Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice

AIM： To determine the effects of allopurinol, an inhibitor of xanthine oxidase, and apocynin, an inhibitor of NADPH oxidase, on oxidant stress and liver injury caused by hepatic ischemia/reperfusion （I/R） procedure in mice.
METHODS： Mice were pretreated with a xanthine oxidase inhibitor, allopurinol, or NADPH oxidase （NOX） inhibitor, apocynin before the hepatic I/R procedure. Then treated or untreated mice underwent the hepatic I/R procedure. The effects on hepatic injury and superoxide anions were determined after starting reperfusion.
RESULTS： A standard warm hepatic I/R procedure led to a marked increase in superoxide anion production as indicated by a superoxide anion tracer, MCLA. At the same time, the procedure caused profound acute liver injury, as indicated by elevated serum alanine aminotransferase and tumor necrosis factor-α levels, reduced liver glutathione levels and elevated malondialdehyde contents, as well as a high apoptotic cell count. All these changes were reversed by the use of apocynin or allopurinol prior to the hepatic I/R procedure.
CONCLUSION： Allopurinol and apocynin exerted protective effects on hepatic ischemia/reperfusion injury. The protection is associated with blocking the generationof superoxide anions during the hepatic I/R procedure by inhibiting xanthine oxidase and NADPH oxidase activity.     

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     

Ankaflavin ameliorates steatotic liver ischemiareperfusion injury in mice

BACKGROUND: It is well-known that steatotic liver is more susceptible to ischemia-reperfusion(I/R) injury during liver transplantation,liver resection and other liver surgeries. The increasing incidence of non-alcoholic fatty liver disease(NAFLD) decreases the availability of liver donors. Although steatotic liver is now accepted as a source of liver for transplantation,NAFLD exacerbates the liver injury after liver surgery. The present study was to investigate the protective role of ankaflavin in steatotic liver I/R injury.METHODS: The model of fatty liver mice was induced with high fat diet in four weeks,ankaflavin or vehicle(saline) was administrated by gavage once a day for one week. The animals were subjected to partial hepatic I/R. Blood samples were collected to measure serum aminotransferases. The liver tissues were used to examine liver steatosis,apoptosis of hepatocytes,hepatic oxidative stress,Kupffer cells and inflammatory cytokines. The effects of ankaflavin on inflammatory cytokines were evaluated in isolated Kupffer cells from the steatotic liver.RESULTS: Ankaflavin reduced liver steatosis in high fat diet mice. Compared with normal mice,I/R induced more damage to the mice with steatosis,such as hepatocyte apoptosis,inflammatory cytokines(TNF-α,IL-6 and IL-1β),serum aminotransferases and thiobarbituric acid reactive substances. Importantly,ankaflavin administration significantly attenuated these changes. In addition,ankaflavin significantly decreased the proliferation of Kupffer cells and the expression of TNF-α,IL-6 and IL-1β protein in isolated Kupffer cells stimulated by TNF-α.CONCLUSION: Ankaflavin has protective effects against I/R injury through anti-inflammatory,anti-oxidant and antiapoptotic mechanisms in fatty livers,these effects are at least partially mediated by inhibiting Kupffer cell functions.     

Protective role of adiponectin in a rat model of intestinal ischemia reperfusion injury

AIM: To determine the potential protective role of adiponectin in intestinal ischemia reperfusion(I/R) injury.METHODS: A rat model of intestinal I/R injury was established. The serum level of adiponectin in rats with intestinal I/R injury was determined by enzymelinked immunosorbent assay(ELISA). The serum levels of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α were also measured by ELISA. Apoptosis of intestinal cells was detected using the terminal deoxynucleotidyl transferase d UTP nick end labeling assay. The production of malondialdehyde(MDA) and superoxide dismutase(SOD) and villous injury scores were also measured.RESULTS: Adiponectin was downregulated in the serum of rats with intestinal I/R injury compared with sham rats. No significant changes in the expression of adiponectin receptor 1 and adiponectin receptor 2 were found between sham and I/R rats. Pre-treatment with recombinant adiponectin attenuated intestinal I/R injury. The production of pro-inflammatory cytokines,including IL-6, IL-1β, and TNF-α, in rats with intestinal I/R injury was reduced by adiponectin pre-treatment. The production of MDA was inhibited, and the release of SOD was restored by adiponectin pre-treatment in rats with intestinal I/R injury. Adiponectin pre-treatment also inhibited cell apoptosis in these rats. Treatment with the AMP-activated protein kinase(AMPK) signaling pathway inhibitor, compound C, or the heme oxygenase 1(HO-1) inhibitor, Snpp, attenuated the protective effects of adiponectin against intestinal I/R injury. CONCLUSION: Adiponectin exhibits protective effects against intestinal I/R injury, which may involve the AMPK/HO-1 pathway.     

