Background
Astrocytes are specialized and most numerous glial cell types in the brain, they play crucial roles in central nervous system (CNS) homeostasis [
1]. Except for providing support to neurons under ischemia-hypoxic condition, astrocytes can also secrete a series of pro-inflammatory and anti-inflammatory cytokines to modify the ambient microenvironment [
2,
3]. Post-ischemic inflammation mediated by astrocytes is a vital contributing factor in the CNS injury [
4‐
10]. Brain ischemia induces damage to astrocytes and stimulates the release of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which are crucial for the pathological processes of brain ischemic injury [
11].
It has been revealed that ischemic injury could trigger endoplasmic reticulum (ER) stress, which subsequently inspires inflammatory response process and cellular damage [
12,
13]. In mild ER stress, the unfolded-protein response (UPR) processes are activated, which is served to synthesis of new chaperons to refold the unfolded protein [
13]. Consequently, this process could compensate damage. However, the intense and prolonged ER stress could activate apoptotic pathways, such as the upregulation of the CEBP homologous protein (CHOP) and the activation of pro-apoptotic protein-caspase-12, and thereby result in cell death. Our previous study revealed that ATP-sensitive potassium (K-ATP) channels were involved in cerebral ischemia injury and post-ischemic inflammation [
14,
15], but the potential molecular mechanisms remain not well understood.
Recently, considerable evidences support microRNAs as an important regulatory molecule of astrocytic functions in ischemic stroke [
16‐
19] and regarded them as potential candidates for stroke therapeutics. MicroRNAs are small molecule non-coding RNAs; various aspects of microRNAs as well as their targets have been reported. By the transcription and/or translation of protein-coding genes, microRNAs exert many biological functions such as regulating ER stress, inflammation, and apoptosis [
20‐
22].
In the present study, we found that oxygen-glucose deprivation (OGD) elevated ER stress and inflammatory injuries in astrocytes. Our further study revealed for the first time that OGD decreased inflammation-associated microRNA 7 (miR-7) but increased the messenger RNA (mRNA) levels of Herpud2, one of the miR-7’s targets. Pretreatment with nicorandil could upregulate miR-7 and alleviate OGD-induced ER stress and inflammatory injuries in astrocytes. These results suggest that miR-7 may be a potential target for the anti-inflammation effect of nicorandil.
Methods
Cell cultures and treatment
Primary culture of astrocytes was prepared as previously described [
23]. Cortices were aseptically separated from 2-day-old C57BL/6 mice pups, minced, and dissociated by trypsinization. Then, they were plated on T75 culture flasks in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and incubated at 37 °C in an incubator supplemented with 5 % CO
2. After 14 days, the flasks were shaken for 1 h at 37 °C to remove the other glia. The astrocytes were then detached by trypsinization and plated in six-well plates at a density of 1 × 10
6 cells/ml. In the drug-treated group, cells were pretreated with nicorandil (a K-ATP channel opener) (TOCRIS, Bristol, UK) for 1 h before OGD and persisted to reoxygenation 24 h. The control group was treated by the vehicle. The experimental groups were divided as follows: (A) control; (B) nicorandil (10 μM); (C) OGD; and (D) OGD plus nicorandil (10 μM). All experimental procedures were approved by IACUC (Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine).
Oxygen-glucose deprivation and reoxygenation model
The protocol was previously described [
15]. After washing twice, astrocytes were immersed in 1-ml deoxygenated custom DMEM without glucose and FBS (GIBCO, CA, USA). Then, they were placed inside an incubator (Thermo scientific, Waltham, MA, USA) for 5 h with a premixed gas (1 % O
2, 94 % N
2, 5 % CO
2). After that, cells were immersed in normal DMEM containing 10 % FBS and transferred to a CO
2 incubator (95 % air and 5 % CO
2) for 24 h. For non-OGD group, cultures were incubated in DMEM containing 5.5-mM D-glucose (GIBCO, CA, USA) and 10 % FBS and placed in 5 % CO
2 in air at 37 °C for 28 h.
