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Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons
European Journal of Pharmacology ] (]]]]) ]]]–]]]
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons Q1
Li-Si Wei a,1, Su Chen a,1, Xian-Ju Huang b, Jing Yao c, Xiang-Ming Liu a,n a
College of Biological & Medical Engineering, South-Central University for Nationalities, Minyuan Road 708, Wuhan, Hubei 430074, China College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, China c School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430030, China b
a r t i c l e i n f o
abstract
Article history: Received 26 May 2012 Received in revised form 20 January 2013 Accepted 31 January 2013
The effects of dragon’s blood and its components cochinchinenin A, cochinchinenin B, loureirin B as well as various combinations of the three components on capsaicin-induced TRPV1 receptor currents were studied in acutely dissociated DRG neurons using both voltage and current whole-cell patch clamp technique. The results indicated that dragon’s blood and its three components concentrationdependently reduce the peak amplitudes of capsaicin-induced TRPV1 receptor currents. There was no significant difference between the effects of dragon’s blood and the combination wherein the three components were present in respective mass fractions in dragon’s blood. The respective concentrations of the three components used alone were all higher than the total concentration of three components used in combination when the percentage inhibition of the peak amplitude was 50%. The proportion of three components was adjusted and the total concentration reduced, the resulting combination still inhibit the currents with a lower IC50 value, and inhibit capsaicin-induced membrane depolarization on current clamp. The combination of three components not only increase the capsaicin IC50 value, but also reduce the capsaicin maximal response. These result suggested that analgesic effect of dragon’s blood may be partly explained on the basis of silencing pain signaling pathways caused by the inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons and could be due to the synergistic effect of the three components. Antagonism of the capsaicin response by the combination of three components is not competitive. The analgesic effect of dragon’s blood was also confirmed using animal models. & 2013 Published by Elsevier B.V.
Keywords: Dragon’s blood Dorsal root ganglion (DRG) neurons Transient receptor potential cation channel Subfamily V Member 1 (TRPV1) Interaction Analgesic drugs
1. Introduction Dragon’s blood from Dracaena cochinchinensis is one of the renowned traditional medicines. It is a multi-component mixture with analgesic activity and has got several therapeutic uses (Gupta et al., 2008; Zhong, 2010). Its molecular targets and active components should be determined with strict scientific method to explore lead compounds for novel analgesic drugs from dragon’s blood. In previous studies, we have observed that modulation of dragon’s blood on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in dorsal root ganglion (DRG) neurons using patch clamp technique. Three compounds, i.e., cochinchinenin A, cochinchinenin B, and loureirin B (Fig. 1) were the active components in dragon’s blood interfering with pain messages. The combination of the three
n
Corresponding author. Tel.: þ86 27 67843892; fax: þ 86 27 67841231. E-mail address: [email protected] (X.-M. Liu). 1 Contributed equally to this work.
components had the same effect as dragon’s blood (Liu et al., 2006). Moreover, extracellular microelectrode recordings were used to indicate that the above combination of the three components was material basis for inhibition of dragon’s blood on evoked discharges of wide dynamic range neurons in spinal dorsal horn of rats (Guo et al., 2008). During the past decade, one relatively new molecular target, TRPV1 receptor has attracted significant attention in the pharmaceutical industry. TRPV1 receptor is widely distributed in the central and peripheral nervous systems. It is activated not only by chemical factors such as capsaicin and acids, but also by a physical factor such as hot temperature (Z42 1C) (Helliwell et al., 1998; Patapoutian et al., 2003). TRPV1 receptor is considered an integrator of noxious stimuli in peripheral nociceptor terminals and therefore may be at a crossroads for pain transmission pathways. The logical strategy for development of novel analgesics is to target the beginning of pain transmission pathway and aim potential treatments directly at nociceptors. The TRPV1 receptor antagonists may deliver broad spectrum efficacy in nociceptive pain via silencing pain signaling pathways (Kym et al., 2009). Therefore, the discovery of new TRPV1
0014-2999/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ejphar.2013.01.052
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Fig. 1. Three compounds of dragon’s blood, cochinchinenin A, cochinchinenin B, and loureirin B cochinchinenin A: 40 -Hydroxy-2,6-dimethoxy dihydrochalcone 17 (C17H18O4). cochinchinenin B: 6-hydroxy-7-methoxy-3-(40 -hydroxybenzyl) 18 chromone (C18H20O5). loureirin B: 1-(4-Hydroxyphenyl)-3-(2,4,6-trimethoxy19 phenyl)propan-1-one (C17H18O4). 20 21 receptor antagonists becomes an attractive destination in the 22 treatment of pain. 23 Nowadays, a number of small molecule TRPV1 receptor 24 antagonists are undergoing clinical trials in patients with inflam25 matory or neuropathic pain. Clinical development of TRPV1 26 receptor antagonists is, however, facing new challenges, some 27 of these antagonists showed worrisome adverse reactions (e.g., 28 hyperthermia) in men, leading to their withdrawal from the 29 clinical trials (Gavva et al., 2008; Gunthorpe and Chizh, 2009; 30 Joshi and Szallasi, 2009). So, it has significance to search for new 31 TRPV1 receptor antagonists among the active components in 32 dragon’s blood, which has displayed very low toxicity and no 33 Q3 apparent side-effects (Zeng et al., 2000). 34 Our previous studies have indicated that two components, 35 cochinchinenin B and loureirin B weakly inhibit capsaicin36 induced TRPV1 receptor current, respectively (Wang et al., 2007, 37 2008). The result suggested that the pharmacological actions of 38 dragon’s blood and the combinations of its active components on 39 TRPV1 receptor currents should be further investigated. Thus we 40 may gain a more comprehensive understanding of the mechanism 41 behind analgesic activity of dragon’s blood and further identify its 42 active components, and provide lead compounds for the devel43 opment of novel analgesic drugs. 44 45 46 2. Materials and methods 47 48 2.1. Preparation of solutions and drugs 49 50 Dragon’s blood, cochinchinenin A, cochinchinenin B, and lour51 eirin B were provided by Guangxi Institute of Traditional Medical 52 and Pharmaceutical Sciences. Dragon’s blood and its three compo53 nents were authenticated and extracted, respectively by Prof. Lu 54 Wenjie. The purity of each component is 98%. The mass fractions of 55 cochinchinenin A, cochinchinenin B and loureirin B in the final 56 products of dragon’s blood are 22%, 11%, and 5%, respectively. 57 An appropriate amount of dragon’s blood was dissolved in 58 external solution to obtain a 0.005% (m/v) concentration. Accord59 ing to the mass fractions of the three components in the final 60 products of dragon’s blood, the solutions of the individual 61 components, the concentrations of which corresponded to the 62 above concentration of dragon’s blood solution, were prepared. 63 The solutions of various combinations of the components were 64 also prepared (Table 1). 65 To explore the possibility of decrease in the total concentra66 tion of the three components used in the combination I without
Table 1 Preparation of solutions and drugs. Components
Solutions Individual
Cochinchinenin A (mmol/l) Cochinchinenin B (mmol/l) Loureirin B (mmol/l)
Combination
1
2
3
A
B
C
I
II
38 / /
/ 19 /
/ / 8
38 19 /
38 / 8
/ 19 8
38 19 8
3.8 1.9 8
influence of the efficacy, another solution of combination II was shown in Table 1. To obtain the concentration–response curves of the individual components and combinations of the three components, the above relevant solutions were diluted and added with 1 mmol/l capsaicin to observe the actions. 2.2. Working solutions for patch clamp experiments External solution used to record capsaicin receptor currents in DRG neurons contained (mmol/l): NaCl 145.0; HEPES 10.0; D-glucose 10.0; KCl 5.0; CaCl2 2.0; MgCl2 1.0. The external solution was adjusted to pH 7.4 with 1 mol/l NaOH. Internal solution contained (mmol/l): KCl 140.0; EGTA 10.0; HEPES 10.0; Na2TAP 10.0; CaCl2 1.0; MgCl2 2.0. The internal solution was adjusted to pH 7.3 with 1 mol/l KOH. Capsaicin was dissolved into stock solution of 10 mmol/l by dehydrated alcohol, and then diluted into 1 mmol/l by external solution described above. Capsazepine, the competitive antagonist of TRPV1 receptor, was prepared at concentration of 10 mmol/ l using the same method. HEPES, EGTA, Na2TAP, capsaicin and capsazepine were obtained from Sigma Company (USA). All other chemicals were of analytical grade unless otherwise stated. 2.3. Whole-cell patch-clamp experiments One-month-old male or female SPF Wistar rats (100–150 g) were provided by Laboratory Animal Center, Tongji Medical College of Huazhong University of Science and Technology. The animals were cared in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China on November 14, 1988. After the rats were decapitated, DRG neurons were dissected and taken out. A volume of 5 ml cell suspension was obtained using enzymatic dissociation (Huang et al., 2007). The suspension was filtrated through 200 mesh gauze and transferred into a 3.5 mm culture dish to keep still. The solution was replaced by the external solution twice after the cells were adhered to the disk. Whole-cell patch clamp recordings were carried out using an EPC-9 amplifier (HEKA, Germany). After 3–5 GO seal formation between a pipette and DRG membrane, the membrane was ruptured and membrane capacitance was compensated (Liu et al., 2006). Only smaller DRG neurons were used for experiments as this kind of cells usually expressed a higher percentage of TRPV1 receptor currents (Bevan and Szolcsa´nyi, 1990; Caterina et al., 1997). TRPV1 receptor currents were evoked by given stimuli at a holding potential. After control currents were recorded, capsaicin (1 mmol/l) was firstly delivered to the cell over 7–10 s using a rapid solution exchange system (DAD-12, ALA, USA). The residual drug was then washed out with the external solution after the peak value emerged. The normal TRPV1 receptor current in DRG was recorded. Then the currents in the presence of drugs were recorded using the same method. Lastly capsaicin (1 mmol/l) was delivered again and the TRPV1 receptor currents were recorded to observe the recovery extent.
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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The intermission of three deliveries should be longer than 30 s (Chen et al., 2004). All the experiments were performed at 22–25 1C.
