Transient receptor potential vanilloid 1 involved in the analgesic effects of total flavonoids extracted from Longxuejie (Resina Dracaenae Cochinchinensis)

Abstract

OBJECTIVE:

To evaluate the analgesic effects of total flavonoids of Longxuejie (Resina Dracaenae Cochinchinensis) (TFDB) and explore the possible analgesic mechanism associated with transient receptor potential vanilloid 1 (TRPV1).

METHODS:

Whole-cell patch clamp technique was used to observe the effects of TFDB on capsaicin-induced TRPV1 currents. Rat experiments in vivo were used to observe the analgesic effects of TFDB. Western blot and immunofluorescence experiments were used to test the change of TRPV1 expression in DRG neurons induced by TFDB.

RESULTS:

Results showed that TFDB inhibited capsaicin-induced TRPV1 receptor currents in acutely isolated dorsal root ganglion (DRG) neurons of rats and the half inhibitory concentration was (16.7 ± 1.6) mg/L. TFDB (2-20 mg/kg) showed analgesic activity in the phase Ⅱ of formalin test and (0.02-2 mg per paw) reduced capsaicin-induced licking times of rats. TFDB (20 mg/kg) was fully efficacious on complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia and capsaicin could weaken the analgesic effects. The level of TRPV1 expressions of DRG neurons was also decreased in TFDB-treated CFA-inflammatory pain rats.

CONCLUSION:

All these results indicated that the analgesic effect of TFDB may contribute to their modulations on both function and expression of TRPV1 channels in DRG neurons.

1. INTRODUCTION

Xuejie (Sanguis Draconis) is a bright red resin that is obtained from different species of four distinct plant genera; Croton, Dracaena, Daemonorops, and Pterocarpus.¹ It has been used as a famous traditional medicine for diverse medical and artistic applications.² Since Peres et al ³ isolated several compounds showing analgesic activity from Croton urucurana Baillon., the analgesic effect of Xuejie (Sanguis Draconis) has begun to attract people's attention. Rao et al ⁴ further indicated that the red viscous sap obtained by making oblique cuts on the bark surface of Croton urucurana Baill. could suppress capsaicin- and cyclophosphamide-induced visceral nociception. Reanmongkol et al ⁵ found that the methanol extract and the methanol fraction of Dracaena loureiri possess analgesic effect.

In Chinese folk medicine, the resin extracted from stems of Dracaena cochinchinensis has mainly been used as Longxuejie (Resina Dracaenae Cochinchinensis).⁶ Its English name is dragon's blood. It is often used clinically as an analgesic and the main chemical constituent is flavonoids.¹ Chen et al ⁷ studied the pharmacological effect of Longxuejie (Resina Dracaenae Cochinchinensis) which is the red resin obtained from the wood of Dracaena cochinchinensis (Lour.) S. C. Chen. The active components of total flavonoids from Longxuejie (Resina Dracaenae Cochinchinensis) had analgesic effect, which may be attributed to inhibition of astrocytic function (like release pro-inflammatory cytokines) and NO release as well as p-cAMP-response element binding protein activation in the spinal dorsal horn. Li et al ⁸ indicated that Longxuejie (Resina Dracaenae Cochinchinensis) (Xishuanbanna Yulin Pharmaceutical Co., Kunming, China) exerted anti-inflammatory and analgesic effects by blocking the synthesis and release of substance P through inhibition of Cyclooxygenase-2 protein induction and intracellular calcium ion concentration.

In fact, many medicinal plants containing flavonoids have been widely used in folk medicine as analgesic. An increasing number of studies indicated that some flavonoids from medicinal plants could be promising candidates for new natural analgesic drugs. Among these flavonoids, some bioactive components with analgesic potential have been identified and reported. It is demonstrated that course treatment with flavonoids derived from Lychnis chalcedonica L. produced a stable pharmacological effect comparable with that of the reference anti-inflammatory drug diclofenac.⁹ Both hesperidin and curcumin attenuate the neuropathic pain induced by partial sciatic nerve ligation in rats.¹⁰ Flavonoids fraction from Tribulus terrestris L. leaves exhibited a better antibacterial, analgesic and anti-inflammatory activities.¹¹ The flavonoids in O. falcata have good anti-inflammatory and analgesic activities,¹² which are comparable to those of a positive drug control (indomethacin).

Our previous experimental results showed that the intraperitoneal injection of Longxuejie (Resina Dracaenae Cochinchinensis) from Dracaena cochinchinensis raised the pain thresholds in hot plate test and tail-flick test, and in writhing test it also leads to a reduction in the number of writhing in comparison with the control group. The analgesic effect of Longxuejie (Resina Dracaenae Cochinchinensis) may be partly explained by the inhibition of its flavonoids on the peak amplitudes of capsaicin-induced transient receptor potential vanilloid 1 (TRPV1) current in dorsal root ganglion (DRG) neurons.¹³ As the activation of TRPV1 results in the release of molecules associated with pain transmission, such as calcitonin gene-related peptide (CGRP) and substance P^14,15 and TRPV1 antagonists may deliver broad spectrum efficacy in nociceptive pain by silencing pain signalling pathways,¹⁶ we hope to study in depth the analgesic effects of total flavonoids of dragon's blood (TFDB) from Dracaena cochinchinensis and the possible analgesic mechanism associated with TRPV1.

