Effect of acupuncture on TNF-alpha, IL-<FONT FACE=Symbol>1b</FONT> and IL-10 concentrations in the peritoneal exudates of carrageenan-induced peritonitis in rats

Efeito da acupuntura sobre a concentração de TNF-alfa, IL-<FONT FACE=Symbol>1b</FONT> e IL-10 no exsudato peritoneal de ratos com peritonite induzida por carragenina

Abstracts

Acupuncture is an ancient and empirical therapeutic procedure known by its efficacy in the treatment of pain. However, the influence of acupuncture on inflammatory process is still poorly understood and additional research is needed. In this work, we investigated the mechanism of action of manual acupuncture on the inhibition of neutrophil migration to the peritoneal cavity induced by the inflammatory stimulus carrageenan in Wistar rats. Previous results from our laboratory showed that this anti-inflammatory effect is not due to endogenous corticoid release. Furthermore, the concentration of IL-<FONT FACE=Symbol>1b</FONT>, but not of TNF-alpha or IL-10 in the carrageenan-induced exudates was reduced in the acupuncture group. Further research will be needed to elucidate the mechanisms involved in the anti-inflammatory action of acupuncture as described here.

acupuncture; inflammation; cytokines; neutrophils; rats; carrageenan

A acupuntura é método terapêutico milenar reconhecido por sua eficácia no tratamento da dor, porém seu efeito sobre processos inflamatórios é ainda pouco conhecido e maiores estudos são necessários. Neste trabalho, é investigado o mecanismo de ação da acupuntura manual sobre a inibição na migração de neutrófilos para a cavidade peritoneal induzida por carragenina em ratos Wistar. Resultados prévios indicam que esse efeito antiinflamatório não depende de hormônios corticóides. Entretanto, as concentrações de IL-<FONT FACE=Symbol>1b</FONT> no exsudato induzido por carragenina foram reduzidas pelo tratamento com acupuntura. Por outro lado os níveis de TNF-alfa e IL-10 não foram afetados pelo tratamento. Mais pesquisas poderão elucidar os mecanismos envolvidos na ação antiinflamatória da acupuntura.

acupuntura; inflamação; citocinas; neutrófilos; ratos; carragenina

PAPERS

CLINIC AND SURGERY

Effect of acupuncture on TNF-a, IL-1b and IL-10 concentrations in the peritoneal exudates of carrageenan-induced peritonitis in rats

Efeito da acupuntura sobre a concentração de TNF-a, IL-1b e IL-10 no exsudato peritoneal de ratos com peritonite induzida por carragenina

Márcia Valéria Rizzo Scognamillo-Szabó^I,1; Gervásio Henrique Bechara^II; Fernando de Queiroz Cunha^III

^IPost-doc fellowship, Department of Veterinary Pathology, Faculty of Agricultural and Veterinary Sciences, São Paulo State University, 14884-902, Jaboticabal, SP, Brazil. E-mail: [e-mail]

^IIPhD in Pharmacology, Full Professor of Animal Pathology, Department of Veterinary Pathology, Faculty of Agricultural and Veterinary Sciences, São Paulo State University, Jaboticabal, SP, Brazil

^IIIPhD in Pharmacology, Full Professor of Pharmacology, Pharmacology Department, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

ABSTRACT

Acupuncture is an ancient and empirical therapeutic procedure known by its efficacy in the treatment of pain. However, the influence of acupuncture on inflammatory process is still poorly understood and additional research is needed. In this work, we investigated the mechanism of action of manual acupuncture on the inhibition of neutrophil migration to the peritoneal cavity induced by the inflammatory stimulus carrageenan in Wistar rats. Previous results from our laboratory showed that this anti-inflammatory effect is not due to endogenous corticoid release. Furthermore, the concentration of IL-1b, but not of TNF-a or IL-10 in the carrageenan-induced exudates was reduced in the acupuncture group. Further research will be needed to elucidate the mechanisms involved in the anti-inflammatory action of acupuncture as described here.

