| Abstract: | Quercetin是一種廣泛存在於蔬果中的類黃酮化物。許多體外實驗顯示，quercetin具有抗氧化及抗發炎等許多生理活性，因此能抑制香菸煙霧中的致癌物質單獨或是合併β-胡蘿蔔素所造成的傷害。然而，quercetin經由phaseⅡ酵素代謝後，是以conjugated metabolites型式而非其aglycone存在於血漿中，且體外研究觀察到的現象常未能真正反應其在體內的重要性。因此，本實驗預計以Balb/c小鼠及蒙古沙鼠做為實驗模式，分為兩部分進行體內試驗，探討quercetin在體內的抗氧化及抗發炎效應。
第一部分
我們研究不同濃度的quercetin（50 mg/kg body wt (LQ)、500 mg/kg body wt (HQ)）對Benzo[a]pyrene (Bap)合併4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)所誘發Balb/c小鼠體內氧化壓力及促發炎激素分泌的影響。動物隨機分為六組（6隻/組）：（1）控制組（2）LQ（3）HQ（4）Bap+NNK（5）Bap+NNK+LQ（6）Bap+NNK+HQ，共為期三個月。實驗期間，quercetin經由管灌餵食，每週二次，Bap（5 mg/kg body wt）及NNK（4 mg/kg body wt）經由腹腔注射每週一次。結果顯示，quercetin管餵1-2小時後，Balb/c小鼠血漿中quercetin glucuronide及sulfate結合的代謝產物總濃度達到最高，LQ及HQ組分別約為2.3及4 μM。LQ顯著地抑制Bap合併NNK造成的內生性DNA傷害; LQ和HQ也能顯著降低Bap合併Bap+NNK+LQ/HQ也顯著的低於Bap合併NNK處理組。且Bap合併NNK顯著地增加肺灌流液及肝臟中TNF-α及IL-1β的分泌， quercetin處理則以劑量依賴效應明顯地降低Bap合併NNK所造成的影響。西方墨點法顯示quercetin抑制JNK及c-Jun蛋白的磷酸化。以上結果顯示，補充quercetin能降低 Bap合併NNK所造成Balb/c小鼠體內的促氧化及促發炎效應。
第二部分
此部分我們探討quercetin是否降低β-胡蘿蔔素在Bap誘發下對於蒙古沙鼠體內的傷害效應。同時我們也將比較quercetin與維生素C合併維生素E（C+E）的效果。實驗期間，沙鼠以管灌單獨餵食β-胡蘿蔔素（10 mg/kg body wt），或是合併quercetin（50 mg/kg body wt (LQ)或100 mg/kg body wt (HQ)）或是合併維生素C（13 mg/kg body wt）+維生素E（92 mg/kg body wt），每週三次，為期六個月。實驗前二個月期間，除控制組以外，其餘組別每週一次經由氣管滴入（intratracheal instillation）Bap。結果顯示，Bap處理組顯著地增加白血球內生性DNA傷害、H2O2-lesion及血漿中TBARs濃度。BC處理明顯地促進Bap所誘發的H2O2-lesion，而LQ或HQ合併BC則明顯的抑制Bap所造成的氧化傷害。Bap處理組其肺部總抗氧化力、穀胱甘含量及超氧歧化、觸及穀胱甘過氧化酵素活性顯著低於控制組。與Bap處理組相比，除超氧歧化外，BC增加了上述測定值的量或活性， LQ合併BC處理組的效應則多與BC組相似，HQ處理組大多顯著地較BC單獨處理組增加，且效果與C+E相似。在發炎指標方面，Bap處理增加了血漿或肺灌流液中IL-6 、IL-1β及TNF-α的分泌，BC則有促進Bap的促發炎激素分泌的趨勢，然而LQ/HQ合併BC處理則具抑制Bap影響的趨勢。與LQ/HQ合併BC組相較下，維生素C合併維生素E的服用在抗發炎的效果與quercetin相似。綜合以上結果顯示，quercetin經由體內代謝後的產物能與β-胡蘿蔔素交互作用，而quercetin的此一效果可能與其代謝產物的抗氧化及抗發炎效果有關。而其確實的機制仍需要進一步的研究。
Quercetin, a flavonoid, is found ubiquitously in vegetables and fruits. Several in vitro studies have shown that quercetin suppresses the harmful effects of cigarette smoking-associated carcinogens alone or in combination with β-carotene through several mechanisms, including antioxidative and anti-inflammatory activity. However, quercetin conjugated metabolites rather than quercetin aglycone is present in human plasma because of its efficient phase II metabolism. Whether those effects of quercetin observed in vitro are significant in vivo is unclear. Therefore, using Balb/c mice and Mongolian gerbils, the thesis work was divided into two parts to study the influence of quercetin in vivo.
