Protection of Veratrum nigrum L. var. ussuriense Nakai alkaloids against ischemia-reperfusion injury of the rat liver

AIM： TO investigate the protective effects and possible mechanisms of Veratrum nigrum L. var. ussuriense Nakai alkaloids （VnA） on hepatic ischemia/reperfusion （I/R） injury in rats.
METHODS： Forty male Wistar rats were randomly divided into four experimental groups （n = 10 in each）： （A） Control group （the sham operation group）; （8） I/R group （pretreated with normal saline）; （C） Small-dose （10 μg/kg） VnA pretreatment group; （D） Large-dose （20 μg/kg） VnA pretreatment group. Hepatic ischemia/ reperfusion （Hepatic I/R） was induced by occlusion of the portal vein and the hepatic artery for 90 min, followed by reperfusion for 240 min. The pretreatment groups were administered with VnA intraperitoneally, 30 min before surgery, while the control group and I/R group were given equal volumes of normal saline. Superoxide dismutase （SOD） activity, myeloperoxidase （MPO） activity and nitric oxide （NO） content in the liver tissue at the end of reperfusion were determined and liver function was measured. The expression of intercellular adhesion molecule-1 （ICAM-1） and E-selectin （ES） were detected by immunohistochemical examinations and Western blot analyses.
RESULTS： The results showed that hepatic I/R elicited a significant increase in the plasma levels of alanine aminotransferase （ALT： 74.53 ± 2.58 IU/L vs 1512.54 ± 200.76 IU/L, P 〈 0.01） and lactic dehydrogenase （LDH： 473.48 ± 52.17 IU/L vs 5821.53 ± 163.69 IU/L, P 〈 0.01）, as well as the levels of MPO （1.97 ± 0.11 U/g vs 2.57 ± 0.13 U/g, P 〈 0.01） and NO （69.37 ± 1.52 μmol/g protein vs 78.39 ± 2.28 μmol/g protein, P 〈 0.01） in the liver tissue, all of which were reduced by pretreatment with VnA, respectively （ALT： 1512.54 ± 200.76 IU/L vs 977.93 ± 89.62 IU/L, 909.81 ± 132.76 IU/L, P 〈 0.01, P 〈 0.01; LDH： 5821.53 ± 163.69 IU/L vs 3015.44 ± 253.01 IU/L, 2448.75 ± 169.4 IU/L, P 〈 0.01, P 〈 0.01; MPO： 2.57 ± 0.13 U/g vs 2.13 ± 0.13 U/g,     

重组人可溶性补体受体1型SCR15-18片段对心肌缺血再灌注损伤的保护作用

目的探讨重组人可溶性补体受体1型SCR15-18片段（sCR1-SCR15-18）对心肌缺血再灌注的保护作用。方法36只SD大鼠随机分为假手术（SO）组,缺血再灌注（I／R）组和sCR1-SCR15-18（sCR1）保护组。建立急性心肌缺血再灌注模型,结扎冠状动脉前立即注射磷酸盐缓冲液（0.1mL／100g）或sCR1-SCR15-18蛋白（15mg／kg）。测定心肌梗塞面积,血清中乳酸脱氢酶（LDH）和肌酸激酶（CK）,心肌组织髓过氧化物酶（MPO）活性,HE染色观察心肌病理改变和免疫组织化学法检测C3c。结果（1）心肌梗死面积：I／R组为（22．9±3．0）％,sCR1保护组为（16．1±3．3）％（P〈0．05）。（2）血清心肌酶CK（U／L）：I／R组为3400．9±534．9,sCR1保护组为2532．5±597．1（P〈0．05）。LDH（U／L）：I／R组为6572．0±476．3,sCR1保护组为5436．2±611．3（P〈0．05）。（3）心肌组织MPO活性（U／g）：I／R组为1．12±0．13,sCR1保护组为0．81±0．14（P〈0．05）。（4）心肌病理改变：I／R组心肌有断裂、坏死,间质肿胀,出血及中性粒细胞浸润,sCR1保护组心肌的以上病理变化明显较I／R组减轻。（5）与I／R组比sCR1保护组梗死区心肌组织C3c的沉积减少。结论sCR1-SCR15-18蛋白对大鼠急性心肌缺血再灌注损伤具有保护作用。     

Pharmacological regulation of postischemic sinusoidal diameters in rats--a new approach for reducing hepatic ischemia/reperfusion injury

