Preparation of plant extract
The roots of Angelica dahurica were purchased from Jiangsu Traditional Chinese Medical Hospital (Nanjing, China). The dried roots weighted 200 g were soaked in water 10 times of their total weight for 1 h, and then heated to refluxing for 2 h. Eight times of water was added for another 1.5 h refluxing after filtering. The filtered extraction solutions were combined and concentrated using a rotary evaporator at 60℃ and then lyophilized. Angelica dahurica extracts (ADE) were then prepared into several desired dose concentration for pharmacological tests.
Analysis of ADE.
The chromatographic separation of ADE was performed on Acquity TM UPLC liquid chromatographic system (Waters, Milford, MA, USA). An Acquity TM UPLC BEH C18 column (50 mm × 2.1 mm, i.d., 1.7 μm) maintained at 30 ℃ with a mobile phase of acetonitrile (A) and 0.1% formic acid aqueous solution (B) using a gradient elution of 0-0.5 min, 5 %A; 0.5~7.5 min, 5 %A～30 %A ; 7.5~9.0 min, 30 %A～40 % A; 9.0~11.0 min, 40 % A～50 % A; 11.0~12.0 min, 50 % A～80%A; 12.0~14.0min, 80%A～80%A; 14.0~15.0min, 80%A～70%A; 15.0~16.0min, 70%A～60%A; 16.0~17.0min, 60%A～50%A; 17.0~18.0min, 50%A～35%A; 18.0~19.0min, 35%A~20%A; 19.0~20.0min, 20%A～5%A .
Mass spectrometric detection was applied with electrospray ionization (ESI) ion source . Mass spectra were acquired in the negative-ion mode over a range of m/z 100–1000. The capillary voltage and cone voltage were kept at 3.0 kV and 30 V, respectively. Cone gas flow (50 L/H) and desolvation gas flow (900 L/H) were kept constant throughout the study. The desolvation temperature and ion source temperature were operated at 400℃ and 150℃, respectively. Scan time was 0.3s, Scan interval time was 0.02s.
Adult male C57BL/6 mice (aged 6-8weeks) from the Beijing Vital River Laboratory Animal Technology were used. All experiments involving animals were approved by the Nanjing University of Chinese Medicine Animal Ethics Committee（approval number：ACU170903）. The animals were housed in Nanjing University of Chinese Medicine Experimental Animal Center with a temperature-controlled colony room (24 ± 2℃), humidity (50%-60%), and a 12 h light/12 h dark cycle (lights on 08:00 to 20:00). Food and water were available and libitum. All behavioral tests were performed between 09:00 and 17:00. All experimental procedures were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine (Nanjing, China). Mice must adapt to laboratory conditions for at least 2 days prior to testing. TRPV1 knock out mice were kindly provided by Dr. Michael J. Caterina (Johns Hopkins University School of Medicine, Baltimore).
CFA-induced chronic inflammatory pain.
C57BL/6 mice received an intraplantar injection of 20μl of CFA (Sigma-Aldrich, USA) to the right hind paw and were divided separately into four groups randomly with the equal number (n=10). Four groups of mice were orally administrated by syringe feeding with distilled water , diclofenac sodium(10mg/kg) or ADE(100mgkg, 600mg/kg) daily for 14 days after CFA injection, respectively.
Von Frey mechanical assay
Mechanical sensitivity in mice were tested using a set of Von Frey filaments (0.008-2 g). Mice were placed on a raised wire mesh grid (6 × 6 mm2 apertures) under plastic chambers. The filament was applied to the plantar surface at a vertical angle for up to 2-3 s from the bottom. Fifty percent withdrawal threshold values were determined using the up-down method. Baseline von Frey measurements were obtained before drug administration or prior to CFA injection and subsequent measurements were taken at 2, 4, 6, 8 h and every two days post-injection until the 14th day. Behavioral assessments were conducted during the light cycle at approximately the same time each day. All behavioral analyses were performed blindly.
