Open Access

Long non‑coding RNA BC168687 small interfering RNA reduces high glucose and high free fatty acid‑induced expression of P2X7 receptors in satellite glial cells

  • Authors:
    • Cheng‑Long Liu
    • Ze‑Yu Deng
    • Er‑Rong Du
    • Chang‑Shui Xu
  • View Affiliations

  • Published online on: February 13, 2018     https://doi.org/10.3892/mmr.2018.8601
  • Pages: 5851-5859
  • Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Purinergic signaling contributes to inflammatory and immune responses. The activation of the P2X purinoceptor 7 (P2X7) in satellite glial cells (SGCs) may be an essential component in the promotion of inflammation and neuropathic pain. Long non‑coding RNAs (lncRNAs) are involved in multiple physiological and pathological processes. The aim of the present study was to investigate the effects of a small interfering RNA for the lncRNA BC168687 on SGC P2X7 expression in a high glucose and high free fatty acids (HGHF) environment. It was demonstrated that BC168687 small interfering (si)RNA downregulated the co‑expression of the P2X7 and glial fibrillary acidic protein and P2X7 mRNA expression. Additionally, HGHF may activate the mitogen‑activated protein kinase signaling pathway by increasing the release of nitric oxide and reactive oxygen species in SGCs. Taken together, these results indicate that silencing BC168687 expression may downregulate the increased expression of P2X7 receptors in SGCs induced by a HGHF environment.

Introduction

Type 2 diabetes mellitus (T2DM) is a prevalent endocrine and metabolic disease. Changes in life style and accelerations in the aging process have contributed to the increasing prevalence of T2DM. It is a chronic non-communicable disease that particularly affects those with cardiovascular or cerebrovascular diseases (13). In addition, diabetic neuropathy may occur, which involves the excessive excitation of primary afferent receptors and central neurons, leading to pain, and other adverse effects (4). The activation of satellite glial cells (SGCs) has been reported to be an essential factor in several experimental models of pain (57). Hyperglycemia and dyslipidemia are hallmark features of pre-diabetes (8,9). Obesity-associated dysregulation of glucose and lipid metabolism has been associated with diabetes, and high blood sugar and free fatty acids (FFA) in serum are thought to contribute to neurological disorder development (10,11). Thus, cell injury inducing a high glucose high free fatty acid (HGHF) environment may effectively model the condition of neurological disorders in T2DM (12,13).

Adenosine 5′-triphosphate (ATP) is an important messenger that is involved in numerous processes, including the transmission of pain signals. It may also act as an acute pro-inflammatory danger signal and a crucial mediator of neuroinflammation. In an environment of inflammation or stress, levels of extracellular ATP (eATP) rapidly approach near millimolar levels and become the main stimulation of pro-inflammatory pathways (14). Subclasses of purinergic 2 (P2) receptors include P2X and P2Y. P2X receptors, particularly the P2X purinoceptor 7 (P2X7), are strongly associated with immunity and inflammation (14). P2X7 receptors are highly expressed in immune cells and are activated as a result of pro-inflammatory cytokine release (15). In SGCs, eATP may activate the P2X7 receptor, thus possibly contributing to the development of chronic inflammatory disease (16).

Long non-coding RNAs (lncRNAs) are non-protein-coding RNA transcripts >200 nucleotides in length. Increasing evidence has highlighted the role of lncRNAs in physiology and disease (17,18). LncRNAs are involved in diverse regulatory processes, including the alteration of chromatin and transcriptional state, nuclear architecture, splicing and mRNA translation (19,20). LncRNA BC168687 is evolutionarily conserved across numerous species and significantly increased levels have been detected in the dorsal root ganglion (DRG) of type 2 diabetic rats (21). Therefore, BC168687 was selected for examination. The present study revealed that lncRNA BC168687 small interfering RNA (siRNA) may downregulate P2X7 receptor expression induced by a HGHF environment in primary cultured SGCs.

Materials and methods

Primary culture

The present study was approved by the Ethical Committee of Nanchang University (Nanchang, China) and animals were treated according to the Guidelines for the Care and Use of Animals (22). Fetal Sprague-Dawley rats (n=6; male; 7–9 g) were obtained from the Laboratory Animal Science Department of Nanchang University (Nanchang, China). All rats were housed in clean, standard metabolic cages and kept at a constant temperature of 37°C with 35–65% humidity. The rats were kept in a 12 h light/dark cycle and had free access to food and water. On the third day, rats were anesthetized using ether. The DRGs of fetal rats were extracted with microforceps and rapidly transferred into Dulbecco's modified Eagle Medium/F12 (DMEM/F12) medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and incubated at 4°C for 30 min prior to the next step. Following the detachment of redundant fibers with ophthalmic forceps, the DRGs were incubated with collagenase type III (0.1 mg/ml; Beijing Solarbio Science and Technology, Ltd., Beijing, China) for 15 min at 37°C. The collagenase was removed by centrifugation at 168 × g for 5 min and DRGs were pre-incubated with 0.25% trypsin-EDTA (0.5 mg/ml; Beijing Solarbio Science and Technology, Ltd.) in a cell incubator for 35–40 min at 37°C. DMEM/F12 containing 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Bei-Haemek, Israel) was subsequently used to terminate enzymatic digestion.