Green tea polyphenols ameliorate non-alcoholic fatty liver disease through upregulating AMPK activation in high fat fed Zucker fatty rats

AIM To investigate protective effects and molecular mechanisms of green tea polyphenols(GTP) on nonalcoholic fatty liver disease(NAFLD) in Zucker fatty(ZF) rats.METHODS Male ZF rats were fed a high-fat diet(HFD) for 2 wk then treated with GTP(200 mg/kg) or saline(5 m L/kg) for 8 wk, with Zucker lean rat as their control. At the end of experiment, serum and liver tissue were collected for measurement of metabolic parameters, alanine aminotransferase(ALT) and aspartate aminotransferase(AST), inflammatory cytokines and hepatic triglyceride and liver histology. Immunoblotting was used to detect phosphorylation of AMP-activated protein kinase(AMPK) acetyl-Co A carboxylase(ACC), and sterol regulatory element-binding protein 1c(SREBP1c). RESULTS Genetically obese ZF rats on a HFD presented with metabolic features of hepatic pathological changes comparable to human with NAFLD. GTP intervention decreased weight gain(10.1%, P = 0.052) and significantly lowered visceral fat(31.0%, P 0.01). Compared with ZF-controls, GTP treatment significantly reduced fasting serum insulin, glucose and lipids levels. Reduction in serum ALT and AST levels(both P 0.01) were observed in GTP-treated ZF rats. GTP treatment also attenuated the elevated TNFα and IL-6 in the circulation. The increased hepatic TG accumulation and cytoplasmic lipid droplet were attenuated by GTP treatment, associated with significantly increased expression of AMPK-Thr172(P 0.05) and phosphorylated ACC and SREBP1c(both P 0.05), indicating diminished hepatic lipogenesis and triglycerides out flux from liver in GTP treated rats. CONCLUSION The protective effects of GTP against HFD-induced NAFLD in genetically obese ZF rats are positively correlated to reduction in hepatic lipogenesis through upregulating the AMPK pathway.     

Antithrombin reduces reperfusion-induced hepatic metastasis of colon cancer cells

AIM:To examine whether antithrombin (AT) couldprevent hepatic ischemia/reperfusion (I/R)-inducedhepatic metastasis by inhibiting tumor necrosis factor(TNF)-α-induced expression of E-selectin in rats.METHODS:Hepatic I/R was induced in rats and miceby clamping the left branches of the portal vein and thehepatic artery.Cancer cells were injected intrasplenically.The number of metastatic nodules was counted on day 7after I/R.TNF-α and E-selectin mRNA in hepatic tissue,serum fibrinogen degradation products and hepatictissue levels of 6-keto-PGF_(1α),a stable metabolite of PGI_2,were measured.RESULTS:AT inhibited increases in hepatic metastasisof tumor cells and hepatic tissue mRNA levels of TNF-αand E-selectin in animals subjected to hepatic I/R.Argatroban,a thrombin inhibitor,did not suppress anyof these changes.Both AT and argatroban inhibitedI/R-induced coagulation abnormalities.I/R-inducedincreases of hepatic tissue levels of 6-keto-PGF_(1α)were significantly enhanced by AT.Pretreatment withindomethacin completely reversed the effects of AT.Administration of OP-2507,a stable PGI_2 analog,showedeffects similar to those of AT in this model.Hepaticmetastasis in congenital AT-deficient mice subjectedto hepatic I/R was significantly increased compared tothat observed in wild-type mice.Administration of ATsignificantly reduced the number of hepatic metastasesin congenital AT-deficient mice.CONCLUSION:AT might reduce I/R-induced hepaticmetastasis of colon cancer cells by inhibiting TNF-α- induced expression of E-selectin through an increasein the endothelial production of PGI_2.These findingsalso raise the possibility that AT might prevent hepaticmetastasis of tumor cells if administered during theresection of liver tumors.     

