Reagents
The commercial paclitaxel injections (Paclitaxel Injection®, Lot: H99404910) were purchased from SichuanTai Ji Group Co., Ltd (Chengdu, China). CSBTA (Yanhuanglian total alkaloids capsules, Lot: 160701) was kindly supplied by Nanjing Zhongshan Pharm Co., Ltd (Nanjing, China). Sodium carboxymethyl cellulose (CMC-Na) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). CSBTA powder was suspended in CMC-Na solution in advance. The quality specification of the CSBTA capsule is controlled by the content of total alkaloids (calculated by dehydrocavidine) and the amount of dehydrocavidine, palmatine, and berberine. Referring to the standard sample of dehydrocavidine, palmatine, and berberine (supplied by National Institutes for Food and Drug Control, Beijing China), the total alkaloids fraction was analyzed as 58% by HPLC-UV analysis.
Animals
Adult male Sprague-Dawley rats (180 ± 20g) were supplied by the Laboratory Animal Center of Nanjing Qinglongshan (Agreement Number: SCXK-zhe-2014-0001). All rats were maintained under the standard conditions in China Pharmaceutical University Laboratory Animal Center under the12 h dark-light cycles’ in the humanized environment (temperature, 23 ± 2 °C, and humidity, 50 ± 10%) with normal chow diet and tap water ad libitum. After acclimation for a week, the rats were used for the following experiments.
Grouping and administration
Rats were randomly divided into 4 groups: blank group (physiological saline & 0.5% CMC-Na solution), paclitaxel group (2 mg/kg paclitaxel & 0.5% CMC-Na solution), 30mg/kg CSBTA group (2 mg/kg paclitaxel & 30 mg/kg CSBTA suspended in CMC-Na solution) and 120 mg/kg CSBTA group (2 mg/kg paclitaxel & 120 mg/kg CSBTA suspended in CMC-Na solution). Except for the blank group, rats were intraperitoneally injected with 2 mg/kg paclitaxel (Paclitaxel Injection®, Tai Ji, Chengdu, China) at 1, 3, 5, and 7 days, and the blank group was intraperitoneally given the corresponding volume of physiological saline [18]. From the first day of the experiment, oral administration of CSBTA with different dose concentrations (30, 120 mg/kg) were given[17], while the blank group and the model group were administered with a corresponding volume of 0.5% CMC-Na solution. During the experimental period, the body weights were recorded daily before drug administration to evaluate the potential effect of CSBTA on body weight changes.
Ahead of the final experiments, the food was deprived overnight (free access to water). After mice were anesthetized, blood was collected from the abdominal aorta, centrifuged at 3000 rpm for 10 min to be harvested plasma for the determination of PGE2, TNF-α, and IL-1β. L4-L6 segment spinal cord, DRG, and plantar skin were harvested and immediately stored at -80 ℃ till analysis.
Mechanical hyperalgesia—Von-Frey test
The mechanical pain threshold of rats was detected by Von-Frey filament according to the "up and down" method initiated by Chaplan[19]. As described previously[17], the rats were individually placed in a transparent plexiglass box with a cover at the top and a reticular structure at the bottom. Every rat was subjected to an adaptive test one week before the formal experiment. After adaptation for 15 min, the mechanical pain threshold was measured when rats were in a quiet state and the movement of walking and scratching disappeared. Von-Frey filament (Stoelting Company, Chicago, USA, stimulus intensity range: 4-180 g) was used to prick the middle area of the ipsilateral posterior toe through the bottom mesh. Initiating from the moderate intensity, the fiber needle vertically pressed the plantar skin until the needle was warped into a "C" shape. If retraction, movement, or lameness of the hind paw appeared, a positive reaction "X" was designated and the corresponding value was recorded. And then, the strength nearest to low-intensity t was used to stimulate, and negative reaction "O" was recorded if there were no response after stimulus for more than 5 s. Sequentially, the closest high-intensity was continued to stimulate till the rats showed positive behavior. Stimulation was conducted every 2 min and repeated six times. Mechanical hyperalgesia was tested by a double-blind test. Followingly, the "X" and "O" sequences could be obtained and the mechanical pain threshold could be finally calculated. The mechanical pain threshold test in each rat was repeated three times to calculate the average response.
