We purchased compound 48/80, probenecid (TRPV2 agonist), capsazepine, gadolinium chloride (TRPVs blocker), 2-aminoethoxydiphenyl borate (2-APB), ruthenium red, phentolamine methanesulfonate salt, metoprolol tartrate salt, anti-dinitrophenyl (DNP)-IgE, DNP-human serum albumin (HSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), bovine serum albumin (BSA), ophthaldialdehyde (OPA), Fura-2/AM, epinephrine assay kit, and evans blue from Sigma Chemical Co. (St. Louis, MO, USA); Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and fetal bovine serum (FBS) from Gibco BRL (Grand Island, NY, USA); TRPV2 and actin antibodies from Santa Cruz Biotechnology (Santacruz, CA, USA); Sarcomeric-α actinin antibody from AB cam (Cambridge, England); Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG and tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC)-conjugated anti-mouse IgG antibodies from Invitrogen (Carlsbad, CA, USA); moxibustion from Gangwhasazabal (Gangwha, Korea).

The original stock of male ICR mice (4 weeks old) and male SD rats (7 weeks old) were purchased from the Dae-Han Experimental Animal Center (Eumsung, Republic of Korea), and the animals were maintained at the College of Korean Medicine, Kyung Hee University. Animal care and experimental procedures were performed under the approval of the animal care committee at Kyung Hee University [Approval no. KHUASP (SE)-13-019]. The animals were housed five to ten per cage in a laminar air-flow room maintained at a temperature of 22 ± 1℃ and a relative humidity of 55 ± 1% throughout the study. No animal was used more than once. The research was conducted in accordance with internationally accepted principles for laboratory animal use and care as found in US guidelines (NIH publication #85-23, revised in 1985).

The mice were given an intraperitoneal (i.p.) injection of compound 48/80 (mast cells degranulator, 8 mg/kg) and then moxibustion was applied (moxa, 10.62 mm long and in the diameter of 3 mm; 1, 2, or 3 times stimulation), indirectly to stimulate at the Shimen (CV5) acupoint on the ventral midline of the mouse and rat for 0, 5, and 8 min. The mice were kept in a holder for the duration of the stimulation time. Mortality was monitored for 30 min after induction of anaphylactic shock. 2-APB (60 mg/kg, i.p.) and probenecid (30, 100, and 300 mg/kg, i.p.) were administered after compound 48/80. Capsazepine (10 mg/kg), gadolinium chloride (10 mg/kg), ruthenium red (50 mg/kg), and adrenergic receptor blockers [mixture of phentolamine 10 mg/kg (nonselective alpha-adrenergic receptor antagonist) and metoprolol 20 mg/kg (selective β₁ receptor blocker)] were intraperitoneally administered for 30 min before compound 48/80 or probenecid (300 mg/kg) treatment. To assess the thermal effect on anaphylaxis, a soldering instrument at the same temperature as moxibustion was used for treatment.

An IgE-dependent cutaneous reaction was generated by sensitizing the skin with an intradermal injection of anti-DNP IgE followed 48 h later with an injection of DNP-HSA into the mouse tail vein. Moxibustion was applied to the CV5 acupoint after DNP-HSA injection and probenecid was administered to some mice 1 h prior to DNP-HSA injection. The amount of dye was determined colorimetrically. The absorbent intensity of the extraction was measured at 620 nm in a spectrofluorometer, and the amount of dye was calculated with the Evans blue measuring-line.

In this study, ear thickness was measured with a digimatic micrometer (Mitutoyo, Japan) under mild anesthesia. The ear swelling response was determined through any increment in thickness above the baseline control values. Compound 48/80 (100 μg/site) was freshly dissolved in saline and injected intradermally into the dorsal aspect of the mouse ear using a microsyringe with a 28-gauge hypodermic needle. To assess any protective effects from moxibustion or probenecid, moxibustion was applied to the CV5 acupoint after compound 48/80 treatment and probenecid was administered to some mice 1 h prior to compound 48/80. The values obtained represent the effect of compound 48/80 rather than the effect of saline injection (physical swelling), since the ear swelling response evoked by physiologic saline returned to baseline thickness within 40 min.

Histamine levels in serum were measured using the OPA spectrofluorometric procedure. The fluorescent intensity was measured at 440 nm (excitation at 360 nm) in a spectrofluorometer.

Epinephrine levels in serum were measured according to the manufacturer’s specification using the Epinephrine Assay Kit (Sigma Chemical Co.).

