Compound Danshen Dripping Pills pretreatment protects the heart from ischemia/reperfusion injury by enhancing autophagic flux

Abstract Compound Danshen Dripping Pills (CDDPs) have been used in clinical treatment to protect the heart from ischemia/reperfusion (IR) injury for many years. However, the underlying mechanism implicated in the protective effects remains to be explored. Here, we determined the effects of CDDPs in Sprague-Dawley rats with the IR model. Cardiac function in vivo was assessed by echocardiography. Transmission electron microscopy, histological and immunohistochemical techniques, Western blotting and recombinant adeno-associated virus 9 transfection were used to illustrate the effects of CDDPs on IR and autophagy. Our results showed that pretreatment with CDDPs decreased the level of serum myocardial enzymes and infarct size in rats after IR. Apoptosis evaluation showed that CDDPs significantly ameliorated the cardiac apoptosis level after IR. Meanwhile, CDDPs pretreatment increased myocardial autophagic flux, with upregulation of LC3B, downregulation of p62, and increased autophagosomes and autolysosomes. Moreover, the autophagic flux inhibitor chloroquine could increase IR injury, while CDDPs could partially reverse the effects. Furthermore, our results showed that the activation of AMPK/mTOR was involved in the cardioprotective effect exerted by CDDPs. Herein, we suggest that CDDPs partially protect the heart from IR injury by enhancing autophagic flux through the activation of AMPK/mTOR.

The multifaceted role of ischemia/reperfusion in sickle cell anemia.

Sickle cell anemia is a unique disease dominated by hemolytic anemia and vaso-occlusive events. The latter trigger a version of ischemia/reperfusion (I/R) pathobiology that is singular in its origin, cyclicity, complexity, instability, perpetuity, and breadth of clinical consequences. Specific clinical features are probably attributable to local I/R injury (e.g., stroke syndromes) or remote organ injury (e.g., acute chest syndrome) or the systematization of inflammation (e.g., multifocal arteriopathy). Indeed, by fashioning an underlying template of endothelial dysfunction and vulnerability, the robust inflammatory systematization no doubt contributes to all sickle pathology. In this Review, we highlight I/R-targeting therapeutics shown to improve microvascular blood flow in sickle transgenic mice undergoing I/R, and we suggest how such insights might be translated into human therapeutic strategies.

Effects of a new thiazolidine compound (GQ-11) on tissue repair process in models of insulin resistance and ischemia-reperfusion / Efeitos de um novo derivado tiazolidínico (GQ-11) no processo de reparo tecidual em modelos de resistência à insulina e isquemia-reperfusão

The thiazolidinediones (TZDs) class comprises drugs with hypoglycemic effects, reducing insulin resistance in peripheral tissues. Our group has demonstrated in preliminary in vivo studies that a new TZD, GQ-11, improves insulin resistance as well as modulates cytokines involved in inflammatory process, suggesting an interesting approach for therapeutic alternatives in tissue repair, especially in metabolic decompensation cases, as insulin resistance and ischemia-reperfusion. In this context, the aim of this study was to investigate GQ-11 effects in tissue repair in three different models: insulin resistance in db/db mice, reconstructed human epidermis (RHE) in glycated collagen matrix and ischemia/reperfusion induced by aorta clamping in Wistar rats. In insulin resistance context, GQ-11 treatment upregulated the expression of anti-inflammatory mediators, such as IL-10, TGF-ß and Arg-1, downregulated the expression of pro-inflammatory cytokines both in db/db mice wounds and in macrophage, besides increasing re-epithelization and collagen deposition. In addition, the treatment also induced keratinocytes proliferation and fibroblasts differentiation in RHE. In ischemia-reperfusion model, the same anti-inflammatory effect was observed along with anti-oxidant properties through regulation of enzymes, such as catalase and GPx, as well as by decreasing TBARS formation. Animals imaging by positron emission tomography (PET) indicated significant less 18F-FDG uptake in animal treated with GQ-11 compared to controls, suggesting decrease of the inflammation process related to reperfusion after aorta clamping. Concluding, the dual PPARα/γ agonist GQ-11 has an important antiinflammatory effect, suggesting a new approach to tissue repair management in diabetes and in prevention of ischemia-reperfusion syndrome post-surgery

