Exosomal OIP5-AS1 attenuates cerebral ischemia-reperfusion injury by negatively regulating TXNIP protein stability and inhibiting neuronal pyroptosis.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) can cause neuronal apoptosis and lead to irreversible brain injury. Numerous lncRNAs have been reported to play important roles in CIRI, but it is unclear whether these lncRNAs can function through exosomes. METHODS: In this study, we utilized the middle cerebral artery occlusion/reperfusion (MCAO/R) animal model and the oxygen-glucose deprivation/ reoxygenation (OGD/R) cell model. RNA sequencing was performed to screen for differentially expressed lncRNAs in M2 microglia-derived exosomes (M2-Exos). RNA pull-down, RNA immunoprecipitation, co-immunoprecipitation and ubiquitination assays were used to explore the molecular mechanism of OIP5-AS1 in alleviating CIRI. RESULTS: M2-Exos could alleviate nerve injury and pyroptosis after CIRI in vitro and in vivo. OIP5-AS1 was found to be significantly up-regulated in M2-Exos and down-regulated in OGD/R neurons, MCAO/R mice and ischemic stroke patients. In MCAO/R mice, OIP5-AS1 could reduce cerebral infarct size, cerebral edema and mNSS scores, and inhibit the expression levels of pyroptosis-related proteins in brain tissue. TXNIP was confirmed to be a reliable binding protein of OIP5-AS1. OIP5-AS1 overexpression significantly attenuated MCAO/R-induced upregulation of TXNIP at the protein level, but not at the mRNA level. OIP5-AS1 promoted the TXNIP degradation process and increased the ubiquitination of TXNIP. ITCH could bind to TXNIP. ITCH overexpression or knockdown did not alter the mRNA level of TXNIP, but negatively regulated TXNIP expression at the protein level. ITCH accelerated the degradation and ubiquitination of TXNIP, which could be attenuated by OIP5-AS1 knockdown. OIP5-AS1 could improve neuronal damage and inhibit neuronal pyroptosis through TXNIP. CONCLUSIONS: M2-Exo-derived OIP5-AS1 can induce TXNIP ubiquitination and degradation by recruiting ITCH, negatively regulate TXNIP protein stability, inhibit neuronal pyroptosis, and attenuate CIRI.

miR-128-3p inhibits the inflammation by targeting MAPK6 in penicillin-induced astrocytes.

OBJECTIVE: Epilepsy causes physical and mental damage to patients. As well known, microRNAs (miRNAs) provide therapeutic target potentials for patients with epilepsy. miR-128-3p was previously reported to be downregulated in temporal lobe epilepsy (TLE) patients, however, its detailed function in epilepsy is unknown. METHODS: Astrocytes function in epilepsy, penicillin-induced astrocytes can be used as a model for seizures in vitro. Currently, the expression levels of mitogen-activated protein kinase 6 (MAPK6), interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) were determined by western blot and reverse transcription-quantitative PCR analyses (RT-qPCR). The expression level of miR-128-3p was evaluated by RT-qPCR. TargetScan 7.1 and dual luciferase reporter assay were used for prediction and verification of interaction between miR-128-3p and MAPK6 3' untranslated region (UTR). Cell viability was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. RESULTS: We found that penicillin-induced decrease in cell viability, and increase of TNF-α/IL-1ß in primary astrocytes. There were lower miR-128-3p and higher MAPK6 in penicillin-treated primary astrocytes. miR-128-3p overexpression rescued penicillin-induced reduction of cell viability, and upregulation of TNF-α/IL-1ß, which was partially abolished by MAPK6 overexpression. CONCLUSION: Altogether, miR-128-3p attenuates penicillin-induced cell injury and inflammation in astrocytes by targeting MAPK6, thus providing a protective role in epilepsy.

Remodeling of occluded internal carotid artery in vessel wall magnetic resonance imaging.

