Heat-shock preconditioning reduces oxidative protein denaturation and ameliorates liver injury by carbon tetrachloride in rats

Membrane lipids and cytosolic proteins are major targets of oxidative injury. This study examined the effect of heat-shock preconditioning associated with the induction of heat-shock protein 72 on liver injury, from the aspect of lipid peroxidation and protein denaturation after carbon tetrachloride (CCl₄) administration in rats - one of the representative oxidative injuries. Male Wistar rats were divided into two groups, group HS (preconditioned by heat exposure) and group C (not preconditioned). Expression of HSP72 in the liver tissue was confirmed by Western blot analysis. After a 48-h recovery period, all rats were given CCl₄ intragastrically. Liver dam-age was assessed by measuring serum liver-related enzyme levels and adenine nucleotide concentration in the liver tissue. Lipid peroxidation and protein denaturation were evaluated by measuring tiobarbituric acid reactive substances (TBARS) and by immunohistochemical staining of 4-hydroxy-2-nonenal(HNE)-modified proteins in the liver. Survival rates of the rats after CCl₄ administration were also compared. Expression of HSP72 was clearly detected in group HS, but not in group C. Heat-shock preconditioning significantly improved the survival rate, suppressed the increase in liver-related enzyme levels and maintained adenosine triphosphate levels (P<0.01 each). HNE-modified proteins - denatured proteins by free radical attack - were significantly less stained in group HS than in group C (P<0.05). However, TBARS levels did not differ between groups. Because heat-shock preconditioning did not alter TBARS levels but reduced HNE-modified proteins in association with the expression of HSP72, it is suggested that HSP72 did not prevent lipid peroxidation but decreased the lipid peroxidation-induced denaturation of proteins. This seemed to be a mechanism of heat-shock preconditioning to ameliorate oxidative liver injury.     

Anti-fibrotic effect of malotilate on liver fibrosis induced by carbon tetrachloride in rats

The effect of malotilate on liver fibrosis of rat induced by carbon tetrachloride (CCl4) was studied. CCl4 was given subcutaneously (1.5 ml/kg) to male Wistar rats twice a week for 11 weeks. Administration of malotilate (100 mg/kg) 5 times a week was started in the 5th experimental week and continued thereafter. The biochemical parameters in serum such as concentration of total protein and transaminase activities were improved by malotilate administration. The livers treated with CCl4 only were atrophic and markedly nodular, and histologically, severe collagen deposition and pseudolobular formation were observed. However, the livers treated with CCl4 and malotilate showed only slight accumulation of collagen fibers although hypertrophic and fatty metamorphosis was observed. Immunocytochemically, type I and type III collagens were stained in livers from rats treated with CCl4 only and rats treated with CCl4 and malotilate, but stainability was rather weak in the latter. The increased amount of hydroxyproline in the liver and that excreted into urine were markedly suppressed by malotilate administration. The levels of protocollagen prolyl hydroxylase activity in the liver were almost the same in CCl4-treated and CCl4 and malotilate-treated rats. However, the levels of collagenolytic enzyme activity were slightly higher in the latter. From these results, anti-fibrotic action of malotilate was clear and involvement of enhanced collagenolytic enzyme activity was suggested.     

褪黑素对大鼠体外循环术后肝脏的保护作用

目的:研究体外循环(CPB)大鼠术前给予外源性褪黑素对于术后肝脏氧化应激损伤的保护作用。方法:选用成年SD大鼠48只,随机分为伪手术组、手术组、低剂量组和高剂量组。伪手术组行类似操作,但不实施CPB。其余3组建立CPB,最大流量[≥100mL/(kg·min)]转流60min。低剂量组和高剂量组分别于转流前在预充液中加入10、20mg/kg的褪黑素。实验过程中记录血压和心率变化,并于转流前全身肝素化后(术前)和转流结束后(术后)进行血气分析。术后24h取肝脏组织,测定丙二醛(MDA)、总一氧化氮合酶(tNOS)、诱导型一氧化氮合酶(iNOS)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、还原性谷胱甘肽(GSH)、谷胱甘肽还原酶(GSSG-R)、谷胱甘肽巯基转移酶(GST)和谷胱甘肽过氧化物酶(GSH-Px)的量或活性。结果:CPB后大鼠血压和心率较术前有改变,但仍在可接受范围。血气分析结果提示,手术组术后动脉氧分压明显升高,pH值、红细胞压积降低较多,余无显著变化。手术组肝脏MDA浓度,tNOS和iNOS活性较伪手术组上升,而GSH浓度,SOD、CAT、GSSG-R、GST、GSH-Px活性的变化则相反。与此同时,在使用褪黑素的两组中,这些指标的变化被明显逆转。结论:褪黑素能有效的抑制CPB后肝脏的氧化应激损伤,同时通过研究发现,不同剂量组的结果无统计学差异。     

