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Original Article

Evaluation of immunomodulatory activity of Immusante® in zebrafish

Anna Venkata Agraz, Mohamed Rafiq, Bhavimani Guru, Madan MN, Suryakanth Dattatreya Anturlikar, Azeemuddin MM, S. N. Manjula.

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Immusante® (IM-133N) a proven polyherbal immunomodulator was evaluated for its immunomodulatory activity in zebrafish models. In the present study, Aeromonas hydrophila (A. hydrophila) is used to develop the experimental model for studying immunomodulatory activity in zebrafish. To determine the A. hydrophila dose for the study, either sex of 30 zebrafish were taken and divided into 5 groups followed by intra-peritoneal injected with 10 µl of 6.0 × 106 , 6.0 × 205 , 6.0 × 105, 6.0 × 104 , and 6.0 × 103 cells/ml of live A. hydrophila suspension, respectively and continuously cultured. Acute toxicity study of Immusante® was done as per the procedures described in OECD 203 guideline. To examine the protective role of Immusante® and to check the survival rate, two zebrafish survival assays were performed. Immusante® was evaluated on A. hydrophila infection in zebrafish to study the effect and differential WBC count was also measured in the zebrafish infected with A. hydrophila. Gene expression analysis was done to check the expression levels of pro-inflammation cytokines. Immusante® at a dose of 200 and 400 mg/L enhanced the survival rate of infected zebrafish and at 400 mg/L, decreased the number of colony forming units (CFUs) of A. hydrophila in the blood and kidney of infected fishes. A significant increase in lymphocyte and decrease in neutrophils was observed in the treatment groups, which are the signs of improvement in non-specific immune response. Immusante® at a dose of 400 mg/L suppressed the pro-inflammatory cytokine’s expression such as IL-1β, IFN-γ, and TNF-α in the kidney of infected zebrafish and prevented the detrimental effects on zebrafish caused due to excessive inflammatory response.

Key words: Immusante®, IM-133N, Immunomodulation, Aeromonas hydrophila, Zebrafish, Proinflammatory cytokines.

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