Exaggerated Liver Injury Induced by Renal Ischemia Reperfusion in Diabetes: Effect of Exenatide

Background/Aim:

This study was designed to investigate the possible effect of exenatide (Glucagon like Peptide-1 receptor agonist) on liver injury (distant organ) induced by renal ischemia reperfusion (IR) in diabetic rats.

Materials and Methods:

In vivo renal IR was performed in both type 2 diabetic and normal rats. Each protocol comprised ischemia for 30 minutes followed by reperfusion for 24 hours and a treatment period of 14 days before induction of ischemia.

Results:

Lipid peroxidation, xanthine oxidase activity, myeloperoxidase activity and nitric oxide level in liver tissue were significantly increased (P < 0.01, P < 0.001, P < 0.001, P < 0.05, respectively), after IR in diabetic rats compared to normal rats. Antioxidant enzymes like glutathione, superoxide dismutase, catalase and glutathione peroxidase were significantly reduced (P < 0.05, P < 0.05, P < 0.01, P < 0.05, respectively), after IR in diabetic rats compared to normal rats. Exenatide treatment significantly normalized (P < 0.01), these biochemical parameters in treated rats compared to diabetic IR rats. Serum creatinine phosphokinase activity and liver function enzymes were also significantly normalized (P < 0.001, P < 0.001, respectively), after administration of exenatide.

Conclusion:

Exenatide exerted protective effect on exaggerated remote organ (liver) injury induced by renal IR in diabetes.     

灯盏细辛对大鼠心肌缺血再灌注心律失常和细胞凋亡的影响

目的观察灯盏细辛(breviscapine)对大鼠缺血/再灌注(I/R)时室性心律失常和心肌细胞凋亡的影响,并与缬沙坦(val-sartan)比较其作用效果。方法40只大鼠随机分为假手术组(Sham组)、I/R组、灯盏细辛组(Bre组)和缬沙坦组(Val组),以穿线结扎左冠状动脉前降支制备大鼠心肌I/R模型,观察各组室性心动过速(VT)、血清肌酸激酶同工酶(CK-MB)、心肌组织总超氧化物歧化酶(TSOD)及丙二醛(MDA)变化。流式细胞凋亡法检测心肌凋亡细胞(AI)及免疫组化法检测心肌P53和细胞色素C(Cyt-C)蛋白表达。结果I/R组VT和CK-MB、MDA均高于Sham组,而TSOD低于Sham组;与I/R组比较,Bre组、Val组VT和、CK-MB、MDA均有降低,而TSOD活性增加。Bre组和Val组AI和P53、Cyt-C蛋白表达水平显著低于I/R组(P<0.01)。结论灯盏细辛可降低VT,下调P53、Cyt-C蛋白表达,抑制细胞凋亡,与缬沙坦有相近的保护效果。     

碧血胶囊对异丙肾上腺素诱导大鼠心肌缺血的保护作用

目的 研究碧血胶囊对异丙肾上腺素(ISO)诱导大鼠心肌缺血的保护作用.方法 应用异丙肾上腺素复制心肌缺血损伤的动物模型,观察组织形态学改变,测定血清丙二醛(MDA)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、肌酸激酶(CK)的活性,以及心电图变化.结果 碧血胶囊中高剂量组显著增高了心重与体重的比值,明显降低了心肌缺血大鼠的MDA、LDH、CK水平,增加SOD水平,且明显改善了心肌组织病变程度,降低了异丙肾上腺素所致心肌缺血大鼠的心电图T波升高,与模型组比较有统计学意义(P<0.05).结论 碧血胶囊对异丙肾上腺素诱导的大鼠心肌缺血具有保护作用.     