Part I
We investigated the preventive effects of quercetin at 50 mg/kg wt body (LQ) or 500 mg/kg body wt (HQ) against benzo[a]pyrene (Bap)+ 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced oxidative stress and the secretion of proinflammatory cytokines in Balb/c mice. The animals were randomly assigned to following six groups (n = 6/group): (1)control, (2) LQ, (3) HQ, (4) Bap+NNK, (5) Bap+NNK+LO, (6) Bap+NNK+HQ. Quercetin was given to animals by gavage 2 times/week for 3 months; whereas Bap+ NNK exposure was performed by intraperitoneal injection 1 time/week. HPLC assay showed that at 1-2 h after oral administration of quercetin, the sum concentration of plasma glucuronidated and sulfated quercetin reached the maximum (about 2.3 and 4 μM at LQ, and HQ group, respectively). LQ significantly reduced Bap+NNK-induced endogenous DNA damage, whereas two doses of qeurcetin significantly reduced Bap+NNK -increased H2O2-lesion in a dose-dependent manner. The TBARs levels in Bap+NNK+LQ and +HQ group were also significantly lower than that of Bap+NNK. In addition, Bap+NNK significantly increased the levels of TNF-α and IL-1βin bronchoalveolar lavage fluid and liver, while quercetin significantly decreased the effects of Bap+NNK in a dose-dependent manner. Such effects of quercetin may be associated with the regulation of c-Jun and JNK activation, because quercetin also decreased the phosphorylation of c-Jun and JNK induced by Bap+NNK. These results suggest that quercetin attenuates the pro-oxidative and pro-inflammatory effects of Bap+NNK in Balb/c mice.
Part II
In this part, we investigated whether quercetin decrease the effects ofβ-carotene induced by Bap in Mongolian gerbils (gerbils). We also compared the effects of quercetin with that of vitamin C (C)+vitamin E (E). The gerbils were given β-carotene (BC; 10 mg/kg body wt) alone or in comebination with quercetin (50 mg/kg body wt for LQ, or 100 mg/kg body wt for HQ) or vitamin C (13 mg/kg body wt) +vitamin E(92 mg/kg body wt) by gavage 3 times/week for 6 months. At the firs 2 months, expect for control group, the gerbils were exposed to Bap by intratracheal instillation 1 time/week. As expected, Bap exposure significantly increased endogenous DNA damage and H2O2-lesion of while blood cells and TBRAs concentration in plasma. BC enhanced the effect of Bap on increase of H2O2-lesion, while LQ/HQ+BC significantly suppressed the effect of Bap mentioned above. The total antioxidant activity, the level of glutathione and the activity of superoxide dimutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in lungs of gerbils in Bap-exposed group were significantly lower than those of control group. Compared with Bap exposure group, BC increased the levels or activity of the parameters mentioned above except for SOD; the effects of LQ/HQ+BC were better than or similar to that of BC alone in these parameters. LQ/HQ+BC also significantly increased the activity of SOD. In addition, Bap exposure significantly or slightly increased the concentration of IL-6, IL-1β and TNF-α in plasma or bronchoalveolar lavage fluid, respectively. BC tended to enhance such effects of Bap while LQ/HQ+BC tended to decreased the effects of Bap. Compared with BC+C+E, LQ/HQ+BC had similar effects on suppressing the proinflammatory effect of BC. These results demonstrated that supplemental quercetin interacts withβ-carotene in vivo. The antioxidative and anti-inflammatory effects of quercetin metabolites may be contributed to such effect of quercetin. Further studies are warranted to investigate the precise mechanisms underlying these effects of quercetin in vivo. |