The percentage inhibition of capsaicin-induced TRPV1 receptor current produced by drug was calculated using Eq. (1): Inhibition% ¼ ½ðIcap Idug Þ=Idug 100%
2.4. Evaluation of analgesic activity of dragon’s blood by using animal models The analgesic activity of dragon’s blood was tested by using hot plate method, tail flick method and acetic acid-induced writhing response in rats. In each test, twenty rats weighing 200–250 g were randomly selected and divided into control group and dragon’s blood group with 10 rats in each group. The rats of control group received an intraperitoneal injection of 2 ml/kg normal saline and the rats of dragon’s blood group received an intraperitoneal injection of 5 mg/kg dragon’s blood (2 ml/kg of injection volume). 2.4.1. Hot plate test Hot plate analgesia meter (Huaibei zhenghua biological equipment Co., Ltd, China) was used. The temperature of the metal surface was kept at (5270.5) 1C. Rats were placed on the hot plate, and the time until either licking of the hind paw or jumping occurred was recorded. Animals were tested before and 30 min after drug administration. A cutoff time of 60 s was set to avoid tissue damage. The maximal possible effect (MPE) was defined as the lack of a nociceptive response during the exposure to the heat stimulus. The percentage of MPE was calculated according to the formula: [(testbaseline)/(cutoff-baseline)] 100, where test and baseline were the latencies obtained before and after drug injection. 2.4.2. Tail-flick test The tail-flick test was carried out in rats using a tail flick analgesia meter (Huaibei zhenghua biological equipment Co., Ltd, China). The tail-flick latency is defined by the time from onset of stimulation to a rapid flick/withdrawal of the tail from a radiant heat source. The heat source was adjusted to produce a baseline tail-flick latency of 3 to 5 s. A cutoff time of 12 s was set to avoid tissue damage. Animals were tested before and 30 min after drug administration. The increase in tail-flick latency was defined as antinociception and calculated as MPE% according to the formula: [(test-baseline)/(cutoff-baseline)] 100, where test and baseline were the latencies obtained before and after drug injection. 2.4.3. Writhing test Thirty minute after an intraperitoneal injection of normal saline or dragon’s blood, the rats were given an intraperitoneal injection of 2% acetic acid solution (2 ml/kg of injection volume). The rats were placed individually into glass beakers and the number of writhing was counted for 15 min. For scoring purposes, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. 2.5. Data analysis Data were analyzed using Pulse Fit software (Version 8.5, HEKA, Germany) and Igor Pro software (Version 4.09, WaveMetrics, USA). Data were expressed as the mean 7S.E.M. The number of cells (n) for each condition was indicated. Unpaired t-test was used to determine the statistical significance of differences between two sets of data. Analysis of variance (ANOVA) with least significant difference (LSD) multiple comparison was used to determine whether there were any significant differences among more than two sets of data. Po0.05 was considered statistically significant.
3
ð1Þ
where Icap is the peak amplitude of TRPV1 receptor currents induced by 1 mmol/l of capsaicin, Idug is the peak amplitude of TRPV1 receptor currents induced by mixed solution of 1 mmol/l of capsaicin and the drug. Drug concentration–response curve was determined by plotting the percentage inhibition of the capsaicin-induced TRPV1 receptor current as a function of the drug concentration. Data were fitted with Hill Eq. (2): Inhibition%=Inhibitionmax % ¼ 1=½1 þðIC50 =cÞh
ð2Þ
where Inhibition% is the percentage inhibition on peak amplitude of capsaicin-induced TRPV1 receptor current produced by drug, Inhibitionmax% is the maximum of Inhibition% also called maximum efficacy. Here the value of Inhibitionmax% was set at 1. c is the drug concentration, h is Hill coefficient and IC50 is the half maximal inhibitory concentration of the drug. 2.7. Assessment of drug interaction Three major interactional effects can occur when two or more drugs interact with each other: additive, antagonistic, and synergistic effects. When the concentration–response curves of drug A and drug B have the same maximal effect Emax and the different Hill coefficients, the concept of dose equivalence lead to the equations of the two different additive isoboles for assessing drug interaction derived by Tallarida (2006). Similarly, based on the concept of dose equivalence, the following equations of the three additive surfaces corresponding to the combined effect Ei produced by three drugs (e.g., cochinchinenin A, cochinchinenin B and loureirin B ) were proposed (Guo et al., 2008) when the concentration–response curves of three drugs have the same Emax and the different Hill coefficients, c ¼ C i C 50 =ðA50 =aÞC 50 =ðB50 =bÞhb =hc
ð3Þ
c ¼ C 50 ½ðAi aÞ=A50 1=ðB50 =bÞhb =ha ha =hc
ð4Þ
c ¼ C 50 ½ðBi bÞ=B50 1=ðA50 =aÞha =hb hb =hc
ð5Þ
where a, b, and c were the concentrations of cochinchinenin A, cochinchinenin B and loureirin B in the combination that produces the specified effect Ei, Ai, Bi and Ci were the concentrations of the three components which produce the same effect Ei when used alone. A50, B50 and C50 were the IC50 value of the three components, respectively. ha, hb and hc were the Hill coefficients of the three components, respectively. In Eq. (3), combination of cochinchinenin A (concentration of a) and cochinchinenin B (concentration of b) yielded the same effect as that of loureirin B whose concentration was (Ci c). In Eq. (4), combination of cochinchinenin B (concentration of b) and loureirin B (concentration of c) yielded the same effect as that of cochinchinenin A whose concentration was (Ai a). In Eq. (5), combination of cochinchinenin A (concentration of a) and loureirin B (concentration of c) yielded the same effect as that of cochinchinenin B whose concentration was (Bi b). The above parameters and concentration– response curves of the three components were obtained, respectively by fitting the Hill Eq. (2) to the corresponding experimental data. Then the values of Ai, Bi and Ci were evaluated according to concentration–response curves. The above parameters, along with Ai, Bi and Ci, were substituted into Eqs. (3)–(5), respectively. In the three-dimensional space whose each axis represents the concentration of one of the three components, the figure describing the positional relationship between the experimental point with coordinates (a, b, c) and three additive surfaces corresponding to the effect Ei was obtained with Matlab software at last.
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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If the experimental point was below all the three additive surfaces, the interactional effect was judged to be synergistic. If the point was among the three additive surfaces, the interactional effect was judged to be additive. If the point was above all the three additive surfaces, the interactional effect was judged to be antagonistic.
3. Results 3.1. Effects of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons At a holding potential of 60 mV, the TRPV1 receptor currents in DRG neurons were evoked by 1 mmol/l of capsaicin without any
voltage stimulation under the mode of voltage clamp. Wash immediately with extracellular fluid after the currents reached a peak. After 30 s washout, the combined solution of 1 mmol/l of capsaicin and 10 mmol/l of capsazepine was ejected on the cell, and record the TRPV1 receptor currents for the second time. The result showed that the currents induced by 1 mmol/l of capsaicin were almost entirely blocked by 10 mmol/l of capsazepine (Fig. 2), consistent with activation of TRPV1 receptor. The effect of dragon’s blood on the TRPV1 receptor current induced by capsaicin (1 mmol/l) was illustrated in Fig. 3A. t-test indicated that the average peak amplitudes of TRPV1 receptor currents were significantly reduced after application of dragon’s blood. The effects of different concentrations of dragon’s blood solutions on capsaicin-induced TRPV1 receptor currents were shown in Table 2. The concentration–response data were fitted to Eq. (2) (Fig. 3B), giving a IC50 value of (0.0000270.000005)% (m/v) and a Hill coefficient of 0.5770.07 (n ¼7).
3.2. Effect of three components used alone on capsaicin-induced TRPV1 receptor currents in DRG neurons
Fig. 2. Effects of capsazepine on capsaicin-induced TRPV1 receptor currents in DRG neurons. 10 mmol/l Capsazepine inhibits rapidly the current induced by 1 mmol/l capsaicin, consistent with activation of TRPV1 receptor. The horizontal bar above each trace indicates period of drug application.
The effects of different concentrations of cochinchinenin A, cochinchinenin B and loureirin B solutions on capsaicin-induced TRPV1 receptor currents were shown in Table 3. The concentration– response data were fitted to Eq. (2), giving IC50 values and Hill coefficients of 46.6473.27 mmol/l and 1.3970.119 (n¼7) for cochinchinenin A, 718.32767.6 mmol/l and 1.4170.166 (n¼7) for cochinchinenin B, and 4.1370.326 mmol/l and 1.7970.242 (n¼ 7) for loureirin B (Fig. 4).
Fig. 3. Effect of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons. (A) Dragon’s blood inhibits rapidly and reversibly the current induced by 1 mmol/l capsaicin. The horizontal bar above each trace indicates period of drug application. (B) Concentration–response curves for inhibition of the capsaicin (1 mmol/l) response by dragon’s blood. Data points, percent change in peak amplitude in the presence of dragon’s blood (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the circle. The data points are fitted with the Hill equation (see Section 2).
