IL-35 alleviates inflammation progression in a rat model of diabetic neuropathic pain via inhibition of JNK signaling

All animal experiments were performed in accordance with the guidelines for Laboratory Animal Care and Use of Henan provincial people’s hospital. Specific pathogen-free (SPF) grade male Sprague-Dawley (SD) rats weighing 200–220 g which were purchased from the Experimental Animal Center of Henan provincial people’s hospital were housed individually in an air-conditioned room maintained at a temperature of 22 °C ± 2 °C and 12−/12-h light/dark cycle and were allowed to eat and drink ad libitum. All mice received adaptive feeding for 3 days prior to the formal experiment. All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of Henan provincial people’s hospital.

For induction of DNP, SD rats were fed a high-sugar and high-fat diet containing 67% commercial rodent diet (Certified Rodent Diet 5002; PMI Feeds, Inc., Richmond, IN, USA), 10% lard, 20% sucrose, 2% cholesterol, and 1% sodium cholate with free access to water. Fasting plasma glucose levels and fasting insulin levels were measured by routine methods, and insulin sensitivity index (ISI) was calculated at 0 and 8 weeks of feeding. After 8 weeks of feeding, the fasting insulin concentration were significantly elevated, while the ISI decreased, which suggested that insulin resistance appeared, the rats were then intraperitoneally administrated with 35 mg/kg STZ (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.9% sterile saline to induce diabetes. Three days later, type I diabetes mellitus was confirmed in STZ-injected rats by measuring fasting plasma glucose concentrations in blood samples obtained from the tail vein. Only rats with blood glucose concentrations greater than 16.7 mmol/L were further used in this study. Two weeks after STZ injection, the eligible rats were again measured for mechanical withdrawal thresholds (MWT), and the rats with pain threshold ≤85% base value were selected for the rat model of type I diabetes neuropathic pain.

Plasma glucose levels were measured using Glucotrend (Roche Diagnostics, Mannheim, Germany), according to the supplier’s instructions.

After standing at room temperature for 5 min, the tail vein blood samples were centrifuged at 4000 rpm for 20 min at 4 °C. The supernatants were collected, which were used to measure insulin concentration by sandwich ELISA kit (Sigma-Aldrich). The ISI was then calculated using the following formula: ISI = 1/(fasting glucose × fasting insulin).

The behavioral testing was conducted between 8:00 AM and 17:00 PM. To quantify mechanical sensitivity of the hind paw, rats were placed in individual plastic boxes (22 cm × 22 cm × 22 cm) on a mesh floor (1 cm × 1 cm) and allowed to acclimate for 15 min. IITC 2390 series electronic von Frey tactile pain measurement instrument (Stoelting, Wood Dale, IL, USA) was perpendicularly applied to the plantar surface of the hind paw with sufficient force. The intensity of the stimulus was measured when the rats lifted or licked their feet. Each trial was repeated 5 times at approximately 10s intervals, and the mean value was used as the force to produce withdrawal responses.

The rats were placed on a glass box with 3 mm thick, thermal radiation was applied to the hind toe using tail flick apparatus (IITC Inc., Woodland Hills, CA, USA), and the time from irradiation to paw withdrawal was recorded. The feet of each rat were tested 5 times, with 5 min time intervals. The values of the last three times were averaged as TWL.

The serum levels of inflammatory cytokines including IL-35, IL-1β, IL-6, TNF-α, and IL-10 were determined using the quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Each blood sample was tested in duplicate.

Prior to western blotting, frozen tissue specimens were incubated in lysis buffer for 30 min before being centrifuged at 12,000 rpm at 4 °C for 20 min. The supernatant was then collected, and the protein concentration was determined using the bicinchoninic acid method. Equal amounts of total protein (35 μg) were resolved by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes and blocked in 5% skim milk in tris buffered saline tween (TBST) at 4 °C overnight. Blots were probed with EBI3 (1:500 dilution, Imgenex, San Diego, CA, USA), JNK and phospho-JNK antibodies (1:1000 dilution; Cell Signaling Technology, Boston, MA, USA), then incubated with horseradish-peroxidase-coupled antibodies (1:2000; Peprotech, Rocky Hill, NJ, USA) at 37 °C for 2 h and visualized on a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA) using an ECL Plus Western Blotting Substrate (Thermo Scientific, Shanghai, China). In addition, β-actin (Sigma-Aldrich) was served as an internal control.

Total RNA was extracted from spinal cord tissues using Trizol reagent (Invitrogen) following the manufacturer’s protocol and reversely transcribed to cDNA using Primer-Script TM one step RT-PCR kit (Takara, Shiga, Japan) for the reverse transcription of mRNAs. The SYBR Green I real time PCR kit (CoWin Bioscience Co., Beijing, China) was used to amplify targets on an ABI 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) and calculated by the 2^−ΔΔCt method. The relative expressions of IL-1β, IL-6, TNF-α, and IL-10 were normalized to GAPDH expression.

Spinal cord segments were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin for transverse paraffin sections. The paraffin sections (4 μm thick) were incubated in 1% Cresyl violet for Nissl staining and observed under a light microscope (Olympus, Tokyo, Japan). The Nissl positive cells were automatically counted at 5 randomly selected fields.

