Systems Pharmacology-Based Study On Material Basis And Molecular Mechanisms Of Herba Ephedrae-Ramulus Cinnamomi Herb Pair On Anti-Inflammation

Background:Herba Ephedrae-Ramulus Cinnamomi(Eph-RC)is a classical herb pair used in Traditional Chinese Medicine.Previous studies have found that Eph-RC herb pair have a certain anti-inflammatory effect and show synergistic characteristics,but the material basis and molecular mechanisms are yet to be elucidated.Objectives:The systems pharmacology was employed to probe the material basis and the molecular mechanisms of Eph-RC herb pair on inflammation,aiming at explaining the multicomponent,multitarget and multipathway effects of anti-inflammatory action from molecular level to pharmacological level,and then to systemic level,providing theoretical basis for its clinical application.Methods:(1)Several acute and chronic inflammation models and analgesic experiments were employed for investigating the anti-inflammatory effect,analgesic effect and its mechanism of Eph-RC herb pair.(2)All ingredients of Eph and RC were collected from database and pertinent literature.ADME evaluation was implemented to screen out the candidate compounds.The target of candidate compounds was predicted and its reliability was verified by molecular docking.Inflammation related potential targets and pathways were identified by the database.The Gene Ontology(GO)biological processes of potential targets were analyzed.The relationship between compounds,targets,pathways and diseases were analyzed by constructing the network between them by Cytoscape 3.3.0.An incorporated"inflammation pathway" was assembled for recognizing the integrative action mechanisms of Eph-RC herb pair for inflammation therapy.(3)MTT was adopted for evaluating the effects of active ingredients in Eph-RC screened by systems pharmacology on the viability of RAW264.7 cells,then the influence of these active ingredients on LPS stimulated RAW264.7 cells releasing NO,PGE2,IL-6 and TNF-?was measured.(4)CCK-8 was employed for inspecting the influence of Eph-RC herb pair on the viability of RAW264.7 cells.Effect of Eph-RC on the mRNA expression of iNOS,TNF-?,IL-6 and COX-2 in LPS stimulated RAW264.7 cells were assessed by qRT-PCR.The key proteins levels involved in MAPKs and NF-?B signaling pathway activation were examined by western blot.Results:(1)Compared with the model group,the inflammatory exudation of Eph-RC groups was significantly lower at a manner depending on dose,and the high dose group was equivalent to the positive control group.Eph-RC at high and medium doses has the pharmacological activity of analgesia,and the high dose group has certain central analgesic effect.(2)A molecular database including 502 compounds in Eph and RC was constructed,of which 47 compounds corresponding to 262 targets were selected as candidate ingredients.And 42 candidate compounds associated with 99 targets and 46 pathways were related to inflammation.These targets have an anti-inflammatory role in various diseases such as cardiovascular,respiratory and immune systems by participating in the biological processes such as inflammation and immune response.Integrated "inflammation pathway" displayed that Eph-RC herb pair regulate the production of inflammatory cytokines and cell proliferation,differentiation and apoptosis by regulating multiple signaling pathways including"MAPK signaling pathway" and "NF-kappa B signaling pathway" for treating inflammation.(3)The concentration of NO,PGE2,TNF-? and IL-6 in the administration groups with safe doses were significantly decreased compared with LPS group,and presented a dose-response relationship.In addtion,the high dosage groups of some ingredients were equivalent to dexamethasone.(4)Compared with LPS group,the expression of iNOS,TNF-?,IL-6 and COX-2 mRNA was significantly inhibited in the Eph-RC group.Western blot results showed that the expression of P-p65,P-I?B-?,P-JNK was significantly reduced,P-ERK increased significantly and P-p38 showed no significant difference in Eph-RC groups at the beginning of inflammation.While the expression of P-p38 and P-ERK decreased significantly in the later stage of inflammation.Conclusion:(1)Eph-RC herb pair has obvious inhibitory effect on acute and chronic inflammation and inflammatory pain,and the mechanism is related to inhibiting generation of PGE2,TNF-?,IL-1? and the expression of iNOS and ICAM-I.(2)42 potential ingredients associated with 99 potential targets and many anti-inflammatory and immune regulation pathways are closely related to inflammation,fully showing the characteristics of multicomponent,multitarget and multipathway of Eph-RC.In addition,these ingredients have the pharmacological mechanism similar to anti-inflammatory western medicine.Some compounds,such as coumarin,?-sitosterol and cianidanol,are common ingredient of Eph and RC.And the different potential bioactive compounds from Eph and RC can associated with the same target or different targets in the same pathway.These illustrate the compatibility mechanism of synergistic effect of Eph and RC.(3)Some vital compounds such as luteolin,coumarin,cinnamaldehyde,ephedrine and pseudoephedrine in Eph-RC have protective effects on the inflammatory response induced by LPS,and may be the material basis.(4)Eph-RC may reduce inflammatory response by regulating the production of inflammatory cytokines and proinflammatory mediators.Eph-RC exerts anti-inflammatory effects by regulating the phosphorylation of p65 and I?B-?in NF-?B signaling pathway and the phosphorylation of p38,ERK and JNK in MAPKs signaling pathway.