Table 2 Effects of dragon’s blood solutions on capsaicin-induced TRPV1 currents. Drug
Concentration (m/v) (%)
Inhibition rate (%) (P value of test) (n¼7)
Dragon’s blood
0.005 0.0005 0.00005 0.000005 0.0000005
90.97 4.7 83.47 4.8 67.47 5.7 29.17 4.7 9.47 3.6
(0.00532) (0.00312) (0.00222) (0.00357) (0.00080)
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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3.3. Effect of combinations of three components on capsaicininduced TRPV1 receptor currents in DRG neurons The total concentration of three components in combination I was 65 mmol/l, among which the proportion of cochinchinenin A, cochinchinenin B and loureirin B was 22:11:5. The total concentration of three components in combination II was 13.7 mmol/l,
Table 3 Effects of three components used alone on capsaicin-induced TRPV1 currents. Components
Cochinchinenin A
Cochinchinenin B
Loureirin B
Concentration (lmol/l)
Inhibition rate (%) (P value of t test) (n¼ 7)
3800 380 190 38 19
73.1 7 5.1 70.2 7 4.8 65.7 7 5.4 30.1 7 2.8 18.2 7 5.3
(0.00312) (0.00212) (0.00562) (0.00807) (0.00030)
19000 3800 750 380 190
80.2 7 5.0 78.3 7 3.4 40.4 7 1.3 25.4 7 3.3 11.7 7 7.3
(0.00448) (0.00712) (0.00021) (0.00905) (0.03807)
83.0 7 4.4 70.7 7 1.4 65.3 7 7.0 44.1 7 2.7 11.9 7 7.6
(0.00782) (0.0183) (0.00176) (0.00137) (0.02676)
200 40 8 4.88 1.44
5
among which the proportion of cochinchinenin A, cochinchinenin B and loureirin B was 2.2:1.1:5. The result of t-test showed that there was no significant difference between the percentage inhibitions of combination I and II on capsaicin-induced TRPV1 receptor currents (P40.05). The two combination solutions were diluted 10, 100 and 1000 times, respectively and the effects of different concentrations of the solutions on capsaicin-induced TRPV1 receptor currents were observed to obtain concentration–response curves. All the solutions showed some degree of inhibition against the currents (Table 4 and Fig. 5). The concentration–response data were fitted to Eq. (2), giving IC50 values and Hill coefficients of 2.5570.43 mmol/l and 0.5370.01 (n¼7) for combination I, 1.0170.29 mmol/l and 0.467 0.06 (n¼7) for combination II (Fig. 5). 3.4. Comparison for the pharmacological effect of dragon’s blood and combinations of its components Three kinds of solutions combined with any two of the components inhibit capsaicin-induced TRPV1 receptor currents in DRG neurons to some extent. The percentage inhibitions of combination A, B, and C solutions on peak amplitudes of TRPV1 receptor currents were (35.2271.5)%, (68.37 75.0)%, and (66.6674.9)% (n ¼7), respectively. ANOVA test demonstrated that there were significant difference among the percentage inhibitions of combination I, A, B, C, and 0.005% (m/v) dragon’s blood solutions on capsaicin-induced TRPV1 receptor currents (F ¼40.71, Po0.01). However, LSD test indicated that there was no significant difference between effects of combination I and 0.005% (m/v) of dragon’s blood (P40.05) (Table 5). For purposes of comparison, the concentration response curves of combination I, II and dragon’s blood were displayed in the same Fig. 6 by converting molarity to percent mass/volume. The IC50 value of combination I and II were (0.00008170.000029)% (m/v) and (0.00003770.000020)% (m/v), respectively. 3.5. Effect of combination II on capsaicin-induced membrane depolarization of DRG neuron under the condition of current clamp
Fig. 4. Concentration–response curves of three components. Data points, percent change in peak amplitude in the presence of cochinchinenin A, cochinchinenin B, and loureirin B, respectively (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the point. Each set of data points is fitted with the Hill equation (see Section 2).
To verify whether the components of dragon’s blood inhibit capsaicin-induced TRPV1 receptor currents, combination II was selected to observe the capsaicin-induced changes in membrane potential of DRG neuron before and after applying the components. Capsaicin evoked a membrane depolarization and elicited action potential firing at 1 mmol/l (Fig. 7, top). Membrane potential recovered back to the normal level after capsaicin washout, the average of resting membrane potential was 57.676.3 mV (n¼7). However, 1 mmol/l capsaicin did not evoke action potential firing in the presence of combination II (Fig. 7, middle), the membrane potential of depolarization was 25.775.4 mV (n¼7). Capsaicin (1 mmol/l) was able to evoke membrane depolarization and action potential firing after washout for 90 s (Fig. 7, bottom). In addition, combination II alone failed to change membrane potential or cause action potential firing in all observed DRG neurons. In summary,
Table 4 Effects of combinations of three components on capsaicin-induced TRPV1 currents in DRG neurons. Combination
Cochinchinenin A (lmol/l)
Cochinchinenin B (lmol/l)
Loureirin B (lmol/l)
Inhibition rate (%) (P value of t test) (n¼7)
Combination I Diluted 10-fold Diluted 100-fold Diluted 1000-fold Combination II Diluted 10-fold Diluted 100-fold Diluted 1000-fold
38 3.8 0.38 0.038 3.8 0.38 0.038 0.0038
19 1.9 0.19 0.019 1.9 0.19 0.019 0.0019
8 0.8 0.08 0.008 8 0.8 0.08 0.008
84.8 7 4.9 59.6 7 4.0 35.5 7 6.1 10.6 7 3.6 79.3 7 6.6 49.1 7 6.6 32.5 7 5.0 9.9 7 4.4
(0.00004) (0.00001) (0.00039) (0.00107) (0.00212) (0.00562) (0.00807) (0.00030)
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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Fig. 5. Effects of combination I and II on capsaicin-induced TRPV1 receptor currents in DRG neurons. (A) Combination I and II inhibit rapidly and reversibly the current induced by 1 mmol/l capsaicin. The horizontal bar above each trace indicates the period of drug application. (B) Concentration–response curves for inhibition of the capsaicin (1 mmol/l) response by combination I and II. Data points, percent change in peak amplitude in the presence of combination I or II (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the point. Each set of data points is fitted with the Hill equation (see Section 2). Table 5 Comparisons for the percentage inhibition of dragon’s blood and that of the combinations. Concentration of individual component in the combination (lmol/l)
Drug
Combination Combination Combination Combination
I A B C
Cochinchinenin A
Cochinchinenin B
Loureirin B
38 38 38 /
19 19 / 19
8 / 8 8
Inhibition rates (%) 9yD yC9 in least square difference (LSD) method
84.81 7 4.9 35.22 7 1.5 68.37 7 5.0 66.66 7 4.9
0.05187 o 0.09468 0.54198 40.09468 0.23910 40.09468 0.22120 40.09468
As Table 5 showed, the differences between the percentage inhibition produced by each combinations and 0.005% (m/v) of dragon’s blood are compared with LSD value (0.09468) at the 5% level, where yD and yC are the percentage inhibition of dragon’s blood and combinations, respectively.
combination II was able to decrease capsaicin evoked depolarization in rat DRG neurons and its effects were partially reversible by washout, these results were consistent with those under voltageclamp condition. 3.6. Effect of combination II on the concentration–response curve of capsaicin
Fig. 6. Comparison of the concentration–response curves for dragon’s blood, combinations I, and combination II. Here the concentration is expressed in the unit of the percent mass/volume. Data points, percent change in peak amplitude in the presence of dragon’s blood, combinations I, and combination II (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the point. Each set of data points is fitted with the Hill equation (see Section 2).
Due to a lower IC50 value of combination II than that of combination I in inhibition of capsaicin-induced TRPV1 receptor currents, effect of combination II on concentration–response curves of capsaicin was investigated. Under the absence and presence of combination II, the peak amplitude of capsaicininduced TRPV1 receptor currents would not increase when the concentration of capsaicin was above 10 mmol/l (P40.05), indicating a maximal effect of capsaicin. Furthermore, all peak amplitudes of capsaicin-induced TRPV1 receptor currents were standardized by the peak amplitude of TRPV1 receptor current induced 0.5 mmol/l capsaicin. Thus the cell-to-cell variability of capsaicin-induced TRPV1 receptor currents were eliminated. Pooled data were used to construct the concentration–response curves for capsaicin in the absence and presence of combination II (Fig. 8). In the absence of combination II, the maximum value of capsaicin-induced standard currents was 2.28 70.20, the IC50 value was 0.6970.06 mmol/l, and the Hill coefficient was 1.5470.23 (n ¼7). In the presence of combination II, the maximum value of capsaicin-induced standard currents was
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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Fig. 7. Effect of combination II on capsaicin-induced membrane potential of DRG neuron. Top, in control, 1 mmol/l capsaicin was applied and induced membrane depolarization enough to evoke action potential firings. The action potential firings were disappeared upon washout. Middle, in the presence of combination II, capsaicin was applied with the same protocol as in control. The capsaicin evoked depolarization was significantly decreased, there is no action potential firing. Bottom, after 1990s washout of combination II and capsaicin, capsaicin was applied again in the same protocol as in control, the effect of capsaicin was partially recovered.
1.64 70.08, the IC50 value was 1.95 70.28 mmol/l, and Hill coefficient was 1.5570.28 (n ¼7). The result indicated that combination II reduced the capsaicin-induced standard currents by 27.9%. 3.7. The effects of combination II inside and outside plasma membrane To explore the binding site of components of dragon’s blood, the effects of combination II (0.38 mmol/l of cochinchinenin A, 0.19 mmol/l of cochinchinenin B, and 0.8 mmol/l of loureirin B) on capsaicin-induced TRPV1 receptor currents was tested when it was applied to the intracellular side and extracellular side, respectively. The inhibitory effect on capsaicin-induced TRPV1 receptor currents was observed after extracellular application of combination II, and the extracellular percentage inhibition was 49.1 76.6% (n ¼7). In contrast, combination II produced no or only a slight effect on capsaicin-induced TRPV1 receptor currents after 20 min intracellular dialysis by pipette solution, and the intracellular percentage inhibition was 2.872.1% (n ¼7). In addition, the percentage inhibition was 51.3175.1% (n¼ 7) when combination II was simultaneously applied to the intracellular side and extracellular side, which similar to the extracellular percentage inhibition (P 40.05). 3.8. Assessment of interaction among the three components The concentrations of the three components that produce the effect of combination I (84.81% inhibition of TRPV1 receptor current) when used alone were determined from the concentration–response curves of the individual components (Fig. 4), and the resulting three concentration values, the concentration values (38, 19, 8) of the three components in combination I, and the parameter values reported above were substituted into Eqs. (3)–(5), respectively. According to the three equations, the additive surface drawings
Fig. 8. Antagonism of the capsaicin response by combination II is not competitive. Concentration–response curves for capsaicin in the absence and presence of combination II: 3.8 mmol/l of cochinchinenin A, 1.9 mmol/l of cochinchinenin B, and 8 mmol/l of loureirin B. All responses are normalized to the peak current (n) induced by 0.5 mmol/l capsaicin. Data points, normalized peak currents (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the circle. Each set of data points is fitted with the Hill equation (see Section 2).
corresponding to 84.81% inhibition were then generated with Matlab software (Fig. 9). To statistically determine the positional relation between the experimental point Q1 (38, 19, 8) and additive surfaces, the coordinate values of theoretical additive points corresponding to the point Q1 were calculated together with the associated 95% confidence intervals. The theoretical additive points are the intersections of the additive surfaces and the ray which
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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Fig. 9. Additive surfaces corresponding to the combined effects produced by cochinchinenin A, cochinchinenin B, and loureirin B. (A) The positional relation between point Q1 and the additive surfaces corresponding to the effect of combination I (EI ¼ 84.81%) and (B) The positional relation between point Q2 and the additive surfaces corresponding to the effect of combination II (EII ¼ 79.26%).
