行政院國家科學委員會補助專題研究計畫成果報告

行政院國家科學委員會補助專題研究計畫成果報告

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Or oxylin A, wogonin, quer cetin 與 r utin 對抑制 LPS,

LPS/IFN- γ與 LTA 誘導之細胞激素，一氧化氮與 PGE2 產 生之研究

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計畫類別：▉個別型計畫 □整合型計畫 計畫編號：NSC90－2320－B－038－027－

執行期間：90 年 08 月 01 日至 91 年 07 月 31 日

計畫主持人：陳 彥 州 共同主持人：

本成果報告包括以下應繳交之附件：

□ 赴國外出差或研習心得報告一份

□ 赴大陸地區出差或研習心得報告一份

□ 出席國際學術會議心得報告及發表之論文各一份 國際合作研究計畫國外研究報告書一份

執行單位：

台北醫學大學生藥學研究所

中 華 民 國 91 年 8 月 16 日

中文摘要

本計劃探討 Oroxylin A, wogonin, quercetin 與 rutin 對 LPS, LPS/IFN-

γ

Abstr act

Flavonoids are distributed widely in the plants, however biological functions of them are still unclear. In the present study, in vitro and in vivo experiments were performed to demonstrate the inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide (NO) and prostaglandin E2 productions in RAW264.7 macrophages, primary peritoneal macrophages, and Balb/c mice, respectively. In vitro results showed that wogonin and quercetin dose dependently suppressed lipopolysaccharide-induced NO production in RAW 264.7 macrophages and primary peritoneal macrophages without a notable cytotoxic effect on both cells, associated with a decrease in inducible nitric oxide synthase (iNOS) protein expression in both cells. Rutin, only at 80

µM, showed the slight but obvious inhibitory effect

production was found. Decrease of iNOS protein, but not cyclooxygenase-2 protein, was detected in liver and lung specimens of lipopolysaccharide-treated Balb/c mice in the presence of rutin, wogonin and quercetin. In conclusion, data obtained both in vitro and in vivo suggest that wogonin and quercetin exert inhibitory activities on lipopolysaccharide-induced NO production through suppression of iNOS expression.

Key Wor ds: rutin, wogonin, quercetin, lipopolysaccharide, nitric oxide, prostaglandin E

緣由與目的

Nitric oxide (NO) as a messenger and effector molecule in a variety of tissues has been demonstrated in several studies (Palmer et al., 1988; Lowenstein et al., 1996). NO has been identified as a neurotransmitter in central nervous system and a potent vasorelaxant in physiologically regulating the blood pressure though modulating muscular tone (Hibbs et al., 1987; Moncada et al., 1992). NO has been defined as a deleterious molecule in the process of inflammation and sepsis (Wheeler and Bernard, 1999). Exposure to outer bacterial toxins such as lipopolysaccharide or lipoteicholic acid stimulated cellular inflammatory responses, and released some factors including NO, prostaglandin E₂, cytokines, tumor necrosis factor-α and eicosanoid mediators to promote inflammatory responses (Penglis et al., 2000; Yamashita et al., 2000). Therefore, agents blocked bacterial toxins-induced NO production might be beneficial in treatment of inflammatory responses. Production of NO was regulated by intracellular nitric oxide synthases, and thee types of nitric oxide synthases including endothelium NO synthase (eNOS), neural NO synthase (nNOS) and inducible NO synthase (iNOS) have been identified. After exposure to endogenous and exogenous stimulators such as bacterial toxin or viral infection, iNOS was able to be induced quantitatively in various

cells such as macrophages, smooth muscle cells and hepatocytes to trigger several deleterious cellular responses, and caused some diseases including inflammation, sepsis and stroke (Duval et al., 1996; Marletta, 1993; Nathan, 1992). Therefore, NO production induced by iNOS may reflect the degree of inflammation, and provides a measure to assess the effect of drugs on the inflammatory process.

Prostaglandin E2 is over-produced at inflammatory sites by activating inducible cyclooxygenase-2, an inducible enzyme in the conversion of arachidonic acid to prostaglandin H₂, prostacyclin and thomboxane A₂ (Fu et al., 1990; Picot et al., 1994). Elevation of cyclooxygenase-2 protein has been demonstrated to be associated with the occurrence of some human diseases such as colon carcinoma (Shao et al., 2000). Specific cyclooxygenase-2 inhibitors effectively attenuated the symptoms of inflammation and reduced cancer occurring rate (Levy, 1997; Wong et al., 1998). Therefore, there is a great interesting to study the effects and usefulness of drugs on cyclooxygenase-2.

Flavonoids were existed as either simple or complex glycosides in many plants (Kuhnau, 1976), and humans have been estimated to consume approximately 1 g flavonoids/day. Our and other previous studies demonstrated that flavonoids exhibit a wide variety of interesting biological activities such as antioxidant and free radical scavenging activities, inhibition of 12-ï-tetradecanoylphorbol 13-acetate-induced tumor promotion, and apoptosis-inducing activity (Mora et al., 1990; Chen et al., 1999). Although flavonoids have been studied extensively, application of flavonoids in treating human diseases is still uncommon because of their higher effective concentration and poor absorptive activity after intake orally. Rutin, wogonin, and quercetin are structurally related flavonoids and existed extensively in the diet.

