Effect of Traumeel S on Cytokine Profile in a Cecal Ligation and Puncture (CLP) Sepsis Model in Rats

Abstract

Background:

Sepsis results in significant morbidity and mortality, with current treatment options limited with respect to efficacy as well as safety. The complex homeopathic remedy Traumeel S has been shown to have both anti-inflammatory and immunostimulatory effects in the in vitro setting.

Objectives:

The objective was to explore the effects of Traumeel S in an in vivo setting, using a cecal ligation and puncture (CLP) sepsis model in rats, evaluating the effects of the medication on cytokine activity.

Design:

Sepsis was induced in 30 rats using accepted CLP methodology. Following the procedure, rats were randomly allocated to receive an intraperitoneal injection of either Traumeel S (n=15) or normal saline (n=15). At 6 hours post-CLP, serum cytokines (interleukin [IL]-1β, tumor necrosis factor-α, IL-6, and IL-10) were evaluated.

Results:

IL-1β levels were significantly higher in the treatment group (p=0.03) with no significant differences found between the groups with respect to the other cytokines tested.

Conclusions:

In contrast to in vitro studies, Traumeel significantly increased IL-1β levels in an in vivo model, without influencing other cytokines. IL-1β is a proinflammatory cytokine that has been shown to have a protective effect in the CLP rat model. Further research is warranted to examine this finding, as well as its clinical implications.

Introduction

S epsis is the leading cause of mortality among critically ill patients, resulting from a cascade of events that are initiated by infection and lead to multiorgan failure and death. Despite major advances in critical care medicine, the mortality of septic patients remains in the range of 30%–50%.^1,2 This is partially due to an incomplete understanding of the complex pathophysiologic mechanisms that induce and propagate the downward spiral of events in the septic patient, with a lack of effective therapies that can slow or stop this process. Despite extensive resources invested in finding a treatment for sepsis, the only clinically relevant therapy to date is recombinant human activated protein C.³ This treatment, however, is of limited benefit and not without potential side-effects, such as an increase in the risk of bleeding.⁴ There is therefore a need for novel approaches in the search for a treatment of sepsis.

Homeopathy is a system of therapy that was founded by the German physician Samuel Hahnemann, more than 200 years ago. It is based on the concept that disease can be treated with highly diluted preparations of compounds that are capable of producing similar symptoms in healthy people (the “Law of Similars”).⁵ While “classic” homeopathy employs highly diluted and individualized remedies, based on the patient's physical complaints and personal traits, “complex” homeopathy uses combinations of homeopathic medicines in lower dilutions that can be administered according to conventional medical indications. It is through the use of the latter that those interested in scientific research of this modality can overcome the challenge presented by the contradicting paradigms between homeopathy and conventional medicine.

Traumeel S is a complex homeopathic remedy used widely throughout Europe and South America for the treatment of trauma, as well as inflammatory and degenerative conditions. It contains lower dilutions of extracts from plants and minerals (from 10⁻¹ to 10⁻⁹; Table 1). Though most of the many clinical studies published on the efficacy of Traumeel are of limited quality, a number of them show an enhancement of the healing process in several orthopedic conditions^6,7 as well as reducing edema,⁸ alleviating inflammatory periodontal disease,⁹ and reducing chemotherapy-induced stomatitis in children undergoing stem cell transplantation.¹⁰ In the in vitro setting, Traumeel S has demonstrated anti-inflammatory properties, inhibiting the proinflammatory mediators interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-8 in resting and activated human immunocytes.¹¹ Lussignoli et al. demonstrated a decrease of IL-6 as well, in a rat paw inflammation model following treatment with Traumeel.¹² The purpose of the current study was to test the effect of Traumeel on cytokine production in an in vivo setting, using a cecal ligation and puncture (CLP) sepsis model in rats, a well-established animal model for the production of inflammatory cytokines.

Table 1.

