肝再生增强因子对缺血再灌注损伤肾脏局部炎性反应的影响

目的 探讨重组人肝再生增强因子( rhALR)对缺血再灌注(IR)肾损伤大鼠模型肾脏局部炎性细胞浸润及炎性因子表达的影响.方法 将SD大鼠按随机数字表法分成假手术组、IR组、rhALR低剂量(100 μg/kg)组及rhALR高剂量(200 μg/kg)组.采用双侧肾蒂夹闭60 min后再灌注建立IR肾损伤动物模型.常规生化法检测血肌酐、尿素氮的水平,HE染色观察肾脏组织学改变,比色法检测肾组织髓过氧化物酶( MPO)的活性,Western印迹法检测肾组织肿瘤坏死因子α(TNF-α)、细胞间黏附分子1(ICAM-1)、单核细胞趋化蛋白1(MCP-1)的蛋白表达.结果 rhALR组的血肌酐和尿素氮显著低于IR组(均P＜ 0.05),肾组织病理损害减轻,rhALR高剂量组较rhALR低剂量组肾功能及肾脏病理改善更明显.IR组大鼠肾组织的MPO活性、TNF-α、ICAM-1、MCP-1的蛋白表达在术后12 h较假手术组显著上升,术后24h有所下降,但仍维持在较高水平(均P＜0.05);rhALR组肾组织MPO活性、肾组织TNF-α、ICAM-1、MCP-1的蛋白表达较IR组显著下降(均P＜0.05),且rhALR高剂量组4者较rhALR低剂量组下降更显著(均P＜0.05).结论 rhALR对IR肾损伤具有保护作用,其作用机制可能与其减少肾脏局部的炎性细胞浸润、抑制炎性因子MCP-1、ICAM-1、TNF-α的表达有关.     

重组人肝再生增强因子对肝纤维化大鼠血清透明质酸浓度的影响

目的探讨重组人肝再生增强因子(hALR)对肝纤维化大鼠血清透明质酸浓度的影响.方法建立四氯化碳中毒性及人血白蛋白免疫损伤性两种大鼠肝纤维化模型.模型完成后予不同剂量hALR(50μg·kg-1·d、10μg·kg-1·d)治疗.在不同的时间点留取大鼠血清标本,测定透明质酸浓度.结果在两种模型中hALR治疗组大鼠血清透明质酸浓度在治疗过程中的不同阶段(治疗1、2个月)均明显低于模型组(四氯化碳模型分别为340.7±32.1、234.1±19.5;人血白蛋白模型分别为366.5±32.3、287.3±30.1).高剂量hALR组大鼠血清透明质酸浓度(四氯化碳模型分别为203.3±15.5、134.1±9.8;人血白蛋白模型分别为218.9±15.8、143.1±8.7)均明显低于低剂量组(四氯化碳模型分别为273.3±13.4、186.6±11.3;人血白蛋白模型分别为276.1±23.4、198.7±11.5).结论重组人肝再生增强因子可降低肝纤维化大鼠血清透明质酸含量.     

重组大鼠肝再生增强因子对急性肾衰竭大鼠肾脏的保护作用

目的 探讨重组大鼠肝再生增强因子(rrALR)对庆大霉素所致急性肾衰竭（ARF）大鼠肾小管上皮细胞及肾功能的保护作用。 方法 雌性Wistar大鼠150只，随机分成5组，每组30只，即健康对照组，ARF模型组，模型+空质粒对照组（空质粒组），模型+rrALR干预组（ALR组）：根据给予rrALR的剂量不同分为ALR1组和ALR2组两个亚组。分别于实验的第4、8、12、16和21天每组随机抽取6只大鼠在留取血、尿标本后，处死大鼠并取肾组织标本。常规生化方法检测各组大鼠BUN、Scr和尿N-乙酰-β-D-葡萄糖苷酶（NAG）酶的变化；PAS染色观察各组大鼠肾组织病理学改变；免疫组化法检测大鼠肾组织中ALR和增殖细胞核抗原（PCNA）的表达；Western印迹法检测肾组织中ALR蛋白的表达量。 结果 ARF大鼠各组BUN、Scr及尿NAG酶水平在第4、8、12、16天时均较对照组显著升高（P < 0.05）。与模型组和空质粒组相比，ALR组BUN、Scr及尿NAG酶水平明显降低（P < 0.05）；肾组织病理损害程度在各时间点明显减轻；而肾组织的ALR蛋白表达增加（P < 0.05）；肾小管上皮细胞增殖活跃；PCNA阳性细胞呈弥漫性分布，增殖指数（PI）明显升高（P < 0.05）。 结论 rrALR对急性损伤的肾小管上皮细胞具有减轻病变和促进再生修复的作用，可明显改善ARF大鼠的肾功能。     