急性肝损伤大鼠肝脏Fas和FasL的表达及其意义

目的 研究急性肝损伤大鼠肝脏Fas和FasL的表达情况，探讨细胞凋亡在中毒性肝损伤发病中的地位及其意义。方法 健康雄性Wistar大鼠35只，随机分为正常对照组和实验组，实验组再分为3、9、16、24、36和48h6个亚组，每组5只。制备四氯化碳中毒性肝损伤动物模型，采用苏木素-伊红（HE）染色，光镜下观察肝组织损伤情况，采用免疫组化方法测定不同时间点肝组织Fas和FasL的表达，采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）观察肝细胞凋亡情况。同时测定各时间点血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和肝组织超氧化物歧化酶（SOD）活性、肝组织丙二醛（MDA）含量变化。结果 Fas和FasL在正常大鼠肝细胞中未见表达，实验组3h后即开始有明显表达，并随时间延长表达相应增强；病理学和TUNEL检测结果均显示肝脏有严重损伤，大量肝细胞发生凋亡。大鼠染毒后血清ALT、AST活性和肝组织MDA含量明显升高，肝组织SOD活性显著降低，与正常对照组比较差异均十分显著（P〈O．05或P〈O．01）。结论 大鼠急性肝损伤时Fas和FasL表达显著增加，和肝细胞凋亡变化相一致，提示Fas／Fasl。系统及介导的细胞凋亡反应在中毒性肝损伤的发病机制中可能占有重要地位。     

熊去氧胆酸联合异甘草酸镁干预四氯化碳诱导小鼠急性肝损伤研究

目的探讨熊去氧胆酸(UDCA)联合异甘草酸镁(Mg IG)对四氯化碳诱导的急性肝损伤小鼠的保护作用及机制。方法 50只小鼠随机分为空白组、模型组、UDCA组,Mg IG组和联合组,每组10只。采用CCl4诱导小鼠急性肝损伤模型,灌胃给予相应药物或生理盐水。测定各组血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST);肝组织匀浆中丙二醛(MDA)含量、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-PX)活性,观察各组肝脏病理检查。结果较空白组,模型组血清ALT、AST,肝组织MDA含量明显升高,SOD、T-AOC、GSH-PX活性降低,肝组织病变程度严重(P0.05,P0.01);与模型组比较,给药组各指标均有所改善,病变程度降低(P0.05,P0.01);联合组血清ALT、AST含量、肝组织MDA含量下降较单独用药组明显(P0.05,P0.01)。结论 UDCA、Mg IG联合对CC14所致小鼠急性化学性肝损伤具有保护作用,其机制可能与其清除自由基、提高抗氧化酶的活性有关。     

Ranitidine reduces ischemia/reperfusion-induced liver injury in rats by inhibiting neutrophil activation