Table 6 Coordinate values of theoretical additive points corresponding to the experimental points Q1 and Q2. coordinate value of Q1 (mmol/l)
Coordinate value of point Q1’s additive points [95% confidence interval] (mmol/l)
Inhibition rate of combination I, EI ¼ 84.81% 38 19 8 Inhibition rate of combination II, EII ¼ 79.26% Coordinate value of Q2 (mmol/l) 3.8 1.9 8
216.2426 [151.170, 281.314] 108.121 [79.459, 118.005] 45.524 [31.825, 59.224]
Coordinate value of point Q2’s additive points [95% confidence interval] (mmol/l) 15.372 15.965 [12.647, 18.096] [13.751, 18.180] 7.686 7.982 [6.323, 9.048] [6.875, 9.090] 32.362 33.612 [26.626, 38.097] [28.949, 38.275]
Table 7 The effects of 5 mg/kg dragon’s blood in three animal models. Group
MPE of hot plate test (%)
MPE of tail-flick test (%)
Number of writhing in writhing test
Control Dragon’s blood
2.6 7 3.1 70.8 7 8.6a
1.77 2.6 81.1 7 7.8a
34.2 74.3 10.4 74.3a
a
233.288 [182.516, 284.06] 116.6443 [91.258, 142.030] 49.113 [38.424, 59.800]
197.46 [158.918, 236.011] 98.732 [75.585, 140.657] 41.571 [33.456, 49.686]
14.987 [12.962, 17.011] 7.493 [6.481, 8.505] 31.552 [27.289, 35.8143]
3.9. Effects of dragon’s blood in three animal models The results of hot plate test, tail flick test and writhing test presented in Table 7 showed that 5 mg/kg Dragon’s Blood significantly raised the pain thresholds in hot plate test and tailflick test, and in writhing test it also lead to a 69.6% reduction in the number of writhing in comparison with the control group.
Compared with control, P o 0.01.
4. Discussion starts at the origin and also passes through the point Q1. A similar calculation was performed for the case of combination II (79.26% inhibition of TRPV1 receptor current) to obtain the coordinate values of theoretical additive points corresponding to the experimental point Q2 (3.8, 1.9, 8) (Table 6). As shown in Table 6, the coordinate values of Q1 and Q2 were all statistically smaller than those of their corresponding theoretical additive points. Therefore, the points Q1 and Q2 were both below the respective corresponding three additive surfaces (Fig. 9), which indicated that interactional effect of the three components in both combination I and II on capsaicin-induced TRPV1 receptor currents were both synergistic.
The present study first observed the inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons using whole-cell patch clamp technique. 0.005% (m/v) solution of dragon’s blood completely suppress capsaicin-induced TRPV1 receptor currents, indicating that it block up the transmission of pain message for primary sensory neurons via direct action on TRPV1 receptor. The effects of three components (cochinchinenin A, cochinchinenin B, and loureirin B) and their combinations on the capsaicin-induced TRPV1 receptor currents in DRG neurons of rats were also observed. The result demonstrated that each individual component and the combinations of any two out of
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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the three components reduce capsaicin-induced TRPV1 receptor currents to some extent, but none of them had an equally inhibition as that of 0.005% (m/v) dragon’s blood. According to the operational definition of the material basis for the efficacy of traditional medicine (Liu et al., 2006), the three individual components and the combinations of two components were regarded as the effective component/combinations but not the corresponding material basis of dragon’s blood. However, the combination of three components prepared in terms of the mass fraction of three components in final products of dragon’s blood, which guaranteed that the quantity of each component in the combination was quite in agreement with that in dragon’s blood, generate similar effect as that of 0.005% (m/v) dragon’s blood. Therefore it was considered that the effective combination containing cochinchinenin A, cochinchinenin B, and loureirin B was the material basis of dragon’s blood for the modulation of capsaicin-induced TRPV1 receptor currents, and this modulation intervene in pain messages and thereby result in pain relief. This remarkable synergistic interaction among three components may explain the mode of their modulation on the capsaicin-induced TRPV1 receptor currents in DRG neurons. The present study also discovered that the effect of loureirin B was stronger than that of cochinchinenin A or cochinchinenin B, suggesting the chief role of loureirin B in the pharmacological effect of the combination. New combination could be made by altering the proportion of the three components, i.e., reducing the relative proportions of cochinchinenin A and cochinchinenin B but increasing the relative proportion of loureirin B. It still generate a strong effect with lower total concentration of the three components. The results showed that the combination II (namely the new combination above-mentioned) had a higher potency than combination I and the components displayed higher synergistic reaction. Thus it should be presumed that the proportion of the combination should be further optimized to obtain more efficient combinations. Our previous study has revealed that dragon’s blood modulate TTX-R sodium currents and the material basis of dragon’s blood for the modulation is the combination of three components, namely cochinchinenin A, cochinchinenin B and loureirin B, which synergistically modulate TTX-R sodium currents (Liu et al., 2006). All these were in accordant with the present study on capsaicin-induced TRPV1 receptor currents. It is noticeable that the IC50 value of dragon’s blood which inhibits TTX-R sodium current is three orders of magnitude higher than that of dragon’s blood which inhibits capsaicin-induced TRPV1 receptor currents, which suggests that in DRG neurons the first target of dragon’s blood is TRPV1 receptor rather than TTX-R sodium channel. The other difference is that although the three components inhibit TTX-R sodium and capsaicin-induced TRPV1 receptor currents, the IC50 value of loureirin B which inhibits TTX-R sodium current was of the same order of magnitude as that of the other two components, whereas the IC50 value of loureirin B inhibiting capsaicin-induced TRPV1 receptor currents was two to three orders of magnitude lower than that of the other two components, which indicated that different mechanisms of dragon’s blood as influence on TTX-R sodium channel and TRPV1 receptor. The addition of combination II do not only make the concentration–response curve of capsaicin-induced TRPV1 receptor currents move rightwards, but also reduce the maximal TRPV1 receptor currents induced by capsaicin, demonstrating that the inhibition of dragon’s blood and its three components were not competitive. The receptor-ligand binding model was used to simulate the interaction of loureirin B and TRPV1 receptor. The result indicated that the interaction of loureirin B and TRPV1 receptor was fitted with the simple model of one ligand and one receptor site. There may be only one binding site on TRPV1 receptor for loureirin B (Wang et al., 2007). In addition, our
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67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 Uncited reference Q2 124 125 Zheng and He (1999). 126 127 128 Acknowledgments 129 130 This work was supported by National Natural Science Founda- 131 tion of China [Grant 30973961]; Young Chenguang Foundation 132 preliminary experiment showed that loureirin B inhibit the inward current induced by extracellular lower pH value of DRG neurons. Since TRPV1 receptor currents was also activated by extracellular lower pH value, and the area of vanillin activation sites on TRPV1 receptor was not the same as proton binding area (Wang and Cao, 2008), it was presumed that the function site of loureirin B would be different from that of capsaicin and H þ . It should bind to TRPV1 receptor on this site and cause the structural change of TRPV1 receptor, decreasing the affinity and activity of excitomotor, thus producing antagonistic effect. The mechanism may be similar to AMG9810, which made TRPV1 receptor in inactive or turn-off condition even vanillin activation sites on TRPV1 receptor had been binded (Gavva et al., 2005). Many evidence indicated that besides capsaicin and resiniferatoxin, capsazepine, the competitive antagonist of TRPV1 receptor, could not antagonize the biological effect of other agonists (Perkins and Campbell, 1992). But the specific agonists, capsaicin and resiniferatoxin were unable to be synthesized in the body of vertebrates. The activation of TRPV1 receptor in the body may mainly depend on the nociceptive thermal stimulus and the conduction of the acid stimulation in tissue. Therefore, TRPV1 receptor antagonist should block up the activation of the receptor induced by thermal stimulus as well as H þ , but not antagonize the binding site of capsaicin (Yan and Zhang, 2002). Unlike capsazepine, the three components of dragon’s blood antagonize TRPV1 receptor without competing with capsaicin for the same site and would not lead to hyperthermia when they are used in the long run. Consequently, it may be a promising candidate in developing new analgesic drugs targeting TRPV1 receptor. The present study shows that combination II is only working outside plasma membrane, this may be because the intracellular application of combination II affects neither the capsaicin response nor the inhibition effect of extracellularly applied combination II on the capsaicin response, which suggests that the binding sites of the three components in combination II are located entirely outside of plasma membrane. This result may provide the clue for further biophysical studies of channel/drug interaction. As for the possible molecular mechanisms of the synergistic effects of the three active compounds, at present we can only propose the following hypotheses: by reason of the different chemical structures of the three components, each component has its own binding site on TRPV1 receptor. When the three components bound to their respective binding sites on TRPV1 receptor, the conformational changes of the binding sites impel the combined inhibition effects of the three components on TRPV1 receptor to be synergistically enhanced. There was no antagonistic interaction could be observed probably because the concentration of each component was too low to bind all of its own sites on TRPV1 receptor. In conclusion, our study further proved that the combination of three components from dragon’s blood resin according to the proportion by weight synergistically contributed to the corresponding analgesia material basis of dragon’s blood resin via the inhibition of capsaicin-induced TRPV1 receptor currents.
