| Summary: | 碩士 === 長庚大學 === 中醫學系天然藥物 === 99 === The elevated expression of various inflammatory proteins such as matrix metalloproteases (MMPs) induced by proinflammatory cytokines has been shown to be implicated in neurodegenerative diseases. MMPs, in particular MMP-9, induced by interleukin-1β (IL-1β), have been demonstrated to be implicated in neuroinflammation such as Alzheimer's disease (AD). Our previous reports have shown that IL-1β-induced proMMP-9 expression is mediated through MAPKs, transactivation growth factor receptors, PI3K/Akt, and AP-1. However, oxidative stress due to NADPH oxidase (Nox) activation and reactive oxygen species (ROS) generation have been shown to trigger intracellular signalings leading to expression of inflammatory proteins and brain injury and inflammation. However, the roles of Nox/ROS in IL-1β-induced MMP-9 expression in rat brain astrocytes (RBA-1) remain unclear. Here, we report that IL-1β-induced MMP-9 expression and cell migration was mediated through Nox activation and ROS production, which were attenuated by N-acetylcysteine (NAC, a scavenger of ROS), the inhibitors of Nox [diphenyleneiodonium chloride (DPI) and apocynin (APO)]. The involvement of Nox in IL-1-induced responses was further confirmed by translocation of Nox subunit (p47phox) to plasma membrane. Further, IL-1β-stimulated Nox/ROS generation leading to MMP-9 expression and cell migration was attenuated by the inhibitors of c-Src (PP1), PDGFR (AG1296), PI3K (LY294992), Akt (SH-5), PKC (GF109203X, Gö6976, rottlerin), Ca2+ chelator (BAPTA-AM, EDTA), CaM (CaMI), CaMKII (KN62), MEK1/2 (U0126), JNK1/2 (SP600125), p38 (SB202190), NF-B (Bay-117280), AP-1 (curcumin and Tanshinone), and p300 (GR343). The involvement of c-Src, PDGFR, Akt, ERK1/2, JNK1/2, and p38 MAPK in Nox/ROS-dependent responses was further confirmed by attenuating the phosphorylation of these kinases in RBA-1 cells pretreated with NAC, DPI, APO, and their respective inhibitors. Furthermore, IL-1β-induced MMP-9 expression was mediated through phosphorylation and degradation of IB and phosphorylation of NF-B (p65) which were attenuated by NAC, DPI, and APO, but not by U0126, SP600125, SB202190, PP1, and AG1296. Moreover, the roles of AP-1 and p300 in IL-1β-induced responses were revealed by phosphorylation of ATF2 and p300, respectively, which were attenuated by NAC, DPI, and APO. These results suggested that in RBA-1 cells, IL-1β-induced MMP-9 expression and cell migration was, at least in part, mediated through Nox/ROS-dependent activation of MAPKs, c-Src-dependent PDGFR/PI3K/Akt/p300, and Ca2+-dependent CaMKII/JNK/AP-1, and NF-B. These results provide new insights into the mechanisms of IL-1β involved in neuroinflammation and neurodegeneration. Increased understanding of these mechanisms underlying up-regulation of proMMP-9 will create opportunities for the development of therapeutic strategies in neurodegenerative diseases.
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