Prophylaxis with carnosol attenuates liver injury induced by intestinal ischemia/reperfusion

AIM： To investigate the possible protective effects of carnosol on liver injury induced by intestinal ischemia reperfusion （I/R）.
METHODS： Rats were divided randomly into three experimental groups： sham, intestinal I/R and carnosol treatment （n = 18 each）. The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h. In the carnosol treatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg carnosol 1 h before the operation. At 2, 4 and 6 h after reperfusion, rats were killed and blood, intestine and liver tissue samples were obtained. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase （AST）, alanine aminotransferase （ALT） and interleukin （IL）-6 were measured. Liver tissue superoxide dismutase （SOD） and myeloperoxidase （IvIPO） activity were assayed. The liver intercellular adhesion molecule-1 （ICAM-1） and nuclear factor κB （NF-κB） were determined by immunohistochemical analysis and western blot analysis.
RESULTS： Intestinal I/R induced intestine and liver injury, characterized by histological changes, as well as a significant increase in serum AST and ALT levels. The activity of SOD in the liver tissue decreased after I/R, which was enhanced by carnosol pretreatment. In addition, compared with the control group, carnosol markedly reduced liver tissue MPO activity and serum IL-6 level, which was in parallel with the decreased level of liver ICAI-1 and NF-κB expression.
CONCLUSION： Our results indicate that carnosol pretreatment attenuates liver injury induced by intestinal I/R, attributable to the antioxidant effect and inhibition of the NF-κB pathway.     

Protective effect of curcumin against liver warm ischemia/reperfusion injury in rat model is associated with regulation of heat shock protein and antioxidant enzymes

AIM： To investigate the hypothesis that the protective effects of curcumin in hepatic warm ischemia/reperfusion （I/R） injury are associated with increasing heat shock protein 70 （Hsp70） expression and antioxidant enzyme activity. METHODS： Sixty Sprague-Dawley male rats were randomly divided into sham, I/R, C ＋ I/R groups. The model of reduced-size liver warm ischemia and reperfusion was used. Curcumin （50 mg/kg） was administered by injection through a branch of superior mesenteric vein at 30 min before ischemia in C ＋ I/R group. Five rats were used to investigate the survival during 1 wk after operation in each group. Blood samples and liver tissues were obtained in the remaining animals after 3, 12, and 24 h of reperfusion to assess serum alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, liver tissue NO2- ＋ NO3-, malondialdehyde （MDA） content, superoxide dismutase （SOD）, catalase （CAT）, nitricoxide synthase （NOS） and myeloperoxidase （MPO） activity, HspT0 expression and apoptosis ratio. RESULTS： Compared with I/R group, curcumin pretreatment group showed less ischemia/reperfusioninduced injury. CAT and SOD activity and Hsp70 expression increased significantly. A higher rate of apoptosis was observed in I/R group than in C ＋ I/R group, and a significant increase of MDA, NO2^- ＋ NO3^- and MPO level in liver tissues and serum transaminase concentration was also observed in I/R group compared to C ＋ I/R group. Curcumin also decreased the activity of inducible NO synthase （iNOS） in liver after reperfusion,but had no effect on the level of endothelial NO synthase （eNOS） after reperfusion in liver. The 7 d survival rate was significantly higher in C ＋ I/R group than in I/R group. CONCLUSION： Curcumin has protective effects against hepatic I/R injury. Its mechanism might be related to the overexpression of Hsp70 and antioxidant enzymes.     

