Animals and cells
Animal experiments procedures were approved by the Animal Care and Use Committee of the Capital Medical University (approval no. AEEI-2014-086). Male Sprague-Dawley (SD) rats weighing 180–220 g were obtained from the Experimental Animal Center at Capital Medical University, Beijing, China (SCXK 2012–0001) and were housed with a 12/12-hour light/dark cycle in a temperature and humidity-controlled environment. All experiments were performed in accordance with relevant guidelines and regulations.
RSC96 cells (CRL-2765) were purchased from the American Type Culture Collection (ATCC, cat no. CRL-2765) and were cultured in DMEM modified to contain 4 mM L-glutamine, 25 mM glucose, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate, 10% FBS and 1% antibiotics at 37 °C in a humidified atmosphere of 5% CO2 and were passaged once every 3 days.
Animal model of DPN and IRE1α siRNA intrathecal injection
Animals were divided into control, DPN, control plus IRE1α siRNA and DPN plus IRE1α siRNA groups. DPN was induced by feeding the animals with high-carbohydrate/high-fat diet and low-dose streptozotocin (STZ, S0130, Sigma). Specifically, rats in two DPN groups were fed with high-carbohydrate/high-fat diet (67% standard diet, 10% lard, 20% sucrose, 2.5% cholesterol and 0.5% cholate) for 4 weeks followed by treating with streptozotocin (35 mg/kg in 0.1 M citric acid buffer, pH 4.5, i.p.) The rats with fasting blood glucose level above 16.7 mM 1-week after STZ injection were considered diabetic and were continued feeding on the high-carbohydrate/high-fat diet for 12 weeks. Fasting blood glucose levels were measured every 2 weeks to monitor the persistence of diabetes and thermal perception thresholds (TPT) were measured every 4 weeks to track the occurrence of peripheral neuropathy.
At 12 weeks after induction of diabetes, intrathecal injection of IRE1 siRNA was carried out. Rats were anesthetized using chloral hydrate (i.p., 300 mg/kg). L4-L5 vertebrae were exposed and a sterile polyethylene tubing (PE-10 catheter) was implanted into the subarachnoid space according to the previously published25. Rats were allowed 24 hours to recover from surgery prior to treatment.
IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v). The final concentration of RNA as an RNA/lipid complex was 0.4 μg/μL. The IRE1α siRNA complex or transfection regent (10 μL) alone was injected in Control IRE1α siRNA transfected (Control + IRE1α siRNA), DPN IRE1α siRNA transfected (DPN + IRE1α siRNA) and Control, DPN groups using a 25 μL microsyringe, respectively. 10 μL saline solution was used to wash the catheter. Injections were given daily for 3 consecutive days24,26. Nerve conduction velocity test and blood, tissue harvest were carried out 24 hour after the last injection.
Cells culture and transfection
RSC96 cells plated at a density of 2 × 105cells/well in 6-well plate were allowed to adhere overnight, and then transfected with IRE1α siRNA according to the manufacturer’s instructions. Briefly, for each transfection, 100 pmols of siRNA and 6 µl transfection reagent were added to 100 µl siRNA transfection medium (sc-36868, Santa Cruz), gently mixed, incubated for 25 minutes at room temperature, then 1.0 ml siRNA transfection medium containing 200 µl siRNA transfection reagent mixture was added to the well. Cells were incubated 8 hours at 37 °C in a CO2 incubator, 1 ml normal growth medium containing 2 times the normal serum and antibiotics concentration (2× normal growth medium) was added without removing the transfection mixture. Cells were incubated for an additional 16 hours and the medium was aspirated and cultured in 25 mM or 150 mM glucose growth medium for 24 hours and 48 hours, respectively.
Cells were treated with 25 mM glucose, 150 mM glucose, 25 mM glucose IRE1α siRNA transfected (25 mM glucose + IRE1α siRNA) and 150 mM glucose IRE1α siRNA transfected (150 mM glucose + IRE1α siRNA). Cells in 25 mM glucose, 150 mM glucose groups were treated with siRNA transfection reagent alone (Supplementary Fig. 1).
Measurement of nerve conduction velocity
Nerve conduction velocity was assessed using Functional Experiment System (BL-420s, Techman, China) as reported previously27. Before the measurement, rats were anesthetized with 10% chloral hydrate (i.p., 300 mg/kg). For measurement of MNCV, stimulation electrode was placed at the notch of sciatic nerve. Sciatic nerve was stimulated with single square wave pluses (1.2 V in intensity, 1 ms in width). For SNCV, recording site was located in the sciatic notch. Stimulation parameters were the same as MNCV methods.
