The Anti-Inflammatory Effects And Mechanism Study Of Sesquiterpene Lactones From Inula Japonica

Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. Several kinds of cells may be involved in the pathogenesis of asthma,including mast cells, macrophages, eosinophils, basophils and neutrophils, etc. Inula japonica Thunb as traditional Chinese medicine has a long history of application.And a variety of pharmacological activities of Inula japonica Thunb like anti-cancer,hepatoprotective and antidiabetic effects have been verified. In our previous study,we have isolated and identified 38 compounds from Inula japonica extracts.6α-isovaleryloxy-1-hydroxy-4αH-1,10-secoeudesma-5（10）,1（13）-dien-12,8β-olide（IVSE） and 1,6-O,O-diacetylbritannilactone（ABLOO） are two sesquiterpene lactones with stronger anti-inflammatory and anti-asthmatic activities in the efficacy prescreen. We take the following study to further investigate their mechanism of action.Part 1:We investigated inhibitory effects of IVSE on nitric oxide（NO） production in lipopolysaccharide（LPS）-stimulated RAW264.7 cells and mechanism of action. The inhibitory effect of IVSE on NO production in LPS-induced RAW264.7 cells was examined using Griess reagent, and the effects of IVSE on the expressions of inducible nitric oxide synthase（iNOS） and its upstream signal proteins including IκB kinase（IKK）/inhibitor kappa B（IκB）-α/nuclear factor κB（NF-κB） and mitogen-activated protein kinases（MAPKs） were investigated by Western blot.Mechanism analysis indicated that IVSE dramatically decreased the expression of iNOS, reduced the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation and degradation of IκB-α, and inhibited the activation of the upstream mediator IKKα/β. Furthermore, our results showed that IVSE inhibited the phosphorylation of MAPKs including extracellular regulated kinases（ERK1/2）, c-Jun N-terminal kinases（JNK） and p38. Furthermore, IVSE inhibited LPS-induced matric metalloproteinases-9（MMP-9） activity.Part 2:（1）We investigated the anti-inflammatory effect of ABLOO on IgE/DNP-HSAinduced leukotriene C4（LTC₄） production and degranulation in bone marrow-derived mast cells（BMMCs）. The level of LTC₄ was measured using enzyme-linked immuno sorbent assay（ELISA） and the level of degranulation was determined byβ-hexosaminidase（β-hex） releasing rate. ABLOO markedly inhibited synthesis of LTC₄ and degranulation by suppressing the activation of cytosolic phospholipase A2（cPLA2） and phospholipase Cγ（PLCγ）.（2） We investigated the anti-asthmatic effects of ABLOO on eotaxin-1 and arachidonate 15-lipoxygenase（ALOX15） mRNA expression in interleukin-4（IL-4）induced A549 cells. The mRNA expression of eotaxin-1 and ALOX-15 were measured by RT-PCR. ABLOO dramatically inhibited IL-4-induced mRNA expression of eotaxin-1 and ALOX-15. Meanwhile ABLOO significantly decreased phosphorylation of STAT6 and JAK3 determined by Western blot and also attenuated IL-4-induced STAT6 transcriptional activity measured by luciferase assay.In conclusion, IVSE significantly suppressed LPS-induced NO production, and ABLOO dramatically reduced IgE/DNP-HSA induced secretion of LTC₄, mast cells degranulation and mRNA expression of eotaxin-1 and ALOX-15 induced by IL-4 in A549 cells. All the significance implies that IVSE and ABLOO have potential for becoming lead compound in treatment of inflammatory disease particularly asthma.And our study provided valid experimental data for the advanced anti-asthma drug develop for IVSE and ABLOO.