香椿之保肝、抗病毒、抗癌之藥理活性及 細胞內NO/cGMP路徑之調節作用

Hepatoprotective, Antiviral and Anticancer Effects of Toona sinensis Roem. and its regulation on intracellular NO/cGMP level.

林信伶(Hsin-Ling Lin)

指導教授 ： 邱慧芬

高雄醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

香椿為中國落葉喬木，本研究將針對香椿進行藥理活性之篩選，包括保肝、抗病毒及抗癌活性之篩選，並對腫瘤細胞生長抑制相關之程式死亡路徑，亦深入地探討對分子相關作用機轉。
在保肝活性之測定以四氯化碳(CCl4)在大白鼠誘發之急性肝炎投與不同劑量之香椿(0.001-10.0 mg/kg)後，發現香椿能隨劑量明顯地降低血清中SGOT及SGPT之值。
同時以光學顯鏡觀察肝臟切片組織變化，發現香椿隨劑量之增加可明顯改善肝炎現象，並與本研究室歷年來所做過數十種中草藥活性成分相比較，發現其具明顯保肝作用。
另外以2.2.15細胞測定香椿抑制B型肝炎病毒顆粒釋出之活性，發現隨著劑量之增加，對HBsAg及HBeAg含量均呈現正相遞減現象，且同劑量時對HBeAg之活性較為顯著。
另外，不同的香椿濃度對人類肝癌細胞(Hep G2)，以MTT及Methylene blue方法測定其對細胞生長之調節或毒殺作用，發現香椿對HepG2之生長有抑制作用，但以濃度1.0 mg/ml為分界，於更高濃度10.0 mg/ml時似有明顯回升現象。其50%抑制生長濃度(IC50)分別為0.517±0.01 mg/ml(MTT)及0.53±0.04 mg/ml (Methylene blue)。
香椿之抗癌活性與細胞週期之關係，則以流式細胞儀(Flow cytometer)測定，發現0.1 mg/ml時香椿即有明顯調節細胞週期作用，在24小時及48小時停滯在S期而在72小時為G1/G0期之停滯作用，隨時間有明顯不同之作用機轉。
香椿之凋亡則以入不同濃度(0.01-1.0 mg/ml)之香椿於HepG2細胞24、48及72小時後，經由原位DNA片段測定法(TUNEL)分析，結果可發現細胞凋亡的數目以0.1mg/ml較為顯著。
香椿對細胞膜上細胞凋亡因子如TNF-α及TNF-R1之調節活性測定，則以不同濃度的香椿(0.01-1.0 mg/ml) 加入HepG2細胞作用72小時後，測定發現香椿0.1 mg/ml對於人類肝癌細胞TNF-α及TNF-R1雖無明顯之作用，但在 1.0 mg/ml卻明顯增加至89.56%及89.32%。
一氧化氮(NO)與肝臟細胞之毒殺作用機轉可能密切相關，我們測定香椿(0.01-1.0 mg/ml)作用24小時後，發現NO之含量有增加現象，且在西方點墨法(Western blot)與免疫組織化學染色法(Immunocytochemistry)測定中亦觀察到一氧化氮合成酶(iNOS)活性之增加，證明NO 之增加可能是香椿抗肝癌活性中細胞凋亡活化機轉之一。
另外，在c-GMP測定結果則發現香椿(0.01-1.0 mg/ml)會隨濃度造成明顯細胞內c-GMP濃度下降之現象，因此NO增加之機轉可能不經由活化c-GMP而是藉由產生過氧自由基離子而發揮細胞毒殺之作用。除此之外，我們亦偵測造成細胞凋亡之下游物質caspase-3之活性，發現香椿具有造成caspase-3活性增加之作用。
本研究之結果顯示香椿為一具有潛力之保肝、抗病毒及抗肝癌新藥並對細胞內NO/cGMP濃度之調節及caspase-3之活化為抗癌活化之重要作用機轉。

關鍵字

cGMP ； 一氧化氮 ； 抗癌 ； 抗病毒 ； 保肝

並列摘要

Toona sinensis (TS) is a broadleaf tree. The leaves and young shoots have been used as a vegetable in China for thousands of years. The leaves and stems of this plant have been used for the treatment of enteritis, dysentery and itch in oriental medicine. Recently, aqueous leaf extracts of TS was used as a folk medicine for lowering blood sugar and blood pressure.
In present study, the leaves extracts of TS on CCl4-induced acute hepatotoxicity were invegtigated, we found TS possessed a significant dose-dependent decline effect on serum SGOT and SGPT levels promotion. Our data have indicated that HBV transfected human hepatoma cells (2.2.15 cells) cultured with different dose of TS (0.01-1.0 mg/ml) showed a significant effect on decreasing the secretion of HBsAg and HBeAg into culture medium of 2.2.15 cell.
We also observed that TS aquous extract (TS) possessed inhibitory effect on tumor cell proliferation (HepG2 cells). Our results have indicated the cytotoxic effect of leaf extracts on various cancer cell lines with MTT and methylene blue colormetric method. The IC50 of TS on hepatoma (HepG2 cells) are 0.53±0.04 mg/ml, 0.52±0.01 mg/ml, separately.
In situ detection of fragmented DNA (TUNEL assay) demonstrated that 0.1 mg/ml casused a more significant effect of HepG2 cells different dosage, TS (0.01-1.0 mg/ml) have in the increased of DNA fragmentation.
The apoptotic signal on membrane TNF-R1 and its ligand TNF-α both were augment by addiation of TS (0.01-1.0 mg/ml).
Nitric oxide (NO) is a muti-regulated molecule in hepatocyte, NO can inhibit the main mediators of cell death include caspase, protease. TS-induced the increase of the intracellular NO level and the when high NO concentration may lead to the formation of toxic reaction products like dinitrogen or peroxynitrite that consequment induced cell apoptosis.
Cyclin GMP produced in response to nitric oxide may regulate the celll proliferation and apoptosis by through phosphorylation of cGMP-dependent protein kinase and realated gene expression. TS decreased the intracellular c-GMP level may be through the inhibition of guanylate cyclase. Meanwhile, TS (0.1 mg/ml) also activated the apoptosis routes final stage effector caspase-3.
In conclusion, our data have indicated that TS is potential as a hepatoprotective, antiviral and antihepatoma new drug and the anticancer mechanism may be through the increase synthase of NO, activation of caspase-3 and decrease of the intracellular cGMP.

並列關鍵字

cGMP ； NO ； Anticancer ； Antiviral ； Hepatoprotective

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