Key words: acupuncture, inflammation, cytokines, neutrophils, rats, carrageenan.

RESUMO

A acupuntura é método terapêutico milenar reconhecido por sua eficácia no tratamento da dor, porém seu efeito sobre processos inflamatórios é ainda pouco conhecido e maiores estudos são necessários. Neste trabalho, é investigado o mecanismo de ação da acupuntura manual sobre a inibição na migração de neutrófilos para a cavidade peritoneal induzida por carragenina em ratos Wistar. Resultados prévios indicam que esse efeito antiinflamatório não depende de hormônios corticóides. Entretanto, as concentrações de IL-1b no exsudato induzido por carragenina foram reduzidas pelo tratamento com acupuntura. Por outro lado os níveis de TNF-a e IL-10 não foram afetados pelo tratamento. Mais pesquisas poderão elucidar os mecanismos envolvidos na ação antiinflamatória da acupuntura.

Palavras-chave: acupuntura, inflamação, citocinas, neutrófilos, ratos, carragenina.

INTRODUCTION

Acupuncture is the insertion of needles in cutaneous specific locations of the body, known as acupoints, for the treatment or prevention of several diseases (SCHOEN, 1993; ERNST & WHITE, 1999). It is a reflex therapy where the nociceptive stimulus given in an area acts on another one (FARBER & TIMO-IARIA, 1994). Although this technique is increasingly used for the treatment of pain and other conditions, the rational basis underlying its use remains unclear. Improvement of knowledge in acupuncture therapeutic mechanism is essential to validating it since acupuncture is difficult to test under double-blind, placebo-controlled conditions (STREITBERGER & KLEINHENZ, 1998; TUKMACHI, 2000; LANGEVIN, et al., 2001; LANGEVIN et al., 2001; SHERMAN et al., 2002). In fact, few reports concerning the effect of acupuncture on inflammatory models are available, although clinical trials claim the success of acupuncture in the treatment of pruritic dermatosis, bronchial asthma, carpal tunnel syndrome, rheumatoid arthritis and other inflammatory disorders in humans and animals (SCOGNAMILLO-SZABÓ & BECHARA, 2001). However, these observations are limited by methodological considerations such as small sample size, placebo effects and lack of control groups (STREITBERGER & KLEINHENZ, 1998; CASIMIRO et al., 2002, MCCARNEY et al., 2004). Therefore, research on the use of acupuncture in experimentally induced inflammatory process will bring new perspectives towards investigations in this area, including on the mechanisms of action of such technique.

The inflammatory response involves a complex set of highly orchestrated events. An initial inflammatory stimulus (chemical or mechanical trauma, microorganism invasion) triggers the release of chemical mediators from plasma or connective tissue cells. Such soluble mediators, acting together or in sequence, amplify the initial inflammatory response and influence its evolution by regulating the subsequent vascular and cellular responses. This is a benign process if there is a suitable regulation of the inflammatory response (TRACEY & WARREN, 2004). The neutrophil migration observed in the inflammatory process is mediated by several chemotactic mediators (BODOLAY et al., 2002). Among them, the cytokines TNF-a and IL-1b have a pivotal role (BURGER, DAYER, 2002). On the other hand, IL-10 down-modulates the neutrophil migration into inflammatory exudates (KOTENKO, 2002).

