An alternative method to reduce anaphylaxis by moxibustion

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  • ABSTRACT

    Epinephrine is a critical drug for patients at risk for anaphylaxis. Here, we suggest moxibustion as an alternative method to reduce anaphylaxis. Moxibustion was applied to the Shimen (CV5) acupoint and found to attenuate compound 48/80-induced mortality. Capsazepine, a transient receptor potential vanilloid (TRPV) 1 antagonist, significantly improved overall survival rates compared to groups treated with moxibustion or 2-aminoethoxydiphenyl borate (an activator of TRPV1, 2, and 3). Probenecid (a TRPV2 agonist) also increased survival rate and reduced histamine levels. Survival rates increased by moxibustion and probenecid were completely inhibited by ruthenium red (a TRPV2 and 3 antagonist) and gadolinium chloride (general TRPV antagonist), respectively. Passive cutaneous anaphylaxis and ear swelling were significantly reduced by moxibustion and probenecid (p < 0.05). In cardiomyocytes, TRPV2 was over-expressed by compound 48/80 and histamine but this increased TRPV2 expression decreased to baseline with moxibustion and probenecid treatment. In addition, intracellular calcium levels increased by compound 48/80 were reduced by probenecid. Overall, these findings suggest that the reduction of anaphylaxis caused by moxibustion could represent a new mechanism of moxibustion related to the regulation of TRPV2 activation and promotion of epinephrine secretion.


  • KEYWORD

    anaphylaxis , moxibustion , transient receptor potential vanilloid2 , epinephrine

