| Summary: | 碩士 === 長庚大學 === 天然藥物研究所 === 95 === Lipopolysaccharide（LPS）is an integral component of Gram-negative bacteria may involved in triggering or exacerbating inflammatory responses and possible increase recruitment of inflammatory cells in the synovium promotes synovial inflammation. LPS has been shown to induce the expression of vascular cell adhesion molecules (VCAM-1) in various cell types and contributes to inflammatory responses. This study demonstrated that in synovial fibroblasts of human rheumatoid arthritis (RASFs), the My-D88 dependent NF-κB and three mitogen-activated protein kinase (MAPK) pathways participate in VCAM-1 expression induced by LPS. Western blotting and RT-PCR analyses showed that LPS increased expression of VCAM-1 mRNA and protein, which was inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), JNK (SP600125) and NF-κB (helenalin). LPS also stimulated phosphorylation of p42/p44 MAPK, p38 and JNK which was attenuated by U0126, SB202190, and SP600125, respectively. Moreover, LPS-stimulated nucleus translocation of NF-κB and degradation of IκB-a was blocked by helenalin not by U0126, SB202190 and SP600125. In addition to MAPKs, intracellular signaling components may be involved in LPS-induced VCAM-1 expression, we further determined whether these signaling pathways involved in VCAM-1 expression. Pretreatment with inhibitors of Src (PP1), PDGFR (AG1296), EGFR (AG1478), and PI3K (LY294002) not only attenuated LPS-induced VCAM-1 protein and mRNA expression but also Src, PDGFR, EGFR and Akt phosphorylation. Moreover, pretreatment with PKC inhibitors: Ro318220, Go 6976 and Rottlerin also blocked LPS-induced VCAM-1 protein and m-RNA expression. Transfection of RASFs with dominant negative mutants of MEK, ERK, p38, JNK, Src, p85, Akt and PKC inhibited LPS-induced VCAM-1 expression. We also found that LPS-induced VCAM-1 promoter activity in a time-dependent manner was attenuated by the inhibitors of U0126, SB202190, SP600125, helenalin, PP1, AG1296, AG1478, and LY294002 revealed by reporter gene assay. Furthermore, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to monolayer of RASFs which was blocked by pretreatment with U0126, SB202190, SP600125 or helenalin, PP1, AG1296, AG1478, and LY294002 prior to LPS exposure or by anti-VCAM-1 antibody. Taken together, these results suggest that LPS-induced VCAM-1 gene expression in RASFs was involved the phosphorylation of p42/p44 MAPK, p38, and JNK, transactivation of receptor tyrosine kinases, and translocation of NF-κB pathways. Results obtained in this study provide more understanding of regulatory mechanisms underlying LPS-induced VCAM-1 expression in RASFs. More impact information of pathophysiological processes of RA affected by LPS and VCAM-1 expression will prove beneficial in the therapeutic against inflammatory disease.
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