Neuroprotective Effects of microRNA-140-5p on Ischemic Stroke in Mice via its Regulation on the TLR4/NF-κB Axis

2020 ◽  
Author(s):  
Song Wenjun ◽  
Tiancheng Wang ◽  
Bei Shi ◽  
Zhijun Wu ◽  
Wenjie Wang ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
Related Factors ◽  
Neuroprotective Effects ◽  
High Level

Abstract Background and aim: Ischemic stroke is one of the main causes of death worldwide and permanent global disability. On the basis of existing literature data, we carried out studies in an effort to explore how miR-140-5p affects ischemic stroke and whether the mechanism relates to toll-like receptor-4 (TLR4) and nuclear factor-kappa B (NF-κB).Methods: Middle cerebral artery occlusion (MCAO) was employed to establish a mouse model of ischemic stroke in vivo, while primary neurons were exposed to oxygen-glucose deprivation (OGD) to set up an ischemic stroke model in vitro. RT-qPCR was performed to detect the miR-140-5p expression and Western blot was employed to detect the expression TLR4, NF-κB, and apoptosis-related factors. Then, based gain-function of experiments using miR-140-5p mimic and TLR4 overexpression plasmid, neurological function score, TTC staining, TUNEL staining, as well as flow cytometry were carried out to evaluate the effects of miR-140-5p and TLR4 on MCAO mice and OGD neurons. Meanwhile, dual-luciferase reporter assay was used to validate the relationship between miR-140-5p and TLR4.Results: miR-140-5p expressed at a low level and TLR4 at a high level in ischemic stroke. It was verified that miR-140-5p targeted TLR4 and downregulated its expression. miR-140-5p overexpression was observed to inhibit the apoptosis of neurons under OGD exposure and restrain the progression of ischemic stroke, while TLR4 overexpression promoted the apoptosis and disease progression. Besides, miR-140-5p overexpression led to a decrease in NF-κB protein level, which was increased by TLR4 overexpression. Conclusion: In conclusion, from our data we conclude that miR-140-5p overexpression may be instrumental for the therapeutic targeting of ischemic stroke by alleviating neuron injury with the involvement of TLR4/NF-κB axis.

scholarly journals Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke

2021 ◽  
Vol 118 (32) ◽  
pp. e2018850118
Author(s):  
Hiroo Takahashi ◽  
Ryo Asahina ◽  
Masayuki Fujioka ◽  
Takeshi K. Matsui ◽  
Shigeki Kato ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cortical Neurons ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Small Gtpase ◽  
Neuroprotective Effects ◽  
Necessary And Sufficient ◽  
Activity Dependent

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

scholarly journals Up-regulation of miR-499a-5p decreases cerebral ischemia/reperfusion injury by targeting PDCD4

2021 ◽  
Author(s):  
Weifeng Shan ◽  
Huifeng Ge ◽  
Bingquan Chen ◽  
Linger Huang ◽  
Shaojun Zhu ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Ischemia Reperfusion Injury ◽  
Cell Model ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Tunel Staining ◽  
Cerebral Ischemia Reperfusion

Abstract MiR-499a-5p was significantly down-regulated in degenerative tissues and correlated with apoptosis. Nonetheless, the biological function of miR-499a-5p in acute ischemic stroke has been still unclear. In this study, we found the plasma levels of miR-499a-5p were significantly down-regulated in 64 ischemic stroke patients and negatively correlated with the National Institutes of Health Stroke Scale score. Then, we constructed cerebral ischemia/reperfusion (I/R) injury in rats after middle cerebral artery occlusion and subsequent reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) treated SH-SY5Y cell model. Transfection with miR-499a-5p mimic was accomplished by intracerebroventricular injection in the in vivo I/R injury model. We further found miR-499a-5p overexpression decreased infarct volumes and cell apoptosis in the in vivo I/R stroke model using TTC and TUNEL staining. PDCD4 was a direct target of miR-499a-5p by luciferase report assay and western blotting. Knockdown of PDCD4 reduced the infarct damage and cortical neuron apoptosis caused by I/R injury. MiR-499a-5p exerted neuroprotective roles mainly through inhibiting PDCD4-mediated apoptosis by CCK-8 assay, LDH release assay and flow cytometry analysis. These findings suggest that miR-499a-5p might represent a novel target that regulates brain injury by inhibiting PDCD4-mediating apoptosis.