Radiant heat assay
Nociceptive thermal sensitivity was measured by focusing a beam of light on the plantar surface of the hind paw to generate radiant heat. Hind paw withdrawal latency was measured by the method of Hargreaves et al.. Mice were placed in elevated chambers on a plexiglas floor and were allowed to acclimated to the testing room for 30 minutes prior to testing. The radiant heat source (Ugo Basile Plantar Test 37370) was applied to the center of the plantar surface of the hind paw with at least 3 min intervals. The average withdrawal latency of the trials was recorded as the response latency. Baseline latency was determined before drug administration or prior to CFA injection.
Cultures of dissociated DRG neurons
Immunostaining of DRG neurons.
Mice were anesthetized with pentobarbital and perfused with 20 ml 0.1M phosphate buffer solution (PBS; pH 7.4; 4℃) followed with 25 ml fixative (4% formaldehyde in PBS; 4℃). Dorsal root ganglia (DRG) of spine levels L4-L6 was dissected from the perfused mice and then post-fixed in fixative at 4℃ for 30 min. DRG was cryoprotected in 30% sucrose for more than 24 hr and were sectioned with a cryostat at 10 µm and mounted on slides. Sections were immediately processed for detection of target protein or stored at −20 ℃ for future use. Sections were incubated in the following solutions: (1) blocking solution (containing 3% fetal bovine serum, 0.1% Triton X-100, and 0.02% sodium azide in PBS) for 1 h at room temperature; (2) primary antibodies(TRPV1 rabbit monoclonal antibody, dilution 1 : 200, proteintech, 22686-1-AP, Neuronal monoclonal antibody, dilution 1 : 500, abcam, ab104225) in blocking solution at 4℃ overnight; (3) PBS, 3 × 10 min each; (4) secondary antibodies （Alexa Fluor 555-labeled Donkey Anti-Rabbit IgG(H+L), 1 : 300, Beyotime, A0453，Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L), 1 : 300, Beyotime, A0423) in blocking solution for 1 h at room temperature; (5) PBS, 3 × 10 min. Tissue sections were examined with a Carl Zeiss Axio Zoom.V16 fluorescence microscope. To calculate the positive cells in DRGs, every two slices were captured and counted. The number of fluorescence positive DRG neurons was counted and calculated.
The ipsilateral L4-L6 DRG neurons were loaded with fura-2-acetomethoxyl ester (Thermo Fisher Scientific) for 25 min in the dark at 37℃ in accord with previous studies. After being washed 3 times with PBS, the glass coverslips were placed into a chamber and perfused with a calcium imaging buffer containing 137mM NaCl, 5.4mM KCl, 1.2mM MgCl2, 1.2mM NaH2PO4, 1mM CaCl2, 10mM glucose, and 20mM HEPES (pH 7.4). Ca2+ influx was detected by Fura-2 excitation at 340 and 380 nm by a high-speed continuously scanning monochromatic light source (Polychrome V, TILL Photonics, Gräfeling, Germany). 100nM capsaicin was added at the indicated time points. Cells were imaged under Olympus IX57 microscope. All calcium imaging assays were performed by an experimenter blind to the groups.
Western blot analysis.
Mice were sacrificed by cervical dislocation under Isoflurane anesthesia 14 days after the ADE and diclofenac sodium administration. DRG neurons from mice were lysed with RIPA Lysis Buffer (Beyotime, P0013B, China) and protein concentrations were determined using a BCA Protein Assay Kit (Pierce). Proteins (50 μg) were separated in 10% SDS PAGE gel, and transferred to PVDF membranes. After 60 minutes blocking, the membranes were incubated with primary antibody (TRPV1 mouse monoclonal antibody, dilution 1: 1000, abcam,ab203103) at 4℃ overnight. After washing with TBST, the membrane was incubated with goat anti-mouse antibody (HRP labeled) diluted with 5% non-fat dried milk in TBST and detected with ECL reagents (Millipore). Blots were scanned with gel and blot imaging system (Biorad，Gel Doc XR+ System）and band densities were compared with Image Lab software. TRPV1 protein levels were normalized against beta-action and all experiments were done for three times.
All statistical calculations were performed using the GraphPad Prism 6.0 software. Data are presented as means ± SEMs. Groups were compared by a two-tailed, unpaired Student’s t test. The designation “n” represents the number of animals analyzed. Differences were considered statistically significant for P < 0.05. Representative data are from experiments that were replicated biologically at least three times with similar results.