In the present study, we first observed the effects of TFDB on capsaicin-induced TRPV1 currents in acutely dissociated rat DRG neurons by using whole-cell patch clamp technique. And then the formalin and capsaicin tests of mice were used to evaluate the short-term analgesic effects of TFDB. The complete Freund's adjuvant (CFA)-induced arthritic rat model was used to evaluate the short and long-term analgesic effects of TFDB. Finally, the effects of TFDB on TRPV1 expression in DRG neurons of CFA-inflammatory pain rats were also observed by using Western blot and immunofluorescence experiments.

2. MATERIALS AND METHODS

2.1. Extraction and preparation of TFDB

Dragon's blood, the ethanol extract of the resinous wood of Dracaena cochinchinensis (Lour.) S. C. Chen, was directly purchased from Pharmaceutical factory, Guangxi Institute of Chinese Medicine and Pharmaceutical Science for this investigation. A voucher specimen (No. DC-20140707) was deposited at the sample of College of Pharmacy, South-Central University for Nationalities, China. The dried powder (300 g) of dragon's blood was subjected to column chromatography (CC) (20 cm × 80 cm) over polyamide gel (100-200 mesh) and eluted with an equal degree of 55% ethanol to yield TFDB fraction (flow rate was 2BV/h), the fraction was dried by vacuum concentration to obtain the TFDB with a yield of 43.2% (w/w, dried extract/dried powder of dragon's blood). CC was performed using silica gel (200-300 mesh, 300-400 mesh, Qingdao Haiyang Chemical Group Co., Qingdao, China) and polyamide gel (100-200 mesh, Shanghai National Medicine Group., Shanghai, China). Thin-layer chromatography was performed on silica gel GF₂₅₄ (Qingdao Haiyang Chemical Group Co., Qingdao, China). MCI was purchased from Mitsubishi Chemical Group Co. (Tokyo, Japan). The stock of TFDB was made up by dimethyl sulfoxide (DMSO) previously. It was diluted in the corresponding solvent (external solution, cell culture medium or normal saline) at a minimum of 1:1000 to a final working concentration.

2.2. Isolation and identification from TFDB

TFDB were purified by semi-preparative high-performance liquid chromatographic (HPLC) to provide various compounds. HPLC analysis was performed for the qualitative determination of TFDB. Semi-preparative HPLC and HPLC were performed on a Diones Ultimate 3000 system equipped with a diode array detector and two C18 columns (250 mm × 4.6 mm, 5 μm, Thermo Fisher Scientific, Piscataway, NJ, USA; 250 mm × 10 mm, 5 μm, YMC Co., Ltd., Kyoto, Japan). The multi-components of TFDB were characterized by HPLC with the detector wave length set at 278 nm, and the mobile phase consisted of water containing acteonitrile (A) and 0.5% (v/v) acetic acid (B). A gradient program was used as follows: 0-10 min, 20%-30% A; 10-30 min, 30% A; 30-50 min, 30%-40% A; 50-65 min, 40% A. The flow rate was 0.8 mL/min and temperature of column was 35℃. The structures of compounds were elucidated by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic analysis. Semi-preparative electrospray ionization mass spectrometry (ESI-MS) was recorded on an Agilent G6230 time of flight (TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were obtained on a Bruker DMX-500 spectrometer (Bruker, Karlsruher, Germany) using TMS as an internal reference. Other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.

2.3. Ethics statement

All experimental procedures involving animals were conducted under a protocol approved by the animal research ethics committee of South-Central University for Nationalities in accordance with "Principles of Laboratory Animal Care" and the declaration of Helsinki.¹⁷

2.4. Preparation of DRG neurons from rats

Cultures of DRG neurons from rats were prepared with a modification of the methods described previously.¹⁸ Sprague-Dawley (SD) rats were provided by Hubei Research Centre of Laboratory Animals [grade specific pathogen free (SPF), SCXK (Hubei) 2015-0018]. Half male and half female SD rats (100-150 g, one month old) were stunned by heavy blow on the head and decapitated. L4-L6 lumbar DRGs were dissected and collected in cold (4 ℃) low-sugar Dulbecco's modified Eagle's medium (L-DMEM, Gibco Grand Island, NY, USA). Ganglia were cleaned and incubated for 30 min in 2 mL warm (37 ℃) high-sugar Dulbecco's modified Eagle's medium (H-DMEM, Gibco, Grand Island, NY, USA) containing 1 mg/mL collagenase I (Sigma, St. Louis, MO, USA) and 0.3 mg/mL trypsin 1:250 (Amresco, Solon, OH, USA). Then ganglia were dissociated mechanically by trituration using fire-polished Pasteur pipettes every 5 min. The digestion was halted by the addition of fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and subsequent centrifugation (3 min, 1000 rpm). The ganglia were resuspended in H-DMEM supplemented with 10% FBS, penicillin and streptomycin (100 U/mL and 100 μg/mL, respectively, Gibco, Grand Island, NY, USA). All steps were operated at (22-25) ℃. Cultured DRG neurons from rats were prepared for patch-clamp recordings.