Ischemia leads to profound endothelin-related constriction of the hepatic microcirculation with resulting disturbances in blood and oxygen supply. The aim of the study was to modulate hepatic microvascular diameters by blocking endothelin receptors with bosentan, and also to find the best possible vessel width (as produced by bosentan) for minimizing ischemia/reperfusion injury.Methods: In an in vivo rat model hepatic ischemia was induced for 30 minutes by crossclamping the hepatoduodenal ligament. The endothelin receptor antagonist (ERA) bosentan was administered before ischemia in stepwise dosages of 0.1, 1.0 and 10 mg/kg bw i.v. and 10 mg/kg bw intraportally (i.p.). Vasoactive effect was assessed by in vivo microscopy. The influence on hepatic oxygen supply and hepatocellular function was evaluated by measuring local tissue pO(2) and AST levels.Results: Because of ischemia sinusoidal diameters were reduced to 76.3 +/- 7.4% compared with values found in sham-operated animals. After administration of 0.1 mg/kg ERA (bosentan) the sinusoids remained constricted (89.7 +/- 9.9%). Blocking endothelin receptors with 1 mg/kg bosentan avoided sinusoidal constriction (99.0 +/- 8.8%, p<0.05) and led to the most effective reduction of AST level peak after 6 h of reperfusion (244.0 +/- 34.2 U/l vs 422.9 +/- 163.3 U/l in untreated ischemia). 10 mg/kg i.v. caused an increase in sinusoidal diameter to 109.1 +/- 6.4% and 10 mg/kg intraportally to 136.8 +/- 19.3% and even an increase in AST levels (618.9 +/- 209.3 U/l). Hepatic ischemia led to a significant decrease of local tissue pO(2) after reperfusion (9.4 +/- 1.2 mm Hg; p<0.05 vs sham: 16.8 +/- 1.8 mm Hg). The greatest improvement in postischemic oxygen supply was found in the 1.0 mg/kg group (12.9 +/- 1.0 mm Hg; p< 0.05 vs ischemia). Venular diameter changed almost to the same extent as sinusoidal diameter. Perfusion rate was significantly increased and sticking of leukocytes in sinusoids and venules was reduced after doses of 1 and 10 mg/kg bw bosentan i.v. (p<0.05).Implications: In this model we were able to regulate the diameters of sinusoids and postsinusoidal venules incrementally. We conclude that the avoidance of constriction, without excessive vasodilatation gives increased perfusion rates with improved hepatic oxygen supply and hepatocellular function.     

Toll样受体参与小鼠肝脏缺血再灌注损伤

目的探讨Toll样受体是否参与小鼠肝脏缺血再灌注损伤及其机制. 方法用Toll样受体缺损小鼠(C3H/Hej,Hej组)和野生型(C3H/Heouj,Heouj组)小鼠复制部分肝脏缺血再灌注损伤模型,于缺血45min,再灌注1h和3h处死动物,检测血清天门冬氨酸氨基转移酶(AST)和血清肿瘤坏死因子α(TNFα)的含量;并以northern blot及髓过氧化物酶(MPO)试验分别检测缺血肝组织TNFα mRNA的表达和MPO的含量. 结果 (1)再灌注1、3h,与假手术组相比,小鼠血浆AST明显升高,但Hej组明显低于Heouj组(661.83U/L±106.09U/L和1215.5U/L±174.03U/L,t＝-6.65,P<0.01;1145.17U/L±132.43U/L和2958.17U/L±186.81U/L,t＝-5.57,P<0.01);(2)再灌注3h时,与假手术组相比,Hej组和Heouj组小鼠血清TNFα浓度明显升高,且前者明显低于后者(152.39pg/ml±43.3pg/ml和249.12pg/ml±51.89pg/ml,t＝-3.13,P<0.05);(3)再灌注1h,除假手术组外,Hej组和Heouj组小鼠缺血肝组织内可见TNFα mRNA的表达,但前者的表达水平明显低于后者,杂交带密度分析显示两者之间差异有显著性 (80.3±28.8与189.4±24.6,t＝-3.25,P<0.05);(4)再灌注3h,与假手术组相比,Hej组和Heouj组小鼠缺血肝组织内MPO含量明显升高,且前者含量明显低于后者(0.059±0.004和0.173±0.025,F＝33.49,P<0.01). 结论 Toll样受体可能通过其介导的炎性通路参与了小鼠肝脏缺血再灌注损伤.     

Gender differences in hepatic ischemic reperfusion injury in rats are associated with endothelial cell nitric oxide synthase-derived nitric oxide

