Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats

NTG (Beijing Yimin Pharmaceutical Co., Ltd, China) was injected subcutaneously (s.c) in the back of rats (10 mg/kg) from a stock of 5.0 mg/ml. Control rats were subcutaneously injected with an equal volume of 0.9 % normal saline (NS) as a vehicle for NTG [3].

R, S-Sulforaphane (SFN) (LKT Laboratories, Inc., St. Paul, MN) was dissolved in sterilized distilled water according to the instructions, and a dose of 5 mg/kg was administered intraperitoneally (i.p) based on previous studies [15, 16]. The vehicle group was also injected intraperitoneally with an equal volume of sterilized water.

First, 60 rats were randomly separated into ten groups according to the different time points (0, 0.5 h, 1 h, 2 h, and 4 h) after NTG/NS injection. TNC tissue samples of the rats were taken for analyzing the expression levels of total cell Nrf2, nuclear Nrf2, HO, and NQO1 using western blot. A group of 18 rats was used to demonstrate the cell localization of Nrf2 in TNC among the groups (Control, NTG 2 h, and NTG 4 h) by immunofluorescence. Second, rats were divided into five groups as follows: 1) Control group (n = 6), rats received NS (s.c) in a volume equal to that of NTG, 2) SFN plus control group (n = 6), rats received SFN (5 mg/kg i.p) 30 min before NS (s.c), 3) NTG group (n = 6), rats received NTG (10 mg/kg s.c), 4) H₂O plus NTG group (n = 6), rats received sterilized distilled water (i.p) 30 min before NTG (10 mg/kg s.c), and 5) SFN plus NTG group (n = 6), rats received SFN (5 mg/kg i.p) 30 min before NTG (10 mg/kg, s.c). Von Frey hair testing was used to evaluate the tactile sensitivity threshold. Western blot was used to detect the c-Fos, nNOS, nuclear Nrf2, HO1, and NQO1 expressions in TNC. Finally, rats were divided into four groups as follows: 1) Control group (n = 6), rats received a subcutaneous injection of NS (s.c) in a volume equal to that of NTG, 2) NTG group (n = 6), rats received NTG (10 mg/kg s.c), 3) H₂O plus NTG group (n = 6), rats received sterilized distilled water (i.p) 30 min before NTG (10 mg/kg, s.c), and 4) SFN plus NTG group (n = 6), rats received SFN (5 mg/kg i.p) 30 min before NTG (10 mg/kg, s.c). Immunofluorescence was performed to evaluate the numbers of c-Fos and nNOS-immunoreactive neurons in TNC.

Tactile sensitivity threshold was evaluated with calibrated (0.008 g, 0.02 g, 0.04 g, 0.07 g, 0.4 g, 0.6 g, 1.0 g, 1.4 g, 2.0 g, 4.0 g, 6.0 g, 8.0 g, 10.0 g, and 15.0 g) von Frey hairs (Stoelting Co., Wood Dale, Illinois, USA) by the up-down method as described previously [17, 18]. Briefly, the rats were accommodated in the testing chambers for a period of 30 min prior to the testing. A series of von Frey hairs with logarithmically incremental stiffness was applied to the periorbital region of the face and middle of the plantar surface of the front paw for 6-8 s at intervals of 30 s between consecutive stimuli. Quick withdrawal or licking of front paw in response to the stimulus or scratching of the periorbital region in response to the stimulus was considered as a positive response. The tactile thresholds to the stimuli of von Frey hairs were analyzed at baseline, 0.5, 1, 2, 3, and 4 h after NTG or NS injection by experimenters who were blinded to each rat group.

Rats were anaesthetized with 10 % chloral hydrate (3 ml/kg, i.p) and then perfused transcardially with 0.9 % saline at 4 °C followed by 4 % paraformaldehyde in phosphate buffered saline (PBS 0.1 mol/L, pH 7.4). Regions from the medulla oblongata to the first cervical cord were immediately isolated, fixed in 4 % paraformaldehyde, cryoprotected in 30 % sucrose, frozen, and serially sectioned 1–5 mm from obex (10 μm-thick transverse sections) on a cryostat (CM1900, Leica, Heidelberg, Germany). Sections were incubated with primary antibody against Nrf2 (rabbit polyclonal antibody, dilution 1:200, Abcam, UK), neuronal nuclei NeuN (mouse monoclonal antibody, dilution 1:500, Millipore, USA), c-Fos (rabbit monoclonal antibody, dilution 1:200, Abcam, UK), and nNOS (rabbit monoclonal antibody, dilution 1:200, CST, USA). After rinsing in PBS (0.01 mol/L, pH 7.4), sections were incubated for 1 h at 25 °C with secondary antibody, Alexa Fluor 488-conjugated anti-rabbit IgG (1:200, Jackson, USA) or Dylight 549-conjugated anti-mouse IgG (1:200, Jackson, USA). Sections were mounted in fluorescent mounting medium (R&D systems, Minneapolis, MN, USA), and signals were detected using a fluorescence microscope (BX51, Olympus, Japan). Negative control sections were incubated with PBS instead of primary antibodies and they showed no positive signals. The number of c-Fos- and nNOS-immunoreactive neurons in TNC confined to a 400 × 300 μm square was determined using Image J software (version 1.4.3.67, NIH). Data from 10 regions sampled from each section (10 sections per rat) were averaged and presented as number per 1.2 × 10⁵ μm² in TNC.