Water was removed for 12 h and the rats were anesthetized with zoletil 50® and rumpun® in a 1:2 ratio solution 1 ml/kg. During anesthesia, one of the external iliac arteries was catheritized by laboratory PolyE polyethylene non-sterile tubing (#598321, Havard Apparatus, USA) and connected to an iWorx 214 (iworx system, USA) with a research grade pressure transducer (Havard Apparatus, USA) using LabScribe2 software (iworx system, USA). Five to thirty min after stabilization, compound 48/80 and probenecid were administered and the blood pressure was recorded. Data gained at 20 Hz.

Using an easy-BLUE^TM RNA extraction kit (iNtRON Biotech, Republic of Korea), total RNA was isolated from organ tissues according to the manufacturer’s specifications. The concentration of total RNA in the final eluate was determined by spectrophotometry. PCR was performed with the following primers: TRPV2(5’ CTA CAG CAA AGC CGA AAA GG 3’; 5’ ACC GCA TGG TGG TTT TAG AG 3’); atrial natriuretic peptide (ANP) 5’ AGG ATT GGA GCC AGA GTG G 3’; 5’ ATA GAT GAA GGC AGG AAG C 3’); WINK4 (5’ TGC CTT GTC TAT TCC ACG GTC TG 3’; 5’ CAG CTG CAA TTT CTT CTG GGC TG 3’); and sodium chloride cotransporter (NCC) (5’ TGA TGC GGA TGT CAT TGA TGG 3’; 5’ AAT GGC AAG GTC AAG TCG 3’). To verify that equal amounts of RNA were used for the reverse transcription and PCR amplification from different experimental conditions, GAPDH (5’ GGC ATG GAC TGT GGT CAT GA 3’; 5’ TTC ACC ACC ATG GAG AAG GC 3’) was used as a loading control. The products were electrophoresed on a 1.0% agarose gel and visualized by staining with ethidium bromide.

Quantitative real-time PCR was performed using a SYBR Green master mix and the detection of mRNA was analyzed using an ABI StepOne real time PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences for the reference gene GAPDH and the genes of interest were as follows: GAPDH (5’ GTT AGT 8 (ADRα2A)(5’ TGT AGA TAA CAG GGT TCA GCG A 3’; 5’ TTC TTT TTC ACC TAC ACG CTC A 3’); beta-1 adrenergic receptor (ADRβ1) (5’ TGG CTT ACT GGC TTG TCTT G 3’; 5’ TTT CCA CTC GGG TCC TTG 3’); and ANP (5’ AGG ATT GGA GCC AGA GTG G 3’; 5’ ATA GAT GAA GGC AGG AAG C 3’). Typical profile times were used the initial step of 95℃ for 10 min was followed by a second step at 95℃ for 10 s for 40 cycles with a melting curve analysis. The levels of target mRNA were normalized to GAPDH levels and compared to the control. Data were analyzed using the ΔΔCT method.

Pregnant mice were euthanized by CO2 asphyxiation. The embryos (12 days old) were isolated from the uterus and then transferred into a dish with ice-cold sterile PBS without Ca²⁺ and Mg²⁺. The embryos were released from the yolk sacs and then transferred into another dish with the same solution as described above. Every single heart was isolated under a microscope using surgical instruments. The heart was incubated at 4℃ for 20 h and subsequently at 37℃ for 15 min in a solution of 0.05% trypsin and 0.02% EDTA (wt/vol) (Biochrom AG). The tissue-trypsin-EDTA-mix was included in 1 ml of DMEM (DMEM high glucose (4.5 g/l) without L-glutamine with 10% fetal calf serum and 1% each of penicillin and streptomycin (Biochrom AG). To dissociate the heart, the tissue was gently pipetted several times. The mix gained by this procedure contains fibroblasts and cardiomyocytes. The cells within the medium from each heart of the same genotype were transferred into a cell culture flask to seed the fibroblast. The cultures were maintained for 1 h at 37℃ in a humidified atmosphere with 5% CO2. The supernatant containing the 9 cardiomyocytes was gently removed, counted, and then plated out at 1×10⁶/ml cardiomyocytes in a 6-well plate coated with fibronectin.

Heart tissue and cardiomyocyte extracts were prepared using a detergent lysis procedure. Samples were heated at 95℃ for 5 min, and briefly cooled on ice. Following centrifugation at 15,000 rpm for 5 min, 50 μg aliquots were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. The resolved proteins were electro-transferred overnight onto nitrocellulose membranes in 25 mM Tris, pH 8.5, 200 mM glycerin and 20% methanol at 25 V. Blots were blocked for at least 2 h with 1 × PBS containing 5% non-fat dry milk and then incubated with the TRPV2 antibodies for 1 h at room temperature. Blots were developed by peroxidase-conjugated secondary antibodies and proteins were visualized by enhanced chemiluminescence procedures (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions.