Morphometric analysis of sciatic nerve
After the conduction velocity test, the left sciatic nerve was isolated and cut into two segments. One segment (2 mm) was fixed in 2.5% glutaraldehyde at 4 °C, and sent to the Electron Microscopy Center of Institute of Capital Medical University for ultrastructure observation.
The other segment (1 cm) was fixed in 10% buffered formalin and processed for paraffin section. Sciatic nerve tissues were cut in slice of 5 μm thick. Sections were stained with Luxol fast blue, and microphotographs were captured using a light microscope (Nikon Eclipse 80i, Japan).
Terminal dUTP nick-end labeling (TUNEL) assay
Apoptotic tissues were labeled using in situ cell death detection kit from Roche Company, according to manufacturer’s protocol. The nerve sections were dewaxed and followed by incubation with proteinase K for 15 min at room temperature. Following digestion, the end-labeling reaction was performed by adding TdT and dUT reaction mix onto the slides at 37 °C for 1 h at humidified chamber. After three PBS washes, converter-POD was added onto sections and incubated at 37 °C for 30 min at humidified chamber followed by reaction with DAB at room temperature for 10 min. Staining was observed using a light microscope.
The nerve sections were dewaxed, antigen repaired and blocked with 3% BSA. One hour later, sections were incubated in the following primary antibodies overnight at 4 °C: anti-IRE1α (sc-390960; 1: 50; Santa Cruz), anti-p-JNK (sc-6254; 1: 50; Santa Cruz) and rabbit anti-Caspase-12 (sc-5627; 1: 50; Santa Cruz). After rinsing, the sections were incubated with the fluorescein (FITC)-conjugated goat anti-mouse IgG (H+L) or FITC-conjugated goat anti-rabbit IgG (H+L) for 1 h at room temperature. Microphotographs were captured using the Nikon Eclipse 80i light microscope7.
Sciatic nerves and RSC96 cells were lysed on ice in the RIPA buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail for 30 min to extract total proteins. The proteins were analyzed with a bicinchoninic acid (BCA) protein assay kit (Biosynthesis, China). 30 μg/lane (sciatic nerve) and 20 μg/lane (RSC96 cell) were used for Western blot analysis as previously described8,27. The primary antibodies were as follows: mouse anti-IRE1α (sc-390960; 1: 1000; Santa Cruz), rabbit anti-P-IRE1α (ab48187; 1: 2000; abcam), mouse anti-XBP1 (sc-8015; 1: 1000; Abcam), mouse anti-GRP78 (sc-376768; 1: 1000; Santa Cruz), mouse anti-GADD153 (sc-7351; 1: 500; Santa Cruz), rabbit anti-Caspase-3 (ab13847; 1: 1000; abcam), rabbit anti-Caspase-12 (om273459; 1: 1000; Omnimabs), mouse anti-Bcl-2 (sc-7382; 1: 1000; Santa Cruz), mouse anti-Bax (sc-7480; 1: 1000; Santa Cruz), mouse anti-p-JNK (sc-6254; 1: 1000; Santa Cruz), and rabbit anti-PGP9.5 (ab108986; 1: 2000; Abcam). Mouse anti-β-actin (TA-09; 1: 20000; Zhongshan Goldenbridge) served as the internal control. The secondary antibodies were goat anti-mouse IgG-HRP (ZB-2305; Zhongshan Goldenbridge) and goat anti-rabbit IgG-HRP (ZB-2301; Zhongshan Goldenbridge). Western Chemiluminescent HRP Substrate and exposed to X-film to form image. The protein bands were quantitated with Image J software27.
An Annexin-FITC Apoptosis Detection Kit (AP101, Multisciences, China) was used to examine apoptosis according to the manufacturer’s instructions8. In brief, cells were added with 500 μL of binding buffer followed by staining with 5 μL of FITC-labeled annexin V and 10 μL of propidium iodide (PI) and incubated at room temperature for 5 min in the dark. BD LSRFortessa™ flow cytometer (BD Biosciences, San Jose, CA, USA) were used to analysis.
Intracellular Ca2+ analysis
Fluo-3 AM (S1056, Beyotime, China) was used to examine intracellular Ca2+ levels according to the manufacturer’s instructions28. Cells were harvested and washed twice with PBS, then loaded with Fluo-3 AM (5 μM) for 30 min at 37 °C in the dark, and was analyzed using BD LSRFortessa™ flow cytometer.
Data were expressed as the mean ± SEM. Statistical analysis was performed by unpaired Student’s t-test or One-way ANOVA followed by least significant difference (LSD) or Tamhane’s T2 analysis using SPSS 17.0. Values of P < 0.05 were statistically significant.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.