2.5. Patch-clamp recordings

The external solution used to record TRPV1 currents in DRG neurons contained (in mmol/L) NaCl 145.0, N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES) 10.0, D-glucose 10.0, KCl 5.0 and MgCl₂ 2.0. The pH was adjusted to 7.4 with 1 mol/L NaOH and the osmolarity was adjusted to (315-325) mOsmol/L with sucrose. The patch pipette solution contained (in mmol/L) KCl 140.0, ethylene glycol tetraacetic acid (EGTA) 10.0, HEPES 10.0, Na₂ATP 10.0, CaCl₂ 1.0 and MgCl₂ 2.0. The pH was adjusted to 7.3 with 1 mol/L potassium hydroxide (KOH) and the osmolarity was adjusted to (305-315) mOsmol/L with sucrose. Capsaicin was dissolved into stock solution of 1 mmol/L by dehydrated alcohol, and then diluted into 1 μmol/L by the external solution. Capsazepine, the competitive antagonist of TRPV1 receptor, was prepared at concentration of 10 μmol/L using the same method. HEPES, EGTA, Na₂TAP, capsaicin and capsazepine were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade unless otherwise stated (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China).

The DRG neuron suspension from adult rat was plated on 5 mm × 5 mm glass coverslips in small Petri dishes (diameter 35 mm). The glass coverslips were treated previously with poly-lysine (1.0 mg/mL) and dried. And then dissociated neurons were cultured at 37℃ in a water-saturated atmosphere with 5% CO₂. After 2 h, a microscopic glass on which several isolated DRG neurons from adult rats attached was taken in a special chamber designed to perfuse the external solution. Patch pipettes were fabricated with glass capillary tubes (World Precision Instruments, LLC., Sarasota, FL, USA) on a 2-stage puller (PC-97, Sutter, Germany). Recording pipettes had resistances of 2-5 MΩ when filled with the patch pipette solution. Only smaller DRG neurons were used for experiments as this kind of cells usually expressed a higher percentage of TRPV1 receptors.¹⁹

An amplifier (EPC-10, HEKA, Stuttgart, Germany) was used to carry out whole-cell patch clamp experiments. After 1-5 GΩ seal formation between a pipette and DRG cell membrane, the membrane was ruptured and the membrane capacitance was appropriately compensated. In voltage-clamp mode, membrane potential was clamped at -60 mV. Signals were sampled at 5-10 kHz and filtered at 2 kHz. Drugs were delivered to the cell by using a rapid solution exchange system (DAD-12, ALA, Farmingdale, NY, USA). The application pipette tip was positioned ∼30 μm away from the recorded neuron. The application of each drug was driven by air pressure came from an air compressor and controlled by the corresponding valve. And then peristaltic pump took away the excess liquid that soaked the recorded neurons from the cell. TRPV1 currents were induced by using the application of 1 μmol/L capsaicin. After the stable TRPV1 currents were recorded, the effects of various concentrations of TFDB (1, 3, 10, 30, 100, 300, 1000 mg/L) were evaluated using co-application with 1 μmol/L capsaicin. Before each recording, the residual drug was washed out with the external solution for at least 30 s. Patch-clamp experimental data were analysed by Pulse Fit software (Version 8.8, HEKA, Stuttgart, Germany) and Igor Pro software (Version 6.1, WaveMetrics, Los Angeles, CA, USA). Inhibition percentages (%) were calculated from the differences between the amplitude of drug-treated current and the drug-untreated current. The concentration-response curve was fitted with Hill's equation.

2.6. Formalin and capsaicin tests

Half male and half female Kunming mice (4-5 weeks old) weighing 18-22 g were purchased from Hubei Research Center of Laboratory Animals [Grade SPF, SCXK (Hubei) 2015-0018] and acclimatized to the laboratory conditions at least one week before experimentation. The mice were housed at a temperature of (22 ± 2) ℃ under a 12 h light/12 h dark cycle (lights on at 7:00 AM, lights off at 7:00 PM) maintained (5 animals per cage) with food and water ad libitum. Separate groups of mice were used for each analgesic test, and the animals were used only once in the experiments.

The procedure of formalin test used was essentially the same as that described by Dubuisson and Dennis.²⁰ The dorsal surface of the right hind paw of mice were injected with 20 μL of 5% formalin. And then the animals were immediately placed in a glass cylinder, 20 cm in diameter. Only licking or biting of the injected paw was defined as a nociceptive response. The amount of time spent on licking or biting of the injected paw was monitored and recorded over a period of 0-5 min (early phase of licking) and 15-30 min (late phase of licking). Totally 50 mice were randomly divided into 5 groups by random number table method. Five groups of 10 mice were respectively treated with the same volume of normal saline (10 mL/kg), aspirin (20 mg/kg) and three doses of TFDB (2, 8, and 20 mg/kg) 30 min before the formalin injection.

The method of capsaicin test followed the procedure of Correa et al.²¹ Capsaicin (20 μL) were injected intraplantarly (1.6 μg per paw) in the ventral surface of the right hind paw of mice. Animals were observed individually for 5 min following capsaicin injection. The amount of time spent on licking the injected paw was recorded with a chronometer and was considered as indicative of nociception. The animal was observed for 5 min, immediately after the capsaicin injection. Six groups of 10 mice were respectively treated with the same volume of normal saline (20 μL per paw), capsazepine (30 μg per paw) and four doses of TFDB (0.02, 0.04, 0.2, 2 mg per paw) 30 min before the capsaicin injection.