We previously reported that ranitidine, an H(2) receptor antagonist, inhibited neutrophil activation in vitro and in vivo, contributing to reduce stress-induced gastric mucosal injury in rats. In this study, we examined whether ranitidine would reduce ischemia/reperfusion-induced liver injury, in which activated neutrophils are critically involved, in rats. We also examined the effect of famotidine, another H(2) receptor antagonist, on leukocyte activation in vitro and after ischemia/reperfusion-induced liver injury in rats to know whether inhibition of neutrophil activation by ranitidine might be dependent on its blockade of H(2) receptors. Ranitidine inhibited the activation of neutrophils in vitro as reported previously, whereas famotidine significantly enhanced it. Ranitidine inhibited the production of tumor necrosis factor-alpha (TNF-alpha) in monocytes stimulated with lipopolysaccharide in vitro, whereas famotidine did not. Although hepatic ischemia/reperfusion-induced increases in hepatic tissue levels of TNF-alpha, cytokine-induced neutrophil chemoattractant, and hepatic accumulation of neutrophils were inhibited by intravenously administered 30 mg/kg ranitidine, these increases were significantly enhanced by 5 mg/kg i.v. famotidine. The decreases in both hepatic tissue blood flow and bile secretion and the increases in serum levels of transaminases seen after reperfusion were significantly inhibited by ranitidine, whereas these changes were more marked in animals given famotidine than in controls. These observations strongly suggested that ranitidine could reduce ischemia/reperfusion-induced liver injury by inhibiting neutrophil activation directly, or indirectly by inhibiting the production of TNF-alpha, which is a potent activator of neutrophils. Furthermore, the therapeutic efficacy of ranitidine might not be explained solely by its blockade of H(2) receptor.     

Expression of Fas antigen and Fas ligand in acute liver injury induced by carbon tetrachloride in rat]

OBJECTIVE: To investigate the dynamics of expression of Fas antigen and Fas ligand (FasL) in a rat model of carbon tetrachloride-induced acute liver injury, and explore the role of apoptosis in liver injury. METHODS: Thirty-five healthy male Wistar rats were randomly divided into normal control group and experiment group, and the latter group was divided into six subgroups: 3, 9, 16, 24, 36 and 48 hours groups with 5 rats in each group. The liver injury was induced by carbon tetrachloride. Sections of liver tissue were stained with hematoxylin and eosin and observed under optical microscope. Fas antigen and FasL in rat liver were determined at different time points with immunohistochemical method. Hepatocytes apoptosis were observed with terminal deoxynucleotidyl-transferase mediated dUTP-biotin nick end labeling (TUNEL) method. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration in the liver tissues were analyzed at the same time point. Serum aspartate aminotransferase (ALT) and alanine aminotransferase (AST) levels were also determined. RESULTS: Fas antigen and FasL were expressed in the liver tissues of control rats. Following carbon tetrachloride challenge, severe liver injury took place in rats as revealed under microscope and a large amount of hepatocytes apoptosis was found. Hepatic Fas and FasL expression were both increased markedly from 3 to 48 hours after carbon tetrachloride challenge in experiment group. Liver MDA concentration and serum ALT and AST were elevated significantly, while SOD activity decreased remarkably in the experiment group compared with control group (P<0.05 or P<0.01). CONCLUSION: Expression of Fas/FasL is remarkably induced in acute liver injury and accords with the changes of hepatocytes apoptosis, which suggests that apoptosis mediated by Fas/FasL may play an important role in the pathogenesis of acute liver injury.     

银杏叶提取物预防大鼠急性肝损害机制的研究

目的 观察银杏叶提取物对大鼠急性肝损害的预防作用及其机制,寻找临床适用的检测指标.方法 四氯化碳诱发大鼠急性肝损害,同时使用银杏叶提取物进行预防,使用生化检测仪检测丙氨酸氨基转移酶和天冬氨酸转氨酶的含量改变;使用比色法检测超氧化物歧化酶和丙二醛的含量变化;采用酶联免疫吸附测定方法 检测肿瘤坏死因子α和转化生长因子β1,的含量变化.结果 银杏叶提取物有效降低了丙氨酸氨基转移酶和天冬氨酸转氨酶的含量;并使超氧化物歧化酶的含量显著升高,与之对应丙二醛的含量显著降低;肿瘤坏死因子α和转化生长因子β1的含量明显降低.结论 银杏叶提取物可以有效预防大鼠急性肝损害,其机制是抑制了氧化应激反应和调节了细胞因子网络.上述检测指标和研究方法 适合在临床推广.     

