic postconditioning on lung ischemia-reperfusion injury in rats and the role of heme oxygenase-1

To investigate the effect of ischemic postconditioning (IPO) on acute lung ischemia-reperfusion (I/R) injury and the protein expression of haeme oxygenase-1 (H0-1), a cytoprotective defense against oxidative injury. Methods: After being anesthetized with chloralhy-drate, forty-eight healthy SD rats were randomly divided into 6 groups (8 in each): sham operation group (S group); I/R group: left lung hilum was clamped for 40 minutes followed by 105 minutes of reperfusion; IPO group: left lung hilum was clamped for40 minutes and postconditioned by 3 cycles of 30 seconds of reperfusion and 30 seconds of reocclusion; Hemin (HM)+ I/R group: hemin, an inducer of HO- 1 was injected intraperitoneally at 40 μmol.kg-1·day-1 for two con-secutive days prior to 40 minutes clamping of left lung hilum; ZnPPIX+IPO group: zinc protoporphyrin Ⅸ, an inhibitor of HO-1 was injected intraperitoneally at 20 mg·kg-1 24 hours prior to 40 minutes clamping of left lung hilum; and HM+S group: HM was administered as in the HM+I/R group without inducing lung I/R. Arterial partial pressure of oxygen (PaO2) and malondialdehyde (MDA) content in serum were assessed. The left lung was removed for determination of wet/dry lung weight ratio and expression of HO-1 protein by immuno-his-tochemical technique and for light microscopic examination. Results: The PaO2 was significantly lower in all the experimental groups compared with sham group (90 mm Hg ±11 mm Hg). However, the values ofPaO2 in IPO (81 mm Hg ±7 mm Hg) and HM+I/R (80 mm Hg±9 mm Hg) were higher than that in I/R (63 mm Hg±9 mm Hg) and ZnPPIX+IPO (65 mm Hg±8 mm Hg) groups (P<0.01). The protein expression of HO-1 in lung tissue was significantly increased in I/R group compared with S group (P<0.01). While the HO-1 protein expression was higher in IPO and HM+I/R groups as compared with I/R group (P<0.05, P<0.01). The lung wet/ dry (W/D) weight ratio and MDA content in serum were significantly increased in I/R group as compared with S or HM+S groups (P<0.01), accompanied by severe lung tissue histological damage, which was attenuated either by IPO or by HM pretreatment (P<0.01, IPO or HM+I/R vs. I/R). The protective effect of IPO was abolished by ZnPPIX. Condusion: Ischemic postconditioning can attenuate the lung ischemia-reperfusion injury through upregulating the protein expression of HO-1 that leads to reduced post-ischemic oxidative damage.     

Transient limb ischemia induces remote preconditioning in liver among rats: the protective role of heme oxygenase-1

BACKGROUND: We have reported the protective role of heme oxygenase-1 (HO-1) in the mechanism of hypoxic preconditioning. We wish to investigate the role of HO-1 in remote preconditioning (RP) against hepatic ischemia/reperfusion (I/R) injury in rats. METHODS: The remote preconditioning was produced by four cycles of 10-min ischemia-reperfusion of the hind limb of rats. Partial hepatic ischemia was produced in the left lobes for 45 min followed by 240 min of reperfusion. Zinc-protoporphyrin IX (ZnPP), a specific inhibitor of HO enzymatic activity, was intra-peritoneally injected 1 hr before the ischemia-reperfusion injury in separate groups of RP rats. Serum alanine transaminase (ALT) levels, expression of hepatic HO-1 protein and mRNA, immunohistochemical staining and HO enzymatic activity were measured. RESULTS: HO-1 was induced in the livers of rats 4 hr after the RP stimuli, and the overexpression persisted for 24 hr. Immunohistochemical staining demonstrated induction of HO-1 in the hepatocytes. The peripheral lymphocytes did not express HO-1 after RP. RP diminished the elevation of serum ALT levels 4 hr after I/R injury (283.7+/-167.4 U L) when compared with controls (1297.7+/-729.3 U L) and RP+ ZnPP pretreated groups (1429.9+/-750.9 U L). The heme oxygenase activity in treated rats also correlated these results (286.8+/-34.3 pmol mg protein hr for the RP group, 156.3+/-27.5 pmol mg protein hr for the RP+ ZnPP pretreated group, and 170.6+/-19.4 pmol mg protein hr for the control group, 144.8+/-7.8 pmol mg protein hr for the control+ ZnPP pretreated group). CONCLUSION: Our results indicated that the induction of HO-1 in remote preconditioning played a protective role against hepatic I/R injury.     

