Animal model and groups
Zucker diabetic fatty (ZDF) rats (a type 2 diabetic animal model) were purchased from Vital River Laboratories (Beijing, China) at 8 weeks of age and were fed with Purina 5008 diet [35]. Lean ZDF rats without leptin receptor mutations [36], were used as the controls. All the rats were housed in a pathogen-free environment with continuous access to food and water on a 12-h light–dark schedule at 21–25 °C. The animals were cared for following the Medical College of Nanchang University Committee on the Care and Use of Animals guidelines.
Fasting plasma glucose (FPG) and postprandial blood glucose (PBG) of the rats were measured. Blood was collected from the tail vein. An oral glucose tolerance test was performed by administering an oral glucose load (2 g/kg glucose by gavage) and by measuring the blood glucose 2 h after dosing. Diabetes was defined as an FPG ≥7.8 mM or PBG ≥11.1 mM after 6 weeks. Then, the rats were randomly divided into four groups: control group, diabetic model group (DM), DM treated with NONRATT021972 siRNA group (DM + NONRATT021972 si), and DM treated with the scrambled siRNA group (DM + NC si). Each group contained 6 rats. The NONRATT021972 siRNA target sequence was TGTGAATCATGGAAATATC. The NONRATT021972 siRNA and negative control (NC si) were purchased from Invitrogen (Carlsbad, CA, USA). The EntransterTM-in vivo transfection reagents were provided by the Engreen Company of Beijing. 320 μl transfection complex consisting of siRNA (NONRATT021972 siRNA or NCsi) was injected into the rats of DM + NONRATT021972 si and DM + NC si groups through sublingual vein according to the reagent’s instructions. Control and DM rats were injected with 320 μl saline. The body weight, blood glucose and behavior of the rats were measured at 1, 4, 6 and 7 weeks after the start of the experiment. After 1 week, the rats were anesthetized with intraperitoneal administration of 50 mg/kg sodium pentobarbital, and the DRG were collected.
Behavior study
Measurement of the mechanical withdrawal threshold (MWT)
Noxious-pressure stimulation was used to evaluate mechanical hyperalgesia. Unrestrained rats were placed inside a clear plastic chamber (22 cm × 12 cm × 22 cm) on a stainless steel mesh floor and allowed to acclimate. Withdrawal responses to mechanical stimulation were determined using von Frey filaments (BME-403, Tianjin) applied through an opening in the stainless-steel mesh floor of the cage (1 cm × 1 cm grid) to an area adjacent to the paw. The filaments were applied in the order of increasing bending force (0.13, 0.20, 0.33, 0.60, 1.30, 3.60, 5.00, 7.30, 9.90, and 20.1 g), with each filament applied 10 times at intervals of 15 s to different parts of the midplantar glabrous skin. The strength of the filaments in the series that evoked at least five positive responses among the ten trials was designated the pain threshold. Values beyond 20.1 g were recorded as 20.1 g.
Measurement of thermal withdrawal latency (TWL)
Noxious heat stimulation was applied by the Thermal Paw Stimulation System (BME-410C, Tianjin), and hyperalgesia was determined using thermal stimulation by Hargreaves’ test. The rats were placed in a transparent, square, bottomless acrylic box (22 cm × 12 cm × 22 cm) on a glass plate. After a 30-min habituation period, the plantar surface of their paws was exposed to a beam of radiant heat. Thermal withdrawal latency (TWL) was taken as an index of the nociceptive threshold. The light beam was turned off automatically when a rat lifted its paw, allowing the measurement of time between the beginning of the light beam and the elevation of the foot. This time was designated as the paw withdrawal latency. The hind paws were tested alternately at 5 min intervals. The cut-off time for heat stimulation was 30 s.
Measurements of sensory nerve conduction velocity
Rats were anesthetized with 10 % chloral hydrate (300 mg/kg) by intraperitoneal injection. Sensory nerve conduction velocity (SNCV) was evaluated on the tail using platinum electrodes adjacent to the nerve to obtain a recording at the end of 1, 4, 6, and 7 weeks after the experiment. An annular stimulus electrode was located in the distal tail nerve. The negative electrode (black) of the stimulus cathode was located proximal to the tail nerve, whereas the positive electrode (red) was located 1 cm distal from the negative electrode. The recording electrode was 10 cm proximal from the stimulus site. The reference electrode was placed between the stimulus electrode and the recording electrode. The stimulus intensity was fixed to 1.2 mA. SNCV was calculated by the following equation: sensory nerve conduction velocity (m/s) = the distance between recording electrode and stimulus electrode (10 cm) × 10/latency (ms).
