Molecular Mechanism And Pharmacology Significance Of Acetylbritannilatone Modulation Inflammatory Response In Vascular Cells

The chronic inflammatory reaction is a basic pathological mechanism that underlies cardiovascular diseases, such as atherosclerosis (AS), restenosis after percutaneous transluminal coronary angioplasty (PTCA) and hypertension. The inflammatory reaction is initiated by a wide range of factors stimulating endothelium, involving aggregation and activation of platelets, adherence of monocytes and releasing of inflammatory mediators, and subsequent transendothelial migration and proliferation of vascular smooth muscle cells (VSMCs). In the pathogenesis of vascular inflammatory diseases,VSMCs, as a major cell component, has abnormal gene expression of over one hundred, resulting in the changes of phenotype and function.Nuclear factorκB (NF-κB) has been shown to regulate a variety of genes involved in inflammatory and immune reactions as well as cell death and proliferation. Comparing sequence shows that inflammatory factor-induced gene expression is trans-activated by NF-κB. Modulation NF-κB activity of vascular cells, blockade of the pathway of inflammatory response and decreasing the releasing of inflammatory mediators in injured blood vessel become the important strategy to studt the novel of anti-cardiovascular disease drug.Inula Britannica is a traditional Chinese medicinal herb that has been used to treat bronchitis and inflammation. Three specific sesquiterpene lactones in Inula britannica-L were isolated and identified, including britannilactone (BL), 1-O-acetylbritannilactone (1-O-ABL), and 1,6-O,O-diacetylbritannilactone (1-O2-ABL). In the present study, our aim was to assess the effects of ABL on LPS-induced VSMC NF-κB activation and cyclooxygenase-2 (COX-2) gene expression to investigate the mechanisms of ABL inhibiting VSMC inflammatory response.1 Acetylbritannilatone (ABL) suppresses VSMC inflammatory response by attenuating NF-κB activationAs a major transcription factor in inflammatory response, NF-κB is involved in the regulation of inflammatory genes. In order to assess the effects of 1-O-ABL on inflammatory response in VSMCs,we investigated the inhibitory effects of ABL on lipopolysaccharide (LPS)-induced NF-κB activation and inflammatory gene expression and the related molecular mechanism in VSMCs.1.1 ABL inhibits LPS-induced nuclear translocation and activation of NF-κBWestern blot and immunostaining analysis showed the level of nuclear NF-κB p65, after induction with LPS for 10 min, was primarily increased up to peak, maintained for 30 min, and then decreased at 1 h. When VSMCs were incubated in the medium containing ABL (5, 10, 20μmol/L), increased in nuclear p65 induced by LPS was declined at an extent in a concentration-dependent manner. These findings suggest that ABL diminished the translocation of NF-κB to nuclei induced by LPS.1.2 ABL inhibits LPS-induced inhibitor ofκB (IκBα) phosphorylation and degradation in VSMCsNF-κB activation depends on the phosphorylation and degradation of IκBα. Western blot showed that LPS triggered IκBαphosphorylation, which was maximal at 10 min and rapidly declined thereafter, along with marked reduction of the level of IκBα. However, pretreatment of cells with ABL before LPS stimulation attenuated the phosphorylation and degradation of IκBαinduced by LPS in a concentration-dependent manner. The level of IκBαreturned to baseline in VSMCs treated with ABL. These results suggest that suppression of NF-κB activation by ABL is via inhibiting phosphorylation and degradation of IκBα.1.3 ABL inhibits LPS-induced the DNA-binding activity of NF-κBIt is necessary for activated NF-κB to bind to NF-κB consensus sequences to initiate the transcription of target genes,as shown by EMSA, stimulation with LPS markedly increased the DNA binding activity of NF-κB, and DNA binding of NF-κB in response to LPS was inhibited by ABL in a concentration-dependent manner. This result indicates that ABL inhibits the DNA binding of NF-κB by a direct modification of active NF-κB in the nucleus.1.4 ABL inhibits LPS-induceded COX-2 expression and prostaglandin E2 (PGE2) productionIn order to evaluate whether ABL-dependent NF-κB inhibition might influence NF-κB-dependent COX-2 expression, Western blot showed that LPS increased the expression