2.7. CFA-induced inflammatory pain models

Adult male SD rats (180-200 g) provided by Hubei Research Center of Laboratory Animals [Grade SPF, SCXK (Hubei) 2015-0018] were housed in cages and given free access to food and water. Animals were maintained on a 12 h light-dark cycle. Inflammatory hyperalgesia was induced by injecting CFA (50%, 0.1 mL) into the plantar surface of the right hind paw of the rats.²² Classical signs of acute inflammation including edema, redness and heat were most intense from days 1 to 3 after injection, and lasted more than 4 weeks. After 48 h of CFA injection, all rats were placed in Plexiglas chambers resting on a temperature-regulated (30 ℃) glass surface for behavioral testing. After 30 min of adaptation to the environment, rats were removed from these chambers for the intraperitoneal injection of drugs and were then returned to their respective chambers. Through the glass surface, a radiant heat source (8 V, 50 W projector bulb) was focused onto the plantar surface of the hind paw. The rat's paw-withdrawal latency to this stimulus was recorded. Each animal's latency score was an average of two trials, which were separated by at least 5 min. In all rats, both the ipsilateral and contralateral hind paws were tested.

Aspirin and normal saline were used for the positive and negative controls, respectively. Three groups of 10 rats were respectively treated with the same volume of normal saline (10 mL/kg), Aspirin (20 mg/kg) and TFDB (20 mg/kg). In order to further verify whether the analgesic effects of TFDB were due to the inhibition on TRPV1, capsaicin, the TRPV1 receptor agonist, was used to resist its analgesic effects caused by inhibiting TRPV1. The fourth group of 10 rats were treated with the same volume of the combination of capsaicin (3 mg/kg) and TFDB (20 mg/kg). The drugs were continuously injected intraperitoneally for 3 days once daily at the above doses 48 h after CFA injection. The rats’ paw-withdrawal latencies of 30, 60, 90 min after the first day of drug administration were observed to confirm the effects of short-term administration on CFA-induced thermal hyperalgesia. The rat's paw-withdrawal latencies after the drug administration on the second and third days were also observed to confirm the effects of long-term administration on CFA-induced thermal hyperalgesia.

2.8. Detection of TRPV1 expression with Western blot and immunofluorescence

After the last day of drug administration, CFA-induced inflammatory pain rats were anesthetized and killed. Right L4-L6 DRG neurons were removed immediately. Half of DRG neurons were lysed with radioimmun-oprecipitation assay (RIPA) buffer (Wuhan goodbio technology CO., Ltd., Wuhan, China) including protease inhibitor cocktail (Roche, Basel, Switzerland) and protein concentrations were determined using a bicinchoninic acid assay (BCA) Protein Assay Kit (Wuhan goodbio technology Co., Ltd., Wuhan, China). Proteins (30-50 μg) were separated in 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Blotting membranes were afterwards blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibody (1:500 mouse monoclonal antibody to TRPV1-C-terminal; 1:10 000 mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase, GAPDH46) overnight at 4 ℃. After 3 western washing buffer (TBS-T) washes, the membranes are incubated with 1:10 000 goat anti-mouse IgG H&L (horse radish peroxidase, HRP) for 60 min at room temperature. After 3 TBS-T washes, membranes were scanned on an Odyssey plate reader (Licor, St Charles, MO, USA) at channel 700 or 800. Analysis of Western blot signals were performed by using Alpha Ease FC (Alpha Innotech, Minneapolis, MN, USA) software. The standardization ratio of TRPV1 to GAPDH band density was used to calculate the change in TRPV1 expression. The above procedure of Western blotting detection was essentially the same as that described by Zhu et al.²³

The other half DRG neurons were post-fixed with 4% paraformaldehyde. According to a defined paraffin-embedding procedure, DRG tissue specimens were embedded in paraffin and sectioned into 7-µm tissue sections. Paraffin embedded sections were deparaffinized with xylene and rehydrated through graded concentrations of alcohol. Antigenic repair was performed using citric acid at pH 6.0 for 2 min. Subsequently, sections were rinsed with tris buffered saline (TBS) at room temperature for 3 times, each time for 5 min. And then sections were incubated with 10% Donkey Serum at room temperature for 20 min. Later, the serum was removed and tissues were incubated overnight with primary antibodies at 4 ℃. The next day, sections were taken out and rewarmed for 15min. Then TBS was used to wash the sections for 3 times, 5 min each time. After that, sections were incubated for 30 min at 37 ℃ with Alexa Fluor 594 donkey anti-rabbit lgG (H + L) secondary antibodies (1:400; Life technologies, Gaithersburg, MD, USA). Then, the sections were washed with TBS again. After washing, cell coverslips were sealed with anti-fluorescence quenching agent and photographed under a fluorescence microscope (Olympus, DP72, Tokyo, Japan). Five random visual field images at × 400 magnification for each sample were selected for measurement. The mean optical density (MOD) of each visual field was calculated by ImageJ software.

2.9. Statistical analysis

Data were expressed as the mean ± standard error of the mean (SEM) and analysed though SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA) and GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA). All statistical analysis were performed using one-way analysis of variance (ANOVA) with Dunnett-t test, or two-way ANOVA with Duncan's multiple comparison. Statistical significance was determined as P < 0.05.