血红素氧合酶-1介导NAD表达在大鼠内毒素急性肺损伤中的作用

目的探讨血红素氧合酶-1(HO-1)介导烟酰胺腺嘌呤二核苷酸(NAD)表达在大鼠内毒素急性肺损伤中的作用。方法清洁级健康雄性SD大鼠40只,体重200~220 g,8周龄,采用随机数字表法分为四组:对照组(C组)、内毒素急性肺损伤组(L组)、HO-1激动剂Hemin+内毒素急性肺损伤组(H组)和HO-1阻断剂ZnPP-IX+内毒素急性肺损伤组(Z组),每组10只。L组、H组和Z组采用尾静脉缓慢注射脂多糖(LPS)5mg/kg溶于生理盐水1 ml,制备大鼠内毒素急性肺损伤模型。H组于模型前1 h腹腔注射Hemin 50mg/kg,用0.1 mol/L NaOH溶液稀释至1 ml。Z组于模型前1 h腹腔注射ZnPP-IX 10μmol/kg,50 mmol/L NaHCO3溶液稀释至1 ml。给予LPS后6 h处死大鼠,采集动脉血行血气分析,留取肺组织,观察病理学结果并行肺损伤评分,计算肺组织湿/干重比(W/D),采用NAD/NADH定量检测试剂盒测定NAD含量,Western blot法检测HO-1蛋白含量。结果与C组比较,L组、H组、Z组pH和PaO2明显降低,PaCO2、肺损伤评分、肺组织NAD含量、HO-1蛋白含量明显升高(P<0.05),肺W/D比值明显增大(P<0.05),肺组织病理损伤明显。与L组比较,H组pH和PaO2明显升高,PaCO2、肺损伤评分明显降低(P<0.05),肺W/D比值明显减小(P<0.05),肺组织NAD含量、HO-1蛋白含量明显升高(P<0.05),肺组织病理损伤减轻;Z组pH和PaO2明显降低,PaCO2、肺损伤评分明显升高(P<0.05),肺W/D比值明显增大(P<0.05),肺组织NAD含量、HO-1蛋白含量明显降低(P<0.05),肺组织病理损伤加重。与H组比较,Z组pH、PaO2明显降低,PaCO2明显升高(P<0.05),Z组肺损伤评分明显升高(P<0.05),肺W/D比值明显增大(P<0.05),肺组织NAD含量明显降低(P<0.05),肺组织病理损伤明显。结论内毒素肺损大鼠通过上调HO-1表达水平而增加肺组织NAD含量,降低了肺组织炎症反应,从而减轻大鼠内毒素急性肺损伤。     

Activation of Nrf2 Pathway by Dimethyl Fumarate Attenuates Renal Ischemia-Reperfusion Injury

BackgroundDimethyl fumarate (DMF) is a novel antioxidant that selectively reduces hydroxyl radicals. This study aimed to investigate the potential role of DMF in the pathogenesis of renal ischemia-reperfusion injury (IRI) and the mechanisms involved.MethodsC57BL/6 wild-type mice were treated with DMF or a vehicle. Subsequently, renal IRI was induced in mice by a model of right kidney nephrectomy and left renal ischemia for 30 minutes followed by reperfusion for 24 hours. Sham operation and phosphate-buffered saline were used as controls. Serum and renal tissues were collected at 24 hours after IRI to evaluate the influence of DMF on the recovery of renal function after IRI. Blood urea nitrogen and serum creatinine levels were measured. Kidney cell apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling-positive staining. Interleukin 6 and tumor necrosis factor α cytokines in the kidney tissues were measured. Indicators of oxidative stress in the kidneys were detected. Finally, Nrf2-deficient mice were used to determine the protective role of the nuclear factor erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) signaling pathways induced by DMF using western blot assay.ResultsDMF significantly attenuated renal dysfunction in mice and showed reductions in the severity of renal tubular injury, cell necrosis, and apoptosis. Moreover, DMF significantly reduced the amount of key inflammatory mediators. Additionally, DMF attenuated the malondialdehyde levels 24 hours after IRI but upregulated the superoxide dismutase activities. Western blot assay showed that DMF significantly increased the protein levels of Nrf2, HO-1, and NQO-1. Importantly, these DMF-mediated beneficial effects were not observed in Nrf2-deficient mice.ConclusionsDMF attenuates renal IRI by reducing inflammation and upregulating the antioxidant capacity, which may be through Nrf2/HO-1and NQO1 signaling pathway.     

Protective effect of hemoglobin—induced heme oxygenase—1 on injured lungs caused by limb ischemia—reperfusion in rats

OBJECTIVE: To determine the role of hemoglobin (HB) induced heme oxygenase-1 (HO-1) in injured lungs caused by limb ischemia-reperfusion (I/R) in rats. METHODS: A rat model of ischemia in the hind limbs was made by clamping the infrarenal aorta with a microvascular clip, and lung injury occurred after reperfusion. To induce the expression of HO-1 in the lungs, Hb was administrated intraperitoneally at 16 hours before reperfusion. Northern blotting and Western blotting were used to detect the expression of HO-1 in the lungs, and the carboxyhemoglobin (COHb) level in arterial blood was assayed. The effect of hemoglobin (Hb) on the injured lungs after limb I/R was determined by measuring the changes of lung histology, polymorphonuclear (PMN) count, malondialdehyde (MDA) content and wet-to-dry weight ratio (W/D). Zinc protoporphyrin (ZnPP), an inhibitor of HO, was used to determine whether HO-1 was induced by Hb after lung injury. RESULTS: Hb led to a significant increase in HO-1 mRNA and protein expression in the lungs, accompanied by the increase of COHb level in arterial blood. Compared with the sham controls, the lung PMN count, MDA content and W/D significantly increased at 4 hours after limb I/R, which reversed by the pretreatment with Hb at 16 hours before reperfusion. ZnPP blocked this protective role of Hb in the injured lungs. CONCLUSIONS: Hb can induce the lung HO-1 expression, which plays an important role in the defense against I/R induced lung injury in rats.