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of Wuhan [Grant 201150431075]; Academic Team of South Central University for Nationalities [Grant XTZ09010]; and Innovative Foundation Project for Students of South-Central University for Nationalities [Grant KYCX110010E]. References Bevan, S., Szolcsa´nyi, J., 1990. Sensory neuron-specific actions of capsaicin: mechanisms and applications. Trends. Pharmacol. Sci. 11, 331–333. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824. Chen, S.C., Chang, T.J., Wu, F.S., 2004. Competitive inhibition of the capsaicin receptor-mediated current by dehydroepiandrosterone in rat dorsal root ganglion neurons. J. Pharmacol. Exp. Ther. 311, 529–536. Gavva, N.R., Treanor, J.J.S., Garami, A., Fang, L., Surapaneni, S., Akrami, A., Alvarez, F., Bak, A., Darling, M., Gore, A., 2008. Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. PAIN 136, 202–210. Gavva, N.R., Tamir, R., Qu, Y., Klionsky, L., Zhang, T., Immke, D., Wang, J., Zhu, D., Vanderah, T.W., Porreca, F., 2005. AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties. J. Pharmacol. Exp. Ther. 313, 474–484. Gunthorpe, M.J., Chizh, B.A., 2009. Clinical development of TRPV1 antagonists: targeting a pivotal point in the pain pathway. Drug. Discov. Today 14, 56–67. Guo, M., Chen, S., Liu, X.M., 2008. Material basis for inhibition of dragon’s blood on evoked discharges of wide dynamic range neurons in spinal dorsal horn of rats. Sci. China, Ser. C Life Sci. 51, 1025–1038. Gupta, D., Bleakley, B., Gupta, R.K., 2008. Dragon’s blood: botany, chemistry and therapeutic uses. J. Ethnopharmacol. 115, 361–380. Helliwell, R.J.A., Mclatchie, L.M., Clarke, M., Winter, J., Bevan, S., McIntyre, P., 1998. Capsaicin sensitivity is associated with the expression of the vanilloid
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Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons Q1
Li-Si Wei a,1, Su Chen a,1, Xian-Ju Huang b, Jing Yao c, Xiang-Ming Liu a,n a
College of Biological & Medical Engineering, South-Central University for Nationalities, Minyuan Road 708, Wuhan, Hubei 430074, China College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, China c School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430030, China b
a r t i c l e i n f o
abstract
Article history: Received 26 May 2012 Received in revised form 20 January 2013 Accepted 31 January 2013
The effects of dragon’s blood and its components cochinchinenin A, cochinchinenin B, loureirin B as well as various combinations of the three components on capsaicin-induced TRPV1 receptor currents were studied in acutely dissociated DRG neurons using both voltage and current whole-cell patch clamp technique. The results indicated that dragon’s blood and its three components concentrationdependently reduce the peak amplitudes of capsaicin-induced TRPV1 receptor currents. There was no significant difference between the effects of dragon’s blood and the combination wherein the three components were present in respective mass fractions in dragon’s blood. The respective concentrations of the three components used alone were all higher than the total concentration of three components used in combination when the percentage inhibition of the peak amplitude was 50%. The proportion of three components was adjusted and the total concentration reduced, the resulting combination still inhibit the currents with a lower IC50 value, and inhibit capsaicin-induced membrane depolarization on current clamp. The combination of three components not only increase the capsaicin IC50 value, but also reduce the capsaicin maximal response. These result suggested that analgesic effect of dragon’s blood may be partly explained on the basis of silencing pain signaling pathways caused by the inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons and could be due to the synergistic effect of the three components. Antagonism of the capsaicin response by the combination of three components is not competitive. The analgesic effect of dragon’s blood was also confirmed using animal models. & 2013 Published by Elsevier B.V.
Keywords: Dragon’s blood Dorsal root ganglion (DRG) neurons Transient receptor potential cation channel Subfamily V Member 1 (TRPV1) Interaction Analgesic drugs
1. Introduction Dragon’s blood from Dracaena cochinchinensis is one of the renowned traditional medicines. It is a multi-component mixture with analgesic activity and has got several therapeutic uses (Gupta et al., 2008; Zhong, 2010). Its molecular targets and active components should be determined with strict scientific method to explore lead compounds for novel analgesic drugs from dragon’s blood. In previous studies, we have observed that modulation of dragon’s blood on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in dorsal root ganglion (DRG) neurons using patch clamp technique. Three compounds, i.e., cochinchinenin A, cochinchinenin B, and loureirin B (Fig. 1) were the active components in dragon’s blood interfering with pain messages. The combination of the three
n
Corresponding author. Tel.: þ86 27 67843892; fax: þ 86 27 67841231. E-mail address: [email protected] (X.-M. Liu). 1 Contributed equally to this work.
components had the same effect as dragon’s blood (Liu et al., 2006). Moreover, extracellular microelectrode recordings were used to indicate that the above combination of the three components was material basis for inhibition of dragon’s blood on evoked discharges of wide dynamic range neurons in spinal dorsal horn of rats (Guo et al., 2008). During the past decade, one relatively new molecular target, TRPV1 receptor has attracted significant attention in the pharmaceutical industry. TRPV1 receptor is widely distributed in the central and peripheral nervous systems. It is activated not only by chemical factors such as capsaicin and acids, but also by a physical factor such as hot temperature (Z42 1C) (Helliwell et al., 1998; Patapoutian et al., 2003). TRPV1 receptor is considered an integrator of noxious stimuli in peripheral nociceptor terminals and therefore may be at a crossroads for pain transmission pathways. The logical strategy for development of novel analgesics is to target the beginning of pain transmission pathway and aim potential treatments directly at nociceptors. The TRPV1 receptor antagonists may deliver broad spectrum efficacy in nociceptive pain via silencing pain signaling pathways (Kym et al., 2009). Therefore, the discovery of new TRPV1
0014-2999/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ejphar.2013.01.052
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Fig. 1. Three compounds of dragon’s blood, cochinchinenin A, cochinchinenin B, and loureirin B cochinchinenin A: 40 -Hydroxy-2,6-dimethoxy dihydrochalcone 17 (C17H18O4). cochinchinenin B: 6-hydroxy-7-methoxy-3-(40 -hydroxybenzyl) 18 chromone (C18H20O5). loureirin B: 1-(4-Hydroxyphenyl)-3-(2,4,6-trimethoxy19 phenyl)propan-1-one (C17H18O4). 20 21 receptor antagonists becomes an attractive destination in the 22 treatment of pain. 23 Nowadays, a number of small molecule TRPV1 receptor 24 antagonists are undergoing clinical trials in patients with inflam25 matory or neuropathic pain. Clinical development of TRPV1 26 receptor antagonists is, however, facing new challenges, some 27 of these antagonists showed worrisome adverse reactions (e.g., 28 hyperthermia) in men, leading to their withdrawal from the 29 clinical trials (Gavva et al., 2008; Gunthorpe and Chizh, 2009; 30 Joshi and Szallasi, 2009). So, it has significance to search for new 31 TRPV1 receptor antagonists among the active components in 32 dragon’s blood, which has displayed very low toxicity and no 33 Q3 apparent side-effects (Zeng et al., 2000). 34 Our previous studies have indicated that two components, 35 cochinchinenin B and loureirin B weakly inhibit capsaicin36 induced TRPV1 receptor current, respectively (Wang et al., 2007, 37 2008). The result suggested that the pharmacological actions of 38 dragon’s blood and the combinations of its active components on 39 TRPV1 receptor currents should be further investigated. Thus we 40 may gain a more comprehensive understanding of the mechanism 41 behind analgesic activity of dragon’s blood and further identify its 42 active components, and provide lead compounds for the devel43 opment of novel analgesic drugs. 44 45 46 2. Materials and methods 47 48 2.1. Preparation of solutions and drugs 49 50 Dragon’s blood, cochinchinenin A, cochinchinenin B, and lour51 eirin B were provided by Guangxi Institute of Traditional Medical 52 and Pharmaceutical Sciences. Dragon’s blood and its three compo53 nents were authenticated and extracted, respectively by Prof. Lu 54 Wenjie. The purity of each component is 98%. The mass fractions of 55 cochinchinenin A, cochinchinenin B and loureirin B in the final 56 products of dragon’s blood are 22%, 11%, and 5%, respectively. 57 An appropriate amount of dragon’s blood was dissolved in 58 external solution to obtain a 0.005% (m/v) concentration. Accord59 ing to the mass fractions of the three components in the final 60 products of dragon’s blood, the solutions of the individual 61 components, the concentrations of which corresponded to the 62 above concentration of dragon’s blood solution, were prepared. 63 The solutions of various combinations of the components were 64 also prepared (Table 1). 65 To explore the possibility of decrease in the total concentra66 tion of the three components used in the combination I without
Table 1 Preparation of solutions and drugs. Components
Solutions Individual
Cochinchinenin A (mmol/l) Cochinchinenin B (mmol/l) Loureirin B (mmol/l)
Combination
1
2
3
A
B
C
I
II
38 / /
/ 19 /
/ / 8
38 19 /
38 / 8
/ 19 8
38 19 8
3.8 1.9 8
influence of the efficacy, another solution of combination II was shown in Table 1. To obtain the concentration–response curves of the individual components and combinations of the three components, the above relevant solutions were diluted and added with 1 mmol/l capsaicin to observe the actions. 2.2. Working solutions for patch clamp experiments External solution used to record capsaicin receptor currents in DRG neurons contained (mmol/l): NaCl 145.0; HEPES 10.0; D-glucose 10.0; KCl 5.0; CaCl2 2.0; MgCl2 1.0. The external solution was adjusted to pH 7.4 with 1 mol/l NaOH. Internal solution contained (mmol/l): KCl 140.0; EGTA 10.0; HEPES 10.0; Na2TAP 10.0; CaCl2 1.0; MgCl2 2.0. The internal solution was adjusted to pH 7.3 with 1 mol/l KOH. Capsaicin was dissolved into stock solution of 10 mmol/l by dehydrated alcohol, and then diluted into 1 mmol/l by external solution described above. Capsazepine, the competitive antagonist of TRPV1 receptor, was prepared at concentration of 10 mmol/ l using the same method. HEPES, EGTA, Na2TAP, capsaicin and capsazepine were obtained from Sigma Company (USA). All other chemicals were of analytical grade unless otherwise stated. 2.3. Whole-cell patch-clamp experiments One-month-old male or female SPF Wistar rats (100–150 g) were provided by Laboratory Animal Center, Tongji Medical College of Huazhong University of Science and Technology. The animals were cared in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China on November 14, 1988. After the rats were decapitated, DRG neurons were dissected and taken out. A volume of 5 ml cell suspension was obtained using enzymatic dissociation (Huang et al., 2007). The suspension was filtrated through 200 mesh gauze and transferred into a 3.5 mm culture dish to keep still. The solution was replaced by the external solution twice after the cells were adhered to the disk. Whole-cell patch clamp recordings were carried out using an EPC-9 amplifier (HEKA, Germany). After 3–5 GO seal formation between a pipette and DRG membrane, the membrane was ruptured and membrane capacitance was compensated (Liu et al., 2006). Only smaller DRG neurons were used for experiments as this kind of cells usually expressed a higher percentage of TRPV1 receptor currents (Bevan and Szolcsa´nyi, 1990; Caterina et al., 1997). TRPV1 receptor currents were evoked by given stimuli at a holding potential. After control currents were recorded, capsaicin (1 mmol/l) was firstly delivered to the cell over 7–10 s using a rapid solution exchange system (DAD-12, ALA, USA). The residual drug was then washed out with the external solution after the peak value emerged. The normal TRPV1 receptor current in DRG was recorded. Then the currents in the presence of drugs were recorded using the same method. Lastly capsaicin (1 mmol/l) was delivered again and the TRPV1 receptor currents were recorded to observe the recovery extent.