OBJECTIVE: The purpose of the present study was to investigate the remodeling pattern of the extracranial occluded internal carotid artery (OICA) by vessel wall magnetic resonance imaging (VWI). METHODS: Thirty-nine atherosclerotic OICAs from 32 consecutive cases underwent 3-Tesla VWI to acquire pre- and post-contrast T1-weighted two-dimensional fluid-attenuated inversion recovery fast spin echo sequences. 25 symptomatic CAs exhibited ipsilateral downstream cerebral ischemia or ophthalmic artery embolism within last three months. The 14 remaining CAs were asymptomatic. Twenty-four CAs from 22 patients with atherosclerosis but no stenosis were recruited as control group. The outer wall area (OWA) was calculated based on the outer contour of the carotid artery drawn on the pre-contrast VWI. Negative remodeling was defined as a lower OWA compared to that of control group. RESULTS: Clinical characteristics including age, sex and vascular risk factors showed no significant difference between the occluded and control group. However, the OWA was lower in the occluded group than in the control group (0.63 versus 0.90 cm2, p = 0.004). For all OICAs, the OWA was larger in symptomatic cases than asymptomatic cases (0.71 versus 0.49cm2, p = 0.025). Using a cutoff value of 0.44, the sensitivity and specificity of OWA for detecting symptomatic OICA were 0.88 and 0.57, respectively. Heterogeneous signal intensity and enhancement were more often observed at the proximal than the distal segment of occlusion (p < 0.001). The inter-observer agreement regarding the evaluation of VWI characteristics was desirable (κ = 0.805 ∼ 0.847). CONCLUSIONS: Negative remodeling is prevalent in OICA, especially in asymptomatic cases.

Adipose-derived mesenchymal stem cells-derived exosome-mediated microRNA-342-5p protects endothelial cells against atherosclerosis.

Exosomes are reported to mediate several disease-related microRNAs (miRNAs) to affect the progression of diseases, including atherosclerosis. Here, we aimed to screen the atherosclerosis-associated miRNAs and preliminarily investigate the potential regulatory mechanism of atherosclerosis. First, the lesion model for human umbilical vein endothelial cells (HUVECs) was favorably constructed. Later, through RNA-sequencing and bioinformatics analyses, miR-342-5p was identified in lesion model for HUVECs. MiR-342-5p overexpression or knockdown evidently promoted or inhibited the apoptosis of HUVECs impaired by H2O2. Mechanistically, PPP1R12B was found to have great potential as a target of miR-342-5p in HUVECs impaired by H2O2, supported by RNA-sequencing and a series of bioinformatics analyses. Meanwhile, the effect of miR-342-5p on PPP1R12B expression in HUVECs' lesion model was explored, revealing that miR-342-5p had an inhibitory role in PPP1R12B expression. Additionally, adipose-derived mesenchymal stem cells (ADSCs) in spindle-like shape and their derived exosomes with 30 to 150 nm diameter were characterized. Furthermore, results showed miR-342-5p was evidently decreased in the presence of ADSCs-derived exosomes. These findings indicated ADSCs-derived exosomes restrained the expression of miR-324-5p in lesion model. Collectively, this work demonstrates an atherosclerosis-associated miR-342-5p and reveals a preliminary possible mechanism in which miR-342-5p mediated by ADSCs-derived exosomes protects endothelial cells against atherosclerosis.

2R,4R-APDC, a Metabotropic Glutamate Receptor Agonist, Reduced Neuronal Apoptosis by Upregulating MicroRNA-128 in a Rat Model After Seizures.

