The Effect Of Emodin On Lipopolysaccharide-Induced Macrophage Oxidative Stress And Inflammation Response

Objective:To explore the effect of emodin on Lipopolysaccharide-induced RAW264.7 cells oxidative stress and inflammation,further to investigate its underlying mechanism.Methods:PartⅠ: First,we detected the toxic effect of emodin on Raw264.7 by using cell proliferation and cytotoxicity assay kit(cell counting kit-8,CKK8)to screen out the optimal concentration of emodin.According to our study aim,We performed three groups as follows:Control group: neither LPS nor emodin administrated;LPS group:giving the same concentration of LPS as in LPS+Emodin group;LPS+Emodin group: giving emodin 15μmmol/L,30 min before LPS(1μg/m L)administrated.After observation for 3h,6h,9h,12 h respectively,Nrf2 m RNA was detected by using real-time fluorescence quantitative-polymerase chain reaction(RT-PCR)and Western Blot was used to determine the expression of Nrf2 total protein and its concentration in nucleus and cytosol.Moreover,the concentrations of CAT,GSH—Px,MDA were detected by using assay kits.And interleukin-6(IL-6)and interleukin-10(IL-10)were determine by enzyme linked immunosorbent assay(ELISA)kits with microplate reader.Part II: We set four groups as follows: Control group:neither LPS nor DEX administrated;LPS group: adding 1μg/m L LPS;LPS+Emodin group:giving emodin15μmmol/L,30 min before LPS(1μg/m L)administrated;LPS+Emodin+ML385(Nrf2inhibitor)group: giving emodin 15μmmol/L,30 min before LPS(1μg/m L)and ML385 administrated.We chose 9h as the indicated time,the expression of Nrf2 total protein and its concentration in nucleus and cytosol in above groups were detected,as well as the concentration of CAT,GSH—Px,MDA,also the levels of IL-6 and IL-10 in culture supernate were detected by the ELISA kits.Results:PartⅠ:Under LPS stimulation,Nrf2 m RNA increased from 3 h and increased significantly at 6 h,9h when compared with control group(P<0.05);and the relative expression level of Nrf2 total protein increased from 3h and reached a peak at 9h,then it decreased at 12h;but its expression in nucleus among varied time didn’t reach a significant difference(p>0.05);however,Nrf2 in cytosol had the same trend as the change of total levels.When compared with LPS group,Nrf2 m RNA expression levels were higher at 6h,9h,12h(P<0.05)and peaked at 6 h in LPS+Emodin group;and the relative level of Nrf2 total protein increased significantly higher than those induced by simple LPS stimulation at 6h,9h,12h(P<0.05),and at 9h,the difference between these two groups was the biggest;and the change of Nrf2 in nucleus had something in common with the total,emodin increased its levels at 9h,12 h when compared to LPS group(P<0.05),and at 9h,the difference between these two groups was the biggest;however,emodin reduced the levels of Nrf2 in cytosol at 9h,12h(P<0.05).Moreover,emodin significantly increased LPS-induced CAT expression level at 9 h,12h(P<0.05),as well as the GSH-px at 6h,9h(P<0.05);but significantly decreased MDA expression at 6h,9h((P<0.05)).What ’s more,emodin pretreatment obviously decreased LPSinduced IL-6 concentration at 6h,9h,12 h,but significantly increased the level of IL-10(P<0.05).Part II: At 9h,The level of Nrf2 protein in total and nucleus in the Nrf2 inhibitor group was lower than that of in corresponding groups without inhibitor(P<0.05).Meanwhile,at 9h,we found CAT concentration and GSH-px in the LPS +Emodin+ ML385 group was significantly lower than that in LPS+Emodin group(2.35±0.51 vs.4.47±0.94mmol/L,64.12±7.40 vs.88.81±7.65U/mgprot,P<0.05),but the MDA concentration was significantly higher(2.48±0.42 vs.1.38±0.11nmol/mgprot,P<0.05).Moreover,the concentration of IL-6 in LPS +Emodin+ML385 group was significantly higher than in LPS +Emodin group(161.82±6.37 vs.132.45±10.42mmol/L,P<0.05),but IL-10 was opposite(435.26±19.87 vs.521.53±36.51mmol/L,P<0.05).Conclusion:Emodin may promote the process of Nrf2 into nucleus and augment the expression of Nrf2 to alleviate macrophage oxidative stress and inflammation induced by LPS.