AIM: This study was designed to examine the hypothesis that gender differences in I/R injury are associated with endothelial cell nitric oxide synthase (eNOS)-derived nitric oxide (NO). METHODS: Wistar rats were randomized into seven experimental groups (12 animals per group). Except for the sham operated groups, all rats were subjected to total liver ischemia for 40 min followed by reperfusion. All experimental groups received different treatments 45 min before the laparotomy. For each group, half of the animals (six) were used to investigate the survival; blood samples and liver tissues were obtained in the remaining six animals after 3 h of reperfusion to assess serum NO, alanine aminotransferase (ALT) and TNF-α levels, liver tissue malondialdehyde (MDA) content, and severity of hepatic I/R injury. RESULTS: Basal serum NO levels in female sham operated (FS) group were nearly 1.5-fold of male sham operated (MS) group (66.7±11.0 μmol/L vs45.3μ10.1 μmol/L, P<0.01). Although serum NO levels decreased significantly after hepatic I/R (P<0.01, vs sham operated groups), they were still significantly higher in female rat (F) group than in male rat (M) group (47.8±8.6 μmol/L vs 23.8±4.7 μmol/L, P<0.01). Serum ALT and TNF-α levels, and liver tissue MDA content were significantly lower in F group than in M group (370.5±46.4 U/L, 0.99±0.11 μg/L and 0.57±0.10 μmol/g vs668.7±78.7 U/L, 1.71±0.18μg/L and 0.86±0.11 μmol/g, respectively, P<0.01). I/R induced significant injury to the liver both in M and F groups (P<0.01 vs sham operated groups). But the degree of hepatocyte injury was significantly milder in F group than in M group (P<0.05 and P<0.01). The median survival time was six days in F group and one day in M group. The overall survival rate was significantly higher in F group than in M group (P<0.05). When compared with male rats pretreated with saline (M group), pretreatment of male rats with 17-β-estradiol (E2) (M+E2 group) significantly increased serum NO levels and significantly decreased serum ALT and TNF-α levels, and liver tissue MDA content after I/R (P<0.01). The degree of hepatocyte injury was significantly decreased and the overall survival rate was significantly improved in M+E2 group than in M group (P<0.01 and P<0.05). The NOS inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) treatment could completely abolish the protective effects of estrogen in both male and female rats. CONCLUSION: The protective effects afforded to female rats subjected to hepatic I/R are associated with eNOS-derived NO.     

Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats

AIM： To investigate the potential protective effect of HO-1 on cirrhotic liver cells in rats.
METHODS： Male Wistar rats included in the current study were randomly divided into 5 groups as follows： normal （N） group; liver cirrhotic （LC） group; sham （S） group; I/R group and I/R ＋ hemin group. The model for inducing liver cirrhosis in rats was established according to a previously published protocol. Following this the segmental hepatic ischemia reperfusion operation was carried out. The rats were treated with 30 l~mol/kg hemin （HO-1 inducer, ferric portoporphyrin IX chloride） i.p. or 0.9% NaCI （control） 24 h and 12 h before hepatic ischemia for 30 min or sham laparotomy. Blood was collected for serum enzymatic measurement 6 and 12 h after reperfusion or sham laparotomy. HO-1, NF-κB and caspase-3 expressions were assessed by immunohistochemical analysis.
RESULTS： The expressions of proteins are inversely correlated to the gray values. HO-1 expression in the I/R ＋ hemin group was increased significantly than I/R group at 6 h and 12 h after hepatic I/R （6 h： 112.0± 8.3 vs 125.1± 5.7, P 〈 0.01; 12 h： 120.8± 11.0 vs 132.4 ± 6.2, P 〈 0.01）. Hemin improved serum manganese superoxide dismutase （MnSOD） （6 h： 131.3 ± 17.6 vs 107.0 ± 13.9, P 〈 0.01; 12 h： 141.4 ：E 12.5 vs 118.3± 10.2, P 〈 0.01）, lessened liver cell injury, decreased caspase-3（6 h： 166.7 ± 8.1 vs 145.5 ± 14.6, P 〈 0.01; 12 h： 172.8± 3.8 vs 148.0 ±6.5, P 〈 0.01） and NF-κB expression （6 h： 150.2 ± 8.6 vs 139.7 ±6.0, P 〈 0.01; 12 h： 151.1 ± 5.9 vs 148.1± 5.3, P 〉 0.05） and serum alanine aminotransferase （ALT） （6 h： 413.3± 104.1 vs 626.8 ±208.2, P 〈 0.01; 12 h： 322.2 ± 98.8 vs 425.8 ± 115.4, P 〈 0.05）, aspartate aminotransferase （AST） （6 h： 665.2 ± 70.1 vs 864.3± 70.4, P 〈 0.01; 12 h： 531.1 ± 98.6 vs 664.4± 115.6, P 〈 0.01）, malondialdehyde （MDA） levels （6 h： 11.1 ± 2.17 vs 13.5 ±2.01, P 〈 0.01; 12 h： 9.36     

Protective effect of N2-mercaptopropionylglycine on rats and dogs liver during ischemia/reperfusion process