Thermal sensitivity—Laser thermal pain meter
PL-200 laser thermal pain meter (PL-200, Taimeng Technology, Chengdu, China) was used to detect heat hyperalgesia to assay latent period of heat-stimulated retraction reflex. As described previously [17], each animal was subjected to an adaptive test one week before the formal experiment. Under constant ambient temperature, rats were placed in an observation box for 15 min in advance to make them accommodative and tranquil. Afterward, the optical source (intensity at 35%) was moved to plantar, and the time (from the initial irradiation) was recorded when thermal pain reaction (contracting claw, licking foot, and scratching) occurred. The cutoff time was 25 s to prevent possible injury from high temperature. The thermal sensitivity test was repeated three times at least 5 min intervals in each rat, and the average was applied for data analysis. Thermal sensitivity was tested by a double-blind test.
Thermal sensitivity—Tail immersion assay
Half an hour after drug administration, rats were subjected to a tail-flick test. As described previously [18], each animal was subjected to an adaptive test one week before the formal experiment. After the rat was no longer struggling, the tail was immersed into a water bath, and time from immersion to tail flick reaction was recorded. The temperature was set at 47 ℃, which was adopted by the tail-flick time lasting 10s for most rats. To prevent the injury from high temperature, the cutoff time was 20 s. Each rat was tested three times with at least 5 min interval, and the average of the three tests was used for data analysis. Thermal sensitivity was tested by a double-blind test.
Thermal sensitivity—Cold hyperalgesia
Similarly, rats were subjected to a cold hyperalgesia test 30 min after drug administration. As described previously [20,21], dry ice was used to assay cold pain threshold of rats, and the results were expressed as the first occurrence time of paw withdrawal, lameness, scratching, or other reactions in rats after cold stimulation. The room’s temperature was kept stable, and rats were placed in an observation box for 15 min to make them quiet. After the dry ice placing on plantar, time from the beginning to the emergence of contracting claw, licking foot, or scratching was recorded. The cutoff time was 90 s to avoid skin cold injury. At least 5 min interval was performed. Each animal was subjected to an adaptive test one week before the formal experiment. Thermal sensitivity was tested by a double-blind test.
TNF-α, IL-1β, and PGE2 in serum and plantar skin tissues
After 12 h of the last dosing, rats in each group were fasted for 10 h (free access to water) and then anesthetized. Blood was rapidly withdrawn from the abdominal aorta to clean EP tubes. Serum was harvested after centrifugation at 3000 rpm for 10 min. The harvested plantar skin tissues from corresponding rats were immediately shaved on the ice. A piece of 1 g tissue was collected and cut into pieces. Subsequently, they were soaked in 5 ml physiological saline after a gentle vortex at 4 ℃ for 2 h. Afterward, the mixtures were centrifuged at 3000 rpm for 10 min at 4 ℃, and the harvested supernatant was stored in -80 ℃ till analysis [17]. The levels of TNF-α, IL-1β, and PGE2 in obtained plantar skin supernatant and serum were separately detected by ELISA (Calvin Biotechnology, Suzhou, China) according to kit instructions.
Primary DRG neurons cell culture
As described previously [22,23], the back skin of rats was quickly cut with surgical scissors to isolate the spine after anesthesia. DRG tissue was extracted and placed in a tube containing MEM medium (Gibco, USA) without fetal bovine serum (FBS, Gibco, USA). Subsequently, an aliquot of 2 ml collagenase A (Roche, USA, 1 mg/ml) was added to digest cells for about 90 min. Then, the digestion solution was replaced by 0.25% trypsin (Gibco, USA) for another 20 min. Finally, the digestion was terminated by the MEM medium containing 10% FBS. MEM medium was mechanically triturated with a 1 ml pipette until the solution turned milky. After filtering through a 70 μm cell strainer, the solution was centrifuged at 1000 rpm for 5 min. After removing the supernatant, the remaining cell pellets were resuspended in MEM medium containing 10% FBS and 1% penicillin and streptomycin mixture (Gibco). Eventually, cell suspensions were divided into a six-well plate (~60,000 per well) covering with poly-D-lysine at the bottom (100 μg/mL, Sigma, USA). After culturing for 24h, MEM medium was replaced with neuronal growth medium (Neurobasal medium: B27 = 50:1, both purchased from Gibco, USA), and 1% other agents including penicillin and streptomycin mixture (Gibco), L-Glutamine (0.1 mg/mL, Sigma-Aldrich), and cytarabine (5 μg/mL, Sigma Aldrich) was added. Medium with supplements was changed every two days.
