探討LCY-2-CHO抑制lipopolysaccharide誘發N9微膠質細胞生成一氧化氮與CHS-111抑制formyl peptide促進大鼠嗜中性白血球產生超氧自由基之機制

1. 微膠質細胞生成的過量一氧化氮對於神經退化疾病扮演著重要的角色。在此研究中，我們發現LCY-2-CHO可抑制由lipopolysaccharide (LPS)/interferon-gamma (IFNg)刺激的N9微膠質細胞或由LPS刺激的N9微膠質細胞、BV-2微膠質細胞與大鼠初代微膠質細胞產生一氧化氮。LCY-2-CHO亦可抑制誘導型一氧化氮合成酶(inducible nitric oxide synthase；iNOS)的蛋白質與mRNA表現量，但不會影響iNOS mRNA的穩定性與iNOS的酵素活性。LCY-2-CHO並不會影響nuclear factor-kappa B (NF-kB)及cyclic AMP response element-binding protein (CREB)的DNA鍵結活性，但是會抑制activator protein-1 (AP-1)、CCAAT/enhancer-binding protein (C/EBP)以及 nuclear factor-interleukin 6 (NF-IL6)的DNA 鍵結能力。在轉錄因子的核轉移作用上，LCY-2-CHO減少細胞核中c-Jun 與 C/EBP-beta的含量，但對於 p50、p65、C/EBP-delta、signal transducer and activator of transcription-1 (STAT-1)，或是IFN regulatory factor-1 (IRF-1)的含量則沒有影響。LCY-2-CHO對LPS/IFNg 所引起N9微膠質細胞的p38 mitogen-activated protein kinase (MAPK)、extracellular signal-regulated kinase (ERK)、c-Jun NH2-terminal kinase (JNK)、STAT-1、 CREB 或是 c-Jun 的磷酸化作用亦沒有影響，但可抑制 C/EBP-beta Ser105和Thr235位置的磷酸化作用，而此一抑制作用與LCY-2-CHO降低C/EBPb的表現量是一致的。綜合而言，LCY-2-CHO 抑制LPS/IFNg 刺激微膠質細胞生成一氧化氮的作用主要是經由抑制AP-1細胞核轉移與C/EBP的蛋白表現量，因而降低 iNOS 的基因與蛋白表現量，進而減少一氧化氮生成。 2. 化合物2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111)可抑制formyl-Met-Leu-Phe (fMLP)刺激大鼠嗜中性白血球產生超氧自由基 (supreoxide；O2•−)的作用。CHS-111並無自體氧化的作用，而且不會對細胞產生毒性。在cell-free系統中，CHS-111不會抑制由acrachidonic acid所引起的NADPH oxidase 活性，亦無法減少由 GTPgS 所促進的 Rac2細胞膜轉移作用。CHS-111可有效地抑制 p40phox、p47phox、p67phox 的細胞膜轉移作用。p47phox serine磷酸化、p47phox - p22phox的連結作用，以及Rac2的活化亦可被CHS-111抑制。此外，p21-activated kinase (PAK)的磷酸化、細胞膜轉移，及酵素活性都受到CHS-111的抑制。CHS-111亦能減少PAK與p47phox 間的交互作用。CHS-111明顯地降低Akt的酵素活性、Akt 與 p47phox的連結作用，以及Akt 與 phospho-PDK1 的細胞膜轉移。然而，p110-gamma的細胞膜轉移作用並不受到 CHS-111 的影響。CHS-111能輕微地抑制Akt Thr308的磷酸化，但不影響 Akt Ser473 的磷酸化。Protein kinase C (PKC) -alpha、-betaI、-betaII、-delta 與-zeta的細胞膜轉移、PKC (betaII Ser660)磷酸化與PKC酵素活性都可受到CHS-111的抑制，而 p38 mitogen-activated protein kinase (MAPK) 與其下游蛋白 MAPK-activated protein kinase-2，以及extracellular signal-regulated kinase 1/2的磷酸化作用不會受到CHS-111的影響。高濃度的 CHS-111 才能抑制由 fMLP 刺激細胞內游離鈣離子濃度增加的作用；但在缺乏細胞外鈣離子存在的情形下，CHS-111 則無法抑制細胞內游離鈣離子的增加。CHS-111可降低細胞內cyclic AMP的量，及輕微增加cyclic GMP的含量。研究結果顯示，CHS-111 抑制大鼠嗜中性白血球產生超氧自由基的作用是經由抑制Rac2-PAK、Akt 與 PKC 的訊息路徑。

1. Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-gamma (IFNg)-stimulated murine microglial N9 cell and in LPS-stimulated N9 cells, BV-2 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-kappa B (NF-kB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor-interleukin 6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBP-beta, but not those of p65, p50, C/EBP-delta, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), STAT-1, CREB or c-Jun in LPS/IFNg-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBP-beta at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBP-beta expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP-beta activation.2. In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl- 3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O2•−) generation, which was not mediated by scavenging the generated O2•− or by a cytotoxic effect. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPgS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40phox, p47phox and p67phox, phosphorylation of serine residues in p47phox, association between p47phox and p22phox, and Rac2 activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47phox were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47phox, moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110-gamma membrane translocation. The membrane recruitment of protein kinase C (PKC) -alpah, -betaI, -betaII, -delta and -zeta, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen- activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2, and extracellular signal-regulated kinase. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca2+ concentration ([Ca2+]i) elevation in the presence but not in the absence of extracellular Ca2+, and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O2•− generation in rat neutrophils through the blockade of Rac2-PAK, Akt and PKC signaling pathways.