血紅素氧化酵素-1 在類黃酮素抑制酯多醣誘導一氧化氮產生與細胞
保護作用之角色
Study the Effect of Heme Oxygenase 1 in the NO Inhibition and
Cytoprotection of Flavonoids
中文摘要
第一章
血紅素氧化酵素(Heme oxygenase-1； HO-1)為一種具有保護作用之蛋白酵素可有效降低自 由基在對細胞所造成傷害。然而類黃酮素化合物(Flavonoids)誘導 HO-1 表現在酯多醣 (Lipopolysaccharide；LPS)誘導一氧化氮(nitric oxide; NO)生成機制並不明確。因此本實 驗以類黃酮素化合物誘導 HO-1 表現來探討抑制 NO 生成作用。實驗結果發現在 RAW264.7 巨 噬細胞中類黃酮素化合物之 3-OH flavone, baicalein, kaempferol 以及 quercetin 不論 LPS 存在與否都明顯誘導 HO-1 mRNA 及蛋白質表現，同時亦可抑制 LPS 誘導 iNOS 表現以及 NO 生成。Hemin 為 HO-1 誘導劑其可明顯誘導 HO-1 表現的同時亦可隨劑量增加而抑制 LPS 誘 導 iNOS 表現以及 NO 生成。然而加入低劑量 hemin (10, 20 及 50 mM)與類黃酮素化合物共 同處理時可隨劑量增加而增強抑制 LPS 誘導 iNOS 表現以及 NO 生成作用。SnPP 為 HO-1 抑 制劑，經過前處理過後可降低類黃酮素化合物抑制 LPS 誘導 NO 生成。同樣實驗以 hesperetin (5,7,3''-trihydroxy-4''-methoxy- flavanone;HT),
naringenin (5,7,4''-trihydroxy flavanone; NE), hesperidin (5,7,3''-trihydroxy-4''-methoxy- flavanone
7-rhamnoglucoside; HD), naringin (5,7,4''-trihydroxy flavanone 7-rhamnoglucoside; NI)來分析 LPS 誘導 NO 生成表現。在 RAW264.7, J774A.1 或以 thioglycolate 誘導 Balb/c 小鼠腹腔活化巨噬細胞中，不具有醣基取代之類黃酮素化合物 HT 及 NE 可明顯誘導 HO-1 表現並抑制 LPS 誘導 NO 生成表現，且 HT 或 NE 與 Hemin 共同處理 具有加成抑制 NO 作用，但此結果並不會表現在有醣基取代之 HD 及 NI。具有醣基取代之 HD 及 NI 分別加入 hesperindinase (HDase)及 naringinase (NIase)將第 7 位碳上 rutinoside 醣基水解後以高效液相層儀分析，其水解後產物分別成為不含醣基結構之 HT 及 NI，並將此反 應物加入細胞培養液內與 LPS 共同處理分析 iNOS 表現以及 NO 生成，結果發現 HD 及 NI 醣 基水解後所得之產物亦可抑制 LPS 誘導 iNOS 表現以及 NO 生成之作用。在送入 HO-1 表現質 體之 RAW264.7/HO-1 轉殖細胞分析 HO-1 表現較原始 RAW264.7 細胞約增強 3 倍，且加入 LPS 或 LTA (lipoteichoic acid)誘導 iNOS 表現以及 NO 生成中也較原始 RAW264.7 降低。 在 HO-1 大量表現之 RAW264.7 細胞加入 3-OH flavone, baicalein, kaempferol,
quercetin, hesperetin, naringenin 以及 LPS 共同處理並分析 NO 含量時發現其抑制 LPS 誘 導 NO 生成中也較原始 RAW264.7 降低。因此我們證實 HO-1 表現在抑制 LPS 誘導 NO 生成 作用具有重要之意義，然而醣基取代之類黃酮素化合物會降低 LPS 誘導 NO 抑制作用。 第二章
在我們先前實驗中已證實類黃酮素化合物誘導 heme oxygenase-1(HO-1)表現對於抑制 lipopolysaccharide (LPS)誘導 inducible nitric oxide synthase (iNOS)以及 nitric oxide (NO)生成表現佔有重大影響因素，但在第 7 位碳上 rutinose 醣基取代對抑制 NO 表現卻是具 有負面的影響。然而類黃酮素化合物及醣基取代部分對自由基造成之保護機制仍不明確，因此在 本實驗部分將探討有關類黃酮素化合物誘導 HO-1 表現對自由基造成細胞毒性之影響。類黃酮 素化合物 baicalein (3,5,7-trihydroxyl flavone; BE)可明顯誘導 HO-1 基因以及蛋白質表現 且加入 cycloheximide 或 actinomycin D 後便會降低 HO-1 表現，然而此現象並不會表現在 具有醣基取代之 baicalin (3,5-dihydroxyl flavone-7-glucuronic acid; BI)。另外我們觀察 baicalein 在 MAPK 表現方面，結果發現 baicalein 可依時間及劑量增加而增強 ERK 磷酸化表 現，但對於 p38 及 JNK 活化並無影響。以 DCHF-DA 分析自由基生成時發現 baicalein 可少量 誘導自由基表現且以 ERK 抑制劑, PD98059 及抗氧化劑, NAC 分別加入細胞可抑制 BE 誘導 HO-1 表現。有趣的是我們將 H2O2 誘導細胞死亡的同時加入 baicalein 後，細胞可顯著的降 低細胞死亡率發生且再分別加入 ERK 抑制劑, PD98059 以及 HO-1 抑制劑, SnPP 也都會降低 baicalein 產生之保護作用。以 H2O2 誘導細胞凋亡現象時發現 baicalein 亦可明顯降低 H2O2 誘導細胞死亡時所產生之 hypodipoloide cells 以及 caspase 3 活性，然而此現象並不會表現 在具有醣基取代之 baicalin。另外以 H2O2 誘導細胞死亡時我們也發現 BE 可增強粒腺體細胞 膜之穩定性並且抑制 cytochrome c 由粒腺體釋放至細胞質中。