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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The intermission of three deliveries should be longer than 30 s (Chen et al., 2004). All the experiments were performed at 22–25 1C.
The percentage inhibition of capsaicin-induced TRPV1 receptor current produced by drug was calculated using Eq. (1): Inhibition% ¼ ½ðIcap Idug Þ=Idug 100%
2.4. Evaluation of analgesic activity of dragon’s blood by using animal models The analgesic activity of dragon’s blood was tested by using hot plate method, tail flick method and acetic acid-induced writhing response in rats. In each test, twenty rats weighing 200–250 g were randomly selected and divided into control group and dragon’s blood group with 10 rats in each group. The rats of control group received an intraperitoneal injection of 2 ml/kg normal saline and the rats of dragon’s blood group received an intraperitoneal injection of 5 mg/kg dragon’s blood (2 ml/kg of injection volume). 2.4.1. Hot plate test Hot plate analgesia meter (Huaibei zhenghua biological equipment Co., Ltd, China) was used. The temperature of the metal surface was kept at (5270.5) 1C. Rats were placed on the hot plate, and the time until either licking of the hind paw or jumping occurred was recorded. Animals were tested before and 30 min after drug administration. A cutoff time of 60 s was set to avoid tissue damage. The maximal possible effect (MPE) was defined as the lack of a nociceptive response during the exposure to the heat stimulus. The percentage of MPE was calculated according to the formula: [(testbaseline)/(cutoff-baseline)] 100, where test and baseline were the latencies obtained before and after drug injection. 2.4.2. Tail-flick test The tail-flick test was carried out in rats using a tail flick analgesia meter (Huaibei zhenghua biological equipment Co., Ltd, China). The tail-flick latency is defined by the time from onset of stimulation to a rapid flick/withdrawal of the tail from a radiant heat source. The heat source was adjusted to produce a baseline tail-flick latency of 3 to 5 s. A cutoff time of 12 s was set to avoid tissue damage. Animals were tested before and 30 min after drug administration. The increase in tail-flick latency was defined as antinociception and calculated as MPE% according to the formula: [(test-baseline)/(cutoff-baseline)] 100, where test and baseline were the latencies obtained before and after drug injection. 2.4.3. Writhing test Thirty minute after an intraperitoneal injection of normal saline or dragon’s blood, the rats were given an intraperitoneal injection of 2% acetic acid solution (2 ml/kg of injection volume). The rats were placed individually into glass beakers and the number of writhing was counted for 15 min. For scoring purposes, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. 2.5. Data analysis Data were analyzed using Pulse Fit software (Version 8.5, HEKA, Germany) and Igor Pro software (Version 4.09, WaveMetrics, USA). Data were expressed as the mean 7S.E.M. The number of cells (n) for each condition was indicated. Unpaired t-test was used to determine the statistical significance of differences between two sets of data. Analysis of variance (ANOVA) with least significant difference (LSD) multiple comparison was used to determine whether there were any significant differences among more than two sets of data. Po0.05 was considered statistically significant.
3
ð1Þ
where Icap is the peak amplitude of TRPV1 receptor currents induced by 1 mmol/l of capsaicin, Idug is the peak amplitude of TRPV1 receptor currents induced by mixed solution of 1 mmol/l of capsaicin and the drug. Drug concentration–response curve was determined by plotting the percentage inhibition of the capsaicin-induced TRPV1 receptor current as a function of the drug concentration. Data were fitted with Hill Eq. (2): Inhibition%=Inhibitionmax % ¼ 1=½1 þðIC50 =cÞh
ð2Þ
where Inhibition% is the percentage inhibition on peak amplitude of capsaicin-induced TRPV1 receptor current produced by drug, Inhibitionmax% is the maximum of Inhibition% also called maximum efficacy. Here the value of Inhibitionmax% was set at 1. c is the drug concentration, h is Hill coefficient and IC50 is the half maximal inhibitory concentration of the drug. 2.7. Assessment of drug interaction Three major interactional effects can occur when two or more drugs interact with each other: additive, antagonistic, and synergistic effects. When the concentration–response curves of drug A and drug B have the same maximal effect Emax and the different Hill coefficients, the concept of dose equivalence lead to the equations of the two different additive isoboles for assessing drug interaction derived by Tallarida (2006). Similarly, based on the concept of dose equivalence, the following equations of the three additive surfaces corresponding to the combined effect Ei produced by three drugs (e.g., cochinchinenin A, cochinchinenin B and loureirin B ) were proposed (Guo et al., 2008) when the concentration–response curves of three drugs have the same Emax and the different Hill coefficients, c ¼ C i C 50 =ðA50 =aÞC 50 =ðB50 =bÞhb =hc
ð3Þ
c ¼ C 50 ½ðAi aÞ=A50 1=ðB50 =bÞhb =ha ha =hc
ð4Þ
c ¼ C 50 ½ðBi bÞ=B50 1=ðA50 =aÞha =hb hb =hc
ð5Þ
where a, b, and c were the concentrations of cochinchinenin A, cochinchinenin B and loureirin B in the combination that produces the specified effect Ei, Ai, Bi and Ci were the concentrations of the three components which produce the same effect Ei when used alone. A50, B50 and C50 were the IC50 value of the three components, respectively. ha, hb and hc were the Hill coefficients of the three components, respectively. In Eq. (3), combination of cochinchinenin A (concentration of a) and cochinchinenin B (concentration of b) yielded the same effect as that of loureirin B whose concentration was (Ci c). In Eq. (4), combination of cochinchinenin B (concentration of b) and loureirin B (concentration of c) yielded the same effect as that of cochinchinenin A whose concentration was (Ai a). In Eq. (5), combination of cochinchinenin A (concentration of a) and loureirin B (concentration of c) yielded the same effect as that of cochinchinenin B whose concentration was (Bi b). The above parameters and concentration– response curves of the three components were obtained, respectively by fitting the Hill Eq. (2) to the corresponding experimental data. Then the values of Ai, Bi and Ci were evaluated according to concentration–response curves. The above parameters, along with Ai, Bi and Ci, were substituted into Eqs. (3)–(5), respectively. In the three-dimensional space whose each axis represents the concentration of one of the three components, the figure describing the positional relationship between the experimental point with coordinates (a, b, c) and three additive surfaces corresponding to the effect Ei was obtained with Matlab software at last.
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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If the experimental point was below all the three additive surfaces, the interactional effect was judged to be synergistic. If the point was among the three additive surfaces, the interactional effect was judged to be additive. If the point was above all the three additive surfaces, the interactional effect was judged to be antagonistic.
3. Results 3.1. Effects of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons At a holding potential of 60 mV, the TRPV1 receptor currents in DRG neurons were evoked by 1 mmol/l of capsaicin without any
voltage stimulation under the mode of voltage clamp. Wash immediately with extracellular fluid after the currents reached a peak. After 30 s washout, the combined solution of 1 mmol/l of capsaicin and 10 mmol/l of capsazepine was ejected on the cell, and record the TRPV1 receptor currents for the second time. The result showed that the currents induced by 1 mmol/l of capsaicin were almost entirely blocked by 10 mmol/l of capsazepine (Fig. 2), consistent with activation of TRPV1 receptor. The effect of dragon’s blood on the TRPV1 receptor current induced by capsaicin (1 mmol/l) was illustrated in Fig. 3A. t-test indicated that the average peak amplitudes of TRPV1 receptor currents were significantly reduced after application of dragon’s blood. The effects of different concentrations of dragon’s blood solutions on capsaicin-induced TRPV1 receptor currents were shown in Table 2. The concentration–response data were fitted to Eq. (2) (Fig. 3B), giving a IC50 value of (0.0000270.000005)% (m/v) and a Hill coefficient of 0.5770.07 (n ¼7).
3.2. Effect of three components used alone on capsaicin-induced TRPV1 receptor currents in DRG neurons
Fig. 2. Effects of capsazepine on capsaicin-induced TRPV1 receptor currents in DRG neurons. 10 mmol/l Capsazepine inhibits rapidly the current induced by 1 mmol/l capsaicin, consistent with activation of TRPV1 receptor. The horizontal bar above each trace indicates period of drug application.
The effects of different concentrations of cochinchinenin A, cochinchinenin B and loureirin B solutions on capsaicin-induced TRPV1 receptor currents were shown in Table 3. The concentration– response data were fitted to Eq. (2), giving IC50 values and Hill coefficients of 46.6473.27 mmol/l and 1.3970.119 (n¼7) for cochinchinenin A, 718.32767.6 mmol/l and 1.4170.166 (n¼7) for cochinchinenin B, and 4.1370.326 mmol/l and 1.7970.242 (n¼ 7) for loureirin B (Fig. 4).
Fig. 3. Effect of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons. (A) Dragon’s blood inhibits rapidly and reversibly the current induced by 1 mmol/l capsaicin. The horizontal bar above each trace indicates period of drug application. (B) Concentration–response curves for inhibition of the capsaicin (1 mmol/l) response by dragon’s blood. Data points, percent change in peak amplitude in the presence of dragon’s blood (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the circle. The data points are fitted with the Hill equation (see Section 2).