As tiazolidinadionas (TZDs) compreendem uma classe de fármacos hipoglicemiantes que reduzem a resistência à insulina pelos tecidos periféricos. Dados preliminares in vivo obtidos em nosso grupo de pesquisa mostraram que um dos novos derivados tiazolidínicos, GQ-11, além de aumentar a resposta à insulina, pode inibir citocinas pró-inflamatórias, o que a torna uma alternativa terapêutica promissora no reparo tecidual, em especial, nos casos de descompensação metabólica como ocorre na resistência à insulina e na isquemia/reperfusão. Nesse contexto, o objetivo deste trabalho foi investigar os efeitos da GQ-11 nas etapas do processo de reparo tecidual em três modelos: resistência à insulina utilizando camundongos db/db, epiderme humana reconstruída em matriz de colágeno glicado e isquemia/reperfusão induzida por clampeamento da aorta em ratos Wistar. No contexto de resistência à insulina, o tratamento com GQ-11 induziu a expressão de mediadores anti-inflamatórios como IL-10, TGF-ß e Arg-1 e diminuiu a expressão de citocinas pró-inflamatórias em lesões de camundongos db/db e em macrófagos, além de aumentar a capacidade de re-epitelização e a deposição de colágeno. Além disso, o tratamento também induziu a proliferação de queratinócitos e a diferenciação de fibroblastos em epiderme humana reconstruída em matriz de colágeno glicado. No modelo de isquemia-reperfusão, o mesmo efeito anti-inflamatório da GQ-11 foi observado ao lado de efeitos anti-oxidantes através da regulação de enzimas como catalase, GPx e diminuição de TBARS. O imageamento dos animais através de tomografia por emissão de pósitrons (PET) demonstrou menor captação de 18F-FDG (18F-fluordesoxiglicose), indicando diminuição do processo inflamatório decorrente da reperfusão pós clampeamento aórtico. Dessa forma, conclui-se que GQ-11, um agonista dual de PPARα/γ, tem efeito anti-inflamatório importante, podendo ser um candidato à fármaco com possível aplicação no reparo tecidual no diabetes e na prevenção da síndrome de isquemia-reperfusão desenvolvida após procedimentos cirúrgicos

Dynamic alteration of the colonic microbiota in intestinal ischemia-reperfusion injury.

BACKGROUND: Intestinal ischemia-reperfusion (I/R) plays an important role in critical illnesses. Gut flora participate in the pathogenesis of the injury. This study is aimed at unraveling colonic microbiota alteration pattern and identifying specific bacterial species that differ significantly as well as observing colonic epithelium change in the same injury model during the reperfusion time course. METHODOLOGY/PRINCIPAL FINDINGS: Denaturing gradient gel electrophoresis (DGGE) was used to monitor the colonic microbiota of control rats and experimental rats that underwent 0.5 hour ischemia and 1, 3, 6, 12, 24, and 72 hours following reperfusion respectively. The microbiota similarity, bacterial diversity and species that characterized the dysbiosis were estimated based on the DGGE profiles using a combination of statistical approaches. The interested bacterial species in the gel were cut and sequenced and were subsequently quantified and confirmed with real-time PCR. Meanwhile, the epithelial barrier was checked by microscopy and D-lactate analysis. Colonic flora changed early and differed significantly at 6 hours after reperfusion and then started to recover. The shifts were characterized by the increase of Escherichia coli and Prevotella oralis, and Lactobacilli proliferation together with epithelia healing. CONCLUSION/SIGNIFICANCE: This study shows for the first time that intestinal ischemia-reperfusion results in colonic flora dysbiosis that follows epithelia damage, and identifies the bacterial species that contribute most.

The effect of methylprednisolone on warm ischemia-reperfusion injury in the liver.

