Effect of nuclear factor kappa B on intercellular adhesionrnolecule-1 expression and neutrophil infiltration in lung injury induced by intestinal ischemia/reperfusion in rats

AIM: To investigate the role of nuclear factor kappa B(NF-κB) in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R), and its effect on intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil infiltration.METHODS: Twenty-four Wistar rats were divided randomly into control, I/R and pyrrolidine dithiocarbamate (PDTC) treatment groups, n = 8 in each. I/R group and PDTC treatment group received superior mysenteric artery (SMA) occluding for 1 h and reperfusion for 2 h. PDTC group was administrated with intraperitoneal injection of 2% 100 mg/kg PDTC 1 h before surgery. Lung histology and bronchia alveolus lung fluid (BALF) protein were assayed. Serum IL-6, lung malondialdehyde (MDA) and myeloperoxidase (MPO) as well as the expression level of NF-κB and ICAM-1 were measured.RESULTS: Lung injury induced by intestinal I/R, was characterized by edema, hemorrhage and neutrophil infiltration as well as by the significant rising of BALF protein. Compared to control group, the levels of serum IL-6 and lung MDA and MPO increased significantly in I/R group (P=0.001). Strong positive expression of NF-κB p65 and ICAM-1 was observed. After the administration of PDTC, the level of serum IL-6, lung MDA and MPO as well as NF-κB and ICAM-1 decreased significantly(P＜ 0.05) when compared to I/R group.CONCLUSION: The activation of NF-κB plays an important role in the pathogenesis of lung injury induced by intestinal I/R through upregulating the neutrophil infiltration and lung ICAM-1 expression. PDTC as an inhibitor of NF-κB can prevent lung injury induced by intestinal I/R through inhibiting the activity of NF-κB.     

Nuclear factor-κB 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-γand 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-y and ICAM-1 [presented as percent of β-actin mRNA(%)] in the NF-κBdecoy 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±12.25for hepatic TNF-α mRNA, 5.58±2.16 vs 50.46±9.35 and6.47±2.53 vs 69.72±13.41 for hepatic IFN-γ mRNA, 6.79±2.83 vs 46.23±8.74 and 5.28±2.46 vs 67.44±10.12for 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-κB 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 vs216.8±17.6 for TNF-α level, 31.5±12.1 vs 102.1±14.5and 40.2±13.5 vs 118.6±16.7 for IFN-γ level after 4 and8 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 vs467.8±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.     

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     

Effect of nuclear factor kappa B on intercellular adhesion molecule-1 expression and neutrophil infiltration in lung injury induced by intestinal ischemia/reperfusion in rats

AIM:To investigate the role of nuclear factor kappa B(NF-κB) in the pathogenesis of lung injury induced byintestinal ischemia/reperfusion (I/R),and its effect onintercellular adhesion molecule-1 (ICAM-1) expressionand neutrophil infiltration.METHODS:Twenty-four Wistar rats weredivided randomly into control,I/R and pyrrolidinedithiocarbamate (PDTC) treatment groups,n=8 ineach.I/R group and PDTC treatment group receivedsuperior mysenteric artery (SMA) occluding for 1 h andreperfusion for 2 h.PDTC group was administrated withintraperitoneal injection of 2% 100 mg/kg PDTC 1 hbefore surgery.Lung histology and bronchia alveoluslung fluid (BALF) protein were assayed.Serum IL-6,lungmalondialdehyde (MDA) and myeloperoxidase (MPO) aswell as the expression level of NF-κB and ICAM-1 weremeasured.RESULTS:Lung injury induced by intestinal I/R,wascharacterized by edema,hemorrhage and neutrophilinfiltration as well as by the significant rising of BALFprotein.Compared to control group,the levels of serumIL-6 and lung MDA and MPO increased significantly in I/Rgroup (P=0.001).Strong positive expression of NF-κBp65 and ICAM-1 was observed.After the administrationof PDTC,the level of serum IL-6,lung MDA and MPOas well as NF-κB and ICAM-1 decreased significantly(P<0.05) when compared to I/R group. CONCLUSION:The activation of NF-kB plays animportant role in the pathogenesis of lung injury inducedby intestinal I/R through upregulating the neutrophilinfiltration and lung ICAM-1 expression.PDTC as aninhibitor of NF-kB can prevent lung injury induced byintestinal I/R through inhibiting the activity of NF-kB.     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats.METHODS: Thirty-two healthy adult Sprague-Dawley (SD)rats were randomly divided into four groups of eight each:normal, NS, model I, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks,the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO)activities assayed. NF-κB p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-αand ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κBp65 and other parameters were analyzed.RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25±1.39 and 6.24±1.04,respectively), which were in accordance with the significantly elevated MPO activity (1.69±0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model Ⅱ group were higher than that of normal control (9.7±1.96 vs1.7±0.15, 84.09±14.52 vs16.03±6.21,77.69±8.09 vs13.41±4.91 P＜0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities,macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152,0.8247; P＜0.05).CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