scholarly journals miR-19a/b-3p promotes inflammation during cerebral ischemia/reperfusion injury via SIRT1/FoxO3/SPHK1 pathway

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Feng Zhou ◽  
Yu-Kai Wang ◽  
Cheng-Guo Zhang ◽  
Bing-Yi Wu
Keyword(s):  
Inflammatory Responses ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Luciferase Assay ◽  
In Vivo Model ◽  
Tunel Staining

Abstract Background Stroke affects 3–4% of adults and kills numerous people each year. Recovering blood flow with minimal reperfusion-induced injury is crucial. However, the mechanisms underlying reperfusion-induced injury, particularly inflammation, are not well understood. Here, we investigated the function of miR-19a/b-3p/SIRT1/FoxO3/SPHK1 axis in ischemia/reperfusion (I/R). Methods MCAO (middle cerebral artery occlusion) reperfusion rat model was used as the in vivo model of I/R. Cultured neuronal cells subjected to OGD/R (oxygen glucose deprivation/reperfusion) were used as the in vitro model of I/R. MTT assay was used to assess cell viability and TUNEL staining was used to measure cell apoptosis. H&E staining was employed to examine cell morphology. qRT-PCR and western blot were performed to determine levels of miR-19a/b-3p, SIRT1, FoxO3, SPHK1, NF-κB p65, and cytokines like TNF-α, IL-6, and IL-1β. EMSA and ChIP were performed to validate the interaction of FoxO3 with SPHK1 promoter. Dual luciferase assay and RIP were used to verify the binding of miR-19a/b-3p with SIRT1 mRNA. Results miR-19a/b-3p, FoxO3, SPHK1, NF-κB p65, and cytokines were elevated while SIRT1 was reduced in brain tissues following MCAO/reperfusion or in cells upon OGD/R. Knockdown of SPHK1 or FoxO3 suppressed I/R-induced inflammation and cell death. Furthermore, knockdown of FoxO3 reversed the effects of SIRT1 knockdown. Inhibition of the miR-19a/b-3p suppressed inflammation and this suppression was blocked by SIRT1 knockdown. FoxO3 bound SPHK1 promoter and activated its transcription. miR-19a/b-3p directly targeted SIRT1 mRNA. Conclusion miR-19a/b-3p promotes inflammatory responses during I/R via targeting SIRT1/FoxO3/SPHK1 axis.

scholarly journals A Probable Dual Mode of Action for Both L- and D-Lactate Neuroprotection in Cerebral Ischemia

2015 ◽  
Vol 35 (10) ◽  
pp. 1561-1569 ◽  
Author(s):  
Ximena Castillo ◽  
Katia Rosafio ◽  
Matthias T Wyss ◽  
Konstantin Drandarov ◽  
Alfred Buck ◽  
...  
Keyword(s):  
Alternative Energy ◽  
Receptor Agonist ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Receptor Expression ◽  
Neuroprotective Effects ◽  
Energy Substrate ◽  
Deprivation Model

Lactate has been shown to offer neuroprotection in several pathologic conditions. This beneficial effect has been attributed to its use as an alternative energy substrate. However, recent description of the expression of the HCA1 receptor for lactate in the central nervous system calls for reassessment of the mechanism by which lactate exerts its neuroprotective effects. Here, we show that HCA1 receptor expression is enhanced 24 hours after reperfusion in an middle cerebral artery occlusion stroke model, in the ischemic cortex. Interestingly, intravenous injection of L-lactate at reperfusion led to further enhancement of HCA1 receptor expression in the cortex and striatum. Using an in vitro oxygen-glucose deprivation model, we show that the HCA1 receptor agonist 3,5-dihydroxybenzoic acid reduces cell death. We also observed that D-lactate, a reputedly non-metabolizable substrate but partial HCA1 receptor agonist, also provided neuroprotection in both in vitro and in vivo ischemia models. Quite unexpectedly, we show D-lactate to be partly extracted and oxidized by the rodent brain. Finally, pyruvate offered neuroprotection in vitro whereas acetate was ineffective. Our data suggest that L- and D-lactate offer neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic (most likely neuronal) cells as well as an energy substrate.

