Long noncoding RNA TUG1 contributes to cerebral ischaemia/reperfusion injury by sponging mir-145 to up-regulate AQP4 expression.

Emerging studies have shown that long noncoding RNA (lncRNA) TUG1 (taurine-up-regulated gene 1) plays critical roles in multiple biological processes. However, the expression and function of lncRNA TUG1 in cerebral ischaemia/reperfusion injury have not been reported yet. In this study, we found that LncRNA TUG1 expression was significantly up-regulated in cultured MA-C cells exposed to OGD/R injury, while similar results were also observed in MCAO model. Mechanistically, knockdown of TUG1 decreased lactate dehydrogenase levels and the ratio of apoptotic cells and promoted cell survival in vitro. Moreover, knockdown of TUG1 decreased AQP4 (encoding aquaporin 4) expression to attenuate OGD/R injury. TUG1 could interact directly with miR-145, and down-regulation of miR-145 could efficiently reverse the function of TUG1 siRNA on AQP4 expression. Finally, the TUG1 shRNA reduced the infarction area and cell apoptosis in I/R mouse brains in vivo. In summary, our results suggested that lncRNA TUG1 may function as a competing endogenous RNA (ceRNA) for miR-145 to induce cell damage, possibly providing a new therapeutic target in cerebral ischaemia/reperfusion injury.

Correction to: Inhibition of T cell immunoglobulin and mucin-1 (TIM-1) protects against cerebral ischemia-reperfusion injury.

Following publication of the original article [1], the authors reported that the given name of Liqing Wang was incorrectly published as Liqiang Wang. The original article has been updated.

Inhibition of T cell immunoglobulin and mucin-1 (TIM-1) protects against cerebral ischemia-reperfusion injury.

BACKGROUND: The T cell Ig domain and mucin domain (TIM)-1 protein expressed on the surface of Th2 cells regulates the immune response by modulating cytokine production. The present study aimed to investigate the role and possible mechanism of TIM-1 in cerebral ischemia-reperfusion injury. METHODS: Western blot was used to detect TIM-1 and apoptosis-related protein expression, whereas TIM-1 mRNA was examined using quantitative real-time reverse transcription PCR. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells and a pathological examination was performed. The migration of neutrophils and macrophages was analyzed by immunohistochemistry. RESULTS: Our results suggest that TIM-1 expression was transiently increased 24 h or 48 h following middle cerebral artery occlusion (MCAO)/reperfusion. The infarct size was markedly increased in MCAO, whereas treatment with a TIM-1-blocking mAb could reduce the infarct size. TIM-1 blocking mAb effectively reduced the number of neutrophils, macrophage functionality, cytokine (i.e., IL-6, IL-1ß, and TNF-α) and chemokine (i.e., CXCL-1 and CXCL-2) production in the brain tissue. The effect of in vitro T cell damage on neurons was significantly reduced following treatment with a TIM-1 blocking mAb or the knockdown of TIM-1 in co-cultured T cells and neurons. CONCLUSION: Take together, these results indicated that TIM-1 blockade ameliorated cerebral ischemia-reperfusion injury. Thus, TIM-1 disruption may serve as a novel target for therapy following MCAO.

miR­29a ameliorates ischemic injury of astrocytes in vitro by targeting the water channel protein aquaporin 4.

Ischemic stroke is the main cause of brain injury and results in a high rate of morbidity, disability and mortality. In the present study, we aimed to determine whether miR­29a played a protective role in oxygen glucose deprivation (OGD) injury via regulation of the water channel protein aquaporin 4 (AQP4). Real­time PCR and western blotting were used to assess miR­29a levels and AQP4 protein levels, respectively. Apoptosis was detected by flow cytometry, and lactate dehydrogenase (LDH) was determined by enzyme­linked immunosorbent assay (ELISA). Overexpression of miR­29a was significantly downregulated in OGD­induced primary astrocytes, and transfection with a miR­29a mimic decreased LDH release and apoptosis, and improved cell health in OGD­induced astrocytes. AQP4 was the target of miR­29a, which suppressed AQP4 expression, and knockdown of AQP4 mitigated OGD­induced astrocyte injury. Furthermore, miR­29a regulated AQP4 expression in OGD­induced astrocytes. AQP4 exacerbated astrocyte injury following ischemic stroke, and knockdown of AQP4 protected OGD/RX­induced primary cultured astrocytes against injury. The effect of miR­29a inhibitor on primary astrocytes was lost following AQP4 knockdown. These findings indicated that miR­29a prevented astrocyte injury in vitro by inhibiting AQP4. Thus, miR­29a may protect primary cultured astrocytes after OGD­induced injury by targeting AQP4, and may be a potential therapeutic target for ischemic injury of astrocytes.

Upregulation of miR-130b protects against cerebral ischemic injury by targeting water channel protein aquaporin 4 (AQP4).

Altered microRNA regulation has been implicated in the pathogenesis of various disorders, including cerebral ischemia/reperfusion injury (I/RI). However, the regulatory mechanism of miR-130b in cerebral ischemia injury has not been reported. In this study, we explored the role of miR-130b in cerebral ischemia injury and investigated its potential mechanism. Levels of miR-130b were quantified by real-time PCR, and the protein level of AQP4 was detected by Western blotting. Cell apoptosis was detected by flow cytometry. In vitro, miR-130b levels in astrocytes were found significantly downregulated after OGD. Overexpression of miR-130b by miR-130b mimic decreased LDH release and apoptosis, but promoted cell health of astrocytes with OGD, thus playing a protective role in astrocyte I/RI. The level of miR-130b was also downregulated in ischemic tissues in MCAO model compared with the sham group, and the expression of miR-130b was gradually downregulated over time after reperfusion. AQP4 was upregulated both in two models, and as the reperfusion went on, AQP4 expression gradually upregulated. Our results indicated knockdown of AQP4 could ameliorate astrocyte injury induced by OGD. Finally, we found that miR-130b regulated astrocyte expression of AQP4, and rescue experiments further proved the protective role of miR-130b was mediated by AQP4 downregulation. Our study demonstrated that miR-130b might exert a neuroprotective effect following cerebral I/RI by regulating AQP4 expression at the post-transcriptional level. Therefore, miR-130b may be a potential therapeutic target for stroke treatment.