BACKGROUND: Liver ischemia-reperfusion (I-R) injury is a well-known cause of morbidity and mortality following liver surgery and transplantation. Further investigation is warranted to identify measures that reduce the untoward sequelae of liver ischemia. METHODS: Male Sprague-Dawley rats (wild-type) and Zucker rats (with hepatic steatosis) were subjected to 75 minutes of 70% hepatic ischemia and 3 hours of reperfusion. The ischemic periods were based on protocols of either continuous clamping (CC) or ischemic preconditioning (IP). Prior to ischemia induction, rats were pretreated with intravenous methylprednisolone (MP; 2 mg/kg) or normal saline. Warm I-R injury was evaluated using serum levels of aspartate aminotransferase (AST), serum interleukin-6 (IL-6), and hematoxylin and eosin staining. RESULTS: Histology, serum IL-6, and AST release revealed that MP treatment provided significant protection as compared with ischemic controls (both CC and IP groups) only in the normal, not steatotic, livers. The inflammatory response was considerably reduced in MP groups with normal livers but not in steatotic livers. In general, the IP groups showed decreased I-R injury compared to the CC group. However, MP was able to further reduce I-R injury only in normal, not steatotic, livers. CONCLUSIONS: MP attenuated the postischemic and inflammatory response in the normal, and not steatotic, livers. MP pretreatment might be effective in reducing warm I-R injury to livers without steatosis. The mechanism of I-R-related hepatocellular damage in steatotic liver is different than in normal liver.

Genomic profiling of kidney ischemia-reperfusion reveals expression of specific alloimmunity-associated genes: Linking "immune" and "nonimmune" injury events.

Increased organ ischemia time leads to delayed graft function (DGF), increased acute rejection (AR), enhanced chronic allograft nephropathy (CAN), and reduced long-term allograft survival. The mechanisms by which IRI predisposes to AR and CAN are unknown. We hypothesized that gene expression profiling of ischemia-reperfusion injury (IRI)-affected kidney would identify how IRI predisposes to AR and CAN. Furthermore, we examined how current immunosuppressive drug molecular targets are altered by IRI. C57BL/6J mice were exposed to 30 (n = 3) or 60 (n = 3) minutes of bilateral kidney ischemia or sham surgery (n = 5). At 36 hour kidney tissue was collected and analyzed using Affymetrix 430MOEA (22626 genes) array and GC-RMA-SAM pipeline. Genes with the false discovery rate (q < 1%) and +/-50% fold change (FC) were considered affected by IRI. Genes coding for histocompatibility and antigen-presenting factors, calcineurin, and mammalian target of rapamycin (mTOR) pathway-associated proteins were selected using Gene Ontology (GO) analysis. GO analysis identified 10 and 17 alloimmunity-related genes affected by IRI induced by 30 and 60 minutes of ischemia, respectively, including Traf6 (FC = 2.99) and H2-D1 (FC = 2.58). We also detected significant IRI genomic responses in calcineurin and mTOR pathways represented by Fkbp5 (FC = 4.18) and Fkbp1a (FC = 2.0), and Eif4ebp1 (FC = 16.8) and Akt1 (FC = 3.64), respectively. These data demonstrated that IRI up-regulates expression of several alloimmunity-associated genes, which can in turn enhance alloimune responses. Our discovery of IRI-induced up-regulation of genes associated with calcineurin and mTOR pathways are consistent with clinical observations that FK506 and Rapamycin can alter the course of DGF. Further validation and dissection of these pathways can lead to novel approaches by which improved management of early "nonimmune" transplant events can decrease susceptibility to more classic "immune" changes and CAN.

Lipid peroxidation in ischemia-reperfusion oxidative injury of the graft preserved in Celsior and University of Wisconsin solutions on a pig pancreas transplantation model.