参芎注射液对大鼠脑缺血再灌注损伤的保护作用

目的观察参芎注射液对脑缺血再灌注大鼠行为学、脑梗死体积和组织病理学的影响,探讨其脑保护作用及机制。方法33只雄性SD大鼠随机分为4组假手术组、对照组、川芎嗪组和参芎组。采用线栓法制作大脑中动脉缺血1h再灌注模型,用Longa5分制评分和氯化三苯四氮唑(TTC)染色法评价行为学和脑梗死体积,再灌注24h时电镜观察缺血边缘区超微结构改变。结果各组在再灌注6h和24h时,Longa评分无显著差异,但参芎组再灌注24h时的Longa评分较再灌注6h时显著改善(P<0·05);川芎嗪组和参芎组的梗死体积较对照组显著缩小(P<0·05),川芎嗪组和参芎组两组比较无显著差异;川芎嗪组、参芎组细胞的损伤程度较对照组显著减轻(P<0·05);在电镜下,参芎组缺血边缘区的神经细胞超微结构改变轻于对照组,优于川芎嗪组。结论参芎注射液对脑缺血再灌注损伤有一定的保护作用,且优于单用川芎嗪。     

菟丝子黄酮对缺血再灌注大鼠心肌细胞凋亡的影响

目的观察菟丝子黄酮对缺血再灌注大鼠心肌细胞凋亡及相关基因表达的影响。方法结扎大鼠冠状动脉左前降支30 min,再灌注2 h建立心肌缺血再灌注模型。将50只SD大鼠随机分为假手术组、缺血再灌注组及菟丝子黄酮低、高剂量组和消心痛组。再灌注结束后检测血清肌酸激酶和乳酸脱氢酶,DNA末端标记法计算凋亡指数,免疫组织化学法和Western Blotting法检测心肌Bcl-2和Bax蛋白表达,计算Bcl-2/Bax值。结果与假手术组比较,缺血再灌注组心肌酶肌酸激酶、乳酸脱氢酶和凋亡指数显著增高(P<0.01),Bcl-2和Bax蛋白表达增强(P<0.01);与缺血再灌注组比较,菟丝子黄酮低、高剂量组及消心痛组能降低血清肌酸激酶、乳酸脱氢酶含量和凋亡指数(P<0.01),增加Bcl-2而减少Bax的表达(P<0.01)。结论菟丝子黄酮能够抑制缺血再灌注损伤大鼠的心肌细胞凋亡,与消心痛效果相近。     

The Protective Effect of Heat Stress Against Reperfusion Arrhythmias in the Rat

Heat stress provides protection against mechanical dysfunction and myocardial necrosis after prolonged ischemia. We have investigated whether this protection extends to reperfusion arrhythmias occurring after a short (non-lethal) ischemic insult. Anaesthetized open chest rats were subjected to a 5-min occlusion of the left coronary artery. The incidence and duration of reperfusion arrhythmias and the duration of sinus rhythm were assessed in the first 5 min of reperfusion. Prior heat stress led to a reduction in the incidence (100-63%, P 0.05) and duration (66.2 ± 15.8 to 9.4 ± 2.9s, P 0.05) of ventricular tachycardia and a non-significant reduction in the incidence (76-50%) and duration (74.3 ± 23.4 to 42.9 ± 24.4s, P = 0.09) of ventricular fibrillation. This resulted in a significant increase in the duration of sinus rhythm (142.1 ± 27.6 to 216.7 ± 24.8s, P 0.05) and reduction in arrhythmia score (P 0.05) in heat stressed rats compared with controls. This protection against reperfusion arrhythmias was associated with a two-fold increase in endogenous catalase activity and expression of the inducible heat stress protein HSP 70. Inhibition of catalase with pre-administered 3-amino triazole resulted in a paradoxical protection in both sham and heat stress hearts. We conclude that heat stress leads to protection against reperfusion arrhythmias; however, we have been unable to resolve whether the changes in catalase activity of HSP expression are the mediators of the demonstrated cardioprotection.     

芪桂益脉灵预处理对缺血再灌注大鼠心肌细胞凋亡基因Fas及Bcl-2表达的影响

目的观察芪桂益脉灵(QGYML)对急性心肌梗死大鼠心肌细胞凋亡及凋亡相关基因表达的影响,探讨其预适应样心肌保护作用。方法取Wistar大鼠56只,随机分为7组,假手术组(8只)大鼠开胸暴露左冠状动脉,但不作结扎,空白对照组(8只)不做处理,其余40只大鼠结扎冠状动脉左前降支造成急性心肌梗死(AMI)模型,造模成功后,分别给QGYML(大、小剂量,即1g/kg、2g/kg和0、6g/kg)、异山梨酯(消心痛)和生理盐水,连续灌服7d。采用免疫组织化学法测定Bcl-2和Fas的表达。结果消心痛组和QGYML大剂量组、小剂量组Bcl-2基因表达均上调,Fas蛋白表达均下调,与缺血再灌注组比较均有统计学意义。结论QGYML能明显抑制AMI后大鼠心肌细胞凋亡,下调Fas蛋白表达,上调Bcl-2蛋白表达,具有抗心肌缺血损伤的作用。     