Cell viability analysis
The MTT assay was used to evaluate the cell viability as previously described [
11]. Primary cultured astrocytes were seeded onto 96-well plates at a density of 5 × 10
4 cells per well. After OGD 5 h and reoxygenation 24 h, the MTT (0.5 mg/ml) was dissolved in the cell medium and incubated at 37 °C for 4 h. Then, we used dimethyl sulfoxide (DMSO) to dissolve the MTT-formazan product. The absorbance was obtained by a Dynatech MR5000 plate counter at a test wavelength of 570 nm.
Real-time PCR for mRNA
Total RNA was extracted with Trizol Reagent (Invitrogen, USA) following the manufacturer’s instructions; 20 million cells were used per milliliter of Trizol. Then, complementary DNA (cDNA) was synthesized from 1 μg total RNA in a 20-μl total volume containing 4-μl mRNA Reversed Transcription Kit (Takara 036A, Japan) and RNase-free water. Reaction was performed in a Thermal Cycler as follows: 42 °C for 15 min, 87 °C for 5 s, and 4 °C forever. PCR amplifications were performed on the ABI 7300 Sequence Detection System (Applied Biosystem, USA); they were performed in a total volume of 20 μl, containing 1 μl cDNA sample and 10 SYBR Green PCR Master Mix (Roche, UK). PCR amplifications were always performed in double wells, using the universal temperature cycles: 10 min at 95 °C, followed by 40 cycles consisting of 15 s at 95 °C and 1 min at 60 °C. The quantification was performed by the comparative Ct (cycle threshold) method, using the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) as internal control. Primers (Sangon Biotech, Shanghai) used for real-time PCR are as listed in Table
1.
Table 1
The primers for PCR amplification
Herpud2 (mouse) | GGCCCAGTGCTGAATGAAGA | CAGCATGGCTCCCATTACCA |
GAPDH (mouse) | CATGGCCTTCCGTGTTCCTA | CCTGCTTCACCACCTTCTTGAT |
miR-7 RT (mouse) | AGCATTCGTCTCGACACAGCAACAAAATC |
MiR-7 (mouse) | TGACTCTGCTGGAAGACTAGTGAT | TAGAGCATTCGTCTCGACACAG |
U6 RT (mouse) | AACGCTTCACGAATTTGCGT |
U6 (mouse) | CTCGCTTCGGCAGCACA | AACGCTTCACGAATTTGCGT |
WT vector for Herpud2 3′UTR (mouse) | cacaactcgagTAAGCTTCTCATGCATATGA | aaggatccGGGCAAGATGCTACTAGCACA |
Mut vector for Herpud2 3′UTR (mouse) | TACTGCAGAAGGAGCTTTATTCATTTCAATTATGTGTA | AGCTCCTTCTGCAGTACTGTTAGCAATGCTATGTTGTTAG |
Real-time PCR for miRNA
Total RNA was isolated with Trizol Reagent (Invitrogen, USA) following the manufacturer’s instruction. Reverse transcription was performed using the Takara MicroRNA ReverseTranscription Kit (Takara 037A, Japan). Total RNA (500 ng) was reverse-transcribed with 2 μl 5×PrimeScript Buffer, 0.5 μl PrimeScript RT Enzyme Mix, 1 μl Specific miRNA RT primer, RNA 1 μl, and 5.5 μl RNase-free water. Reverse transcription reaction was performed in a thermal cycler as follows: 42 °C for 15 min, 87 °C for 5 s, and 4 °C forever. PCR amplifications were performed on the ABI 7300 Sequence Detection System (Applied Biosystems, USA); they were performed in a total volume of 20 μl, containing 1 μl cDNA sample and 10 SYBR Green PCR Master Mix (Roche, UK). PCR amplifications were always performed in double wells, using the universal temperature cycles: 10 min at 95 °C, followed by 40 cycles consisting of 15 s at 95 °C and 1 min at 60 °C. The expression of miR-7 was normalized using U6 as the internal control. Measurements were normalized to U6 (ΔCt) and comparisons calculated as the inverse log of the ΔΔCT to give the relative fold change for miR-7 level. Primers (Shanghai GenePharma Co., Ltd, Shanghai) used for real-time PCR are listed in Table
1.
Plasmids
The 3′UTRs of Herpud2 mRNA harboring the predicted miR-7a binding sequences were PCR amplified from mouse genomic DNA and cloned into Bam HI and Xho I of the pLUC-Report luciferase vector (Shenzhen Kangbio Biological Technology Co., Ltd, Shenzhen, Guangdong) to generate the Herpud2-3′UTR reporter construct. Mutagenesis of predicted targets with a mutation of 7 bp from the site of perfect complementarity was performed using a site-directed Mutagenesis Kit (Takara). The primers are listed in supplementary Table
1.
