original paper

Roeland Van Wijk, PhD and Fred AC Wiegant, PhD

Roeland Van Wijk and Fred AC Wiegant are from the Department of Molecular Cell Biology, Faculty of Biology, Utrecht University, Utrecht, the Netherlands.

The similia principle is considered to be the essence of homeopathy. This article describes a research program for study of the similia principle in cultured mammalian cells. This systematic program with its rather simple research model was set up ultimately to contribute to the design of studies of the similia principle with more complex organisms such as humans. With respect to application of the similia principle, the concepts of self-defense and self-recovery are central. At the cellular level, self-defense and recovery largely depend on the availability of proteins with a cell-protective function, most notably, stress or heat shock proteins. To study the similia principle, we use four lines of research to examine the processes of self-defense. First, stimulation of self-defense in disturbed and disordered cells is studied by using low doses of an agent homologous or identical to the disturbing agent. The second line of research deals with the specificity of this stimulation: Is cellular selfdefense after exposure to toxicant A also effectively stimulated in an analogous or heterologous way by low doses of other toxicants such as B or C? The third line of research involves the duration of low-dose sensitivity of disordered cells for homologous stimulations, in particular, the desensitization of cells toward these homologous stimulations. The fourth line of research deals with whether—according to the similia principle—the state of desensitization can be overruled by heterologous condition(s) that induce an analogous pattern of protector proteins (ie, a pattern closely resembling the damage-induced pattern) and thus effectively stimulate cellular defense and recovery. (Alternative Therapies in Health and Medicine. 1997;3(2):33-38)

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he essence of homeopathy is the stimulation of biological defense and recovery mechanisms by the use of compounds according to the similia principle. The similia principle suggests that any state of disturbance that is not corrected spontaneously (and leads to a state of “disease”) can be corrected by minute doses of compounds that at a higher dose can produce effects closely resembling the symptoms of the disease being treated or by minute amounts of the compounds that actually caused the disease. When cells are damaged, a mechanism is switched on to withstand the disturbing effects and to stimulate recovery. Increased understanding of such a mechanism would strengthen the foundation of the similia principle.1,2 The basis of this mechanism that regulates the transition between life and death has been the subject of much research for many decades. As a tentative approach to characterizing the essence of the living state, it was proposed that life is a process of being an “organizing entity,” which means an open system that (re)structures and (re)organizes itself. Defense and recovery are biological phenomena that encompass the way in which components of the system are temporally, spatially, and hierarchically rearranged. To study the similia principle, one must understand how to intervene in, adjust, or stimulate these recovery processes. This approach requires quantitative experimentation and evaluation methods (eg, mathematical modeling) and thus requires parameters to study the damage-induced recovery mechanisms experimentally. This article describes the phases of a research program to study the similia principle in cultured mammalian cells. Understanding of the mechanisms of cellular protection and recovery from damage in non–lethally injured cells at the molecular level has increased greatly in recent years. These processes appear to be largely dependent on the availability of proteins with a cell-protective function, most notably stress or heat shock proteins.3-5 When working with cells as a model system, it is crucial to determine what actually occurs in cases of disorder and especially the way the defense and recovery mechanisms are regulated to prevent further infliction of injury or cell


