Animal experiments
Male Sprague-Dawley rats (n = 108, 200 ~ 220 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. The rats were subjected to a 12 h/12 h light-dark cycle (light: 8 am to 8 pm; dark: 8 pm to 8 am) at room temperature (23 ± 1 °C) and fed with standard food and water for at least one week. All the animal experiments were approved by the Animal Protection and Use Committee of Hunan Provincial People’s Hospital and strictly followed the “Guidelines for the Use and Management of Laboratory Animals” issued by the National Institutes of Health (NIH).
The rats were subjected to sciatic nerve ligation operation after being anesthetized using pentobarbital sodium (40 mg/kg). An incision was made at the biceps femoris in the left thigh to expose the sciatic nerve. Four sterile 4 − 0 chromic gut sutures (ethicon, Somerville, NJ), 1 mm apart, were loosely tied to the proximal end of the trifurcation of the sciatic nerve so long as each suture could detect small twitches of the hind limb. After ligation, the muscle and skin were immediately stitched. Rats that received sciatic nerve ligation were established as chronic constriction injury (CCI) models (Model group). Rats in sham group were sham-operated (no nerve ligation was performed). Rats in Control group were left totally untreated (no surgery or drug treatment).
After model establishment, rats were subjected to corresponding treatments and accordingly grouped into DEX group (intraperitoneally injected with 5 µg/kg DEX for post-operational 7 days, Nhwa Pharmaceutical, Jiangxi, China), MCC950 group (intraperitoneally injected with 50 µg/kg NLRP3 antagonist MCC950 for post-operational 7 days, MedChemExpress, Shanghai, China), Nigericin group (intraperitoneally injected with 1 mg/kg NLRP3 activator Nigericin for post-operational 7 days, MedChemExpress, Shanghai, China), DEX + ML385 group (intraperitoneally injected with 5 µg/kg DEX and 30 mg/kg Nrf2 inhibitor ML385 for post-operational 7 days, Selleck, Shanghai, China), DEX + Nigericin group (intraperitoneally injected with 5 µg/kg DEX and 1 mg/kg Nigericin for post-operational 7 days) and MCC950 + ML385 group (intraperitoneally injected with 50 µg/kg MCC950 and 30 mg/kg ML385 for post-operational 7 days). The animal groups and treatment are listed in Table 2. Rats subjected to behavioral tests received drug treatment for post-operational 14 days. Experiment timetable is shown in Fig. 1.
Table 2
Corresponding treatment strategy for rats in each group
Groups
|
Treatment
|
Behavioral tests
|
H&E staining
|
TUNEL
|
ELISA
|
Gene expression
|
Control group (n = 6)
|
Untreated (no surgery or drug treatment)
|
√
|
√
|
√
|
√
|
√
|
Model group (n = 6)
|
CCI models
|
√
|
√
|
√
|
√
|
√
|
Sham group (n = 6)
|
Sham-operated, no nerve ligation was performed
|
√
|
√
|
√
|
√
|
√
|
DEX group (n = 6)
|
Intraperitoneally injected with 5 µg/kg DEX for post-operational 7 days
|
√
|
√
|
√
|
√
|
√
|
MCC950 group (n = 6)
|
Intraperitoneally injected with 50 µg/kg MCC950 for post-operational 7 days
|
√
|
√
|
√
|
√
|
√
|
Nigericin group (n = 6)
|
Intraperitoneally injected with 1 mg/kg Nigericin for post-operational 7 days
|
√
|
√
|
√
|
√
|
√
|
DEX + ML385 group (n = 6)
|
Intraperitoneally injected with 5 µg/kg DEX and 30 mg/kg ML385 for post-operational 7 days
|
√
|
√
|
√
|
√
|
√
|
DEX + Nigericin group (n = 6)
|
Intraperitoneally injected with 5 µg/kg DEX and 1 mg/kg Nigericin for post-operational 7 days
|
√
|
√
|
√
|
√
|
√
|
MCC950 + ML385 group (n = 6)
|
Intraperitoneally injected with 50 µg/kg MCC950 and 30 mg/kg ML385 for post-operational 7 days
|
√
|
√
|
√
|
√
|
√
|
Note: CCI, chronic constriction injury; DEX, dexmedetomidine |
Animal behaviors
Rats were subjected to behavioral tests 0, 1, 3, 7 and 14 after operation. These rats were accordingly assigned into Control, Sham, Model, DEX, MCC950, Nigericin, DEX + ML385, MCC950 + ML385 and DEX + Nigericin groups (n = 6 in each group). The behavioral tests were conducted from 9 am to 12 am in a noiseless environment. The rats were placed in a wire cage for 30 minutes to adjust to the environment before experiment. Mechanical withdrawal threshold (MWT) of the rats was measured every 5 min for three times (each time for 2 s) using automated dynamic plantar aesthesiometer (Ugo Basile, Varese, Italy). Von Frey filaments were vertically placed on the inner plantar surface of rat's right hind paw and increasing pressure was put on to bend the filaments (from 0.6, 1, 2, 4, 6, 8, 10, 15, 25 to 60 g; break force = 60 g). The minimum retraction force (G) of rat's right hind paw was recorded and the average value of the power that induced a reliable retreat was recorded as threshold. Quick withdrawal or licking of paws in response to stimuli was considered a positive reaction.