Kupffer细胞在肝移植缺血再灌注损伤中的双重作用

Kupffer细胞足定居于肝内的巨细胞,在月十移植缺血再灌注损伤中发挥着重要的作用,门静脉恢复血流后刺激Kupffer细胞激活,释放活性氧族、多种炎性介质和细胞因子,对肝脏造成损伤.另一方面又可上调HO-1的表达,保护肝脏缺血再灌注损伤,因此,Kupffer细胞在肝移植缺血再灌注损伤中发挥着双重效应.     

Ischemic post-conditioning attenuates renal ischemic reperfusion injury via down-regulation of toll-like receptor 4 in diabetic rats

Background: Ischemia/reperfusion (I/R) injury, which is commonly seen in the field of renal surgery or transplantation, is a major cause of acute renal failure (ARF). The ischemic ARF in diabetic rats is much more severe than that in the normal rats exposed to as same ischemic time. Ischemic post-conditioning (IPO) is a phenomenon by which intermittent interruptions of blood flow in the early phase of reperfusion can protect organs from I/R injury. To determine whether the renal protection effect of IPO mediates by toll-like receptor 4 (TLR4) signaling pathway in diabetic rats.

Methods: Streptozotocin-induced diabetic rats were randomly divided into three groups: sham operation group, I/R group, and IPO group. Except sham operation group, rats were subjected to 30?min of renal ischemia, both with and without treatment with IPO, then reperfusion 24?h. Light microscope and transmission electronic microscope were used to observe structural changes of renal tubule. RT-PCR was used to measure TLR4 and tumor necrosis factor-alpha (TNF-α) mRNA expression level, renal TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) protein expression was detected by Western blot.

Results: The results demonstrated that IPO markedly decreased renal ischemic injury caused by I/R and inhibited the proinflammatory expression levels of TLR4, TNF-α, and NF-κB, all of which up-regulated by I/R in diabetic rats.

Conclusion: Taken together, our results suggest that proper IPO may have protective effect on the ischemic injury mediated by renal I/R, which might be associated with inhibition of TLR4 signaling pathway in diabetic rats.     

Augmenter of liver regeneration attenuates inflammatory response in the postischemic mouse liver in vivo

Background

Augmenter of Liver Regeneration (ALR), a protein synthesized in the liver is suggested to be protective against oxidative stress–induced cell death. Hepatic ischemia–reperfusion (I/R) injury is triggered by reactive oxygen species. Here, we tested the hypothesis that ALR attenuates hepatic I/R injury in vivo.

Methods

C57BL6 mice were subjected to warm hepatic ischemia for 90 min. Either recombinant ALR (100 μg/kg) or vehicle were administered to mice prior ischemia. During reperfusion, neutrophil and CD4+ T cell migration and sinusoidal perfusion were analyzed using intravital microscopy. Alanine aminotransferase–aspartate aminotransferase (plasma) and caspase-3 (tissue) activities were determined as markers of hepatocellular necrotic and apoptotic injury.