The DRGs were blown gently using sterile disposable pipettes before being passed through a cell strainer (aperture, 70 µm; 200 mesh). Glial cells (5×105 cells/ml) were inoculated on polylysine-coated coverslips into 24-well plates to obtain cell climbing slides. SGCs were purified from glial cells by replacing the medium twice every 24 h. The purified SGCs were sustained in DMEM/F12 containing 10% FBS (Biological Industries), 100 U/ml penicillin and 100 mg/ml streptomycin sulfate at 37°C in a humidified incubator with 5% CO2. To imitate hyperglycemia and dyslipidemia, 40 mM D-glucose (Beijing Solarbio Science and Technology, Ltd.) and 0.60 mM FFAs were added to DMEM/F12 medium. FFAs were a mixture of oleate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and palmitate (Sigma-Aldrich, Merck KGaA) at a 2:1 ratio (w/w) (23,24). In addition, 20 mM D-Mannitol (Beijing Solarbio Science and Technology) was added into DMEM/F12 as an isotonic control.

SGCs were divided into five groups: Control, HGHF (40 mM D-glucose and 0.60 mM FFAs), HGHF+BC168687 small interfering RNA (siRNA), HGHF+negative control siRNA (NCsi) and HGHF+empty vector control (VD; Entranster-R4000; Engreen Biosystem, Ltd., Beijing, China). SGCs were treated with HGHF for 72 h. When the cells reached 70–80% confluence, siRNAs (50 nM) were transfected into SGCs in 24-well plates for further experimentation.

siRNA transfection

The following BC168687 siRNA and negative control siRNA sequences were synthesized (Novobio Scientific, Inc., Shanghai, China) and used: BC168687-rat-159 (5′-GAGAUUAUUAAGGUGUACUTT-3′), BC168687-rat-1172 (5′-GACGGUUGAUACUGACUCUTT-3′), BC168687-rat-2400 (5′-GUUGGAUCCUUCUCAAUCATT-3′) and negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′). The three different BC168687 siRNA duplexes were transfected into SGCs using the Entranster-R4000 (Engreen Biosystem, Ltd.) according to the manufacturer's protocol. After 48 h, the expression levels of BC168687 were evaluated by reverse transcription quantitative-polymerase chain reaction (RT-qPCR).

Cell viability test

The viability of SGCs was analyzed with the TransDetect Cell Counting kit (CKK; Beijing TransGen Biotech, Co., Ltd., Beijing, China). A suspension of 100 µl SGCs (5×103 cells/ml) from each group was placed onto a 96-well microplate. Each group was tested in triplicate. Following culture of SGCs at the different concentrations of D-glucose and FFA for 72 h, 10% CCK diluent was added to each well. Cells were subsequently maintained in a cell incubator for 2 h. A wavelength of 450 nm was used to detect the absorbance using a multimode plate reader. The data was analyzed with GraphPad Prism v6.0 (GraphPad Software Inc., La Jolla, CA, USA).

RT-qPCR

RNA was extracted with Transzol Up (Beijing TransGen Biotech, Ltd.) and reverse transcribed at 37°C for 1 h using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The concentration of cDNA for each group was detected to be ~4×103 ng/µl using the NanoDrop2000 (Thermo Fisher Scientific, Inc.). The primer sequences used (Sangon Biotech Co., Ltd., Shanghai, China) were as follows: BC168687 forward, 5′-GGACAAGTCCTTAGCCATGC-3′ and reverse, 5′-CAACACCGTTGGATCCTTCT-3′; P2X7 forward, 5′-GCACGAATTATGGCACCGTC-3′ and reverse, 5′-CCCCACCCTCTGTGACATTC-3′; and β-actin forward, 5′-CCTAAGGCCAACCGTGAAAAGA-3′ and reverse, 5′-GGTACGACCAGAGGCATACA-3′. RT-qPCR was performed using the SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd., Dalian, China) and the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Inc.). The thermo cycling conditions were as follows: 95°C for 30 sec, 60°C for 15 sec, 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. The melting curve was used to determine the amplification specificity and results were analyzed using the StepOnePlus Real-Time PCR system. The average threshold cycle (Cq) value for the target minus the average value for β-actin was used to calculate the ∆Cq value (∆Cq=Cq target-Cq reference). The ∆∆Cq value was calculated as follows: ∆Cq test sample-∆Cq calibrator sample. The relative quantity (RQ) of the gene expression was calculated using the following equation: 2−ΔΔCq (21).

Immunocytochemistry

Immunocytochemistry was performed with the SPlink Detection kit (cat no. SP-9001; OriGene Technologies, Inc., Beijing, China) and the working solutions provided by the manufacturer were used if not otherwise specified. Cell climbing slides (diameter, 8 mm) were removed from DMEM/F12 and washed three times in PBS and subsequently fixed in 4% paraformaldehyde (Beijing Solarbio Science and Technology, Ltd.) for 15 min at room temperature. Following three washes with PBS, slides were blocked with normal goat serum at 37°C for 30 min. The slides were washed in PBS and incubated with P2X7 primary antibody (cat no. APR-004-AO; 1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C. Slides were washed in PBS and incubated with Biotin labeled goat anti-rabbit IgG polymer secondary antibody for 15 min at 37°C. Slides were washed again with PBS prior to incubation with alkaline phosphatase-labeled streptavidin for 15 min at 37°C. Slides were subsequently stained with 3,3′-diaminobenzindine solution (OriGene Technologies, Inc.) at room temperature for 10 min and sealed by neutral balsam (OriGene Technologies, Inc.). The expression of P2X7 receptors was visualized with a fluorescence inverted microscope (magnification, ×200) and the integrated optical density (IOD) of the P2X7 receptors was calculated using Image-Pro Plus v6.0 (Media Cybernetics Inc., Rockville, MD, USA).