Acupuncture treatment reduces both the number of inflammatory cells and volume of exudate in several inflammatory models, such as carrageenan injection in the air pouch of rats, turpentine in rabbit ears, carrageenan-induced paw oedema, carrageenan-induced pleurisy, Freund’s adjuvant-induced rheumatoid arthritis, sepsis and chemical peritonitis in rats (SIN et al., 1983; SIN, GWEE & LOH, 1984; SIN, 1986; KENDALL, 1989; QINGLAN, 1991; CECCHERELI et al., 1999; KWON, et al., 2001; KWON, et al., 2002; ZHANG et al., 2002; SCOGNAMILLO-SZABÓ et al., 2004; SCOGNAMILLO-SZABÓ et al., 2004). Supporting these data, clinical trials confirm the efficacy of acupuncture treatment on allergic rhinitis, pruritic dermatosis, bronchial asthma, eczema, rheumatoid arthritis and other disorders (TOWNSEND et al., 1999; TUMACHI, 1999; CASIMIRO et al., 2002; PETTI et al., 2002; STEURER-STEYA; RUSSIB & STEURERC, 2002; BIELORY & HEIMALL, 2003; MADSEN et al., 2003; MCCARNEY et al., 2004).

Previous results demonstrated that acupuncture significantly suppressed neutrophil migration to the peritoneal cavity induced by intraperitoneal carrageenan injection in rats. It was found that the effect of acupuncture was not mediated by endogenous corticoids, as the administration of mifepristone (RU-486, an antagonist of the glucocorticoid receptor) did not interfere with it. Providing there was no difference in neutrophil migration between sham-acupuncture and acupuncture treated rats, it was clear that the anti-inflammatory effect of acupuncture was not due to the nonspecific effect of needling (SCOGNAMILLO-SZABÓ et al., 2004). The present study investigated the effect of acupuncture treatment on cytokine concentration in the peritoneal exudates of carrageenan-induced peritonitis rats.

MATERIALS AND METHODS

Animals

Twelve male Wistar rats weighing 180-200g were used throughout the experiment. The animals were housed in cages in temperature controlled rooms and received water and food ad libitum. All experimental protocols were approved by the Animal Care and Use Committee of University of São Paulo in accordance with NIH guidelines on animal care.

Experimental peritonitis

Peritonitis was induced through an intraperitoneal injection of carrageenan in pyrogen-free saline (30mg mL^-1 cavity^-1). Negative control animals received 1mL i.p. of the vehicle. All experiments were performed in the mornings between 8:00 AM and 11:00 AM, in order to avoid circadian interference (CHEN & HE, 1989; STORCH et al., 2002). The inflammatory exudate was harvested 4h after carrageenan injection.

Treatments

Manual acupuncture and sham-acupuncture were performed twice: 10 minutes and two hours after inflammatory stimulus. Detailed description of treatments are shown on table 1.

Neutrophil migration into the peritoneal cavity

Neutrophil migration was assessed 4h hours after carrageenan injection. The animals received an over-dose of sub-cutaneous anaesthetic (2.5% tribromoethanol) and the cells present in the peritoneal cavity were harvested after local administration of 10mL of phosphate-buffered saline (PBS) containing 1mM EDTA followed by a gentle belly massage. Total cell counts were performed with a cell counter (Coulter AcT series analyzer; Coulter Corp., Miami, Fla.) and differential cell counts were carried out on cytocentrifuge slides (Cytospin 3, Shandon Southern Products, Astmoore, United Kingdom) stained by the May-Grunwäld Giemsa (Rosenfeld) method. The results are expressed as the number of neutrophils per cavity.

Cytokine measurements

The concentrations of tumor necrosis factor-alpha (TNF-a), interleukin-1b (IL-1b), and interleukin-10 (IL-10) in the peritoneal exudates were determined by using a double-ligand enzyme-linked immunossorbent assay (ELISA). Briefly, flat-bottomed 96-well microtiter plates were coated with 100mL per well of antibody specific to one of the above cytokines at a dilution of 2mg mL^-1 of coating buffer and incubated overnight at 4ºC. Afterwards, the plates were washed and nonspecific binding was blocked for 120min at 37ºC with 1mL calf normal serum. Nondiluted samples and standards were loaded in plates and incubated at room temperature for 4h.