Table 2 Effects of dragon’s blood solutions on capsaicin-induced TRPV1 currents. Drug
Concentration (m/v) (%)
Inhibition rate (%) (P value of test) (n¼7)
Dragon’s blood
0.005 0.0005 0.00005 0.000005 0.0000005
90.97 4.7 83.47 4.8 67.47 5.7 29.17 4.7 9.47 3.6
(0.00532) (0.00312) (0.00222) (0.00357) (0.00080)
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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3.3. Effect of combinations of three components on capsaicininduced TRPV1 receptor currents in DRG neurons The total concentration of three components in combination I was 65 mmol/l, among which the proportion of cochinchinenin A, cochinchinenin B and loureirin B was 22:11:5. The total concentration of three components in combination II was 13.7 mmol/l,
Table 3 Effects of three components used alone on capsaicin-induced TRPV1 currents. Components
Cochinchinenin A
Cochinchinenin B
Loureirin B
Concentration (lmol/l)
Inhibition rate (%) (P value of t test) (n¼ 7)
3800 380 190 38 19
73.1 7 5.1 70.2 7 4.8 65.7 7 5.4 30.1 7 2.8 18.2 7 5.3
(0.00312) (0.00212) (0.00562) (0.00807) (0.00030)
19000 3800 750 380 190
80.2 7 5.0 78.3 7 3.4 40.4 7 1.3 25.4 7 3.3 11.7 7 7.3
(0.00448) (0.00712) (0.00021) (0.00905) (0.03807)
83.0 7 4.4 70.7 7 1.4 65.3 7 7.0 44.1 7 2.7 11.9 7 7.6
(0.00782) (0.0183) (0.00176) (0.00137) (0.02676)
200 40 8 4.88 1.44
5
among which the proportion of cochinchinenin A, cochinchinenin B and loureirin B was 2.2:1.1:5. The result of t-test showed that there was no significant difference between the percentage inhibitions of combination I and II on capsaicin-induced TRPV1 receptor currents (P40.05). The two combination solutions were diluted 10, 100 and 1000 times, respectively and the effects of different concentrations of the solutions on capsaicin-induced TRPV1 receptor currents were observed to obtain concentration–response curves. All the solutions showed some degree of inhibition against the currents (Table 4 and Fig. 5). The concentration–response data were fitted to Eq. (2), giving IC50 values and Hill coefficients of 2.5570.43 mmol/l and 0.5370.01 (n¼7) for combination I, 1.0170.29 mmol/l and 0.467 0.06 (n¼7) for combination II (Fig. 5). 3.4. Comparison for the pharmacological effect of dragon’s blood and combinations of its components Three kinds of solutions combined with any two of the components inhibit capsaicin-induced TRPV1 receptor currents in DRG neurons to some extent. The percentage inhibitions of combination A, B, and C solutions on peak amplitudes of TRPV1 receptor currents were (35.2271.5)%, (68.37 75.0)%, and (66.6674.9)% (n ¼7), respectively. ANOVA test demonstrated that there were significant difference among the percentage inhibitions of combination I, A, B, C, and 0.005% (m/v) dragon’s blood solutions on capsaicin-induced TRPV1 receptor currents (F ¼40.71, Po0.01). However, LSD test indicated that there was no significant difference between effects of combination I and 0.005% (m/v) of dragon’s blood (P40.05) (Table 5). For purposes of comparison, the concentration response curves of combination I, II and dragon’s blood were displayed in the same Fig. 6 by converting molarity to percent mass/volume. The IC50 value of combination I and II were (0.00008170.000029)% (m/v) and (0.00003770.000020)% (m/v), respectively. 3.5. Effect of combination II on capsaicin-induced membrane depolarization of DRG neuron under the condition of current clamp
Fig. 4. Concentration–response curves of three components. Data points, percent change in peak amplitude in the presence of cochinchinenin A, cochinchinenin B, and loureirin B, respectively (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the point. Each set of data points is fitted with the Hill equation (see Section 2).
To verify whether the components of dragon’s blood inhibit capsaicin-induced TRPV1 receptor currents, combination II was selected to observe the capsaicin-induced changes in membrane potential of DRG neuron before and after applying the components. Capsaicin evoked a membrane depolarization and elicited action potential firing at 1 mmol/l (Fig. 7, top). Membrane potential recovered back to the normal level after capsaicin washout, the average of resting membrane potential was 57.676.3 mV (n¼7). However, 1 mmol/l capsaicin did not evoke action potential firing in the presence of combination II (Fig. 7, middle), the membrane potential of depolarization was 25.775.4 mV (n¼7). Capsaicin (1 mmol/l) was able to evoke membrane depolarization and action potential firing after washout for 90 s (Fig. 7, bottom). In addition, combination II alone failed to change membrane potential or cause action potential firing in all observed DRG neurons. In summary,
Table 4 Effects of combinations of three components on capsaicin-induced TRPV1 currents in DRG neurons. Combination
Cochinchinenin A (lmol/l)
Cochinchinenin B (lmol/l)
Loureirin B (lmol/l)
Inhibition rate (%) (P value of t test) (n¼7)
Combination I Diluted 10-fold Diluted 100-fold Diluted 1000-fold Combination II Diluted 10-fold Diluted 100-fold Diluted 1000-fold
38 3.8 0.38 0.038 3.8 0.38 0.038 0.0038
19 1.9 0.19 0.019 1.9 0.19 0.019 0.0019
8 0.8 0.08 0.008 8 0.8 0.08 0.008
84.8 7 4.9 59.6 7 4.0 35.5 7 6.1 10.6 7 3.6 79.3 7 6.6 49.1 7 6.6 32.5 7 5.0 9.9 7 4.4
(0.00004) (0.00001) (0.00039) (0.00107) (0.00212) (0.00562) (0.00807) (0.00030)
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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L.-S. Wei et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]
Fig. 5. Effects of combination I and II on capsaicin-induced TRPV1 receptor currents in DRG neurons. (A) Combination I and II inhibit rapidly and reversibly the current induced by 1 mmol/l capsaicin. The horizontal bar above each trace indicates the period of drug application. (B) Concentration–response curves for inhibition of the capsaicin (1 mmol/l) response by combination I and II. Data points, percent change in peak amplitude in the presence of combination I or II (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the point. Each set of data points is fitted with the Hill equation (see Section 2). Table 5 Comparisons for the percentage inhibition of dragon’s blood and that of the combinations. Concentration of individual component in the combination (lmol/l)
Drug
Combination Combination Combination Combination
I A B C
Cochinchinenin A
Cochinchinenin B
Loureirin B
38 38 38 /
19 19 / 19
8 / 8 8
Inhibition rates (%) 9yD yC9 in least square difference (LSD) method
84.81 7 4.9 35.22 7 1.5 68.37 7 5.0 66.66 7 4.9
0.05187 o 0.09468 0.54198 40.09468 0.23910 40.09468 0.22120 40.09468
As Table 5 showed, the differences between the percentage inhibition produced by each combinations and 0.005% (m/v) of dragon’s blood are compared with LSD value (0.09468) at the 5% level, where yD and yC are the percentage inhibition of dragon’s blood and combinations, respectively.
combination II was able to decrease capsaicin evoked depolarization in rat DRG neurons and its effects were partially reversible by washout, these results were consistent with those under voltageclamp condition. 3.6. Effect of combination II on the concentration–response curve of capsaicin
Fig. 6. Comparison of the concentration–response curves for dragon’s blood, combinations I, and combination II. Here the concentration is expressed in the unit of the percent mass/volume. Data points, percent change in peak amplitude in the presence of dragon’s blood, combinations I, and combination II (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the point. Each set of data points is fitted with the Hill equation (see Section 2).
Due to a lower IC50 value of combination II than that of combination I in inhibition of capsaicin-induced TRPV1 receptor currents, effect of combination II on concentration–response curves of capsaicin was investigated. Under the absence and presence of combination II, the peak amplitude of capsaicininduced TRPV1 receptor currents would not increase when the concentration of capsaicin was above 10 mmol/l (P40.05), indicating a maximal effect of capsaicin. Furthermore, all peak amplitudes of capsaicin-induced TRPV1 receptor currents were standardized by the peak amplitude of TRPV1 receptor current induced 0.5 mmol/l capsaicin. Thus the cell-to-cell variability of capsaicin-induced TRPV1 receptor currents were eliminated. Pooled data were used to construct the concentration–response curves for capsaicin in the absence and presence of combination II (Fig. 8). In the absence of combination II, the maximum value of capsaicin-induced standard currents was 2.28 70.20, the IC50 value was 0.6970.06 mmol/l, and the Hill coefficient was 1.5470.23 (n ¼7). In the presence of combination II, the maximum value of capsaicin-induced standard currents was
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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7
Fig. 7. Effect of combination II on capsaicin-induced membrane potential of DRG neuron. Top, in control, 1 mmol/l capsaicin was applied and induced membrane depolarization enough to evoke action potential firings. The action potential firings were disappeared upon washout. Middle, in the presence of combination II, capsaicin was applied with the same protocol as in control. The capsaicin evoked depolarization was significantly decreased, there is no action potential firing. Bottom, after 1990s washout of combination II and capsaicin, capsaicin was applied again in the same protocol as in control, the effect of capsaicin was partially recovered.
1.64 70.08, the IC50 value was 1.95 70.28 mmol/l, and Hill coefficient was 1.5570.28 (n ¼7). The result indicated that combination II reduced the capsaicin-induced standard currents by 27.9%. 3.7. The effects of combination II inside and outside plasma membrane To explore the binding site of components of dragon’s blood, the effects of combination II (0.38 mmol/l of cochinchinenin A, 0.19 mmol/l of cochinchinenin B, and 0.8 mmol/l of loureirin B) on capsaicin-induced TRPV1 receptor currents was tested when it was applied to the intracellular side and extracellular side, respectively. The inhibitory effect on capsaicin-induced TRPV1 receptor currents was observed after extracellular application of combination II, and the extracellular percentage inhibition was 49.1 76.6% (n ¼7). In contrast, combination II produced no or only a slight effect on capsaicin-induced TRPV1 receptor currents after 20 min intracellular dialysis by pipette solution, and the intracellular percentage inhibition was 2.872.1% (n ¼7). In addition, the percentage inhibition was 51.3175.1% (n¼ 7) when combination II was simultaneously applied to the intracellular side and extracellular side, which similar to the extracellular percentage inhibition (P 40.05). 3.8. Assessment of interaction among the three components The concentrations of the three components that produce the effect of combination I (84.81% inhibition of TRPV1 receptor current) when used alone were determined from the concentration–response curves of the individual components (Fig. 4), and the resulting three concentration values, the concentration values (38, 19, 8) of the three components in combination I, and the parameter values reported above were substituted into Eqs. (3)–(5), respectively. According to the three equations, the additive surface drawings
Fig. 8. Antagonism of the capsaicin response by combination II is not competitive. Concentration–response curves for capsaicin in the absence and presence of combination II: 3.8 mmol/l of cochinchinenin A, 1.9 mmol/l of cochinchinenin B, and 8 mmol/l of loureirin B. All responses are normalized to the peak current (n) induced by 0.5 mmol/l capsaicin. Data points, normalized peak currents (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the circle. Each set of data points is fitted with the Hill equation (see Section 2).
corresponding to 84.81% inhibition were then generated with Matlab software (Fig. 9). To statistically determine the positional relation between the experimental point Q1 (38, 19, 8) and additive surfaces, the coordinate values of theoretical additive points corresponding to the point Q1 were calculated together with the associated 95% confidence intervals. The theoretical additive points are the intersections of the additive surfaces and the ray which
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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Fig. 9. Additive surfaces corresponding to the combined effects produced by cochinchinenin A, cochinchinenin B, and loureirin B. (A) The positional relation between point Q1 and the additive surfaces corresponding to the effect of combination I (EI ¼ 84.81%) and (B) The positional relation between point Q2 and the additive surfaces corresponding to the effect of combination II (EII ¼ 79.26%).
