Abstract
To date, there is no good evidence that intestine epithelial cells (IEC) affected by ischemia/reperfusion (I/R) injury are able to cause cortical neuron injury directly. Additionally, it remains unclear whether the neuronal damage caused by I/R injured IEC can be affected by therapeutic hypothermia (TH, 32 °C). To address these questions, we performed an oxygen–glucose deprivation (OGD) affected IEC-6-primary cortical neuron coculture system under normothermia (37 °C) or TH (32 °C) conditions. It was found that OGD caused hyperpermeability in IEC-6 cell monolayers. OGD-preconditioned IEC-6 cells caused cortical neuronal death (e.g., decreased cell viability), synaptotoxicity, and neuronal apoptosis (evidenced by increased caspase-3 expression and the number of TUNEL-positive cells), necroptosis (evidenced by increased receptor-interacting serine/threonine-protein kinase-1 [RIPK1], RIPK3 and mixed lineage kinase domain-like pseudokinase [MLKL] expression), and pyroptosis (evidenced by an increase in caspase-1, gasdermin D [GSDMD], IL-1β, IL-18, the apoptosis-associated speck-like protein containing a caspase recruitment domain [ASC], and nucleotide oligomerization domain [NOD]-like receptor [NLRP]-1 expression). TH did not affect the intestinal epithelial hyperpermeability but did attenuate OGD-induced neuronal death and synaptotoxicity. We also performed quantitative real-time PCR to quantify the genes encoding 84 exosomal microRNAs in the medium of the control-IEC-6, the control-neuron, the OGD-IEC-6 at 37 °C, the OGD-IEC-6 at 32 °C, the neuron cocultured with OGD-IEC-6 at 37 °C, and the neurons cocultured with OGD-IEC-6 at 32 °C. We found that the control IEC-6 cell s or cortical neurons are able to secrete a basal level of exosomal miRNAs in their medium. OGD significantly up-regulated the basal level of each parameter for IEC-6 cells. As compared to those of the OGD-IEC-6 cells or the control neurons, the OGD-IEC-6 cocultured neurons had significantly higher levels of 19 exosomal miRNAs related to apoptosis, necroptosis, and/or pyroptosis events. Our results identify that I/R injured intestinal epithelium cells can induce cortical neuron death via releasing paracrine mediators such as exosomal miRNAs associated with apoptosis, necroptosis, and/or pyroptosis, which can be counteracted by TH.
Highlights
To date, there is no good evidence that intestine epithelial cells (IEC) affected by ischemia/reperfusion (I/R) injury are able to cause cortical neuron injury directly
Our results identify that I/R injured intestinal epithelium cells can induce cortical neuron death via releasing paracrine mediators such as exosomal miRNAs associated with apoptosis, necroptosis, and/ or pyroptosis, which can be counteracted by therapeutic hypothermia (TH)
We performed an in vitro study to ascertain whether the mediators released from intestinal epithelial cell line No 6 (IEC-6) cells preconditioned with oxygen–glucose deprivation (OGD) cause primary cortical neuron death under therapeutic hypothermia or normothermia conditions
Summary
There is no good evidence that intestine epithelial cells (IEC) affected by ischemia/reperfusion (I/R) injury are able to cause cortical neuron injury directly It remains unclear whether the neuronal damage caused by I/R injured IEC can be affected by therapeutic hypothermia (TH, 32 °C). It remains unclear whether the brain injury caused by I/R injured intestine epithelial cells, like rat heart, can be affected by therapeutic hypothermia (TH)[6] To address these questions, we cocultured the oxygen–glucose deprivation (OGD)-infected intestinal epithelial cell lines (IEC-6) with rat primary cortical neuronal cells in an in vitro coculture system under normothermia (37 °C) or TH (32 °C) conditions. We performed quantitative real-time reverse transcription PCR (qRT-PCR) to quantify the genes encoding 84 exosomal microRNAs (miRNAs)[9] in the cocultured medium of the control IEC-6, the control neurons, the OGD-IEC-6 at 37 °C, the OGD-IEC-6 at 32 °C, the neurons cocultured with OGD-IEC-6 at 37 °C, and the neurons cocultured with OGD-IEC-6 at 32 °C
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