依达拉奉药物后处理与缺血后处理对急性缺血再灌注心肌保护作用的对比实验研究

目的:探讨新型自由基清除剂依达拉奉的药物后处理是否对急性心肌缺血再灌注损伤有保护作用,与缺血后处理对比,并分析其可能机制.方法:建立大鼠急性心肌缺血再灌注模型.40只大鼠随机分为5组(每组8只):①假手术组;②缺血再灌注组;③缺血后处理组;④依达拉奉主动脉根部注射组(依达拉奉1组);⑤依达拉奉股静脉注射组(依达拉奉2组).进行心电及血流动力学监测,测定血清肌酸激酶MB同工酶(CK-MB)、丙二醛(MDA)活性及超氧化物歧化酶(SOD)活力,采用伊文思蓝-TYC染色确定梗死及缺血的心肌范围.采用Western blot印迹检测心肌组织中Bax和Be1-2蛋白的表达.结果:与缺血再灌注组相比,缺血后处理组、依达拉奉1组和依达拉奉2组的心肌梗死面积显著减少,CK-MB、MDA的水平显著降低,SOD活力增加(P＜0.05～0.01),差异均有统计学意义.上述指标缺血后处理组、依达拉奉1组和依达拉奉2组组间比较差异无统计学意义(P＞0.05).缺血后处理组、依达拉奉1组心肌组织Be1-2蛋白表达分别是缺血再灌注组的1.8倍和1.7倍,Bax蛋白的表达分别是缺血再灌注组的0.52倍和0.54倍(P均＜0.05),差异均有统计学意义.结论:对于急性缺血的心肌,在再灌注前注射依达拉奉进行药物后处理可减轻心肌缺血再灌注损伤,保护强度与机械性缺血后处理接近,有效清除氧自由基、提高机体抗氧化应激的能力以及降低凋亡指数(Bax/Bc1-2)是二者的共同机制;主动脉根部一冠状动脉注射途径给药的保护效果不差于静脉注射途径,可能是一种临床上适宜的药物后处理方式.     

脑心通对大鼠脑缺血再灌注损伤后血管内皮生长因子表达的保护作用

目的探讨脑心通对大鼠脑缺血再灌注损伤后血管内皮生长因子（VEGF）表达的影响。方法将120只大鼠随机分为大鼠脑缺血6h开始用药组（A组），脑缺血12h开始用药组（B组），正常脑缺血再灌注损伤组（C组）及假手术对照组（D组），每组30例。每组大鼠在脑缺血6h再灌注12h、24h、48h、72h及7d时进行神经功能评分，然后采用免疫组织化学和原住杂交的方法观察大鼠脑缺血再灌注不同时间点VEGF的表达。结果脑缺血6h时，各组间神经功能评分无统计学意义，脑缺血再灌注不同时间点对各组进行神经功能评分发现，A组、B组大鼠神经功能评分明显低于C组；在缺血再灌注不同时间点免疫组织化学和原位杂交结果为，A组大鼠VEGF的表达明显高于B组和C组。结论脑心通促进了VEGF的表达，其通过促进VEGF的表达参与了脑缺血再灌注损伤的保护机制。     