同樣實驗部分將類黃酮素化合 物中不具有醣基取代之 querectin 與含有醣基取帶之 rutin 及 quercitrin 來比較自由基誘導細 胞死亡所產生之保護作用，結果發現不具有醣基取代之 querectin 可明顯誘導 HO-1 表現並且 降低 H2O2 誘導細胞死亡，然而此類黃酮素化合物誘導 HO-1 表現之保護作用同樣也不會表現 在具有醣基取代的 rutin 及 quercitrin。另外，將 HO-1 基因送入 RAW264.7 細胞可發現其所 誘導 HO-1 表現較 neo-RAW264.7 細胞所誘導之 HO-1 表現約高出 3 倍，且分別以 H2O2 測 試自由基生成量及細胞死亡表現時發現 HO-1 大量表現 RAW264.7 所誘導自由基生成量較 neo-RAW264.7 細胞少。在分析細胞存活時亦發現以 H2O2 誘導細胞死亡在 HO-1 大量表現 RAW264.7 較 neo-RAW264.7 細胞有較高的存活率。另外，將 HO 代謝產物包括 Fe+2, Fe+3, biliverdin, bilirubin 及 carbon monoxide (CO) 進行 NO 抑制作用及細胞保護作用分析，發 現除 CO 具有誘導 HO-1 表現並抑制 LPS 誘導 NOS 及 NO 生成及細胞保護作用外，其他 HO 之代謝產物對 NO 抑制及細胞保護作用並不具任何之影響。由以上實驗我們證實不具有醣基取 代之類黃酮素化合物如 baicalein 及 quercetin 較具醣基取代之類黃酮素化合物表現出細胞保 護作用，而此誘導 HO-1 表現之保護機制與 ERK 活化表現有之密不可分之相關性。 英文摘要 Abstracts 1
Heme oxygenase-1 (HO-1) has been shown to protect cells from oxidative stress- induced damage, however the effect of HO-1 on lipopolysaccharide (LPS)-induced NO production is unclear. In the present study, flavonoids including 3-OH flavone, baicalein, kaempferol, and quercetin induce HO-1 gene expression at the protein and mRNA levels in the presence or absence of LPS in RAW264.7 macrophages. This
effect was associated with suppression of LPS-induced NO production and inducible nitric oxide synthase (iNOS) protein expression. Hemin induced HO-1 protein expression and this was associated with the suppression of LPS-induced NO production and iNOS protein expression in a dose-dependent manner. However, Hemin, at the doses of 10, 20 and 50 mM, dose-dependently stimulated the flavonoid (50 mM)-induced HO-1 protein expression, and enhanced their inhibitory effects on LPS-induced NO production and iNOS protein expression. The HO-1 inhibitor, tin protoporphyrin (SnPP, 10 mM), attenuated the inhibitory activities of the indicated flavonoids on LPS-induced NO production. Similar results were found in the
inhibitory effect of hesperetin (HT), hesperidin (HD), naringenin (NE), and naringin (NI) on LPS-induced NO production in macrophages. Both HT and NE, but not their glycosides HD or NI, induced HO-1 expression in according with inhibition of LPS-induced NO production and iNOS expression in RAW264.7, J774A.1, and thioglycolate-elicited peritoneal macrophages. Removal of rutinose at C7 of HD and NI by enzymatic digestion using respective hesperidinase (HDase) and naringinase (NIase), caused them to exhibit inhibitory activity on LPS-induced NO production, according to the production of the aglycones, HT and NE, by HPLC analysis.
Transfection of a HO-1 vector in macrophages (HO-1/RAW264.7) resulted in a 3-fold increase in HO-1 protein compared with that the parental RAW264.7 cells. NO
