Leukemia Inhibitory Factor Receptor Is Involved in Apoptosis in Rat Astrocytes Exposed to Oxygen-Glucose Deprivation

Abstract

Leukemia inhibitory factor (LIF) and leukemia inhibitory factor receptor (Lifr) protect CNS cells, specifically neurons and myelin-sheath oligodendrocytes, in conditions of oxygen-glucose deprivation (OGD). In the case of astrocyte apoptosis resulting from reperfusion injury following hypoxia, the function of the Lifr remains to be fully elucidated. This study established models of in vivo ischemia/reperfusion (I/R) using an in vitro model of OGD to investigate the direct impact of silencing the Lifr on astrocyte apoptosis. Astrocytes harvested from newborn Wistar rats were exposed to OGD. Cell viability and apoptosis levels were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and annexin V/propidium iodide (PI) staining assays, respectively. Apoptosis was further investigated by the TdT-mediated dUTP nick-end labelling (TUNEL) assay. A standard western blotting protocol was applied to determine levels of the protein markers Bcl2, Bax, p-Akt/Akt, p-Stat3/Stat3, and p-Erk/Erk. The cell viability assay (MTT) showed that astrocyte viability decreased in response to OGD. Furthermore, blocking RNA to silence the Lifr further reduces astrocyte viability and increases levels of apoptosis as detected by annexin V/PI double staining. Likewise, western blotting after Lifr silencing demonstrated increased levels of the apoptosis-related proteins Bax and p-Erk/Erk and correspondingly lower levels of Bcl2, p-Akt/Akt, and p-Stat/Stat3. The data gathered in these analyses indicate that the Lifr plays a pivotal role in the astrocyte apoptosis induced by hypoxic/low-glucose environments. Further investigation of the relationship between apoptosis and the Lifr may provide a potential therapeutic target for the treatment of neurological injuries.

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“…Apoptosis was confirmed and quantified in tissue sections using the TUNEL assay. Tissue sections were stained with fluorescein-conjugated TUNEL (Click-iT™ TUNEL Alexa Fluor™ 594 Imaging Assay, Thermo Fisher Scientific, USA) according to the manufacturer's instructions [21]. The number of TUNEL-positive cells was counted in 30 sections per slide.…”