Results

Hepatic I/R led to dramatic enhancement of neutrophil and CD4+ T cell recruitment in hepatic microvessels, sinusoidal perfusion failure, and strong elevation of aspartate aminotransferase–alanine aminotransferase and caspase-3 activities. During early reperfusion (60 min), the pretreatment with ALR improved postischemic perfusion failure (P < 0.05) and attenuated liver enzyme activities. Recruitment of CD4+ T cells, but not of neutrophils was attenuated. After 240 min of reperfusion, the protective effect of ALR was stronger, since the liver enzyme activity, perfusion failure, and leukocyte influx were significantly attenuated. As shown by the measurement of caspase-3 activity, postischemic apoptosis was reduced in the ALR-treated group.

Conclusions

Our in vivo data show that ALR has a therapeutic potential against postischemic liver injury. As a mechanism, we suggest a direct protective effect of ALR on apoptotic and necrotic death of hepatocytes and an attenuation of inflammatory cell influx into the postischemic tissue.     

Toll样受体2/4在糖尿病大鼠肾缺血再灌注损伤时的表达及肾康注射液的保护作用

目的探讨糖尿病(DM)大鼠肾缺血再灌注损伤(IRI)的可能机制及肾康注射液(SKI)的保护作用。 方法采用高糖高脂饮食诱导6周联合小剂量链脲佐菌素的方法建立DM大鼠模型,将造模成功的SD大鼠随机分为糖尿病肾缺血再灌注组(DMIR)、糖尿病肾缺血再灌注SKI治疗组(DMIR＋SKI)、糖尿病假手术组(DMS)和正常对照组(NCS)。DMIR＋SKI组大鼠应用SKI[6.0 g/(kg·d)]干预8周,其它组用等量生理盐水。应用右肾切除联合左肾动脉夹闭45 min后再灌注24 h的方法诱导肾IRI。最后,抽血、留取肾组织。ELISA法检测大鼠血清高迁移率族蛋白(HMGB1)水平;免疫组化方法检测肾组织HMGB1、Toll样受体(TLR)2和TLR4的表达;免疫印迹法和实时定量PCR法检测肾组织TLR2、TLR4、髓样分化因子88 (MyD88),核因子-κBp56 (NF-κBp56)的表达。 结果DMIR组大鼠血清HMGB1水平,肾小管HMGB1、TLR2和TLR4的表达以及肾组织TLR2、TLR4、MyD88和NF-κBp56的蛋白及mRNA水平均明显高于DMS组(P<0.01);经过SKI干预治疗8周,DMIR＋SKI组大鼠的上述指标均明显低于DMIR组(P<0.01)。 结论SKI可通过抑制TLR2/4炎症反应通路,减轻DM大鼠肾IRI炎症反应而起到肾脏保护作用。     

Novel roles of lipopolysaccharide and TLR4/NF-κB signaling pathway in inflammatory response to liver injury in Budd-Chiari syndrome

BACKGROUNDBudd-Chiari syndrome (BCS) is an uncommon disorder characterized by obstruction of hepatic venous outflow. To date, the exact mechanism underlying hepatic injury derived from the hepatic venous outflow obstruction in BCS remains largely unknown.AIMTo assess the role of NF-κB-mediated inflammation in BCS-induced liver injury in humans and rats.METHODSA total of 180 rats were randomly assigned into nine groups, including four BCS model groups (1, 3, 6 and 12 wk), four sham-operated groups (1, 3, 6 and 12 wk), and a control group. Lipopolysaccharide (LPS) levels in each group were detected by the Tachypleus Amebocyte Lysate assay. The mRNA and protein levels of TLR4, NF-κB, tumor necrosis factor (TNF)-α, interleukin (IL)-2 and interferon (IFN)-γ were quantified. In addition, 60 patients with BCS and 30 healthy controls were enrolled, and their blood samples were analyzed.RESULTSHepatic and plasma LPS levels were significantly increased in rats. The mRNA and protein expression levels of TLR4, NF-κB and inflammatory cytokines (TNF-α, IL-2 and IFN-γ) in liver tissues were significantly higher in the BCS model groups compared with the other two groups. In addition, the model groups (1, 3, 6 and 12 wk after BCS induction) showed significant differences in the levels of LPS, TLR4, NF-κB, TNF-α, IL-2 and IFN-γ. Notably, there was a significant correlation between the LPS concentrations and mRNA and protein levels of TLR4, NF-κB and inflammatory cytokines. Importantly, it was revealed that the levels of LPS, TLR4, NF-κB and inflammatory cytokines were significantly greater in chronic BCS patients than healthy controls and acute BCS patients.CONCLUSIONLPS level is markedly elevated in BCS, in turn activating the TLR4/NF-κB signaling pathway, leading to induction of inflammatory cytokines (TNF-α, IL-2 and IFN-γ) in response to BCS-induced liver injury.     