Western blot analysis

SGCs total protein was extracted with the Mammal Cell Protein Extraction reagent (Wuhan Boster Biological Technology, Ltd., Wuhan, China). Protein concentrations were detected with a multimode plate reader using a Bradford Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Supernatant samples containing 20 µg of protein were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked in 5% bovine serum albumin (Beijing Solarbio Science and Technology, Ltd.) at room temperature for 2 h and subsequently incubated with the following primary antibodies overnight at 4°C: rabbit anti-P2X7 (cat. no. APR-004-AO; dilution, 1:500; Alomone Labs), mouse anti-glial fibrillary acidic protein (GFAP; cat. no. 644701; dilution, 1:200; BioLegend, Inc., San Diego, CA, USA), rabbit anti-phosphorylated-extracellular signal-related kinase 1/2 (ERK1/2) (Thr202/Thr204) (cat. no. D13.24.4E; dilution, 1:1,000; Cell Signaling Technology, Inc. Danvers, MA, USA), rabbit anti-ERK1/2 (cat. no. 137F5; dilution, 1:1,000; Cell Signaling Technology, Inc.), and mouse anti-β-actin antibody (cat. no. TA-09l; dilution, 1:800; OriGene Technologies, Inc.). The following day membranes were washed three times with TBST and incubated with the following secondary antibodies: Peroxidase-conjugated goat anti-rabbit IgG (1:2,000; cat no. ZB-2301; OriGene Technologies, Inc.) and goat anti-mouse IgG (1:2,000; cat no. ZB-2305; OriGene Technologies, Inc.) for 2 h at room temperature. Bands were visualized using the Enhanced Chemiluminescent reagent kit (Wuhan Boster Biological Technology, Ltd.) and the IOD was calculated using Image-Pro Plus v6.0 (Media Cybernetics Inc.).

Double immunofluorescence

Cell climbing slides (diameter, 8 mm) were removed from DMEM/F12 and washed three times with PBS. Slides were fixed with 4% paraformaldehyde (Beijing Solarbio Science and Technology, Ltd.) for 15 min at room temperature. Slides were washed three times in PBS and subsequently blocked with normal goat serum (OriGene Technologies, Inc.) at the working solution provided by the manufacturer for 1 h in a thermostatic water bath at 37°C, prior to incubation with rabbit anti-P2X7 (cat. no. APR-004-AO; dilution, 1:200; Alomone Labs) and mouse anti-GFAP (cat. no. 644701; dilution, 1:200; BioLegend, Inc.) overnight at 4°C. Slides were then washed three times with PBS, and incubated with fluorescent goat anti-mouse fluorescein isothiocyante (1:200; cat no. ZF-0311; OriGene Technologies, Inc.) and goat anti-rabbit tetramethylrhodamine isothiocyanate secondary antibodies (1:200; cat no. ZF-0313; OriGene Technologies, Inc.) in the dark at 37°C for 1 h. Slides were washed three times with PBS and subsequently stained with DAPI (cat. no. AR1176; dilution, 1:1,000; Wuhan Boster Biological Technology, Ltd.) at 37°C for 60 sec. Slides were washed three times with PBS, sealed with anti-fluorescent quencher (Wuhan Boster Biological Technology, Ltd.) and visualized with a fluorescence inverted microscope.

Measurement of eATP

The release of ATP from SGCs was measured using an ATPlite 1 step luminescence assay system kit (10 ml; PerkinElmer, Inc., Waltham, MA, USA). A suspension of 100 µl SGCs from each group was placed onto a 96-well microplate. Each group was tested in triplicate. The substrate vial and buffer solution were equilibrated at room temperature. The lyophilized substrate solution was then mixed with the buffer and left to stand at room temperature for 5 min. The reconstituted reagent (100 µl) was subsequently added into each well containing the 100 µl suspension. The 96-well microplates were agitated for 2 min at 168 × g at 37°C and the luminescence was measured using a multimode plate reader.

Measurement of intracellular nitric oxide (NO) and ROS

Intracellular NO was measured using the nitrate reductase method (21). The NO concentration in each group was calculated according to the formula provided in the Nitric Oxide Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The IOD values of each group were calculated using a multimode plate reader.

Intracellular ROS levels were inspected with the ROS Assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Following the removal of DMEM/F12 from SGCs, diluted 2′,7′-dichlorofluorescin diacetate (DCFH-DA; 1:1,000; 10 µM; cat no. S0033, Beyotime Institute of Biotechnology) was added. Sample were placed into 24-well plates and incubated for 20 min at 37°C, then subsequently washed three times with serum-free DMEM/F12. The fluorescence density was detected at an excitation wavelength of 488 nm and an emission wavelength of 525 nm with the multimode plate reader.

Statistical analysis

Results are presented as the mean ± standard error. GraphPad Prism v6.0 (GraphPad Software Inc.) and Image-Pro Plus v6.0 (Media Cybernetics Inc.) were used to perform the statistical tests. The unpaired Student's t-test was used when comparing two groups and the one-way analysis of variance with the Bonferroni correction was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

Screening of high D-glucose and FFAs concentrations

SGC viability was measured with CCK. The results indicated that the viability of SGCs in a D-glucose environment significantly decreased at D-glucose concentrations of ≥40 mM: Control, 0.49±0.02; isotonic control, 0.50±0.02; 10 mM D-glucose; 0.48±0.02; 20 mM D-glucose, 0.48±0.01; 40 mM D-glucose, 0.45±0.02; 80 mM D-glucose, 0.43±0.02; and 160 mM D-glucose 0.31±0.00 (Fig. 1A).