BACKGROUND: Graft pancreatitis (GP) is one the main technical problems associated with pancreas transplant (PT). It occurs in 20% of patients representing a risk factor for thrombosis and cause of graft loss. GP is related to oxidative effects from oxygen-derived free radicals (OFR) in ischemia-reperfusion injury. We evaluated lipid peroxidation by the OFR in the PT of pig organs preserved with either Celsior or Wisconsin solutions. METHODS: In Landrace pigs we performed 24 pancreas allografts, which were preserved 18 or 24 hours: 12 with Celsior solution (CS) and 12 with Wisconsin solution (UW). No immunosuppression was administered. The oxidative effects were determined by quantification of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) and of the carbonyl groups of proteins in our pancreatic tissue samples and measured at different times: (A) baseline in the donor, (B) after perfusion of the graft, (C) after the ischemia period, and (D) 30 minutes after ischemia-reperfusion of the graft. RESULTS: The MDA and 4-HDA values were similar in conditions A, B, and C, but showed an extraordinary increase after ischemia-reperfusion in D, among both the 18- or 24-hour preserved grafts and in the same proportion with CS and UW. The carbonyl groups of the proteins rose in conditions B and C (cold ischemia), but less so in state D (reperfusion). CONCLUSIONS: The oxidative injury of a pancreatic graft preserved for 18 or 24 hours occurs during reperfusion, with an extraordinary intensity, but similarly with CS and UW, an observation that may help to explain graft pancreatitis.

Topical hepatic hypothermia attenuates pulmonary injury after hepatic ischemia and reperfusion.

BACKGROUND: Prolonged periods of hepatic ischemia are associated with hepatocellular injury and distant organ dysfunction in experimental models. Neutrophils (PMN) and tumor necrosis factor (TNF)-alpha have been implicated, mostly because of their local deleterious effects on the hepatocyte after hepatic ischemia and reperfusion (I/R) injury. We hypothesize that topical hepatic hypothermia (THH) reduces ischemia and reperfusion-induced hepatic necrosis, PMN infiltration, TNF-alpha release, and consequent acute pulmonary injury. STUDY DESIGN: Sprague-Dawley rats (250 to 300g) were evenly divided into three groups: 90 minutes of normothermic (37 degrees C) partial hepatic ischemia (normothermic I/R), 90 minutes of hypothermic (25 degrees C) partial hepatic ischemia (hypothermic I/R), and sham laparotomy (without ischemia). There were six animals in each experimental group per time point unless otherwise specified. Hepatic necrosis and PMN infiltration were evaluated and scored on hematoxylin and eosin-stained liver specimens 12 hours after reperfusion. Serum TNF-alpha levels were determined by ELISA at 0 minutes, 15 minutes, 30 minutes, 1 hour, and 12 hours postreperfusion. Pulmonary PMN infiltration and vascular permeability were measured by myeloperoxidase activity and Evans blue dye extravasation, respectively, to quantitate pulmonary injury 12 hours after reperfusion. RESULTS: Normothermic I/R results in a significant increase in TNF-alpha at 15 and 30 minutes (p < 0.005), PMN infiltration (p < 0.001), and hepatic necrosis (p < 0.001), compared with sham. Institution of THH reduced peak serum TNF-alpha levels by 54% at 15 minutes (p < 0.005) and by 73% at 30 minutes (p < 0.001) postreperfusion compared with normothermic I/R. Similarly, hepatic PMN infiltration and necrosis at 12 hours were reduced by 60% (p < 0.05) and 47% (p < 0.05), respectively. Myeloperoxidase activity and Evans blue extravasation (measures of acute lung injury) were reduced by 42% and 39%, respectively, with institution of THH compared with animals undergoing normothermic I/R (p < 0.001). CONCLUSIONS: These results demonstrate that THH protects the liver from ischemia and reperfusion-induced necrosis and PMN infiltration. In addition, THH reduces the serum levels of TNF-alpha and associated pulmonary injury. These data suggest that the ischemic liver is a potential source of inflammatory mediators associated with hepatic ischemia and reperfusion-induced pulmonary injury.

[Staging mesenteric ischemia based on histopathologic findings in the animal model]. / Stadieneinteilung der mesenterialen Ischämie aufgrund histopathologischer Befunde im Tiermodell.

With a minimal invasive access, a segmental intestinal ischemia of different duration was established in 25 pigs. After 24 h of reperfusion, the bowel was resected and examined by light microscopy. Considering these results, we developed a histopathological grading of intestinal ischemia to distinguish between viable and necrotic segments.