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  • [Fig. 1.] Moxibustion was applied to the Shimen (CV5) acupoint.
    Moxibustion was applied to the Shimen (CV5) acupoint.
  • [Table 1.] Survival (%) by time course of moxibustion after compound 48/80 treatment in mice
    Survival (%) by time course of moxibustion after compound 48/80 treatment in mice
  • [Fig. 2.] Effect of moxibustion on anaphylaxis. Mice were given an intraperitoneal injection of the mast cell degranulator, compound 48/80 (8 mg/kg). Moxibustion was post-applied indirectly to stimulate at the Shimen (CV5) acupoint. (a) Survival (%) was monitored for 30 min after induction of anaphylactic shock (n = 9). (b) The soldering instrument was used for post-treatment at the same temperature as moxibustion (n = 7). Survival (%) was monitored for 30 min. (c) Epinephrine (30 and 300 μg/kg) was post-stimulated after 5 min of compound 48/80 treatment (n = 7). Com 48/80, compound 48/80; M, moxibustion; Epi, epinephrine.
    Effect of moxibustion on anaphylaxis. Mice were given an intraperitoneal injection of the mast cell degranulator, compound 48/80 (8 mg/kg). Moxibustion was post-applied indirectly to stimulate at the Shimen (CV5) acupoint. (a) Survival (%) was monitored for 30 min after induction of anaphylactic shock (n = 9). (b) The soldering instrument was used for post-treatment at the same temperature as moxibustion (n = 7). Survival (%) was monitored for 30 min. (c) Epinephrine (30 and 300 μg/kg) was post-stimulated after 5 min of compound 48/80 treatment (n = 7). Com 48/80, compound 48/80; M, moxibustion; Epi, epinephrine.
  • [Fig. 3.] Stimulation temperature of moxibustion and the soldering instrument. (a) Moxibustion and (b) Soldering instrument.
    Stimulation temperature of moxibustion and the soldering instrument. (a) Moxibustion and (b) Soldering instrument.
  • [Fig. 4.] Activation of TRPV2 by moxibustion in anaphylaxis. (a) Capsazepine pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and moxibustion. (b) Capsazepine pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and a soldering instrument. (c) Capsazepine pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and 2-APB (60 mg/kg). (d) Gadolinium chloride pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and probenecid (30, 100, and 300 mg/kg). (a-d lower panel) Histamine in serum was analyzed by the histamine assay. #p < 0.05, significantly different from the normal group; *p < 0.05, significantly different from the compound 48/80 group. N, normal mice; Com 48/80, compound 48/80; Moxa, moxibustion; CZP, capsazepine; Solder, soldering instrument; Pro, probenecid; Gd, gadolinium chloride.
    Activation of TRPV2 by moxibustion in anaphylaxis. (a) Capsazepine pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and moxibustion. (b) Capsazepine pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and a soldering instrument. (c) Capsazepine pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and 2-APB (60 mg/kg). (d) Gadolinium chloride pre-treatment (10 mg/kg) was given 30 min before stimulation with compound 48/80 and probenecid (30, 100, and 300 mg/kg). (a-d lower panel) Histamine in serum was analyzed by the histamine assay. #p < 0.05, significantly different from the normal group; *p < 0.05, significantly different from the compound 48/80 group. N, normal mice; Com 48/80, compound 48/80; Moxa, moxibustion; CZP, capsazepine; Solder, soldering instrument; Pro, probenecid; Gd, gadolinium chloride.
  • [Fig. 5.] Effect of moxibustion and probenecid in allergic animal models. (a) Mice were intradermally injected with 100 ng of anti-DNP-IgE. Probenecid (300 mg/kg) was administered intraperitoneally for 1 h prior to challenge with 1 mg/ml of antigen (DNP-HSA) containing 4% Evans blue (1:1) via the tail vein after 48 h and then moxibustion applied after 5 min. The amount of effused dye from PCA skin was evaluated. (b) Probenecid (300 mg/kg) was intraperitoneally administered 1 h prior to compound 48/80 application. 20 μl of compound 48/80 was intradermally applied before moxibustion. All data represents the mean ± S.D. #p < 0.05, significantly different from the normal group; *p < 0.05, significantly different from the compound 48/80 group or IgE-DNP-HSA group. n = 6.
    Effect of moxibustion and probenecid in allergic animal models. (a) Mice were intradermally injected with 100 ng of anti-DNP-IgE. Probenecid (300 mg/kg) was administered intraperitoneally for 1 h prior to challenge with 1 mg/ml of antigen (DNP-HSA) containing 4% Evans blue (1:1) via the tail vein after 48 h and then moxibustion applied after 5 min. The amount of effused dye from PCA skin was evaluated. (b) Probenecid (300 mg/kg) was intraperitoneally administered 1 h prior to compound 48/80 application. 20 μl of compound 48/80 was intradermally applied before moxibustion. All data represents the mean ± S.D. #p < 0.05, significantly different from the normal group; *p < 0.05, significantly different from the compound 48/80 group or IgE-DNP-HSA group. n = 6.
  • [Fig. 6.] Effect of moxibustion and probenecid on epinephrine secretion. A drenergic receptor blockers (mixture of phentolamine 10 mg/kg and metoprolol 20 mg/kg) and ruthenium red (50 mg/kg) were given 30 min before compound 48/80, moxibustion, or probenecid treatment. (a) Epinephrine in serum was analyzed using an Epinephrine Assay Kit. (b) Mortality (%) was monitored for 20 min. *p < 0.05, significantly different from the compound 48/80 group. n = 10. Com 48/80, compound 48/80; Blockers, adrenergic receptor blockers.
    Effect of moxibustion and probenecid on epinephrine secretion. A drenergic receptor blockers (mixture of phentolamine 10 mg/kg and metoprolol 20 mg/kg) and ruthenium red (50 mg/kg) were given 30 min before compound 48/80, moxibustion, or probenecid treatment. (a) Epinephrine in serum was analyzed using an Epinephrine Assay Kit. (b) Mortality (%) was monitored for 20 min. *p < 0.05, significantly different from the compound 48/80 group. n = 10. Com 48/80, compound 48/80; Blockers, adrenergic receptor blockers.
  • [Fig. 7.] Moxibustion suppresses heart TRPV2 levels in a compound 48/80-induced anaphylaxis model. (a) The mRNA expressions of TRPV2, ANP, WINK4, and NCC in various organ tissues from both compound 48/80 and compound 48/80 + moxibustion groups were analyzed using reverse transcription-PCR. The expression was normalized to GAPDH mRNA expression. (b) The expression of TRPV2 protein was analyzed by Western blot analysis in heart tissue from mice under anaphylaxis. (c) The TRPV2+ (green) cells from heart tissues were examined with a confocal laser-scanning microscope (scale bar = 50 μm). Results are representative of three independent experiments. M, marker; N, normal mice; compound 48/80; Moxibustion, compound 48/80 + moxibustion.
