Effects Of 1-o-acetylbritannilactone On Expression Of Proinflammatory Gene In Vascular Cells And The Related Mechanism

Vascular inflammation is a basic pathological mechanism that underlies cardiovascular diseases, such as atherosclerosis(AS), restenosis after percutaneous transluminal coronary angioplasty (PTCA). The inflammatory reaction involves the complex interaction between inflammatory cells and vascular cells(including endothelial cells and smooth muscle cells), and between inflammatory mediators and vascular cells. The expression of vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1(ICAM-1)on the surface of endothelial cells is elevated in response to inflammatory stimuli, which promote the adhesion of circulating leukocytes to vascular endothelium. Both nitric oxide(NO) and prostaglandin E2 (PGE2) are known as important mediators of acute and chronic inflammation. Their syntheses are catalyzed by nitric oxide synthase(NOS) and cyclo-oxygenase(COX)respectively. Inducible nitric oxide synthase(iNOS) and cyclo-oxygenase-2(COX-2) are upregulated in response to inflammatory and proinflammatory stimuli, and their products can influence many aspects of the inflammatory cascade. Nuclear factor-κB(NF-κB) acts as a central mediator of the immune response and controls the expression of various genes involved in inflammation and proliferation, such as ICAM-1,VCAM-1, iNOS and COX-2. NF-κB is located in cytoplasm in an inactive form in association with the inhibitor I-κBα.In response to a number of stimuli including inflammatory cytokines and bacterial lipopolysaccharide(LPS), I-κBαis phosphorylated, ubiquitinated, and degraded by a proteosome-dependent pathway allowing active NF-κB to translocate to the nucleus where it can activate genes expression. therefore, ICAM-1, VCAM-1, iNOS and COX-2 expression, and NF-κB activation can used as biomarker for the screening of anti-inflammatory activity. 1-o-acetylbritannilatone(ABL) was a new active compound isolated from Inula Britannica-F. It was founded that ABL had a potent anti-inflammatory activity in preliminary expriment . ABL reduced LPS/IFN-γ-induced iNOS, COX-2 protein, as well as mRNA expression in RAW 264.7, by affecting the activation of transcription factor NF-κB. But now, effects of ABL on vascular cells is not clear. Based on preliminary experiment, we investigated the effects of ABL on adhesion molecule ICAM-1,VCAM-1 and inflammatory mediator iNOS and COX-2 expression in vascular cells, and the related molecular mechanism. 1 Effects of ABL on ICAM-1,VCAM-1 and COX-2 expression in ECVs activated with LPS Endothelial dysfunction is the first step in the development of AS and vascular restenosis. The adhesion of circulating leukocytes to vascular endothelium is critical to inflammatoty responses. To elucidate the effects of ABL on endothelium cells, we detected effects of ABL on the expression of ICAM-1,VCAM-1 and COX-2 in ECVs activated with LPS. The results are as follows: 1.1 Total RNA was extracted from ECV treated with LPS for 18 hours. Compared with the control ( 0 h), the level of COX-2 mRNA was robustly increased by 1.40-fold at 18 hours, ECVs were exposed to ABL (12.5, 25, 50μmol/L) for 1h and then treated with 100ng/ml LPS for 18 hours.The level of mRNA was reduced by 35.85%, 42.46% and 53.46%, respectively , compared with control (cell was induced by LPS at 18h). Western blot showed that ABL could decrease expression of COX-2 induced by LPS. ABL had no effects on cells viability of ECV at concentration of 50μmol/L, assessed by MTT method. Thus, these inhibitory effects were not the result of cytotoxicity. 1.2 After ECV was pretreated with ABL (12.5, 25, 50μmol/L)for 1h, and then treated with 100ng/ml LPS for 18 hours. The level of ICAM-1 protein was reduced by 9.77%, 40.87% and 46.83% respectively, compared with control.1.3 We found that ABL decreased expression of VCAM-1 protein stimulated by LPS. Western blot showed that after ECV was pretreated with ABL (12.5, 25, 50μmol/L) for 1h, expression of VCAM-1 protein induced by LPS was reduced by 1.16%, 2.0% and 36.5% respectively, compared with control. 