This study aimed to study the protective effect of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a selective metabotropic glutamate receptor agonist, against hippocampal neuronal apoptosis induced by seizures in a rat model of pilocarpine-induced epilepsy. The Morris water maze test was used to assess the spatial memory abilities of epileptic rats with or without 2R,4R-APDC treatment. TUNEL assay was performed to examine neuronal apoptosis in hippocampus. Western blot was conducted to evaluate changes in the levels of caspase-3 and caspase-9 in hippocampus. Real-time PCR was used to determine the levels of microRNA-128 (miR-128) in hippocampus. The results of the Morris water maze test showed that the 2R,4R-APDC treatment reduced the escape latencies and swimming lengths of rats after seizures. The TUNEL assay showed that 2R,4R-APDC significantly counteracted seizure-induced cell apoptosis. The western blot confirmed this finding, demonstrating that the levels of cleaved caspase-3 and cleaved caspase-9 were potently decreased by 2R,4R-APDC in rat hippocampus after seizures. In addition, 2R,4R-APDC upregulated miR-128 expression levels in the hippocampus. A miR-128 mimic or inhibitor decreased or increased the percentage of TUNEL-positive cells in rats after seizures and 2R,4R-APDC treatment, respectively. The levels of both cleaved caspase-3 and cleaved caspase-9 were decreased in hippocampus exposed to the miR-128 mimic, whereas they were markedly increased in miR-128 inhibitor-treated hippocampus. In conclusion, 2R,4R-APDC protected hippocampal cells from cell apoptosis after seizures, possibly by upregulating miR-128.

Cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated acute epileptic neuronal death in vitro.

Intense poly(ADP-ribose) polymerase-1 (PARP-1) activation was implicated as a major cause of caspase-independent cell death in the hippocampal neuronal culture (HNC) model of acute acquired epilepsy (AE). The molecular mechanisms are quite complicated. The linkage among neuronal death, cellular nicotinamide adenine dinucleotide (NAD) levels, apoptosis-inducing factor (AIF) translocation, SIRT1 expression and activity were investigated here. The results showed that PARP-1 over-activation caused by Mg²âº-free stimuli led to cellular NAD depletion which could block AIF translocation from mitochondria to nucleus and attenuate neuronal death. Also, SIRT1 deacetylase activity was reduced by Mg²âº-free treatment, accompanied by elevated ratio of neuronal death, which could be rescued by NAD repletion. These data demonstrated that cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated epileptic neuronal death in the HNC model of acute AE.

Benazepril, an angiotensin-converting enzyme inhibitor, alleviates renal injury in spontaneously hypertensive rats by inhibiting advanced glycation end-product-mediated pathways.

1. Advanced glycation end-products (AGE) and their receptors (RAGE) have been implicated in renal damage in diabetes. The aim of the present study was to investigate the effects of benazepril, an angiotensin-converting enzyme inhibitor (ACEI), on the formation of AGE, the expression RAGE and other associated components in the oxidative stress pathway in spontaneously hypertensive rats (SHR). 2. Groups of SHR were treated with or without 10 mg/kg per day benazepril for 12 weeks. Systolic blood pressure (SBP) and angiotensin (Ang) II levels were evaluated in SHR and control Wistar-Kyoto (WKY) rats. Renal function was investigated by determining levels of proteinuria and glomerulosclerosis. Furthermore, reactive oxygen species (ROS) in the rat renal cortex were analysed using an H(2)O(2)-based hydroxyl radical-detection assay and the renal content of AGE, RAGE, NADPH oxidase p47phox, nuclear factor (NF)-kappaB p65, phosphorylated (p-) NF-kappaB p65, vascular cell adhesion molecule (VCAM)-1 and transforming growth factor (TGF)-beta1 was determined by immunohistochemistry, quantitative real-time polymerase chain reaction and western blot analysis. 3. Treatment with benazepril inhibited the formation of AngII, reduced SBP and alleviated renal lesions in SHR compared with both untreated SHR and control WKY rats. Benazepril treatment significantly suppressed the accumulation of AGE and expression of RAGE in the kidney of SHR. In addition, benazepril treatment reduced the upregulation of NADPH oxidase p47phox, ROS generation and NF-kappaB p65, p-NF-kappaB p65, VCAM-1 and TGF-beta1 expression in the kidney of SHR compared with both untreated SHR and control WKY rats. 4. The results of the present study provide new insights into the regulation by the renin-angiotensin system of AGE-RAGE, oxidative stress and nephropathy, increasing our understanding of the role of the RAS in nephropathy.