3. RESULTS

3.1. Isolated fractions and compounds of TFDB

The powdered TFDB (240 g) were subjected to CC over silica gel (200-300 mesh) and eluted with a gradient of CH₂Cl₂-MeOH (9: 1, 8: 2, 7: 3, v/v) to yield 3 fractions (Fractions 1-3). Fraction 1 (10.87 g) was subjected to a CC over silica gel (300-400 mesh) with cyclohexane-acetone (9: 1, 8: 2, 7: 3, 0: 1, v/v) to yield 5 sub-fractions (Fractions 1.1-1.5). Fraction 1.3 (2 g) was subjected to CC over silica gel (300-400 mesh) with cyclohexane-acetone (6:4, v/v) to yield 5 sub-fractions (Fractions 1.3.1-1.3.5). Fraction 1.3.1 (300 mg) was purified by semi-preparative HPLC using MeCN-H₂O (55: 45, v/v, 278 nm) to provide compounds 1 (6.6 mg), 2 (2.7 mg), 3 (3.0 mg). Fraction 1.3.2 (350 mg) was purified by semi-preparative HPLC using MeCN-H₂O (55: 45, v/v, 278 nm) to provide compounds 4 (9.0 mg), 5 (10.8 mg), 6 (12.5 mg), 7 (5.5 mg), and 8 (8.7 mg). Fraction 4 (42.72 g) was subjected to CC over silica gel (200-300 mesh) and eluted with a gradient of CH₂Cl₂-MeOH (9: 1, 8: 2, 7: 3, 6: 4, 0: 1, v/v) to yield 5 fractions (Fractions 4.1-4.5). Fraction 4.2 (8 g) was subjected to CC over silica gel (300-400 mesh) and eluted with a gradient of CH₂Cl₂-MeOH-H₂O (9: 1: 0.1, 8: 2: 0.2, v/v) to yield 3 fractions (Fractions 4.2.1-4.2.3). Fractions 4.2.2 was subjected to MCI with MeOH-H₂O (3: 7, 4: 6, v/v) to yield 4 sub-fractions (Fractions A.1-A.4). Fraction A.1 (1 g) was subjected to CC over silica gel (300-400 mesh) and eluted with a gradient of CH₂Cl₂-MeOH (9: 1, 8: 2, v/v) to yield 3 sub-fractions (Fractions A.1.1-A.1.3). Fraction A.1.1 (200 mg) and Fraction A.1.2 (100 mg) were purified by semi-preparative HPLC using MeCN-H₂O (40: 60-65: 35, v/v, 40 min, 278 nm) to yield compounds 9 (28.7 mg), 10 (10.5 mg), 11 (31.2 mg), and 12 (8.5 mg).

3.2. Structures and contents of the compounds from TFDB

Twelve compounds are Kumatakemin B (1), Resveratrol (2), Loureirin D (3), 7,4'-Dihydroxydihydrohomoiso-flavone (4), Loureirin C (5), 4,4'-Dihydroxy-2,6-dime-thoxydihydrochalcone (6), 7,4'-Dihydroxyflavan (7), 3,4'-dihydroxy-5-methoxystilbene (8), Cochinchinenin B (9), Loureirin A (10), Loureirin B (11) and Pterostilbene (12). Their structures were shown in Figure1A. Twelve compounds from TFDB were identified by comparing their retention time and measuring the contents with those of the identified compounds (Figure 1B). The contents of 1-12 from TFDB were 4.65%, 3.49%, 0.28%, 1.38%, 2.71%, 3.06%, 0.27%, 4.48%, 3.15%, 2.62%, 4.17% and 9.16%, respectively.

Figure 1. Chemical structures of 1-12 and the typical HPLC chromatographic profile of TFDB.

A: chemical structures of 1-12 from TFDB. Twelve compounds are Kumatakemin B (1), Resveratrol (2), Loureirin D (3), 7,4'-Dihydroxydihydrohomoisoflavone (4), Loureirin C (5), 4,4'-Dihydroxy-2,6-dimethoxydihydrochalcone (6), 7,4'-Dihydroxyflavan (7), 3,4'-dihydroxy-5-methoxystilbene (8), Cochinchinenin B (9), Loureirin A (10), Loureirin B (11) and Pterostilbene (12); B: typical HPLC chromatographic profile of TFDB. The typical HPLC chromatographic profile of TFDB. HPLC peaks: 1 (11.633 min); 2 (12.093 min); 3 (14.460 min); 4 (17.073 min); 5 (19.760 min); 6 (21.413 min); 7 (24.827 min); 8 (27.147 min); 9 (28.387 min); 10 (50.587 min); 11 (52.080 min); 12 (62.320 min). HPLC: high performance liquid chromatography; TFDB: total flavonoids of dragon's blood.