血管肽酶抑制剂对心力衰竭大鼠心肌细胞凋亡和心室重构的影响

目的研究心肌细胞凋亡和心室重构在心力衰竭发展中的变化及血管内肽酶抑制剂的干预作用,探讨血管内肽酶抑制剂治疗心力衰竭的机制。方法选用雄性SD大鼠,随机分为假手术组,心肌梗死组(MI组)和心肌梗死后Omapatrilat(OMA)干预组(MI OMA组)。经冠脉前降支结扎术建立心肌梗死模型,术后24 h MI OMA组大鼠给予Omapatrilat 40 mg/kg.d,饮水给药,假手术组和MI组大鼠饲普通饮水。术后4、6周检测大鼠非心肌梗死区心肌细胞凋亡指数;Western蛋白印迹检测心肌细胞凋亡加速基因Bax和凋亡抑制基因Bcl-2的蛋白表达水平;VG染色测胶原容积分数(CVF);压力传感器记录左室血流动力学改变。结果(1)术后非心肌梗死区心肌细胞凋亡指数,MI组及MI OMA组均显著高于假手术组(4周,3.8%±1.0%,2.6%±1.0%vs 0.1%±0.1%,P<0.01;6周,4.1%±1.3%,2.5%±0.9%vs 0±0,P<0.01),但MI OMA组显著低于MI组(4周,P<0.05;6周,P<0.01);(2)与假手术组相比,MI组和MI OMA组术后的Bax蛋白表达量均显著增高(4周,321.3%±17.5%,174.6%±13.9%vs 100.0%±8.3%,P<0.01;6周,362.3%±21.0%,193.9%±19.6%vs 104.7%±10.6%,P<0.01),但MI OMA组显著低于MI组(P<0.01);MI组和MI OMA组的Bcl-2蛋白表达量均低于假手术组(4周,79.3%±7.9%,86.6%±4.9%vs 100.0%±5.9%,P<0.01;6周,76.6%±9.3%,84.7%±3.4%vs 97.1%±6.0%,P<0.01),而MI OMA组高于MI组(P<0.05);(3)MI OMA组非梗死区CVF明显低于MI组(4周,10.5%±0.7%vs 14.7%±1.18%,P<0.01;6周,11.9%±1.4%vs 15.7%±1.1%,P<0.01),但仍显著高于假手术组(4周,10.5%±0.7%vs 3.9%±1.1%,P<0.01;6周,11.9%±1.4%vs 3.9%±1.2%,P<0.01);(4)MI组和MI OMA组大鼠4周和6周的心功能明显降低(与假手术组比,P<0.05),而MI OMA组大鼠的心功能显著改善(与MI组比,P<0.01)。结论血管肽酶抑制剂可通过调节凋亡相关基因Bax和Bcl-2的蛋白表达,减少心肌细胞凋亡,抑制心肌间质纤维化,从而减轻慢性心力衰竭阶段的心室重构,进而改善心功能。     

Apoptosis of human cholangiocarcinoma cells induced by ESC-3 from Crocodylus siamensis bile

AIM: To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells.METHODS: ESC-3 was isolated from crocodile bile by Sephadex LH-20 and RP-18 reversed-phase column. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was conducted to determine the effects of ESC-3 on the proliferation of human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1). Giemsa staining, Hoechst 33258 and acridine orange/ethidium bromide staining showed the morphological changes of Mz-ChA-1 cells exposed to ESC-3 at different concentrations. Flow cytometry with regular propidium iodide (PI) staining was performed to analyze the cell cycle distribution of Mz-ChA-1 cells and to assess apoptosis by annexin v-fluorescein isothiocyanate (V-FITC)/PI staining. Rh123 staining was used to detect the alteration of mitochondrial membrane potential (ΔΨm). The protein levels of Bax, Bcl-2, Cdk2, cytochrome c and caspase-3 were further confirmed by Western blotting.RESULTS: ESC-3 significantly inhibited the growth of three human cholangiocarcinoma cell lines and arrested Mz-ChA-1 cell cycle at G0/G1 phase. Mz-ChA-1 cells showed typical apoptotic morphological changes after treated with ESC-3 (10 μg/mL) for 48 h. Cell death assay indicated that Mz-ChA-1 cells underwent apoptosis in a dose-dependent manner induced by ESC-3. In addition, ESC-3 treatment could downregulate the protein level of Bcl-2 and upregulate the Bax, leading to the increase in the ratio of Bax to Bcl-2 in Mz-ChA-1 cells. Meanwhile, cytochrome c was released from the mitochondria into the cytosol, which subsequently initiated the activation of caspase-3. All these events were associated with the collapse of the mitochondrial membrane potential.CONCLUSION: ESC-3, the active ingredient of crocodile bile, induced apoptosis in Mz-ChA-1 cells through the mitochondria-dependent pathway and may be a potential chemotherapeutic drug for the treatment of cholangiocarcinoma.     