of COX-2 protein by 6.8-fold. The pretreatment with ABL (5μmol/L) decreased COX-2 expression by 62 %, and pretreatment with ABL (10,20μmol/L) decreased COX-2 expression to baseline in a concentration-dependent manner. This result was in good agreement with the immunostaining and RT-PCR analysis. Enzyme-linked immunosorbent assay (ELISA) showed that ABL inhibited PGE2 synthesis in a concentration-dependent manner in LPS-stimulated VSMCs. The results showed that ABL could inhibit COX-2 expression and activity.1.5 ABL inhibits the expression of NF-κB-dependent adhesion moleculesThe expression of vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1(ICAM-1) is elevated in response to inflammatory stimuli, which promote the adhesion of circulating leukocytes to vascular endothelium. An important character of inflammatory response is cell chemotaxis. The release of adhesion molecules is the premise for cell migration. VSMCs treated with LPS for 16 hours increased the level of ICAM-1 and VCAM-1 protein by 4.4-fold, 3.8-fold, respectively. VSMCs treated with ABL could decrease expression of ICAM-1 and VCAM-1 induced by LPS, but ABL had no inhibitory effects on IκBαexpression. These results suggest that ABL inhibit or decrease inflammtory response of VSMCs.2 Acetylation enhances sesquiterpene lactone anti-inflammatory actionComponent analysis showed that other two homologues, britannilactone (BL), 1.6-O2-ABL, were isolated and identified from Inula Britannica-L.. The difference among BL, 1-O-ABL and 1.6-O2-ABL is the number of their acetyl side groups in molecule. The aim of this work was to investigate the role of acetyl side groups in the biological activity and anti-inflammatory mechanisms of sesquiterpene lactone. It is not clear whether the anti-inflammatory activity of ABL depends on the acetyl side groups in structure. In the present study, the anti-inflammatory effects of three sesquiterpene lactones were compared in RAW 264.7 macrophages to definite the structural basis of anti-inflammatory.2.1 Acetylation enhances the inhibitory effects of sesquiterpene lactone on inducible nitric oxide synthase (iNOS) expression and activityWe determined the effect of three homologues on the production of NO in RAW264.7 cells exposed to LPS/IFN-γ. The pretreatment with 1-O-ABL, 1-O2-ABL decreased NO production by 28% and 33%, respectively. BL did not show the activity of inhibitory iNOS activity. Western analysis showed that pretreatment with 1-O-ABL, 1-O2-ABL, but not BL, decreased iNOS expression by 54% and 95%,respectively. This result was in good agreement with the RT-PCR analysis. Their inhibiting activity was positive correlation with the acetyl side groups number, and the acetyl structure may augment the anti-inflammatory effects.2.2 Acetylation enhances the inhibitory effects of sesquiterpene lactone COX-2 expression and activityThree homologues inhibited PGE2 synthesis in LPS/IFN-γ-stimulated RAW264.7 cells, and the inhibiting activity was, in turn, 1,6-O2-ABL >1-O-ABL >BL. Their inhibiting activity was associated with the acetyl side groups number. Western blot analysis showed that all three homologue inhibited LPS/IFN-γ-induced COX-2 protein expression. BL, 1-O-ABL and 1,6-O2-ABL decreased COX-2 expression by 23%, 67%, 75%, respectively. The inhibitory activity of 1.6-O2-ABL was the strongest. These findings suggest that ABL anti-inflammatory activity depends on the number of acetyl side group.2.3 Acetylation enhances the inhibitory effects of sesquiterpene lactone on NF-κB activationTo determine the anti-inflammation activity mechanism of ABL with different acetyl side group number, we determined the effects of three homologues on NF-κB activation. When RAW264.7 cells were respectively incubated in the medium containing different ABL (10μmol/L) for 60 min, increase in nuclear p65 induced by LPS/IFN-γwas declined at a extent, and the inhibitory effects of were stronger than that of 1-O-ABL and BL. EMSA showed that, in ABL treated cells, NF-κB activity was significantly inhibited in a acetyl-dependent manner especially.2.4 Acetyl side groups enhance inhibitory effects of ABL on degradation and phosphorylation of IκBαPretreatment of cells with three ABL before LPS/IFN-γstimulation, IκB-αphosphorylation induced by LPS/IFN-γwas decreased at a extent. The effect of 1,6-O2-ABL was the strongest, and 