The possible structural changes in the adrenal gland cortex after induction of hepatic ischemia–reperfusion injury in male albino rats: Light and electron microscopic study

The adrenal gland is an important endocrine gland in the body that secrets the adrenal hormones. One of the important clinical issues is the hepatic ischemia–reperfusion (IR) injury. Liver IR injury results in many distant organs dysfunctions such as lung, kidney, intestine, pancreas, and myocardium. The aim of the present study was to investigate the possible remote effects of hepatic IR on the structure of the adrenal cortex. Twenty healthy males, Sprague–Dawley albino rats aged 6–8 weeks were randomly divided into two groups (10 rats each): the sham control group (SC-group) and the ischemia–reperfusion group (IR-group). Sera were estimated for the following: aspartate transaminase (AST), alanine transaminase (ALT), lactic dehydrogenase (LDH), and corticosterone levels. Also oxidative markers such as malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α), and the antioxidative enzyme, catalase were measured. Adrenal glands were processed for light and transmission electron microscopic study. The results showed a significant increase in serum liver enzymes (AST, ALT, and LDH), corticosterone, MDA, and TNF-α levels and a significant decrease in serum levels of catalase in IR-group compared with SC-group. Adrenal cortical tissue of IR-group showed the loss of normal appearance. Some cells of zona glomerulosa and most of the zona fasciculata cells appeared swollen and degenerated with highly vacuolated cytoplasm. Other cells were shrunken with deeply acidophilic cytoplasm and pyknotic nuclei. Degenerated mitochondria with disrupted cristae, lipid droplets were confluent and dilated smooth endoplasmic reticulum were seen. Few zona reticularis cells had the dark nucleus and cytoplasmic vacuolations. In the different zones, blood capillaries were markedly congested and some inflammatory cells infiltrations were observed. Liver IR affected the structure of the adrenal cortex.     

Exosomes from human-bone-marrow-derived mesenchymal stem cells protect against renal ischemia/reperfusion injury via transferring miR-199a-3p

Renal ischemia/reperfusion (I/R) injury is the main reason for acute kidney injury (AKI) and is closely related to high morbidity and mortality. In this study, we found that exosomes from human-bone-marrow-derived mesenchymal stem cells (hBMSC-Exos) play a protective role in hypoxia/reoxygenation (H/R) injury. hBMSC-Exos were enriched in miR-199a-3p, and hBMSC-Exo treatment increased the expression level of miR-199a-3p in renal cells. We further explored the function of miR-199a-3p on H/R injury. miR-199a-3p was knocked down in hBMSCs with a miR-199a-3p inhibitor. HK-2 cells cocultured with miR-199a-3p-knockdown hBMSCs were more susceptible to H/R injury and showed more apoptosis than those cocultured with hBMSCs or miR-199a-3p-overexpressing hBMSCs. Meanwhile, we found that HK-2 cells exposed to H/R treatment incubated with hBMSC-Exos decreased semaphorin 3A (Sema3A) and activated the protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK) pathways. However, HK-2 cells cocultured with miR-199a-3p-knockdown hBMSCs restored Sema3A expression and blocked the activation of the AKT and ERK pathways. Moreover, knocking down Sema3A could reactivate the AKT and ERK pathways suppressed by a miR-199a-3p inhibitor. In vivo, we injected hBMSC-Exos into mice suffering from I/R injury; this treatment induced functional recovery and histologic protection and reduced cleaved caspase-3 and Sema3A expression levels, as shown by immunohistochemistry. On the whole, this study demonstrated an antiapoptotic effect of hBMSC-Exos, which protected against I/R injury, via delivering miR-199a-3p to renal cells, downregulating Sema3A expression and thereby activating the AKT and ERK pathways. These findings reveal a novel mechanism of AKI treated with hBMSC-Exos and provide a therapeutic method for kidney diseases.     

