Effects of Lectin Subunit Alpha on the Expression of Staphylococcus aureus-stimulated Inflammatory Cytokines Mediated

doi:10.13560/j.cnki.biotech.bull.1985.2017-0731

Abstract

Abstract: The attempt of this work is to investigate the anti-inflammatory activity of Musca domestica lectin subunit alpha（Lsa）toward Staphylococcus aureus. The well-constructed pLEX-Lsa and pLEX were transfected into RAW264.7 cells with recombinant lentiviruses,then the stably-expressed RAW-pLEX-Lsa and RAW-pLEX were obtained after screening,and RAW-pLEX was as a negative control. S. aureus was used to stimulate RAW-pLEX-Lsa cells and RAW-pLEX cells,then both un-stimulated cells and stimulated cells for 3 h,6 h,12 h,and 24 h were collected,and the cell culture supernatants of 6 h un-stimulated and stimulated cells were also collected. Real-time quantitative reverse transcription PCR（RT-PCR）was performed to analyze the mRNA expression of TNF-a,IL-1β,NFκB-1 and NFκB-2,and the protein level of TNF-a and IL-1β in the supernatant were detected by ELISA. The results of RT-PCR showed that the stimulation of RAW-pLEX-Lsa by S. aureussignificantly down-regulated the mRNA expression of TNF-a transcript variant 1（TNF-a-tv-1）at 6 h（P<0.05）and TNF-a transcript variant 2（TNF-a-tv-2）at 6 h and 12 h（P < 0.05）,compared with the RAW-pLEX cells. The stable transfection of Lsa in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of IL-1β-T at 3 h,6 h and 12 h（P < 0.05）and the mRNA expression of IL-1β at 3 h and 6 h（P < 0.05）,compared with the RAW-pLEX cells,while the mRNA expression of NFκB-1 and NFκB-2 in RAW-pLEX-Lsa had insignificant decreasing. The results of ELISA demonstrated that the protein level of TNF-a in the supernatants of RAW-pLEX-Lsa cells at 6 h after stimulation by S. aureus significantly down-regulated（P < 0.05）. In conclusion,Lsa possesses potent anti-inflammatory activity,and stable transfection of Lsa in RAW264.7 cells inhibits the expression and production of TNF-α and IL-1β. However,the inhibition of TNF-α and IL-1β is not resulted from blocking the activation of NF-κB signal pathway.

Key words: lectin, RAW264.7, inflammatory cytokines, NFκB, transcript variant

WEI Rui-yang, BAI Jie, XU Bin, XI Yan-yan, WANG Lin-yi, WANG Gai-li, FU Chen, WEI Feng-xian, LI Shao-yu. Effects of Lectin Subunit Alpha on the Expression of Staphylococcus aureus-stimulated Inflammatory Cytokines Mediated[J]. Biotechnology Bulletin, 2018, 34(1): 215-222.

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