The Study On The Molecular Mechanism And Structure-Activity Relationship Of Rhubarb Anthraquinone Inhibiting The Inflammatory In LPS-stimulated Macrophages

ObjectiveWe aimed to quantitatively evaluate the molecular mechanism and dose-effective relationship of the anti-inflammatory effect of rhein,emodin and aloe-emodin in vitro based on TLR4 mediated MyD88-/TRIF-dependent signaling pathway and combined with pharmacodynamic model and median effect equation.Furthermore,we sought to clarify the scientific intention of the relationship between anti-inflammatory activity and molecular structure of rhubarb anthraquinones.Methods1.MTT assay was used to detect the effect of rhein,emodin,and aloe-emodin on the activity of RAW 264.7 macrophages,thereby clarifying the vitro safe concentration range of the three drugs and determining the experimental concentration.2.The pre-dosing protocol were conducted in the study.Rhein（80μM,40μM,20μM）,emodin（80μM,40μM,20μM）,aloe-emodin（20μM,10μM,5μM）were pre-incubated with RAW 264.7 macrophages for 30 min.After this,LPS were added at the1μg/mL final concentration and the time were marked as“0”time point.After 0,5,15,30,60,90,and 120 min,the cell lysate was used to detect the expression of IKKβand TBK1 protein and the phosphorylated protein by Western blot.After 0,10,30,60,120,and 240 min,the cell lysates were taken for detecting NFκB p65,PI3K,IRF3protein expression and the phosphorylated protein by Western blot.After 0,1,2,4,8,12 and 24 h,the cell lysate was used to detect the expression of iNOS protein by Western blot,the cell supernatant was taken to detect the NO concentration by Griess kit and the IL-6、TNF-α、IFN-β、IFN-γconcentration by Elisa kit.3.The pharmacodynamic model was established by Monolix?2016R1 software to quantitatively capture the anti-inflammatory dynamics and molecular mechanism of rhein,emodin and aloe-emodin in vitro.At the same time,three drugs were set in seven concentration groups at 4 h and 24 h for testing concentration of IL-6 and NO,respectively,and the median effect equation was calculated.Combining the pharmacodynamic model with the median effect equation,the in vitro anti-inflammatory dose-effect relationship and potency of the three compounds were confirmed.4.We analyzed and compared the molecular hydrophobicity coefficient（logP）,polar/non-polar desolvation energy,topological polar surface area,molecular surface electrostatic potential,and excess free energy penetrating phospholipid bilayer of rhein,emodin,and aloe-emodin,in association with pharmacodynamic studies,we revealed the anti-inflammatory structure-activity relationship of rhubarb anthraquinones.Results1.The 0.1μM¹00μM of rhein and emodin,and 0.1μM⁴0μM aloe-emodin were the safe concentration range in RAW 264.7,which were lower than IC₅₀.Therefore,the experimental concentrations of rhein and emodin were determined to be 80,40,and 20μM,respectively,the experimental concentrations of aloe-emodin were 20,10,and 5μM.2.LPS activates MyD88-dependent signaling pathway and TRIF-dependent signaling pathway in RAW 264.7 cells,and the upstream protein expression and downstream terminal factor production have their own unique dynamic changes.IKK-βrapidly phosphorylates and reaches the highest expression level after 5 min of LPS stimulation.The phosphorylation level of NF-κB p65 was detected at 10 min after LPS stimulation,and then gradually decreased,phosphorylated NF-κB p65 level increased again at 120min,and phosphorylated NF-κB p65 level showed an oscillating curve trend.PI3K increased rapidly after LPS stimulation 60 min and increased with time.The phosphorylated TBK1 was detected to increase in levels 30 min after LPS stimulation and continued to increase over time.The phosphorylation level of IRF3 was significantly increased 60 min after LPS stimulation,and it entered the plateau phase,and phosphorylated IRF3 level tended to be stable within 60-240 min.The expression level of iNOS protein was significantly increased at 8 h after LPS stimulation,and continued to increase within 8 h to 24 h.The NO concentration in the supernatant of the cells continued to increase from 8 h to 24 h after LPS stimulation.The concentration of IL-6 in the supernatant of the cells increased significantly within 2 h⁸ h after LPS stimulation,and stabilized at 8 h²4 h.The TNF-αin the supernatant of the cells increased sharply within 0 h² h after LPS stimulation,and its concentration decreased slightly and stabilized at 4 h²4 h.No IFN-γor IFN-βwas detected in the cell supernatant.3.Rhein,emodin and aloe-emodin could inhibit the activation of MyD88-dependent signaling pathway and TRIF-dependent signaling pathway induced by LPS,and can down-regulate the phosphorylation of IKKβ、NFκB p65、PI3K、TBK1、IRF3 in the dose-dependent manner,and inhibit iNOS protein expression and the production of NO and IL-6.On the other hand,it was observed that three rhubarb anthraquinones slightly up-regulate the secretion of TNF-α,which is related to the bidirectional regulation of IKKβ.4.The results of the median effect equation showed that the inhibition rates of aloe-emodin on IL-6 and NO production were stronger than that of emodin and rhein at the same concentration.The IC₅₀ of Rhein,emodin and aloe-emodin inhibiting LPS stimulation of RAW 264.7 macrophages to generate IL-6 was 100.92μM,39.81μM,18.84μM,while the IC₅₀ for inhibiting NO production was 53.12μM,46.80μM,17.83μM.The pharmacodynamic model showed that the pharmacodynamic model of rhubarb anthraquinones treatment of this in vitro inflammation was a dual-target inhibition model,which inhibited NF-κB p65 phosphorylation and iNOS protein expression.The logarithmic linear inhibition coefficients of NF-κB p65 phosphorylation were 0.0125,0.0208,and 0.0410.The logarithmic linear inhibition coefficients of iNOS protein expression were 0.0142,0.0587,and 0.0763,respectively.Apparently,the rhubarb anthraquinones inhibited the expression of iNOS protein more strongly than the inhibition of NF-κB p65 phosphorylation（about twice）.Both the median effect equation and the pharmacodynamic model showed that the anti-inflammatory mechanisms of the three bismuth compounds were the same in vitro,but they had different potency:aloe-emodin>emodin>rhein.However,due to solubility limitations,aloe-emodin is less effective than emodin and rhein.5.The results of structure-activity relationship analysis showed that the hydrophobic structure of the three rhubarb anthraquinones was the main cause of the difference in anti-inflammatory potency.The receptors that bind to emodin,emodin,and aloe-emodin have hydrophobic pockets.The molecular structure of aloe-emodin has the strongest hydrophobic effect,so its binding ability to receptors is stronger than that of emodin and rhein,thus showing more strong anti-inflammatory capabilities.ConclusionRhubarb anthraquinones（rhein,emodin,and aloe-emodin）can inhibit inflammation in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner,and the mechanism of action is dual-target inhibition（NF-κB p65 and iNOS）.At the same dose,the anti-inflammatory effect of aloe-emodin is stronger than that of emodin and rhein.The difference in the anti-inflammatory ability of the three anthraquinones is mainly due to the difference in hydrophobic interaction caused by the molecular structure branch.The receptor that binds to the terpenoid has a hydrophobic pocket,and the hydrophobic effect of the aloe-emodin molecular structure is stronger than that of emodin and rhein,so it has stronger anti-inflammatory potency.