BACKGROUND: N2-mercaptopropionylglycine is a powerful super oxide synthesis inhibitor and has been tested as a preventive agent of metabolic and structural hepatic damage in the ischemia/reperfusion process. AIM: To analyze some effects of N2-mercaptopropionylglycine administration to animals of two species submitted to normothermic liver ischemia/reperfusion. MATERIAL AND METHODS: Twenty-two rats and 22 dogs were divided into four groups: group I: rats that received intravenous saline 0.9%; group II: rats that received 100 mg/kg of N2-mercaptopropionylglycine; group III: dogs that received saline intravenous 0.9% and group IV: dogs that received 100 mg/kg N2-mercaptopropionylglycine. RESULTS: Ten minutes after the saline or drug administration, each group was submitted to left lobe liver ischemia for 25 minutes followed by reperfusion. Biochemical studies 24 hours after reperfusion revealed a significantly lower elevation of transaminases in animals of groups II (AST = 271 +/- 182; ALT = 261 +/- 161 ) and IV (AST = 101 +/- 45; ALT = 123 +/- 89) when compared to the controls group: I (AST = 2144 +/- 966; ALT = 1869 +/- 1040 00) and III (AST = 182 +/- 76.51; ALT = 277 +/- 219), respectively. Histology study demonstrated a significantly minor aggression to animals of groups II and IV when compared to groups I and III, respectively. CONCLUSION: These results suggest a significant release of free radicals of oxygen in the process and that N2-mercaptopropionylglycine may have a significant protective effect on liver parenchyma when submitted to ischemia/reperfusion.     

缺血后处理对高血脂大鼠缺血/再灌注心肌Caspase-3活性的影响

目的：观察缺血后处理对高血脂大鼠缺血再灌注心肌Caspase（半胱氨酸天冬氨酸酶）-3活性的影响。方法：选择高血脂SD大鼠48只,随机分为3组：假手术组（开胸后穿线做套环,但不收紧结扎线）、缺血再灌注组（收紧结扎线缺血40min,放松结扎线再灌注240min）、缺血后处理组（缺血40min后,再灌注10s,缺血10s,连续3个循环,然后再灌注240min）,每组16只。再灌注结束后自右颈动脉采血,测定血清肌酸激酶（CK）活性,再灌注心肌凋亡程度,及Caspase-3活性,并进行比较分析。结果：再灌注结束后,①缺血后处理组和缺血再灌注组CK活性明显高于假手术组[（745.26±62.18）U/L比（926.38±76.49）U/L比（237.67±21.82）U/L],缺血后处理组明显低于缺血再灌注组（P均〈0.05）;②心肌凋亡细胞计数：假手术组未见明显细胞凋亡（〈5%）,缺血后处理组心肌细胞凋亡率明显低于缺血再灌注组[（11.7±2.6）%比（20.8±3.4）%,P〈0.05];③缺血后处理组缺血区心肌组织Caspase-3活性明显低于缺血再灌注组[（545.79±98.25）μmol PNA/mg比（739.83±113.57）μmol PNA/mg,P〈0.01]。结论：缺血后处理可以减轻高血脂大鼠缺血再灌注损伤,其心肌保护作用可能与降低Caspase-3活性有关。     

Effect of ischemic preconditioning on P-selectin expression in hepatocytes of rats with cirrhotic ischemia-reperfusion injury

AIM: To investigate the effects and mechanisms of ischemic preconditioning (IPC) on the ischemia/reperfusion (I/R) injury of liver cirrhosis in rats and the effect of IPC on P-selectin expression in hepatocytes.METHODS: Forty male SD rats with liver cirrhosis were randomly divided into sham operation group (SO group),ischemia/reperfusion group (I/R group), ischemic preconditioning group (IPC group), L-Arginine preconditioning group (APC group), L-NAME preconditioning group (NPC group), eight rats in each group. Hepatocellular viability was assessed by hepatic adenine nucleotide level and energy charge (EC) determined by HPLC, ALT, AST and LDH in serum measured by auto- biochemical analyzer and bile output.The expression of P-selectin in the liver tissue was analyzed by immunohistochemical technique. Leukocyte count in ischemic hepatic lobe was calculated.RESULTS: At 120 min after reperfusion, the level of ATP and EC in IPC and APC groups was higher than that in I/R group significantly. The increases in AST, ALT and LDH were prevented in IPC and APC groups. The livers produced more bile in IPC group than in I/R group during 120 min after reperfusion (0.101±0.027 versus 0.066±0.027 ml/g liver,P=0.002). There was a significant difference between APC and I/R groups, (P=0.001). The leukocyte count in liver tissues significantly increased in I/R group as compared with SO group (P＜0.05). The increase in the leukocyte count was prevented in IPC group. Administration of L-arginine resulted in the same effects as in IPC group. However,inhibition of NO synthesis (NPC group) held back the beneficial effects of preconditioning. Significant promotion of P-selectin expression in hepatocytes in the I/R group was observed compared with the SO group (P＜0.01). IPC or L-arginine attenuated P-selectin expression remarkably (P＜0.01). However, inhibition of NO synthesis enhanced Pselectin expression (P＜0.01). The degree of P-selectin expression was positively correlated with the leukocyte counts infiltrating in liver (r=0.602, P=0.000).CONCLUSION: IPC can attenuate the damage induced by I/R in cirrhotic liver and increase the ischemic tolerance of the rats with liver cirrhosis. IPC can abolish I/R induced leukocyte adhesion and infiltration by preventing postischemic P-selectin expression in the rats with liver cirrhosis via a NO-initiated pathway.     