Dual luciferase target validation assays
HEK 293T cells were plated at a density of 2 × 104 cells/well in 96-well plates 1 day before transfection. When cells were grown to 50 % confluence in 96-well plates, they were co-transfected with 0.2 μg plasmid DNA, 0.15 μg sensor reporter gene, and 0.45 μg miR-7 mimics or miRNA negative control (NC) (Gemma Pharmaceutical Technology Co., Ltd, Shanghai) using Fugene (Roche) according to the manufacturer’s instructions. After 48 h, cells were lysed and assayed using dual luciferase reporter assay system (Promega) according to the manufacturer’s protocol. Results were displayed as relative luciferase activity, while Renilla luciferase activity was normalized to firefly luciferase activity.
Western blotting
The cytosolic and nuclear proteins in samples were extracted according to the KEYGEN protein extraction kit (Nanjing, Jiangsu, China). Protein concentrations were determined using the Beyotime BCA Kit (Beyotime Biotechnology, Shanghai). The supernatants (30 μg protein) were separated by Tris-glycine SDS-PAGE, transferred to PVDF membranes (Millipore, USA) with the electrophoretic transfer system (Trans-blot Semi-dry Transfer Cell, Bio-Rad, Hercules, CA, USA), and blocked with 5 % nonfat dry milk in Tris-HCl buffer saline (TBS, pH 7.4) containing 0.1 % Tween 20 (TBS-T) for 1 h at room temperature. Then, the PVDF membranes were incubated with primary antibody against glucose-regulated protein 78 (GRP78) (1:1000, CST, Boston, USA), CHOP (1:1000, CST, Boston, USA), Caspase-12 (1:800, CST, Boston, USA), H3 (1:800, Bioworld Technology, USA), nuclear factor κB (NF-κB) (1:800, Bioworld Technology, USA), phosphorylated IκB kinase a/β (pIKK a/β) (1:800, CST, Boston, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, CST, Boston, USA) overnight at 4 °C. After being washed in TBS-T, the membranes were incubated with corresponding secondary antibody for 1 h at room temperature. Finally, visualization of the signal was performed by enhanced chemiluminescence (Ultra-Lum, Claremont, CA, USA). Quantification of bands was made by scanned densitometric analysis and Image J analysis system.
Enzyme-linked immunosorbent assay
After 24 h reoxygenation, we collected the medium from astrocytes. Release of the pro-inflammatory cytokines (TNF-α and IL-1β) from the cellular supernatant was performed using specific enzyme-linked immunosorbent assays (ELISAs) (R&D Systems, UK) according to manufacturers’ guidelines.
Statistical analysis
Data are shown as mean ± S.E.M. Unless stated otherwise, all statistical quantitative assessments were carried out and performed in a blinded manner: for two groups, paired t test, for three or more groups, one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls tests. Differences were considered significant for P < 0.05.
Discussion
Dysfunctions of reactive astrocytes are essential for the pathological processes of many CNS diseases, such as stroke, Parkinson’s disease, and Alzheimer’s disease [
3,
24‐
26]. Accumulating studies suggest that ER stress and inflammation in astrocytes are important mechanisms involved in brain injury [
27‐
29]. In the present study, our results also proved that both ER stress and inflammation are responsible for OGD-induced injury in astrocytes. Opening K-ATP channels could protect against OGD-induced inflammatory damage in astrocytes.
The endoplasmic reticulum is an important subcellular compartment and dispensable for cell survival; particularly, it plays an irreplaceable role in folding and processing cellular proteins. At the early stage of ER stress, unfolded-protein response (UPR) was initiated and demonstrated by accumulation of unfolded proteins including GRP78 and HERP [
30]. It has been proved the pivotal roles of GRP78 in dysfunction of astrocytes during ischemic stroke [
31], but little is mentioned about HERP. There are two types of HERP, such as HERP1 and HERP2, which are respectively encoded by Herpud1 and Herpud2 gene. HERP2 is constitutively expressed in cells, whereas HERP1 is highly induced by ER stress [
32]. Both of them are essential for ER stress; they mainly participate in the process of ER-associated degradation and mediate ER stress-induced inflammation. Deficiency of HERP could attenuate ER stress-induced inflammatory reaction in atherosclerosis [
33]. Most of the scientists focus on their functions but pay little attention to their regulation. MicroRNAs are proved to regulate gene expression at the post-transcriptional level, via degradation or translational inhibition of their target mRNAs [
34]. In the present study, we found that miR-7 targeted the 3′UTR of Herpud2 (Fig.
