Introduction

Itch, an unpleasant sensation that evokes a desire to scratch1, is one of the important physiological functions that humans and animals acquired during their long-term evolution. It includes acute and chronic itch; acute itch serves us well in guarding against environmental threats

Experimental procedures

Animals

C57BL/6 male mice (8–10 weeks) were used for behavioral testing (Experimental Animal Center, Nan**g University of Chinese Medicine, Nan**g, China). The study was performed in accordance with relevant guidelines and regulations of the Institutional Animal Care and Use Committee of the Nan**g University of Chinese Medicine. All experimental protocols were approved by the International Association for the Study of Pain.

AEW and AD treatment

Mice were housed in a temperature-controlled animal room (22 ± 2 °C) under a 12-h light/dark cycle, with free access to food and water. They were acclimated to the testing environment for 30 min before the initiation of behavioral tests. And the animal behaviors were analyzed by investigators who were blind to genotype and animal treatment condition.

To experimentally induce dry skin, we treated the nape of the neck of mice with acetone-ether-water (AEW), as reported previously58. Animals were shaved at the nape of the neck in the first three days before starting treatment. A mixture of acetone and diethylether (1:1) was applied to the shaved area for 15 s, followed immediately by distilled water for 30 s. The control group just used cotton wetted by water for 45 s instead. The animals were treated twice daily.

For AD model, animals were shaved at the nape of the neck and abdomen fur, 150 μL 0.5% DNFB dissolved in an acetone: olive oil mixture (4:1) was applied into the abdomen for sensitization (day −4). From day 0 to day 11, 0.2% DNFB dissolved in an acetone: olive oil mixture (4:1) (model group) and olive oil (control group) were applied to challenge the shaved area of neck (50 μL) and three times a week.

Behavioral test

For the chronic itch test, scratching behaviors in model mice with AEW or AD were surveyed for 30 min after molding treatment. On the 7th day or 12th day of treatment, we adopted subcutaneous injection of MT (30 mg/kg, 50 μL, dissolved in saline) or saline at the treated area. Then the scratching behaviors were observed for another 30 min. A bout of scratching was defined as a continuous scratching movement with a hind paw directed at the treated site or drug injection site. The bouts were obtained from the number of subtractions after injection.

For an acute experimental test, different concentrations of MT (3, 6, 15, 30 mg/kg) were treated with the nape of the neck by subcutaneous injection or intraperitoneal injection for 30 min. Then, the histamine (200 mM), chloroquine (8 mM), or compound 48/80 (13 mM) was injected into the same area of the neck and the scratching behaviors were observed for another 30 min.

Culture of dissociated DRG neurons

DRG neurons were collected in ice cold DH10 medium (90% DMEM/F-12, 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, Gibco). Dissociated DRG neurons were then digested for 30 min at 37 °C in a protease solution (5 mg/mL dispase, 1 mg/mL collagenase type I in HBSS without Ca2+ or Mg2+, Gibco). After digestion, DRGs were triturated to free neurons and then neurons were collected by centrifugation (1000 rpm, 5 min). Cells were plated on the coverslips with poly-D-lysine (0.5 mg/mL, Sigma, Aldrich, USA) and laminin (10 mg/mL, Sigma, Aldrich, USA) coated, and 1‰ neuron growth factor (NGF, dissolved in DH10) was added into the media. All these cells were incubated at 37 °C, 5% CO2 for 16–18 h before the calcium imaging experiment.

Whole-cell voltage-clamp recordings from DRG neurons

DRG neurons were identified by inverted microscopy (ZEISS, Axio Oberver D1, Germany). Coverslips were transferred into a chamber with the extracellular solution. Whole-cell current clamp and voltage-clamp recording experiments were performed at room temperature (23–25 °C) using a Multi-clamp 700B amplifier and Digital 1440 with pClamp10 software (Molecular Devices, USA).

Signals were sampled at 20 kHz and filtered at 2 kHz. The patch pipettes were pulled from borosilicate glass capillaries using a P-97 micropipette puller (Sutter Instrument) and had a resistance of 3–5 MΩ for patch-clamp recordings. The series resistance was routinely compensated at 60–80%. The resting membrane potential was recorded for each neuron under the current-clamp mode after stabilization (within 3 min). Neurons whose seal resistance were below 1 GΩ after breaking the cell membrane and whole-cell recording formation were excluded from analysis. The liquid junction potential was 8 mV and corrected. A single intact action potential was induced by a series of depolarizing current steps, each of 2 ms duration, increments of 50 pA through the recording electrode. The internal solution contained the following (in mM): KCl 135, MgATP 3, Na2ATP 0.5, CaCl2 1, EGTA 2, glucose 5, with pH adjusted to 7.38 using KOH, and osmolarity adjusted to 300 mOsm with sucrose. The external solution contained the following (in mM): NaCl 140, KCl 4, CaCl2 2, MgCl2 2, HEPES 10, glucose 5, with pH adjusted to 7.4 using NaOH, and osmolarity adjusted to 310 mOsm with sucrose.