The viability of SGCs in a FFA environment was also significantly decreased at FFA concentrations ≥0.60 mM: Control, 0.49±0.02; isotonic control, 0.49±0.01; 0.15 mM, 0.48±0.01; 0.30 mM, 0.47±0.00; 0.60 mM, 0.46±0.01; 1.2 mM, 0.44±0.01; and 2.4 mM, 0.44±0.02 (Fig. 1B). Based on the aforementioned results, 40 mM D-glucose and 0.6 mM FFA were selected as the final concentrations to produce a HGHF environment.

P2X7 receptor and lncRNA BC168687 expression in SGCs in a HGHF environment

The relative expression level of BC168687 was determined using RT-qPCR (Fig. 2A) and the expression of P2X7 receptors was analyzed with immunocytochemistry (Fig. 2B and C). The results indicated that the relative expression levels of BC168687 and P2X7 were higher in the HGHF environment compared with the control group (Fig. 2A and B; P<0.01).

BC168687 siRNA screening and P2X7 mRNA expression in SGCs

Following the transfection of siRNAs into SGCs for 48 h, duplexes from three different siRNA sequences were screened for effective BC168687 silencing (Fig. 3A). The relative level of BC168687 and P2X7 mRNA expression in SGCs was determined using RT-qPCR (Fig. 3B). The results revealed that the relative level of BC168687 was significantly decreased by siRNAs 1172 (P<0.05) and 2400 (P<0.001): Control, 1.00±0.00; BC168687-159si, 0.83±0.08; BC168687-1172si, 0.75±0.19; BC168687-2400si, 0.56±0.04. Thus, BC168687-2400 was selected as the targeting siRNA of BC168687.

The relative mRNA expression of P2X7 in each group was as follows: Control, 1.00±0.00; HGHF, 2.47±0.44; HGHF+BC168687si, 1.06±0.29; HGHF+NCsi, 2.60±0.64; and HGHF+VD, 2.23±0.31. The variance was statistically significant (P<0.01). Compared with the control group, P2X7 mRNA expression in the HGHF group was significantly increased. P2X7 mRNA expression in the HGHF+BC168687si group was significantly lower compared with the HGHF group (P<0.01). No significant differences were observed among the HGHF, HGHF+NCsi and HGHF+VD group. Based on the results obtained, it was concluded that BC168687 siRNA was able to attenuate the upregulation of P2X7 mRNA induced in a HGHF environment.

BC168687 siRNA downregulates the expression of P2X7 and GFAP in SGCs

Following transfection of the siRNAs into SGCs for 72 h, P2X7 and GFAP expression was detected by western blot analysis (Fig. 4A and B). The relative expression of P2X7 in each group was as follows: Control, 0.50±0.02; HGHF, 0.65±0.01; HGHF+BC168687si, 0.43±0.03; HGHF+NCsi, 0.63±0.02 and HGHF+VD 0.67±0.01. The variance analysis was statistically significant between the HGHF+BC168687si and HGHF group (P<0.001). The relative expression of GFAP in each group was as follows: Control, 0.53±0.08; HGHF, 0.85±0.06; HGHF+BC168687si, 0.59±0.10; HGHF+NCsi, 0.93±0.05; and HGHF+VD, 0.84±0.04. Expression of P2X7 protein and GFAP in the HGHF group was signficantly increased compared with the control group (P<0.01). Compared with the HGHF group, the P2X7 protein and GFAP expression levels were signficantly decreased in the HGHF+BC168687si group (P<0.01). No significant differences were observed among the HGHF, HGHF+NCsi and HGHF+VD groups. Therefore, BC168687 siRNA may attenuate the upregulation of the P2X7 receptor and GFAP induced by a HGHF environment in SGCs.

P2X7 and GFAP co-expression is induced by a HGHF environment in SGCs

Immunofluorescence was used to detect the co-expression of P2X7 receptor and GFAP in SGCs (25). The co-expression quantities of the P2X7 receptors and GFAP in the five groups was detected following 72 h of siRNA transfection, based on the co-localization of P2X7 and GFAP in SGCs (Fig. 5). Compared with the control group, the P2X7 receptor and GFAP co-expression quantities were increased in the HGHF group. The co-expression quantities of the HGHF+BC168687si group were decreased compared with the HGHF group. No apparent difference was observed among the HGHF, HGHF+NCsi and HGHF+VD groups. Therefore, it was inferred that BC168687 siRNA may reduce the P2X7 receptor upregulation induced by a HGHF environment.

BC168687 siRNA reduces the upregulation of p-ERK1/2 expression induced by a HGHF environment in SGCs

The expression level of phosphorylated-ERK1/2 protein in SGCs was detected by western blot analysis. The relative expression levels of p-ERK1/2 protein in each group were as follows: Control, 0.50±0.02; HGHF, 0.58±0.02; HGHF+BC168687si, 0.52±0.10; HGHF+NCsi, 0.65±0.03; and HGHF+VD, 0.60±0.01. Compared with the control group, the p-ERK1/2 protein expression in the HGHF group was signficantly increased. The expression level of p-ERK1/2 protein in the HGHF+BC168687si group was significantly decreased compared with the HGHF group (Fig. 6A; P<0.01). No significant difference was observed among the HGHF, HGHF+NCsi and HGHF+VD groups. Based on the results obtained, it was concluded that BC168687 siRNA was able to reduce the upregulation of p-ERK1/2 signalling induced by a HGHF environment in SGCs.

