Authors: Jie Song Na Li Yang Xia Zhong Gao SaFeng Zou YuHui Yan ShaoHeng Li Yue Wang YaKun Meng JingXian Yang TingGuo Kang
Publish Date: 2016/07/07
Volume: 60, Issue: 1, Pages: 115-129
Abstract
Mechanical trauma injury is a severe insult to neural cells Subsequent secondary injury involves the release of inflammatory factors that have dramatic consequences for undamaged cells leading to normal cell death after the initial injury The present study investigated the capacity for arctigenin ARC to prevent secondary effects and evaluated the mechanism underlying the action of microRNA miRNA199a and miRNA16 in a mechanical trauma injury MTI model using SHSY5Y cells in vitro SHSY5Y cells are often applied to in vitro models of neuronal function and differentiation Recently miRNAs have been demonstrated to play a crucial role in NFκB and cholinergic signaling which can regulate inflammation The cell model was established by scratchinduced injury of human SHSY5Y cells which mimics the characteristics of MTI A cell counting kit8 CCK8 terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and immunocytochemistry were used to measure cell viability Enzymelinked immunosorbent assay ELISA was used to evaluate the inflammatory cytokine and cholinesterase CHE content The lactate dehydrogenase LDH content was measured to assess the degree of cell injury The mRNA levels were measured by RTPCR to analyze ARC’s mechanism of action miRNA inhibitors and mimics were used to inhibit and strengthen the expression of miRNAs Protein expression was detected by western blotting analysis ARC treatment reduced the TNFα and IL6 levels as well as the number of TUNEL+ apoptotic SHSY5Y cells surrounding the scratch and increased the IL10 level compared to the controls ARC attenuated the increase of the cell damage degree and LDH content induced by scratching indicating increased cell survival Mechanistic studies showed that ARC upregulated the miRNA16 and miRNA199a levels to reduce upstream protein IKKα and IKKβ expression and inhibit NFκB signaling pathway activity moreover the increased miRNA199a suppresses cholinesterases to increase cholinergic signaling resulting in decreased expression of proinflammatory cytokines ARC treatment confers protection for SHSY5Y cells through positive regulation of miRNA expression thereby reducing the inflammatory response In turn these effects accelerate injury repair in the scratchinduced injury model These results might provide insights into the pharmacological role of ARC in antiinflammation and neuroprotection in neural cells
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