Contents of Traumeel S ^a

Active components ^b	Concentration (μL)	Dilution ^c
Arnica montana	2.2	D 2
Calendula officinalis	2.2	D 2
Atropa belladonna	2.2	D 2
Aconitum napellus	1.32	D 2
Bellis perennis	1.1	D 2
Hypericum perforatum	0.66	D 2
Echinacea angustifolia	0.55	D 2
Echinacea purpurea	0.55	D 2
Symphytum officinale e radice	2.2	D 6
Chamomilla recutita	2.2	D 3
Achillea millefolium	2.2	D 3
Mercurius solubilis (mixture of Mercurius-amido-nitrate (NH₂Hg₂NO₃) and Mercurius-oxide (Hg₂O)	2.2	D 6
Hepar sulfuris (mixture of calcium-polysulfites and calcium sulfate)	2.2	D 6
Hamamelis virginiana	2.2	D 1

Contents of each 2.2-mL injectable ampoule of Traumeel S (manufacturer's information).

Inactive ingredients: Isotonic sodium chloride solution.

D is the expression of the decimal dilution of the stem solution (i.e., D1=10⁻¹, D2=10⁻², D3=10⁻³, etc.).

Materials and Methods

Animals

Thirty male Sprague-Dawley rats, each weighing 275–350 g (Harlan Biotech Israel Limited, Rechovot, Israel) were used in this study. All rats were housed in a controlled environment, with room temperatures maintained at 22°C±2°C and light/darkness cycles of 12 hours, for at least 7 days before the experiments. The rats had free access to food and water, and were not fasted before surgery. The study protocol was approved by the Institutional Ethics Committee for Animal Care and Use (authorization codes: IL-30-5-2004, IL-29-5-06, initially from the Ben Gurion University of the Negev, Beer Sheba, Israel; and MD-07-11006-4, subsequently from the Hadassah University Hospital, Hebrew University, Jerusalem, Israel). Care of the animals was in full compliance with the Guide for the Care and Use of Laboratory Animals set forth by this committee.

Study medication

Two (2) 2.2-mL ampoules (4.4 mL) of Traumeel S (Biologische Heilmittel Heel GmbH, Baden-Baden, Germany) were used for each rat in the treatment group as the study medication. Traumeel S is manufactured according to the European Union Guidelines on Good Manufacturing Practice for Medicinal Products and in accordance with the German Homeopathic Pharmacopoeia. The medications were prepared with physical and microbiologic controls in accordance with the European Pharmacopoeia specifications (Table 1). Normal saline (0.9%), 4.4 mL, of identical color, odor, and viscosity served as placebo medication in the control group rats.

Study procedure

All 30 rats were anesthetized using 80 mg/kg of intraperitoneal ketamine (Pfizer Healthcare, Ireland, PA) and 12 mg/kg of xylasine (Phoenix Scientific, St. Joseph, MO). Sepsis was induced using CLP methodology: The abdominal cavity was opened through a midline incision, with the cecum then identified, mobilized, and gently exteriorized. After ligation, just distal to the ileocecal valve (>35% of cecal length ligated) with 1–0 silk, the cecum was punctured through both sides of the bowel (“through and through”), in the antimesenteric aspect, with a 19-gauge needle and gently squeezed to ensure patency of the holes, extruding a small amount of feces (one drop) from the perforation site. The cecum was then returned to the peritoneal cavity and the abdominal wall closed in two layers. This methodology induces a protracted, polymicrobial form of sepsis.^13,14 The surgeon performing the CLP was blinded as to the treatment allocation.

After closing the abdominal wall, each rat received a 4.4-mL intraperitoneal injection of either Traumeel (treatment group; n=15) or 0.9% normal saline (control group; n=15), with rats allocated to the treatment or control group in a random sequence. Fluid resuscitation with 5 mL of 0.9% saline was then administered subcutaneously in both groups, and postoperative analgesia administered with subcutaneous 20 mg/kg tramadol hydrochloride (Gruenenthal GmbH, Aachen, Germany).¹⁵ At this point, the study rats were placed in their cages in a lateral decubitus position with a slightly elevated head and watched by the investigators until awakening. The awakening was spontaneous, usually occurring after 20–30 minutes when the rats started to move and rise on their feet.