Impaired renal organic anion transport 1 (SLC22A6) and its regulation following acute myocardial infarction and reperfusion injury in rats

Acute kidney injury (AKI) is a high frequent and common complication following acute myocardial infarction (AMI). This study examined and identified the effect of AMI-induced AKI on organic anion transporter 1 (Oat1) and Oat3 transport using clinical setting of pre-renal AKI in vivo. Cardiac ischaemia (CI) and cardiac ischaemia and reperfusion (CIR) were induced in rats by 30-min left anterior descending coronary artery occlusion and 30-min occlusion followed by 120-min reperfusion, respectively. Renal hemodynamic parameters, mitochondrial function and Oat1/Oat3 expression and function were determined along with biochemical markers. Results showed that CI markedly reduced renal blood flow and pressure by approximately 40%, while these parameters were recovered during reperfusion. CI and CIR progressively attenuated renal function and induced oxidative stress by increasing plasma BUN, creatinine and malondialdehyde levels. Correspondingly, SOD, GPx, CAT mRNAs were decreased, while TNFα, IL1β, COX2, iNOS, NOX2, NOX4, and xanthine oxidase were increased. Mitochondrial dysfunction as indicated by increasing ROS, membrane depolarisation, swelling and caspase3 activation were shown. Early significant detection of AKI; KIM1, IL18, was found. All of which deteriorated para-aminohippurate transport by down-regulating Oat1 during sudden ischaemia. This consequent blunted the trafficking rate of Oat1/Oat3 transport via down-regulating PKCζ/Akt and up-regulating PKCα/NFκB during CI and CIR. Thus, this promising study indicates that CI and CIR abruptly impaired renal Oat1 and regulatory proteins of Oat1/Oat3, which supports dysregulation of remote sensing and signalling and inter-organ/organismal communication. Oat1, therefore, could potentially worsen AKI and might be a potential therapeutic target for early reversal of such injury.     

Inhibition of miR-181b-5p protects cardiomyocytes against ischemia/reperfusion injury by targeting AKT3 and PI3KR3

Ischemic heart disease (IHD) is the most occurring cardiovascular-associated disease, which is a primary leading cause of cardiac disability and death worldwide. Myocardial ischemia/reperfusion injury (MI/RI) has been linked to IHD-induced cardiomyocytes apoptosis and tissue damage. The clinical studies have indicated that pathophysiologic mechanisms of MI/RI are associated with reactive oxygen species generation, calcium overload, energy metabolism disorder, neutrophil infiltration, and others. However, the genetic mechanism of MI/RI remains unclear. In this study, we successfully established the reproducing abnormal heart observed in rat, of IHD-induced MI/RI post operation. By using these rats, we illustrated that expression of miR-181b-5p was increased not only in both hypoxia/reoxygenation-cultured H9C2 but also heart of myocardial ischemia/reperfusion (MI/R) rat. Suppression of the miR-181b-5p cardiomyocytes apoptosis and rescued myocardial infarction. Additionally, our data indicated that miR-181b-5p negatively regulates the expression of AKT3 and PIK3R3 through directly binding with its 3′-untranslated region. More importantly, suppression of miR-181b-5p protects the cardiomyocytes apoptosis and tissue damage from MI/R via regulation of PIK3R3 and AKT3. Hence, our study indicates that miR-181b-5p is essential for MI/RI via regulation of PI3K/Akt signaling pathway and could be a potential therapeutic target in IHD.     

Glaucocalyxin B protects against oxygen-glucose-deprivation/reperfusion-induced neuronal injury in PC-12 cells

