| Background:For a long time, so many scholars concentrate on studying the ischemia/reperfusion injury. However, the progress in clinical treatment is very limited. Here, we evaluate the potential effects of irisin and other interested therapeutic targets in the treatment of I/R injury through in vivo and in vitro researches. The results will be descripted in three parts.Part I. The effects and mechanism of irisin in myocardial ischemia reperfusion injuryBackground:Limb remote ischemic preconditioning (RIPC) is an effective means for protection against ischemia/reperfusion (I/R) induced injury to multiple organs. Many studies are focused on identifying endocrine mechanisms that underlie the cross-talk between muscle and RIPC-mediated organ protection. Here we report that RIPC releases irisin, a myokine derived from the extracellular portion of fibronectin domain-containing 5 protein (FDNC5) in skeletal muscle, to facilitate protection against injury to the heart.Objects:To discover the effects and mechanism of irisin, a myokine on acute myocardial ischemia/reperfusion injury and H9c2 cells cultured under anoxia and reoxygenation.Methods:1. For in-vivo studies, acute ischemia/reperfusion injury was induced in a rat acute myocardial I/R model by a 45min ischemia and 24h reperfusion. Irisin was administrated just before reperfusion. Echocardiology was performed to determine cardiac structure and function in anesthesied rats before execution. Blood samples were collected to check cardiac damage markers. TTC-staining was used to evaluate myocardial infarct size. TUNEL assay wasy used to evaluate the cell apoptotic rate in tissue. The hearts were harvested to check the apoptosis, oxidative stress levels.2. For in-vitro studies, H9c2 cell was used for the experiments, the cell viability and LDH release in supernatant of H9c2 cell culture medium were use to evaluate the effects of irisin pretreatment on hypoxia/reoxygenation injuries in H9c2 cells. Immunofluorescent staining was performed to assess the location of irisin in subcelluar organelle. DCFH-DA was used in the measurement of intracellular ROS generation. Mitochondiral membrane potential was measured to check the mitochondrial function. Western blotting was applied to detect the expression and co-localization of SODs and irisin. Transmission electron microscope was used to observe the ultrastructure changes of subcelluar organelles.3. Western blotting and ELISA were applied to detect the concentration of serum irisin after RIPC stimulation. IHC staining was performed to locate the distribution of irisin in myocardial tissue.Results:1. Animal model studies show that intravenous administration of exogenous irisin markedly reduced the myocardial infarction size after injured by a 45min ischemia and 24h reperfusion at the dose of 0.5 μg, 1μg and 5μg/kg, downregulated the serum level of cardiac troponin I (CTnI), creatine kinase (CK) and lactate dehydrogenase (LDH) activity, compared with vehicle treated I/R group (P<0.05). Irisin also restored the left ventricular ejection fraction (LVEF) in I/R treated rats, compared with vehicle treated I/R group, and, was bloked by boiled or neutralized with specific antibody before administration (P<0.05). Apoptosis of myocardium, expression and activity of key protein (casepase 3 and bax) in the apoptotic signal pathways, and oxidative stress indicator (MDA, MPO) were decreased, and the expression of anti-apoptic protein (bcl-2) and anti-ROS enzyme (SOD) recoverd in irisin treated I/R group, compared with vehicle treated group (P<0.05).2. Irisin pretreatment prevented A/R induced cell death and preserved cell integrity in a dose dependent manner, and inhibited intracellular ROS generation. Irisin treatment increased Co-localized of irisn and mitochondrial SOD2, also increased mitochondrial membrane potential against A/R injury in myocardial cytes. Electron microscopy showed that irisin can increase the number of mitochondria in cells, attenuated mitochondrial ultrastructure damage caused by A/R injury.3. RIPC restored the serum irisin concentration and increased the expression of irisin in injured area in myocardial tissues after I/R injury.Part II. Protective Effects of Aliskiren on Ischemia-Reperfusion-Induced Renal Injury in RatsBackground:The system and local RAAS activation have been envolved in