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. 2018 May 22;9:1091. doi: 10.3389/fimmu.2018.01091

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Copyright © 2018 Kumar, Baruah, Nguyen, Smagghe, Vossen and Bossier.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Phloroglucinol-generated prooxidant activity mediates the protective response in brine shrimp larvae. (A) Survival (%) of brine shrimp larvae pretreated with antioxidant enzymes, phloroglucinol, or in combination with both (antioxidant enzymes and phloroglucinol) after 48 h of challenge with Vibrio parahaemolyticus MO904. The larvae were pretreated with antioxidant enzymes (catalase, 10 mg/l and superoxide dismutase (SOD), 75 units), phloroglucinol (30 µM), and mixture of antioxidant enzymes and phloroglucinol for 2 h, rinsed to wash away the compound, and then allowed to recover for 2 h. The larvae were subsequently challenged with V. parahaemolyticus at 10⁷ cells/ml of rearing water. Non-pretreated larvae that were either challenged with V. parahaemolyticus (positive control) and those not challenged with V. parahaemolyticus (negative control) served as controls. Error bars represent the standard error of five replicates; different letters indicate significant differences (P < 0.001). (B) Induction of heat shock protein (Hsp70) protein in brine shrimp larvae. The brine shrimp larvae were pretreated with antioxidant enzymes, phloroglucinol, or in combination with both (antioxidant enzymes and phloroglucinol) as described in Figure 2A. Non-pretreated larvae were maintained as control. (a) Protein extracted from different treatment groups was resolved on an SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane and probed with an antibody to brine shrimp Hsp70. Molecular mass standards (M) in kilodaltons are shown on the left. The induction of Hsp70 in HeLa cells was regarded as 1. (b) Quantitative analysis of Hsp70 levels in the brine shrimp larvae is presented relative to Hsp70 production in HeLa cells. Error bars represent the standard error of three replicates; different letters indicate significant differences (P < 0.001). (C) Effect of phloroglucinol on lipid peroxidation in brine shrimp larvae. Lipid peroxidation is expressed as malondialdehyde (MDA) content in each sample. The larvae were pretreated with phloroglucinol (30 µM) for 2 h, rinsed to wash away the compound, and then allowed to recover for 2 h. The larvae were subsequently collected and analyzed for MDA/g of Artemia. Non-pretreated larvae were maintained as control. Error bars represent the standard error of two independent samples; asterisks represent significant difference between the control and the treatment, *P < 0.001.

Phloroglucinol-generated prooxidant activity mediates the protective response in brine shrimp larvae. (A) Survival (%) of brine shrimp larvae pretreated with antioxidant enzymes, phloroglucinol, or in combination with both (antioxidant enzymes and phloroglucinol) after 48 h of challenge with Vibrio parahaemolyticus MO904. The larvae were pretreated with antioxidant enzymes (catalase, 10 mg/l and superoxide dismutase (SOD), 75 units), phloroglucinol (30 µM), and mixture of antioxidant enzymes and phloroglucinol for 2 h, rinsed to wash away the compound, and then allowed to recover for 2 h. The larvae were subsequently challenged with V. parahaemolyticus at 10⁷ cells/ml of rearing water. Non-pretreated larvae that were either challenged with V. parahaemolyticus (positive control) and those not challenged with V. parahaemolyticus (negative control) served as controls. Error bars represent the standard error of five replicates; different letters indicate significant differences (P < 0.001). (B) Induction of heat shock protein (Hsp70) protein in brine shrimp larvae. The brine shrimp larvae were pretreated with antioxidant enzymes, phloroglucinol, or in combination with both (antioxidant enzymes and phloroglucinol) as described in Figure 2A. Non-pretreated larvae were maintained as control. (a) Protein extracted from different treatment groups was resolved on an SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane and probed with an antibody to brine shrimp Hsp70. Molecular mass standards (M) in kilodaltons are shown on the left. The induction of Hsp70 in HeLa cells was regarded as 1. (b) Quantitative analysis of Hsp70 levels in the brine shrimp larvae is presented relative to Hsp70 production in HeLa cells. Error bars represent the standard error of three replicates; different letters indicate significant differences (P < 0.001). (C) Effect of phloroglucinol on lipid peroxidation in brine shrimp larvae. Lipid peroxidation is expressed as malondialdehyde (MDA) content in each sample. The larvae were pretreated with phloroglucinol (30 µM) for 2 h, rinsed to wash away the compound, and then allowed to recover for 2 h. The larvae were subsequently collected and analyzed for MDA/g of Artemia. Non-pretreated larvae were maintained as control. Error bars represent the standard error of two independent samples; asterisks represent significant difference between the control and the treatment, *P < 0.001.