Recombinant rat TNF-a, IL-1b, and IL-10 standard curves were used to calculate cytokine concentrations. The plates were thoroughly washed and the appropriated biotinylated polyclonal or monoclonal anticytokine antibodies were added. The plates were washed 1h later, avidin-peroxidase (diluted 1:5000) was added to each well for 15min, and each plate was thoroughly washed again. In the sequence, substrate (0.4mg of o-phenylenediaminc [OPD] for 1mL of substract buffer) was added, the reaction was stopped with H₂SO₄ (1M), and the optical density was measured in an ELISA plate scanner (Spectra Max 250, Molecular Devices) at 490nm. The results were expressed as picograms of TNF-a, IL-1b, and IL-10 per mL of supernatant, comparing the optical density in the samples with standard curves.

RESULTS

It was observed that the concentration of IL-1b in the peritoneal exudates of carrageenan injected rats was inhibited by the acupuncture. However the concentration of TNF-a and IL-10 in the exudates was not affected by the acupuncture (Figure 1).

DISCUSSION

Controlled clinical trials claim acupuncture efficacy in the treatment of inflammatory process, e.g., arthritis (TOWNSEND et al., 1999; TUMACHI, 1999; Tukmachi, 2000; WHO, 1996; XUE et al., 2003). However, experimental studies concerning acupuncture and the inflammatory process are rare (SIN et al., 1984; KENDALL, 1989; ZHAO & ZHU, 1990; QINGLAN, 1991; ZHAO & ZHU, 1992; Ma, 1992; SCOGNAMILLO-SZABÓ et al., 2004; SCOGNAMILLO-SZABÓ et al., 2004; Sin, et al. 1984), highlighting the importance of a better understanding of mechanisms involved on acupuncture anti-inflammatory effects.

Among experimental models, carrageenan-induced peritonitis is largely used in the study of acute inflammatory process. Although it is a good model to use, there are no references to its use in the investigation of the anti-inflammatory mechanisms of action of the acupuncture.

There are several evidences demonstrating that the cytokines and chemokines mediate the neutrophil migration into the inflammatory site (SCHEIN, 2002). On the other hand, anti-inflammatory cytokine, such as IL-10, down-modulates the process (KOTENKO, 2002). In order to investigate whether the acupuncture inhibits the production of the chemotactic cytokines or increases the production of the anti-inflammatory cytokines, the effect of acupuncture on the IL-1b, TNF-a and IL-10 concentration in the exudates of carrageenan-injected rats was investigated. IL-1b and TNF-a mediate the neutrophil migration observed in several experimental models and also in human inflammatory diseases. On the other hand, IL-10 inhibits the neutrophil migration induced by several inflammatory stimuli and also by chemotactic mediators (LAICHALK et al., 1996; NAGANO et al., 2002).

Our results showed that the production of IL-1b, but not TNF-a and IL-10 was partially inhibited by acupuncture treatment. Further studies need to be done to clarify whether the effect of acupuncture treatment is a consequence of the inhibition of IL-1b production and also the possible participation of other chemotactic cytokines and/or chemokines.

In conclusion, these data show a measurable and significative interference of acupuncture on the inflammatory process. Since previous results indicate that anti-inflammatory effect of acupuncture does not involve either the release of corticoids nor the nonspecific effect of needling, it seems that the anti-inflammatory effect is due to specificity of the stimulated area: the acupoint, as charted by the ancient Chinese Medicine. The fact that the acupuncture inhibited the production of IL-1b points out the importance to investigating the role of cytokines on the anti-inflammatory effect of acupuncture.

ACKNOWLEDGMENTS

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, no 99/07104-7 to M.V.R.S.-S.), Brazil. We thank, Giuliana Bertozi for ELISA, Ana Kátia dos Santos, Ieda Regina dos Santos Schivo and Sérgio Roberto Rosa for technical assistance.

