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The Metabolomic Profiles of Sera of Mice Infected with Plasmodium berghei and Treated by Effective Fraction of Naja naja oxiana Using 1H Nuclear Magnetic Resonance Spectroscopy

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Abstract

Background

The use of venom fractions from the Iranian cobra could be useful adjunct treatments of malaria with chloroquine. A metabolomic investigation with 1HNMR spectroscopy was conducted on an effective fraction tested earlier using Plasmodium berghei as an experimental murine model.

Purpose

We sought to ascertain both safety and anti-parasitic effects of experimental therapies.

Methods

After purification of the venom fractions, 25 mice were infected, then treated for 4 days with 0.2 ml of 5 mg/kg, 2.5 mg/kg and 1 mg/kg of the effective fraction, chloroquine, and a drug vehicle. An ED50 was obtained using Giemsa staining and real-time PCR analysis. The toxicity tests inspecting both liver and kidney tissues were performed.

Results

A clear inhibitory effect on parasitaemia was observed (with 75% inhibition with 5 mg/kg and 50% reduction when 2.5 mg/kg dosage used). ED50 obtained 2.5 mg/kg. The metabolomics were identified as differentiation of aminoacyl-t-RNA biosynthesis, valine, leucine, isoleucine biosynthesis and degradation pathways were observed.

Conclusion

Upon therapeutic effects of cobra venom fraction, further optimization of dose-dependent response of pharmacokinetics would be worthwhile for further exploration in adjunct experimental venom therapies.

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Acknowledgements

We wish to acknowledge Iran National Science Foundation (INSF), Pasteur Institute of Iran and Iran University of Medical Sciences for jointly funding this Project.

Funding

This work was supported by Iranian University of Medical Sciences [Code: 9223651202].

Author information

Authors and Affiliations

  1. Department of Medical Parasitology, School of Medicine-International Campus, Iran University of Medical Sciences, Tehran, Iran

    Fateme Hajialiani

  2. Department of Medical Biotechnology, Laboratory of Venom and Bio Therapeutics Molecules, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Delavar Shahbazzadeh

  3. Department of Parasitology, Faculty of Para Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

    Fatemeh Maleki

  4. Department of Laboratory Science, Islamic Azad University, Babol Branch, Babol, Iran

    Taher Elmi

  5. Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

    Fatemeh Tabatabaie

  6. Biochemistry Department, Pasteur Institute of Iran, Pasteur Avenue, Tehran, Iran

    Zahra Zamani

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  1. Fateme Hajialiani
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Contributions

FH, ZZ, FT and TE performed experiments and analyzed the data. FT and FM searched the literature and designed the project. FT, ZZ and DS have participated in drafting the manuscript and supervised the research. FT and ZZ wrote the final manuscript. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Fatemeh Tabatabaie or Zahra Zamani.

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Ethical Approval

This study was approved by the Ethics Committee of Iran University of Medical Sciences in accordance with guidelines. The designated approval code is: IR.IUMS.REC 1395.9223651202.

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11686_2021_456_MOESM1_ESM.docx

Supplementary file1 Supplement Fig.1 (A). The standard curve of cloned gene 18S rRNA sequence. (B) The red points show different concentration of standard gene, and the blue point show samples extracted from the mice for the ED50 test. Supplement Fig. 2 (A). The melt curve of the 18S rRNA gene synthesized and cloned into the PUC57 from samples extracted from the mice for the ED50 test. (B). The amplification of the 18S rRNA gene synthesized and cloned into the PUC57, and the samples extracted from the mice for the ED50 test. Supplement Fig.3. The histological sections of the liver for toxicity assay. Hematoxylin and Eosin staining with magnification of 100x, 400x and 420x. Number 1: A liver cell; Number 2: Central vein; Number 3: A macrophage or Kupffer cell. Negative control group: Hepatic lobules, the central vein, portal areas and hepatocytes were completely normal without hyperemia, edema or inflammation. (B) Positive control group: A slight degree of hyperemia was observed in this group. The invasion of the inflammatory cells, i.e., macrophages, was also visible in the liver parenchyma (shown with arrows). Explanation: The so-called Kupffer cells or macrophages in the liver tissue contain triangular or oblong nuclei from a histological perspective; nevertheless, the macrophages in the present histological section appeared to have recently entered the liver tissue from blood, and did not resemble Kupffer cells in terms of the appearance of their cytoplasm and nuclei. (C) ED50 effective fraction: Hyperemia was not observed in this group. The number of macrophages were also lower, and most of them appeared with a general structure of a Kupffer cell (shown with arrows). Supplement Fig.4. Histological sections of the kidney in the toxicity assay. Hematoxylin and Eosin staining with a magnification of upper row:40x, middle row:100x and lowest row:400x. Number 1: Renal corpuscle: Number 2: Proximal convoluted tubule; Number 3: Distal convoluted tubule. Negative control group: The histo-architecture of the cortex and center of the kidney was normal with no breakdown, hyperemia, edema and inflation. (B) Positive control group: In this group, the epithelial disruption of renal tubes was observed in some segments along with their release into the central lumen as well as sediments in proximal and distal convoluted tubules (shown with arrows). (C) ED50 effective fraction group: The entire histo-architecture of the kidney was normal in this group. Supplement Fig.5. Histological sections of the spleen in the toxicity assay. Hematoxylin and Eosin staining with a magnification of upper row 40x, middle row 100x and lower row 400x. Number 1: The white pulp of the spleen; Number 2: The red pulp of the spleen; Number 3: Lymphoid cells. Negative control group: The general histology of the spleen, including the white pulp comprising lymphatic cells and the red pulp comprising red blood cells, was normal with no hyperemia, edema, and inflation. (B) Positive control group: A significant influx of lymphatic cells, especially macrophages, was observable in this group. In some cases, the macrophages in this group were joined together forming giant cells (shown by arrows). (C) ED50 effective fraction: The entire histo-architecture of the spleen was normal in this group. Supplement Fig.6 (A). Score plot from chemometric analysis of spectra from ED50 of effective fraction and negative controls. (B). Score plot of spectra from ED50 of effective fraction and positive controls.Supplement Fig.7 (A). VIP scores from PLS-DA in ED50 of effective fractions and positive fractions showing levels of important components. (B). VIP scores from PLS-DA in ED50 of effective fractions and negative fractions showing levels of important components. (DOCX 1199 kb)

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Hajialiani, F., Shahbazzadeh, D., Maleki, F. et al. The Metabolomic Profiles of Sera of Mice Infected with Plasmodium berghei and Treated by Effective Fraction of Naja naja oxiana Using 1H Nuclear Magnetic Resonance Spectroscopy. Acta Parasit. 66, 1517–1527 (2021). https://doi.org/10.1007/s11686-021-00456-7

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