Anti-inflammatory Activity And Mechanisms Of Magnolol

The diseases caused by Gram-negative bacteria are very common in veterinarymedicine. LPS is one of the major virulence factors of Gram-negative bacteria thatcan cause damage to the host. LPS induces inflammatory response mainly byactivating TLR4signal transduction pathway. PPAR-γ plays an important role in theregulation of TLR4signal transduction pathway. Therapeutic targeting of TLR4signal pathway for inflammatory and infectious disease is not only a novel idea inveterinary clinical practice, but also an important pathway to development of novelanti-inflammatory drugs. In this study, the anti-inflammatory activity of magnolol, ahydroxylated biphenyl compound isolated from the traditional Chinese medicineMagnolia officinalis was studied. Firstly, the anti-inflammatory effect of magnololwas detected in LPS-induced acute lung injury. Secondly, the anti-inflammatorymechanism of magnolol was studied in LPS-stimulated RAW264.7cells. Thirdly, themolecular targets of the anti-inflammatory actions of magnolol was identified inLPS-stimulated RAW264.7cells. In the last, the therapeutic effect of magnolol wereexamined in LPS-induced mouse mastitis.First, the anti-inflammatory effects of magnolol was evaluated in LPS-inducedacute lung injury in mice. All mice were randomly divided into six groups: controlgroup, LPS group, Magnolol (5,10and20mg/kg)+LPS group, DEX+LPS group.Male BALB/c mice were pretreated with dexamethasone or magnolol1h beforeintranasal instillation of LPS.7h after LPS administration, the myeloperoxidase inlung tissues, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavagefluid (BALF) were determined. The levels of tumor necrosis factor-α (TNF-α),interleukin-6(IL-6), interleukin-1β (IL-1β) in the BALF were measured by ELISA.The results showed that magnolol markedly attenuated the histological alterations inthe lung; reduced the number of total cells, neutrophils, and macrophages in theBALF; decreased the W/D ratio of lungs in the BALF; down-regulated the level ofpro-inflammatory mediators, including TNF-α, IL-1β and IL-6, caused by LPS. Takentogether, our results suggest that magnolol has an anti-inflammatory effects against the LPS-induced acute lung injury.Secondly, the anti-inflammatory mechanism of magnolol were detected inLPS-stimulated RAW264.7cells. The potential cytotoxicity of magnolol wasevaluated by the MTT assay and the result showed that cell viabilities were notaffected by the magnolol at concentrations used (15,30,60μg/ml). The expression ofproinflammartory cytokines were determined by ELISA and reversetranscription-PCR. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein,p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) andToll-like receptor4(TLR4) were determined by Western blot. The result showed thatthe purity of magnolol used in this study was100%. Magnolol inhibited theexpression of TNF-α, IL-6and IL-1β in LPS-stimulated RAW264.7cells in adose-dependent manner. Western blot analysis showed that magnolol suppressedLPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK andP38. Magnolol could significantly down-regulated the expression of TLR4stimulating by LPS. The results suggest that magnolol exerts an anti-inflammatoryproperty by down-regulated the expression of TLR4up-regulated by LPS, therebyattenuating TLR4mediated the activation of NF-κB and MAPK signaling and therelease of pro-inflammatory cytokines.Thirdly, molecular targets of the anti-inflammatory actions of magnolol wasidentified. The binding affinity and activating of PPAR-γ were detected by PPAR-γligand binding assays and Western blot. Meanwhile, to evaluate the involvement ofPPAR-γ in the activation of TLR4and to determine whether the effects of magnololon TLR4expression is dependent on PPAR-γ activation, GW9662, the specificPPAR-γ antagonist was added into the medium30min before magnolol incubation.Then the cells were stimulated by LPS and TLR4expression was detected. The resultsshowed that magnolol could bind and activate PPAR-γ. Furthermore, magnolol couldsignificantly down-regulated the expression of TLR4stimulating by LPS and theinhibition effects of TLR4by magnolol can be reversed by GW9662, a specificantagonist for PPAR-γ. These results suggest that magnolol activate PPAR-γ, therebyattenuating TLR4expression and TLR4mediated NF-κB and MAPK activation andthe release of pro-inflammatory cytokines.In the last, the therapeutic effect of magnolol were examined in LPS-inducedmouse mastitis. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. All mice were randomly divided into six groups:control group, LPS group, Magnolol (5,10and20mg/kg)+LPS group, DEX+LPSgroup. Magnolol was administered intraperitoneally1h before and12h afterinduction of LPS. MPO activity and the level of TNF-α, IL-1β and IL-6in mousemammary tissues were detected. The results showed that magnolol significantlyattenuated infiltration of neutrophilic granulocyte, activation of MPO; down-regulatedthe level of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6inLPS-induced mastitis. In conclusion, these results indicated that magnolol exertedbeneficial effects on experimental mastitis induced by LPS and may be a promisingpotential therapeutic reagent for mastitis treatment.