Recebido para publicação 22.06.04

Aprovado em 11.08.04

• BIELORY, L; HEIMALL, J. Review of complementary and alternative medicine in treatment of ocular allergies. Current Opinion in Allergy and Clinical Immunology, v.3, n.5, p.395-399, 2003.
• BODOLAY, E. et al. Angiogenesis and chemokines in rheumatoid arthritis and other systemic inflammatory rheumatic diseases. Journal of Cellular and Molecular Medicine, v.6, p.357-376, 2002.
• BURGER, D.; DAYER, J.M. Cytokines, acute-phase proteins, and hormones: IL-1 and TNF-alpha production in contact-mediated activation of monocytes by T lymphocytes. Annals of the New York Academy of Science, v.966, p.464-473, 2002.
• CASIMIRO, L. et al. Acupuncture and electroacupuncture for the treatment of RA. Cochrane Database Systematic Reviews, v.3, 2002, CD003788.
• CECCHERELI F. et al. The effects of parachlorophenylalanine and naloxone on acupuncture and electroacupuncture modulation of capsaicin-induced neurogenic edema in the rat hind paw. A controlled blind study. Clinical and Experimental Rheumatology, v.17, p.655-662, 1999.
• CHEN, X.; HE, Z. A quantitative study of circadian variations in mast cell number in different regions of the mouse. Acta Anatomica, v.136, p.222-225, 1989.
• ERNST, E.; WHITE, A. (Ed). Acupuncture, a scientific appraisal Oxford : Butterworth-Heinemann, 1999. 162p.
• FARBER, P.L; TIMO-IARIA, C. Acupuntura e sistema nervoso. Jornal Brasileiro de Medicina, v.67, n.5-6, p.125-31, 1994.
• KENDALL, D.E. A scientific model for acupuncture. Part I. American Journal of Acupuncture (California), v.17, n.3, p.251-268, 1989.
• KOTENKO, S.V. The family of IL-10-related cytokines and their receptors: related, but to what extent? Cytokine Growth Factor Review, v.13, p.223-240, 2002.
• KWON, Y.B. et al. The water-soluble fraction of bee venom produces antinociceptive and anti-inflammatory effects on rheumatoid arthritis in rats. Life Sciences, v.31, p.191-204, 2002.
• KWON, Y.B. et al. Bee venom injection into an acupuncture point reduces arthritis associated oedema and nociceptive responses. Pain, v.90, p.271-280, 2001.
• LAICHALK, L.L. et al. Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity. FEMS Immunology and Medical Microbiology, v.15, n.4, p.181-187, 1996.
• LANGEVIN, H.M. et al. Mechanical signaling through connective tissue: a mechanism for the therapeutic effect of acupuncture. FASEB Journal, v.15, p.2275-2282, 2001.
• LANGEVIN, H.M. et al. Biomechanical response to acupuncture needling in humans. Journal of Applied Physiology, v.91, p2471-2478, 2001.
• MA, H. The effect of acupuncture in the content of substance P in serum of gravida during delivery (Resumo). Chen Tzu Yen Chiu, v.17, n.1, p.65-66, 1992.
• MADSEN, H. et al. Use of complementary/alternative medicine among pediatric patients. European Journal of Pediatry, v.162, p.334 341, 2003.
• MCCARNEY, R. et al. Acupuncture for chronic asthma. Cochrane database systematic reviews V.1, 2004, CD000008.
• NAGANO, S. et al. Molecular mechanisms of lipopolysaccharide-induced cyclooxygenase-2 expression in human neutrophils: involvement of the mitogen-activated protein kinase pathway and regulation by anti-inflammatory cytokines. International Immunology, v.14, n.7, p.733-740, 2002.
• PETTI, F.B. et al. Study on cytokines IL-2, IL-6, IL-10 in patients of chronic allergic rhinitis treated with acupuncture. Journal of Traditional Chinese Medicine, v.22, n.2, p.104-111, 2002.
