MiR-320 regulates cardiomyocyte apoptosis induced by ischemia–reperfusion injury by targeting AKIP1

Background

MicroRNAs play important roles in regulation of the cardiovascular system. The purpose of this study was to investigate microRNA-320 (miR-320) expression in myocardial ischemia-reperfusion (I/R) injury and the roles of miR-320 in cardiomyocyte apoptosis by targeting AKIP1 (A kinase interacting protein 1).

Methods

The level of miR-320 was detected using quantitative real-time polymerase chain reaction (qRT-PCR), and cardiomyocyte apoptosis was detected via terminal dUTP nick end-labeling assay. Cardiomyocyte apoptosis and the mitochondrial membrane potential were evaluated via flow cytometry. Bioinformatics tools were used to identify the target gene of miR-320. The expression levels of AKIP1 mRNA and protein were detected via qRT-PCR and Western blot, respectively.

Results

Both the level of miR-320 and the rate of cardiomyocyte apoptosis were substantially higher in the I/R group and H9c2 cells subjected to H/R than in the corresponding controls. Overexpression of miR-320 significantly promoted cardiomyocyte apoptosis and increased the loss of the mitochondrial membrane potential, whereas downregulation of miR-320 had an opposite effect. Luciferase reporter assay showed that miR-320 directly targets AKIP1. Moreover, knock down and overexpression of AKIP1 had similar effects on the H9c2 cells subjected to H/R.

Conclusions

miR-320 plays an important role in regulating cardiomyocyte apoptosis induced by I/R injury by targeting AKIP1 and inducing the mitochondrial apoptotic pathway.

     

Sphingolipid therapy in myocardial ischemia–reperfusion injury

Sphingolipids are known to play a significant physiological role in cell growth, cell differentiation, and critical signal transduction pathways. Recent studies have demonstrated a significant role of sphingolipids and their metabolites in the pathogenesis of myocardial ischemia–reperfusion injury. Our laboratory has investigated the cytoprotective effects of N,N,N-trimethylsphingosine chloride (TMS), a stable N-methylated synthetic sphingolipid analogue on myocardial and hepatic ischemia–reperfusion injury in clinically relevant in vivo murine models of ischemia–reperfusion injury. TMS administered intravenously at the onset of ischemia reduced myocardial infarct size in the wild-type and obese (ob/ob) mice. Following myocardial I/R, there was an improvement in cardiac function in the wild-type mice. Additionally, TMS also decreased serum liver enzymes following hepatic I/R in wild-type mice. The cytoprotective effects did not extend to the ob/ob mice following hepatic I/R or to the db/db mice following both myocardial and hepatic I/R. Our data suggest that although TMS is cytoprotective following I/R in normal animals, the cytoprotective actions of TMS are largely attenuated in obese and diabetic animals which may be due to altered signaling mechanisms in these animal models. Here we review the therapeutic role of TMS and other sphingolipids in the pathogenesis of myocardial ischemia–reperfusion injury and their possible mechanisms of cardioprotection.     

ADSCs-exo attenuates hepatic ischemia–reperfusion injury after hepatectomy by inhibiting endoplasmic reticulum stress and inflammation

