ABSTRACT
This study pertains to the new approach for the development of hybrid peptide LL-37Tα1 and its biomedical applications. A linear cationic hybrid peptide, LL-37Tα1 was derived from two parental peptides (LL-37 and Tα1) recognized as potent anti-endotoxin without any hemolytic or cytotoxic activity. We successfully cloned the gene of hybrid peptide LL-37Tα1 in PpICZαA vector and expressed in the Pichia pastoris. The recombinant peptide was purified by Ni-affinity column and reverse-phase high performance liquid chromatography (RP-HPLC) with an estimated molecular mass of 3.9 kDa as determined by SDS-PAGE and mass spectrometry. We analyzed the LPS neutralization by limulus amebocyte lysate (LAL) activity and the results indicate that the hybrid peptide LL-37Tα1 directly binds endotoxin and significantly (p < 0.05) neutralizes the effect of LPS in a dose-dependent manner. Lactate dehydrogenase (LDH) assay revealed that LL-37Tα1 successfully reduces the LPS-induced cytotoxicity in mouse RAW264.7 macrophages. Moreover, it significantly (p < 0.05) decreased the levels of nitric oxide, proinflammatory cytokines including TNF-α, IL-6, IL-1ß, and diminished the number of apoptotic cells in LPS-stimulated mouse RAW264.7 macrophages. Our results suggest that the P. pastoris expression system is cost-effective for commercial production of the immunomodulatory and anti-inflammatory hybrid peptide (IAHP) LL-37Tα1 and the peptide may serve as effective anti-endotoxin/anti-inflammatory agent with minimal cytotoxicity.
Subject(s)
Anti-Inflammatory Agents/metabolism , Antimicrobial Cationic Peptides/metabolism , Fish Proteins/metabolism , Immunologic Factors/metabolism , Pichia , Animals , Antimicrobial Cationic Peptides/genetics , Chromatography, High Pressure Liquid , Cloning, Molecular , Cytokines/metabolism , Fish Proteins/genetics , Inflammation Mediators/metabolism , L-Lactate Dehydrogenase/metabolism , Lipopolysaccharides/metabolism , Mice , Nitric Oxide/metabolism , RAW 264.7 Cells , CathelicidinsABSTRACT
Objective To study the effects of the active components of eight kin ds of Chinese herbs on cytochrome P450 enzymes (CYP) 1A1,2E1,3A4 and 3A5 mRNA expression. Methods The mRNA expression levels of four CYP enzymes were determin ed by real-time quantitative reverse-transcriptase polymerase chain reaction. Results Baicalin,baicalein and artemisinin induced CYP1A1 expression at differe nt concentrations. Compared with baicalin and baicalein,the effect of artemisin in was weaker. The expression of CYP3A4 gene was significantly obvious after ind uced by sodium aescinate,baicalein and artemisinin. Conclusion HepG2 cells shou ld be an appropriate in-vitro system for investigating potential human CYP indu cing agents. CYP1A1 and CYP3A4 expression could be significantly induced by baic alin,baicalein,artemisinin and sodium aescinate,which would supply the eviden ce for the interaction of herbal medicine and western medicine based on cytochro me P450 and toxicology.