Effect of BC168687 siRNA on ATP levels in SGCs induced by a HGHF environment

As a proinflammatory mediator released from SGCs, ATP contributes to the initiation and maintainence of neuropathic pain (26). The results revealed that the concentrations of ATP (pM) in each group were as follows: Control, 63.33±11.55; HGHF, 140±17.32; HGHF+BC168687si, 76.67±5.77; and HGHF+NCsi, 176.67±35.12. ATP levels in the HGHF group were signficantly increased compared with the control group (Fig. 6B; P<0.01) and the levels of ATP in the HGHF+BC168687si group were significantly decreased compared with the HGHF group (P<0.01). There was no significant difference between the HGHF and HGHF+NCsi groups.

Effect of BC168687 siRNA on NO and ROS levels in SGCs induced by a HGHF environment

NO and ROS are oxidative injury factors released from SGCs that are also considered to contribute to the initiation and maintainence of neuropathic pain (2729). The results revealed that the concentrations (µM) of NO in each group were as follows: Control, 16.11±3.47; HGHF, 41.67±2.89; HGHF+BC168687si, 23.33±2.89; and HGHF+NCsi, 46.67±2.89 (Fig. 7A). The variance was statistically significant between the HGHF+BC168687si and HGHF group (P<0.01). Intracellular ROS levels were measured by fluorescence density. The results of the ROS assay kit were as follows: Control, 2,655±243.98; HGHF, 3,394±141.74; HGHF+BC168687si, 2,807±58.03; and HGHF+Ncsi, 3,642±213.18 (Fig. 7B). NO and ROS levels in the HGHF group were significantly increased compared with the control group (P<0.01). NO and ROS levels in the HGHF+BC168687 si group were signficantly decreased compared with the HGHF group (P<0.01). There was no signifcant difference between the HGHF and HGHF+NCsi groups.

Discussion

Compared with short-chain ncRNAs, including microRNAs, siRNAs and Piwi-interacting RNAs, lncRNAs account for the majority of ncRNAs that regulate biological mechanisms and processes (30,31). They participate in the regulation of transcription and intracellular signal transduction pathways, including those involved in organism development (32). Therefore, dysregulated lncRNA expression may contribute to the development of numerous human diseases (3335). P2X7 receptors are expressed in SGCs and studies have demonstrated that P2X7 receptors contribute to neuropathic pain (3639). High levels of glucose and FFAs have been identified as a primary cause of nervous system dysfunction in diabetes (8,13). NcRNAs lack the ability to encode proteins, but possess regulatory functions, and are involved in almost all physiological and pathological processes (30,31,4042). The present study demonstrated that BC168687 expression in SGCs in a HGHF environment group was significantly increased compared with the control group. P2X7 receptor expression was also upregulated in SGCs in a HGHF environment, inferring the involvement of BC168687 in pathological processes mediated by P2X7 receptors in SGCs.

The P2 receptor family is comprised of ligand-gated ion channel P2X receptors and G-protein coupled P2Y receptors (43). Autocrine release of ATP by glial cells activates P2X7 receptors and may amplify pain signals through a cascade reaction (4447). Thus, inhibiting P2X7 receptors may relieve inflammatory and chronic neuropathic pain (37,39). The present study demonstrated that a HGHF environment increased ATP release in SGCs and BC168687 siRNA was able to decrease this release. P2X7 mRNA and protein expression in SGCs in the HGHF group was significantly increased compared with the control group. Expression of P2X7 mRNA and protein was significantly decreased in the HGHF+BC168687si group compared with the HGHF group, suggesting that BC168687 is associated with the upregulation of P2X7 receptors observed in the HGHF group.

The increasing incidence of T2DM along with its comorbidities makes it urgent to understand the pathogenesis and regulatory mechanisms of the disease. The specific involvement of lncRNAs in diabetes is unclear (48,49). Diabetes is a chronic inflammatory disease, and the expression P2X7 receptors may be upregulated by inflammatory injury (25,50). In an inflammatory state, ATP can be released from sensory neurons and SGCs in an autocrine or a paracrine fashion and activate P2 receptors (7,51). Excessive P2X7 receptor excitation by ATP can promote the opening of plasma membrane pores, and may increase the release of pro-inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor-α (37,52). These cytokines further induce glial cells to release pro-inflammatory mediators and exacerbate neuronal damage (14,45).

Oxidative stress is one of the important factors leading to diabetic neuropathy. Along with ATP, NO and ROS are released from glial cells and contribute to chronic neuropathic pain in diabetes (27,53,54). The present study indicated that HGHF significantly increased the release of NO and ROS, and these levels were decreased in the HGHF+BC168687si group. This suggests that BC168687 contributes to the pathological processes involving P2X7 receptors, leading to neuropathic and peripheral inflammatory pain.

The mitogen-activated protein kinase (MAPK) signaling pathway is involved in cell proliferation, differentiation and adaptation, and may also contribute to the development of neuronal injury and disease. The MAPK family contains p38 MAPKs, ERK and c-Jun N-terminal kinases (55,56). The signaling pathways of MAPKs are crucial to signal transduction between neurons and glial cells, both of which are also essential for persistent pain (57,58). However, different MAPKs have distinct actions in glial cells following injury (58). Active ERK1/2 signaling occurs between the nucleus and the cytoplasm, and ROS is able to influence the ERK MAPK signaling pathway through phosphorylation (57,59). In the present study, an upregulation of p-ERK1/2 signaling was observed in SGCs in the HGHF group. Thus, it may be inferred that the ERK MAPK signaling pathway is involved in the aberrant activation of SGCs in a HGHF environment. Overall, it was concluded that BC168687 may be involved in the increased expression of P2X7 receptors in SGCs in a HGHF environment, and BC168687 siRNA may have the potential to alleviate diabetic neuropathic pain mediated by P2X7. These findings suggest BC168687 siRNA as a novel treatment for P2X7 associated diseases including diabetic neuropathic pain. Further research to elucidate the specific mechanisms of BC168687 siRNA are required.