Hepatic ischemia–reperfusion (I/R) injury commonly occurs during liver surgery. Exosomes from adipose-derived stem cells (ADSCs-exo) induce a hepatoprotective effect during hepatic I/R injury. This study aimed to investigate the possible mechanism by which ADSCs-exo attenuates hepatic I/R injury in rats. Rats were randomly divided into four groups: Sham, I30R + PH, ADSCs, and ADSCs-exo groups. Liver tissues were collected immediately after 24 h of reperfusion for further analyses. The content of inflammatory factors in liver tissue was detected using enzyme-linked immunosorbent assay. The pathological changes in liver tissue were analyzed using HE staining. Transmission electron microscopy was used to visualize the ultrastructural changes of hepatocytes. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to detect the expression of endoplasmic reticulum stress (ERS)-related genes and proteins. Liver histomorphology and hepatocyte ultrastructure changes improved after ADSCs-exo treatment. Moreover, ADSCs-exo treatment significantly downregulated tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 levels while upregulating IL-10 levels. Western blot analysis suggested that the protein expressions of GRP78, p-PERK, p-eIF2α, p-IRE1α, XBP1s, ATF-6, ATF-4, CHOP, p-JNK, cleaved-Caspase-3, cleaved Caspase-9, and cleaved Caspase-12 significantly decreased after ADSCs-exo treatment. RT-qPCR results demonstrated that mRNA expression of GRP78, IRE1α, XBP1, ATF-6, ATF-4, CHOP, JNK, Caspase-3, Caspase-9, and Caspase-12 markedly reduced after ADSCs-exo treatment. In conclusion, ADSCs-exo protects against hepatic I/R injury after hepatectomy by inhibiting ERS and inflammation. Therefore, ADSCs-exo can be considered as a viable option for the treatment of hepatic I/R injury.     

Emerging roles of microRNAs in intestinal ischemia/reperfusion–induced injury: a review

Journal of Physiology and Biochemistry - Intestinal ischemia/reperfusion (II/R) injury is a serious pathological phenomenon in underlying hemorrhagic shock, trauma, strangulated intestinal...     

Intravenous immunoglobulin is effective in alleviating hepatic ischemia–reperfusion injury: a rat model study

Molecular Biology Reports - Hepatic ischemia–reperfusion injury (I/R) is an important factor affecting the prognosis of patients undergoing liver surgery. This study aimed to explore the...     

Effect of taurine on ischemia–reperfusion injury

Taurine is an abundant β-amino acid that regulates several events that dramatically influence the development of ischemia–reperfusion injury. One of these events is the extrusion of taurine and Na⁺ from the cell via the taurine/Na⁺ symport. The loss of Na⁺ during the ischemia–reperfusion insult limits the amount of available Na⁺ for Na⁺/Ca²⁺ exchange, an important process in the development of Ca²⁺ overload and the activation of the mitochondrial permeability transition, a key process in ischemia–reperfusion mediated cell death. Taurine also prevents excessive generation of reactive oxygen species by the respiratory chain, an event that also limits the activation of the MPT. Because taurine is an osmoregulator, changes in taurine concentration trigger “osmotic preconditioning,” a process that activates an Akt-dependent cytoprotective signaling pathway that inhibits MPT pore formation. These effects of taurine have clinical implications, as experimental evidence reveals potential promise of taurine therapy in preventing cardiac damage during bypass surgery, heart transplantation and myocardial infarction. Moreover, severe loss of taurine from the heart during an ischemia–reperfusion insult may increase the risk of ventricular remodeling and development of heart failure.     

The protective role of curcumin in myocardial ischemia–reperfusion injury

Coronary artery disease (CAD) is a well-known pathological condition that is characterized by high morbidity and mortality. The main pathological manifestation of CAD is myocardial injury due to ischemia–reperfusion (I–R). Currently, no efficacious treatment of protecting the heart against myocardial I–R exists. Hence, it is necessary to discover or develop novel strategies to prevent myocardial-reperfusion injury to improve clinical outcomes in patients with CAD. A large body of experimental evidence supports cardioprotective properties of curcumin and the ability of this phytochemical to modify some cardiovascular risk factors. However, the detailed effects of curcumin in myocardial I–R injury are still unclear and there is a lack of evidence concerning which curcumin regimen may be ideal for myocardial I–R injury. This paper presents a brief review of the pathophysiology of myocardial I–R injury and the mechanisms of action of curcumin in reducing myocardial I–R injury.     