中性粒细胞在肺血栓栓塞症犬肺缺血-再灌注损伤中的作用

目的观察中性粒细胞(PMN)在肺血栓栓塞症(PTE)犬肺缺血-再灌注(I/R)损伤中的作用。方法 15只犬按随机数字表法随机分为3组:假手术组、缺血组、再灌注组。对 PTE 犬行取栓术,分别观察肺组织普通病理、组织匀浆髓过氧化物酶(MPO)含量及超微结构;Annexin V-FITC 凋亡检测试剂盒标记后用流式细胞仪检测再灌注前及再灌注后2、4、6 h 外周血 PMN 的凋亡率。结果再灌注6 h 后,再灌注组肺泡腔 PMN 计数明显高于假手术组及缺血组[(31±11)、(1±1)及(8±4)/10个高倍镜视野],肺组织匀浆 MPO 含量分别为(11.7±1.6)、(8.3±2.1)及(9.1±0.5)μg/L;缺血组肺泡腔内少许炎细胞渗出,再灌注组肺泡腔内见大量以 PMN 为主的炎细胞渗出;电镜下,再灌注后PMN 紧紧黏附于肺泡间隔,间隔内含水肿液及空泡样变性等;再灌注6 h 后外周血 PMN 凋亡率为(3.0±2.5)%,明显低于再灌注前的(7.4±4.5)%;术后4 h,再灌注组外周血 PMN 凋亡率为(4.8±2.6)%,明显低于缺血组的(9.3±2.0)%;6 h 组结果类似[(3.0±2.5)%,(8.0±1.6)%]。结论在该 PTE 犬 I/R 模型中,再灌注后 PMN 活性增强、凋亡减少;PMN 的参与是该模型肺 I/R 损伤的重要细胞学机制之一。     

Effects of Wy14643 on hepatic ischemia reperfusion injury in rats

AIM： To investigate the effects and possible mechanisms of Wy14643 on hepatic ischemiareperfusion （I/R） injury in rats. METHODS： Thirty male Sprague-Dawley rats weighing 220-280 g were randomly divided into five experimental groups： sham group （G1, n = 6）： a sham operation was performed （except for liver I/R）, I/R-untreated group （G2, n = 6）： rats underwent liver ischemia for 90 min followed by reperfusion for 4h; and I/R ＋ Wy14643 groups （G3, G4, G5; n = 6）： after the same surgical procedure as in group 2, animals were pretreated with Wy14643 at the dose of 1, 5 and 10 mg/kg 1 h before ischemia, respectively. Hepatic ischemia-reperfusion （I/R） was induced by clamping blood supply to the left lateral and median lobes of the liver for 90 min, and atraumatic clamp was removed for 4 h reperfusion. Blood samples and liver tissues were obtained at the end of reperfusion to assess serum and hepatic tissue homogenate aminotransferase （ALT）, aspartate aminotransferase （AST）, myeloperoxidase （MPO）, serum interleukin- 1β（IL-1β） and tumor necrosis factor alpha （TNF-α）, as well as activity of superoxide dismutase （SOD） and content of malondialdehyde （MDA） in the hepatic tissue homogenate. RESULTS： Hepatic I/R induced a significant increase in the serum levels of ALT, AST, TNF-α, IL-1β and MPO, as well as the levels of ALT, AST and MDA in the liver tissue homogenate, which were reduced by pretreatment with Wy14643 at the dose of 1, 5 and 10 mg/kg, respectively. The activity of SOD in the liver tissue homogenate was decreased after hepatic I/R, which was enhanced by Wy14643 pretreatment. In addition, serum and liver tissue homogenate ALT and AST in the Wy14643 10 mg/kg group were lower than in the Wy14643 1 mg/kg and 5 mg/kg groups, respectively. CONCLUSION： Wy14643 pretreatment exerts significant protection against hepatic I/R injury in rats. The protective effects are possibly associated with enhancement of anti-oxidant and inhibition inflammation res     

丝裂原激活蛋白激酶信号通路在肝脏缺血预处理细胞保护效应中的作用

目的 研究p44/42丝裂原激活蛋白激酶(MAPK)信号通路在肝脏缺血预处理细胞保护效应中的作用。方法 建立体内和体外肝脏缺血预处理模型,应用蛋白激酶C(PKC)抑制剂和MAPK抑制剂,通过检测p44/42MAPK磷酸化水平,细胞活力,血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的活性变化,同时观察光镜细胞形态学损害,对相关数据进行统计学处理。 结果 体内和体外的缺血预处理两部分实验都观察到相似的结果。和缺血再灌注组比较,预处理组的p44/42 MAPK磷酸化水平显著增高,肝细胞结构损伤改变较小,血清ALT、AST水平显著降低,缺血再灌注组的ALT、AST水平分别为(762.8±130.5)U/L和(820.9±111.3)U/L,预处理组的ALT、AST水平分别为(281.0±35.6)U/L和(407.7±73.7)U/L;和缺血预处理组相比,PKC抑制剂组和丝裂原蛋白激酶(MEK)抑制剂组相应的观察指标呈相反的变化,p44/42 MAPK磷酸化激活显著减少,肝组织细胞结构出现较明显的损伤改变,血清ALT、AST水平显著升高,PKC抑制剂组和MEK抑制剂组的ALT、AST水平分别为(645.61±90.4)U/L、(678.6±136.5)U/L和(466.2±82.8)U/L、(732.9±91.1)U/L。 结论 肝脏缺血预处理细胞保护作用中,p44/42 MAPK通路起到至关重要的作用。     

