Abstract: Background and Objective: Curcuma longa (C. longa) extract has been reported as a potent anti-inflammatory agent. The main objective of this study was to evaluate the effectiveness of Curcuma longa extract to decrease Tumor Necrosis Factor-alpha (TNF-α) and cells damage prevention in early pregnant mice with acute toxoplasmosis. Materials and Methods: This study evaluated 20 early pregnant mice. The mice were divided into five groups (G1-G5). G1-G4 were injected with 10 tachyzoites of Toxoplasma gondii and G5 was not injected. Three days later, G1 and G2 were intervened with C. longa extract dose of 125 mg and 500 mg kg–1/day, respectively. G3 was a positive control and G4 was a negative control. G5 was not intervened. The TNF-α level was examined serially (before and 3 days post tachyzoites injection and 3 and 7 days post-intervention). The placental mice were taken 7 days after intervention for histopathology examination. Results: The TNF-α level increased significantly 3 days after tachyzoites injection (p<0.05) and TNF-α level decreased significantly 3 and 7 days after curcuma longa intervention compared to the negative control (p<0.05). Hemorrhagic and necrotic cells were not found in the group intervened with C. longa extract but it found in the positive and negative control groups were 75 and 100%, respectively. Conclusion: Curcuma longa is effective to suppress TNF-α level and prevent placental cells damage in early pregnant mice with acute toxoplasmosis.
INTRODUCTION
Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii 1,2. It can cause abortion if the infection happens during early pregnancy3,4. The abortion caused by Toxoplasma gondii (T. gondii) not only caused by direct effect of parasites but can occur due to excessive pro-inflammatory reaction. Toxoplasma gondii will trigger antibody (anti-toxoplasma IgG-IgM anti-body)5,6 and cellular immunity (TNF-α)7,8. The excessive TNF-α levels reported can cause intravascular thrombosis, ischemic and necrotic cells. The increased of TNF-α levels due to Toxoplasma gondii infection in early pregnancy can lead to abortion9.
Serranti et al.10 reported that until now, there has been no ideal drug for toxoplasmosis therapy in pregnant women10. Spiramycin is still the drug of choice but it has been proven unable to eradicate infection in the fetus. Early pregnancy with acute T. gondii infection may be treated with spiramycin11. The therapy with spiramycin will kill the parasite because it works as a bactericidal but spiramycin unable to inhibit the inflammatory process. The abortion caused by T. gondii infection is not only due to the direct effects of the parasite but it can also result from excessive inflammatory responses leading to abortion9,12.
Curcuma longa has been shown to have potent anti-inflammatory13-15 and anti-bacterial, anti-parasites but more likely to be anti-inflammatory15-17. Curcumin is the main content of C. longa, it has been shown to suppress TNF-α13-15. There was no study conducted which evaluate the C. longa effect on the toxoplasmosis18. Hence, this study aimed to determine the effectiveness of C. longa extract compared to spiramycin, especially in preventing cell damage on early pregnant mice with acute toxoplasmosis by analyzing levels of TNF-α and histopathology examination of placental tissue.
MATERIALS AND METHODS
This study was carried out from February, 2017-September, 2017 in Molecular Biology and Immunology Laboratory, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia.
Subjects: The study was conducted on 20 Balb/c female mice that were conditioned into 1-3 day(s) pregnancy that fulfill inclusion criteria (mice age 11-13 weeks, weigh 16- 20 g, active movement, no physical defect and willing to eat and drink). The early pregnant mice were divided into five groups (G1-G5) randomly, 4 mice each group. G1-G4 were injected with 10 tachyzoites of T. gondii RH strain intra-peritoneal and G5 without infection. At the end of the study, the mice were sacrificed then buried in specified places. This research has been approved by the animal ethics research committee of the Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia (Number: 535/114.8.4.5.31/PP36-KOMITEK/20160), dated 2 May, 2016.
Preparation and modeling of experimental animals: Mice were conditioned into pregnancy by bringing together female and male mice in one cage. Every morning, signs of pregnancy were evaluated in the form of a plug in the vagina (reddish and swollen vagina). If plug is found, the mice were considered pregnant and the gestational age is day 0. The C. longa extract use was obtained by maceration and the curcuminoid compound was evaluated using KLT densitometry and spectrophotometry. Curcuminoid level in C. longa extract obtained were at 25.5%.Tachyzoites used in this study were from the T. gondii strain RH (strain pathogen; SRA: ERS670498).