Role of microRNA-195 in cardiomyocyte apoptosis induced by myocardial ischaemia–reperfusion injury

This study aims to investigate microRNA-195 (miR-195) expression in myocardial ischaemia–reperfusion (I/R) injury and the roles of miR-195 in cardiomyocyte apoptosis though targeting Bcl-2. A mouse model of I/R injury was established. MiR-195 expression levels were detected by real-time quantitative PCR (qPCR), and the cardiomyocyte apoptosis was detected by TUNEL assay. After cardiomyocytes isolated from neonatal rats and transfected with miR-195 mimic or inhibitor, the hypoxia/reoxygenation (H/R) injury model was established. Cardiomyocyte apoptosis and mitochondrial membrane potential were evaluated using flow cytometry. Bcl-2 and Bax mRNA expressions were detected by RT-PCR. Bcl-2, Bax and cytochrome c (Cyt-c) protein levels were determined by Western blot. Caspase-3 and caspase-9 activities were assessed by luciferase assay. Compared with the sham group, miR-195 expression levels and rate of cardiomyocyte apoptosis increased significantly in I/R group (both P<0.05). Compared to H/R + negative control (NC) group, rate of cardiomyocyte apoptosis increased in H/R + miR-195 mimic group while decreased in H/R + miR-195 inhibitor group (both P<0.05). MiR-195 knockdown alleviated the loss of mitochondrial membrane potential (P<0.05). MiR-195 overexpression decreased Bcl-2 mRNA and protein expression, increased BaxmRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). While, downregulated MiR-195 increased Bcl-2 mRNA and protein expression, decreased Bax mRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). Our study identified that miR-195 expression was upregulated in myocardial I/R injury, and miR-195 overexpression may promote cardiomyocyte apoptosis by targeting Bcl-2 and inducing mitochondrial apoptotic pathway.     

NDUFV1 attenuates renal ischemia–reperfusion injury by improving mitochondrial homeostasis

Impaired mitochondrial function and dysregulated energy metabolism have been shown to be involved in the pathological progression of kidney diseases such as acute kidney injury (AKI) and diabetic nephropathy. Hence, improving mitochondrial function is a promising strategy for treating renal dysfunction. NADH: ubiquinone oxidoreductase core subunit V1 (NDUFV1) is an important subunit of mitochondrial complex I. In the present study, we found that NDUFV1 was reduced in kidneys of renal ischemia/reperfusion (I/R) mice. Meanwhile, renal I/R induced kidney dysfunction as evidenced by increases in BUN and serum creatinine, severe injury of proximal renal tubules, oxidative stress, and cell apoptosis. All these detrimental outcomes were attenuated by increased expression of NDUFV1 in kidneys. Moreover, knockdown of Ndufv1 aggravated cell insults induced by H₂O₂ in TCMK-1 cells, which further confirmed the renoprotective roles of NDUFV1. Mechanistically, NDUFV1 improved the integrity and function of mitochondria, leading to reduced oxidative stress and cell apoptosis. Overall, our data indicate that NDUFV1 has an ability to maintain mitochondrial homeostasis in AKI, suggesting therapies by targeting mitochondria are useful approaches for dealing with mitochondrial dysfunction associated renal diseases such as AKI.     

Ebselen ameliorates renal ischemia–reperfusion injury via enhancing autophagy in rats

Renal ischemia–reperfusion (I/R) injury is one of the most common causes of chronic kidney disease (CKD). It brings unfavorable outcomes to the patients and leads to a considerable socioeconomic burden. The study of renal I/R injury is still one of the hot topics in the medical field. Ebselen is an organic selenide that attenuates I/R injury in various organs. However, its effect and related mechanism underlying renal I/R injury remains unclear. In this study, we established a rat model of renal I/R injury to study the preventive effect of ebselen on renal I/R injury and further explore the potential mechanism of its action. We found that ebselen pretreatment reduced renal dysfunction and tissue damage caused by renal I/R. In addition, ebselen enhanced autophagy and inhibited oxidative stress. Additionally, ebselen pretreatment activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. The protective effect of ebselen was suppressed by autophagy inhibitor wortmannin. In conclusion, ebselen could ameliorate renal I/R injury, probably by enhancing autophagy, activating the Nrf2 signaling pathway, and reducing oxidative stress.