肝移植术后早期乳酸清除率与ALT和AST的相关性及对移植肝损伤的评估研究

目的分析肝移植术后早期乳酸清除率(early lactate clearance rate,ELCR)和初期肝特异性氨基转移酶水平变化的特点及相关性,探讨ELCR对移植肝损伤的评估价值。方法回顾性分析301例原位肝移植(orthotopic liver transplantation,OLT)病例资料,针对其术后转入重症监护室的初期ALT、AST、动脉血乳酸水平以及术后6 h动脉血乳酸水平计算ELCR,并与ALT、AST及AST/ALT比值进行相关性分析。结果 ELCR与ALT、AST呈中度负相关(r分别为-0.668、-0.602,P均0.001),而与AST/ALT比值无相关性(r=0.077)。经统计分析构建的3个ELCR等级分别为Ⅰ级25%、Ⅱ级为25%~50%、Ⅲ级≥50%。Ⅰ级、Ⅱ级和Ⅲ级对应ALT水平分别为(1670.20±897.41)U/L、(937.87±350.06)U/L和(556.16±173.56)U/L,AST为(2835.98±1549.06)U/L、(1642.87±1106.00)U/L和(827.44±260.92)U/L,3组间ALT和AST水平差异有统计学意义(P均0.001)。随着ELCR等级增大,ALT和AST值降低。结论 ELCR可方便快速地反映肝移植后初期肝缺血-再灌注损伤程度,其不同等级可对肝移植术后初期移植肝功能进行初步评估。     

Protection against hepatic ischemia/reperfusion injury via downregulation of toll-like receptor 2 expression by inhibition of Kupffer cell function

AIM: To elucidate the mechanism of liver protection by inhibition of Kupffer cells (KCs) function. METHODS: All the animals were randomly divided into three groups. Blockade group (gadolinium chloride solution (GdCl3) injection plus ischemia/reperfusion (I/R) injury): GdCl3 solution was injected once every 24 h for 2 d via the tail vein before I/R injury. Non-blockade group (saline solution injection plus I/R injury): saline instead of GdCl3 as a control was injected as in the blockade group. Sham group: saline was injected without I/R injury. Liver samples were collected 4 h after blood inflow restoration. The blockade of the function of KCs was verified by immunostaining with an anti-CD68 mAb. Toll-like receptor 2 (TLR2) was immunostained with a goat antimouse polyclonal anti-TLR2 antibody. Membrane proteins were extracted from the liver samples and TLR2 protein was analyzed by Western blot. Portal vein serum and plasma were taken respectively at the same time point for further detection of the levels of tumor necrosis factor-α (TNF-α) and alanine aminotransferase (ALT), an indicator of liver function. RESULTS: Compared to non-blockade group, CD68+ cells significantly reduced in blockade group (OPTDI, optical density integral): 32.97±10.55 vs 185.65±21.88, P<0.01) and the liver function impairment was relieved partially (level of ALT: 435.89±178.37 U/L vs 890.21±272.91 U/L, P<0.01).The expression of TLR2 protein in blockade group significantly decreased compared to that in non-blockade group (method of immunohistochemistry, OPDTI: 75.74±17.44 vs 170.58±25.14, P<0.01; method of Western blot, A value: 125.89±15.49 vs 433.91±35.53, P<0.01). The latter correlated with the variation of CD68 staining (r= 0.745, P<0.05). Also the level of portal vein TNF-a decreased in blockade group compared to that in non-blockade group (84.45±14.73 ng/L vs 112.32±17.56 ng/L, P<0.05), but was still higher than that in sham group (84.45±14.73 ng/L vs 6.07±5.33 ng/L, P<0.01). CONCLUSION: Inhibition of the function of KCs may protect liver against I/R injury via downregulation of the expression of TLR2.     