Intervention of experimental animals: Three days after injection of tachyzoites, G1 and G2 were each given C. longa extract dose of 125 and 500 mg kg–1/day, respectively, G3 (positive control) was given spiramycin dose of 60 mg kg–1/day and G4 (negative control/placebo) was given 0.2 mL distilled water. Each intervention was administered orally using cannulas for 7 days. G5 was not infected nor intervened with.
Taking and examination of sample: Blood samples were taken from the tail vein serially (1 day before tachyzoites injection, 3 days after tachyzoites injection, 3 and 7 days after the intervention). Examination of anti-toxoplasma IgG-IgM antibody levels and TNF-α level were examined using Enzyme-Linked Immunosorbent Assay (ELISA).
Seven days after the intervention, the mice were sacrificed by breaking the cervical vertebrae and then surgery was performed to remove the uterus containing the placenta. Examination of placental histopathology in all samples was done to study and assessed the degree of damage of placental tissue in the form of the congestive capillary, extracellular edema, hemorrhagic and necrotic cells, all of which were histopathology findings.
Statistical analysis: The data obtained were statistically analyzed using paired t-test to determine the TNF-α level before and after intervention. One-way ANOVA test was conducted to determine the difference of TNF-α level between the five groups. Histopathology presentation was reported in percentage and chi-square test was performed to identify cells damage between groups. The p<0.05 is used.
RESULTS
The results of TNF-α level are reported in Table 1. The TNF-α level increased significantly 3 days after tachyzoites injection (p<0.05). Three and 7 days after intervention with C. longa extract dose of 125 mg kg–1/day, C. longa dose of 500 mg kg–1/day and spiramycin dose of 60 mg kg–1/day, the TNF-α level decreased significantly (p<0.05) (Table 2). The decrease of TNF-α level among the three groups (G1-G3) was not significant (Table 3 and 4). The TNF-α level in negative control (G4) increased significantly during the study, while in the non-infected group (G5), TNF-α level during the study fluctuated insignificantly (p>0.05) (Table 2).
The results of the histopathology presentation are shown in Fig. 1.
| Table 1: | TNF-α level before and after the injection of tachyzoites and post-intervention |
Groups G1-G4: Early pregnant mice were injected tachyzoites. Interventions: G1 and G2 (mice given C. longa extract dose of 125 and 500 mg kg–1/day, respectively), G3 (positive control and G4 (negative control/placebo), each group is administered for 7 days, G5 (normal group). Blood sampling times: A (1 day before tachyzoites injection), B (3 days after tachyzoites injection), C (3 days after the intervention) and D (7 days after the intervention) |
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| Table 2: | Statistic analysis of TNF-α levels before and after intervention |
Groups: G1 and G2 (mice given C. longa extract dose of 125 and 500 mg kg–1/day respectively), G3 (positive control), G4 (negative control/placebo), G5(Normal group). Tumor Necrosis Factor (TNF), TNFA (TNF-α levels 1 day before tachyzoites injection), TNFB (TNF-α levels 3 days after tachyzoites injection), TNFC (TNF-α days 3 after the intervention) and TNFD (TNF-α levels 7 days after the intervention) (p<0.05) |
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| Fig. 1: | Histopathology presentation of placental tissue sort by group (G1-G5). G1-G4 were injected 10 tachyzoites to intra-peritoneal space. Interventions: G1 and G2 (mice given C. longa extract dose of 125 and 500 mg kg–1/day respectively), G3 (positive control), G4 (negative control/placebo), 7days each and G5 (normal group) |
| Table 3: | Statistic analysis of TNF-α levels after 3 days intervention among five groups |
TNFC: TNF-" levels after 3 days intervention, p<0.05 |
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Hemorrhagic and necrotic cells were not present in the group intervened with C. longa extract dose of 125 mg kgG1/day and dose of 500 mg kgG1/day but in the group intervened with spiramycin, 75% of the samples obtained had hemorrhagic and necrotic cells and in the negative control group, the hemorrhagic and necrotic features were obtained in all samples (100%). The occurrence of hemorrhagic and necrotic cells between C. longa and spiramycin intervention were significantly different (p<0.05).