     

Tle1 attenuates hepatic ischemia/reperfusion injury by suppressing NOD2/NF-κB signaling

ABSTRACT

Liver damage induced by ischemia/reperfusion (I/R) remains a primary issue in multiple hepatic surgeries. Innate immune-mediated inflammatory responses during the reperfusion stage aggravate the injury. Nevertheless, the detailed mechanism of hepatic I/R has not been fully clarified yet. Our research focuses on the role of Transducin-like enhancer of split-1 (Tle1) in the liver I/R injury and the relation between Tle1 and Nucleotide-binding oligomerization domain 2 (NOD2). To answer these questions, we constructed mouse models of I/R and cell models of hypoxia/reoxygenation (H/R). We found decreased Tle1 accompanied by increased NOD2 during reperfusion. Mice pro-injected with Tle1-siRNA emerged aggravated liver dysfunction. Repression of Tle1 had a significant impact on NOD2 and downstream NF-κB signaling in vitro. However, alteration of NOD2 failed to affect the expression of Tle1. To conclude, our study demonstrates that Tle1 shelters the liver from I/R injury through suppression of NOD2-dependent NF-κB activation and subsequent inflammatory responses.     

Role of CC-chemokine receptor 5 on myocardial ischemia–reperfusion injury in rats

The expression level of CC-chemokine receptor 5 (CCR5) is enhanced post inflammatory stimulations and might play a crucial role on inflammatory cells infiltration post myocardial ischemia. The purpose of this study was to evaluate the role of CCR5 on myocardial ischemia–reperfusion (I/R) injury in rats. Adult male rats were randomized to sham group, I/R group (I/R, 30 min coronary artery occlusion followed by 2-h reperfusion), ischemic preconditioning (I/R + Pre), CCR5 antibody group [I/R + CCR5Ab (0.2 mg/kg)], and CCR5 agonist group [I/R + CCR5Ago, RNATES (0.1 mg/kg)], n = 12 each group. The serum level of creatine kinase (CK) and tumor necrosis factor α (TNF-α) were measured by ELISA. Myocardial infarction size and myeloperoxidase (MPO) activity were determined. Myocardial protein expression of CCR5 and intercellular adhesion molecule-1 (ICAM-1) were evaluated by Western blotting and immunohistochemistry staining, respectively. Myocardial nuclear factor-kappa B (NF-κB) activity was assayed by electrophoretic mobility shift assay. Myocardial CCR5 protein expression was significantly reduced in I/R + Pre group (P < 0.05 vs. I/R) and further reduced in I/R + CCR5Ab group (P < 0.05 vs. I/R + Pre). LVSP and ±dP/dt _max were significantly lower while serum CK and TNF-α as well as myocardial MPO activity, ICAM-1 expression, and NF-κB activity were significantly higher in I/R group than in sham group (all P < 0.05), which were significantly reversed by I/R + Pre (all P < 0.05 vs. I/R) and I/R + CCR5Ab (all P < 0.05 vs. I/R + Pre) while aggravated by I/R + CCR5Ago (all P < 0.05 vs. I/R). Our results suggest that blocking CCR5 attenuates while enhancing CCR5 aggravates myocardial I/R injury through modulating inflammatory responses in rat heart.     

The CD39-adenosinergic axis in the pathogenesis of renal ischemia–reperfusion injury

Hypoxic injury occurs when the blood supply to an organ is interrupted; subsequent reperfusion halts ongoing ischemic damage but paradoxically leads to further inflammation. Together this is termed ischemia–reperfusion injury (IRI). IRI is inherent to organ transplantation and impacts both the short- and long-term outcomes of the transplanted organ. Activation of the purinergic signalling pathway is intrinsic to the pathogenesis of, and endogenous response to IRI. Therapies targeting the purinergic pathway in IRI are an attractive avenue for the improvement of transplant outcomes and the basis of ongoing research. This review aims to examine the role of adenosine receptor signalling and the ecto-nucleotidases, CD39 and CD73, in IRI, with a particular focus on renal IRI.     