The Similia Principle as a Therapeutic Strategy



death. This article thus focuses on the research strategies for determining which cellular components play a role in defense and on the integration of this information. The chosen research strategy is on complex systems, which requires the analysis of nonlinear interactions in a network. The task of discovering the nature of the defense process during interventions according to the similia principle is the next topic of research. Next, the postulated, precise kinetic and interactive behavior of defense proteins must be tested and it must be shown that the results of the various experiments, when fitted into a quantitative model, are in accord with the observed changes. DAMAGE AND DEFENSE Proteotoxicity The so-called stress response, which started as a molecular curiosity in fruit flies in the early 1960s, now constitutes an active area of research in molecular cell biology.3 The heat shock proteins, one of the most highly conserved group of proteins characterized so far, are implicated as being essential components in a number of diverse biological processes, in particular cellular protection. Following the nomenclature first used for fruit flies, the various heat shock proteins in animal cells are named on the basis of their mode of induction and apparent molecular mass. Hence their designation as hsp70 or grp78, for example, refers to heat shock proteins of 70 kD and glucose-regulated proteins of 78 kD, respectively. A survey of these proteins is presented in the Table. Earlier work was perplexing in that many different agents were able to lead to similar changes in gene expression. However, in the past 15 years, a variety of observations have provided support for the so-called abnormal protein hypothesis suggested to explain the induction of the heat shock response.6 This aspect of toxicity at the level of proteins has been termed proteotoxicity.7 When cells have been exposed to heat shock or to toxic substances such as cadmium and arsenite, or to oxidative stresses, many proteins exhibit deleterious changes in structure. These changes then interfere with the functional interactive capabilities of these proteins. The risk is also high that these abnormal protein molecules aggregate not only with other damaged proteins but also with still functional proteinaceous cellular structures. The damage at the level of proteins lies at the basis of cellular damage and eventually cellular death. Stress Proteins in Cell Protection To understand cellular recovery, one must study the mechanisms that are activated when proteins are damaged and determine how the cell copes with these damaged, life-threatening molecules. Some work on in vitro refolding of a small number of proteins led to the long-lasting impression that folding of newly synthesized polypeptides is an intrinsic feature of their primary structure, independent of other factors. However, the relatively high protein concentration in the cytosol gives rise to misfolding followed by aggregation. To deal with this kind of problem, a set

Major stress protein families and their functions Hsp family hsp100 hsp90 Stress protein hsp100 hsp90 (hsp84) Functions in cell metabolism and cellular stress defense Heat and ethanol tolerance Stabilization of proteins; maintenance of the inactive protein form during transport Essential for cell viability hsp70 grp94 (grp95) hsp70 (hsp73/hsc70) Role in protein folding? Molecular chaperone; required for protein assembly, protein translocation, secretion and import into organelles (Thermo)tolerance Promotes survival in extreme temperatures grp78 (BiP) Maturation of proteins for excretion protein translocation in mitochondria Molecular chaperone that facilitates folding of monomeric proteins and assembly of oligomeric protein complexes; mainly in mitochondrial matrix Metabolism of haem Contributes to thermotolerance and cytoskeletal stabilization Protein denuration




small hsps

hsp32 (haem oxygenase) hsp27


Adapted from Parsell and Lindquist4 (1993)

of stress proteins, collectively called chaperones, ensure that polypeptides fold and assemble properly in the cell. The stress proteins are also named according to their function. Examples include chaperone proteins (which form complexes with proteinaceous and other, more intricate, cellular structures in order to prevent premature or deleterious interactions between proteins), binding proteins (which form complexes with receptor proteins to regulate their activity, ie, a receptor function), or protector proteins (which have crucial functions in the mechanism of cell defense and recovery). In this respect, the protector proteins seem crucial in many ways in the normal functioning of proteins under adverse conditions and in the refolding of structurally damaged proteins.4



The Similia Principle as a Therapeutic Strategy

Pattern of Protector Proteins is Damage Specific A variety of different protector proteins have been found. Interestingly, the pattern of protector proteins that is induced appears to be damage specific.8-10 Under different deleterious conditions, qualitatively different patterns of protector proteins seem to be induced. Although an explanation for such a complex regulation is lacking, the patterns do suggest some differentiation in the induction of the type of damage-induced protection mechanisms. Because these protector proteins are essential to survive threatening conditions and because their induction occurs at concentrations of various toxic compounds that are not yet lethal for the cells, the damage-specific pattern of induction may be considered as the cellular equivalent of the homeopathic remedy picture. The Problem of Regulation of Availability of Protector Proteins A simple model for the regulation of protector proteins in defense after cell damage is depicted in Figure 1. The quantity of free protector proteins available in the cell decreases after cell damage. As long as these protector proteins are available, damage is reduced to a minimum. However, when a shortage of protector proteins arises in the case of an overload of damage, the abnormal protein molecules can complex with other cell structures. Cell damage and death can then be avoided only by production of increasing amounts of new protector proteins. The