NF-kB在大鼠肝缺血再灌注损伤中的活化及意义

目的探讨NFkB在肝缺血再灌注损伤过程中的作用。方法选择健康雌性Wistar大鼠24只,随机分为手术对照组,肝缺血90min组,肝缺血90min/再灌注120min组,每组8只。常规方法观察肝脏组织学改变,检测血清酶学水平和肝组织中髓过氧化物酶(MPO)含量,采用sABC免疫组织化学方法测定肝组织中NFkB的活化程度。结果手术对照组肝组织形态正常,无NFkB活化,肝功能酶学和MPO正常水平;缺血组动物肝细胞索排列紊乱,肝小叶变形,肝细胞和内皮细胞普遍水肿变性,NFkB呈中重度活化,血清酶学和MPO水平升高(P<0.01);肝缺血/再灌注组肝组织在缺血组改变基础上合并中央区局灶性肝细胞坏死,血窦内微血栓形成,汇管区中性粒细胞浸润,NFkB活化最为明显,血清酶学和MPO升高最为显著(P<0.01)。结论肝缺血再灌注时,NFkB被活化,使中性粒细胞组织浸润,对肝脏缺血再灌注损伤病理过程起到重要的作用。     

In vivo suppressive effect of nuclear factor-κB inhibitor on neutrophilic inflammation of grafts after orthotopic liver transplantation in rats

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC), a novel nuclear factor-κB (NF-κB) inhibitor, on expression of multiple inflammatory mediators and neutrophilic inflammation of cold preserved grafts after rat liver transplantation and its significance.METHODS: Orthotopic liver transplantation (OLT) was performed after 24 h of cold storage using University of Wisconsin solution with varied concentrations of PDTC. We determined the time course of NF-κB activation and expression of multiple inflammatory signals, such as tumor necrosis factor-α (TNF-α), cytokine-inducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1(ICAM-1) by ElISA methods. Serum alanine aminotransferase (ALT), intrahepatic myeloperoxidase (MPO)/WBC (ameasure of neutrophil accumulation) and Mac-1 expression (a measure of circulating neutrophil activity) were also evaluated.RESULTS: PDTC decreased NF-κB activation induced by prolonged cold preservation in a dose dependent manner (from 20 mmol/L to 60 mmol/L), diminished TNF-α, CINC, ICAM-1 proteins in the grafts, and reduced the expression of increases in plasma TNF-α levels induced by prolonged cold preservation. Neutrophilic inflammation of the graft was significantly suppressed after preservation with PDTC (P<0.05). The total neutrophil accumulation in PDTC (40 mmol/L) group (7.04±0.97) was markedly reduced compared to control group (14.07±1.31) (P<0.05). Mac-1expression was significantly reduced in PDTC (40 mmol/L)group (181±11.3%) compared with the control group (281±13.2%) (P<0.05) at 6 h after reperfusion. Furthermore,PDTC inhibited the increased serum ALT levels after liver transplantation.CONCLUSION: PDTC can inhibit B NF-κB activation and expression of the inflammatory mediators, which are associated with improved graft viability via inhibiting intrahepatic neutrophilic inflammation. Our study suggests that a therapeutic strategy directed at inhibition of NF-κB activation in the transplanted liver might be effective in reducing intrahepatic neutrophilic inflammation, and would be beneficial to cold preserved grafts.     