Oxidative stress has been implicated in the development of cerebral ischemia/reperfusion (I/R) injury. Glaucocalyxin B (GLB), one of five ent-kauranoid diterpenoids, was reported to possess neuroprotective activity. However, the effect of GLB on oxygen-glucose-deprivation/reperfusion (OGD/R)-induced cell injury in PC-12 cells has not been explored. PC-12 cells was treated with various concentrations of GLB (0, 2.5, 5 and 10 μM), and cell viability was detected using the MTT assay. PC-12 cells were pretreated with the indicated concentration of GLB (2.5-10 μM, 2 hours pretreatment), and were maintained under OGD for 3 hours, followed by 24 hours of reoxygenation. Cell viability was assessed using the MTT assay. The levels of superoxide dismutase, malondialdehyde, and glutathione peroxidase were detected using commercially available ELISA Kits. Intracellular reactive oxygen species level was measured using the fluorescent probe 2′,7′-dichlorofluorescein diacetate. The levels of Bcl-2, Bax, p-Akt, Akt, p-mTOR, mTOR were detected using Western blot. Our results revealed that GLB significantly protected PC12 cells against OGD/R-induced cell injury. In addition, GLB efficiently inhibited oxidative stress and cell apoptosis in OGD/R-stimulated PC-12 cells. Mechanistic studies revealed that pretreatment with GLB could induce the activation of Akt/mTOR signaling pathway resulting in protection of OGD-treated PC12 cells. In conclusion, our data indicate for the first time that GLB protects against OGD/R-induced neuronal injury in PC-12 cells. The mechanism of the protective effect of GLB is partially associated with activation of the Akt/mTOR signaling pathway. Thus, GLB may be a potential agent for protection against cerebral I/R injury.     

Microvesicles-mediated communication between endothelial cells modulates,endothelial survival,and angiogenic function via transferring of miR-125a-5p

Endothelial cells (ECs) released microvesicles (EMVs) could modulate the functions of target cells by transferring their microRNAs (miRs). We have reported that miR-125a-5p protected EC function. In this study, we determined whether EMVs provided beneficial effects on ECs by transferring miR-125a-5p. Human brain microvessel ECs were transfected with miR-125a-5p mimic or miR-125a-5p short hairpin RNA to obtain miR-125a-5p overexpressing ECs and miR-125a-5p knockdown ECs, and their derived EMVs. For the functional study, ECs or hypoxia/reoxygenation injured ECs were coincubated with various EMVs. The survival and angiogenic function of ECs were measured. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used for measuring the levels of phosphoinositide 3-kinase (PI3K), phosphorylation-Akt (p-Akt)/Akt, p-endothelial nitric oxide synthase (p-eNOS), cleaved caspase-3, and miR-125a-5p. PI3K inhibitor was used for pathway analysis. EMVs promoted the proliferation, migration, and tube formation ability of ECs, and alleviated the apoptotic rate of ECs. These effects were associated by an increase in p-Akt/Akt and p-eNOS, and a decrease in cleaved caspase-3 could be abolished by LY294002. Overexpression or downregulation of miR-125a-5p in EMVs promoted or inhibited those effects of EMVs. EMVs could enhance the survival and angiogenic function of ECs via delivering miR-125a-5p to modulate the expression of PI3K/Akt/eNOS pathway and caspase-3.     

Stomatin-like protein-2 relieve myocardial ischemia/reperfusion injury by adenosine 5′-monophosphate-activated protein kinase signal pathway

Previous studies have shown that stomatin-like protein-2 (SLP-2) could regulate mitochondrial biogenesis and function. The study was designed to explore the contribution of SLP-2 to the myocardial ischemia and reperfusion (I/R) injury. Anesthetized rats were treated with SLP-2 and subjected to ischemia for 30 minutes before 3 hours of reperfusion. An oxygen-glucose deprivation/reoxygenation model of I/R was established in H9C2 cells. In vivo, SLP-2 significantly improved cardiac function recovery of myocardial I/R injury rats by increasing fractional shortening and ejection fraction. SLP-2 pretreatment alleviated infarct area and myocardial apoptosis, which was paralleled by decreasing the level of cleaved caspase-3 and the ratio of Bax/Bcl-2, increasing the content of superoxide dismutase and reducing oxidative stress damage in serum. In addition, SLP-2 increased the level of ATP and stabilized mitochondrial potential (Ψm). The present in vitro study revealed that overexpression with SLP-2 reduced H9C2 cells apoptosis, accompanied by an increased level of ATP, the ratio of mitochondrial DNA/nuclear DNA, activities of complex II and V, and decreased the production of mitochondrial reactive oxygen species. Simultaneously, SLP-2 activated the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway in myocardial I/R injury rats and H9C2 cells. This study revealed that SLP-2 mediates the cardioprotective effect against I/R injury by regulating AMPK signaling pathway.