Investigation of the Effect of Tarantula cubensis Extract on Acute Phase Response

Background: Tarantula cubensis alcoholic extract is used to accelerate wound healing and to relieve edema in many animal species. In addition, it may be useful for many infectious diseases. Considering to these effects, it is believe that these effects may be on immune system. Cytokines (tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interleukin-10 and interferon gamma) secreted by immune cells and acute phase proteins (haptoglobin, alpha 1 acid glycoprotein, serum amyloid A) secreted by liver play role in acute phase response. The aim of the present study was to determine the effect of Tarantula cubensis alcoholic extract on cytokine and acute phase protein levels in sheep. Materials, Methods & Results: Tarantula cubensis alcoholic extract (6 mL/sheep, subcutaneously, single dose) was administered to 6 healthy sheep. Blood samples were obtained before (0 h) and after treatments at 2, 4, 8, 12, 24 and 48 h. Then, blood samples were centrifuged to obtain serum samples. Acute phase cytokines such as serum tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interleukin-10, interferon gamma and acute phase proteins such as haptoglobin, alpha 1 acid glycoprotein and serum amyloid-A concentrations were determined with commercially available kits on ELISA reader. Administration of Tarantula cubensis alcoholic extract caused fluctuations in tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interleukin-10, interferon gamma levels in sheep. In addition, levels of haptoglobin, alpha 1 acid glycoprotein, serum amyloid A showed fluctuations. But, these fluctuations in acute phase cytokines and acute phase proteins were not statistically significant (P > 0.05). Discussion: Tarantula cubensis alcoholic extract, homeopathic medicine, is used trauma, retentio secundinarium, tendinitis, bluetongue, foot and mouth, metritis and arthritis in many animal species including sheep. Cytokines, secreted against various stimulus including infectious diseases, play role in wound healing and in the regulation of the immune system. In current study, administration of Tarantula cubensis alcoholic extract lead to fluctuations in tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interleukin-10 and interferon gamma levels, but these changes were not statistically significant (P > 0.05). Non-statistical fluctuations in cytokines result from inadequate immunological response of sheep against to Tarantula cubensis alcoholic extract. Also, use of molecular analysis techniques may be changed these results. Acute phase proteins are significantly secreted from the liver during the acute phase response. In current study, administration of Tarantula cubensis alcoholic extract in sheep caused non-statistifical fluctuations on haptoglobin, alpha 1 acid glycoprotein and serum amyloid A levels (P > 0.05). Tumor necrosis factor alpha and interleukin-1 beta stimulate synthesis of interleukin-6. Interleukin-6 provides synthesis of acute phase proteins in liver. Non-statistical fluctuations in acute phase proteins result from inadequate stimulus of IL-6. In conclusion, it may be stated that administration of Tarantula cubensis alcoholic extract has no distinctive effect on the acute phase response. However, when Tarantula cubensis alcoholic extract is administered repeated times or other acute phase parameters are evaluated, different results may be observed.


INTRODUCTION
Theranekron D6 is veterinary product and includes Tarantula cubensis alcoholic extract (TCAE).It is prepared from the spider Tarantula cubensis.In veterinary medicine, TCAE is often used as demarcation provider, epithelialization accelerator and edema relieving effects in traumatic or necrotic disorders and various infectious diseases [2].
Cytokines are soluble glycoproteins.They play role in immune system regulation, inflammatory response, cell division/differentiation, wound healing and hematopoiesis [3,11,16].Proteins changed significantly in the blood levels as the immune system's response at early (acute) period in cases such as infection, inflammation, trauma and stress are given acute phase proteins name.In particular, they are synthesized by interleukin (IL)-6 in the liver and developing response takes 1-2 days.Essential tasks are return to live normal function, to limit microbial growth and to regulate hemostasis as well as many biological effects.Haptoglobin, alpha 1 acid glycoprotein (A1AG) and serum amyloid A (SAA) are acute phase proteins and increases in inflammation [4,9,18,21].
Cytokines and acute phase proteins are necessary for the functionality of immune system and concentrations of these rise within hours of blood concentrations in infection [3,9,19,21].It has been hypothesized that cytokines and acute phase proteins have been increased by TCAE.
The main objective of this study was to determine the effect of single dose TCAE on tumor necrosis factor alpha (TNFα), IL-1 beta, IL-6, IL-10, interferon gamma (IFNγ), haptoglobin, A1AG and SAA levels in sheep.

Statistical analysis
The data were evaluated by ANOVA and Tukey post hoc test (SPSS 19.0).P < 0.05 was considered as statistically significant difference.

DISCUSSION
TCAE is evaluated a homeopathic medicine in the veterinary field and recommended in many animal species, including sheep.Product is used primarily as demarcation provider, epithelialization accelerator and edema relief in necrotic or traumatic situations.In addition, it is used in infectious diseases and cancer [1,2,5].It is stated that the absence of significant side effects of TCAE in sheep or horse [6,15].
Cytokines are secreted especially immune cells against various warnings including infectious diseases, and play role involved in wound healing and regulation of the immune system [11,16].
IL-6 allows the synthesis of acute phase protein from liver.Synthesis of IL-6 has stimulated by TNFα and IL-1 [14,19].A significant effect on the measured cytokines can not be seen due to not enough immunological response to the TCAE application or not be researched with molecular techniques.
As with changes in levels of cytokines, acute phase proteins levels not determined statistically change.This state can result from the not enough stimulation of the synthesis IL-6 by TCAE, because IL-6 starts acute phase proteins synthesis from liver.

CONCLUSION
In conclusion, it may be stated that TCAE application has no distinctive effect on the acute phase cytokines or proteins.However, when TCAE is administered repeated times or other acute phase parameters are evaluated, different results may be observed  Funding.This study was supported by SUBAK (15401030).
Ethical approval.The study protocol was approved by Ethical Committee of Faculty of Veterinary Medicine, Selcuk University, Turkey (No.2014/27).

Declaration of interest.
The authors report no conflicts of interest.The authors alone are responsible for the content and writing of the paper.