Mechanism of combined use of vitamin D and puerarin in anti-hepatic fibrosis by regulating the Wnt/β-catenin signalling pathway

AIM To reveal the protective mechanism of the combined use of vitamin D and puerarin in the progression of hepatic fibrosis induced by carbon tetrachloride(CCl4).METHODS Eight-week-old male Wistar rats were randomly divided into a normal control group(C group), a CCl4 group(CCl4 group), a vitamin D group(V group), a puerarin group(P group), and a combined group of vitamin D and puerarin(V + P group), each of which contained ten rats. In this way, we built a rat model of CCl4-induced hepatic fibrosis with intervention by vitamin D, puerarin, or a combination of the two. After eight weeks, the mice were sacrificed to collect serum and liver specimens. Blood was collected to detect the hyaluronic acid(HA). We also measured hydroxyproline(Hyp) and prepared paraffin sections of liver. After Sirius red staining, the liver specimens were observed under a microscope. RT-PCR and western blot analysis were adopted to detect the mRNA and the proteinlevels of Collagen I, Collagen III, Wnt1, and β-catenin in the liver tissues, respectively.RESULTS Hepatic fibrosis was observed in the CCl4 group. In comparison, hepatic fibrosis was attenuated in the V, P, and V + P groups: the HA level in blood and the Hyp level in liver were reduced, and the mRNA levels of Collagen I, Collagen III, Wnt, and β-catenin in liver were also decreased, as well as the protein levels of Wnt1 and β-catenin. Among these groups, the V + P group demonstrated the greatest amelioration of hepatic fibrosis.CONCLUSION The combined application of vitamin D and puerarin is capable of alleviating CCl4-induced hepatic fibrosis of rats. As to the mechanism, it is probably because the combined use is able to silence the Wnt1/β-catenin pathway, suppress the activation of hepatic stellate cells, and reduce the secretion of collagen fibers, therefore improving the anti-hepatic fibrosis effect.     

Steatotic livers are susceptible to normothermic ischemiareperfusion injury from mitochondrial Complex-Ⅰ?dysfunction

AIM: To assess the effects of ischemic preconditioning(IPC, 10-min ischemia/10-min reperfusion) on steatotic liver mitochondrial function after normothermic ischemia-reperfusion injury(IRI).METHODS: Sixty male Sprague-Dawley rats were fed8-wk with either control chow or high-fat/high-sucrose diet inducing 60% mixed steatosis. Three groups(n = 10/group) for each dietary state were tested:(1) the IRI group underwent 60 min partial hepatic ischemia and 4 h reperfusion;(2) the IPC group underwent IPC prior to same standard IRI; and(3) sham underwent t h e s a m e s u r g e r y w i t h o u t I R I o r I P C. H e p a t i c mitochondrial function was analyzed by oxygraphs. Mitochondrial Complex-Ⅰ, Complex-Ⅱ enzyme activity, serum alanine aminotransferase(ALT), and histological injury were measured.RESULTS: Steatotic-IRI livers had a greater increase in ALT(2476 ± 166 vs 1457 ± 103 IU/L, P 0.01) and histological injury following IRI compared to the lean liver group. Steatotic-IRI demonstrated lower Complex-Ⅰ?activity at baseline [78.4 ± 2.5 vs 116.4 ± 6.0 nmol/(min.mg protein), P 0.001] and following IRI [28.0 ± 6.2 vs 104.3 ± 12.6 nmol/(min.mg protein), P 0.001]. Steatotic-IRI also demonstrated impaired Complex-Ⅰ?function post-IRI compared to the lean liver IRI group. Complex-Ⅱ activity was unaffected by hepatic steatosis or IRI. Lean liver mitochondrial function was unchanged following IRI. IPC normalized ALT and histological injury in steatotic livers but had no effect on overall steatotic liver mitochondrial function or individual mitochondrial complex enzyme activities. CONCLUSION: Warm IRI impairs steatotic liver Complex-Ⅰ?activity and function. The protective effects of IPC in steatotic livers may not be mediated through mitochondria.     

