Abstract
ABSTRACT Objective Increasing evidence suggests that long-noncoding RNAs can exert neuroprotective effects in cerebral ischemia-reperfusion injury. Levels of the long noncoding RNA ANRIL (ANRIL) are reportedly altered in ischemic stroke (IS) patients, but its role in IS requires further clarification. This study was designed to explore the mechanistic function of ANRIL in IS. Methods In vitro, HT22 cells was treated with an oxygen-glucose deprivation/reperfusion (OGD/R). In vivo, brain ischemia/reperfusion was induced by 60-minute transient middle cerebral artery occlusion/reperfusion (MCAO/R) IS model in C57/BL6 mice. Additionally, cells were transfected with si-ANRIL, pcDNA3.1-ANRIL, pcDNA3.1-NF-κB, or appropriate negative controls, and si-ANRIL and pcDNA3.1-NF-κB were administered into the lateral ventricles in MCAO/R model mice. Cell viability and apoptosis were detected via MTT and flow cytometry assays. mRNA and protein expression of NF-κB were detected via qRT-PCR and Western blotting. IL-1β, IL-6, TNF-a, and iNOS levels were detected via ELISA. In addition, infarcted area and neuronal injury were evaluated via TTC, Nissl, and immunofluorescent staining. Results We found that ANRIL knockdown increased cell viability and reduced apoptosis in vitro. Additionally, we found that ANRIL knockdown decreased p-P65, P65, IL-1β, IL-6, TNF-a, and iNOS levels, whereas these effects were reversed by NF-κB overexpression both in vitro and in vivo. Conclusion our results suggest that ANRIL knockdown attenuates neuroinflammation by suppressing the expression of NF-κB both in vitro and vivo model of IS, sugguesting that ANRIL might be a potentially viable therapeutictarget to diminish neuroinflammation in IS patients.
Highlights
Ischemic stroke (IS) is one of the most common causes of death and disability in the world, with more than two million young adults (18–50 years old) suffering from IS episodes each year (Ekker et al 2018; Hankey 2017)
Neurological deficits scores was elevated in middle cerebral artery occlusion/ reperfusion (MCAO/R) mice, and reduced by si-ANRI (Figure. 4B).We observed a significant increase in infarcted area (30.6%) in MCAO/R model animals, while the infarcted area was reduced by 12.9% upon si
We found that fall latency was significantly reduced in MCAO/R model animals relative to sham controls in a rota-rod test, whereas this phenotype was significantly improved in animals in the MCAO/R + si-ANRIL treatment group, and NF-κB overexpression reversed these effects (Figure. 5H)
Summary
Ischemic stroke (IS) is one of the most common causes of death and disability in the world, with more than two million young adults (18–50 years old) suffering from IS episodes each year (Ekker et al 2018; Hankey 2017). Inflammation and oxidative stress occurring secondary to IS can cause irreversible neuronal damage, with unrestrained neuroinflammation having the potential to exacerbate this damage (Chamorro et al 2016; Q Yang et al 2019). The pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) begin to be produced rapidly within 6 h of ischemia, potentially causing neuronal injury (Bonaventura et al 2016). These neurons are highly sensitive to irreversible damage caused by ischemia and hypoxia (Shi et al 2019). The therapeutic options available to treat IS are limited, and novel therapies capable of preventing IS-associated neuroinflammatory damage are urgently needed
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