Cell viability assay
The cell suspension was injected into a 96-well plate and cultured in a 5% CO2, 37 ℃ incubator. After three days, CSBTA group was treated with 0, 0.05, 0.5, 5, 50 μg/ml of CSBTA, and paclitaxel groups were given 0, 1, 10, 100, 500, 1000nM paclitaxel (A4393, APExBIO, USA). Five days later, Cell Counting Kit-8 (CCK-8) solution (Dojindo, Japan) was added. After incubation for 4 hours, absorbance at 450 nm was measured with a microplate reader.
Drug treatment
After cultured three days, primary DRG neuron cells were equally divided into following five groups: blank group (phosphate buffer saline), paclitaxel group (300 nM paclitaxel), 25 μg/ml CSBTA group (300 nM paclitaxel + 25 μg/ml CSBTA), 50 μg/ml CSBTA group (300 nM + 50 μg/ml CSBTA) and PKCε inhibitor group (300 nM paclitaxel + 100 nM Staurosporine(A8192, APExBIO, USA)). The co-incubation lasted 5 days and cells were processed as the preparation procedure.
Western blot analysis
As described previously [17], the spinal cord and DRG were lysed in lysis buffer (RIPA: PMSF: phosphatase inhibitor = 100:1:1) at 1:5 (mg·μL-1) and vigorously crushed by glass homogenizer on ice. An appropriate aliquot of lysis buffer was added and cells were scrapped by cell scraper on ice. The homogenates were immediately centrifuged at 12000 rpm for 20 min at 4 ℃. Protein quantification was performed according to the procedure of the Bicinchoninic Acid Protein Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Subsequently, extracted protein samples (6×loading buffer: supernatant=1:2, v/v) were boiled for 15 min and stored at -80℃ till analysis.
Loading protein (50 μg) was electrophoresed and separated on a 10% SDS-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (0.22 μm, Pall, USA). PVDF membrane was blocked in 5% skimmed milk in TBST buffer (Tris-HCl, 5 mM, pH 7.6, NaCl 136 mM, 0.05% Tween-20) at room temperature for 2 h, followed by incubation with different primary antibodies—p38 (1:1000, Abcam, USA), p-p38 (1:1000, Cell Signaling Technology, USA), PKCε (1:1000, Abcam, USA) and TRPV1 (1:200, Alomone, Israel) in TBST at 4 ℃ overnight. After washed with TBST three times, membranes were further incubated with horseradish peroxides (HRP)-conjugated secondary antibody (1:5000 dilution in TBST) for 2 h at room temperature. Antibody binding was detected by enhanced chemiluminescence (ECL). Imagines were acquired by Bio-Rad and quantified by densitometry analysis using Image J software.
Real-time qPCR analysis
Referring to the manufacture’s instruction, spinal cord and DRG tissue weighing 100 mg were homogenized in 1 ml Trizol reagent (Invitrogen, USA) on ice to extract total RNA. While cells were lysed with 1ml Trizol reagent per well to extract total RNA [24]. RT-PCR was proceeded according to HieffTM First Strand cDNA Synthesis SuperMix Kit instruction. β-actin was used as an endogenous control. Quantitative PCR was performed in 20 μl reactions using HieffTM qPCR SYBR Green Mester Mix (No Rox) kit. Primer sequences are listed in Table 1. Relative quantities of the candidate genes and β-actin mRNA were detected by the Bio-Rad image system (Bio-Rad, USA) and calculated by the comparative threshold cycle (Ct) method.
Statistical analysis
Data were expressed as a mean ± SD (standard deviation) of at least three independent experiments. Statistical analysis was performed using GraphPad Prism software, version 6.0 (San Diego, CA, USA). The statistical significance was analyzed using unpaired Student’s t-test, or one-way ANOVA followed by Dunnett’s multiple comparison test. A value of p < 0.05 was considered to be statistically significant.