Effect of cold-ischemia time on nuclear factor-κB activation and inflammatory response in graft after orthotopic liver transplantation in rats

AIM: To study the mechanism and effect of nuclear factorκB (NF-κB) activation and inflammatory response on the extended cold-preserved graft injury after orthotopic liver transplantation (OLT).METHODS: OLT was performed in rats with varying time of cold ischemia grafts (6, 18 and 24 h in University Wisconsin solution at 4 ℃). We determined the time of NF-κB activation and expression of tumor necrosisfactor-α (TNF-α), cytokineinducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1 (ICAM-1) within 6 h after reperfusion.Serum alarming aminotransferase (ALT), neutrophil sequestration, circulating neutrophil CD11b and L- selectin expression were also evaluated.RESULTS: The accumulation of neutrophils in the graft was significantly increased in the 18 h and 24 h cold-ischemia groups within 0.5 h after reperfusion, compared with the 6 h group. But the strongly activated neutrophils was slightly increased at 2 h after reperfusion and remained at high levels 4 h after reperfusion, which was synchronized with the common situation of recipients after transplantation.Prolonged cold-preservation did not affect neutrophil accumulation and activation. NF-κB activation preceded the expression of TNF-α, CINC, and ICAM-1 in the liver, which was significantly increased with prolonged cold preservation.In prolonged cold preserved grafts, prominently elevated NF-κB activation occurred at 0.5 h and 1 h, compared with that at 2 h after reperfusion, which was consistent with greatly increased intrahepatic TNF-α response.CONCLUSION: NF-κB activation is correlated with the expression of TNF-α, CINC, and ICAM-1 in vivo in OLT rats.Extended cold preservation of grafts might up-regulate TNF-α,CINC, and ICAM-1 expression in the grafts, most probably through elevated NF-κB activation, and might contribute to neutrophil infiltration in the grafts after reperfusion. Elevated NF-κB activity is harmful to inflammatory response in the grafts, and inhibited NF-κB activity might protect against early graft injury after liver transplantation.     

Hepatic preconditioning of doxorubicin in stop-flow chemotherapy:NF-κB/IκB-α pathway and expression of HSP72

AIM: To provide hepatic protection through administration of doxorubicin before stop-flow chemotherapy (SFC) and to investigate the expression of heat shock protein 72 (HSP72) and role of nuclear factor kappa B (NF-κB) in this effect. METHODS: The hepatic preconditioning of doxorubicin was established in a porcine model by injection of doxorubicin (1 mg/kg) before SFC. The experimental animals were randomized into two groups: groups receiving doxorubicin (DOX) and normal saline (NS). Serial serum and tissue samples were taken from both groups to evaluate the protection of doxorubicin. Western blot and immunoprecipitation were applied to detect the expression of HSP72, NF-κB p65 protein, inhibitor κB-α (IκB-α) and phosphorylated IκB-α as well. The expression of tumor necrosis factor α (TNF-α) was estimated by semiquantitative RT-PCR. And the extent of the hepatic injury was estimated with the level of serum aminotransferases. RESULTS: An abundance production of HSP72 in porcine liver was observed after 24 h of intravenous administration of doxorubicin, but without any change in the expression of NF-κB p65 subunit in cytoplasm. NF-κB p65 subunit accumulated in nuclei at the end of SFC and reached its highest level at 30 min after the restoration of the abdominal circulation and decreased gradually during the 6 h after SFC in NS group, while there was little change in DOX group. There was also a slight decrease of IκB-α at 30 min after the restoration of the abdominal circulation in NS group accompanying with the appearance of phosphorylated IκB-α. The expression of TNF-α was significantly higher in NS group than that in DOX group (average 1.40±0.17 vs 0.62±0.22, P<0.01) at serial time points after SFC. Serum ALT and AST levels of NS group were higher after 24 h than those of DOX group (93.2±7.8 IU/L vs 53.3±13.9 IU/L, 217.0±29.4 IU/L vs 155.0±15.6 IU/L for ALT and AST respectively, P<0.05) and after 48 h than those of DOX group (66.6±18.1 IU/L vs 43.3±16.7 IU/L, 174.4±21.3 IU/L vs 125.7±10.5 IU/L for ALT and AST respectively, P<0.05). CONCLUSION: Doxorubicin renders the liver to be tolerant to the hepatic influence in SFC in a porcine model through the NF-κB/IκB-αpathway with the expression of HSP72.     