缺血预处理对肝硬化大鼠肝脏缺血再灌注损伤保护作用的研究

目的探讨缺血预处理(IP)对硬化肝脏缺血再灌注损伤的保护作用及其可能机制。方法Wistar雄性大鼠制成肝硬化模型。分两组:IP组离断肝周韧带,消除侧枝循环,用Pringle's法阻断肝门15min,然后恢复血供,关腹;C组只予以开、关腹。48 h后,再次Pringle's法阻断肝门30 min,恢复血供。用Western blotting法测IP后6、24、48 h肝组织中热休克蛋白70(HSP70)表达的水平;测再灌注1、3 h血清生化酶(ALT、AST、LDH)及再灌注3h肝组织中谷胱苷肽(GSH)水平;行病理学观察。结果IP后6 h,两组均有微量HSP70表达,至24-48hIP组HSP70表达显著增强,呈高水平;而C组中在各时点HSP70均无表达增强。再灌注1h,IP组的ALT、AST、LDH水平显著低于C组(P<0.01或P<0.05);再灌注3h,IP组的ALT、AST水平明显低于C组(P<0.01)而其肝组织中的GSH水平明显高于C组(P<0.05)。术后一周生存率IP组(93.10%)明显高于C组(73.33%)(P<0.05)。缺血再灌注后1、3h,IP组的肝细胞损伤明显轻于C组。结论在硬化大鼠肝脏中,IP启动内源性保护机制,诱导HSP70的大量表达,而HSP70通过增加或提高GSH的产生及其活性,清除自由基,减轻硬化肝脏缺血再灌注损伤。     

Ischemic preconditioning ameliorates ischemia- and reperfusion-induced intestinal epithelial hyperpermeability in rats

We hypothesized that ischemic preconditioning (IPC) would ameliorate ischemia (I) and reperfusion (R)-induced intestinal mucosal hyperpermeability and that this effect would be diminished by lowering local adenosine concentrations using adenosine deaminase (ADA). The small intestine of anesthetized rats (group 1; n = 6) was divided into six 10-cm segments (A1-F1) each perfused by a different set of mesenteric branches. Segments D1-F1 were subjected to 3 cycles of IPC (2 min I/5 min R). Segments A1, B1, and C1 were excised at baseline, after 60 min of I (160), and after 60 min of I followed by 60 min of R (160/R60), respectively. Segment D1 was excised immediately after the last cycle of IPC, E1 was excised at 160 after IPC, and F1 was excised at 160/R60 after IPC. In group 2 (n = 6), the intestine was divided into five 10-cm vascularly isolated segments (A2-E2). Segment A2 was resected at baseline. The lumen of the remaining segments was filled with ADA (32 U/50 cm). Segment B2 was removed at the end of the experiment having been exposed to ADA for 150 min (ADA150). Segments C2, D2, and E2 were subjected to IPC. Segment C2 was excised immediately thereafter. Segments D2 and E2 were excised at 160 and 160/R60, respectively. Intestinal permeability to fluorescein isothiocyanate-labeled dextran (molecular weight 4000 D) was assessed ex vivo by using an everted gut sac method. IPC ameliorated intestinal hyperpermeability induced by 160 (43.0+/-7.6 vs. 70.4+/-8.3 nLmin/cm2; P = 0.024) and 160/R60 (20.2+/-3.7 vs. 69.5+/-10.8 nL/min/cm2; P= 0.003). IPC prevented ischemia-induced reduction in villus height. Treatment with ADA partially reversed the protective effect of IPC on the changes in permeability and villus height induced by I/R. We conclude that IPC partially protects against mucosal barrier dysfunction in rats subjected to mesenteric I/R. Adenosine is a mediator of IPC in the gut mucosa, but other factors also may be important.     

Octacosanol attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in rats intoxicated with carbon tetrachloride

We examined whether octacosanol, the main component of policosanol, attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in rats intoxicated with carbon tetrachloride (CCl(4)). In rats intoxicated with CCl(4) (1 ml/kg, i.p.), the activities of serum transaminases increased 6 h after intoxication and further increased at 24 h. In the liver of CCl(4)-intoxicated rats, increases in lipid peroxide (LPO) concentration and myeloperoxidase activity and decreases in superoxixde dismutase activity and reduced glutathione (GSH) concentration occurred 6 h after intoxication and these changes were enhanced with an increase in xanthine oxidase activity and a decrease in catalase activity at 24 h. Octacosanol (10, 50 or 100 mg/kg) administered orally to CCl(4)-intoxicated rats at 6 h after intoxication attenuated the increased activities of serum transaminases and the increased hepatic myeloperoxidase and xanthine oxidase activities and LPO concentration and the decreased hepatic superoxide dismutase and catalase activities and GSH concentration found at 24 h after intoxication dose-dependently. Octacosanol (50 or 100 mg/kg) administered to untreated rats decreased the hepatic LPO concentration and increased the hepatic GSH concentration. These results indicate that octacosanol attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in CCl(4)-intoxicated rats.     