落新妇甙对热缺血再灌注损伤肝脏肿瘤坏死因子α与白细胞介素-10表达的影响

Objective To investigate the effects of astilbin on the expressions of TNF α and IL-10 during liver warm ischemia-reperfusion injury. Methods C57BL/ 6 mice were randomly divided into 4 groups (n = 8): sham-operated group (Sham), model control group(I/R), low dosage of astilbin treatment group (10 mg/kg) and high dosage of astilbin (40 mg/kg) treatment group. The treatment group mice were intraperitoneally injected with 10 or 40 mg/kg astilbin 24 hours and one hour before Ischemia, the hepatic ischemia-reperfusion model were thus established. After jn90 of min ischemia and 6 h reperfusion of the partial hepatic lobe, the expressions of TNF α and IL-10 in liver tissues collected from the experimental groups were detected by Western blot and semiquantitative RT-PCR. Results The expression of TNF a protein in liver tissues gradually decreased in treatment groups (low and high dosages of astilbin treatment groups) as compared to the I/R model control group. Similar results were observed in the mRNA expressions of these genes as determined by semiquantitative RT-PCR (P < 0.05 for low dosage group; P < 0.01 for high dosage group). Compared with the I/R model control group, the expression of IL-10 was increased in both treatment groups (low dosage group P < 0.05; large dosage group P < 0.01). Conclusion Treatment with astilbin decreases TNF α expression but induces IL-10 expression in liver during warm ischemia-reperfusion injury.     

落新妇甙对热缺血再灌注损伤肝脏肿瘤坏死因子α与白细胞介素-10表达的影响

Objective To investigate the effects of astilbin on the expressions of TNF α and IL-10 during liver warm ischemia-reperfusion injury. Methods C57BL/ 6 mice were randomly divided into 4 groups (n = 8): sham-operated group (Sham), model control group(I/R), low dosage of astilbin treatment group (10 mg/kg) and high dosage of astilbin (40 mg/kg) treatment group. The treatment group mice were intraperitoneally injected with 10 or 40 mg/kg astilbin 24 hours and one hour before Ischemia, the hepatic ischemia-reperfusion model were thus established. After jn90 of min ischemia and 6 h reperfusion of the partial hepatic lobe, the expressions of TNF α and IL-10 in liver tissues collected from the experimental groups were detected by Western blot and semiquantitative RT-PCR. Results The expression of TNF a protein in liver tissues gradually decreased in treatment groups (low and high dosages of astilbin treatment groups) as compared to the I/R model control group. Similar results were observed in the mRNA expressions of these genes as determined by semiquantitative RT-PCR (P < 0.05 for low dosage group; P < 0.01 for high dosage group). Compared with the I/R model control group, the expression of IL-10 was increased in both treatment groups (low dosage group P < 0.05; large dosage group P < 0.01). Conclusion Treatment with astilbin decreases TNF α expression but induces IL-10 expression in liver during warm ischemia-reperfusion injury.     

N-acetylcysteine inhibits activation of toll-like receptor 2 and 4 gene expression in the liver and lung after partial hepatic ischemia-reperfusion injury in mice

BACKGROUND:Toll-like receptor 2 and 4(TLR2/4)may play important roles in ischemia-reperfusion(I/R)injury, and N-acetylcysteine(NAC)can prevent the generation of reactive oxygen species(ROS)induced by I/R injury.This study aimed to investigate the changes in TLR2/4 gene expression in the liver and lung after I/R injury with or without NAC pretreatment. METHODS:BALB/c mice were used in a model of partial hepatic I/R injury and randomly assigned to a sham-operated control group(SH),a hepatic ischemia/ reperfusion group(I/R)or a NAC pretreated,hepatic I/R group(I/R-NAC).The levels of TNF-αin the portal vein and plasma alanine aminotransferase(ALT)were measured at 1 and 3 hours after reperfusion.The lung wet-to-dry ratio was measured,and the expression of TLR2/4 mRNA and protein in the liver and lung were assessed with RT-PCR and Western blotting at the same time points. RESULTS:Compared with the I/R group,the expression of TLR2/4 mRNA and protein in the liver and lung in the I/R-NAC group was decreased at the same time point (P<0.05).The levels of portal vein TNF-αand plasma ALT increased continuously in the I/R group at 1 and 3 hours of reperfusion compared with the SH group;however,they declined significantly in the group pretreated with NAC (P<0.05).The extent of lung edema was relieved in the I/ R-NAC group compared with the I/R group(P<0.05).CONCLUSIONS:TLR2/4 was activated in the liver and lung in the process of partial hepatic I/R injury.NAC inhibited the activation of TLR2/4 and the induction of TNF-α resulting from I/R injury via modulating the redox state, thus it may mitigate liver and lung injury following partial hepatic I/R in mice.     