scholarly journals Restoration of Polyamine Metabolic Patterns inIn VivoandIn VitroModel of Ischemic Stroke following Human Mesenchymal Stem Cell Treatment

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Tae Hwan Shin ◽  
Geetika Phukan ◽  
Jeom Soon Shim ◽  
Duc-Toan Nguyen ◽  
Yongman Kim ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cell Model ◽  
Glucose Deprivation ◽  
Human Mesenchymal Stem Cell ◽  
Artery Occlusion ◽  
Control Group ◽  
Stem Cell Treatment ◽  
Cerebral Artery Occlusion

We investigated changes in PA levels by the treatment of human bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in ischemic stroke in rat brain model and in cultured neuronal SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD). In ischemic rat model, transient middle cerebral artery occlusion (MCAo) was performed for 2 h, followed by intravenous transplantation of hBM-MSCs or phosphate-buffered saline (PBS) the day following MCAo. Metabolic profiling analysis of PAs was examined in brains from three groups: control rats, PBS-treated MCAo rats (MCAo), and hBM-MSCs-treated MCAo rats (MCAo + hBM-MSCs). In ischemic cell model, SH-SY5Y cells were exposed to OGD for 24 h, treated with hBM-MSCs (OGD + hBM-MSCs) prior to continued aerobic incubation, and then samples were collected after coculture for 72 h. In thein vivoMCAo ischemic model, levels of some PAs in brain samples of the MCAo and MCAo + hBM-MSCs groups were significantly different from those of the control group. In particular, putrescine, cadaverine, and spermidine in brain tissues of the MCAo + hBM-MSCs group were significantly reduced in comparison to those in the MCAo group. In thein vitroOGD system,N1-acetylspermidine, spermidine,N1-acetylspermine, and spermine in cells of the OGD + hBM-MSCs group were significantly reduced compared to those of OGD group.

scholarly journals Metformin Protects against Oxidative Stress Injury Induced by Ischemia/Reperfusion via Regulation of the lncRNA-H19/miR-148a-3p/Rock2 Axis

2019 ◽  
Vol 2019 ◽  
pp. 1-18 ◽  
Author(s):  
Jing Zeng ◽  
Long Zhu ◽  
Jing Liu ◽  
Tao Zhu ◽  
Zhaohui Xie ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Ischemia Reperfusion ◽  
Luciferase Reporter ◽  
Neuroprotective Effects ◽  
Stress Injury ◽  
Oxidative Stress Injury

Previous studies have shown that metformin not only is a hypoglycemic agent but also has neuroprotective effects. However, the mechanism of action of metformin in ischemic stroke is unclear. Oxidative stress is an important factor in the pathogenesis of cerebral ischemia-reperfusion injury. It has been reported that metformin is associated with stroke risk in the clinical population. This study is aimed at investigating the effect and mechanism of metformin in an experimental model of oxidative stress induced by ischemia/reperfusion (I/R) in vivo and oxygen glucose deprivation/reperfusion (OGD/R) in vitro. Metformin (100, 200, and 300 mg/kg) was administered intraperitoneally immediately after induction of cerebral ischemia. The indicators of oxidative stress selected were antioxidant enzyme activities of catalase, malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and glutathione peroxidation enzyme (GSHPx). First, we demonstrated that metformin can significantly alleviate acute and chronic cerebral I/R injury and it has a strong regulatory effect on stroke-induced oxidative stress. It can reduce the elevated activities of MDA and NO and increase the levels of GSHPx and SOD in the cerebrum of mice and N2a cells exposed to I/R. Furthermore, real-time PCR and western blot were used to detect the expression of long noncoding RNA H19 (lncRNA-H19), microRNA-148a-3p (miR-148a-3p), and Rho-associated protein kinase 2 (Rock2). The direct interaction of lncRNA-H19, miR-148a-3p, and Rock2 was tested using a dual luciferase reporter assay. lncRNA-H19 altered OGD/R-induced oxidative stress by modulating miR-148a-3p to increase Rock2 expression. The expression of lncRNA-H19 and Rock2 could be downregulated with metformin in vivo and in vitro. In conclusion, our study confirmed that metformin exerts neuroprotective effects by regulating ischemic stroke-induced oxidative stress injury via the lncRNA-H19/miR-148a-3p/Rock2 axis. These results provide new evidence that metformin may represent a potential treatment for stroke-related brain injury.