Acknowledgements

The present study was supported by The Youth Science Foundation of the Educational Department of Jiangxi Province (grant no. GJJ14146), The Cultivating Foundation of Young Scientists (Star of Jinggang) of Jiangxi Province (grant no. 20153BCB23033) and The Innovation Foundation of the Graduate School of Nanchang University (grant no. cx2016361).

References

1 

Szuszkiewicz-Garcia MM and Davidson JA: Cardiovascular disease in diabetes mellitus: Risk factors and medical therapy. Endocrinol Metab Clin North Am. 43:25–40. 2014. View Article : Google Scholar : PubMed/NCBI

2 

Rathmann W and Giani G: Global prevalence of diabetes: Estimates for the year 2000 and projections for 2030. Diabetes Care. 27:2568–2569. 2004. View Article : Google Scholar : PubMed/NCBI

3 

Katsiki N, Purrello F, Tsioufis C and Mikhailidis DP: Cardiovascular disease prevention strategies for type 2 diabetes mellitus. Expert Opin Pharmacother. 18:1243–1260. 2017. View Article : Google Scholar : PubMed/NCBI

4 

Baron R: Peripheral neuropathic pain: From mechanisms to symptoms. Clin J Pain. 16 2 Suppl:S12–S20. 2000. View Article : Google Scholar : PubMed/NCBI

5 

Wu JR, Chen H, Yao YY, Zhang MM, Jiang K, Zhou B, Zhang DX and Wang J: Local injection to sciatic nerve of dexmedetomidine reduces pain behaviors, SGCs activation, NGF expression and sympathetic sprouting in CCI Rats. Brain Res Bull. 132:118–128. 2017. View Article : Google Scholar : PubMed/NCBI

6 

Ji RR, Chamessian A and Zhang YQ: Pain regulation by non-neuronal cells and inflammation. Science. 354:572–577. 2016. View Article : Google Scholar : PubMed/NCBI

7 

Hanani M: Role of satellite glial cells in gastrointestinal pain. Front Cell Neurosci. 9:4122015. View Article : Google Scholar : PubMed/NCBI

8 

Xu H, Wu B, Jiang F, Xiong S, Zhang B, Li G, Liu S, Gao Y, Xu C, Tu G, et al: High fatty acids modulate P2×(7) expression and Il-6 release via the P38 Mapk pathway in Pc12 cells. Brain Res Bull. 94:63–70. 2013. View Article : Google Scholar : PubMed/NCBI

9 

Namekawa J, Takagi Y, Wakabayashi K, Nakamura Y, Watanabe A, Nagakubo D, Shirai M and Asai F: Effects of high-fat diet and fructose-rich diet on obesity, dyslipidemia and hyperglycemia in the Wbn/Kob-Lepr(Fa) rat, a new model of type 2 diabetes mellitus. J Vet Med Sci. 79:988–991. 2017. View Article : Google Scholar : PubMed/NCBI

10 

Ruan X: Long Non-Coding Rna central of glucose homeostasis. J Cell Biochem. 117:1061–1065. 2016. View Article : Google Scholar : PubMed/NCBI

11 

Kornfeld JW, Baitzel C, Könner AC, Nicholls HT, Vogt MC, Herrmanns K, Scheja L, Haumaitre C, Wolf AM, Knippschild U, et al: Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b. Nature. 494:111–115. 2013. View Article : Google Scholar : PubMed/NCBI

12 

Fan B, Gu JQ, Yan R, Zhang H, Feng J and Ikuyama S: High glucose, insulin and free fatty acid concentrations synergistically enhance perilipin 3 expression and lipid accumulation in macrophages. Metabolism. 62:1168–1179. 2013. View Article : Google Scholar : PubMed/NCBI

13 

Singh H, Brindle NP and Zammit VA: High glucose and elevated fatty acids suppress signaling by the endothelium protective ligand angiopoietin-1. Microvasc Res. 79:121–127. 2010. View Article : Google Scholar : PubMed/NCBI

14 

Burnstock G: P2× ion channel receptors and inflammation. Purinergic Signal. 12:59–67. 2016. View Article : Google Scholar : PubMed/NCBI

15 

Baudelet D, Lipka E, Millet R and Ghinet A: Involvement of the P2×7 purinergic receptor in inflammation: An update of antagonists series since 2009 and their promising therapeutic potential. Curr Med Chem. 22:713–729. 2015. View Article : Google Scholar : PubMed/NCBI

16 

Faria RX, Freitas HR and Reis RAM: P2×7 receptor large pore signaling in avian muller glial cells. J Bioenerg Biomembr. 49:215–229. 2017. View Article : Google Scholar : PubMed/NCBI

17 

Kwok ZH and Tay Y: Long noncoding RNAs: Lincs between human health and disease. Biochem Soc Trans. 45:805–812. 2017. View Article : Google Scholar : PubMed/NCBI

18 

Sun M and Kraus WL: From discovery to function: The expanding roles of long noncoding RNAs in physiology and disease. Endocr Rev. 36:25–64. 2015. View Article : Google Scholar : PubMed/NCBI

19 

Wu Z, Liu X, Liu L, Deng H, Zhang J, Xu Q, Cen B and Ji A: Regulation of lncRNA expression. Cell Mol Biol Lett. 19:561–575. 2014. View Article : Google Scholar : PubMed/NCBI

20 

Taylor DH, Chu ET, Spektor R and Soloway PD: Long non-coding RNA regulation of reproduction and development. Mol Reprod Dev. 82:932–956. 2015. View Article : Google Scholar : PubMed/NCBI

21 

Liu C, Tao J, Wu H, Yang Y, Chen Q, Deng Z, Liu J and Xu C: Effects of LncRNA BC168687 SiRNA on diabetic neuropathic pain mediated by P2X7 receptor on SGCs in DRG of rats. Biomed Res Int. 2017:78312512017. View Article : Google Scholar : PubMed/NCBI

22 

Jones-Bolin S: Guidelines for the care and use of laboratory animals in biomedical research. Curr Protoc Pharmacol Appendix. 4:Appendix 4B2012.

