Oryzanol Inhibits Lipopolysaccharide-Induced Inflammatory Cytokines Expression In Macrophage And Its Molecular Mechanism

China’s annual rice yield is more than 1000 million tons in recent years, and rice bran is the main by-product of the rice processing. Oryzanol is one of the main active components in rice bran. It is very important to study and develop its physiological function of the active ingredient in rice, which will highly add economic value. Recent studies showed that oryzanol has anti-inflammatory biological activity, but the molecular mechanism of anti-inflammatory function is not clear. Lipopolysaccharide (LPS)-induced inflammatory model of RAW264.7 macrophage is commonly used to evaluate anti-inflammatory function. In this study, the inflammatory model is used to study the anti-inflammatory molecular mechanism of oryzanol.The effect of oryzanol on LPS-induced cell activity and apoptosis:it sets control group, oryzanol group (10,20 and 40μg/ml), LPS group and LPS+(10,20 and 40μg/ml) oryzanol protection group. After treatment for 24h and MTS for 2h, OD values were measured. Oryzanol has no significant inhibitory effect on RAW246.7 in the 10-40 μg/mL range (P>0.05). Hoechst33342 dye was used to stain treated cells and fluorescence microscopy was used to observe if it exist apoptosis. The results showed that 10-40 μg/mL oryzanol and/or 1μg/mL LPS does not promote cell apoptosis. Therefore, the concentrations of 10-40 μg/mL oryzanol were selected to use our study.The effect of oryzanol on the mRNA expression of cytokines:groups were divided as described before, it sets control group, oryzanol group (10,20 and 40μg/ml), LPS group and LPS+(10,20 and 40μg/ml) oryzanol protection group. Compared with the control group, Cox-1, Cox-2, IL-1, IFN-y, TNF-α and iNOS expression of LPS group were significantly increase (P< 0.01). Compared with LPS group, Cox-2, IL-1, IFN-γ, TNF-α and iNOS expression of oryzanol protective group obviously decrease (P< 0.05), and COX-1 gene expression had no significant difference (P> 0.05).The effect of oryzanol on protein expression of inflammatory factors:compared with the control group, IL-1, TNF-α and iNOS protein expression increased significantly in LPS group; After oryzanol treatments, the protein expressions of inflammatory factors decrease significantly, and with dose dependent manner.The effect of oryzanol on nuclear translocation of transcription factors:compared with control group, the percentage of NFκB nuclear translocation is obviously increase by Western blotting of p65; and the percentage of AP-1 nuclear translocation is obviously increase by Western blotting of c-Jun. Compared with LPS group, the AP-1 and NFκB translocation of oryzanol protective groups was significantly reduced, and p65 protein expression variation has a significant difference (P<0.05), the c-Jun protein expression variation with a very significant difference (P<0.01). Oryzanol can inhibit AP-1 and NFκB nuclear translocation.The effect of oryzanol on DNA-binding activities and transcript acitivies of transcription factors:the negative control group was not detected DNA-binding oligonucleotide probe; compared with control group, LPS treatments significantly increase the DNA-binding activities of AP-1 and NFκB; compared with LPS group, oryzanol treatments results in decrease of the DNA-binding activities in a dose-dependent manner. And oryzanol can reduce the transcriptional activity of nuclear AP-1 and NFκB, with dose dependent manner. Compared with control group, LPS stimulates the transcript activities of AP-1 and NFκB, and oryzanol inhibits the the transcript acitivies of AP-1 and NFκB.It exist single or multiple of AP-1 and NFκB binding sites (cis acting elements) in the gene promoter of Cox-2, IL-1, IFN-y, TNF-a and iNOS. Transcription factor activations of AP-1 and NFκB are the key factor to regulate the expressssion of inflammatory factors. Taken together, comprehensive analysis of the data showed that the molecular mechanisms of oryzanol in the anti-inflammatory effect:oryzanol reduced the nuclear translocation of AP-1 and NFκB and results in the decrease of DNA-binding activities of AP-1 and NFκB; and then oryzanol reduce the transcript activities of AP-1 and NFκB. Finally oryzanol reduce the expression of IFN-y, TNF-a, iNOS and contribute its anti-inflammatory function.