急性肺血栓栓塞患者血清酶学及肌钙蛋白I的变化

目的观察急性肺血栓栓塞（PTE）患者血清酶学及肌钙蛋白Ⅰ（TnI）的变化，了解其与估测肺动脉收缩压、右心运动功能及预后的关系。方法519例PTE患者来自北京24家医院参与的国家“十五”科技攻关课题——肺栓塞规范化诊治方法的研究。根据2001年5月中华医学会呼吸病学分会制定的《肺血栓栓塞症的诊断与治疗指南（草案）》的诊断标准确定大面积、次大面积、非大面积肺栓塞患者。大面积、次大面积肺栓塞患者采用溶栓治疗，非大面积肺栓塞患者采用抗凝治疗。按中心随机方法将患者分组，应用尿激酶和重组组织型纤溶酶原激活剂及普通肝素和低分子肝素。结果（1）大面积肺栓塞患者治疗前血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、肌酸激酶（CPK）、乳酸脱氢酶（LDH）水平[（74±33）、（88±40）、（157±75）、（419±264）U／L]明显高于次大面积肺栓塞患者[（52±21）、（43±18）、（75±30）、（284±176）U／L]和非大面积肺栓塞患者[（38±13）、（35±11）、（78±24）、（239±178）U／L]；（2）非大面积肺栓塞患者应用普通肝素治疗7d后血清ALT[（84±39）U／L]明显高于应用低分子肝素患者[（67±26）U／L]；（3）519例患者中45例肺动脉收缩压≥80mmHg（1mmHg=0．133kPa），治疗前存在右心运动功能减弱169例，预后不良48例。大面积肺栓塞患者中17例（41．5％）AIJT升高，次大面积中76例（45．5％），非大面积中26例（54．5％）。大面积肺栓塞患者中24例（54．4％）LDH升高，次大面积中68例（40．2％），非大面积中15例（30．8％）；（4）39例血清TnI≥0．07μg／L的患者中右心功能减弱24例（63．3％），预后不良者8例（24．2％）。结论（1）急性PTE患者可出现血清ALT、ASF、CPK、LDH水平升高；（2）非大面积肺栓塞患者应用抗凝治疗，普通肝素较低分子肝素更易引起血清ALT升高；（3）血清ALT、LDH以及TnI的升高与PTE患者的肺动脉收缩压、右心运动功能及预后密切相关，其变化可能有助于对急性肺栓塞患者进行危险分层。     

Effects of ischemic preconditioning on cyclinD1 expression during early ischemic reperfusion in rats

AIM: To observe the effect of ischemic preconditioning on cyclinD1 expression in rat liver cells during early ischemic reperfusion. METHODS: Fifty-four SD rats were randomly divided into ischemic preconditioning group (IP), ischemia/ reperfusion group (IR) and sham operation group (SO). The IP and IR groups were further divided into four sub-groups (n - 6). Sham operation group (SO) served as the control group (n = 6). A model of partial liver ischemia/reperfusion was used, in which rats were subjected to liver ischemia for 60 min prior to reperfusion. The animals in the IP group underwent ischemic preconditioning twice for 5 min each time prior to the ischemia/reperfusion challenge. After 0, 1, 2, and 4 h of reperfusion, serum and liver tissue in each group were collected to detect the level of serum ALT, liver histopathology and expression of cyclinDi mRNA and protein. Flow cytometry was used to detect cell cycle as the quantity indicator of cell regeneration. RESULTS: Compared with IR group, IP group showed a significantly lower ALT level in 1 h to 4 h sub-groups (P < 0.05). Proliferation index(PI) indicated by the S-phase and G2/M-phase ratio [(S G2/M)/(G0/G1 S G2/M)] was significantly increased in IP group at 0 and 1 h (26.44±7.60% vs 18.56±6.40%,41.87±7.27% vs 20.25±6.70%, P < 0.05). Meanwhile, cyclinDi protein expression could be detected in IP group. But in IR group, cyclinDi protein expression occurred 2 h after reperfusion. The expression of cyclinDi mRNA increased significantly in IP group at 0 and 1 h (0.568±0.112 vs 0.274±0.069, 0.762±0.164 vs 0.348±0.093, P < 0.05). CONCLUSION: Ischemic preconditioning can protect liver cells against ischemia/reperfusion injury, which may be related to cell proliferation and expression of cyclinD1 during early ischemic reperfusion.     

β-catenin基因在小鼠肝脏缺血再灌注损伤中的保护作用及机制

目的研究β—catenin基因对小鼠肝脏缺血再灌注损伤的保护作用及机制。方法建立特异性敲除β—catenin基因小鼠及部分肝脏缺血再灌注损伤模型,检测部分肝脏缺血再灌注后血清学指标ALT、AST及肝脏病理学改变,应用实时荧光定量聚合酶链反应（RT—PCR）技术测定β—catenin。一组和野生型组小鼠再灌注6h后肝组织内缺氧诱导因子（HIF）10t、肿瘤坏死因子（TNF）α、白细胞介素（IL）1β的mRNA表达水平。分析数据组间比较采用t检验或方差分析。结果β—catenin^-／-组小鼠再灌注后病理损伤较野生型（WT）对照组严重,血清中ALT、AST水平明显高于WT组,（8178．61±78．76）vs（891．83±23．73）U／L,t=24．296;（7348．94±141．99） vs （873．50±20．27）,t=27．854,P均〈0．001。β—catenin^-／-组肝组织HIF-1α的mRNA表达水平显著低于WT组,（0．31±0．04） vs （1．004-0．22）,t=3．949,P〈0．01。而TNFα、IL-1β的mRNA表达水平显著高于WT组（3．14±0．37）V8（1．00±0．19）,t=3．676;（3．72±0．33） vs （1．00±0．24）,t=4．017,P均〈0．01。结论β—catenin基因缺失可通过影响HIF-1α表达加重肝脏缺血再灌注损伤。     

