Background Recent studies in various animal models have suggested that anesthetics such as propofol, when administered early in life, can lead to neurotoxicity. These studies have raised significant safety concerns regarding the use of anesthetics in the pediatric population and highlight the need for a better model by which to study anesthetic-induced neurotoxicity in humans. Human embryonic stem cells (hESCs) are capable of differentiating into any cell type and represent a promising model to study mechanisms governing anesthetic-induced neurotoxicity. Methods Cell death in hESC-derived neurons was assessed using TUNEL staining and microRNA (miR) expression was assessed using quantitative reverse transcription polymerase chain reaction (qRTPCR). miR-21 was overexpressed and knocked down using a miR-21 mimic and antagomir, respectively. Sprouty 2 was knocked down using a small interfering RNA and the expression of the miR-21 targets of interest was assessed by Western blot. Results Propofol dose and exposure time-dependently induced significant cell death (n = 3) in the neurons and downregulated several microRNAs, including miR-21. Overexpression of miR-21 and knockdown of Sprouty 2 attenuated the increase in TUNEL-positive cells following propofol exposure. In addition, miR-21 knockdown increased the number of TUNEL-positive cells by 30% (n = 5). Finally, activated Signal Transducer and Activator of Transcription 3 (STAT3) and protein kinase B (Akt) were downregulated and Sprouty 2 was upregulated following propofol exposure (n = 3). Conclusions These data suggest that: (1) hESC-derived neurons represent a promising in vitro human model for studying anesthetic-induced neurotoxicity, (2) propofol induces cell death in hESC-derived neurons and (3) the propofol-induced cell death may occur via a STAT3/miR-21/Sprouty2-dependent mechanism.
MicroRNAs (miRNAs or miRs) are endogenous, small RNA molecules that suppress expression of targeted mRNA. miR-21, one of the most extensively studied miRNAs, is importantly involved in divergent pathophysiological processes relating to ischemia/reperfusion (I/R) injury, such as inflammation and angiogenesis. The role of miR-21 in renal I/R is complex, with both protective and pathological pathways being regulated by miR-21. Preconditioning-induced upregulation of miR-21 contributes to the protection against subsequent renal I/R injury through the targeting of genes such as the proapoptotic gene programmed cell death 4 and interactions between miR-21 and hypoxia-inducible factor. Conversely, long-term elevation of miR-21 may be detrimental to the organ by promoting the development of renal interstitial fibrosis following I/R injury. miR-21 is importantly involved in several pathophysiological processes related to I/R injury including inflammation and angiogenesis as well as the biology of stem cells that could be used to treat I/R injury; however, the effect of miR-21 on these processes in renal I/R injury remains to be studied.
Background The role of microRNA-21 in isoflurane-induced cardioprotection is unknown. We addressed this issue using microRNA-21 knockout mice and explored the underlying mechanisms. Methods C57BL/6 and microRNA-21 knockout mice were echocardiographically examined. Mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in vivo or ex vivo in the presence or absence of 1.0 minimum alveolar concentration of isoflurane administered before ischemia. Cardiac Akt, eNOS, and nNOS proteins were determined by Western blot. Opening of the mitochondrial permeability transition pore (mPTP) in cardiomyocytes was induced by photoexcitation-generated oxidative stress and detected by rapid dissipation of tetramethylrhodamine ethyl ester fluorescence using a confocal microscope. Results Genetic disruption of miR-21 gene did not alter phenotype of the left ventricle, baseline cardiac function, area at risk, and the ratios of p-Akt/Akt, p-eNOS/eNOS, and pnNOS/nNOS. Isoflurane decreased infarct size from 54±10% in control to 36±10% (P<0.05, n=8 mice/group), improved cardiac function after reperfusion, and increased the ratios of p-Akt/AKT, p-eNOS/eNOS, and p-nNOS/nNOS in C57BL/6 mice subjected to ischemia/reperfusion injury. These beneficial effects of isoflurane were lost in microRNA-21 knockout mice. There were no significant differences in time of the mPTP opening induced by photoexcitation-generated oxidative stress in cardiomyocytes isolated between C57BL/6 and microRNA-21 knockout mice. ISO significantly delayed mPTP opening in cardiomyocytes from C57BL/6 but not microRNA-21 knockout mice. Conclusions Isoflurane protects mouse hearts from ischemia/reperfusion injury by a microRNA-21-dependent mechanism. The Akt/NOS/mPTP pathway is involved in the microRNA-21-mediated protective effect of isoflurane.
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