Anti-radiation damage effect of polyethylenimine as a toll-like receptor 5 targeted agonist

Hu, Zhiqiang; Xing, Yaling; Qian, Yuanyu; Chen, Xiaojuan; Tu, Jian; Ren, Lening; Wang, Kai; Chen, Zhongbin

Toll-like receptor 5 (TLR5) belongs to the TLR family and plays a pivotal role in the natural immune system. TLR5 is mainly expressed in epithelial cells, mononuclear cells, macrophages and dendritic cells. It mediates immune response and inflammation formation, and activates downstream NF-κB signaling pathways. To our knowledge, the only natural agonist available is a bacterial flagellum protein, flagellin [8, 9] . Flagellin is present as a soluble protein in certain bacteria, such as Salmonella, and can be recognized by the natural immune system of fruit flies, plants and mammals. Based on the structure of flagellin, Burdelya and colleagues [10] have successfully produced CBLB502 peptide by using genetic engineering technique. CBLB502 peptide offers protection against radiation injury both in vitro and in vivo, and can inhibit the process of normal cell apoptosis [10] . Therefore, it can prevent normal tissues from receiving radiation damage while maintaining the sensitivity of tumors to radiation. This function mainly occurs through the NF-κB signaling pathway [11] . Polyethyleneimine (PEI) is a compound that is derived mainly from industrial sewage. It has linear, branching and extra-branching types. The application of PEI in biological medicine has attracted considerable attention, since PEI can be used as a carrier in gene therapy [12, 13] , and has shown anti-fungal effect in in vitro experiments [14] . Recently, it has been reported that PEI has a high-density ionic charge that can selectively activate the intracellular TLR5 receptor, promoting generation and secretion of downstream TLR5 signaling pathway factors, such as keratinocyte chemoattractant (KC) and IFN-inducible protein 10 (IP-10) in mice [13] . However, there have been no reports published investigating the protective effects of PEI against radiation injury.We hypothesize that the high-density ionic charge of PEI activates the hTLR5 receptor-mediated NF-κB pathway, and provides an anti-radiation effect. This paper aimed to investigate whether polyethylenimine could activate the hTLR5 mediated NF-κB pathway and if so, whether polyethylenimine provides an anti-radiation effect as a TLR5 targeted agonist in a mouse model. . Protease inhibitor cocktail was purchased from Sigma. ECL chemiluminescence reagents were purchased from Biomed, China. V5 antibody was purchased from MBL Company (Tokyo, Japan). Restrictive endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). The anti-β actin antibody, horseradish peroxidase-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, MTT cell proliferation and cytotoxicity detection kit, and dual luciferase assay kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Restriction enzymes, DNA ligase and Taq enzyme were purchased from TaKaRa Bio INC (Shiga, Japan). DNA purification kit was purchased from Bio-med Company (Beijing, China). Plasmids pcDNA3.1-V5/HisB vector was purchased from Invitrogen. NF-kB luciferase reporter plasmid (NF-κB-Luc) was kindly donated by Huazhong Agricultural University, Wuhan, China. Renilla-luciferase (TK-PRL), used as an internal control, was purchased from Promega.