Sib Sankar Giria, c, 1, Shib Sankar Senb, 1, Jin Woo Juna, V. Sukumaranc,*, Se Chang Parka,*
a Lab of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Gwanak-ro, Seoul, South Korea
b School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
c Dept. of Biotechnology, Periyar Maniammai University, Thanjavur, Tamil Nadu, India
1 These authors contributed equally to this work.
*Corresponding author : V. Sukumaran, Se Chang Park
Abstract
The present study investigated the protective effects of leucine against lipopolysaccharide (LPS)-induced inflammatory responses in Labeo rohita (rohu) in vivo and in vitro. Primary hepatocytes, isolated from the hepatopancreas, were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of leucine against LPS-induced inflammation were studied. Finally, we investigated the efficiency of dietary leucine supplementation in attenuating an immune challenge induced by LPS in vivo. Exposure of cells to 10-25 ¥ìg mL-1 of LPS for 24 h resulted in a significant production of nitric oxide and release of lactate dehydrogenase to the medium, whereas cell viability and protein content were reduced (p < 0.05). LPS exposure (10 ¥ìg mL-1) increased mRNA levels of the pro-inflammatory cytokines TNF-¥á, IL-1¥â and IL-8 in vitro (p < 0.05). However, pretreatment with leucine prevented the LPS-induced upregulation of TNF-¥á, IL-1¥â and IL-8 mRNAs by downregulating TLR4, MyD88, NF-¥êBp65, and MAPKp38 mRNA ____expression____. Interestingly, mRNA ____expression____ of the anti-inflammatory cytokine, IL-10, which was increased by LPS treatment, was further enhanced (p < 0.05) by leucine pretreatment. The enhanced ____expression____ of IL-10 might inhibit the production of other pro-inflammatory cytokines. It was found that leucine pretreatment attenuated the excessive activation of LPS-induced TLR4-MyD88 signaling as manifested by lower level of TLR4, MyD88, MAPKp38, NF-¥êBp65 and increased level of I¥êB-¥á protein in leucine pre-treatment group. In vivo experiments demonstrated that leucine pre-supplementation could protect fish against LPS-induced inflammation through an attenuation of TLR4-MyD88 signaling pathway. Taken together, we propose that leucine pre-supplementation decreases LPS-induced immune damage in rohu by enhancing the ____expression____ of IL-10 and by regulating the TLR4-MyD88 signaling pathways.
Keywords : Leucine; Lipopolysaccharide; Hepatocyte; Labeo rohita; IL-10; TLR4 signaling