Original articleProtective effects of isorhamnetin on N2a cell against endoplasmic reticulum stress-induced injury is mediated by PKCε
Graphical abstract
Introduction
The endoplasmic reticulum stress (ERS), caused by perturbations of calcium (Ca2+) homeostasis and accumulation of unfolded and misfolded proteins in the ER lumen [1], [2], [3], is involved in numerous pathologies including cancer [4], [5], atherosclerosis [6], hypertension [7], obesity [8], diabetes [9], and neurodegenerative diseases [10]. Once in stress, a highly conserved signal transduction pathway called the unfolded protein response (UPR) is triggered by three main ER transmembrane stress sensors, protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6), to restore ER homeostasis [2], [11], [12]. In the physiological state, the three main sensors interacts with the ER chaperone glucose-regulated protein 78 kDa (GRP78) to stay in an inactive condition. When the ER stress is too severe or prolonged, the UPR induces an apoptotic response [13], [14]. On the one hand, Ca2+ effluxes from the ER increase cytosolic Ca2+ levels, which results into the activation of the executioner caspases like caspase 3 leading to apoptosis. On the other hand, the overload of Ca2+ in cytosolic induce the generation of ROS by evoke influx of Ca2+ in the nuclei and mitochondria. Meanwhile, ATP depletion as a result of the accumulation of misfolded proteins may stimulate mitochondrial oxidative phosphorylation to increase ATP and ROS production [15], [16], [17].
Protein kinase C epsilon (PKCε), a members of the PKC family characterized as a calcium-independent and phorbol ester/diacylglycerol-sensitive serine/threonine kinase, is abundantly expressed in neurons and has been found that it participates in various effects in neurons [18]. The increasing evidence have confirmed that PKCε signaling is involved in alleviate Ca2+ depletion and ROS producton in ER and inhibit apoptosis [19], [20].
Isorhamnetin (Iso; 3-Methylquercetin; Quercetin-3′-methyl ether, C16H12O7) is a plant flavonoid abundant in herbal medicinal plants Hippophae rhamnoides L. and has gained extensive attention due to its wide range of biological and pharmacological properties, including anti-inflammatory, anti-cancer, anti-ischemia reperfusion injury and reduce blood pressure and endothelial dysfunction [21], [22], [23], [24], [25]. It has been reported that Iso has effective protective effects against oxidative stress-induced cellular apoptosis and protects the brain against ischemic injury in mice [26], [27].
Thus, we hypothesized that Iso can attenuate ER stress injury through the PKCε signal pathway. The current investigation was to elucidate this possibility in cultured N2a cells.
Section snippets
Chemicals and reagents
Isorhamnetin (Iso), Thapsigargin (TG), εV1-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Fluo-3/AM, MTT, propidium iodide (PI) and DCFH-DA were purchased from Beyotime Biotechnology (Shanghai, China). Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s Modified Eagles Medium (DMEM) were purchased from Biological Industries (BI) (northern Kibbutz Beit Haemek, Israel). Trypsin and Phosphate Buffered Saline (PBS, pH 7.4) were from Beijing solarbio science
Effects of pretreatment with iso on the expression of GRP78 protein in N2a cells suffered ERS injury
To verify the optimal concentration and pretreatment time of Iso on GRP78 protein (ERS marker) expression was evaluated by Western blotting analysis. N2a cells were pretreated with or without Iso (10, 20 or 40 μM) for 3, 6 and 12 h, respectively before induction of ERS. the expression of protein GRP78 induced by Iso pretreatment were decreased, particularly, the expression of GRP78 protein in group, pretreated with Iso for 3 h before experiencing to ERS, was decrease than those 6, and 12 h (Fig. 1
Discussion
Neuronal apoptosis is the common feature of several neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. capitalize apoptosis can be triggered by three different pathways: Mitochondrial caspase apoptopic signaling pathway, death receptor signaling pathway and endoplasmic reticulum stress (ERS) signaling pathway. ERS results in Ca2+ release from the ER to the cytosol, which induces ROS burst, and eventually induces neuronal apoptosis [32], [33],
Conclusions
we report here that Iso can elicit protective effect against ERS injury by decreasing GRP78 protein, attenuating cytosolic Ca2+ overload, decreasing ROS generation, increasing viability and inhibiting apoptosis in N2a cells. Importantly, we identified that PKCε is responsible for the isoinduced protective effect against ERS in N2a cells.
Conflict of interest
The authors declare that there is no conflict of interest associated with this study.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grants 81561019), the Natural Science Foundation Project of Jiangxi Province (grants 20161BAB205214), the Science and Technology Foundation of the Education Department of Jiangxi Province (grants GJJ150759), and a Jinggangshan University grant (grants JZB15006).
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Contributed equally to this work.