Animals
A total of 70 healthy male Sprague-Dawley rats (8 weeks old; weight, 200–250 g) were purchased from the Experimental Animal Center of Suzhou Aiermaite Technology Co., Ltd. (Suzhou, China). The rats were housed in a specific pathogen-free animal facility, at 22–24°C, 50–60% humidity and a 12 h light/dark cycle. The rats had free access to food and water prior to experimentation. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Weifang People's Hospital (Weifang, China).
CCI model construction
A total of 70 male Sprague-Dawley rats were randomly divided into the following five groups (n=14 rats/group): The control group (Ctrl), the ozone and gardenoside treatment control group (Ctrl + Ozo&Gar), the sham surgery group (Sham), the CCI model group (CCI) and the ozone and gardenoside treatment CCI model group (CCI + Ozo&Gar). The rats in each group were treated as follows. After environmental adaptation for ≥5 days, two groups (CCI and CCI + Ozo&Gar groups; total number of rats, 28) were used to generate the CCI model. For 24 h prior to surgery, these rats were provided only water, with no food. The CCI model is well characterized for the study of neuropathic pain (14,15). Briefly, rats were anesthetized with 10% chloral hydrate (350 mg/kg, intraperitoneal), fixed in the supine position and were subjected to an aseptic operation. The right legs were shaved and sterilized with 70% ethanol and betadine antiseptic solution. Following exposure of the thigh muscle, the sciatic nerve was separated and loosely ligated with sterile 4-0 catgut thread at four consecutive sites, with an interval of ~1 mm. The nerve was carefully ligated, so as not to completely block peripheral blood flow in the nerves. In addition, a sham surgery was performed in the Sham group, in which the sciatic nerve was exposed, but not ligated. Finally, the wound was sutured in layers and was cleaned with iodine, followed by an intramuscular injection of penicillin (40,000 U/mouse). Rats were then maintained in the same manner as the rats in the control group. The two control groups (Ctrl and Ctrl + Ozo&Gar) did not undergo surgery.
Drug application
The rats in the Ctrl + Ozo&Gar and CCI + Ozo&Gar groups were administered an intravenous injection of 30 µg/ml ozone (Chengdu Must Biotechnology Co., Ltd., Chengdu, China) and 300 µmol/l gardenoside (Chengdu Must Biotechnology Co., Ltd.). The rats in the Ctrl, Sham and CCI groups were administered the same volume of saline. The drugs were delivered intravenously once daily for 14 days, beginning on day 1 after CCI. Specimens were collected from the L4-L5 DRG on the 15th day for analysis.
Evaluation of pain behavior
The five groups of rats were raised in separate cages after the operation. MWT and TWL were measured prior to the operation (day 0), and 1, 3, 5, 7, 9, 11 and 14 days after the operation. The time of each daily measurement, the room temperature and other conditions were similarly maintained across all measurements.
Detection of mechanical hyperalgesia
BME-403 Von Frey fine thread (Beijing Jinuotai Technology Development Co. Ltd., Beijing, China) was used to measure MWT. The rats were maintained in a transparent plexiglass box (20×30×30 cm); the bottom of the plexiglass box was made of wire netting (1×1 cm). The rats were maintained in the plexiglass box for 15 min prior to the measurement. The bending forces of the wires inserted through the wire netting used were as follows: 0.13, 0.20, 0.33, 0.60, 1.30, 3.60, 5.00, 7.30, 9.90 and 20.1 g, and tolerance of bending forces >20.1 g were recorded as 20.1 g. The interval between two stimulations was 20 sec or until the stimulation-induced reactions, such as licking feet and paw withdrawal. The test was stopped once paw withdrawal was induced. Each test was performed from the smallest to the largest bending force until the frequency of withdrawal was ≥50%. From this, the MWT value was determined. The test was repeated three times and the mean was calculated.
Detection of thermal hyperalgesia
Thermal hyperalgesia was measured using an automatic thermalgia stimulator system (BME-410C; Institute of Bioengineering, Chinese Academy of Sciences, Beijing, China) to irradiate the paws. The plexiglass box was placed on a glass plate (3 mm thick), and the rats were maintained in the box for 30 min prior to the experiment. The time it took for the rats to withdraw their paws from the thermal stimulus was considered the TWL. The cut-off time was 30 sec, in order to prevent tissue damage. Each rat was measured three times and the mean value was taken as the threshold value.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
To measure the mRNA expression levels of P2X3 and P2X7 receptors, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (350 mg/kg) and the L4 and L5 DRG was harvested on the 15th day post-surgery, and was used for total RNA isolation using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Subsequently, 1,000 ng total RNA was used as a template for RT using the Applied Biosystems RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer instructions. β-actin was used as the internal reference gene. The primer sequences are listed in Table I. The SYBR-Green master kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) PCR products were amplified using the following cycling parameters: 94°C for 5 min, followed by 35 cycles at 94°C for 45 sec, 53°C for 30 sec and 72°C for 40 sec, and a final step at 72°C for 5 min. Calculated relative expression of the target gene expression was calculated with the 2−ΔΔCq method (16).
Western blot analysis
For western blot analysis, rats were deeply anesthetized with diethyl ether and were sacrificed by decapitation. The L4 and 5 DRG was rapidly removed and lysed with 20 mM Tris-HCl buffer (pH 8.0), containing 1% NP-40, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% β-mercaptoethanol, 0.5 mM dithiothreitol, and a mixture of proteinase and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Protein concentration was determined using the bicinchoninic acid protein assay method with bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China) as a standard. β-actin served as an internal control. Protein samples (60 µg/lane) from DRG were separated by 8% SDS-PAGE and were then electrotransferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing Tween 20 (50 mM Tris, 150 mM NaCl and 0.1% Tween 20 v/v, pH 7.4) for 2 h at room temperature, and were then incubated at 4°C overnight with P2X3 (cat no. sc-12215) and P2X7 (cat no. sc-25698) receptor antibodies (1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and β-actin antibody (cat no. sc-130656; 1:1,000; Santa Cruz Biotechnology, Inc.). The membrane was subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (cat no. sc-2005; Santa Cruz Biotechnology, Inc.) overnight at 4°C. The signal was detected using the Amersham enhanced chemiluminescence system (Amersham; GE Healthcare, Chicago, IL, USA). The relative expression levels of P2X3 and P2X7 receptors were densitometrically semi-quantified using Image-Pro Plus (Media Cybernetics, Inc., Rockville, MD, USA), and were calculated according to the reference bands of β-actin.
Statistical data analysis
Statistical analysis was performed using SPSS statistical software, version 19.0 (IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation. Experiments were repeated three times. Statistical analyses were performed using one-way analysis of variance followed by a least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.