replenishment of these protector proteins starts with activation of associated protector protein gene promoters on the cell’s DNA. This highly specific activation occurs by binding of specific DNA-binding factors, called heat shock transcription factors (HSFs), on these DNA sites.11 The binding of the HSF to the promoter on the cell’s DNA is the signal that triggers transfer of information from DNA into messenger RNA (mRNA), leading eventually to synthesis of new protector proteins. Whether or not these DNA-binding factors interact with the DNA depends on the existing quantity of protector proteins in the cell. The genome is specifically activated to trigger this synthesis of additional protector proteins only when the quantity of protector proteins falls below a certain threshold. Normally, at least one type of protective protein, hsp70, and a DNA-binding factor, HSF, form a complex that provides the basis for this regulation. If protector proteins are required to neutralize abnormal proteins, this complex then dissociates, releasing HSF, which then binds to the promoters and induces production of mRNA, with the ensuing synthesis of new protector proteins. When sufficient new protector proteins have been produced, that is, when the level of these proteins is raised above the threshold value, hsp70 will again form a complex with HSF molecules, uncoupling the HSF from DNA, with a concomitant halt in production of mRNA. In terms of systems theory, one might say that this is the autoregulation loop that forms the basis of damage-induced recovery processes. Mathematical Modeling as a Future Step in Understanding the Regulation of Defense Defense and recovery are not simple and singular phenomena but are determined by the kinetic parameters of the autoregulation loop as illustrated in Figure 2. Insight into the effectiveness of this loop for cellular defense can therefore be obtained only from studies involving the parameters mentioned in this mathematical model. This mathematical model of defense and recovery processes has only recently started with the development of a model of hsp70 regulation in the cell.12 So far, five main blocks, each describing one or more processes involved in the functions of hsp70 and its synthesis, have been developed.1,12 These blocks constitute the basic structure of a mathematical model for the regulation of hsp70 in cells. The major processes included in the model so far are (1) denaturation of proteins after an increase in temperature and the binding of hsp70 to denatured or nascent proteins; (2) interaction of hsp70 with HSF and the activation of HSF not bound to hsp70; (3) interaction of activated HSF with a specific HSF binding sequence (heat shock element, HSE) on DNA; (4) the relationship between HSE and transcriptionally active HSF and the level of hsp70mRNA actively engaged in synthesis of hsp70; and (5) translation of viable hsp70mRNA into its protein. In the model constructed so far, the output of this regulation is the level of free, that is, unbound, hsp70 in the cell. Future steps in developing this mathematical model concern the inclusion of lethality in order to predict stress-dependent survival and


Compensation cycle HSF (inactive)


Pool of free HSPs Protector protein HSF (active)

Denatured protein


HSP FIGURE 1 Basic processes of damage and defense at the cellular level. Proteotoxicity is illustrated as a change in protein conformation. Protector proteins interact with these abnormal proteins. After depletion of the pool of free HSPs, supplementation of these protector proteins occurs by way of the compensation cycle.

The Similia Principle as a Therapeutic Strategy



2.0 Relative synthesis of hsp70




SIMILIA PRINCIPLE According to homeopathic insights, recovery can be stimulated by all kinds of substances, provided that they are applied in a specific way. Of all substances, the one most suitable for stimulation of recovery is the one that can produce the artificial situation of disorder that most closely resembles the disordered state. In other words, this concept hinges on the resemblance between symptoms of the disturbed system and the symptoms caused by the applied substance in a healthy system. Application of Low Doses of Homologous Damaging Conditions The similia principle in its most elementary form can easily be tested at the cellular level by determination of the extent to which recovery is stimulated by a small dose of a substance that in the first instance—at a higher dose—is responsible for deregulating the system or leading to sickness. The relevant question for this overview is whether this stimulation will be translated into increased synthesis of protector proteins and increased survival of cells. This hypothesis, representing homologous sensitization, has been studied by a step-down heating protocol in which the initial treatment with high heat is immediately followed by a second treatment at lower doses. When these step-down heating conditions were used, an enhanced synthesis of protector proteins was indeed seen.13-15 Other studies show an enhanced occurrence of thermotolerance when mild step-down heatings were used.16 The next relevant question is whether this response is a general occurrence and can thus also be observed after stressor conditions other than heat shocks. To this end, a damage-inducing treatment of cells with arsenite or cadmium was followed by low doses of arsenite or cadmium, respectively. When this step-down treatment with arsenite or cadmium was used, cells exhibited an enhanced synthesis of various protector proteins and development of tolerance.17,18 The lower doses had no effect on synthesis of protector proteins or development of tolerance in cells that were not pretreated (ie, healthy cells). Figure 2 shows, schematically, the transient induction of hsp70. The synthesis of this protein is further enhanced when a low stressor dose is applied during the so-called posttreatment period. The level of tolerance that is achieved is also higher when the low dose is applied after treatment as compared to cells that received a pretreatment only. The Specificity of the Low-Dose Effect With respect to the specificity of the defense-enhancing effect of low doses of an analogous stressor, some recent studies examined induction of protector proteins in sensitized cells by low doses of analogous but potentially damaging conditions. So far, experiments have examined the effects of pretreatments given with either a heat shock, sodium arsenite, or cadmium chloride. After these pretreatments, the cultures were exposed to low doses of heat, arsenite, or cadmium or to control conditions.