the I/R injury. It has been reported that the direct rennin inhibitor (Direct rennin inhibitor, DRI, like aliskiren) exhibits a protective effect of on ischemia-reperfusion (I/R) injury in many important organs, like the heart and brain. Whether or not aliskiren shows a simular effect in renal I/R injury is not known. Therefore, we evaluated the effect of DRI in kidney in this study. On the other hand, we considered the usage of aliskiren can also be looked as a positive control in the experiments of renal I/R injury intervention in rat model.Objects:To evaluate the effects and mechanism of aliskiren on acute renal ischemia/reperfusion injury.Methods:For in-vivo studies, renal ischemia was induced by occluding the left renal pedicle, and maintained ischemia for 45 min, the reperfusion lasted for 24h. Aliskiren was administrated 15 min before ischemia. Blood samples and the kidneys were collected to check for renal function, pathological changes, angiotensin II (Ang II), apoptosis, oxidative stress and inflammation levels.Results:Compared with the sham rats, serum creatinine (SCR) and blood urea nitrogen (BUN) were significantly increased in I/R rats, accompanied by histopathological damage of the kidney, including tubular cell swelling, desquamation, and cast formation. There were more apoptotic cells and leukocyte infiltration in I/R rats than in the sham rats. Pretreatment with aliskiren ameliorated I/R induced renal injury, i.e. reduced SCR and BUN levels, ameliorated renal histopahological changes, attenuated the apoptosis of cells and leukocyte infiltration in kidney, and decreased the serum AngⅡ levels. I/R injury also decreased superoxide dismutase (SOD) and glutathione (GSH-reduced form) levels, which were blocked with the aliskiren pretreatment.Part Ⅲ. Upregulation the linkage of Dopamine D3 Receptor-Ga12 attenuates renal ischemia/reperfusion injuryBackground:It is well known that G protein coupled receptors (G protein coupled receptors GPCRs) play critical roles in many diseases. Many of the new medicine discovered every year around the world are come from the work on the GPCRs. The targets in the research against the I/R injury is similar. But GPCRs have so many members, that the research on GPCRs will cost too many resources. While the numbers of G protein, the mediators of the GPCRs, are limited. Dopamine receptor family is a member of the GPCRs, and was divided into different subtypes according to the different binding downstream G proteins. Dopamine D1-like receptors are coupled with Gas, and Dopamine D2-like receptors are mainly coupled with Gai/o subtypes. Our previous works are mainly focus on the role of dopamine receptors in the mechanisms of primary hypertension. Recently, we have revealed that the dopamine D1-like receptors and D3 receptor can also bind with G protein subfamily, Ga12/13, and this increased connection can downregulate the reabsorption of water and sodium, decrease the blood pressure. The dopamine D3 receptor is one of the important members of the dopamine D2-like receptors. Our previous study found that there are interactions existing between dopamine D3 receptor and AT1R and endothelin receptors in the kidney, inhibitting AT1R and promoting the function of endothelin receptor ETB (ETB and ETA are antagonistic to each other in the regulation of renal water and sodium reabsorption). The renal dopamine D3 receptor inhibits reabsorption of sodium, diastolic blood vessels, lowers blood pressure. In the kidney, the enhanced linkage of dopamine D3 receptor with G alphaal2/13 is also contributed to the inhibition of Na+/H+ exchanger function, involved in blood pressure regulation, lowering blood pressure. What’s more, inhibition of ATiR, endothelin receptor and Na+/H+exchanger is against renal I/R injury. Therefore, the dopamine D3 receptor is likely one important intrinsic role against I/R injury in the kidney. As a member of limited 4 subfamiles of G proteins, Ga12/13 are connected with many existing GPCRs in the kidney. Recent studies show that activated Ga12 subunit and increased activity can play important roles in the traumatic signals transferring in the renal I/R injury, enhance the I/R induced damages, enhance the cellular oxidative stress responses, disrupt the renal tubular epithelial cell junctions, impair the renal function. Therefore, we