After anaesthetizing as described above, the TNC was rapidly dissected, 1–5 mm from obex, homogenized in tissue lysates (Pierce, Rockford, IL, USA) with protease inhibitor cocktail (Merck, Darmstadt, Germany), and the protein concentration was determined using BCA reagent (Pierce, Rockford, IL, USA). Equal amounts of protein extracts were separated electrophoretically on 10 % sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Temecula, CA, USA). After blocking with 5 % skimmed milk for 1 h, the membranes were incubated with primary antibody against Nrf2 (rabbit polyclonal antibody, dilution 1:1000, Abcam, UK), HO1 (rabbit monoclonal antibody, dilution 1:1000, Abcam, UK), NQO1 (rabbit polyclonal antibody, dilution 1:1000, Abcam, UK), c-Fos (rabbit monoclonal antibody, dilution 1:500, Abcam, UK), nNOS (rabbit monoclonal antibody, dilution 1:1000, CST, USA), β-actin (mouse monoclonal antibody, dilution 1:5000, Sigma, USA), and fibrillarin (mouse monoclonal antibody, dilution 1:4000, Abcam, UK). For the negative control, rabbit primary antibody was replaced by normal serum. This was followed by adding horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (dilution 1:5000, Jackson, USA). An enhanced chemiluminescence kit (Millipore, Temecula, USA) was used for the visualization of the bands. Densitometric analysis was performed using Image J software.

To investigate the changes in the antioxidant system in rats treated with NTG, we analyzed Nrf2 expression in the nuclear and total cell fractions of TNC from rat models (Fig. 1). The subcutaneous administration of NTG (10 mg/kg) significantly increased Nrf2 levels in the total cell and nuclear fractions. This increase began within 0.5 h and persisted for 4 h after NTG injection. The control group with NS injection showed no statistical difference at the different time points. Moreover, immunofluorescence analysis (Fig. 2) showed that Nrf2 was located only in the neuronal cytoplasm of control group. Whereas, both nucleus and cytoplasm of neurons in the NTG group shared an obvious Nrf2 existence. We further analyzed the protein levels of two typical Nrf2-regulated phase II enzymes, HO1 and NQO1, in TNC of the rat models (Fig. 3). The expression of these two proteins also increased within 0.5 or 1 h, and persisted for 4 h after NTG exposure.

To determine the possible mechanism of Nrf2 effect, we used sulforaphane, a small-molecule inducer of this factor, to regulate the Nrf2/ARE pathway in this study. We performed immunoblotting to analyze the nuclear Nrf2, HO1, and NQO1 expressions. As shown in Fig. 4, sulforaphane treatment significantly increased nuclear Nrf2 level in NTG-treated rats (Fig. 4b). Moreover, it resulted in a significant increase in HO1 and NQO1 levels in these animals, compared to those in the control samples (Fig. 4c and d). Meanwhile, these protein expression levels were also increased in the sulforaphane plus control group compared to those in the control group.

Increased neuronal nitric oxide synthase (nNOS) and pro-oncogene c-Fos expressions in TNC are considered as the markers of TGVS activation [19, 20]. To check whether Nrf2 was involved in NTG-induced TGVS activation, we measured the expression levels of nNOS and c-Fos in NTG-treated samples at 4 h after sulforaphane pretreatment. As shown in Fig. 5, sulforaphane significantly reversed NTG-induced increase in nNOS and c-Fos expression. It also significantly reduced the number of nNOS- and c-Fos-immunoreactive neurons in TNC (Fig. 6).

We further investigated the efficacy of sulforaphane pretreatment on tactile sensitivity with calibrated von Frey hairs. We observed that subcutaneous administration of NTG significantly reduced the periorbital sensory and front paw withdrawal thresholds compared to that seen in the control group, which began 0.5 h after NTG injection (Fig. 7). Withdrawal thresholds of the front paw were significantly elevated in sulforaphane-pretreatment rats from 0.5–4 h after NTG injection (Fig. 7a). For periorbital tactile threshold analysis, an obvious increase was observed in the sulforaphane group from 1–4 h after NTG injection, as compared to the vehicle group (Fig. 7b).