Anti-inflammation And Anti-inflammatory/Antioxidant Linkage Mechanism Of Flavonoids Based On NF-?B Signaling Pathway

Flavonoids are widely found in fruits and vegetables,which play a crucial role in body's resistance to inflammatory reactions and oxidative stress.Flavonoids through dietary intake are beneficial to human health.The effects of flavonoids on macrophage inflammatory reaction have largely connected with NF-kappa B signaling pathway.Exploring flavonoids'structure-activity relationship on the NF-?B signaling pathway will be beneficial to analyse and screen high anti-inflammatory flavonoids.The linkage mechanism of flavonoids'anti-inflammatory/antioxidant activity may be a result of their effect on IKK?and Nrf2.However,it has not been confirmed yet.In this study,four parts of research were conducted,results are listed as follows:1 Flavonoids'structure-activity relationship of downstream products COX-2 mRNA was analyzed.Fluorescence quantitative PCR method was used to indirectly detect the intracellular mRNA level of Cyclooxygenase-2?COX-2?.Compared with flavonoid glycosides,flavonoid aglycones showed stronger inhibitory activity against mRNA of COX-2.For C-glycosylflavones,the glycosyl substitution at C8 was more beneficial to the inhibition mRNA of COX-2 than glycosyl substitution at C6.C4'methoxyl-substituted flavonoids showed strong COX-2 mRNA inhibitory activity.C2-C3double bond may be not a benifical structure for inhibiting COX-2 mRNA.2 QSAR was applied to further analyze the structural characteristics of flav onoids for COX-2 mRNA inhibition.A 2D-QSAR model was formed to analyze the structural characteristics of flavonoids inhibiting COX-2 mRNA.2D-QSAR model established with high q² and R²_pred?0.836 and 0.638,respectively?indicated that the model has a strong prediction ability internally and externally.Low value vsurf_DD12?considered good molecular balance?,no C2-C3 double bond,and high value SMR_VSA5?considered small molecular polarity?contributed to an ideal structure for flavonoids in suppressing COX-2 mRNA in inflammatory RAW264.7 cell model.It indicated that flavanone and flavones with high balance,high molar refractivity would be efficient to inhibit COX-2mRNA,which would be potential COX-2 inhibitors.3 Inhibiton and structure-activity relationship with NF-?B signaling pathways were analyzed based on flavonoids'COX-2 mRNA inhibition.Attention was focused on IKK??I?B??P65 so as to explain structure-activity relationship of flavonoids on NF-?B signaling pathways.The anti-inflammatory effect on cytokine such as TNF-?,IL-6 was evaluated.Results are as follows:1)The structure-activity relationship of flavonoids on the regulation of the NF-?B signaling pathway were mainly as follows:for flavonoid aglycone,hydroxyl substitution at C3'was not beneficial to inhibit the activity of P65.Hydroxyl substitution at C5 and C7 did not affect the regulatory activities of flavonoids on IKK?,I?B,and P65.Polymethoxylated substitution at C5,C6,C7 and C8 resulted in no inhibitory activity against IKK and I?B,but significantly inhibited the expression and nuclear importing activity of P65,while had little effect on IKK?and I?B.Glycosylated substitution made flavonoids have lower NF-?B inhibitory activity only show inhibiton on P65.2)Through inhibiting IKK?-I?B-P65 signal transduction,apigenin suppressed the expression of TNF-?,IL-6,which played a strong activity of anti-inflammation.The anti-inflammatory/antioxidant activity linkage mechanism of flavonoids was studied.Flavonoids with a strong inhibition on IKK?phosphorylation were chosen as research objects,Nrf2,reactive oxygen species?ROS?and antioxidant enzymes HO-1 were evaluated.Results indicated that:1)flavone,apigenin activated the transcriptional activity of Nrf2.And flavone,apigenin could not only significantly promote the expression of Nrf2 in cells?P<0.01?,but also significantly promote nuclear importing of Nrf2?P<0.01?,which led to the upregulation of HO-1 or a lower ROS levels?P<0.05?.2)In THP-1 macrophages stimulated by LPS,apigenin and flavone showed double activities of anti-inflammatory/antioxidant by inhibiting IKK?,repressing NF-?B inflammatory signaling pathway while activating Keap1-Nrf2 antioxidant signaling pathway,promoting Nrf2's nuclear transcription activity simultaneously.In conclusion,flavonoids generally have a good COX-2 enzyme mRNA inhibitory activity.However,glycoside substitution and C2-C3 double bond are not conducive to inhibite COX-2 mRNA of flavonoids.Favanones and flavonoids with better molecular balance and higher molecular refractive have better activity of inhibiting COX-2 mRNA,which are potential COX-2 inhibitors.Apigenin can down-regulate the expression of TNF-a,Il-6?P<0.05?and other cytokines by inhibiting IKK?-I?B?-P65 signal axis.Apigenin and flavone,IKK?inhibitors,can also activate the Nrf2 conducted a double activity of anti-inflammatory/antioxidant.