0.0 0 2 4 Time (hours) 60 B 6 8

Relative survival (%)



0 C x X X-x

FIGURE 2 Schematic presentation of the effect of a low dose of a homologous stressor on the synthesis of hsp70 (A) and on development of tolerance (B). When the stress condition is followed by a low dose of the same stressor, an enhancement of hsp70 synthesis (filled triangles) and of tolerance development is observed in comparison with the effect of the stress condition alone (open circles). A low dose of the stressor does not influence the control cells (open triangles). C=control, x=low-dose stressor, X=stressor, X-x=stressor followed by low-dose stressor.

changes in cellular sensitivity to stressors. To sum up, the integrity of the cellular systems, their defense and recovery, depends not only on the available protective proteins but also on the speed of activation of the total rescue mechanism. Future developments in this research are directed at the damage dependency and differentiation of the mentioned production process. A quantitative experimental analysis and a mathematical evaluation are considered essential for this study and for understanding any interventions in this production process, particularly interventions according to the similia principle.



The Similia Principle as a Therapeutic Strategy

The stimulating effect on the synthesis of various protector proteins was then studied. This application of either heat shock, arsenite, or cadmium at subliminal conditions to already disordered cells early in their recovery yielded a different degree of synthesis of protector proteins. Only incubation of damaged cells with low doses of homologous conditions resulted in this extra synthesis of protector proteins.1 Additionally, the degree of stimulation by low doses of stressors depended on the degree of similarity between the two stressors (Figure 3): The more similar the induced pattern of protector proteins by two stressors, the larger the stimulatory action. These observations confirm the specificity of the low-dose effect and are now extended by incorporation of a broader range of stress conditions in the experimental protocol. Specific Desensitization A particular aspect of the cellular response to stressors is that the cells show a biphasic change in their sensitivity to a stressor. An initially enhanced sensitivity (sensitization) toward a second application of the same stressor is followed by a reduced sensitivity (desensitization or tolerance). This phenomenon is represented schematically in Figure 4. This biphasic change in sensitivity is observed after heat treatments and after treatments with cadmium 18 or arsenite. 19 In particular, the change in sensitivity can be seen after the period of stressorinduced synthesis of protector proteins. This state of refractori-

6 5 LD50 (arbitrary units) 4 3 2 1 0 0 1 2 3 4 5 6 Time (hours)

6 5 4 3 2 1 0 HSP inducibility (arbitrary units)

FIGURE 4 Schematic presentation of the change in sensitivity (filled squares) and HSP inducibility (open squares) at various times after stressor application. Closed circles=control level.