远端缺血后处理对兔心脏缺血/再灌注损伤的保护作用及其机制研究

目的:观察远端肢体缺血后处理对兔心脏缺血/再灌注损伤的作用,并探讨其可能机制。方法:36只健康新西兰大白兔随机分为6组(每组6只):假手术组、缺血对照组、缺血后处理组(MPostC组)、远端肢体缺血后处理组(RPostC组)、缺血后处理+5-羟葵酸组(MPostC+5-HD组)、远端肢体缺血后处理+5-羟葵酸组(RPostC+5-HD)。结扎左冠状动脉左室支45 min,再灌注120 min造模,结扎双侧髂外动脉5 min骨骼肌短暂缺血。于缺血前、后及再灌注1 h、2 h观察心功能指标和肌酸激酶(CK)、乳酸脱氢酶(LDH)活力及心肌梗死范围。结果(:1)再灌注1 h、2 h后,MPostC组、RPostC组心功能指标较缺血对照组明显改善(P0.05)。MPostC+5-HD组、RPostC+5-HD组分别较MPostC组、RPostC组改善不明显(P0.05)(;2)再灌注2 h时,MPostC组、RPostC组的CK、LDH活力均显著低于缺血对照组(P均0.05),MPostC+5-HD组、RPostC+5-HD组的CK、LDH活力分别较MPostC组、RPostC组明显增高(P均0.05)(;3)缺血对照组的缺血范围[缺血区重量/左心室重量(AAR/LVg),缺血区面积/左心室面积(AAR/LVs)]与其他5组比较差异均无统计学意义(P0.05);MPostC组、RPostC组梗死范围[梗死区重量/缺血区重量(AN/AARg)、梗死区面积/缺血区面积(AN/AARs)]均较缺血对照组明显降低(P均0.05),MPostC+5-HD组、RPostC+5-HD组分别与MPostC组、RPostC组比,梗死范围均增加(P均0.05)。MPostC+5-HD组、RPostC+5-HD组、缺血对照组的梗死范围组间差异无统计学意义(P0.05)。结论:经典的缺血后处理和远端肢体缺血后处理对心脏缺血/再灌注损伤均有明显的保护作用,其共同的机制可能为线粒体ATP敏感钾通道(MitoKATP)的激活。     

川芎嗪联合左旋精氨酸对缺血—再灌注损伤兔心肌线粒体功能的影响

目的探讨川芎嗪联用左旋精氨酸对心肌缺血-再灌注损伤时心肌细胞线粒体功能的影响。方法选用日本大耳白兔50只,随机分为正常对照组(A组)、心肌缺血-再灌注组(B组)、心肌缺血—再灌注 川芎嗪治疗组(C组)、心肌缺血—再灌注 左旋精氨酸治疗组(D组)和心肌缺血—再灌注 川芎嗪 左旋精氨酸治疗组(E组)。观察心肌线粒体呼吸功能、Ca2 浓度、丙二醛浓度、超氧化物歧化酶活性和心肌组织三磷酸腺苷(ATP)和能荷的变化。结果与A组比较,B组线粒体呼吸控制率、Ⅲ态呼吸速率和超氧化物歧化酶明显降低,Ⅳ态呼吸速率、Ca2 浓度和丙二醛显著升高,心肌组织ATP和能荷明显降低。与B组比较,C组、D组和E组线粒体呼吸控制率、Ⅲ态呼吸速率和超氧化物歧化酶明显升高,Ⅳ态呼吸速率、Ca2 浓度、丙二醛显著降低,心肌组织ATP和能荷明显增高;且与A组比较,E组上述指标均无明显差异。结论川芎嗪联用左旋精氨酸可通过降低氧自由基水平和减轻钙超载,而改善缺血—再灌注损伤心肌的线粒体功能。     