3.3. TFDB inhibits capsaicin-induced TRPV1 currents in DRG neurons

At a holding potential of -60 mV, the application of 1 μmol/L capsaicin evoked some inward currents ranging from 0.5 to 8 nA in medium and small DRG neurons (diameter < 30 μm) under the mode of voltage-clamp. After the currents reached a peak, DRG neurons were washed out immediately with the external solution for 30 s. And then with the co-application of 1 μmol/L capsaicin and 10 μmol/L capsazepine, a common characteristic of capsaicin-induced TRPV1 currents that they could be almost completely blocked by 10 μM capsazepine was observed (Figure 2A). The peak amplitudes of capsaicin-induced TRPV1 currents were reduced after the application of 1, 3, 10, 30, 100 μg/mL TFDB. TFDB reversibly decreased the amplitude of capsaicin-induced TRPV1 currents in a concentration-dependent manner (Figure 2B). Only TFDB at 1 μg/mL has no inhibitory effect on TRPV1 currents (P > 0.05). Other concentrations of TFDB can produce different degrees of inhibitory effects on TRPV1 currents (P < 0.05). In order to determine the maximum efficacy of TFDB, the inhibition rates of 300 and 1000 μg/mL TFDB on capsaicin-induced TRPV1 currents were also observed. The relationship of concentration and response was fitted with Hill's equation. Unfortunately, the maximum inhibition rate of TFDB on capsaicin-induced TRPV1 currents can only reach (61.5 ± 1.4) μg/mL, which indicated that the modulation of TRPV1 by TFDB may also be manifested in other aspects. The half inhibitory concentration (IC₅₀) of TFDB was (16.7 ± 1.6) μg/mL (Figure 2C).

Figure 2. Effects of TFDB on capsaicin-induced TRPV1 currents in DRG neurons.

A: effects of capsazepine on capsaicin-induced TRPV1 currents in DRG neurons. Capsazepine (10 μmol/L) inhibited rapidly the current induced by 1 μmol/L capsaicin, consistent with activation of TRPV1 receptor. B: concentration-response curves for the inhibition on the capsaicin-induced TRPV1 currents of TFDB. Data points represented the percent change in peak amplitude in the presence of TFDB (each concentration n = 7, one-way analysis of variance with Dunnett-t test, P < 0.05). The data points are fitted with Hill's Equation. C: effect of TFDB on capsaicin-induced TRPV1 currents in DRG neurons. TFDB (1, 3, 10, 30, 100 μg/mL) inhibited rapidly and reversibly the current induced by 1 μmol/L capsaicin. CAP: capsaicin; CPZ: capsazepine; TFDB: total flavonoids of dragon's blood; TRPV1: transient receptor potential vanilloid 1; DRG: dorsal root ganglion.

3.4. Analgesic effects of TFDB in formalin and capsaicin tests

In the formalin test, the time spent in licking the injected paw with formalin in the first phase and the second phase was (54 ± 4) s and (81 ± 4) s respectively in control mice. The results depicted in Figure 3A showed that TFDB and aspirin did not reduce the time spent in licking the injected paw in the first phase (P > 0.05). However, TFDB caused marked and dose-related inhibition in the second phase of formalin-induced licking (Figure 3B). Aspirin was also anti-nociceptive in the second phase. Results showed TFDB could relieve formalin-induced acute inflammatory pain.

Figure 3. Effects of TFDB in different pain models (formalin test, capsaicin test and CFA-induced inflammation pain rats).

A: effect of TFDB against formalin-induced licking in the first phase in mice; B: effect of TFDB against formalin-induced licking in the second phase in mice; C: effect of TFDB against capsaicin-induced licking in mice. D: short term analgesic effect of TFDB on CFA-induced inflammatory pain rats. Paw-withdrawal latencies before and after 48 h CFA administrated. After CFA-induced inflammatory pain model rats were successfully established, paw-withdrawal latencies after 30, 60 and 90 min drug administrated; E: Long term analgesic effect of TFDB on CFA-induced inflammatory pain rats. Paw-withdrawal latencies before and after 48 h CFA administrated. After CFA-induced inflammatory pain model rats were successfully established, paw-withdrawal latencies after 1, 2 and 3 d drug administrated. TFDB: total flavonoids of dragon's blood; CFA: complete freunds adjuvant; SEM: standard error of the mean; ANOVA: analysis of variance. Each column or point represented the mean of the values obtained in 10 mice and the error bars indicate the SEM. Statistical analysis was performed using one-way ANOVA with Dunnett-t test. ^aP < 0.01 vs control, ^bP < 0.05 vs control.

In the capsaicin test, the time spent in licking the injected paw in 5 min was (39.9 ± 3.4) s in control mice. Capsazepine resulted in a significant inhibition of capsaicin-induced pain in mice (P < 0.05). TFDB decreased the time spent in licking the injected paw with capsaicin in a dose-dependent manner (Figure 3C). Therefore, the results showed TFDB could relieve capsaicin-induced acute neuropathic pain.

Effects of TFDB on CFA-induced inflammation pains Before CFA administrated, the paw-withdrawal latency of rats was (5.1 ± 0.5) s, and after 48 h CFA administrated, the paw-withdrawal latency of rats was (12.4 ± 1.7) s. The comparison of data before and after CFA injection (P < 0.001) showed that the CFA-induced inflammation pain model was successful.

On the first day of administration of normal saline, there was no difference in the paw-withdrawal latency of rats before and after the administration of 30, 60 and 90 min (P > 0.05). On the first day of administration of Aspirin, it showed the analgesic efficacy after the administration of 30, 60 and 90 min (P < 0.001). TFDB also showed a similar analgesic effect with aspirin (P < 0.001). On the first day of administration of the combination of capsaicin and TFDB, it only showed the analgesic efficacy after the administration of 60 and 90 min (P < 0.01). After 30min drug administration, there was no difference in the paw-withdrawal latency of rats between normal saline group and the combination group (P > 0.05). After 60 min (P < 0.05) and 90 min (P < 0.001) drug administration, there was significant difference in the paw-withdrawal latency of rats between TFDB group and the combination group. The above results showed that TFDB can produce long-term analgesic effect, which can be partially offset by capsaicin (Figure 3D). After the administration of 90 min, TFDB could increase the paw-withdrawal latency by 80.7% ± 9.6%. Therefore, on the second and third day of administration, the observation of paw-withdrawal latencies was performed 90 min after the administration.