2.0 Relative synthesis of hsp70

ness (or tolerance) is also seen in the induction of synthesis of protector proteins by a second heat shock20-22 and by fractionated arsenite17,23 or cadmium treatments.18 The consequence of this temporal refractoriness is that homologous sensitization is only transient. These observations led to the conclusion that low doses of homologous substances are effective only during the early period of recovery. The next questions in the research program are whether this desensitization later in cellular recovery is a stressor-specific phenomenon and to what extent a heterologous reinduction remains possible. Heterologous Reinduction Recently, the specificity of desensitization of protector protein induction was tested with heat shock, sodium arsenite, and cadmium chloride used as primary and secondary inducers of protector proteins.23 The data suggest that the degree, but not the pattern, of reinduction of protector proteins is influenced by the type of stressor used in the pretreatments. Thus, stimulation of synthesis of protector proteins after cadmium as the secondary stressor is severely inhibited in cadmium-pretreated cells, whereas reinductions after arsenite and heat shock are specifically inhibited in cells pretreated with arsenite and heat shock, respectively. In summary, homologous sensitization is transient, and homologous desensitization develops specifically. Apparently, a common denominator regulates the coordinate expression of a group of protector proteins. Heterologous reinduction can still occur but, in that case, the pattern of protector proteins induced by the secondarily applied stressor shows a stressor specificity that is dependent only on the second treatment and is independent of any pretreatment.


0 After Before 0 as As cd 0 as Cd cd

FIGURE 3 Schematic presentation of the specificity in stimulation of HSP synthesis by low doses of stressors. A high dose induces a certain level of HSPs, which is further enhanced when subsequently incubated with a low dose of the same stressor, but not with a low dose of stressor conditions that were not similar. A low dose exerts no effects on the level of hsp70 found in control conditions (black bars).

The Similia Principle as a Therapeutic Strategy


Future Research Aimed at Further Understanding of the Similia Principle The possible implication of the results mentioned so far is that the increased production of protector proteins at a later time after an injury, that is, during the period of homologous tolerance, occurs only after application of a heterologous damaging compound. When we consider the pattern of synthesized protector proteins as the molecular symptoms of recovery, the specificity of the similia principle reveals itself in the degree of relatedness between the effects of the disturbing compound on the pattern of synthesis of these protector proteins. We must search for analogous damaging conditions that induce patterns of protector proteins similar to those generated by the primary stressor without being identical to this primary stressor. For this step, different compounds must be tested at the cellular level. Another possibility is to look for conditions that induce the whole range of protector proteins, such as, for instance, the use of a mixture of stressors in low doses (Figure 5). Then, as a result of a concerted action, the synthesis of all protector proteins will be enhanced by an overruling or circumvention of the lack of induction of some protector proteins due to stressor specificity. In fact, from this perspective, nature seems to use fever (low hyperthermic temperatures) to provide organisms with a means of stimulating protection and recovery mechanisms after damage, as suggested by preliminary data.1 Significance of Fundamental Studies Studies on the similia principle at the cellular level are without doubt significant for our understanding of the stimulation of recovery processes at the cellular level. It is apparent that the regulation of recovery can be stimulated in a specific way by

application of low doses of damaging conditions. The selection of condition can be based on the molecular symptoms of the cellular defense process. In this respect, the application of low doses according to the similia principle warrants further study. It should now also be easier to understand the role of these cellular recovery processes in an organ’s functionality after exposure of an intact organism to stressful conditions. Therefore, the similia principle can be expected to manifest itself as a general biological phenomenon. This offers exciting opportunities for developing new avenues for therapeutic interventions in biomedicine.
C A van der Mast, PhD, is gratefully acknowledged for critical reading of the manuscript. This work was supported by the HomInt organization, Karlsruhe, Germany.