production mediated by LPS or lipoteichoic acid (LTA) in HO-1 over-expressed RAW264.7 cells (HO-1/RAW264.7) was significant less than that in parental
RAW264.7 cells. And, 3-OH flavone, baicalein, kaempferol, quercetin, hesperetin and naringenin showed a more significant inhibition on LPS-induced NO production in HO-1/RAW264.7 cells than in parental RAW264.7 cells. These data provide scientific evidence to indicate that HO-1 possesses the ability to inhibit LPS-induced NO production and is involved in the inhibitory mechanism of flavonoids; a negative effect of glycosylation on NO inhibition by flavonoids was proposed.
Abstract 2
Our previous study demonstrated that heme oxygenase-1 (HO-1) is involved in flavonoid inhibition of NO production induced by lipopolysaccharide (LPS) in macrophages. If the role of HO-1 in flavonoid protection of free radical
(ROS)-induced cell death is still unclear. Here, we showed that baicalein (3,5,7-trihydroxyl flavone; BE) but not its glycosides, baicalin (3,5-dihydroxyl flavone-7-glucuronic acid; BI), induces HO-1 gene expression at both the protein and mRNA levels, which was blocked by the addition of actinomycin D or cycloheximide. Induction of HO-1 gene expression by BE occurred in parallel with ERK (but not JNK or p38) protein phosphorylation and a slight but significant increase in
(but not SB203580 or SP600125), and a chemical antioxidant NAC, (N-acetyl cysteine), attenuated BE-induced HO-1 gene expression. Interestingly, BE but not BI prevented cells from H2O2-induced apoptosis, which was reversed by adding the inhibitor, SnPP, or PD98059. BE also inhibited the occurrence of hypodiploid cells, PARP and pro-caspase 3 cleavage and caspase 3 activity. In addition, BE maintained the mitochronrial membrances potential by DiOC6 assay and prevented cytochrome c releasing from mitochrondrial to cytosol by H2O2 treatment. Similar results showing differential protective effects of quercetin and its glycosides, rutin and quercitrin on H2O2-induced apoptosis via alternative HO-1 gene expression were also identified. HO-1-overexpressing (HO-1/RAW264.7) cells exhibited less apoptosis induced by H2O2 compared with that in neo-control (neo-RAW264.7) cells by reducing the intracellular peroxide level. However, carbon monoxide (CO), but not Fe+2, Fe+3, biliverdin or bilirubin, inhibited LPS-induced iNOS and NO production and expressed cytoprotective effect with HO-1 induction. Results of the present study suggested that HO-1 induced by flavonoids exhibited protective effect on H2O2-induced cells death in macrophages, and the negative role of glycoside was explored.