Table 6 Coordinate values of theoretical additive points corresponding to the experimental points Q1 and Q2. coordinate value of Q1 (mmol/l)
Coordinate value of point Q1’s additive points [95% confidence interval] (mmol/l)
Inhibition rate of combination I, EI ¼ 84.81% 38 19 8 Inhibition rate of combination II, EII ¼ 79.26% Coordinate value of Q2 (mmol/l) 3.8 1.9 8
216.2426 [151.170, 281.314] 108.121 [79.459, 118.005] 45.524 [31.825, 59.224]
Coordinate value of point Q2’s additive points [95% confidence interval] (mmol/l) 15.372 15.965 [12.647, 18.096] [13.751, 18.180] 7.686 7.982 [6.323, 9.048] [6.875, 9.090] 32.362 33.612 [26.626, 38.097] [28.949, 38.275]
Table 7 The effects of 5 mg/kg dragon’s blood in three animal models. Group
MPE of hot plate test (%)
MPE of tail-flick test (%)
Number of writhing in writhing test
Control Dragon’s blood
2.6 7 3.1 70.8 7 8.6a
1.77 2.6 81.1 7 7.8a
34.2 74.3 10.4 74.3a
a
233.288 [182.516, 284.06] 116.6443 [91.258, 142.030] 49.113 [38.424, 59.800]
197.46 [158.918, 236.011] 98.732 [75.585, 140.657] 41.571 [33.456, 49.686]
14.987 [12.962, 17.011] 7.493 [6.481, 8.505] 31.552 [27.289, 35.8143]
3.9. Effects of dragon’s blood in three animal models The results of hot plate test, tail flick test and writhing test presented in Table 7 showed that 5 mg/kg Dragon’s Blood significantly raised the pain thresholds in hot plate test and tailflick test, and in writhing test it also lead to a 69.6% reduction in the number of writhing in comparison with the control group.
Compared with control, P o 0.01.
4. Discussion starts at the origin and also passes through the point Q1. A similar calculation was performed for the case of combination II (79.26% inhibition of TRPV1 receptor current) to obtain the coordinate values of theoretical additive points corresponding to the experimental point Q2 (3.8, 1.9, 8) (Table 6). As shown in Table 6, the coordinate values of Q1 and Q2 were all statistically smaller than those of their corresponding theoretical additive points. Therefore, the points Q1 and Q2 were both below the respective corresponding three additive surfaces (Fig. 9), which indicated that interactional effect of the three components in both combination I and II on capsaicin-induced TRPV1 receptor currents were both synergistic.
The present study first observed the inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons using whole-cell patch clamp technique. 0.005% (m/v) solution of dragon’s blood completely suppress capsaicin-induced TRPV1 receptor currents, indicating that it block up the transmission of pain message for primary sensory neurons via direct action on TRPV1 receptor. The effects of three components (cochinchinenin A, cochinchinenin B, and loureirin B) and their combinations on the capsaicin-induced TRPV1 receptor currents in DRG neurons of rats were also observed. The result demonstrated that each individual component and the combinations of any two out of
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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the three components reduce capsaicin-induced TRPV1 receptor currents to some extent, but none of them had an equally inhibition as that of 0.005% (m/v) dragon’s blood. According to the operational definition of the material basis for the efficacy of traditional medicine (Liu et al., 2006), the three individual components and the combinations of two components were regarded as the effective component/combinations but not the corresponding material basis of dragon’s blood. However, the combination of three components prepared in terms of the mass fraction of three components in final products of dragon’s blood, which guaranteed that the quantity of each component in the combination was quite in agreement with that in dragon’s blood, generate similar effect as that of 0.005% (m/v) dragon’s blood. Therefore it was considered that the effective combination containing cochinchinenin A, cochinchinenin B, and loureirin B was the material basis of dragon’s blood for the modulation of capsaicin-induced TRPV1 receptor currents, and this modulation intervene in pain messages and thereby result in pain relief. This remarkable synergistic interaction among three components may explain the mode of their modulation on the capsaicin-induced TRPV1 receptor currents in DRG neurons. The present study also discovered that the effect of loureirin B was stronger than that of cochinchinenin A or cochinchinenin B, suggesting the chief role of loureirin B in the pharmacological effect of the combination. New combination could be made by altering the proportion of the three components, i.e., reducing the relative proportions of cochinchinenin A and cochinchinenin B but increasing the relative proportion of loureirin B. It still generate a strong effect with lower total concentration of the three components. The results showed that the combination II (namely the new combination above-mentioned) had a higher potency than combination I and the components displayed higher synergistic reaction. Thus it should be presumed that the proportion of the combination should be further optimized to obtain more efficient combinations. Our previous study has revealed that dragon’s blood modulate TTX-R sodium currents and the material basis of dragon’s blood for the modulation is the combination of three components, namely cochinchinenin A, cochinchinenin B and loureirin B, which synergistically modulate TTX-R sodium currents (Liu et al., 2006). All these were in accordant with the present study on capsaicin-induced TRPV1 receptor currents. It is noticeable that the IC50 value of dragon’s blood which inhibits TTX-R sodium current is three orders of magnitude higher than that of dragon’s blood which inhibits capsaicin-induced TRPV1 receptor currents, which suggests that in DRG neurons the first target of dragon’s blood is TRPV1 receptor rather than TTX-R sodium channel. The other difference is that although the three components inhibit TTX-R sodium and capsaicin-induced TRPV1 receptor currents, the IC50 value of loureirin B which inhibits TTX-R sodium current was of the same order of magnitude as that of the other two components, whereas the IC50 value of loureirin B inhibiting capsaicin-induced TRPV1 receptor currents was two to three orders of magnitude lower than that of the other two components, which indicated that different mechanisms of dragon’s blood as influence on TTX-R sodium channel and TRPV1 receptor. The addition of combination II do not only make the concentration–response curve of capsaicin-induced TRPV1 receptor currents move rightwards, but also reduce the maximal TRPV1 receptor currents induced by capsaicin, demonstrating that the inhibition of dragon’s blood and its three components were not competitive. The receptor-ligand binding model was used to simulate the interaction of loureirin B and TRPV1 receptor. The result indicated that the interaction of loureirin B and TRPV1 receptor was fitted with the simple model of one ligand and one receptor site. There may be only one binding site on TRPV1 receptor for loureirin B (Wang et al., 2007). In addition, our
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67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 Uncited reference Q2 124 125 Zheng and He (1999). 126 127 128 Acknowledgments 129 130 This work was supported by National Natural Science Founda- 131 tion of China [Grant 30973961]; Young Chenguang Foundation 132 preliminary experiment showed that loureirin B inhibit the inward current induced by extracellular lower pH value of DRG neurons. Since TRPV1 receptor currents was also activated by extracellular lower pH value, and the area of vanillin activation sites on TRPV1 receptor was not the same as proton binding area (Wang and Cao, 2008), it was presumed that the function site of loureirin B would be different from that of capsaicin and H þ . It should bind to TRPV1 receptor on this site and cause the structural change of TRPV1 receptor, decreasing the affinity and activity of excitomotor, thus producing antagonistic effect. The mechanism may be similar to AMG9810, which made TRPV1 receptor in inactive or turn-off condition even vanillin activation sites on TRPV1 receptor had been binded (Gavva et al., 2005). Many evidence indicated that besides capsaicin and resiniferatoxin, capsazepine, the competitive antagonist of TRPV1 receptor, could not antagonize the biological effect of other agonists (Perkins and Campbell, 1992). But the specific agonists, capsaicin and resiniferatoxin were unable to be synthesized in the body of vertebrates. The activation of TRPV1 receptor in the body may mainly depend on the nociceptive thermal stimulus and the conduction of the acid stimulation in tissue. Therefore, TRPV1 receptor antagonist should block up the activation of the receptor induced by thermal stimulus as well as H þ , but not antagonize the binding site of capsaicin (Yan and Zhang, 2002). Unlike capsazepine, the three components of dragon’s blood antagonize TRPV1 receptor without competing with capsaicin for the same site and would not lead to hyperthermia when they are used in the long run. Consequently, it may be a promising candidate in developing new analgesic drugs targeting TRPV1 receptor. The present study shows that combination II is only working outside plasma membrane, this may be because the intracellular application of combination II affects neither the capsaicin response nor the inhibition effect of extracellularly applied combination II on the capsaicin response, which suggests that the binding sites of the three components in combination II are located entirely outside of plasma membrane. This result may provide the clue for further biophysical studies of channel/drug interaction. As for the possible molecular mechanisms of the synergistic effects of the three active compounds, at present we can only propose the following hypotheses: by reason of the different chemical structures of the three components, each component has its own binding site on TRPV1 receptor. When the three components bound to their respective binding sites on TRPV1 receptor, the conformational changes of the binding sites impel the combined inhibition effects of the three components on TRPV1 receptor to be synergistically enhanced. There was no antagonistic interaction could be observed probably because the concentration of each component was too low to bind all of its own sites on TRPV1 receptor. In conclusion, our study further proved that the combination of three components from dragon’s blood resin according to the proportion by weight synergistically contributed to the corresponding analgesia material basis of dragon’s blood resin via the inhibition of capsaicin-induced TRPV1 receptor currents.
Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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Please cite this article as: Wei, L.-S., et al., Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.052i
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