1. Van Wijk R, Wiegant FAC. Cultured Mammalian Cells in Homeopathy Research: The Similia Principle in Self-recovery. Utrecht, the Netherlands: Utrecht University, 1994:1-230. 2. Van Wijk R, Ooms H, Wiegant FAC, et al. A molecular basis for understanding the benefits from subharmful doses of toxicants: an experimental approach to the concept of hormesis and the homeopathic similia law. Environ Management Health. 1994;5:13-25. 3. Nover L. Heat Shock Response. Boca Raton, Fla: CRC Press; 1991:1-499. 4. Parsell DA, Lindquist S. Heat shock proteins and stress tolerance. In: Morimoto RI, Tissières A, Georgopoulos C, eds. The Biology of Heat Shock Proteins and Molecular Chaperones. New York, NY: CSHL Press; 1994:457-494. 5. Welch WJ. How cells respond to stress. Sci Am. May 1993:34-41. 6. Georgopoulos C, Welch WJ. Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol. 1993;9:601-634. 7. Hightower LE. Heat shock, stress proteins, chaperones and proteotoxicity. Cell. 1991;66:191-197. 8. Sanders BM. Stress proteins in aquatic organisms: an environmental perspective. Crit Rev Toxicol. 1993;23:49-75. 9. Wiegant FAC, Souren JEM, Van Rijn J, Van Wijk R. Stressor-specific induction of heat shock proteins in rat hepatoma cells. Toxicology. 1994;94:143-159. 10. Ovelgönne JH, Bitorina M, Van Wijk R. Stressor-specific activation of heat shock genes in H35 rat hepatoma cells. Toxicol Appl Pharmacol. 1995;135:100-109. 11. Morimoto RI. Cells in stress: transcriptional activation of heat shock genes. Science. 1993;259:1409-1410. 12. Peper A, Grimbergen CA, Spaan JAE, Souren JEM, Van Wijk R. A mathematical model of the hsp70 regulation in the cell. J Cell Biochem. 1995;19B[suppl]:202. 13. Schamhart DHJ, Zoutewelle G, van Aken H, Van Wijk R. Effects on the expression of heat shock proteins by step-down heating and hypothermia in rat hepatoma cells with a different degree of heat sensitivity. Int J Hyperthermia. 1992;8:701-716. 14. Delpino A, Gentile FP, Di Modugno F, Benassi M, Mileo AM, Mattei E. Thermosensitization, heat shock protein synthesis and development of thermotolerance in M-14 human tumor cells subjected to step-down heating. Radiat Environ Biophys. 1992;31:323-332. 15. Ovelgönne JH, Van Wijk R. Modulation of hsp68 gene expression after heat shock in thermosensitized and thermotolerant cells is not solely regulated by binding of HSF to HSE. Int J Hyperthermia. 1995;11:719-732. 16. Van Wijk R, Ovelgönne JH, de Koning E, Jaarsveld Van Rijn I, Wiegant FAC. Mild stepdown heating causes increased transcription levels of hsp68 and hsp84 mRNA and enhances thermotolerance development in Reuber H35 hepatoma cells. Int J Hyperthermia. 1994;10:115-125. 17. Ovelgönne JH, Wiegant FAC, Souren IEM, Van Rijn I, Van Wijk R. Enhancement of the stress response by low concentrations of arsenite in arsenite-pretreated H35 hepatoma cells. Toxicol Appl Pharmacol. 1995;132:146-155. 18. Wiegant FAC, Van Rijn I, Van Wijk R. Enhancement of the stress response by minute amounts of cadmium in sensitized Reuber H35 hepatoma cells. Toxicology. (in press). 19. Wiegant FAC, Souren IEM, Van Rijn I, Van Wijk R. Arsenite-induced sensitization and self-tolerance of Reuber H35 hepatoma cells. Cell Biol Toxicol. 1993; 9:49-59. 20. Laszlo A. The relationship of heat-shock proteins, thermotolerance, and protein synthesis. Exp Cell Res. 1988;178:401-414. 21. Li GC, Mak JY. Re-induction of HSP70 synthesis: an assay for thermotolerance. Int J Hyperthermia. 1989;5:389-403. 22. Tuijl MIM, Cluistra S, van der Kruijssen CMM, Van Wijk R. Heat-induced unresponsiveness of heat shock gene expression is regulated at the transcriptional level. Int J Hyperthermia. 1993;9:125-136. 23. Wiegant FAC, Spieker N, Van der Mast CA, Van Wijk R. Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins? J Cell Physiol. 1996;169:364-372.

Stimulating Damage conditions y x


z2 z3




y Time (hours)

FIGURE 5 Stimulation of recovery of damaged cells by three types of treatment. Upon damage by stressor X, a specific pattern of HSPs (indicated by the solid bars) is observed. In the time after damage, the cells show a differential sensitivity in time toward these treatments. These treatments include a homologous condition (isopathy, X followed by x); a singular heterologous but similar condition (X followed by y), and a composite of heterologous conditions (X followed by z1 through z3).



The Similia Principle as a Therapeutic Strategy