Heart tissues were immediately fixed with 4% formaldehyde and embedded in paraffin. After dewaxing and dehydration, sections were blocked with bovine serum albumin followed by a 60 min incubation with anti–rabbit TRPV2 and anti-mouse sarcomeric-α actinin (cardiomyocytes marker) at a concentration of 1 μg/ml. The secondary antibodies, FITCconjugated anti-rabbit or TRITC-conjugated anti-mouse IgG (invitrogen), were added to the incubation for 30 min. The mounting medium containing 4’, 6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) was used to counterstain DNA. All specimens were examined with a confocal laser-scanning microscope. Sections were coded and randomly analyzed by two blinded observers (Jeong H-J and Nam S-Y).

To measure the time-dependent levels of intracellular calcium, cardiomyocytes (1 × 10⁵) were pretreated with Fura-2/AM in IMDM containing 10% FBS for 30 min and then harvested. After washing twice with calcium free medium (containing 0.5 mM EGTA, an extracellular calcium chelator), approximately 100 μl of the cell suspension were placed into a 96-well plate and treated with compound 48/80, histamine (100 μM), or probenecid (100 μM). The kinetics of the intracellular calcium levels were recorded every 10 s at 440 nm (excitation at 360 nm) in a spectrofluorometer.

The experiments shown are a summary of the data from at least three experiments. Statistical analyses were performed using SPSS statistical software (SPSS 11.5, Armonk, NY, USA). Treatment effects were analyzed by an independent t-test and one way ANOVA with Tukey’s post hoc test. p < 0.05 was used to indicate significance.

To examine the contribution of moxibustion on anaphylactic reactions, an in vivo model of systemic anaphylactic reaction was applied using compound 48/80 (8 mg/kg) as a systemic fatal anaphylaxis inducer. After the injection of compound 48/80, the mice were monitored for 30 min, and then the mortality rate was determined. Moxibustion was poststimulated 1 (M × 1), 2 (M × 2), and 3 (M × 3) times after 0, 5, and 8 min of compound 48/80 treatment at the Shimen (CV5) acupoint (Fig. 1). As shown in Fig. 1a, an oral administration of saline as a control after compound 48/80 treatment induced a fatal reaction in 100% of mice in each group. However, moxibustion improved overall survival rates compared with the saline control. The most effective moxibustion treatment was two applications at 5 or 8 min after compound 48/80 treatment (Table 1 and Fig. 2). To specifically assess the thermal effect on anaphylaxis, a soldering instrument (200℃ indirect, Fig. 3) was applied at the same temperature as moxibustion. The soldering instrument reduced compound 48/80-induced mortality but moxibustion was more effective than the soldering instrument on anaphylaxis (Fig. 2b). Epinephrine (positive control) reduced compound 48/80-induced mortality (Fig. 2c).

TRPV1 is well known for its contributions to acute thermal nociception and injury-elicited hyperalgesia (Caterina et al., 2000; Davis et al., 2000). TRPV1 channels are activated by heat, acid, and a large number of chemical stimuli (Hu et al., 2004). To investigate the role of TRPV1 on the inhibitory effects of moxibustion, the effect of capsazepine (a TRPV1 antagonist) on anaphylaxis was examined. Interestingly, capsazepine significantly improved overall survival rates compared with the moxibustion or soldering instrument stimulation groups (Fig. 4a and b, upper). Histamine release was significantly reduced in moxibustion and moxibustion plus capsazepine groups compared with the compound 48/80 group (Fig. 4a, lower panel, p < 0.05). In mice treated with a soldering instrument, however, capsazepine did not affect histamine release (Fig. 4b, lower). The effect of 2-APB (an activator of TRPV1, TRPV2, and TRPV3) was applied to confirm the relationship between TRPVs and moxibustion on anaphylaxis. 2-APB alone did not affect mortality. However, when capsazepine and 2-APB were administered for 30 min, the mortality rate and histamine release induced by compound 48/80 were significantly reduced (Fig. 4c, p < 0.05). Thus, we studied the role of TRPV2 (a molecule sensitive to temperatures above 52℃) on compound 48/80-induced anaphylaxis. Probenecid (a TRPV2 agonist) increased the survival rate and significantly reduced histamine release in a dose-dependent manner (Fig. 4d, p < 0.05). However, the increased survival rate increased due to probenecid was completely inhibited by treatment with gadolinium chloride (a TRPV blocker) (Fig. 4d).