Impact of endogenous hydrogen sulfide on toll-like receptor pathway in renal ischemia/reperfusion injury in rats

Abstract

In this study, we investigated the impact of endogenous hydrogen sulfide (H₂S) on toll-like receptors (TLRs)-mediated inflammatory response and apoptosis in renal ischemia–reperfusion injury (IRI). Twenty-four male Wistar rats were randomly divided into four groups: sham, IR, IR?+?propargylglycine (PAG) and IR?+?hydroxylamine (HA). After right nephrectomy, rats were given saline for the sham and IR group, PAG for the IR?+?PAG group and HA for the IR?+?HA group, through the left renal artery for 20?min. Five minutes after drug administration, all rats except sham underwent 45?min of left renal ischemia followed by 24?h of reperfusion. Kidneys were harvested for histological and biochemical evaluation. Levels of TLRs, downstream signaling molecules and pro-inflammatory cytokines were determined by Western blot or immunohistochemistry. Hematoxylin and eosin (H&E) stained renal sections were used for histological grading of renal injury. Apoptotic cells were detected by TUNEL assay. Compared to the sham group, rats in the IR group showed higher renal levels of TLR-2, TLR-4, nuclear NF-κB p65, phosphorylated ASK1, phosphorylated TRAF2, IL-1β, IL-6, IL-18 and TNF-α (p?<?0.05), and exhibited acute kidney injury (p?<?0.05) and apoptosis (p?<?0.05). Compared to the IR group, rats receiving PAG or HA showed significantly higher levels of TLR-2, TLR-4, nuclear NF-κB p65, phosphorylated ASK1, phosphorylated TRAF2, IL-1β, IL-6, IL-18 and TNF-α (p?<?0.01), more severe acute kidney injury (p?<?0.05) and increased apoptosis (p?<?0.01). Thus, inflammatory response and apoptosis mediated by TLRs are involved in renal IRI. Inhibition of endogenous H₂S significantly activated inflammatory response and apoptosis, and thus promoted renal IRI.     