1,6-O2-ABL blocked LPS/IFN-γ-induced phosphorylation of IκBα. These results indicate that the acetylation enhances the activity of ABL inhibiting inflammation but does not change drug binding effective site. Three homologus had the same active element,but the inhibitory activity was different,which depended the acetyl side groups number.3 ABL inhibits phosphorylation and degradation of IκBαvia canonical NF-κB activation pathwayIn the present study, we used 1,6-O2-ABL, which inhibitory effects is strongest, to investigate the effects of ABL on LPS/IFN-γ-induced activation of IκB kinase (IKK) and canonical/alternative pathway of NF-κB activation, to assess the mechanism of ABL inhibiting signaling pathway of inflammatory response.3.1 ABL inhibits IKKβphosphorylation but no effect on phosphorylation of IKKαTo determine the upstream mechanism of ABL inhibiting NF-κB activation, the phosphoryation of IKKαand IKKβwere analysed by Western blot. Stimulation of RAW264.7 cells with LPS/IFN-γtriggered IKKαand IKKβ phosphorylation. When cells were pretreated with ABL, the phosphorylation of IKKβbut not IKKαwas abolished. This result was in good agreement with the immunoprecipitation analysis.3.2 ABL inhibits the IKKβ-dependent NF-κB activation but not IKKαThe pcDNA3.1-IKKβtransfected RAW264.7cells increased the expression level of IKKβby 10-fold,but no effects on the phosphorylation of IKKβand IκBα. Treatment with LPS/IFN-γin transfected pcDNA3.1-IKKβRAW264.7 cells increased the level of phosphorylation of IKKβand IκBα, resulting in cascade effect of IκBαphosphorylation. In pretreatment the transfected cells with ABL, LPS/IFN-γ-induced IKKβphosphorylation was attenuated, and the level of IκBαphosphorylation was markedly decreased. The results suggest that ABL inhibits the IKKβ-dependent NF-κB activation. IKKαphosphorylation relies on the upstream activation of NIK, ABL did not exert any inhibition on the phosphorylation of NIK.4 ABL inhibits PDGF-stimulated VSMC proliferation and injury-induced neointimal hyperplasia4.1 ABL inhibits PDGF-stimulated VSMC proliferationTreatment with PDGF increased cell proliferation by 4.6-fold. Pretreatment with ABL (5,10,20μmol/L) decreased PDGF-stimulated cell proliferation by 23%,67%,75%, respectively. When quiescent cells were treated with ABL in the absence of PDGF, no significant difference was observed in proliferation activity.4.2 ABL attenuates PDGF-stimulated tyrosine phosphorylation of Erk1/2, AktPDGF triggered Erk1/2 and Akt phosphorylation, which was increased at 30 min, maximal at 1 h and declined thereafter. However, pretreatment of cells with ABL before PDGF stimulation attenuated the phosphorylation of Erk1/2 and Akt in a concentration-dependent manner. The results suggest that ABL inhibit PDGF-stimulated proliferation signaling pathway.4.3 ABL decreases PDGF-stimulated tyrosine phosphorylation of PDGF- receptorTo determine the upstream mechanism of ABL inhibitory Erk1/2 and Akt activation, the phosphoryation of PDGF-receptor were analysed by immunoprecipitation. PDGF increased phosphotyrosine levels of PDGF-receptor, which was decreased by ABL in a concentration-dependent manner. Neither treatment with PDGF nor ABL changed the protein levels of PDGF receptor.4.4 ABL decreases the injury-induced Erk1/2 and Akt phosphorylationAt 3 days after balloon injury, level of Erk1/2 and Akt phosphorylation increased. However, following ABL treatment, the level of phosphorylated Erk1/2 and Akt was reduced by 52% and 62%, respectively. These results suggest that ABL suppresses neointimal hyperplasia via attenuation of proliferation signaling cascade.Conclusions1 ABL suppreses NF-kB activation via inhibition of IKKβand IκBαphosphorylation and degradation, leading to a subsequent blockade of the nuclear translocation of p65.2 BL, 1-O-ABL and 1,6-O2-ABL are protent anti-inflammatory agent, the activity was, in turn, 1.6-O2-ABL >1-O-ABL >BL. The acetylation may result in the stronger anti-inflammation action.3 ABL is a potent single compound to modulate the inflammatory response in RAW264.7 cells and VSMCs.4 ABL attenuates PDGF-stimulated tyrosine phosphorylation of Erk1/2, Akt via inhibiting the phosphorylation of PDGF- receptor, further inhibting cell proliferation.5 ABL attenuates neointima formation via inhibiting injury-induced Erk1/2, Akt phosphorylation.