In situ hybridization (ISH)
The rats were anesthetized. The DRG were dissected immediately and fixed in 4 % paraformaldehyde (PFA) for 2 h at room temperature. Then, they were transferred to 15 % sucrose dissolved in 4 % PFA overnight. Tissues were sectioned at 15 mm. Diethyl pyrocarbonate (DEPC) water was used for all of the solutions and appliances of ISH. After deparaffinization and hydration, the slides were digested with 20 μg/ml proteinase K (Promega, Madison) and incubated at 37 °C for 5 min. The slides were stored for 4 h at 42 °C in the pre-hybridization solution in a humidified chamber and then, covered with sense or antisense probes (2.6 ng/μl) that were also denatured (46 °C for 30 min) and incubated at 46 °C for 18 h. After this period, the slides were washed three times with stringency wash solution 1 (50 % formamide, 2X SSC) and 2 (2X SSC) at 46 °C for 5 min. The slides were washed twice in MABT (maleic acid buffer containing Tween 20) for 30 min at 25 °C, transferred to a humidified chamber and covered with 200 μl of blocking buffer added to each section (MABT + 2 % BSA, 1 % blocking reagent (Roche)) for 1 h at 25 °C. Immunodetection was performed using anti-DIG alkaline phosphatase conjugated to antibody (Roche, Basel) diluted in blocking solution (1:1000). After incubation for 1 h in a humidified chamber, the slides were washed five times with MABT for 10 min at 25 °C each wash. The slides were washed twice for 10 min with pre-staining buffer (100 mM Tris pH 9.5, 100 mM NaCl) and incubated in the dark at 37 °C with NBT/BCIP developing solution (Roche, Basel). The reaction was stopped by incubating with PBS (16 h at 4 °C). Finally, the sections were counterstained with Bismarck Brown Y 0.5 % and dehydrated. The slides were mounted with a cover glass with erv-mount resin (Easy Path, São Paulo). Hybridization with sense riboprobes were used as a negative control. The presence of lncRNA in different tissues of the control and DM group was semi-quantified according to the ISH evaluation method proposed by Henke [37].
Quantitative real-time PCR
Total RNA was isolated from the DRG using the TRIzol Total RNA Reagent (Beijing Tiangen Biotech CO.). The reverse transcription reaction was completed using a RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Glen Bernie, MD, USA) following the manufacturer’s instructions. The primers were designed with the Primer Express 3.0 software (Applied Biosystems), and the sequences were as follows: NONRATT021972, sense 5'-TAGGATGAGTACCAGTCAGGT-3′; anti-sense 5′-TTTTTGGTTTTTTGACAGGG-3′, and β-actin, sense 5′-GCTCTTTTCCAGCCTTCCTT-3′; anti-sense: 5′-CTTCTGCATCCTGTCAGCAA-3′. P2X7, forward 5′-CTTCGGCGTGCGTTTTG-3′, reverse 5′-AGGACAGGGTGGATCCAATG-3′. Quantitative PCR was performed using SYBR® Green MasterMix in an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA, USA). The thermal cycling parameters were 95 °C for 30 s, followed by 40 cycles of amplification at 95 °C for 5 s and 60 °C for 30 s [38]. The amplification specificity was determined by the melting curve, and the results were processed by the software within the ABI7500 PCR instrument.
Immunohistochemistry
Immunohistochemistry was performed as follows. The rats’ DRG were removed and placed in 4 % paraformaldehyde for 2 h at room temperature. The DRG were washed in 0.1 M PBS prior to incubation in 20 % sucrose in PBS overnight. Then, 12-μm-thick sections were cut using a cryostat (Leica) and mounted onto the slides. The sections were washed in PBS and incubated in blocking solution containing 3 % bovine serum albumin (BSA) in PBS with 0.3 % Triton X-100 for 30 min at room temperature. Primary antibodies against glial fibrillary acidic protein (GFAP) (chicken anti-GFAP, Abcam) and P2X7 (rabbit anti-P2X7, Abcam) were diluted 1:100 in PBS containing 1 % BSA and incubated overnight at 4 °C. The sections were washed in PBS and incubated with the secondary antibody [goat anti-rabbit TRITC (tetraethyl rhodamine isothiocyanate) (Jackson ImmunoResearch Inc., West Grove, PA, USA) and goat anti-chicken FITC (Beijing Zhongshan Biotech CO.)] diluted 1:200 in PBS for 45 min at 37 °C. Finally, the sections were washed in PBS. Controls omitted the primary antibody. The sections were imaged using a fluorescence microscope (Olympus, Tokyo, Japan). Data were collected from six animals in each group. Five fields randomly selected that contained approximately 50 neurons each were analyzed from each animal and averaged.