Effect of notoginsenoside R1 on hepatic microcirculation disturbance induced by gut ischemia and reperfusion

AIM： To assess the effect of notoginsenoside R1 on hepatic microcirculatory disturbance induced by gut ischemia/reperfusion （I/R） in mice. METHODS： The superior mesenteric artery （SMA） of C57/BL mice was ligated for 15 min to induce gut ischemia followed by 30-rain reperfusion. In another set of experiments, R1 was continuously infused （10 mg/kg per hour） from 10 min before I/R until the end of the investigation to study the influence of R1 on hepatic microcirculatory disturbance induced by gut I/R. Hepatic microcirculation was observed by inverted microscopy, and the vascular diameter, red blood cell （RBC） velocity and sinusoid perfusion were estimated. Leukocyte rolling and adhesion were observed under a laser confocal microscope. Thirty and 60 min after reperfusion, lactate dehydrogenase （LDH）, alanine aminotransferase （ALl＇） and aspartate transaminase （AST） in peripheral blood were determined. The expression of adhesion molecules CD11b/CD18 in neutrophils and tumor necrosis factor- alpha （TNF-α）, interleukin-6 （IL-6） and monocyte chemotactic protein-1 （MCP-1） in plasma were evaluated by flow Oltometry. E-selectin and intercellular adhesion molecule-1 （ICAM-1） in hepatic tissue were examined by immunofluorescence.RESULTS： After gut I/R, the diameters of terminal portal venules and central veins, RBC velocity and the number of perfused sinusoids were decreased, while the leukocyte rolling and adhesion, the expression of E-selectin in hepatic vessels and CD18 in neutrophils, IL-6, MCP-1, LDH, ALT and AST were increased. R1 treatment attenuated these alterations except for IL-6 and MCP-1. CONCLUSION： R1 prevents I/R-induced hepatic microcirculation disturbance and hepatocyte injury, The effect of R1 is related to its inhibition of leukocyte rolling and adhesion by inhibiting the expression of E-selectin in endothelium and CD18 in neutrophils.     

Melatonin protects liver from intestine ischemia reperfusion injury in rats

AIM： To investigate the protective effect of melatonin on liver after intestinal ischemia-reperfusion injury in rats. METHODS： One hundred and fifty male Wistar rats, weighing 190-210 g, aged 7 wk, were randomly divided into melatonin exposure group, alcohol solvent control group and normal saline control group. Rats in the melatonin exposure group received intraperitoneal （IP） melatonin （20 mg/kg） 30 min before intestinal ischemia-reperfusion （IR）, rats in the alcohol solvent control group received the same concentration and volume of alcohol, and rats in the normal saline control group received the same volume of normal saline. Serum samples were collected from each group 0.5, 1, 6, 12, and 24 h after intestinal IR. Levels of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were measured with an auto-biochemical analyzer. Serum TNF-α was tested by enzyme-linked immunosorbent assay （ELISA）. Malondialdehyde （MDA） in liver was detected by colorimetric assay. Pathological changes in liver and immunohistochemical straining of ICAM-1 were observed under an optical microscope. RESULTS： The levels of ALT measured at various time points after intestinal IR in the melatonin exposure group were significantly lower than those in the other two control groups （P 〈 0.05）. The serum AST levels 12 and 24 h after intestinal IR and the ICAM-1 levels （%） 6, 12 and 24 h after intestinal IR in the melatonin exposure group were also significantly lower than those in the other two control groups （P 〈 0.05）. CONCLUSION： Exotic melatonin can inhibit the activity of ALT, AST and TNF-α, decrease the accumulation of MDA, and depress the expression of ICAM-1 in liver after intestinal IR injury, thus improving the liver function.     

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.     