scholarly journals MLIF Modulates Microglia Polarization in Ischemic Stroke by Targeting eEF1A1

2021 ◽  
Vol 12 ◽  
Author(s):  
Yulan Liu ◽  
Shanshan Deng ◽  
Zhibing Song ◽  
Qian Zhang ◽  
Yuchen Guo ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Macrophage Polarization ◽  
Elongation Factor ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Human Neuroblastoma ◽  
Associated Proteins ◽  
Bv2 Microglia

Monocyte locomotion inhibitory factor (MLIF) is a heat-stable pentapeptide from Entamoeba histolytica. Our previous study found that MLIF protects against ischemic stroke in rats and mice and exerts a neuroprotection effect in human neuroblastoma SH-SY5Y cells. Microglia/macrophage polarization has been proven to be vital in the pathology of ischemic stroke. Nevertheless, whether MLIF is able to modulate microglia/macrophage polarization remains unclear. We performed middle cerebral artery occlusion (MCAO) on C57BL/6J male mice and induced cultured BV2 microglia by oxygen-glucose deprivation (OGD), respectively. Immunfluorescence was utilized to detect the M1/2 markers, such as CD206 and CD16/32. qPCR and ELISA were used to detect the signature gene change of M1/2. The MAPK and NF-κB pathway associated proteins were measured by Western blot. To identify the protein target of MLIF, a pull-down assay was performed. We found that MLIF promoted microglia transferring from a “sick” M1 phenotype to a “healthy” M2 phenotype in vivo or in vitro. Furthermore, we proved that eukaryotic elongation factor 1A1 (eEF1A1) was involved in the modulation of microglia/macrophage polarization. Knocking down eEF1A1 by siRNA exhibited the M1 promotion effect and M2 inhibition effect. Taken together, our results demonstrated MLIF modulated microglia/macrophage polarization by targeting eEF1A1 in ischemic stroke.

scholarly journals Mir-184 Contributes to Brain Injury Through Targeting PPAP2B Following Ischemic Stroke in Male Rats

2021 ◽  
Vol 14 ◽  
Author(s):  
Huajun Yang ◽  
Yifan Zhang ◽  
Hongqun Chen ◽  
Yingwu Zhu ◽  
Yuan Li ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Brain Injury ◽  
Infarct Volume ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Cerebral Infarct ◽  
Male Rats ◽  
Luciferase Reporter ◽  
Pcr Analysis

Our previous study revealed that miR-184 expression is significantly altered in the brain following ischemic stroke in rats. However, it is unknown whether this alteration in miR-184 expression contributes to brain injury after ischemic stroke. Here, we aim to address the potential of miR-184 to impact nerve injury following ischemia and reperfusion. Rats received ICV injection of miR-184 adenovirus or empty vector and were subjected to right middle cerebral artery occlusion (MCAO) to establish an ischemic stroke model. We cultured SH-SY5Y cells under oxygen-glucose deprivation/reoxygenation (OGD/R) and transfected them with miR-184 lentivirus to explore the primary mechanisms. To evaluate miR-184 expression, neurological function deficits, the cerebral infarct volume, cell viability, and apoptosis, qRT-PCR analysis of miR-184 expression, the modified neurological severity score (mNSS) system, TTC staining, the CCK-8 assay, flow cytometry, and dual-luciferase reporter assays were utilized. We found that miR-184 expression was downregulated and that the cerebral infarct volume and mNSSs were increased following ischemic stroke; however, increasing the level of miR-184 alleviated brain damage. Overexpression of miR-184 resulted in increased viability and reduced apoptosis of SH-SY5Y cells following OGD/R in vitro. We identified the phosphatidic acid phosphatase type 2B (PPAP2B) gene as a direct target gene of miR-184. In summary, our results reveal that attenuation of miR-184 levels in ischemic stroke contributes to ischemic injury through targeting PPAP2B mRNA-mediated apoptosis, which may be a promising therapeutic target for ischemic stroke.