Suppressive effects of 171β-estradiol on hepatic fibrosis in CCI4-induced rat model

AIM: To investigate the pathway via which 17β-estradio(β-Est) exerts suppressive effects on rat hepatic fibrosis.METHODS: In vivo study was done in CCI4-induced female hepatofibrotic rats. Fibrosis-suppressive effect of β-Est(20 μg/kg&#183;d) was evaluated in intact and ovariectomized rat models. Six weeks after the treatment, all the rats were sacrificed and specimens of serum or liver tissue were collected for the studies. Serum liver enzymes,fibrosis markers and estradiol levels were determined by standard enzymatic methods, ELISA and RIA, respectively.Degrees of fibrosis and areas of hepatic stellate cells(HSCs) positive for alpha-smooth muscle actin (a-SMA) in the liver were determined by van Gieson (VG) stain and immunohistochemistry. In vitro studies, HSCs were isolated by a combination of pronase-collagenase perfusion and density gradient centrifugation. First-passage HSCs were randomly divided into 10 groups, and different concentrations of β-Est, 2-hydroxyestradiol (2OHE) or 2-methoxyestradiol(2MeOE) were separately added to the cell groups. After incubation for 72 h, the degree of cell proliferation, collagen production, ~-SMA or estrogen receptor (ER) expression was determined by MTT assay, ELISA and immunohistochemistry,respectively.RESULTS: β-Est treatment reduced aspartate aminotransfer-ase (AST), alanine aminotransferase (ALT), hyaluronic acid(HA) and type IV collagen (C IV) in sera, suppressed hepatic collagen content, decreased the areas of HSCs positive for α-SMA significantly in both intact and ovariectomized female hepatofibrotic rats. There was a negative correlation between the percentage of fibrotic area of liver tissue and the serum estradiol level; the calculated correlation coefficient was -0.57 (P&lt;0.01). β-Est and its metabolites concentration-dependently (10^-9 mol/L-10^-7 mol/L) inhibited HSC proliferation and collagen synthesis. At the concentration of 10^-7 mol/L, they could inhibit α-SMA expression. The order of potency was 2MeOE&gt;2OHE&gt;β-Est.CONCLUSION: β-Est may suppress hepatic fibrosis probably via its biologically active metabolites.     

Resveratrol attenuates oxidative stress and histological alterations induced by liver ischemia/reperfusion in rats

AIM： To investigate the effects of resveratrol on liver ischemia/reperfusion （I/R） injury in rats. METHODS： A total of 40 male Sprague-Dawley rats weighing 240-290 g were randomized into four groups of ten： （1） controls： data from unmanipulated animals; （2） sham group： rats subjected to the surgical procedure, except for liver I/R, and given saline; （3） I/R group： rats underwent liver ischemia for 45 rain followed by reperfusion for 45 rain; （4） I-R/Resveratrol group： rats pretreated with resveratrol （10 umol/L, iv）. Liver tissues were obtained to determine antioxidant enzyme levels and for biochemical and histological evaluation. RESULTS： Plasma aminotransferase activities were higher in the I/R group than in the I-R/Resveratrol group. Malondialdehyde levels and the hepatic injury score decreased, while superoxide dismutase, catalase, and glutathione peroxidase levels increased in group 4 compared to group 3. In group 4, histopathological changes were significantly attenuated in resveratroltreated livers. CONCLUSION： These results suggest that resveratrol has protective effects against hepatic I/R injury, and is a potential therapeutic drug for ischemia reperfusionrelated liver injury.     

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.     

Experimental study of osthole on treatment of hyperlipidemic and alcoholic fatty liver in animals

AIM: To evaluate the effects of osthole on fatty liver, and investigate the possible mechanism. METHODS: A quail model with hyperlipidemic fatty liver and rat model with alcoholic fatty liver were set up by feeding high fat diet and alcohol, respectively. These experimental animals were then treated with osthole 5-20 mg/kg for 6 wk, respectively. Whereafter, the lipid in serum and hepatic tissue, and coefficient of hepatic weight were measured. RESULTS: After treatment with osthole the levels of serum total cholesterol (TC), triglyceride (TG), lower density lipoprotein-cholesterol (LDL-C), coefficient of hepatic weight, and the hepatic tissue contents of TC and TG were significantly decreased. The activity of superoxide dismutase (SOD) in liver was improved. In alcohol-induced fatty liver rats, the level of malondialdehyde (MDA) in liver was decreased. In high fat-induced fatty liver quails, glutathione peroxidase (GSH-PX) in liver was significantly improved. The histological evaluation of liver specimens demonstrated that the osthole dramatically decreased lipid accumulation. CONCLUSION: These results suggested that osthole had therapeutic effects on both alcohol and high fat-induced fatty liver. The mechanism might be associated with its antioxidation.     