Assessment of hepatic ischemia-reperfusion injury by simultaneous measurement of tissue pO2, pCO2, and pH

INTRODUCTION: The objective of this study was to determine whether the simultaneous measurement of tissue pH, pCO(2), and pO(2) with a multiple-parameter fiberoptic sensor (Paratrend 7) can be used for continuous monitoring of hepatic microperfusion in a pig model of hepatic ischemia given endothelin(A) receptor antagonist (ET(A)-RA) or isotonic saline. METHODS: Fourteen anesthetized swine were subjected to 2 h of hepatic vascular exclusion. The animals were randomized into two groups: control group (n = 7, saline solution iv) and therapy group (n = 7, ET(A)-RA). For evaluation of ischemia-reperfusion injury, the data of the multiple-parameter sensor (pO(2para), pCO(2para), and pH(para)) were compared with partial oxygen pressure in tissue (p(ti)O(2)), laser Doppler flow, and systemic hemodynamic, metabolic data, and time course of transaminases. RESULTS: In the control group 30 and 60 min after reperfusion, the following values were measured: p(ti)O(2): 34.0 +/- 8.6 / 36.3 +/- 7.0 mm Hg (P < 0.05 vs. preop.: 49.8 +/- 12.1 mm Hg), laser Doppler area: 133.3 +/- 23.2 / 156.4 +/- 15.4 (P < 0.05 vs. preop.: 215.9 +/- 14.8). Animals in the therapy group revealed significantly improved values (p(ti)O(2): 54.0 +/- 8.6 / 58.1 +/- 7.8 mm Hg, laser Doppler: 210.2 +/- 38.5 / 225.2 +/- 21.3; P < 0.05). Using the Paratrend, also an improvement in the therapy group was seen 30 and 60 min after reperfusion. The values showed a strong correlation with p(ti)O(2) (r = 0.895; P < 0.05) and laser Doppler flow (r = 0.807; P < 0.05). In the treatment group, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutamate dehydrogenase (GLDH) were reduced 6 and 18 h after reperfusion, respectively, indicating hepatoprotection by the therapy (P < 0.05 vs. control). CONCLUSIONS: The Paratrend sensor offers the opportunity to study postischemic organ hemodynamics through the simultaneous measurement of interstitial pH, pCO(2), and pO(2) in a small tissue region. This method offers a prognostic tool for the study of the effects of experimental vasoactive therapy on liver microcirculation and perspectives for continuous monitoring of human liver microperfusion after liver surgery and trauma.     

Melatonin treatment protects against ischemia/reperfusion-induced functional and biochemical changes in rat urinary bladder

Reactive oxygen metabolites play important roles in ischemia/reperfusion (I/R) injury in several systems. The aim of this study was to investigate the role of melatonin against I/R injury of the rat urinary bladder. The abdominal aorta was clamped to induce ischemia for 30 min, then the animals were subjected to 60 min of reperfusion. Melatonin (10 mg/kg, i.p.) or the vehicle (control 1% alcohol i.p.) was administered before I/R. After decapitation, the bladder was removed and the tissue was either used for functional studies or stored for measurement of products of lipid peroxidation (LP), glutathione (GSH) levels and myeloperoxidase activity (MPO). Bladder strips were suspended in oxygenated Tyrode's buffer at 37 degrees C and isometric contractions to carbachol (CCh; 10(-8)-10(-4) m) were recorded. In the I/R group, the contractile responses of the bladder strips were lower than those of the control group (P < 0.01-0.001) and were reversed by treatment with melatonin (P < 0.05-0.001). LP which was higher in I/R group compared with control (27.68 +/- 1.69 and 10.59 +/- 1.27 nmol/g, respectively; P < 0.001) was partially reversed by melatonin (19.01 +/- 1.85 nmol/g; P < 0.01). Similarly, GSH showed a decrease in the I/R group compared with controls (0.27 +/- 0.03 and 0.43 +/- 0.04 micromol/g, respectively; P < 0.05) and melatonin prevented this effect completely (0.45 +/- 0.04 micromol/g; P < 0.05). MPO activity in the I/R group (4.19 +/- 0.08 U/g) was significantly higher than that of the control group (1.41 +/- 0.08 U/g; P < 0.001) and melatonin treatment reduced MPO levels compared with I/R alone (3.16 +/- 0.07; P < 0.001). Melatonin almost completely reversed the low contractile responses of rat urinary bladder strips to CCh and prevented oxidative tissue damage following I/R.