DISCUSSION
In this study, 3 days after the injection of tachyzoites, the anti-toxoplasma IgG-IgM antibody levels increased significantly. Increased levels of anti-toxoplasma IgG-IgM antibodies in this study proves that mice in this study have occurred acute toxoplasmosis. Simanjuntak et al .6 reported that injecting 10 tachyzoites intra-peritoneal in mice during early pregnancy caused the anti-toxoplasma IgM antibody level increased significantly 24 h after the tachyzoites injection and IgG antibody level increased significantly 72 h after the injection.
| Table 4: | Statistic analysis of TNF-" levels after 7 days intervention among five groups |
TNFD: TNF-α levels after 7 days intervention, p<0.05 |
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The elevated level of anti-toxoplasma IgM antibody after T. gondii infection is a marker that can be considered as an acute infection19,20.
In this study, TNF-α levels increased significantly after 3 days injections of 10 tachyzoites T. gondii strains of RH. Some previous investigators also reported elevated TNF-α levels in T. gondii 21-24. Increased level of TNF-α commences with the detection of toxoplasma parasite protein/glycosylphosphatidylinositol (GPI)-anchored by toll-like receptors (TLRs). TLRs especially TLR2 and TLR4 that are activated by GPI will trigger macrophages to produce TNF-α and IL-1221. The T. gondii infection also specifically stimulates TNF type 1 receptors (p55), so TNF-α can work as a pro-inflammator25.
Seven days after intervention with C. longa extract, the TNF-α levels decreased significantly compared with the negative control group. The decreased in levels of TNF-α in this study occurred because curcumin which is the main component of C. longa, can inhibit the production and action of TNF-α in various ways15. It primarily suppresses the expression of TNF-α through the down-regulation of NF-κB, causing TLR2 and TLR 4 being unable to induce macrophages to produce TNF-α25. Curcumin will also bind to myeloid differentiation protein-2 (MD-2). This MD2-curcumin compound will inhibit MyD 88, consequently inhibiting TLR2, TLR 4, which results in inhibiting TLRs that stimulates macrophages to produce TNF-α26.
The excessive TNF-α level can cause cells damage such as ischemic, hemorrhagic and necrotic cells9. If the TNF-α excess levels happen in early pregnancy it can cause abortion9. This study discovered that hemorrhagic and necrotic cells is not found in intervention groups with C. longa extract dose of 125 and 500 mg kg–1/day but in intervention groups with spiramycin dose of 60 mg–1/day (positive control), it was found 75% and intervention groups with 0.2 mL distilled water it was found 100%. The different incidence of hemorrhagic and necrotic cells among groups intervened with C. longa and spiramycin although TNF-α levels in these two groups did not differ significantly, the researchers suspected to be associated with antioxidant effects possessed by curcuma but need to be further proven.
CONCLUSION
Curcuma longa extract dose of 125 mg kg–1/day for 7 days was effective to decrease the TNF-α levels and it was effective to prevent the hemorrhagic and necrotic cells of placental tissue of early pregnant mice with acute toxoplasmosis. Curcuma longa extract dose of 125 mg kg–1/day can be considered as an alternative therapy in early pregnant women with acute toxoplasmosis but further research is needed for the effectiveness and toxicity of the first trimester pregnancy.
SIGNIFICANCE STATEMENT
This study discovers that C. longa extract dose of 125 mg kg–1/day during 7 days effective to decrease TNF-α level and to prevent cells damage placental tissue in early pregnancy with acute toxoplasmosis. This investigation will facilitate the researchers to obtain an alternative drug for early pregnancy with acute toxoplasmosis that many researchers were not able to explore.
ACKNOWLEDGMENTS
We thanks Prof. Irawan Yusuf, Dr. Andi Mardiah Tahir, Prof. Nurpudji A. Taslim, Prof. Nusratuddin, Dr. Burhanuddin, Hasnawati Ph.D and the laboratory staffs of Molecular Biology and Immunology Laboratory, Faculty of Medicine, Hasanuddin University, Makassar and also to Phytochemistry Laboratory, Faculty of Pharmacy, Hasanuddin, Makassar, for their help in this study.