人脂肪干细胞来源外泌体对急性肝衰竭大鼠炎症反应的抑制作用及相关机制

目的:研究人脂肪干细胞来源外泌体(ADSCs-Exos)对急性肝衰竭大鼠炎症反应的抑制作用及相关机制。方法:脂肪组织来自整形手术中无菌切除的医疗废弃物,分离培养人脂肪干细胞(ADSCs),收集培养上清,采用密度梯度离心法提取外泌体(Exos)。45只SD大鼠随机分为健康组(10只)和造模组(35只),健康组大鼠未做特殊处理,造模组大鼠采用一次性D-Gal/LPS诱导法建立急性肝衰竭模型,28只大鼠建模成功,随机分为疾病组(9只)、Exos组(9只)和阳性对照组(10只)。Exos组大鼠尾静脉注射ADSCs-Exos 100μg,阳性对照组大鼠尾静脉注射甘草酸二铵注射液15 mg/kg,健康组与疾病组大鼠尾静脉注射200μl PBS溶液,尾静脉注射1次/周,持续12周。观察实验大鼠肝组织病理变化,测定实验大鼠血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、白介素-10(IL-10)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,肝组织Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子κB(NF-κB)蛋白相对表达量。结果:与健康组比较,疾病组、Exos组、阳性对照组大鼠血清AST、ALT、IL-6、TNF-α水平均升高,IL-10水平均降低(P<0.05);与疾病组比较,Exos组、阳性对照组大鼠血清AST、ALT、IL-6、TNF-α水平均降低(P<0.05),IL-10水平均升高(P<0.05);与阳性对照组比较,Exos组大鼠血清AST、ALT、IL-6、TNF-α水平均升高(P<0.05),IL-10水平降低(P<0.05)。疾病组大鼠肝组织有大片坏死灶,并伴有明显门静脉充血及凝血现象;阳性对照组大鼠肝组织有少量小面积、散在坏死灶,少量多型核白细胞浸润;Exos组大鼠肝组织坏死灶数量减少、面积缩小,门静脉充血及凝血现象改善。与健康组比较,疾病组、Exos组、阳性对照组大鼠肝组织TLR4、MyD88、NF-κB蛋白相对表达量均升高(P<0.05);与疾病组比较,Exos组、阳性对照组大鼠肝组织TLR4、MyD88、NF-κB蛋白相对表达量均降低(P<0.05);与阳性对照组比较,Exos组大鼠肝组织TLR4、MyD88、NF-κB蛋白相对表达量均升高(P<0.05)。结论:ADSCs-Exos可改善急性肝衰竭大鼠肝功能,抑制炎症反应,减轻肝组织病理变化,推测其作用机制与抑制TLR4信号通路有关。     

Nigella sativa relieves the deleterious effects of ischemia reperfusion injury on liver

AIM： To determine whether Nigella sativa prevents hepatic ischemia-reperfusion injury to the liver. METHODS： Thirty rats were divided into three groups as sham （Group 1）, control （Group 2）, and Nigella sativa （NS） treatment group （Group 3）. All rats underwent hepatic ischemia for 45 min followed by 60 min period of reperfusion. Rats were intraperitoneally infused with only 0.9% saline solution in group 2. Rats in group 3 received NS （0.2 mL/kg） intraperitoneally, before ischemia and before reperfusion. Blood samples and liver tissues were harvested from the rats, and then the rats were sacrifi ced. Serum aspartate aminotransferase （AST）, alanine aminotransferase （ALT）, and lactate dehydrogenase （LDH） levels were determined. Total antioxidant capacity （TAC）, catalase （CAT）, total oxidative status （TOS）, oxidative stress index （OSI） and my-eloperoxidase （MPO） in hepatic tissue were measured. Also liver tissue histopathology was evaluated by light microscopy. RESULTS： The levels of liver enzymes in group 3 weresignifi cantly lower than those in the group 2. TAC in liver tissue was significantly higher in group 3 than in group 2. TOS, OSI and MPO in hepatic tissue were signifi cantly lower in group 3 than the group 2. Histological tissue damage was milder in the NS treatment group than that in the control group. CONCLUSION： Our results suggest that Nigella sativa treatment protects the rat liver against to hepatic ischemia-reperfusion injury.     