1.4 ECV was cultured in 96-well plate, after pretreated with ABL (25, 50μmol/L) for 1h, and then treated with 100μg/ml LPS for 24 hours. Lymphocytes were added. Adhesion assay indicated that adhesion rate decreased with increasing ABL concentration. At two dose of ABL, there was a significant difference (p<0.05), compared with control. Adhesion rate was decreased by 8% and 14%,respectively。In summary, ABL can significantly inhibit expression of ICAM-1,VCAM-1 and COX-2 in ECVs activated with LPS, and inhibit LPS –induced cell-cell adhesion between ECVs and lymphocytes. 2 The effects of ABL on expression of iNOS ,COX-2 induced by LPS in VSMC The expression of iNOS and COX-2 is mostly associated with pathological states, such as atherosclerosis and restenosis after PTCA, It has been demonstrated that expression of iNOS in response to inflammatory and proinflammatory mediator may promote cellular proliferation. COX-2 expression is increased by proinflammatory mediator stimuli that are implicated in the development of atherosclerosis. To further study the effects of ABL on inflammatory mediator , we investigated the effects of ABL on expression of iNOS ,COX-2 induced by LPS in VSMC. 2.1 In order to evaluate the iNOS inhibitory capacity of ABL, nitrite accumulation was examined in culture medium of LPS-stimulated VSMC. LPS was added to the culture media after cells were pretreated with ABL( 25, 50μmol/L) for 1h, cells were incubated for 24h, NO production in culture supernatant was spectrophotometrically evaluated by measuring nitric oxidative product of NO. The results suggested that ABL inhibited nitrite accumlation in a concentration-dependent manner. Upon treatment with ABL, NO production was reduced by 30 and 90% (p<0.05), respectively.2.2 We found that ABL significantly reduced iNOS synthesis. After VSMCs were incubated with 50μmol/L ABL for 1h, and then treated with LPS for an additional 12 h, RT-PCR analysis showed that although hardly detected in unstimulated VSMC by RT-PCR, iNOS mRNA was significantly expressed after stimulation with LPS for 24 h. And the presence of ABL in LPS-stimulated cell culture markedly decreased iNOS expression. Western blot also showed that ABL decreased the expression level of iNOS protein. 2.3 RT-PCR analysis was performed with VSMC pretreated with the ABL( 50μmol/L) for 1h and then incubated with LPS for an additional 12 hours. The level of COX-2 mRNA was markedly reduced, compared with control. Western blot result coincided with the RT-PCR analysis.The study suggested that ABL inhibits LPS-mediated COX-2 expression. 2.4 Expression of iNOS in balloon-injured arteries was detected with immuno-histochemistry. The expression of iNOS was detected in the neointima at day 3,14 after balloon injury, the positive stain for iNOS focused in neointima at day14. The expression pattern of iNOS showed that balloon injury induced iNOS expression during neointima formation. iNOS,COX-2 activity were increased in vascular wall after injury, the levels of iNOS, COX-2 decreased significantly in the rats treated with ABL, compared with the model. The findings suggested that ABL inhibited iNOS,COX-2 activation in balloon-injuried arteries. All above results indicated that ABL significantly inhibited expression of iNOS and NO production, and also ABL significantly inhibited expression of COX-2 in VSMC stimulated by LPS. ABL could effectively reduced iNOS and COX-2 expression in neointima in balloon-injuried arteries. 