The effects of moxibustion and probenecid were examined in various allergic animal models. Moxibustion and probenecid significantly reduced the PCA reaction, a common murine anaphylaxis model (Fig. 5a, p < 0.05). In addition, the effect of moxibustion and probenecid were tested on the compound 48/80-induced ear swelling response. Compound 48/80 significantly induced an ear swelling response, but the ear swelling response was significantly reduced by moxibustion and probenecid (Fig. 5b, p < 0.05).

Epinephrine typically used in the treatment for anaphylaxis. Thus, the effect of moxibustion and probenecid were investigated to determine their ability to increase epinephrine secretion in anaphylaxis. As shown in Fig. 6a, epinephrine levels were significantly increased by moxibustion or probenecid (p < 0.05). The role of the epinephrine signaling pathway on the regulatory effect of moxibustion and probenecid in anaphylaxis was investigated. The mice were monitored for 20 min, and then the mortality rate was determined. As shown in Fig. 6b, mice administered the adrenergic receptor blockers died more quickly than compound48/80-treated mice. In addition, increased survival rates from moxibustion and probenecid were partially decreased by application of the adrenergic receptor blockers (Fig. 6b). Ruthenium red completely abolished moxibustioninduced survival (Fig. 6b). Anaphylaxis drastically decreases blood pressure, resulting in hypotension. Okazaki et al. reported that moxibustion increases blood pressure (Okazaki et al., 1990). To investigate the role of TRPV2 on blood pressure, blood pressure was measured in probenecid-administered mice. The decreased blood pressure from treatment with compound 48/80 was increased by probenecid administration (data not shown).

As moxibustion has anti-anaphylactic effect, potentially through activating TRPV2 and by increasing blood pressure through epinephrine, TRPV2 expression and blood pressurerelated factors were investigated in various organs. In previous studies, TRPV2 mRNA was detected in various organs. In this study, TRPV2 expression was investigated in the heart, kidney, spleen, hypothalamus, and ear tissues. The expression of TRPV2 mRNA was only increased through compound 48/80 treatment in heart tissue. Moxibustion decreased the expression of TRPV2 mRNA in heart tissue (Fig. 7a), however, the expression of TRPV2 mRNA by moxibustion in kidney, spleen, and hypothalamus were unchanged compared with the compound 48/80-treated mice. In agreement with the TRPV2 mRNA data, a reduction of TRPV2 protein in the heart of the moxibustion group compared to the compound 48/80 group (Fig. 7b) was observed. Histological analyses also revealed that moxibustion suppressed compound 48/80-induced TRPV2 protein expression in heart tissue (Fig. 7c). In contrast, levels of ANP (a powerful vasodilator) decreased by compound 48/80 were increased by moxibustion in heart tissue (Fig. 7a). Moxibustion had no significant effects on the mRNA expression of WINK4 and NCC (blood pressure-related factors) (Fig. 7a).

To determine the TRPV2-expressing cells in heart tissue, fibroblast and cardiomyocytes were isolated from heart tissue. TRPV2 mRNA was constitutively expressed in cardiomyocytes but not fibroblasts (Fig. 8a). Compound 48/80 markedly increased TRPV2 protein expression (Fig. 8b). The role of histamine released by compound 48/80 was assessed as well. Histamine also increased TRPV2 expression. However, probenecid decreased compound 48/80 or histamine-induced TRPV2 expression in cardiomyocytes (Fig. 8b). The immunocytochemical analysis of the cardiomyocytes also revealed that TRPV2 was highly expressed in the compound 48/80 group but decreased in probenecid treated group (Fig. 8c). Moxibustion also decreased TRPV2 protein expression in cardiomyocytes of heart tissue (Fig. 9). Epinephrine increases blood pressure (Weissgerber, 2008). Therefore, the effect of epinephrine on compound 48/80-indued TRPV2 over-expression was investigated. Epinephrine significantly decreased compound 48/80- or histamine-induced TRPV2 expression through the modulation of adrenergic receptors (ADRα2A and ADRβ1) in cardiomyocytes (Fig. 10a-c, p < 0.05). Epinephrine also significantly decreased ANP expression (Fig. 10b, p < 0.05). Ruthenium red did not affect the TRPV2 expression (Fig. 10a).

TRPV2 is a moderately calcium-selective cationic channel and increases in intracellular Ca²⁺ are sufficient for lowering blood pressure (Murata et al., 2004). Thus, the regulatory effect of probenecid on Ca²⁺ influx in compound 48/80-stimulated cardiomyocytes was examined. Stimulation with compound 48/80 increased Ca²⁺ release from intracellular stores (in 0.5 mM EGTA containing media), whereas probenecid decreased the level of intracellular Ca²⁺ increased by compound 48/80 (Fig. 11). Probenecid alone increased the level of intracellular Ca²⁺.