2c). Our data showed that OGD could downregulate miR-7 (Fig.
2a) and upregulate mRNA level of Herpud2 (Fig.
2b); it motivated ER stress via upregulating ER stress proteins including GRP78, CHOP, and Caspase-12 (Fig.
4a‐
d). Thus, our study suggested that miR-7 might be an important upstream sign that modulate ER-associated degradation. MiR-7 is a proven small molecule that implicates in inflammation in various diseases [
35], such as neurodegenerative disease and glioma [
11,
36‐
38]. Our study revealed that miR-7 was also involved in OGD-induced ER stress and inflammatory responses in astrocytes.
ER stress is one of the most important acute-phase responses that mediate inflammatory responses. ER stress and inflammation interconnected through various mechanisms including activation of NF-κB signaling pathway [
39]. The present data showed that OGD induced increase of pro-inflammatory cytokines including TNF-α and IL-1β. Inflammation induced by OGD was mediated by activating pro-inflammatory transcription factor NF-κB, which was the central mediator of pro-inflammatory pathway. Activated IKK phosphorylates inhibitor of κB, initiating inhibitor of κB and thereby leading to NF-κB activation, activated NF-κB then migrate to the nucleus, and facilitated transcription of pro-inflammatory cytokines such as TNF-α and IL-1β, which were transcribed by NF-κB [
39]. In the present study, results demonstrated that OGD promoted production of TNF-α and IL-1β, which was mediated by phosphorylation of IKKα/β and activation of nuclear NF-κB. Opening K-ATP channel could inhibit OGD-induced inflammatory responses. These results suggested that K-ATP channels were an important regulator for OGD-induced inflammation.
The K-ATP channels widely express and act as an important sensor of energetic metabolism. Ischemia or OGD leads to cellular energy loss, which contributes to the dysfunction of K-ATP channels. In turn, dysfunction of K-ATP channels results in the intolerance of cells to stress. Particularly, K-ATP channels are abundant in the endoplasmic reticulum; dysfunction of them is responsible for ER stress pathway [
40]. Beyond that, it is proved that K-ATP channels are pivotal for CNS inflammation, especially in glia. K-ATP channel knockdown increased glial reaction and production of inflammatory cytokines in ischemic brain [
14]. K-ATP channel-deficient mice showed more susceptible to infection [
41]. All of these suggest that K-ATP channels are pivotal for ER stress and inflammation. In the present study, pretreatment with nicorandil (a K-ATP channels’ opener) could rescue the reduction of miR-7 induced by OGD (Fig.
2d). Meanwhile, nicorandil could attenuate ER stress (Fig.
4) as well as decrease pro-inflammatory cytokine (TNF-α and IL-1β) production (Fig.
5). These results suggested that K-ATP channel’s opener alleviated OGD-mediated ER stress and inflammation via regulating miR-7 and thereby protected astrocytes against OGD-induced damage.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China (No. 81273495, No. 81473197, and No. 81402906), Major Project of Jiangsu Provincial Department of Education (No. 12KJA310002), the 333 Talent Project (BRA2014055), and Jiangsu Province National Natural Science Foundation of China (No. BK20151566).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YFD was responsible for determining the expression of the miR-7 and Herpud2 in astrocytes by real-time PCR, collecting data and drafting the manuscript. ZZC mainly charged with expressions of proteins involved in ER stress and inflammatory responses by western blotting. ZZ and DDY participated in cell culture, preparing the OGD model and ELISA assay of TNF-α and IL-1β and carried out photographs of images, assessment of LDH release, and the statistical analysis. JJ performed the dual luciferase target validation assays. XLS conceived the designed the study, performed the statistical analysis and helped to draft the manuscript. All authors read and approved the final manuscript.