Western blotting
DRG tissues from rats were sampled for western blot analysis. Total protein was extracted by homogenizing the DRG sample by mechanical disruption in lysis buffer (50 mmol/L Tris–Cl, pH 8.0, 150 mmol/L NaCl, 0.1 % sodium dodecyl sulfate (SDS), 1 % Nonidet P-40, 0.02 % sodium deoxycholate, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/ml aprofinin, and 1 % protease inhibitor) and incubating on ice for 50 min. The lysates were centrifuged at 12,000 × g for 10 min at 4 °C. The supernatants were collected to measure the protein concentrations using a bicinchoninic acid assay reagent kit and then stored at −20 °C for later use. The supernatants were diluted with sample buffer (250 mmol/L Tris–Cl, 200 mmol/L dithiothreitol, 10 % SDS, 0.5 % bromophenol blue, and 50 % glycerol) and denatured by heating at 95 °C for 10 min. Supernatant samples containing 20 μg protein were loaded onto 10 % SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The polyvinylidene fluoride membranes were blocked for 1 h at room temperature in 5 % nonfat dried milk in buffer containing 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.05 % Tween-20. The membranes were incubated with the primary antibodies anti-rabbit P2X7 (1:1000, Abcam), anti-chicken GFAP (1:1000, Abcam), or anti-mouse TNF-α (1:1000, Affinity) overnight at 4 °C, followed by incubation with an HRP-conjugated secondary antibody (1:5000, Beijing Zhongshan Biotech CO.) at room temperature. The membranes were stripped and incubated with mouse anti-β-actin (Sigma-Aldrich) to verify equal loading of the proteins in the western blot analysis. The densities of the bands were determined using Image J Software.
Rapid prediction of the interaction between RNA and protein
Bioinformatics technology can be applied to rapidly predict the interaction between RNA and proteins (fast predictions of RNA and protein interactions and domains, CatRAPID) [20]. We used the CatRAPID online algorithm forces to evaluate the interaction of the NONRATT021972 RNA and the protein (P2X7 receptor) based on the secondary structure, hydrogen bonding and molecular inter-atomic forces. This method can be used to locate RNA targets.
Isolation of DRG non-neurons
The rats were anesthetized with urethane (1.2 g/kg, intraperitoneally, i.p.). The DRG were isolated from the rats and transferred immediately into Dulbecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4 and 340 mosmol/kg. After removal of the attached nerves and surrounding connective tissues, the DRGs were minced with dissecting spring scissors and incubated with trypsin (0.5 mg/ml; type III, Sigma), collagenase (1.0 mg/ml; type IA, sigma) and DNase (0.1 mg/ml; type IV, Sigma) in 5 ml of DMEM at 35 °C in a shaking bath for 35–40 min. Then, soybean trypsin inhibitor (1.25 mg/ml; type II-S, Sigma) was added to stop the enzymatic digestion. The isolated non-neuronal cells (satellite glial cells) were transferred into a 35-mm culture dish and kept stationary for 30 min [39]. The experiments were performed at room temperature (20–30 °C).
HEK 293 cell culture and transfection
HEK 293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum and 1 % penicillin and streptomycin at 37 °C in a humidified atmosphere containing 5 % CO2. The cells were transiently transfected with the human pcDNA3.0-EGFP-P2X7 plasmid and NONRATT021972 siRNA using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. When the HEK293 cells were 70–80 % confluent, the cell culture medium was replaced with OptiMEM two hours prior to transfection. The transfection media were prepared as follows: (a) 4 μg of DNA or siRNA diluted into 250 μl final volume of OptiMEM; (b) 10 μl of Lipofectamine2000 diluted into 250 μl final volume of OptiMEM; and (c) the Lipofectamine-containing solution was mixed with the plasmid-containing solution and incubated at RT for 20 min. Subsequently, 500 μl of the cDNA/Lipofectamine complex solution was added to each well. The cells were incubated for 6 h at 37 °C in 5 % CO2. After incubation, the cells were washed in MEM containing 10 % FBS and incubated for 24–48 h. The GFP fluorescence was evaluated as a reporter for the transfection efficiency. Whole-cell patch clamp recordings were performed 1–2 days after transfection [40].
Electrophysiological recordings
Electrophysiological recording was performed using a patch/whole cell clamp amplifier (Axopatch 200B) [39]. A micropipette was filled with an internal solution (in mM) containing KCl (140), MgCl2 (2), HEPES (10), EGTA (11), and ATP (5); the solution’s osmolarity was adjusted to 340 mosmol/kg with sucrose and its pH was adjusted to 7.4 with KOH. The external solution (in mM) contained NaCl (150), KCl (5), CaCl2 (2.5), MgCl2 (1), HEPES (10), and D-glucose (10); the solution’s osmolarity was adjusted to 340 mosmol/kg with sucrose, and its pH was adjusted to 7.4 with NaOH. The resistance of the recording electrodes was in the range of 1 to 4 MΩ, with 3 MΩ being optimal. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GΩ), and then more negative pressure was applied to rupture it to establish a whole-cell mode. The holding potential (HP) was set to −60 mV. A-438079 (a selective antagonist of P2X7 receptor, Tocris Bioscience, Ellis-ville, MO), BzATP, and ATP (Sigma Company) were dissolved in the external solution and delivered by gravity flow from an array of tubules (500 μm O.D., 200 μm I.D.) connected to a series of independent reservoirs. The distance from the tubule mouth to the examined cell was approximately 100 μm. Rapid solution exchange was achieved by shifting the tubules horizontally with a micromanipulator.
Statistical analysis
Significant differences were evaluated using one-way ANOVA followed by Dunnett’s or Tukey’s tests. The statistical analyses were performed using GraphPad Prism version 5.0. The results were expressed as the mean ± SD. to show variation between groups. Differences were considered significant when p˂0 .05.