Nuclear factor-KB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-κB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-κB decoy ODNs or scrambled ODNs. NF-κB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-κB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-α, IFN-γ and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-κB activation in liver graft was induced in a time-dependent manner, and NF-κB remained activated for 16 h after graft reperfusion. NF-κB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-κB decoy ODNs significantly suppressed NF-κB activation as well as mRNA expression of TNF-α, IFN-γ and ICAM-1 in the liver graft. The hepatic NF-κB DNA binding activity [presented as integral optical density (IOD) value] in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16±0.78 vs 36.78 ±6.35 and 3.06±0.84 vs 47.62± 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-α, IFN-γ and ICAM-1 [presented as percent of p-actin mRNA (%)] in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31 ±3.48 vs 46.37±10.65 and 7.46± 3.72 vs 74.82±12.25 for hepatic TNF-a mRNA, 5.58±2.16 vs 50.46±9.35 and 6.47±2.53 vs 69.72±13.41 for hepatic IFN-y mRNA, 6.79 ±2.83 vs 46.23±8.74 and 5.28±2.46 vs 67.44±10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-κB decoy ODNs almost completely abolished the increase of serum level of TNF-α and IFN-γ induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-α and IFN-γ in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7±13.6 vs 176.7±15.8 and 48.4±15.1 vs 216.8±17.6 for TNF-α level, 31.5±12.1 vs 102.1±14.5 and 40.2±13.5 vs 118.6±16.7 for IFN-γ level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-κB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17±0.07 vs 1.12 ±0.25 and 0.46±0.17 vs 1.46±0.32 for hepatic MPO content, 71.7±33.2 vs 286.1±49.6 and 84.3±39.7 vs 467.8±62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P< 0.001). CONCLUSION: The data suggest that NF-κB decoy ODNs protects against I/R injury in liver graft by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.     

Endogenous leptin fluctuates in hepatic ischemia/reperfusion injury and represents a potential therapeutic target

AIM: To evaluate the role of leptin in the internal disorders during hepatic ischemia/reperfusion injury. METHODS: A rat model of 70% hepatic ischemia/reperfusion injury was established, with groups of shamoperation (Sham), 60 min ischemia/60 min reperfusion (I60’R60’), I60’R150’, I60’R240’ and I60’R360’. Serum leptin was detected by a self-produced radioimmunoassay; serum glucose, total anti-oxidation capacity, myeloperoxidase, alanine transaminase and diamine oxidase were determined by relevant kits, while histologicalalterations and protein levels of leptin in the lung, liver and duodenum were examined by hematoxylin-eosin staining and immunohistochemistry. Spearman’s rank correlation between leptin and other variables or grading of tissue impairment were analyzed simultaneously. RESULTS: Serum leptin in I60’R360’ was significantly higher than in Sham and I60’R240’ groups (both P < 0.05), serum glucose in I60’R360’ was higher than in Sham and I60’R150’ (both P < 0.05), and serum total anti-oxidation capacity in I60’R240’ and I60’R360’ were higher than in Sham (both P < 0.05) and I60’R150’ groups (both P < 0.01). Serum myeloperoxidase in groups of I60’R240’ and I60’R360’ were lower than in I60’R150’group (both P < 0.05), serum alanine transaminase in the four reperfusion groups were higher than in the Sham group (all P < 0.05), while serum DAO in I60’R360’ was lower than in I60’R60’ (P < 0.05). Histological impairment in the lung, liver and duodenum at the early phase of this injury was more serious, but the impairment at the later phase was lessened gradually. Protein levels of leptin in the lung in the four reperfusion groups were significantly lower than in the Sham group (all P < 0.01), decreasing in the order of I60’R150’, I60’ R60’, I60’R360’ and I60’R240’; the levels in the liver in I60’R60’ and I60’R240’ were higher than in the Sham group (both P < 0.01), while the levels in I60’R240?