| Antimicrobial peptides(AMPs)have broad activity to directly kill bacteria,and have low toxicity for mammal cells.One of the most notable features of AMPs is that it rarely induces bacterial resistance.Therefore AMPs have been considered as one of the best potential sources for the development of a new class of antibiotics to be used in the future.Cecropin is an antimicrobial peptide.Lectin playing an anti-bacterial and anti-inflammatory role and antimicrobial peptides are characterized as immune factors.House flies,Musca domestica L.(Diptera: Muscidae),are ubiquitous and transmit more than 100 human and animal diseases,however,they can thrive without causing infection.House flies must have a sound and efficient immune defense mechanism,which is an important resource for people to study natural anti-inflammatory substances.The trial was conducted to evaluate the antiinflammatory functions of Musca domestica cecropin-A2(Mdc-A2)and lectin subunit alpha(Lsa)toward Staphylococcus aureus(S.aureus).1)The anti-inflammatory functions of Mdc-A2 toward S.aureus.RAW264.7 cells were transfected with recombinant lentiviruses introducing p LEX-Mdc-A2 into the RAW264.7 cell line(RAW-Mdc-A2).The RAW264.7 cell line with empty p LEX(RAW-p LEX)was produced in the same manner as a negative control.Real-time quantitative reverse transcription PCR(Real-Time PCR)was performed to analyze the m RNA expression of TNF-α,IL-1β,NFκB-1 and NFκB-2 in S.aureus-stimulated RAW-Mdc-A2 cells and RAW-p LEX cells in untreated cells and cells treated for 3 h,6 h,12 h and 24 h.Real-Time PCR was performed to analyze the m RNA expression of TNF-α,NFκB-1 and NFκB-2 stimulated by Lipoteichoic acid(LTA).Production of TNF-α was detected by enzyme-linked immunosorbent assay(ELISA).Colony counts were used to calculate the number of CFU per m L of cell culture supernatants.The results showed that compared to RAW-p LEX cells,stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S.aureus significantly downregulated the m RNA expression of TNF-α transcript variant 1(TNF-α-tv-1)at 6 h and 12 h and the m RNA expression of TNF-α transcript variant 2(TNF-α-tv-2)at 3 h,6 h and 12 h.Compared to RAW-p LEX cells,stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S.aureus significantly down-regulated the m RNA expression of IL-1β-T at 3 h,6 h and 12 h as well as the m RNA expression of IL-1β at 3 h and 6 h.The expression and production of TNF-α and bacterial burden of cell culture supernatants were significantly down-regulated in RAW-Mdc-A2 cells stimulated by S.aureus,and the expression and production of TNF-α were significantly down-regulated in RAWMdc-A2 cells stimulated by LTA.Compared to RAW-p LEX cells,stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S.aureus significantly down-regulated the m RNA expression of NFκB-1 at 3 h,6 h and 12 h as well as the m RNA expression of NFκB-2 at 6 h.Additionally,stable transfection of Mdc-A2 in RAW264.7 cells stimulated by LTA significantly down-regulated the m RNA expression of NFκB-1.2)The anti-inflammatory functions of Lsa toward S.aureus.RAW264.7 cells were transfected with recombinant lentiviruses to obtain RAW264.7 cell line with RAW-p LEX-Lsa.And RAW264.7 cell line with empty p LEX(RAW-p LEX)was produced in the same manner as a negative control.Real-Time PCR was performed to analyze the m RNA expression of TNF-α,IL-1β,NFκB-1 and NFκB-2 in S.aureus-stimulated RAW-p LEX-Lsa cells and RAW-p LEX cells in untreated cells and cells treated for 3 h,6 h,12 h and 24 h,and the cell culture supernatants were collected in untreated cells and cells treated for 6 h.Production of TNF-α and IL-1β were detected by ELISA.The results showed that compared with the RAW-p LEX cells,stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S.aureus significantly down-regulated the m RNA expression of TNF-α-tv-1 at 6h(P<0.05),and the m RNA expression of TNF-α-tv-2 at 6 h and 12 h(P<0.05).Compared with the RAW-p LEX cells,stable transfection of Lsa in RAW264.7 cells stimulated by S.aureus significantly down-regulated the m RNA expression of IL-1β-T at 3 h,6 h and 12 h,and the m RNA expression of IL-1β at 3 h and 6 h.And compared with the RAW-p LEX cells,stable transfection of Lsa in RAW264.7 cells stimulated by S.aureus did not down-regulated the m RNA expression of NFκB-1 and NFκB-2.The production of TNF-α was significantly down-regulated in RAW-p LEX-Lsa cells stimulated by S.aureus.Conclusions1)Mdc-A2 possesses potent anti-inflammatory activity and potent antimicrobial activity.Additionally,Mdc-A2 executes strong anti-inflammatory activity by blocking the activation of NF-κB signaling pathways.2)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,inhibition of TNF-α and IL-1β is not caused by blocking the activation of NF-κB signaling pathways. |
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