橙皮素对H2O2诱导H9c2心肌细胞凋亡的抑制效应研究

目的 探讨橙皮素(HES)对H₂O₂诱导的H9c2心肌细胞凋亡有何影响。方法 采用H₂O₂建立H9c2心肌细胞氧化应激损伤模型,实验分为四组：正常对照组(Control组),H₂O₂损伤组(H₂O₂组),单纯橙皮素处理组(HES组),橙皮素预处理 H₂O₂组(HES H₂O₂组)。H₂O₂(400 μM)处理2 h建立心肌细胞氧化应激损伤模型,HES H₂O₂组于建模前1 h加入40 μM橙皮素。采用CCK-8法确定H9c2细胞活性;DCFH-DA 探针检测细胞活性氧簇(ROS)水平,流式细胞术检测心肌细胞凋亡,分光光度计检测Caspase-3活性。结果 橙皮素预处理可明显改善H₂O₂诱导的H9c2心肌细胞活性降低,并且呈浓度依赖性;给予40 μM橙皮素预处理后ROS的产生明显减少,Caspase-3活性显著下降,心肌细胞凋亡率下降到30%左右。结论 橙皮素对氧化应激诱导的心肌细胞凋亡具有抑制效应。     

急性心肌梗死延迟再灌注后心肌细胞凋亡与bcl-2和bax基因mRNA表达的研究

目的:探讨犬实验性心肌梗死延迟再灌注(LR)后对梗死周边缺血区心肌细胞凋亡及凋亡相关基因bcl-2和baxmRNA表达的影响。方法:健康成年杂交犬28只全麻下常规开胸暴露冠状动脉后随机分为3组:假手术(SHAM)组(8只),急性心肌梗死(AMI)组(10只),LR组(10只)。SHAM组仅行左冠状动脉前降支下穿过丝线而不结扎冠状动脉,AMI组行左冠状动脉前降支高位永久结扎,LR组在高位结扎左冠状动脉前降支6h后松解结扎线予以再灌注6h。共有23只犬模型制作成功。各组犬均于术后12h处死,采集心肌标本。使用脱氧脲核苷酸缺口末端标记法检测心肌细胞凋亡,计算心肌细胞凋亡指数(AI)。逆转录聚合酶链反应法检测心肌细胞中bcl-2和baxmRNA表达水平,以β-actin作为内参照。结果:LR组心肌细胞AI较AMI组明显减少(P<0.05)。与SHAM组相比,bcl-2和baxmRNA在AMI组和LR组的表达均升高(P<0.01),但bcl-2mRNA在该2组间差异无统计学意义(P>0.05);而baxmRNA在AMI组的表达较LR组明显增高(P<0.05)。结论:AMI后LR可以减少梗死周边缺血区心肌细胞凋亡,其机制可能与降低心肌细胞baxmRNA的表达有关。     

血红素氧合酶对大鼠肝脏缺血再灌注损伤细胞凋亡及相关基因的影响

目的通过应用血红素氧合酶（HO）的诱导剂氯高血红素和抑制剂锌原卟啉（ZnPP），探讨HO对大鼠肝脏缺血再灌注（IR）细胞凋亡及其相关基因的影响。方法将96只Sprague—Dawley大鼠采用钳夹法制备肝脏IR模型，随机分为假手术组、IR组、氯高血红素组和ZnPP组，检测再灌注0、1．5、4h和8h各个时间点大鼠肝脏功能以及病理学改变，流式细胞法测定肝细胞凋亡率、TUNEL法观察再灌注后4h大鼠肝细胞的凋亡情况，Western blot法检测再灌注后8h Bcl-2和Caspase-3的表达。结果在IR组各时间点均可见ALT和AST增高，病理学检查可见肝细胞肿胀，肝窦变窄，嗜中性粒细胞浸润和片状坏死等变化，肝组织中细胞凋亡率明显升高，Bcl-2的表达减少，而Caspase-3的表达增加。在氯高血红素组再灌注后1．5、4h和8h ALT和AST值明显降低，肝脏病理学改善，凋亡细胞减少及细胞凋亡率降低，肝脏Bcl-2的表达增加，Caspase-3的表达减少。ZnPP组则显示与之相反的结果。结论HO在肝脏IR损伤中具有保护作用，这种保护作用可能与抑制细胞凋亡有关。