H&E staining
Spinal cords (L4 ~ L6) were extracted from rats of the Control, Sham, Model, DEX, MCC950, Nigericin, DEX + ML385, MCC950 + ML385 and DEX + Nigericin groups (n = 6 in each group) 7 days after operation. The spinal cords were fixed in 4% paraformaldehyde and then dehydrated in 30% sucrose at 4 °C overnight. The spinal cords were embedded in paraffin and cut into 5 µm sections (10 slices of each spinal cord) for hematoxylin and eosin staining. Finally, the spinal cord sections were observed using a microscope (Olympus ABX50, Tokyo, Japan). The scoring criteria of spinal cord injury are as follows: 0 = no lesion; 1 = gray matter contained 1 ~ 5 eosinophilic neurons; 2 = gray matter contained 5 ~ 10 eosinophilic neurons; 3 = gray matter contained more than 10 eosinophilic neurons; 4 = infarction of less than 1/3 of the gray matter area; 5 = infarction of 1/3 to 1/2 of the gray matter area; 6 = infarction of more than 1/2 of the gray matter area.
TUNEL staining
Spinal cords (L4 ~ L6) were extracted from rats of the Control, Sham, Model, DEX, MCC950, Nigericin, DEX + ML385, MCC950 + ML385 and DEX + Nigericin groups (n = 6 in each group) 7 days after operation. Ten slices were obtained from each spinal cord, and sections with a thickness of about 5 µm were dewaxed with xylene and dehydrated using gradient alcohol. After that, the spinal sections were treated with proteinase K for 30 min and immersed in blocking solution at room temperature for 10 min. TUNEL detection solution (Beyotime Biotechnology, Shanghai, China) was then added for 60 min of incubation at 37 °C in the dark. Finally, the spinal sections were washed with PBS for three times and observed using a microscope. Apoptosis of 15 randomly selected areas of each spinal cord section was assessed. Apoptosis rate (%) = number of brown cells/total number of cells × 100%.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from L4-L6 segments of the spinal cords of the rats from the Control, Sham, Model, DEX and DEX + ML385 groups (n = 6 in each group) 7 days after operation using Trizol (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. Then the RNAs were reversely transcribed into cDNAs through Reverse Transcription System (Promega, WI, USA). The expressions of the genes were detected using LightCycler 480 (Roche, IN, USA) in accordance with the instructions of the fluorescence quantitative PCR kit (SYBR Green PCR kit, Takara Bio, Inc., Otsu, Japan). The reaction conditions were as follows: 5 min of pre-degeneration at 95℃, followed by 40 cycles of 10 s of degeneration at 95℃, 10 s of annealing at 60℃ and 20 s of expansion at 72℃. The relative quantitation was performed using comparative 2−∆∆Ct method, with GAPDH applied as internal reference. The primer sequences are shown in Table 1.
Table 1
Name
|
Sequence
|
Nrf2
|
F: 5'- AGGTTGCCCACATTCCCAAA-3'
|
R: 5'- AGTGACTGAAACGTAGCCGA-3'
|
GAPDH
|
F: 5'-TTCTGGGATACACGGAGCAC-3'
|
R: 5'-TACCAGCACCAGCGTCAAAG-3'
|
Note: F, forward; R, reverse |
Western blot
The L4-L6 segments of the spinal cords extracted from rats of the Control, Sham, Model, DEX, MCC950, Nigericin, DEX + ML385, MCC950 + ML385 and DEX + Nigericin groups (n = 6 in each group) 7 days after operation were mixed with RIPA buffer containing protease inhibitor and phosphatase inhibitor. Then the segments were centrifuged at 13000 rpm for 30 min at 4℃ for obtaining of total proteins or nucleoprotein using nuclear protein extraction kit (Solarbio Science and Technology Corporation, Beijing, China). The concentration of proteins was measured using a BCA kit. Then the proteins, separated by SDS-PAGE, were transferred onto a PVDF membrane for co-incubation with primary antibodies (abcam, Cambridge, UK) of GAPDH (1:10000, ab181602), Nrf2 (1:100, ab137550), NLRP3 (1:500, ab214185) and HO-1 (#82206, 1:1000, Cell signaling technology, Danvers, USA) at 4℃ overnight. After being washed with PBST for three times, the membrane was incubated with the secondary antibodies of goat anti rabbit IgG (1:5000, Beijing ComWin Biotech Co.,Ltd., Beijing, China) or goat anti rat IgG (1:2000, ab205719) at room temperature for 30 min. Finally, the membrane was subjected to washing with PBST for four times and allowed for color development with ECL. The brands were then detected using chemiluminescence imaging system (GE Healthcare, Beijing, China).
ELISA
The whole blood of rats from the Control, Sham, Model, DEX, MCC950, Nigericin, DEX + ML385, MCC950 + ML385 and DEX + Nigericin groups (n = 6 in each group) was mixed with EDTA and then centrifuged at 1000 g for 10 min at 4℃ to remove the yellow supernatant. The expressions of TNF-α, IL-1β, IL-6 and IL-10 were detected using a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
Statistical analysis
The statistics were analyzed by GraphPad Prism 7.0. T was used to compare differences between two groups. One-way analysis of variance was applied for multi-group comparison. P < 0.05 was considered statistically significant.