A PPAR-γ ligand, 15-deoxy-Δ12,14-prostaglandin J2, inhibited gastric mucosal injury induced by ischemia–reperfusion in rats

Abstract

Introduction: Recent studies have demonstrated the anti-inflammatory action of 15-deoxy-Δ12,14-prostaglandin J₂ (15d-PGJ₂), a derivative of the PGD₂ metabolic pathway. Acute inflammation, including neutrophil activation, plays a critical role in the pathogenesis of ischemia–reperfusion (I/R). The aim of the present study was to determine the effect of 15d-PGJ₂ on I/R-induced gastric mucosal injury in rats.

Methods: Gastric mucosal damage was induced in male Wistar rats by clamping the celiac artery for 30 min followed by reperfusion. 15d-PGJ₂ (0.01–1.0 mg/kg) was given to the rats intraperitoneally 1 h before the vascular clamping. The area of gastric mucosal erosions (erosion index) was measured. Thiobarbituric acid reactive substances (TBARS) and tissue-associated myeloperoxidase (MPO) activity were measured in the gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. The expression of tumor necrosis factor-α (TNF-α) in gastric mucosa was measured by ELISA. In addition, to elucidate whether the protective effects of 15d-PGJ₂ are related to the activation of the PPAR-γ receptor, we also investigated the effects of a PPAR-γ antagonist, GW9662.

Results: After 60 min of reperfusion, the area of gastric erosion index had significantly increased from the mean basal levels. The increase in the erosion index was significantly inhibited by pretreatment with 15d-PGJ₂ in a dose-dependent manner. On the other hand, GW9662 reversed the protective effect of 15d-PGJ₂. The concentration of TBARS and MPO activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with 15d-PGJ₂ significantly reduced these increases. The TNF-α content was significantly higher in the I/R group than in the sham-operated group. However, the increase in TNF-α was significantly inhibited by pretreatment with 15d-PGJ₂.

Conclusions: 15d-PGJ₂ significantly inhibited the severity of acute gastric mucosal injury induced by I/R in rats through PPAR-γ-dependent mechanisms. This effect may be due, in part, to a reduction in the infiltration of neutrophils into the gastric mucosa, possibly via the inhibition of inflammatory cytokine.     

Investigate of AQP gene expression in the liver of mice after ischemia–reperfusion

Ischemia–reperfusion (IR) injury usually occurs during liver transplantation. Aquaporins (AQPs) are transmembrane channels that facilitate water permeability through cell membranes and are essential for the regulation of water homeostasis. Changes in the AQPs expression have been correlated with several inflammatory diseases. Less is known about AQPs expression in hepatic ischemia reperfusion injury. To clarify the roles of AQPs in IR injury, in this current study we examined the gene expression patterns of AQP1, 8 and 9 in the liver after IR injury. Male balb/c mice were exposed to partial (70%) hepatic ischemia for 65 min and then randomized into five groups of reperfusion [0 h (A), 8 h (B), 1 day (C), 3 days (D), and 7 days (E)]. A surgical group was also selected as the sham group. Serum and liver tissue samples were collected for evaluation of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver histopathology. Real time PCR was performed to evaluate the AQPs expression. I/R injury resulted in a significant increase in ALT and AST (p?<?0.05) compared to sham mice in each group. The gene expression of AQPs was significantly increased in the IR group compared with the sham group (p?<?0.05). AQP8 and AQP1 after 8 h (group B) showed the highest gene expression in comparison with other groups, but the highest level of AQP9 gene expression was observed after 1 day (group C). Pathologic changes in the liver after reperfusion were confirmed the IR. In the IR group cytoplasmic vacuolization, inflammatory cell infiltration and focal necrosis were detected. In conclusion, our findings indicated that the damage caused by ischemia–reperfusion in the liver can change the expression of AQP genes, which can interfere with hepatocellular homeostasis and their function. Upregulation of AQP1, 8 and 9 could contribute to the development of hepatocellular swelling after hepatic IR injury.     

