Anti-inflammatory Effect Of DHA And Quercetin And Their Role In NFκB And MAPK Signaling Pathway

Quercetin(QE) is known as one of the flavonoids compound that widely existed in fruits and vegetables. While DHA is an important Ω-3poly unsaturated fatty acid which shows a lot benefits for human health. Both DHA and QE are proved to be anti-inflammation, antioxidant bioactivities, and shows cardiovascular protection effect. Thus, we hypothesis that DHA and QE may show enhanced or synergestic anti-inflammatory effect in combination. In this paper in vitro and in vivo inflammation model was established to evaluate the anti-inflammatory effect of DHA and QE individually and in combination, as well as their effect on NF-κB and MAPK signaling pathway. Firstly, an in vitro inflammation model was conducted by LPS induced RAW264.7 macrophages. The inflammatory mediators such as NO、PGE2、i NOS and COX-2 were used as marker of inflammation. Griess reagent and ELISA methods were applied to analysis the anti-inflammatory effect of DHA and QE along and together. Secondly, two models were carried out according to the manner of LPS that induce inflammation and ELISA method was used to evaluate the effect of DHA and QE on cytokines production. Thirdly, Western blot was used to study the effect of DHA and QE on NF-κB and MAPK signaling pathway. Fourthly, the effect of DHA and quercetin on NF-κB and MAPK gene expression was evaluated by RT-PCR. Finally, in vivo LPS induced rat model was applied to study the anti-inflammatory effect of DHA and QE. The main findings are as following:1. The in vitro model was established according to the time and dose that LPS was added to cell to induce inflammation. The results indicated that 0.1μg/m L LPS and 24 h cooperation was applied in this research. The dose of DHA and quercetin was selected according to the result of MTT. Thus, we choose 30μM and 20μM as the concentration of DHA and QE in the following experiment. DHA and QE along and in combination, show inhibitory effect on the production of inflammatory mediators such as NO, PGE2, i NOS and COX-2. The combination of DHA and QE showed enhanced inhibitory effect.2. DHA and QE along and in cooperation show inhibitory effect on the production of TNF-α、IL-1β、IL-6 in both models established in the study. Enhanced effect was observed when DHA and QE in combination. Upon the in vitro model established in previous chapter, two models which established according to the method that LPS was applied together with DHA/QE/DHA+QE or after 4h of pretreatment of them. There was little effect of DHA and QE on the production of IL-10 even in combination.3. DHA and QE can inhibit the activation of NF-κB and MAPK signaling pathway separately or in combination. Individually, DHA and QE can inhibit the phosphorylation of IκB、p50 and p65, as well as the degradation of IκB. Likewise, DHA can inhibit phosphorylation of ERK 、 JNK and p38 while QE only inhibit JNK and p38 phosphorylation. DHA and QE in combination shows synergistic inhibitory effect on the phosphorylation of p50、p65、ERK and JNK according to Bliss independent model. DHA and QE show little inhibitory effect on the expression of p50、p65、JNK and p38 but in combination the effect was obvious(P<0.05). DHA and QE together showed enhanced inhibitory effect on the phosphorylation and degradation of IκB.4. The combination of DHA and QE can down regulate the expression of p50、p65、ERK、JNK and P38 m RNA, while up regulate the m RNA expression of IκB. In contrast, DHA and QE individually has no effect on the IκB, p50, p65, ERK, JNK and P38 m RNA expression in LPS induced RAW264.7 macrophages.5. The in vivo experiment turned out that high doses of DHA or QE can inhibit the production of TNF-α in the plasma, and down regulate the expression of inflammatory cytokines gene expression such as i NOS, IL-6 and TNF-α. The antioxidant ability was up regulated by DHA or QE as well. In contrast the low doses of DHA showed little effect on anti-inflammatory effect and antioxidant activation while the inhibitory effect of QE was statistically significant. The combination of DHA and QE showed stronger inhibitory effect on the expression of TNF-α in the plasma, and stronger up regulation effect on the antioxidant ability in liver, spleen and plasma. Meanwhile, the combination also showed enhanced inhibitory effect on the m RNA expression of i NOS, IL-6 and TNF-α. The high dose combination and low dose combination showed no significant difference on the anti-inflammatory and antioxidant effect.