After 48 h of CFA injection, CFA-induced inflammatory paws displayed a hyperactive response and maintained it for 3 d (Figure 3E). Aspirin treatment plays a role in the development of inflammation pain. When the rats were injected aspirin, thermal hyperalgesia were attenuated (P < 0.001). TFDB treated rats displayed a decreased withdrawal to thermal stimuli, suggesting that it had a curative effect on CFA-induced inflammation pain (P < 0.001). Three days after continuous administration, TFDB showed a stronger analgesic effect than aspirin (P < 0.05). On the first day of drug administration, the analgesic effects of TFDB could be mostly weakened by 1 mmol/L capsaicin (P < 0.05). On the second day (P < 0.01) and third day (P < 0.001) of drug administration, the attenuation effect of capsaicin on the analgesic effect of TFDB decreased gradually.

3.5. Regulation of TRPV1 expression in DRG neurons of CFA-induced inflammation pain rats by TFDB

TRPV1 expression levels in DRG neurons of CFA-induced inflammation pain rats which were given 3-day intraperitoneal injection of TFDB were significantly lower than those with injection of normal saline (P < 0.001). On the contrary, there was no difference in TRPV1 expression levels in DRG neurons of CFA-induced inflammation pain rats between 3-d intraperitoneal injection of aspirin group and normal saline (P > 0.05) (Figure 4A, 4B). The results of immunofluorescence and Western blot were consistent (Figure 4C, 4D). The above results showed that CFA-induced hyperalgesia was accompanied by TRPV1 up-regulation in DRG neurons but TFDB could eliminate the up-regulation of TRPV1 expression in DRG neurons.

Figure 4. Effects of TFDB on TRPV1 expression in DRG neurons of CFA-induced inflammation pain rats.

A: an example of Western blot images for TRPV1 and GAPDH of all drug administration groups; B: standardization ratio of TRPV1 to GAPDH band densities in DRG neurons of various drugs-treated CFA-induced inflammation pain rats (Six batches for all the groups). C: fluorescence optical intensity of TRPV1 protein (Five random visual field images at × 400 magnification for each sample); D: Immunofluorescence showed the expression of TRPV1 receptor protein in DRG neurons. Scale bar = 50 μm; D1: DRG neurons immunofluorescence image of normal saline group; D2: DRG neurons immunofluorescence image of aspirin group; D3: DRG neurons immunofluorescence image of TFDB group. DRG neurons were derived from three groups of rats. Three groups of 10 rats were respectively treated with the same volume of normal saline (10 mL/kg), aspirin (20 mg/kg) and TFDB (20 mg/kg). TFDB: total flavonoids of dragon's blood; TRPV1: transient receptor potential vanilloid 1; DRG: dorsal root ganglion; CFA: complete freunds adjuvant; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ANOVA: analysis of variance; SEM: standard error of the mean. Data were expressed as the mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett-t test. ^aP < 0.001 vs control.

4. DISCUSSION

TRPV1 receptor is identified to be a member of the transient receptor potential (TRP) family of cation channels.²⁴ Studies on isolated cells demonstrated that capsaicin and other natural substances, as well as some mediators of inflammation and ischemia, activated TRPV1.²⁵ TRPV1, expressed by both peptidergic and non-peptidergic DRG neurons at the origin of C and A_y primary afferent fibers,^18,19 is considered an important marker for nociceptors.²⁶ Functionally, TRPV1 is important for thermal nociception and inflammatory hyperalgesia and allodynia and has also been implicated in neuropathic pain.^27,28 TRPV1 selective antagonists are particularly potent and efficacious in preclinical pain models associated with low pH and thermal hyperalgesia such as acute and chronic inflammation.²⁹

In this study, we observed that TFDB could block the capsaicin-induced TRPV1 currents in DRG neurons, which suggested that some compounds of TFDB should be the TRPV1 antagonists. Consistent with the above results, dragon's blood and its three components also could block the capsaicin-induced TRPV1 currents in DRG neurons.¹³ The three components are cochinchinenin A, cochinchinenin B and loureirin B. Two of the components (cochinchinenin B and loureirin B) can be found in the 12 compounds isolated from the TFDB (Figure 1). By calculating and comparing the contents of cochinchinenin B and loureirin B in TFDB, the IC₅₀ for blocking the capsaicin-induced TRPV1 currents of cochinchinenin B, loureirin B and TFDB, it could be found that loureirin B played an important role in antagonizing TRPV1 receptors among the compounds of TFDB, but it was not excluded that other potential compounds of TFDB may antagonize TRPV1 or may be ineffective against TRPV1.