• QINGLAN, W. Studies of the regulatory effects of acupuncture on Yin-Yang equilibrium. In: World Veterinary Congress, 24, 1991, Rio de Janeiro. Anais... Rio de Janeiro: 1991. p.59.
• SCHEIN, C.H. The shape of the messenger: using protein structure information to design novel cytokine-based therapeutics. Current Pharmacology Design, v.8, p.2113-2129, 2002.
• SCHOEN, A.M. Introduction to veterinary acupuncture: scientific basis and clinical applications. In: Annual Convention the American Association of Equine Practioners, 39., 1993, California. Proceedings... San Diego, 1993. p.39.
• SCOGNAMILLO-SZABÓ, M.V.R.; BECHARA G.H. Acupuntura: bases científicas e aplicações. Ciência Rural, v.31, n.6, p.1091-1099, 2001.
• SCOGNAMILLO-SZABÓ, M.V.R. et al. Effect of Various Acupuncture Treatment Protocols Upon Sepsis In Wistar Rats. Annals of New York Academy of Sciences (in press, 2004).
• SCOGNAMILLO-SZABÓ, M.V.R. et al. Efeito inibitório da acupuntura sobre a migração de neutrófilos para a cavidade peritoneal de ratos. Ars Veterinária (no prelo, 2004)
• SHERMAN, K.J. et al. Description and validation of a nonivasive acupuncture procedure. Journal of Alternative and Complementary Medicine, v.8, p.11-19, 2002.
• SIN, Y.M. Effect of different waveforms on acute pleurisy during electrical acupuncture. American Journal of Acupuncture, v.14, n.1, p.39-42, 1986.
• SIN, Y.M. et al. Electric acupuncture on carrageenan-pleurisy: comparative study using various body regions for stimulation. American Journal of Acupuncture, v.12, n.4, p.355-358, 1984.
• SIN, Y.M. et al. Effect of electric acupuncture stimulation on acute inflammation. American Journal of acupuncture, v.11, p.359-362, 1983.
• STEURER-STEYA, C. et al. Complementary and alternative medicine in asthma do they work? Swiss Medicine Weekly, v.132, p.338344, 2002.
• STORCH, K.F. et al. Extensive and divergent circadian gene expression in liver and heart. Nature, v.417, n.6884, p.78-83, 2002.
• STREITBERGER, K.; KLEINHENZ, J. Introducing a placebo needle into acupuncture research. The Lancet, v.352, p.364-365, 1998.
• TOWNSEND, S. et al. The effect of acupuncture on patients with rheumatoid arthritis: a randomized, placebo-controlled cross over study. Rheumatology, v.38, p864-869, 1999.
• TRACEY, K.J.; WARREN, H.S. An inflammatory issue. Nature, v.429, n.6987, p.35-36, 2004.
• TUKMACHI, E. Acupuncture treatment of osteoarthritis. Acupuncture Medicine, v.17, p.65-67, 1999.
• TUKMACHI, E. Acupuncture and rheumatoid arthritis. Rheumatology, v.39, p.1153-1154, 2000.
• WHO (World Health Organization) (1996). Acupuncture: review and analysis of reports on controlled clinical trials, 87p.
• XUE, CCL. et al. Effect of adding a Chinese herbal preparation to acupuncture for seasonal allergic rhinitis: randomized double-blind controlled trial. Hong Kong Medicine Journal, v.9, n.6, p.427-434, 2003.
• ZHANG, Y.Q. et al. Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats. Pain, v.99, p.525-535, 2002.
• ZHAO, F.; ZHU, L. [Therapeutic effects of electroacupuncture on acute experimental arthritis in rats]. Chen Tzu Yen Chiu, v.15, n.3, p.197-202, 1990.
• ZHAO, F.; ZHU, L. [Effect of electroacupuncture on neurogenic inflammation]. Chen Tzu Yen Chiu, v.17, n.3, p.207-211, 1992.

1

Autor para correspondência.

Publication Dates

• Publication in this collection
  13 Jan 2005
• Date of issue
  Feb 2005

History

• Accepted
  11 Aug 2004
• Received
  22 June 2004