Upon awakening, the study rats were given access to food and water ad libitum. At 6 hours, animals were then anesthetized with closed-chamber isoflurane, and blood was drawn via intracardiac puncture. Samples were placed immediately into ice after withdrawal, followed by cooled centrifuge (at 4°C). Immediately thereafter, rats were euthanized with a lethal dose of intraperitoneally administered pentobarbitone sodium 200 mg/kg, as per guidelines of the Institutional Animal Care and Use committee. Blood samples were centrifuged (3000 rpm for 3 minutes), and serum was removed and stored at −20°C for later measurement of cytokines.

Cytokine analysis

Cytokines were analyzed at the Immunology, Tumor Diagnosis and Cytokine Standardization Laboratory, at the Department of Oncology, Hadassah Hebrew University Medical Center, Jerusalem. Serum levels of rat inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α,) were measured by a solid-phase rat enzyme-linked immunosorbent assay (Quantikine, R&D Systems, Minneapolis, MN). This assay employs the quantitative “sandwich” enzyme immunoassay technique. A monoclonal antibody specific for the interleukin molecule had been precoated onto the polystyrene microtiter plate. Standards and samples were introduced into the wells, and the interleukin present was bound by the immobilized antibody. Assay sensitivity was determined using a minimal detected dose of ≥5 pg/mL for IL-1β and IL-6, ≥10 pg/mL for IL-10, and ≥14 pg/mL for TNF-α in a standard rat-specific test serum.

After washing away any unbound proteins, the second enzyme-linked polyclonal or monoclonal antibody specific for the interleukin was added to the wells in order to “sandwich” the interleukin immobilized during the first incubation. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of interleukin bound in the initial step. The color development was stopped and the intensity of the color was measured using a spectrometer. A curve was prepared, plotting the optical density versus the concentration of the given interleukin in the standard wells. By comparing the optical density of the samples to this standard curve, the concentration of the interleukin in unknown samples was then determined.¹⁶

Statistical analysis

Cytokine levels were compared using Student's t tests, with a Pearson correlation analysis used to test for univariate linear relationships between the cytokines in each group. All reported p-values were two-tailed, and p-values of less than 0.05 were considered statistically significant. All analyses were performed using SAS software, version 9 (SAS Institute, Cary, NC).

Results

Three (3) of the 30 study rats died immediately following surgery: 2 from the treatment group and 1 from controls. At 6 hours post-CLP, IL-1β levels in the surviving rats were significantly higher in the Traumeel group when compared with controls (p=0.03), though TNFα, IL-6, and IL-10 levels were not significantly different between the two groups (Table 2). In each group separately, there was a strong statistically significant positive correlation between IL-10 and TNFα (r=0.82, p=0.001 in the Traumeel group; r=0.77, p=0.002 in controls) as well as a positive correlation between IL-1β and TNFα (r=0.62, p=0.023 in the Traumeel group; r=0.71, p=0.005 in controls). IL-1β and IL-10 were also positively correlated in the univariate analysis, but this relationship disappeared after controlling for TNFα. No correlation was found between IL-6 and the other cytokines (results not shown).

Table 2.

Mean Cytokine Levels in Traumeel S and Control (Saline)–Treated Rats

	Control (n=14)	Traumeel S (n=13)	p-Value ^a
IL-1ß (mean±SD) pg/mL	756.1±809	1875.0±1467	0.03
TNF (mean±SD) pg/mL	14.7±19	42.9±63	0.14
IL-6 (mean±SD) pg/mL	4242.3±2778	3847.8±936	0.62
IL-10 (mean±SD) pg/mL	133.6±140	201.5±260	0.41

Two-sided t-test.

