The Effects Of ?-carotene On Inflammatory Cytokines And NF-?B Signaling Pathway In LPS-induced RAW264.7 Cells

The occurrence and development of inflammation are closely related to the intestinal inflammation of piglet weaning and many diseases.The steroids and nonsteroidal traditional drugs are commonly used as anti-inflammatory drugs to cure inflammatory diseases,which produce side-effects in human body.Thus,to find a new type of effective substance with minimal side effects is very important to cure inflammatory diseases.As a natural carotenoid,?-carotene is an important precursor of vitamin A and has a variety of biological effects including antioxidant,enhancing gap junction intercellular communication and promoting immunity.Moreover,as an important transcription factor,NF-?B can induced inflammatory cytokines when RAW264.7 cells are stimulated LPS.Thus,inflammation model of LPS-stimulated RAW264.7 cells was set up to study the possible effects of ?-carotene on the inflammatory cytokines and relationship with NF-?B pathway,and reveal the anti-inflammatory activities and possible mechanisms of ?-carotene.Firstly,to determine the most suitable concentration and time of ?-carotene and LPS,the cells viability was measured by MTT.To build inflammation model and to ensure the effects of ?-carotene on the inflammatory cytokines,the mRNA relative expression and production of IL-1?,IL-6 and TNF-? were detected by quantitative RT-PCR and ELISA.Then,the relative expression of NF-?B p65 mRNA and protein was measured by quantitative RT-PCR and Western Blot to research the effect of ?-carotene on NF-?B signaling pathway.Finally,to analyse the effects of LPS+PDTC+?-carotene group and LPS+PDTC groupon inflammatory cytokines and NF-?B p65 by above methods.The results are as follows:In the study of ?-carotene on the inflammatory cytokines,the most suitable concentration and time of ?-carotene are 20,40,80,160 ?mol/L and 3 hours,the most suitable concentration and time of LPS are 5 ?g/mL and 24 hours.RAW264.7 cells were treated with 5 ?g/mL of LPS for 24 h and then stimulated with different concentrations of ?-carotene for 3 h,which significantly reduced the mRNA relative expression of IL-1?,IL-6 and TNF-?(P<0.01),although the effect of ?-carotene on the production of inflammatory cytokines was not all significant,there was a tendency toward lower levels.These results indicated that ?-carotene caused the inhibition of inflammatory cytokines in LPS-induced RAW264.7 cells.In the study of ?-carotene on NF-?B signaling pathway,RAW264.7 cells were induced by 5 ?g/ml of LPS and different concentration of PDTC that can inhibit the expression of NF-?B specifically together for 24 h,which significantly suppressed the mRNA and protein relative expressions of NF-?B p65 compared to LPS group(P<0.01),significantly inhibited the mRNA relative expression of IL-1?,IL-6 and TNF-?(P<0.01),and reduced the production of inflammatory cytokines.These results indicated that the suppression of NF-?B p65 contributed to inhibit the expression and production of inflammatory cytokines in LPS-induced RAW264.7 cells.In the study of NF-?B signaling pathway on specificity,addition of ?-carotene significantly reduced the mRNA and protein relative expressions of NF-?B p65 in LPS-induced RAW264.7 cells(P<0.05),which suggested that ?-carotene can inhibit the expression and production of inflammatory cytokines through suppressing the expression of NF-?B p65 in LPS-induced RAW264.7 cells.The inhibition of inflammatory cytokines in LPS+PDTC+?-carotene group was more obvious than that in LPS+PDTC group,but there was little difference about the expression of NF-?B p65 between those two groups.These results comprehensively indicated that NF-?B signaling pathway was not unique one by which ?-carotene inhibited expression and production of inflammatory cytokines.In summary,?-carotene inhibits the expression and production of key inflammatory cytokines,which is regulated through NF-?B signaling pathway.