缺血预处理和肢体远隔缺血后处理对大鼠心肌缺血/再灌注损伤中炎症反应影响的比较

目的 比较缺血预处理(ischemic preconditioning,IPC)和肢体远隔缺血后处理(limb remote ischemic postconditioning,LRIPOC)对大鼠心肌缺血/再灌注损伤(ischemia/reperfusion injury,I/RI)中炎症反应的影响. 方法 雄性SD大鼠80只,体重250 g～350 g,采用随机数字表法将其随机分为4组(每组20只):假手术组(S组)、缺血/再灌注组(I/R组)、IPC组和LRIPOC组.监测缺血/再灌注期间的心率(HR)和平均动脉压(MAP),并计算HR和收缩压乘积(rate pressure product,RPP)作为心肌氧耗指数.各组随机取10只大鼠,于再灌注30、60、120 min时采集颈静脉血样,采用ELISA法检测血清心肌肌钙蛋白(cardiac troponin I,cTnI)、磷酸肌酸激酶同工酶(creatine kinase-MB,CK-MB)、肿瘤坏死因子(tumor necrosis factor,TNF)-α、高迁移率组蛋白-1(high mobility group box-1 protein,HMGB-1)、细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)、白介素(interleukin,IL)-1、IL-6和IL-10的浓度;于再灌注120 min颈静脉采血后,采用伊文蓝和TTC双重染色法测定心肌梗死体积.各组随机取10只大鼠,于再灌注120 min处死后分别取缺血区和非缺血区心肌组织,采用ELISA法检测心肌TNF-α 、HMGB-1、ICAM-1、IL-1、IL-6和IL-10含量. 结果 I/R组、IPC组和LRIPOC组的心肌梗死体积值分别是(72±9)％、(36±13)％和(57±9)％,3组的血清cTnI浓度分别是(0.99±0.14)(0.37±0.08)、(0.54±0.07) μg/L,而3组的血清CK-MB浓度分别是(110±13)、(38±8)、(45±6) μg/L.与I/R组比较,IPC组和LRIPOC组心肌梗死体积、血清cTnI和CK-MB浓度显著降低,IPC组再灌注30、60、120 min时血清TNF-α 浓度、再灌注60、120 min时血清HMGB-1浓度、再灌注120 min时血清ICAM-1、IL-1和IL-6浓度显著降低,缺血区心肌组织内TNF-α 、HMGB-1、ICAM-1、IL-1和IL-6含量显著降低,非缺血区心肌组织内TNF电、ICAM-1、IL-1和IL-6含量显著降低(P＜0.05);LRIPOC组再灌注30、60、120 min时血清TNF-α 浓度、再灌注120 min时血清HMGB-1、ICAM-1、IL-1和IL-6浓度显著降低,缺血区与非缺血区心肌组织内 TNF-α、HMGB-1、ICAM-1、IL-1和IL-6的含量显著降低(P＜0.05).与IPC组比较,LRIPOC组的心肌梗死体积、血清cTnI和CK-MB浓度显著升高,再灌注60 min时血清TNF-α 浓度、再灌注120 min时血清HMGB-1和ICAM-1浓度显著升高,缺血区心肌组织内TNF-α 、ICAM-1、IL-1和IL-6含量显著升高,非缺血区心肌组织内ICAM-1、IL-1和IL-6含量显著升高(P＜0.05). 结论 IPC减轻大鼠心肌I/RI中炎症反应的作用强于LRIPOC,这可能是IPC对心肌I/RI保护作用强于LRIPOC的原因之一.     

The lectin-like domain of thrombomodulin is a drug candidate for both prophylaxis and treatment of liver ischemia and reperfusion injury in mice

Ischemia and reperfusion injury (IRI) can occur in any tissue or organ. With respect to liver transplantation, the liver grafts from donors by definition experience transient ischemia and subsequent blood reflow. IRI is a problem not only in organ transplantation but also in cases of thrombosis or circulatory disorders such as mesenteric ischemia, myocardial, or cerebral infarction. We have reported that recombinant human soluble thrombomodulin (rTM), which is currently used in Japan to treat disseminated intravascular coagulation (DIC), has a protective effect and suppresses liver IRI in mice. However, rTM may not be fully safe to use in humans because of its inherent anticoagulant activity. In the present study, we used a mouse liver IRI model to explore the possibility that the isolated lectin-like domain of rTM (rTMD1), which has no anticoagulant activity, could be effective as a therapeutic modality for IRI. Our results indicated that rTMD1 could suppress ischemia and reperfusion-induced liver damage in a dose-dependent manner without concern of associated hemorrhage. Surprisingly, rTMD1 suppressed the liver damage even after IR insult had occurred. Taken together, we conclude that rTMD1 may be a candidate drug for prevention of and therapy for human liver IRI without the possible risk of hemorrhage.     