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,     

外源性H2S通过核因子-κB介导的炎性反应通路减轻大鼠局灶性脑缺血再灌注损伤

目的 探讨外源性硫化氢(hy drogen sulfide,H2S)对脑缺血再灌注后脑损伤和炎性反应的影响.方法 48只雄性SD大鼠随机分为假手术组、缺血再灌注(ischemia/rep erfusion,I/R)组、H2S 30 ppm组和H2S 60 ppm组(1 ppm=1 mg/L),每组12只.采用大脑中动脉闭塞法建立大鼠局灶性脑缺血2h再灌注24 h模型.再灌注24 h后应用胶带剔除实验进行神经功能评价,应用2,3,5-三苯基氯化四氮唑染色法测定脑梗死体积百分比,采用酶联免疫吸附法测定缺血脑组织白细胞介素(interleukin,IL)-1β和IL-6水平,采用蛋白质印迹法测定缺血脑组织诱导型一氧化氮合酶(induciblenitric oxide synthase,iNOS)和细胞间黏附分子-1(intercellular cell adhesion molecule-1,ICAM-1)的表达及核因子-κB(nuclear factor-κB,NF-κB)的转位激活情况.结果 与I/R组比较,吸入H2S能以剂量依赖性方式缩短胶带剔除所需的时间[I/R组与H2S 30 ppm组和H2S 60 ppm组比较:180 s对130(113～157)s对110(87～138) s;P ＜0.05],缩小脑梗死体积(48.8％±9.1％对23.3％±5.1％对17.3％±3.5％;P＜0.05)],下调缺血脑组织IL-1β[(39.53±6.02)pg/mg蛋白对(30.17±3.46)pg/mg蛋白对(22.69±6.09)pg/mg蛋白;P＜0.05]和IL-6[(54.65±10.68)pg/mg蛋白对(37.89±4.54) pg/mg蛋白对(27.00±3.08)pg/mg蛋白;P＜0.05]表达水平,显著降低NF-κB胞核/胞质比[4.40±1.05对3.07±0.82对2.30±0.60;P＜ 0.05)],抑制iNOS(4.22±0.67对3.14±0.90对2.08±0.35;P ＜0.05)和ICAM-1(5.45±1.08对3.45±0.67对2.21±0.39;P＜0.05)]表达.结论 吸入外源性H2S能以剂量依赖性方式缩小脑缺血再灌注后的脑梗死体积和改善神经功能,其机制可能与抑制NF-κB激活,下调其下游的iNOS和ICAM-1表达以及降低IL-1 β和IL-6水平有关.     

抵抗素对脂肪变性肝细胞核因子κ B和肿瘤坏死因子α表达的影响

目的 探讨抵抗素(resistin)对脂肪变性肝细胞脂质代谢及核因子κB (NF-κ B)、肿瘤坏死因子(TNF)α表达的影响. 方法 选用软脂酸诱导L02细胞脂肪变性模拟非酒精性脂肪性肝病(NAFLD)体外模型.分C组(正常对照组),P组(模型组:软脂酸20μg/ml)及分别加入重组人抵抗素50μg/L的CR组和PR组.各组给予相应处理72 h后,观察细胞的脂肪变并测定细胞培养上清液中甘油三酯(TG)、ALT、AST及γ谷氨酰转移酶(GGT)的浓度;采用半定量RT-PCR及Western blot法检测细胞胰岛素受体底物2(IRS-2)、NF-κB、TNFα的基因及蛋白表达水平.根据资料不同应用方差分析或t检验进行统计学分析,P＜ 0.05为差异具有统计学意义. 结果 P组、CR组及PR组细胞培养上清液中TG、ALT、AST、GGT的浓度明显高于C组.分别与C组比较,P组、CR组及PR组NF-κ B mRNA相对表达量0.54±0.04、0.60±0.04及1.00±0.06,与C组的0.10±0.01比较,t值分别为17.64,22.03,26.06;TNFαmRNA相对表达量0.58±0.04、0.61±0.06及1.05±0.09,与C组的0.33±0.06比较,t值分别为5.67,5.38,11.64,相对表达量明显升高,而IRS-2 mRNA (t值分别为8.19,9.23,20.93)明显降低,且P值均＜0.05,差异有统计学意义.P组与CR组间比较,t值分别为1.75,0.58,2.14,P值均＞0.05,差异无统计学意义.蛋白质表达变化趋势与mRNA变化趋势相一致.结论 抵抗素可能通过NF-κ B依赖途径对肝细胞脂肪变性胰岛素抵抗发挥促炎作用,从而促进NAFLD的发生与发展.