Quercetin was derived from rutin through hydrolization by the glucosidase activity in the gastrointestinal tract (Manach et al., 1997), and showed a wide range of biological activities including inhibition of Na⁺/K⁺-ATPase, protein kinase C, tyrosine kinase… etc (Kang and Liang, 1997). Our recent studies demonstrated that quercetin and wogonin showed the significant inhibitory activities on lipopolysaccharide-induced NO production though blocking iNOS gene expressions in RAW264.7 macrophages (Chen et al., 2000; Chen et al., 2001a;

Chen et al., 2001b). However, in vivo action of these compounds on lipopolysaccharide-induced NO production was still undefined. A series of experiments using RAW264.7 macrophages, primary peritoneal macrophages and Balb/c mice to demonstrate the differential inhibitory activities of rutin, wogonin, and querecetin on lipopolysaccharide-induced NO and prostaglandinE₂ productions were involved in this study.

結果與討論

Inhibition of lipopolysacchar ide-Induced NO and prostaglandin E

Productions by r utin, wogonin, and quercetin in RAW 264.7 Macrophages

The chemical structures of rutin, wogonin, and quercetin are shown in Fig. 1. The effects of rutin, wogonin, and quercetin on lipopolysaccharide-induced NO production in RAW 264.7 macrophages were investigated by measuring the accumulated nitrite, estimated by the Griess reaction, in the culture medium. Rutin, wogonin, and quercetin at 80 µM did not interfere with the reaction between nitrite and Griess reagents (data not shown). Un-stimulated macrophages, after 12 h of incubation in culture medium, produced background levels of nitrite (Fig. 2).

When the cells were incubated with indicated compounds alone, the concentration of nitrite in the medium was maintained at a background level similar to that in the un-stimulated samples.

After treatment with lipopolysaccharide (50 ng/ml) for 12 h, nitrite concentrations in the medium increase remarkably by about 20 folds (~30 µM). When RAW 264.7 macrophages were treated with different concentrations of wogonin and quercetin together with lipopolysaccharide (50 ng/ml) for 12 h, significant concentration-dependent inhibition of nitrite production was detected (Fig. 2A). Examination of cytotoxicity of rutin, wogonin and quercetin in RAW 264.7 macrophages by thiazolyl blue (MTT) assay indicated that all thee

compounds, even at 80

µM, did not affect viability of RAW 264.7 cells (data not shown).

Therefore, inhibition of lipopolysaccharide-induced nitrite production by wogonin and quercetin was not the result of possible cytotoxic effect on these cells. On analysis of prostaglandin E2 production by ELISA, un-stimulated RAW 264.7 macrophages in culture medium for 12 h produced basal amount of prostaglandin E2 in the medium as a control. After treatment with lipopolysaccharide (50 ng/ml) for 12 h, the medium concentration of prostaglandin E₂ elevated significantly, compared with control group. This increase was inhibited by co-treatment of cells with different concentrations (40 and 80

µM) of wogonin

Inhibitor y activities of r utin, wogonin and quercetin on lipopolysacchar ide-induced NO and prostaglandin E

productions in thioglycollate-elicited per itoneal macrophages

Upon lipopolysaccharide (5

µg/ml) treatment for 12 h, nitrite production in medium was

Alter native inhibitor y effects of r utin, quercetin and wogonin on lipopolysacchar ide-induced iNOS and cyclooxygenase-2 proteins by wester n blotting

µg/ml for primary peritoneal macrophages) treatment for 12 h, iNOS and

µ

In vivo inhibitor y effects of r utin, wogonin, and quercetin on lipopolysacchar ide-induced NO and prostaglandin E

productions in Balb/c mice

In vitro data in RAW264.7 and primary peritoneal macrophages indicated that wogonin and quercetin are potent inhibitors on lipopolysaccharide-induced NO and prostaglandin E2

productions. Furthermore, in vivo experiments were performed in Balb/c mice. Fig. 5A showed time dependent induction of NO production in the serum of lipopolysaccharide (10 mg/kg, i.v.)-treated Balb/c mice, and the maximal induction of NO was detected at post-treatment for 24 h. Pretreatment of L-arginine analog L-NAME (4 mg/kg, i.v.) for 1 h inhibited lipopolysaccharide-induced NO production (Fig. 5B). The same animal model was used to study the in vivo effects of rutin, wogonin, and quercetin on lipopolysaccharide-induced NO production. Results of Fig. 6 appeared that intravenous injection of Balb/c mice with rutin, wogonin, and quercetin (6 mg/kg for rutin, 3 mg/kg for wogonin, and 3 mg/kg for quercetin, i.v.) with or without L-NAME (4 mg/kg, i.v.) for 1 h followed by lipopolysaccharide (10 mg/kg, i.v.) treatment was able to decrease

lipopolysaccharide-induced NO production in the serum. However, no significant inhibition of rutin, wogonin and quercetin on lipopolysaccharide-induced prostaglandin E2 production was found in vivo (Data not shown). And, L-NAME did not show the synergistic inhibitory effect on lipopolysaccharide-induced NO production with rutin, wogonin, and quercetin. Western blot was performed to identify the expressions of iNOS and cyclooxygenase-2 proteins in the liver and lung specimens of Balb/c mice under various treatments. Results of Fig. 7 showed that injection of lipopolysaccharide (10 mg/kg, i.v.) for 24 h induced iNOS and cyclooxygenase-2 proteins in the liver and lung specimens of Balb/c mice (n=3). And, pre-treatment of Balb/c mice with rutin, wogonin, and quercetin significantly inhibited lipopolysaccharide-induced iNOS protein expressions in the liver and lung specimens, however cyclooxygenase-2 protein remained unchanged in both tissues (n=3).

成果自評

此結果已發表於 European Journal of Pharmacology (2002)與 Biochemical Pharmacology (2001)。詳細內容請參閱 Eur J Pharmacol 2002(446):187-194.

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