Daphnetin, a natural coumarin derivative, provides the neuroprotection against glutamate-induced toxicity in HT22 cells and ischemic brain injury.

Daphnetin (DAP), a coumarin derivative, has been reported to have multiple pharmacological actions including analgesia, antimalarial, anti-arthritic, and anti-pyretic properties. It is unclear whether DAP has neuroprotective effects on ischemic brain injury. In this study, we found that DAP treatment (i.c.v.) reduced the infarct volume at 24 h after ischemia/reperfusion injury and improved neurological behaviors in a middle cerebral artery occlusion mouse model. Moreover, we provided evidences that DAP had protective effects on infarct volume in neonate rats even it was administrated at 4 h after cerebral hypoxia/ischemia injury. To explore its neuroprotective mechanisms of DAP, we examined the protection of DAP on glutamate toxicity-induced cell death in hippocampal HT-22 cells. Our results demonstrated that DAP protected against glutamate toxicity in HT-22 cells in a concentration-dependent manner. Further, we found that DAP maintained the cellular levels of glutathione and superoxide dismutase activity, suggesting the anti-oxidatant activity of DAP. Since DAP has been used for the treatment of coagulation disorder and rheumatoid arthritis for long time with a safety profile, DAP will be a promising agent for the treatment of stroke.

A natural coumarin derivative esculetin offers neuroprotection on cerebral ischemia/reperfusion injury in mice.

Previous studies have demonstrated that a natural coumarin compound esculetin (Esc) possesses antioxidant, anti-tumor, and anti-inflammation activities and rescues cultured primary neurons from NMDA toxicity. In this study, we investigated the neuroprotective effects of Esc on cerebral ischemia/reperfusion (I/R) injury in a middle cerebral artery occlusion model in mice. Esc (20 µg) was administered intracerebroventricularly at 30 min before ischemia. We found that Esc significantly reduced infarct volume and decreased neurological deficit scores after 75 min of ischemia and 24 h of reperfusion. Post-treatment of Esc still provided neuroprotection even when Esc was administered after 4 h of reperfusion. Our data also indicated that intraperitoneal administration of Esc showed protective effects on cerebral I/R injury in a dose-dependent manner. We further explored the protective mechanisms of Esc on cerebral I/R injury and found that Esc decreased cleaved caspase 3 level, a marker of apoptosis. Finally, our data demonstrated that Esc exerted its anti-apoptotic activity by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax, two apoptosis-related proteins. Because of its clinical use as an anticoagulant and its safety profile, Esc may have a therapeutic potential for the treatment of stroke in the future clinical trials.

Carvacrol, a food-additive, provides neuroprotection on focal cerebral ischemia/reperfusion injury in mice.

Carvacrol (CAR), a naturally occurring monoterpenic phenol and food additive, has been shown to have antimicrobials, antitumor, and antidepressant-like activities. A previous study demonstrated that CAR has the ability to protect liver against ischemia/reperfusion injury in rats. In this study, we investigated the protective effects of CAR on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that CAR (50 mg/kg) significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. This neuroprotection was in a dose-dependent manner. Post-treatment with CAR still provided protection on infarct volume when it was administered intraperitoneally at 2 h after reperfusion; however, intracerebroventricular post-treatment reduced infarct volume even when the mice were treated with CAR at 6 h after reperfusion. These findings indicated that CAR has an extended therapeutic window, but delivery strategies may affect the protective effects of CAR. Further, we found that CAR significantly decreased the level of cleaved caspase-3, a marker of apoptosis, suggesting the anti-apoptotic activity of CAR. Finally, our data indicated that CAR treatment increased the level of phosphorylated Akt and the neuroprotection of CAR was reversed by a PI3K inhibitor LY-294002, demonstrating the involvement of the PI3K/Akt pathway in the anti-apoptotic mechanisms of CAR. Due to its safety and wide use in the food industry, CAR is a promising agent to be translated into clinical trials.

Antioxidant activity of 7,8-dihydroxyflavone provides neuroprotection against glutamate-induced toxicity.

Glutamate, an excitatory neurotransmitter in the central nervous system, plays an important role in neurological disorders. Previous studies have shown that excess glutamate can cause oxidative stress in a hippocampal HT-22 cell line. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, is a selective tyrosine kinase receptor B (TrkB) agonist that has neurotrophic effects in various neurological diseases such as stroke and Parkinson's disease. In this study, we found that there is no TrkB receptor in HT-22 cells. Despite this, our data demonstrate that 7,8-DHF still protects against glutamate-induced toxicity in HT-22 cells in a concentration-dependent manner, indicating that 7,8-DHF prevents cell death through other mechanisms rather than TrkB receptors in this cell model. We further show that 7,8-DHF increases cellular glutathione levels and reduces reactive oxygen species (ROS) production caused by glutamate in HT-22 cells. Finally, our data demonstrate that 7,8-DHF protects against hydrogen peroxide and menadione-induced cell death, suggesting that 7,8-DHF has an antioxidant effect. In summary, although 7,8-DHF is considered as a selective TrkB agonist, our results demonstrate that 7,8-DHF can still confer neuroprotection against glutamate-induced toxicity in HT-22 cells via its antioxidant activity.