23 

Hirose H, Lee YH, Inman LR, Nagasawa Y, Johnson JH and Unger RH: Defective fatty acid-mediated beta-cell compensation in Zucker diabetic fatty rats. pathogenic implications for obesity-dependent diabetes. J Biol Chem. 271:5633–5637. 1996. View Article : Google Scholar : PubMed/NCBI

24 

Xu H, He L, Liu C, Tang L, Xu Y, Xiong M, Yang M, Fan Y, Hu F, Liu X, et al: LncRNA NONRATT021972 SiRNA attenuates P2X7 receptor expression and inflammatory cytokine production induced by combined high glucose and free fatty acids in PC12 Cells. Purinergic Signal. 12:259–268. 2016. View Article : Google Scholar : PubMed/NCBI

25 

Liu S, Zou L, Xie J, Xie W, Wen S, Xie Q, Gao Y, Li G, Zhang C, Xu C, et al: LncRNA NONRATT021972 siRNA regulates neuropathic pain behaviors in type 2 diabetic rats through the P2X7 receptor in dorsal root ganglia. Mol Brain. 9:442016. View Article : Google Scholar : PubMed/NCBI

26 

Inoue K: Neuropharmacological study of ATP receptors, especially in the relationship between Glia and Pain. Yakugaku Zasshi. 137:563–569. 2017.(In Japanese). View Article : Google Scholar : PubMed/NCBI

27 

Laursen JC, Cairns BE, Kumar U, Somvanshi RK, Dong XD, Arendt-Nielsen L and Gazerani P: Nitric oxide release from trigeminal satellite glial cells is attenuated by glial modulators and glutamate. Int J Physiol Pathophysiol Pharmacol. 5:228–238. 2013.PubMed/NCBI

28 

Gwak YS, Hulsebosch CE and Leem JW: Neuronal-Glial interactions maintain chronic neuropathic pain after spinal cord injury. Neural Plast. 2017:24806892017. View Article : Google Scholar : PubMed/NCBI

29 

Chung MK, Asgar J, Lee J, Shim MS, Dumler C and Ro JY: The role of Trpm2 in hydrogen peroxide-induced expression of inflammatory cytokine and chemokine in rat trigeminal ganglia. Neuroscience. 297:160–169. 2015. View Article : Google Scholar : PubMed/NCBI

30 

Taft RJ, Pang KC, Mercer TR, Dinger M and Mattick JS: Non-Coding RNAs: Regulators of disease. J Pathol. 220:126–139. 2010. View Article : Google Scholar : PubMed/NCBI

31 

Ponting CP, Oliver PL and Reik W: Evolution and functions of long noncoding RNAs. Cell. 136:629–641. 2009. View Article : Google Scholar : PubMed/NCBI

32 

Fitzgerald KA and Caffrey DR: Long noncoding RNAs in innate and adaptive immunity. Curr Opin Immunol. 26:140–146. 2014. View Article : Google Scholar : PubMed/NCBI

33 

Lutz BM, Bekker A and Tao YX: Noncoding RNAs: New players in chronic pain. Anesthesiology. 121:409–417. 2014. View Article : Google Scholar : PubMed/NCBI

34 

Wu P, Zuo X, Deng H, Liu X, Liu L and Ji A: Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases. Brain Res Bull. 97:69–80. 2013. View Article : Google Scholar : PubMed/NCBI

35 

Ma B, Gao Z, Lou J, Zhang H, Yuan Z, Wu Q, Li X and Zhang B: Long noncoding RNA MEG3 contributes to cisplatininduced apoptosis via inhibition of autophagy in human glioma cells. Mol Med Rep. 16:2946–2952. 2017. View Article : Google Scholar : PubMed/NCBI

36 

Kobayashi K, Yamanaka H and Noguchi K: Expression of ATP receptors in the rat dorsal root ganglion and spinal cord. Anat Sci Int. 88:10–16. 2013. View Article : Google Scholar : PubMed/NCBI

37 

Inoue K: P2 receptors and chronic pain. Purinergic Signal. 3:135–144. 2007. View Article : Google Scholar : PubMed/NCBI

38 

Skaper SD, Debetto P and Giusti P: The P2×7 purinergic receptor: From physiology to neurological disorders. FASEB J. 24:337–345. 2010. View Article : Google Scholar : PubMed/NCBI

39 

Sperlágh B, Vizi ES, Wirkner K and Illes P: P2×7 receptors in the nervous system. Prog Neurobiol. 78:327–346. 2006. View Article : Google Scholar : PubMed/NCBI

40 

Amaral PP, Clark MB, Gascoigne DK, Dinger ME and Mattick JS: lncRNAdb: A reference database for long noncoding RNAs. Nucleic Acids Res. 39(Database Issue): D146–D151. 2011. View Article : Google Scholar : PubMed/NCBI

41 

Maass PG, Luft FC and Bähring S: Long non-coding RNA in health and disease. J Mol Med (Berl). 92:337–346. 2014. View Article : Google Scholar : PubMed/NCBI

