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

林慧宜(Hui-Yi Lin)

指導教授 ： 陳彥州

臺北醫學大學/藥學院/藥學研究所/博士(2004年)

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。

關鍵字

血紅素氧化酵素-1 ； 類黃酮素 ； 一氧化氮 ； 細胞保護作用

並列摘要

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 intracellular peroxide levels by DCHF-DA assay. A specific ERK inhibitor, PD98059 (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.

並列關鍵字

Heme Oxygenase 1 ； Flavonoids ； Nitric Oxide ； Cytoprotection

參考文獻

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