Involvement of tumor necrosis factor-α receptor 1 and tumor necrosis factor-related apoptosis-inducing ligand-(TRAIL) receptor-2/DR-5, but not Fas, in graft injury in live-donor liver transplantation

The pathway leading to cell death in clinical liver transplantation is not known. Eight liver transplant recipients and eight donors were enrolled in this study. Postoperative serum levels of alanine transferase had significantly increased in the recipients compared with those in the donors. Mild centri-lobular necrosis was observed in only liver tissues taken from the recipients. Tumor necrosis factor (TNF)-R1 and death receptor 5 expression levels had increased in liver tissues taken from the recipients. There were no changes in the levels of Fas/Fas ligand expression in liver tissues from either the donors or recipients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression was down-regulated in donor liver after hepatectomy and liver allograft after implantation. The results suggest that, although ischemic liver injury was not serious, due to the short ischemia time, TNF and TRAIL signals are associated with liver ischemic injury in live-donor liver transplantation but Fas signal is not     

Omega-3 fatty acids are protective in hepatic ischemia reperfusion injury in the absence of GPR120 signaling

BackgroundA single dose of IV fish oil (FO) before hepatic ischemia reperfusion injury (HIRI) increases hepatocyte proliferation and reduces necrosis in wild type (WT) mice. It has been suggested that the GPR120 receptor on Kupffer cells mediates FO's ability to reduce HIRI. The purpose of this study was to determine whether GPR120 is required for FO to reduce HIRI.MethodsSixty-four (n = 8/group) adult male WT (C57BL/6) and GPR120 knockout (KO) mice received IV FO (1 g/kg) or saline 1 h prior to HIRI or sham operation. Mice were euthanized 24 h postoperatively for analysis of hepatic histology, NFκB activity, and serum alanine transaminase (ALT) levels.ResultsFO pretreated livers had less necrosis after HIRI than saline pretreated livers in both WT (mean ± SEM 25.9 ± 7.3% less, P = 0.007) and KO (36.6 ± 7.3% less, P < 0.0001) mice. There was no significant difference in percent necrosis between WT-FO and KO-FO groups. Sham groups demonstrated minimal necrosis (0–1.9%). Mean [95% CI] ALT after HIRI was significantly higher (P = 0.04) in WT-Saline mice (1604 U/L [751–3427]) compared to WT-FO (321 U/L [150–686]) but was not significantly higher in KO-Saline mice compared to KO-FO. There were no differences in ALT between WT-FO and KO-FO mice who underwent HIRI or between groups who underwent sham surgery. There were no differences in NFκB or IKKβ activation among groups as measured by Western blot analysis.ConclusionsIV FO pretreatment was able to reduce HIRI in GPR120 KO mice, suggesting the hepatoprotective effects of FO are not mediated by GPR120 alone.     

Intracellular organelles in health and kidney disease

Subcellular organelles consist of smaller substructures called supramolecular assemblies and these in turn consist of macromolecules. Various subcellular organelles have critical functions that consist of genetic disorders of organelle biogenesis and several metabolic disturbances that occur during non-genetic diseases e.g. infection, intoxication and drug treatments. Mitochondrial damage can cause renal dysfunction as ischemic acute renal injury, chronic kidney disease progression. Moreover, mitochondrial dysfunction is an early event in aldosterone-induced podocyte injury and cardiovascular disease due to oxidative stress in chronic kidney disease. Elevated production of reactive oxygen species could be able to activate NLRP3 inflammasome representing new deregulated biological machinery and a novel therapeutic target in hemodialysis patients. Peroxisomes are actively involved in apoptosis and inflammation, innate immunity, aging and in the pathogenesis of age related diseases, such as diabetes mellitus and cancer. Peroxisomal catalase causes alterations of mitochondrial membrane proteins and stimulates generation of mitochondrial reactive oxygen species. High concentrations of hydrogen peroxide exacerbate organelles and cellular aging. The importance of proper peroxisomal function for the biosynthesis of bile acids has been firmly established. Endoplasmic reticulum stress-induced pathological diseases in kidney cause glomerular injury and tubulointerstitial injury. Furthermore, there is a link between oxidative stress and inflammations in pathological states are associated with endoplasmic reticulum stress. Proteinuria and hyperglycemia in diabetic nephropathy may induce endoplasmic reticulum stress in tubular cells of the kidney. Due to the accumulation in the proximal tubule lysosomes, impaired function of these organelles may be an important mechanism leading to proximal tubular toxicity.