IL, interleukin; TNF, tumor necrosis factor; SD, standard deviation.

Discussion

The effects of homeopathic remedies on physiologic systems remain controversial, largely due to the conflicting paradigms of therapy and the extremely high dilutions of compounds. Yet, while many believe that the homeopathic medications are incapable of having any effect whatsoever, others have attributed significant toxicity to these treatments.^17,18 In the present study, the authors found that the complex homeopathic medication Traumeel induced an immunomodulatory effect, causing a significant elevation of IL-1β in an in vivo CLP rat model of sepsis. This is a largely accepted animal model for mimicking the pathophysiologic derangements in human sepsis, and is the model used in evaluating efficacy and safety of potential treatments for this leading cause of mortality among critical care patients.¹⁹ Serum cytokine levels parallel physiologic derangements observed in septic patients, and are used in commonly applied scoring systems and prediction models, thus serving as a logical target for therapeutic interventions.^20,21 The authors chose to sample the cytokines at 6 hours post-CLP, as this time point is usually considered the peak of the septic process and its production of cytokines in this model.²²

The significant increase in IL-1β levels, as well the positive correlation between this cytokine with TNFα and IL-10 levels, suggest that Traumeel is somehow involved in a broader activation of key cytokine immunomodulators. This increase, however, is in contrast to in vitro studies in which Traumeel was shown to lead to a decrease in levels of pro-inflammatory cytokines, including IL-1β.^11,12 There may be a number of reasons for this, the main one possibly residing in the fact that in vivo and in vitro studies might be inherently different in the immune response to sepsis. As mentioned, in the present study, blood was drawn at 6 hours post-CLP—an interval considered to be the peak of sepsis in this model²²—while in the in vitro studies, levels were checked from 24 to 72 hours post-CLP.

The clinical significance of the increased IL-1β elicited by Traumeel in the study group is controversial. Although IL-1 is considered pro-inflammatory and is associated with negative outcomes in critically ill patients,^20,21,23,24 its role in infection remains controversial. Studies have found both an enhancing effect on host resistance and a protective role of the IL-1 (both α and β) receptor antagonist.^25

–31 In a murine sepsis model, a combined pretreatment regimen including IL-1 and TNFα was shown to significantly reduce mortality rates.^32,33 IL-1α, a closely related cytokine, has been found to reduce intestinal permeability and bacterial translocation in a burn and sepsis model.²⁹ It is also possible that exposure of immunocytes to Traumeel in the in vivo environment responded differently than those following in vitro exposure to the medication. A concurrent contradictory effect of a drug, at once both enhancing and inhibiting various systems, is not unknown in conventional science. The term “hormesis” refers to a dose–response phenomenon characterized by simultaneous low-dose stimulation and high-dose inhibition, resulting in either a J-shaped or an inverted U-shaped dose response.³⁴ This phenomenon had been observed in almost all biologic system types, and with almost all toxicants.^35

–38

There are a number of limitations in this study that need to be addressed in future research. While sepsis reflects a complex series of pathophysiologic mechanisms that induce and propagate a downward spiral of events, this study only evaluated four cytokines: TNFα, IL-1β, IL-6, and IL-10. Furthermore, the ultimate purpose of research such as this in using the CLP model to quantify and manipulate cytokine production is to show efficacy in reducing morbidity and mortality, as well as safety. Nevertheless, it was not the authors' intention to address these issues in the current study, but rather to search for scientifically quantifiable effect of a homeopathic remedy. Indeed, the discovery that Traumeel does result in a significant immunomodulatory response, as well as previous clinical research showing possible benefits of homeopathy in the treatment of sepsis,³⁹ should encourage further studies to examine these effects, as well as their clinical implications. The increased IL-1β levels observed in the current study might suggest that Traumeel beneficially affects the septic process in a manner shown in prior studies.^{25

–33}

Footnotes

Disclosure Statement

No competing interests exist.

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