Activated protein C reduces stress-induced gastric mucosal injury in rats by inhibiting the endothelial cell injury

Summary. Background and objective: Activated protein C (APC) is a natural anticoagulant with anti‐inflammatory activity. APC inhibits neutrophil activation through inhibition of tumor necrosis factor (TNF)‐α production. Such anti‐inflammatory activity of APC has recently been shown to be critical in the treatment of patients with severe sepsis. We previously demonstrated that activated neutrophils play a crucial role in the development of stress‐induced gastric mucosal injury. Thus, inhibition of neutrophil activation by APC should reduce endothelial cell damage, maintain gastric blood flow, and lessen gastric mucosal injury. In the present study, we examined this possibility by using a rat model of water‐immersion restraint stress (WIRS)‐induced gastric mucosal injury. Methods and results: Gastric mucosal injury was observed 4 h after WIRS, without increases in gastric mucosal levels of either myeloperoxidase activity or TNF‐α, but with significant increases in plasma levels of TNF‐α 1 h after WIRS. Intravenous administration of APC (100 µg kg^?1) significantly reduced WIRS‐induced gastric mucosal injury by inhibiting decrease in gastric mucosal blood flow. Administration of APC also inhibited both the decrease in gastric tissue levels of 6‐keto‐prostaglandin F_1α and the increase in gastric mucosal micorvascular permeability in animals subjected to WIRS. Furthermore, APC inhibited WIRS‐induced increases in plasma levels of TNF‐α. Neither active site‐blocked factor Xa, which is a selective inhibitor of thrombin generation, nor active site‐blocked APC had any effect on these events. Intraperitoneal administration of anti‐rat TNF‐α antibody produced effects similar to those of APC. Conclusions: The observations in the present study strongly suggest that APC reduces stress‐induced gastric mucosal injury by inhibiting the decrease in gastric mucosal blood flow through attenuation of the activated neutrophil‐induced endothelial cell injury via inhibition of TNF‐α production. In addition, we show that serine protease activity of APC, rather than its anticoagulant activity, is critical for the protective mechanism(s) by which TNF‐α production could be inhibited.     

Ischemic preconditioning reduces intestinal epithelial apoptosis in rats

Recent experimental studies have described protective effect of ischemic preconditioning (IPC) on ischemia-reperfusion (I/R) injury of the intestine. We hypothesize that to reach a new point of view on the effect of IPC in intestinal barrier function, the relationship between I/R-induced mucosal injury and apoptosis must first be clarified. The present study was undertaken to investigate the role of IPC on intestinal apoptosis and probable contributions of bcl-2 expression to this process. We also investigated the effect of intestinal IPC on ileal malondyaldihyde levels. Forty-four male Wistar rats were randomized into four groups each consisting of 11 rats: sham-operated control, I/R group (30 min of superior mesenteric artery occlusion), IPC-I/R group (10 min of temporary artery occlusion prior before an ischemic insult of 30 min), and IPC alone group (10 min of preconditioning). Twenty-four hours later, ileum samples were obtained. Ileal malondyaldihyde levels were increased in the I/R group (31.9 +/- 18.8 vs. 106.8 +/- 39.8) but not in the IPC alone and IPC-I/R groups (38.1 +/- 13.6 and 44.7 +/- 12.7; P < 0.01). The number of apoptotic cells was significantly lower in IPC-I/R group than that of I/R group, and these findings were further supported by DNA laddering and M30 findings. Diminished bcl-2 expression observed in the ileal specimens of I/R group was prevented by IPC. Our results indicate that IPC may provide a protective effect on ileal epithelium and that this effect is probably the result of a significant increase in the expression of bcl-2 after the insult. The reversal of apoptosis by IPC might help preserving the vitality of intestinal structures that have a critical function, cessation of which often leads to multiorgan dysfunction syndrome.