Cardiac proteomic responses to ischemia–reperfusion injury and ischemic preconditioning

Cardiac ischemia and ischemia–reperfusion (I/R) injury are major contributors to morbidity and mortality worldwide. Pathological mechanisms of I/R and the physiological mechanisms of ischemic preconditioning (IPC), which is an effective cardiac protective response, have been widely investigated in the last decade to search for means to prevent or treat this disease. Proteomics is a powerful analytical tool that has provided important information to identify target proteins and understand the underlying mechanisms of I/R and IPC. Here, we review the application of proteomics to I/R injury and IPC to discover target proteins. We analyze the functional meaning of the accumulated data on hundreds of proteins using various bioinformatics applications. In addition, we review exercise-induced proteomic alterations in the heart to understand the potential cardioprotective role of exercise against I/R injury. Further developments in the proteomic field that target specialized proteins will yield new insights for optimizing therapeutic targets and developing a wide range of therapeutic agents against ischemic heart disease.     

Ablation of cereblon attenuates myocardial ischemia–reperfusion injury

Cereblon (CRBN) was originally identified as a target protein for a mild type of mental retardation in humans. However, recent studies showed that CRBN acts as a negative regulator of AMP-activated protein kinase (AMPK) by binding directly to the AMPK catalytic subunit. Because AMPK is implicated in myocardial ischemia–reperfusion (I–R) injury, we reasoned that CRBN might play a role in the pathology of myocardial I–R through regulation of AMPK activity. To test this hypothesis, wild-type (WT) and crbn knockout (KO) mice were subjected to I–R (complete ligation of the coronary artery for 30 min followed by 24 h of reperfusion). We found significantly smaller infarct sizes and less fibrosis in the hearts of KO mice than in those of WT mice. Apoptosis was also significantly reduced in the KO mice compared with that in WT mice, as shown by the reduced numbers of TUNEL-positive cells. In parallel, AMPK activity remained at normal levels in KO mice undergoing I–R, whereas it was significantly reduced in WT mice under the same conditions. In rat neonatal cardiomyocytes, overexpression of CRBN significantly reduced AMPK activity, as demonstrated by reductions in both phosphorylation levels of AMPK and the expression of its downstream target genes. Collectively, these data demonstrate that CRBN plays an important role in myocardial I–R injury through modulation of AMPK activity.     

Cellular and molecular mechanisms of 17β-estradiol postconditioning protection against gastric mucosal injury induced by ischemia/reperfusion in rats

AimsTo investigate the protective effects of 17β-estradiol postconditioning against ischemia/reperfusion (I–R)-induced gastric mucosal injury in rats.Main methodsThe animal model of gastric ischemia/reperfusion was established by clamping of the celiac artery for 30 min and reperfusion for 30 min, 1 h, 3 h, 6 h, 12 h or 24 h. 17β-estradiol at doses of 5, 50 or 100 μg/kg (rat) was administered via peripheral veins 2 min before reperfusion. In a subgroup of rats, the estrogen receptor antagonist fulvestrant (Ful, 2 mg/kg) was intravenously injected prior to 17β-estradiol administration. Histological and immunohistochemical methods were employed to assess the gastric mucosal injury index and gastric mucosal cell apoptosis and proliferation. The malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, xanthine oxidase (XOD) activity and hydroxyl free radical (–OH) inhibitory ability were determined by colorimetric assays. Subsequently, the expression of Bcl-2 and Bax in rat gastric mucosa was examined by western blotting.Key findings17β-estradiol dose-dependently inhibited gastric I–R (GI–R) injury, and 17β-estradiol (50 μg/kg) markedly attenuated GI–R injury 1 h after reperfusion. 17β-estradiol inhibited gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation in addition to increasing SOD activity and –OH inhibitory ability and decreasing the MDA content and XOD activity. The Bax protein level increased 1 h after GI–R and was markedly reduced by intravenous administration of 17β-estradiol. In contrast, the level of Bcl-2 protein decreased 1 h after GI–R and was restored to normal levels by intravenous administration of 17β-estradiol. These effects of 17β-estradiol were inhibited by pretreatment with fulvestrant.Significance17β-estradiol postconditioning should be investigated further as a possible strategy against gastric mucosal injury.