Apoptotic cells in the spinal cord tissues were detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Paraffin-embedded sections were deparaffinized with xylene, rehydrated with graded ethanol and treated with 2% hydrogen peroxide in phosphate-buffered saline (PBS) at room temperature for 5 min to block the endogenous peroxidase activity. After washed with PBS, sections were incubated with terminal deoxynucleotidyl transferase enzyme and TUNEL reaction mixture for 1 h at in the dark. Slides were counterstained with 0.05% diaminobenzine (DAB) and counted under a microscope (Olympus).

Data were expressed as mean ± standard deviation (SD) and were analyzed using SPSS 22.0 (IBM, Armonk, NY, USA). Two or more data sets were compared using Student’s t-test or one-way analysis of variance, with P < 0.05 being considered statistically significant.

As shown in Table 1, compared to the control group, the concentration of serum glucose in DNP group was significantly elevated after 8 weeks of feeding with high-sugar and high-fat diet (P < 0.05), but was lower than the diagnostic criteria for diabetes (> 16.7 mmol/L). In addition, DNP rats displayed higher insulin levels and lower ISI compared to the control group (P < 0.05).

Compared with the DNP group, intrathecal injection of SP600125 and IL-35 recombinant protein markedly decreased the fasting insulin levels, and treatment with JNK activator anisomycin could partially revert insulin levels (Fig. 1a; P < 0.05). No significant differences were observed in the basic value of MWT and TWL among all groups. However, compared with the control group, the MWT and TWL in the other groups were significantly decreased after 14 days of intraperitoneal injection of STZ. DNP rats also exhibited lower MWT and TWL as relative to the control group (P < 0.05). Conversely, the values of the MWT and TWL were increased at 1, 3, 7 and 14 days after administrating SP600125 and recombinant IL-35 into subarachnoid space in DNP group. At the end of the 2rd week, MWT and TWL were significantly lower in IL-35 + Ani groups than in IL-35 group (Fig. 1b; P < 0.05). Serum IL-35 levels were downregulated in DNP and SP group, but were highly expressed in IL-35 and IL-35 + Ani group as relative to the control group (Fig. 1c; P < 0.05). Moreover, we found that the levels of three pro-inflammatory factors IL-1β (Fig. 1d; P < 0.05), IL-6 (Fig. 1e; P < 0.05) and TNF-α (Fig. 1f; P < 0.05) in rat serum were also significantly increased, while the production of anti-inflammatory cytokine IL-10 (Fig. 1g; P < 0.05) was reduced in DNP group. Intrathecal injection of SP600125 or IL-35 recombinant protein markedly reduced the levels of IL-1β (Fig. 1d; P < 0.05), IL-6 (Fig. 1e; P < 0.05) and TNF-α (Fig. 1f; P < 0.05) and enhanced the levels of IL-10 (Fig. 1g; P < 0.05). In contrast, anisomycin administration partly overturned the anti-inflammatory effect of IL-35. Collectively, these findings elucidated that IL-35 treatment inhibited inflammatory response in a rat model of DNP.

Reports show that the protein levels of p-JNK increase in the spinal cord in a rat model of DNP. Therefore, we investigated whether IL-35 modulated inflammation in DNP progression via mediating JNK signaling. As expected, the protein levels of IL-35 in spinal cord were notably decreased in DNP and SP groups, but were increased in IL-35 and IL-35 + Ani groups compared to the control group (Fig. 2a; P < 0.05). We found that the protein levels of p-JNK were significantly increased in the spinal cord of DNP rats compared with the control group of rats. The 2 week treatment of IL-35 or SP600125 significantly inhibited the increase in the protein levels of p-JNK. Anisomycin further increased JNK phosphorylation (Fig. 2b; P < 0.05). Furthermore, qRT-PCR analysis showed that IL-35 or SP600125 injection could effectively exerted an inhibitory effect on IL-1β (Fig. 2c; P < 0.05), IL-6 (Fig. 2d; P < 0.05) and TNF-α (Fig. 2e; P < 0.05) secretion and IL-10 downregulation (Fig. 2f; P < 0.05) in the spinal cord induced by DNP. Activation of JNK by anisomycin further triggered the inflammatory response by upregulating IL-1β (Fig. 2c; P < 0.05), IL-6 (Fig. 2d; P < 0.05) and TNF-α (Fig. 2e; P < 0.05) and decreasing the mRNA levels of IL-10 (Fig. 2f; P < 0.05). Neuron survival in the spinal cord was further evaluated by Nissl staining. DNP group exhibited necrotic neurons and marked loss of Nissl granules in spinal cord tissues. Conversely, the injection of SP600125 or IL-35 recombinant protein markedly increased Nissl bodies, while anisomycin led to a decrease in the number of neurons and Nissl bodies (Fig. 3a). These results indicate that IL-35 administration might improve the histological changes of spinal cord and restore neuron function after DNP induction. Furthermore, the apoptotic cells in spinal cord tissues were significantly increased in the DNP group, while IL-35 or SP600125 injection reduced the number of TUNEL-positive cells, which were partly reversed by anisomycin (Fig. 3b; P < 0.05). Taken together, we concluded that IL-35 attenuated DNP through inhibiting neuroinflammation and neuroapoptosis by inhibition of JNK pathway.