For voltage-gated calcium (Cav) current recording, the intracellular pipette solution contained (in mM): CsCl 135, CaCl2 1, MgCl2 2, MgATP 1.5, Na2GTP 0.3, EGTA 11, HEPES 10, with pH of 7.3 and osmolarity of 310 mOsm. The total calcium currents were evoked in response to depolarization steps to different testing potentials from −70 mV to +50 mV in 10 mV increments with a duration of 300 ms, preceded by a 500 ms prepulse of −90 mV. LVA Ica was evoked at −40 mV (20 ms) from a holding potential of −80 mV, and HVA Ica was evoked at 10 mV (20 ms) from a holding potential of −60 mV or −80 mV, repeated every 20 s. We used neurons with HVA Ica (10 mV)/Ica (−40 mV) > 1 for drug testing to limit the potential contamination of small HVA currents from residual LVA currents. The voltage dependence of current activation was obtained by depolarizing from −80 mV to +30 mV in 10 mV increments with a duration of 80 ms, preceded by a 100 ms prepulse of −70 mV, and estimated using a modified Boltzmann function to fit normalized I-V data: G/Gmax = 1/(1 + exp ((V1/2 act − Vm)/κ)), where G is conductance, Vm is the test potential, V1/2 act is the mid-point of activation, and κ is the slope factor. Steady-state inactivation relationships (or availability curves) were obtained by depolarizing to −10 mV for 50 ms after a prepulse of 500 ms depolarizing steps from −80 mV to +20 mV in 10 mV increments and estimated by fitting averaged data to a standard Boltzmann function: I/Imax = 1 + exp ((Vm − V1/2 inact)/κ), where Imax is the maximal current recorded at −30 mV, V1/2 inact is the midpoint of steady-state inactivation, and κ is the slope.

Whole-cell voltage-clamp recordings from spinal cord slices

To prepare spinal cord slices, we first performed a laminectomy in adult (4-week-old) C57BL/6 mice that were deeply anesthetized with CO2, then the lumbosacral segment of the spinal cord was rapidly removed and placed in ice-cold, low-sodium Krebs solution (in mM: NaCl 95, KCl 2.5, NaHCO3 26, NaH2PO4-2H2O 1.25, MgCl2 6, CaCl2 1.5, glucose 25, sucrose 50, saturated with 95% O2/5% CO2). The tissue was trimmed and mounted on a tissue slicer (Vibratome VT1200, Leica Biosystems, Buffalo Grove, IL). Transverse slices (400 µm) with attached dorsal roots were prepared and incubated in normal Krebs solution (in mM: NaCl 125, KCl 2.5, NaHCO3 26, NaH2PO4-2H2O 1.25, MgCl2 1, CaCl2 2, glucose 25, saturated with 95% O2/5% CO2). The slices recovered at 34 °C for 40 min and then at room temperature for an additional hour before being used for experimental recordings. For electrophysiology recording, slices were transferred to a low-volume recording chamber, which was perfused with normal Krebs solution at a rate of 2 mL/min and bubbled with a continuous flow of 95% O2/5% CO2. Whole-cell patch-clamp recording of lamina II cells was carried out under oblique illumination with an Olympus fixed-stage microscope system (FV1200, Olympus, Japan). Data were acquired by a Multi-clamp 700B amplifier and Digital 1550 with pClamp10 software (Molecular Devices, USA). We fabricated thin-walled glass pipettes (Sutter Instruments, Sarasota, FL) that had a resistance of 3–6 MΩ and were filled with internal solution (in mM): K-gluconate 120, KCl 20, MgCl2 2, EGTA 0.5, Na2ATP 2, Na2GTP 0.5 and HEPES 20. To minimize inhibitory postsynaptic current contamination of EPSC recording, all recordings were made in the presence of SR95531 (10 μM) and strychnine (5 μM) in the external solution, to block GABAA and Glycine receptor, respectively. A seal resistance of at least 1 GΩ and an access resistance of 20–35 MΩ were considered acceptable. Spontaneous EPSCs (sEPSCs) were recorded at a holding potential of −70 mV. For evoked EPSC (eEPSC), we delivered paired-pulse test stimulation to the dorsal root entry zone consisting of two synaptic volleys (150–500 µA, 0.1 ms) 400 ms apart at a frequency of 0.05 Hz. To study membrane currents elicited by exogenous transmitters, L-glutamate was applied from glass pipettes to the soma of the recorded neuron by short (10 s) pressure pulses.

