The Cytoprotection Role Of Delta Opioid Receptor In Hypoxia/reoxygenation Injury Model:via Nucleus Translocation Of Transcriptor Factor Nrf2

ABSTRACT:Ischemic/hypoxia reperfusion injury is that the tissue or organ develops to a more serious damage when blood flow is subsequently reintroduced following a period of ischemia or hypoxia.This phenomenon is involved in various disease states including myocardial infarction, stroke, solid organ and stem cell transplantation. A number of molecular mechanisms related to ischemic/hypoxia reperfusion has been well documented including inflammatory, neutrophil, and oxidative stress. Among them, oxidative stress, which elevates excessive reactive oxygen species (ROS) and decreases detoxifying activities of intracellular antioxidants within5min after reperfusion is considered as a major and early event. The nuclear factor erythroid2(NFE2)-related factor2(Nrf2) is the master transcription factor controls a web of antioxidant pathways at basal and induced level to counterbalanced the physiological and pathophysiological outcomes of oxidant stress. Nrf2was sequestered by Kelch-like ECH associating protein1(Keapl) in the cytoplasm at normal state. Once the insults such as reactive oxygen species or electrophilic attack the Nrf2-Keapl complex, dissociating Nrf2from Keap1, stabilized Nrf2then translocates to the nuclei and upregulates a large number of phase Ⅱ detoxification and antioxidant enzymes expression, which are governed by a cis-acting regulatory element termed the antioxidant response element (ARE). It is noted that Keapl dissociated with Nrf2is at least partly mediated by PKC pathway conducted phophorylation of40position serine of Nrf2. Reoxygenation-specific activation of Nrf2were shown by several studies, however, there is no direct evidence showing cytoprotective effect mediated by Nrf2at these pathophysiological state.Intricate antioxidant systems to maintain the cellular redox homeostatis which balanced by Nrf2can be divided into certain categories:1) Phase Ⅱ enzymes, which containing glutathione S-transferases (GSTs), NAD(P)H quinone oxidoreductase (NQO1), heme oxygenase-1(HO-1), UDP-glucuronyl transferase1A6.2) Noncatalytic antioxidant proteins, such as thioredoxin (Trx), glutaredoxin (Grx), peroxiredoxin (Prx)10and metallothioneins (MTs);3) Rate-limiting enzyme of GSH generating, glutamate cysteine ligase (GCL). GCL is composed for a catalytic (GCLC) and modifier (GCLM) subunit and both of them are regulated by Nrf2, and so on. Since Nrf2has been considered as the major transcription factor to so many proteins that protect body from oxidative stress, it has rapidly arisen as a key regulator of oxidation-reduction equilibrium under both physiological and pathophysiological state.Opioid receptors containing Mu (μ), kappa (K), delta (δ), and opioid receptor like-1(ORL1) are belonged to G-protein coupled receptor (seven-transmembrane protein) superfamily. These receptors sense to their ligands endorphins, dynorphin, enkephalin and nociceptin receptively, which are released out of the cell and activate inside inhibitory G protein signal transduction pathways result in cellular responses. Opioid receptors are widely distributed in central and peripheral nervous system and have been used for pain management and related disorder since last century. In addition to pain treatment, our previous works observed that activation of delta opioid receptor (DOR) can promote cortical neurons survival against glutamate and hypoxia-induced injury. Furthermore, DOR was identified as a "pivot" in delayed and rapid hypoxia preconditioning (HPC) mediated neuroprotective effect. Furthermore, another important mechanism underlies the DOR-mediated neuroprotective role is that DOR maintains ionic homeostasis of hypoxic/ischemic disruption. DOR signal pathway attenuates Na+influx through the membrane and Na+channels and inhibits the acculmation of intracellular Ca2+, whereas decreasing the excessive leakage of intracellular K+. It is noted that a PKC-dependent but PKA-independent signal transduction pathway have been reported to be involved in all the mechinary of protection. These studies highlight that DOR participant in oxygen-regulated protective mechanism. But whether DOR has protection effect in H/R has remained unclear.Because protein kinase C (PKC) is the downstream signaling pathway of delta opioid receptor whereas PKC mediated phosphorylation of Nrf2at Ser40leads to Nrf2functional elevation by nuclear translocation. On the basis of these, we predicted that DOR and Nrf2may have a relationship and DOR might have a cytoprotective role in H/R through coordinating Nrf2. To test this hypothesis, we administrated specific agonist and antagonist of DOR to HEK293t cell line and primary cultured astrocyte of SD rat to determine their underlying relationship. So, the main results of our research are:1. The agonist of DOR, UFP512, induced Nrf2accumulation in HEK293t nucleus and increased its target gene expression, which is completely abolished by naltrindole the antagonist of DOR.2. The elevation of Nrf2activity induced by delta opioid receptor system is mediated by Protein Kinase C rather than PI3K signal transduction pathway.3. DOR system play an crucial protection role in hypoxia/reoxygenation stress model in HEK293t cell, which can be removed by Nrf2siRNA transfection.4. In the hypoxia/reoxygenation injury model, the agonist of DOR, UFP 512, induced Nrf2accumulation in nucleus and increased its target gene expression in both HEK293t, which is completely abolished by the antagonist of DOR naltrindole.5. In the hypoxia/reoxygenation injury model, the agonist of DOR, UFP512, induced Nrf2accumulation in nucleus and increased its target gene expression in rat primary cultured astrocytes, which is completely abolished by the antagonist of DOR naltrindole.6. The Nrf2inducer tBHQ increase DOR protein rather than mRNA expression.Above all, our study reported DOR system play a crucial protection role in hypoxia/reoxygenation model in both HEK293t cell and primary cultured astrocytes. The underlying mechanism of this phenonmenon is that DOR activation can induce Nrf2accumulation in nucleus and increased its target anti-oxdative gene expression. The protein kinase C rather than PI3K signal transduction pathway is involved in Nrf2translocation mediated by DOR. Finally, we find out Nrf2inducer tBHQ can induce DOR protein but not mRNA expression.