scholarly journals Circular RNA FLNA acts as a sponge of miR-486-3p in promoting lung cancer progression via regulating XRCC1 and CYP1A1

2021 ◽  
Author(s):  
Jiongwei Pan ◽  
Gang Huang ◽  
Zhangyong Yin ◽  
Xiaoping Cai ◽  
Enhui Gong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Lung Cancer Cells ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Related Factors

AbstractSignificantly high-expressed circFLNA has been found in various cancer cell lines, but not in lung cancer. Therefore, this study aimed to explore the role of circFLNA in the progression of lung cancer. The target gene of circFLNA was determined by bioinformatics and luciferase reporter assay. Viability, proliferation, migration, and invasion of the transfected cells were detected by CCK-8, colony formation, wound-healing, and transwell assays, respectively. A mouse subcutaneous xenotransplanted tumor model was established, and the expressions of circFLNA, miR-486-3p, XRCC1, CYP1A1, and related genes in the cancer cells and tissues were detected by RT-qPCR, Western blot, or immunohistochemistry. The current study found that miR-486-3p was low-expressed in lung cancer. MiR-486-3p, which has been found to target XRCC1 and CYP1A1, was regulated by circFLNA. CircFLNA was located in the cytoplasm and had a high expression in lung cancer cells. Cancer cell viability, proliferation, migration, and invasion were promoted by overexpressed circFLNA, XRCC1, and CYP1A1 but inhibited by miR-486-3p mimic and circFLNA knockdown. The weight of the xenotransplanted tumor was increased by circFLNA overexpression yet reduced by miR-486-3p mimic. Furthermore, miR-486-3p mimic reversed the effect of circFLNA overexpression on promoting lung cancer cells and tumors and regulating the expressions of miR-486-3p, XRCC1, CYP1A1, and metastasis/apoptosis/proliferation-related factors. However, overexpressed XRCC1 and CYP1A1 reversed the inhibitory effect of miR-486-3p mimic on cancer cells and tumors. In conclusion, circFLNA acted as a sponge of miR-486-3p to promote the proliferation, migration, and invasion of lung cancer cells in vitro and in vivo by regulating XRCC1 and CYP1A1.

scholarly journals LINGO-1 shRNA protects the brain against ischemia/reperfusion injury by inhibiting the activation of NF-κB and JAK2/STAT3

Human Cell ◽  
2021 ◽  
Author(s):  
Jiaying Zhu ◽  
Zhu Zhu ◽  
Yipin Ren ◽  
Yukang Dong ◽  
Yaqi Li ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Mice Model ◽  
Down Regulation

AbstractLINGO-1 may be involved in the pathogenesis of cerebral ischemia. However, its biological function and underlying molecular mechanism in cerebral ischemia remain to be further defined. In our study, middle cerebral artery occlusion/reperfusion (MACO/R) mice model and HT22 cell oxygen–glucose deprivation/reperfusion (OGD/R) were established to simulate the pathological process of cerebral ischemia in vivo and in vitro and to detect the relevant mechanism. We found that LINGO-1 mRNA and protein were upregulated in mice and cell models. Down-regulation LINGO-1 improved the neurological symptoms and reduced pathological changes and the infarct size of the mice after MACO/R. In addition, LINGO-1 interference alleviated apoptosis and promoted cell proliferation in HT22 of OGD/R. Moreover, down-regulation of LINGO-1 proved to inhibit nuclear translocation of p-NF-κB and reduce the expression level of p-JAK2 and p-STAT3. In conclusion, our data suggest that shLINGO-1 attenuated ischemic injury by negatively regulating NF-KB and JAK2/STAT3 pathways, highlighting a novel therapeutic target for ischemic stroke.

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