Calcium imaging

For calcium imaging experiments, the cells were loaded with Fura-2-acetomethoxyl ester (molecular Probes, Eugene, OR, USA) in HBSS solution for 30 min in the dark at room temperature. After washing 3 times, the glass coverslips were placed into a chamber and perfused with normal solution (in mM): NaCl 140, KCl 5, HEPES 10, CaCl2 2, MgCl2 2, glucose 10 and pH 7.4 with NaOH to adjust. A high-speed, continuously scanning, monochromatic light source (Polychrome V, Till Photonics, Gräfeling, Germany) was used for excitation at 340 and 380 nm, enabling us to detect changes in intracellular free calcium concentration.

MTT assay

To investigate the effect of MT on the survival of cells, cell viability of DRG neurons or HEK293 cultured were tested by the MTT assay. Cells at a density of 5 × 103 cells/well were seeded into each well in black well 96-well plates (Sigma, Aldrich, USA). After being cultured for 24 hours, cells were incubated by MT at 0.01, 0.1, 1 and 10 mM concentrations or pure DMSO. After another 24 hours, 10 µL tetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 1 mg/ml) (Sigma, Aldrich, USA) was added and cells were cultured at 37 °C for 4 hours. And then the medium was removed and DMSO (100 µL) was added. The optical density as the parameter of cell viability was measured at 570 nm with a microplate reader (Multiskan EX, Thermo, Ventana, Finland).

HPLC-MS/MS assay

Twelve C57BL/6 mice were divided into two groups: One group of animals was shaved at the nape of the neck and the MT 30 mg/kg was injected by subcutaneous injection; the other group was injected MT 30 mg/kg with intraperitoneal injection manner. Blood samples were collected from mandibular vein at 0.5, 1, 2 and 4 hours after MT treatment. Then, the samples were centrifuged at 3000 rpm for 5 min and the plasma was isolated. The plasma samples (10 μL) were added to the acetonitrile solution (200 μL) containing 10 ng/mL dexamethasone and the mixture (1 μL) was prepared for HPLC-MS/MS test. The following modular HPLC-MS/MS system was used: Agilent 1200 HPLC-MS/MS instrument (Agilent, USA). The analytical column (2.1 × 150 mm, 2.5 µm) was packed with Kromasil C18 (2.5 µm). The mobile phase was composed of acetonitrile and water (50:50). The flow rate was 0.6 mL/min and the temperature was 40 °C. The mass spectrometry conditions were electrospray ionization with multi-channel response monitor manner. The test gas pressure: atomized gas 50 psi, heating auxiliary gas 50 psi, curtain gas 35 psi, collision gas 25 psi. Cluster voltage was 80 V, collision voltage was 10 V and collision pool outlet voltage was 17.5 V. Before blood MT concentration test, standard control plasma samples at 1, 2, 10, 30, 100, 300, 1000, 2700 and 3000 ng/mL were prepared by adding to the blank plasma with atorvastatin. The control sample was tested with HPLC-MS/MS as mentioned above and we could get a standard ion peak-concentration curve. Then, through comparing ion peak of MT plasma and standard plasma, the MT plasma concentration could be achieved.

Data analysis

Electrophysiological data were analyzed and fitted using Clampfit (Axon Instruments, Foster City, CA) and Origin Pro 8 (Origin Lab, USA) software. All data were analyzed by ANOVA or two-tailed student’s t-test, and expressed as means ± standard errors of the means (SEM). The statistical significance was set at P < 0.05.

Significance

Chronic pruritus is a disease that is often refractory to the current available medications and seriously compromises the life quality of patients. As a traditional Chinese medicine, Sophorae Flavescentis Radix (SFR) has been widely used in treatment of chronic pruritus. To further develop and rationally use SFR in the treatment of pruritus patients, the antipruritic mechanism of MT, we studied a major active component of SFR. We found that MT had an anti-pruritus effect similar to SFR in the mouse models of acute and chronic pruritus. It was further proved that the anti-pruritus effect of MT was by inhibiting the presynaptic N-type calcium channel. These findings would provide an important reference and guidance for the clinical application of MT.