It has been reported mice lacking TRPV1 receptors exhibit reduced thermal noxious response and capsaicin-induced paw licking.³⁰ Injection of the capsaicin selective antagonist, either capsazepine or ruthenium red (50-500 pmol/paw), in association with capsaicin, dose-dependently attenuated capsaicin-induced nociception.³¹ As TFDB could block the TRPV1 receptor, it is not surprising that TFDB effectively attenuated capsaicin-induced nociception. However, the co-injection of capsazepine or ruthenium red (50-500 pmol/paw), together with formalin, concentration-dependently attenuated the early phase of formalin response and caused partial but significant, though non-concentration dependent, inhibition of the late phase of the formalin-induced pain.³¹ This result was not consistent with that TFDB did not affect the paw licking of mice in the first phase of the formalin test. The possible reason is that the nociceptors involved in the first phase of the formalin test are not completely blocked by TFDB. Although the behavioral symptoms of this phenomenon (pain which induces licking or biting responses) are similar regardless of the kind of chemical used, it is assumed that these two substances have distinct mechanisms of action. Capsaicin is an agonist of TRPV1 channels, whereas formalin not only stimulates transient receptor potential ankyrin type 1 (TRPA1) channels but it has also other pronociceptive mechanisms.^32,33

The observation results of Sałat et al ³⁴ are consistent with our experimental results. Nonselective TRPV1 antagonist capsazepine and selective TRPV1 antagonist SB-366791 did not have antinociceptive properties either in the early or in the late phase of the formalin test. The similar experimental result also appeared on other flavonoids as TRPV1 antagonists. Treatment with the water-soluble flavonone hesperidin methyl chalcone (HMC) inhibited the second phase of formalin (1.5%, 25 μL)-induced paw flinching and licking, but not the first phase.³⁵ Formalin induces a biphasic nociceptive response. The first phase of formalin test (neurogenic phase, 0-5 min) depends on TRPA1 channels activation in primary nociceptive neurons,^36,37 while the second phase (10-30 min) is associated with the release of local endogenous mediators responsible for sensitization of primary and spinal sensory neurons and subsequent inflammatory reactions.^38,39 Similar to HMC, the TFDB inhibition of the second phase of formalin test is consistent with its inhibition on acetic acid-induced writhing, which are also dependent on the release of inflammatory cytokines.⁴⁰ Thus, it is plausible that TFBD exhibits its protective effect in the acetic acid-induced writhing and second phase of formalin test by targeting inflammatory cytokines production without affecting TRPA1 channels. Our results proved that TFDB mainly relieves the inflammatory pain.

This is also confirmed by the analgesic effect of TFDB on CFA-induced inflammatory pain model rats. It is well known that thermal hyperalgesia occurs within several days after CFA injection, i.e. in the acute phase of inflammatory pain. In the present study, we observed that after 48 h of the CFA injection, thermal hyperalgesia exhibited and lasted for 3 d in the CFA inflammatory rats relative to the control rats, which was consistent with the previous report.⁴¹ TFDB respectively showed good short-term and long-term analgesic effects after the first administration and for three days of administration. Furthermore, as the TRPV1 antagonist, their effects were in close agreement with the phenotype observed in TRPV1 knockout mice when challenged with inflammatory agents such as CFA.^27,42 Eriodictyol, a flavonoid antagonist of the TRPV1 receptor, also reduced the thermal hyperalgesia and mechanical allodynia elicited by CFA paw injection.⁴⁰ These all indicated that the analgesic effect of TFDB on inflammatory pain should be closely related to TRPV1 receptor.

It has been reported that TRPV1 expression increased following CFA injection from day 1 to 21 and a shift of TRPV1 expression from small to medium DRG neurons over the observation period was seen, which suggested that TRPV1 could play an important role in the early stage, but not the late stage, of CFA inflammatory nociception.²² The action period of TFDB was just the active phase of TRPV1 in the CFA-induced inflammatory response. Results of Western blot and immunofluorescence experiments showed TRPV1 expression in the DRG neurons of the TFDB-treated CFA-induced inflammation pain rats in the third day of TFDB administration decreased compared to the normal saline-treated CFA-induced inflammation pain rats, which further confirmed the above conclusion. TRPV1 can be regulated by changing the open probability of the channel (Po) and/or by controlling the available number of channels (N) in the plasma membrane of cells. Both these processes can result in a decrease of nociceptor excitability and in the reduction of pain.⁴³

From the comprehensive analysis of all experimental results, TFDB can not only interfere with the activation of TRPV1 receptor, but also induce a long-term down-regulation of TRPV1 expression. Since TFDB is a mixture of several flavonoids, it is certain that one or more flavonoids will have analgesic effects as specific antagonists of TRPV1. The expression of TRPV1 has been shown to be unregulated during nerve injury-induced thermal hyperalgesia and diabetic neuropathy.^44,45 Therefore, the down-regulation of TRPV1 receptor expression by flavonoids in TFDB may be related to the signal pathway specifically involved in the inflammatory pain process. It can be speculated that the antagonistic effect of TFDB on TRPV1 receptor may also be related to these signal pathways. The extraction and separation of flavonoid monomer chemical components from TFDB and the study of their effects on TRPV1 activation related signal pathways in inflammatory pain are likely to have profound implications for drug development and clinical applications in the management of nociceptive and neuropathic pain conditions.

Funding Statement

Supported by High Level Talents Project of Affiliated Hospital of Youjiang Medical University for Nationalities: Study of Soft-Du’an Capsule’s Mechanism and Efficacy of Regulating TRPV1 Pashways in Relieving Oral and Maxillofacial Trigeminal Neuralgia (No. YYFYR20213002); Innovative Group Project of Natural Science Foundation of Hubei Province: Study on the Mechanisms of Pain Signal Transduction and Drug Analgesia (No. 2020CFA025)