3 The effects of ABL on NF-κB activation in vascular cell induced with LPS In above two parts, we demonstrated that ABL can significantly inhibited expression of ICAM-1,VCAM-1,iNOS and COX-2 in vascular cells. The activation of NF-κB is critical for the induction of these molecules by LPS .We further examined effects of ABL on NF-κB activation.3.1 Western blot analysis was performed to examine the translocation of the subunit p65 into the nucleus. The results showed that LPS treatment caused to translocation of the subunit p65 into the nucleus, p65 expression was increased by 4.24-fold at 1 hour after LPS stimulation, compared with control. However, the amount of p65 translocation into nucleus in response to LPS was significantly reduced in the presence of ABL(50μmol/L), approach to control. Immuno-histochemistry analysis confirmed that ABL inhibited p65 translocation. 3.2 EMSA result showed that treatment with LPS after 1 h, the DNA binding activity of NF-κB was markedly increased, DNA binding of NF-κB in response to LPS was inhibited by addition of ABL. 3.3 We determined the effect of ABL on LPS-mediated degradation of I-κBαby Western bloting. Treatment with LPS led to a rapid disappearance of I-κBαwith 1 h, reduced by 27%, however , LPS induced degradation of I-κBαwas attenuated in the presence of ABL(25,50μmol/L), the level of I-κBαreach to 80% and100%, respectively, compared with control. 3.4 Expression of P65 and I-κBαin VSMC was detected with immunohistochemistry. Treatment with LPS after 1h, the positive stain for p65 was increased in nucleus; I-κBαin cytoplasm was reduced. However, by addition of 50μmol/L ABL, the positive stain for p65 LPS-induced became weaker in nucleus, I-κBαin cytoplasm increased. The results implied that in ECV ABL not only markedly inhibited the translocation of NF-κB into nucleus, but also inhibted the DNA binding of actived NF-κB. ABL attenuated the degradation of I-κBα. In VSMC, ABL markedly inhibited the translocation of NF-κB into nucleus and the degradation of I-κBα. 4 Effects of ABL on VSMC proliferation In third part we demonstrated ABL inhibited NF-κB activation in vascular cells. The contribution of ABL to VSMC proliferation is unknown.We investigated effects of ABL on VSMC proliferation. 4.1 VSMCs were exposed to ET 48 h, the ability of VSMC proliferation wasobviously increased by 1.21-fold,compared with control. pretreated with ABL(12.5,40,50,75μmol/L)for 1 h, VSMC proliferation ET-stimulated was decreased by 2%,4%,7%和7.5%, respectively. 4.2 We established an intimal hyperplasia model by denuding the rat aorta endothelium with balloon catheter. Compared with smooth intima and orderly arranged medial VSMCs of aorta in the normal rats, The progressive and widespread intimal thickening and lumen stenosis were observed at day 14. The morphological changes suggested that migration and proliferation of VSMCs were main causes for neointimal formation after denuding the endothelium. The rats treated with ABL had significantly less neointimal thickening than model. The intimal area/medial area(I/M)declined in the treatment group(0.61±0.09 versus 0.13±0.03,p<0.05),The results suggested that the ABLcould inhibited the proliferation of the intima and the stenosis of the lumen after the injury of artery. The above results suggested ABL significantly inhibited proliferation induced by ET,and attenuated neointimal formation after denuding the endothelium. Conclusions 1 ABL significantly inhibits LPS-induced expression of ICAM-1,VCAM-1, iNOS and COX-2 in vascular cells, and decreases LPS-induced adhesion between ECV and lymphocyte. 2 ABL significantly inhibits LPS-stimulated expression of iNOS and NO production, ABL significantly inhibits expression of COX-2 in VSMC stimulated by LPS. ABL can effectively reduces iNOS and COX-2 expression in neointima after denuding the endothelium. 3 In ECV ABL not only markedly inhibits the translocation of NF-κB into nucleus, but also inhibits the DNA binding of active NF-κB. ABL prevents the degradation of I-κBα. In VSMC, ABL markedly inhibits the translocation of NF-κB into nucleus and the degradation of I-κBα. 4 ABL can significantly inhibits proliferation induced by ET, and attenuate...