    Moxibustion suppresses heart TRPV2 levels in a compound 48/80-induced anaphylaxis model. (a) The mRNA expressions of TRPV2, ANP, WINK4, and NCC in various organ tissues from both compound 48/80 and compound 48/80 + moxibustion groups were analyzed using reverse transcription-PCR. The expression was normalized to GAPDH mRNA expression. (b) The expression of TRPV2 protein was analyzed by Western blot analysis in heart tissue from mice under anaphylaxis. (c) The TRPV2+ (green) cells from heart tissues were examined with a confocal laser-scanning microscope (scale bar = 50 μm). Results are representative of three independent experiments. M, marker; N, normal mice; compound 48/80; Moxibustion, compound 48/80 + moxibustion.
  • [Fig. 8.] Probenecid suppresses compound 48/80-induced TRPV2 expression in cardiomyocytes. Embryonic cardiomyocytes and fibroblasts from the heart were analyzed using quantitative real-time PCR analysis for TRPV2. (a) The mRNA expression of TRPV2 in fibroblast and cardiomyocytes. The cells were pretreated with ruthenium red (2 μM) for 20 min before compound 48/80, histamine (100 μM), or probenecid (100 μM) stimulation. The expression of TRPV2 protein was analyzed using Western blot analysis (b) and immunocytochemistry analysis (c). The TRPV2+ (FITC) and sarcomeric-α actinin+ (TRITC) cells were examined with a confocal laser-scanning microscope. Representative photomicrographs were examined at 60 × magnification. (Scale bar = 10 μm). Com 48/80, compound 48/80, Com + Pro, compound 48/80 + probenecid; Com + Pro + RR, compound 48/80 + probenecid + ruthenium red; His + Pro, histamine + probenecid.
    Probenecid suppresses compound 48/80-induced TRPV2 expression in cardiomyocytes. Embryonic cardiomyocytes and fibroblasts from the heart were analyzed using quantitative real-time PCR analysis for TRPV2. (a) The mRNA expression of TRPV2 in fibroblast and cardiomyocytes. The cells were pretreated with ruthenium red (2 μM) for 20 min before compound 48/80, histamine (100 μM), or probenecid (100 μM) stimulation. The expression of TRPV2 protein was analyzed using Western blot analysis (b) and immunocytochemistry analysis (c). The TRPV2+ (FITC) and sarcomeric-α actinin+ (TRITC) cells were examined with a confocal laser-scanning microscope. Representative photomicrographs were examined at 60 × magnification. (Scale bar = 10 μm). Com 48/80, compound 48/80, Com + Pro, compound 48/80 + probenecid; Com + Pro + RR, compound 48/80 + probenecid + ruthenium red; His + Pro, histamine + probenecid.
  • [Fig. 9.] Moxibustion suppresses compound 48/80-induced TRPV2 expression in cardiomyocytes of heart tissue. The expression of TRPV2 protein was analyzed using immunohistochemistry analysis. The TRPV2+ (FITC) and sarcomeric-α actinin+ (TRITC) cells were examined with a confocal laserscanning microscope. The merged image indicates the colocalization of cardiomyocytes and TRPV2. Representative photomicrographs were examined at 60 × magnification. S-ACTININ, sarcomeric-α actinin. (scale bar = 50 μm).
    Moxibustion suppresses compound 48/80-induced TRPV2 expression in cardiomyocytes of heart tissue. The expression of TRPV2 protein was analyzed using immunohistochemistry analysis. The TRPV2+ (FITC) and sarcomeric-α actinin+ (TRITC) cells were examined with a confocal laserscanning microscope. The merged image indicates the colocalization of cardiomyocytes and TRPV2. Representative photomicrographs were examined at 60 × magnification. S-ACTININ, sarcomeric-α actinin. (scale bar = 50 μm).
  • [Fig. 10.] Epinephrine suppresses compound 48/80-induced TRPV2 expression in cardiomyocytes. Embryonic cardiomyocytes from the heart were isolated and the cells pretreated with ruthenium red (2 μM) for 20 min before compound 48/80, histamine (100 μM), or epinephrine (5 nM) treatment. (a) The expression of TRPV2 protein was analyzed using Western blot analysis. (b) Quantitative real-time PCR analysis for TRPV2, ANP, ADRα2A, and ADRβ1. Data represents the mean ± S.D. (c) Immunocytochemistry analysis. The TRPV2+ (FITC) and sarcomeric-α actinin+ (TRITC) cells were examined with a confocal laser-scanning microscope. The merged image indicated the colocalization of cardiomyocytes and TRPV2. Representative photomicrographs were examined at 60 × magnification. (scale bar = 10 μm). His + Epi, histamine + epinephrine. #p < 0.05, significantly different from the blank group; *p < 0.05, significantly different from the histamine group.
    Epinephrine suppresses compound 48/80-induced TRPV2 expression in cardiomyocytes. Embryonic cardiomyocytes from the heart were isolated and the cells pretreated with ruthenium red (2 μM) for 20 min before compound 48/80, histamine (100 μM), or epinephrine (5 nM) treatment. (a) The expression of TRPV2 protein was analyzed using Western blot analysis. (b) Quantitative real-time PCR analysis for TRPV2, ANP, ADRα2A, and ADRβ1. Data represents the mean ± S.D. (c) Immunocytochemistry analysis. The TRPV2+ (FITC) and sarcomeric-α actinin+ (TRITC) cells were examined with a confocal laser-scanning microscope. The merged image indicated the colocalization of cardiomyocytes and TRPV2. Representative photomicrographs were examined at 60 × magnification. (scale bar = 10 μm). His + Epi, histamine + epinephrine. #p < 0.05, significantly different from the blank group; *p < 0.05, significantly different from the histamine group.
  • [Fig. 11.] Probenecid decreases intracellular calcium levels induced by compound 48/80 in cardiomyocytes. Cardiomyocytes were treated with compound 48/80, histamine (100 μM), or probenecid (100 μM). The kinetics of intracellular calcium was measured every 10 s for 500 s. Each datum represents the mean ± S.D. of three independent experiments. Blank, unstimulated cells.
    Probenecid decreases intracellular calcium levels induced by compound 48/80 in cardiomyocytes. Cardiomyocytes were treated with compound 48/80, histamine (100 μM), or probenecid (100 μM). The kinetics of intracellular calcium was measured every 10 s for 500 s. Each datum represents the mean ± S.D. of three independent experiments. Blank, unstimulated cells.