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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 (1–3). 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 (5–7). 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).

Figure 1. Effect of increasing (A) D-glucose and (B) FFA concentrations on cell viability as detected by the TransDetect Cell Counting kit. *P<0.05, **P<0.01, ***P<0.001 vs. control group. ns, no significant difference; OD, optical density; FFA, free fatty acid.

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).

Figure 2. HGHF upregulates BC168687 and P2X7 receptor expression in primary cultured SGCs. (A) The relative expression levels of BC168687 and (B) P2X7 receptors in HGHF-induced SGCs was determined by reverse transcription quantitative-polymerase chain reaction. (C) P2X7 receptor expression in SGCs was visualized by immunocytochemistry. The arrows indicate the positive SGCs. Scale bar, 50 µm. **P<0.01 vs. control group. P2X7, P2X purinoceptor 7; HGHF, high glucose and high free fatty acids; SGC, satellite glial cell; lncRNA, long noncoding RNA; IOD, integrated optical density.

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.

Figure 3. BC168687 siRNA and P2X7 mRNA expression in SGCs was determined by reverse transcription quantitative-polymerase chain reaction. (A) Three different BC168687 siRNAs were transfected in order to determine the most effective silencing sequence. BC168687 was selected. (B) P2X7 mRNA expression in the HGHF group was increased compared with the control group and P2X7 mRNA expression in the BC168687si group was decreased compared with the HGHF group. *P<0.05, **P<0.01 vs. control group. ##P<0.01 vs. HGHF group. siRNA, small interfering RNA; SGC, satellite glial cell; P2X7, P2X purinoceptor 7; HGHF, high glucose and free fatty acids; NCsi, negative control siRNA; ns, no significant difference; lncRNA, long noncoding RNA; VD, empty vector control.

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.

Figure 4. BC168687 siRNA downregulates the expression of P2X7 and GFAP induced by HGHF in SGCs. (A) Representative western blot analysis and the relative expression levels of P2X7 and (B) GFAP. β-actin from the same sample was used as an internal control. The expression of P2X7 protein and GFAP in the HGHF group was significantly increased compared with the control group. The expression levels of P2X7 protein and GFAP in the BC168687si group significantly decreased compared with the HGHF group. **P<0.05, ***P<0.001 vs. Control group. ##P<0.05, ###P<0.001 vs. HGHF group. siRNA, small interfering RNA; P2X7, P2X purinoceptor 7; GFAP, glial fibrillary acidic protein; HGHF, high glucose and high free fatty acids; SGC, satellite glial cell; NCsi, negative control siRNA; IOD, integrated optical density; VD, empty vector control.

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.

Figure 5. P2X7 and GFAP co-expression was increased in SGCs in a HGHF environment. The co-expression of P2X7 receptor and GFAP in the HGHF group was increased compared with the control group, whereas the co-expression value in the BC168687si group was decreased compared with the HGHF group. The green signal indicates GFAP staining with fluorescein isothiocyante. The red signal indicates P2X7 staining with tetramethylrhodamine isothiocyanate; the blue signal indicates nucleus staining with DAPI. The arrows indicate the positive SGCs. Scale bar, 20 µm. P2X7, P2X purinoceptor 7; GFAP, glial fibrillary acidic protein; HGHF, high glucose and high free fatty acids; SGC, satellite glial cell; NCsi, negative control siRNA; VD, empty vector control.

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.

Figure 6. BC168687 siRNA reduced the level of p-ERK1/2 protein and ATP induced by HGHF in SGCs. (A) p-ERK1/2 protein expression levels in the HGHF group were increased compared with the control group and expression levels in the BC168687si group were decreased compared with the HGHF group (B) ATP levels in HGHF group were increased compared with the control group. ATP levels in the BC168687si group were decreased compared with the HGHF group. **P<0.01 vs. control group; ##P<0.01 vs. HGHF group. siRNA, small interfering RNA; p-, phosphorylated; ERK, extracellular signal-related kinase; ATP, adenosine 5′-triphosphate; HGHF, high glucose and high free fatty acids; SGC, satellite glial cell; NCsi, negative control siRNA; IOD, integrated optical density; VD, empty vector control.

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 (27–29). 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.

Figure 7. BC168687 siRNA reduced NO and ROS in SGCs in a HGHF environment. The levels of (A) NO and (B) ROS in HGHF group were increased compared with the control group. Levels of NO and ROS in the BC168687si group were decreased compared with the HGHF group. **P<0.01 vs. control group; ##P<0.01 vs. HGHF group. siRNA, small interfering RNA; NO, nitric oxide; ROS, reactive oxygen species; SGC, satellite glial cell; HGHF, high glucose and high free fatty acids; NCsi, negative control siRNA.

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 (33–35). P2X7 receptors are expressed in SGCs and studies have demonstrated that P2X7 receptors contribute to neuropathic pain (36–39). 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 (30,31,40–42). 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 (44–47). 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