speculated that the connection level of dopamine D3 receptor and Ga12 may play important roles in against renal I/R injury. In this study, we intend to use rat renal I/R injury model in vivo, NRK-52E cell (a renal epithelial cell line widely used in reseaches.) hypoxia/reoxygenation model in vitro, to investigate whether the activation of dopamine D3 receptor receptor, and the increased linkage with Ga12 will contribute to the protection of renal I/R injury.Objects:To investigate the effects and mechanism of cross talk between dopamine D3R and Gα12 stimulated by PD128907, a dopamine D3R agonist, on acute renal ischemia/reperfusion injury and NRK-52E cells under hypoxia/reoxygenation (H/R) injuries.Methods:1. For in-vivo studies, ischemia/reperfusion injury was induced in a rat renal unilateral clamping model use in Part Ⅱ. PD128907 (0.2 mg/kg, IV), a D3R agonist, or vehicle, was administered 15 min before clamping (or hypoxia) in both the in-vivo or in-vitro studies. Blood samples and the kidneys were collected to check for renal function, apoptosis and oxidative stress levels, and co-linkage of D3R and Ga12.2. For in-vitro studies, cell viability and LDH release in supernatant of culture medium were use to evaluate the effects of PD128907 pretreatment on H/R and CS.RW injuries in Gα12 transfected NRK52E cells.Results:1. In the rat renal unilateral clamping model, pretreatment with PD128907 protected against renal I/R injury and increased survival rate during a long-term follow-up after seven days. A decrease in the generation of ROS, apoptosis and inflammation may be involved in the D3R-mediated protection since pretreatment with PD128907 increased renal glutathione and superoxide dismutase levels and decreased MDA levels in the I/R group. The increase in cytokines (TNF-a, IL-1β,and IL-10) and MPO in I/R injured kidney was also prevented with a simultaneous decrease in the apoptosis of the epithelial cells and expression of apoptosis biomarkers in kidney harvested one day after I/R injury. The increase in the co-immunoprecipitation between D3R and Ga12 with D3R stimulation paralleled the observed renal protection from I/R injury.2. Moreover, in vitro studies showed that transient overexpression of Ga12 in the NRK52E cells attenuated the protective effect of PD 128907 on H/R injury. The protective effect of PD 128907 might be of significance to renal transplantation, since cold storage/ rewarming induced injury increased LDH release and decreased cell viability in NRK52E cells. Conversely, in the presence of PD 128907, the increased LDH release and decreased cell viability were reversed.Conclusion:1. Limb remote ischemic preconditioning (RIPC) is an effective means for protection against ischemia/reperfusion (I/R) induced injury to multiple organs. Many studies are focused on identifying endocrine mechanisms that underlie the cross-talk between muscle and RIPC-mediated organ protection. Here we report that RIPC releases irisin, a myokine derived from the extracellular portion of fibronectin domain-containing 5 protein (FDNC5) in skeletal muscle, to facilitate protection against injury to the heart. Application of brief periods of ischemia pre-conditioning stimulates release of irisin from skeletal muscle into circulation and transfer of irisin to heart that receives I/R injury. Irisin enters cardiomyocytes through lipid raft-mediated endocytosis and targets onto mitochondria. Interaction between irisin and superoxide dismutase allows for prevention of I/R-induced oxidative stress and preservation of mitochondrial function. Animal model studies show that intravenous administration of exogenous irisin produces dose-dependent protection against I/R-induced injury to the heart via improvement of mitochondrial function. These results demonstrate that irisin is a myokine that facilitates RIPC-mediated cardio-protection. Targeting the action of irisin in mitochondria presents a novel therapeutic intervention for myocardial infarction.2. Aliskiren pretreatment exerts a protective effect on ischemia/reperfusion injury in the kidney, via amelioration of oxidative stress, and reduction in leukocyte infiltration and cellular apoptosis.3. Activation of D3R, by decreasing Ga12-induced renal damage, may exert a protective effect from I/R injury. |
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