42 

Simionescu-Bankston A and Kumar A: Noncoding RNAs in the regulation of skeletal muscle biology in health and disease. J Mol Med (Berl). 94:853–866. 2016. View Article : Google Scholar : PubMed/NCBI

43 

Jarvis MF and Khakh BS: ATP-gated P2X cation-channels. Neuropharmacology. 56:208–215. 2009. View Article : Google Scholar : PubMed/NCBI

44 

Caseley EA, Muench SP, Fishwick CW and Jiang LH: Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists. Biochem Pharmacol. 116:130–139. 2016. View Article : Google Scholar : PubMed/NCBI

45 

Volonté C, Apolloni S, Skaper SD and Burnstock G: P2X7 receptors: Channels, pores and more. CNS Neurol Disord Drug Targets. 11:705–721. 2012. View Article : Google Scholar : PubMed/NCBI

46 

Wei L, Caseley E, Li D and Jiang LH: ATP-induced P2X receptor-dependent large pore formation: How much do we know? Front Pharmacol. 7:52016. View Article : Google Scholar : PubMed/NCBI

47 

Dubyak GR: Go it alone no more-P2X7 joins the society of heteromeric ATP-gated receptor channels. Mol Pharmacol. 72:1402–1405. 2007. View Article : Google Scholar : PubMed/NCBI

48 

Wu H, Yang L and Chen LL: The diversity of long noncoding RNAs and their generation. Trends Genet. 33:540–552. 2017. View Article : Google Scholar : PubMed/NCBI

49 

Yan B, Yao J, Liu JY, Li XM, Wang XQ, Li YJ, Tao ZF, Song YC, Chen Q and Jiang Q: lncRNA-MIAT regulates microvascular dysfunction by functioning as a competing endogenous RNA. Circ Res. 116:1143–1156. 2015. View Article : Google Scholar : PubMed/NCBI

50 

Peiró C, Lorenzo Ó, Carraro R and Sánchez-Ferrer CF: IL-1β inhibition in cardiovascular complications associated to diabetes mellitus. Front Pharmacol. 8:3632017. View Article : Google Scholar : PubMed/NCBI

51 

Kojima S, Ohshima Y, Nakatsukasa H and Tsukimoto M: Role of ATP as a key signaling molecule mediating radiation-induced biological effects. Dose Response. 15:15593258176906382017. View Article : Google Scholar : PubMed/NCBI

52 

Lee JH, Zhang Y, Zhao Z, Ye X, Zhang X, Wang H and Ye J: Intracellular ATP in balance of pro- and anti-inflammatory cytokines in adipose tissue with and without tissue expansion. Int J Obes (Lond). 41:645–651. 2017. View Article : Google Scholar : PubMed/NCBI

53 

Blum E, Procacci P, Conte V and Hanani M: Systemic inflammation alters satellite glial cell function and structure. A possible contribution to pain. Neuroscience. 274:209–217. 2014. View Article : Google Scholar : PubMed/NCBI

54 

Mima A: Inflammation and oxidative stress in diabetic nephropathy: New insights on its inhibition as new therapeutic targets. J Diabetes Res. 2013:2485632013. View Article : Google Scholar : PubMed/NCBI

55 

Liu Y, Wang Z, Xie W, Gu Z, Xu Q and Su L: Oxidative stress regulates mitogenactivated protein kinases and c-Jun activation involved in heat stress and lipopolysaccharideinduced intestinal epithelial cell apoptosis. Mol Med Rep. 16:2579–2587. 2017. View Article : Google Scholar : PubMed/NCBI

56 

Ji RR, Berta T and Nedergaard M: Glia and pain: Is chronic pain a gliopathy? Pain. 154 Suppl 1:S10–S28. 2013. View Article : Google Scholar : PubMed/NCBI

57 

Ponnusamy M, Liu N, Gong R, Yan H and Zhuang S: ERK pathway mediates P2×7 expression and cell death in renal interstitial fibroblasts exposed to necrotic renal epithelial cells. Am J Physiol Renal Physiol. 301:F650–F659. 2011. View Article : Google Scholar : PubMed/NCBI

58 

Ji RR, Gereau RW IV, Malcangio M and Strichartz GR: MAP kinase and pain. Brain Res Rev. 60:135–148. 2009. View Article : Google Scholar : PubMed/NCBI

59 

Son Y, Kim S, Chung HT and Pae HO: Reactive oxygen species in the activation of MAP kinases. Methods Enzymol. 528:27–48. 2013. View Article : Google Scholar : PubMed/NCBI

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Volume 17 Issue 4

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APA
Liu, C., Deng, Z., Du, E., & Xu, C. (2018). Long non‑coding RNA BC168687 small interfering RNA reduces high glucose and high free fatty acid‑induced expression of P2X7 receptors in satellite glial cells. Molecular Medicine Reports, 17, 5851-5859. https://doi.org/10.3892/mmr.2018.8601
MLA
Liu, C., Deng, Z., Du, E., Xu, C."Long non‑coding RNA BC168687 small interfering RNA reduces high glucose and high free fatty acid‑induced expression of P2X7 receptors in satellite glial cells". Molecular Medicine Reports 17.4 (2018): 5851-5859.
Chicago
Liu, C., Deng, Z., Du, E., Xu, C."Long non‑coding RNA BC168687 small interfering RNA reduces high glucose and high free fatty acid‑induced expression of P2X7 receptors in satellite glial cells". Molecular Medicine Reports 17, no. 4 (2018): 5851-5859. https://doi.org/10.3892/mmr.2018.8601