穿心蓮內酯抗發炎和解毒效應研究

Study the antiinflammatory and detoxification effects of andrographolide

呂佳陽(Chia-Yang Lu)

指導教授 ： 劉凱莉

中山醫學大學/健康管理學院/營養學研究所/博士(2013年)

https://doi.org/10.6834/CSMU.2013.00074

摘要

根據行政院衛生署所公布101年國人十大死因統計，癌症、心血管疾病、慢性肝病及肝硬化仍是造成國人死亡的主要疾病。近年來，食療和保健養生觀念日盛，食物及中草藥中各種植化素(phytochemicals)所具有的生理功能也愈發被重視。穿心蓮(Andrographis paniculata)是東南亞國家常用的傳統中草藥，被用於治療感冒、咽喉腫痛、發燒、感染和腹瀉。本草綱目紀載穿心蓮性寒味苦、具清熱解毒、消腫止痛等功效。穿心蓮內酯(andrographolide)是穿心蓮中最具生理活性的雙萜類之一。研究證實，穿心蓮內酯具有抗發炎、抗氧化和抗肝毒性等作用，本研究將針對穿心蓮內酯抗肝毒性及抗發炎能力做進一步探討。

Pi屬麩胱甘肽硫轉移酶(the pi class of glutathione S-transferase, GSTP)為Phase II生物轉換系統中重要成員之一，具有解毒能力。先前研究證實，穿心蓮內酯可以上調大鼠初代肝細胞GSTP表現，但其調控機制尚未完全釐清。因此，論文第一部分探討穿心蓮內酯透過何種訊息路徑上調大鼠初代肝細胞GSTP表現。實驗將穿心蓮內酯溶於dimethyl sulfoxide (DMSO)，以大鼠初代肝細胞為實驗模式(DMSO於培養液中最終濃度為0.1%)，利用西方墨點法、siRNA、免疫沉澱法和electrophoretic mobility shift assay (EMSA)進行實驗。結果顯示，處理40 μM穿心蓮內酯引發最大量Akt磷酸化發生在30分鐘，最大量c-jun磷酸化發生在3小時，細胞預處理20 μM PI3K抑制劑(wortmannin和LY294002) 1小時或siPI3K抑制穿心蓮內酯磷酸化c-jun及GSTP蛋白質表現，EMSA結果發現wortmannin和LY294002預處理或siPI3K可以減弱穿心蓮內酯誘發AP-1與DNA結合；進一步搭配免疫沉澱結果顯示，穿心蓮內酯增加細胞核內c-fos和c-jun二聚體形成，但加入c-jun或c-fos抗體則會減弱核蛋白與DNA序列結合。因此，結論是穿心蓮內酯可能透過活化PI3K/Akt路徑，磷酸化c-jun，增加細胞核內AP-1含量及其與基因啟動子序列結合，進而向上調控GSTP基因轉錄。

氧化壓力被認為是造成老化、發炎、粥狀動脈硬化、癌症和糖尿病的主要危險因子之一。NADPH oxidase是細胞內活性氧分子(reactive oxygen species, ROS)的重要來源。論文第二部份利用EA.hy926 endothelial-like cells為實驗模式，透過西方墨點法、膜蛋白分離法、real-time PCR、shRNA、胞內ROS含量測定、胞內GSH含量測定及單核球黏附試驗，探討穿心蓮內酯是否減緩腫瘤壞死因子α (TNFα)誘發細胞氧化壓力，進而抑制發炎反應。結果顯示，1 ng/ml TNFα處理細胞5分鐘後磷酸化Src且在15分鐘後活化NADPH oxidase次單位p47phox和p67phox，造成這兩個次單位轉移到細胞膜，1 ng/ml TNFα處理細胞6小時後能提升ICAM-1表現；細胞預處理7.5 μM穿心蓮內酯16小時抑制TNFα誘發Src磷酸化；細胞預處理7.5 μM穿心蓮內酯16小時或NADPH oxidase抑制劑(2 mM apocynin, APO和5 μM diphenyleneiodonium, DPI) 1小時，抑制TNFα誘發p47phox和p67phox膜轉移及ICAM-1表現。利用shp47phox靜默p47phox表現，顯示shp47phox抑制TNFα誘發p65核轉移、ICAM-1基因表現及HL-60細胞黏附作用。7.5 μM穿心蓮內酯處理細胞24小時誘發血基質氧化酶(heme oxygenase 1, HO-1)和麩胺酸-半胱胺酸連接酶修飾次單位(glutamate cysteine ligase modifier subunit, GCLM)基因表現，誘發屬於時間依賴型，7.5 μM穿心蓮內酯同時也增加胞內GSH濃度且降低TNFα誘發ROS生成。利用shGCLM抑制穿心蓮內酯誘發GCLM表現和減少胞內GSH含量逆轉穿心蓮內酯抑制TNFα誘發HL-60細胞黏附作用；作用類似shGCLM，shHO-1也會部分逆轉穿心蓮內酯抑制TNFα誘發HL-60細胞黏附作用。細胞處理7.5 μM穿心蓮內酯2小時磷酸化ERK及Akt；細胞預處理20 μM ERK抑制劑(PD98059)和PI3K抑制劑(LY294002) 1小時，再處理7.5 μM穿心蓮內酯16小時，結果顯示LY294002抑制穿心蓮內酯誘發HO-1和GCLM表現及增加胞內GSH含量；PD98059卻無類似效果，證實穿心蓮內酯透過活化PI3K/Akt路徑誘發HO-1及GCLM表現。7.5 μM穿心蓮內酯處理細胞2小時促進Nrf2核轉移及c-jun磷酸化，預處理LY294002抑制穿心蓮內酯誘發Nrf2核轉移和c-jun磷酸化；利用shNrf2及shJUN靜默Nrf2和c-jun表現減少穿心蓮內酯誘發HO-1和GCLM基因表現。綜合上述，穿心蓮內酯抑制TNFα誘發發炎反應至少部分和它誘發胞內抗氧化酵素HO-1與GCLM基因表現，並抑制NADPH oxidase活化、減少胞內ROS生成有關。穿心蓮內酯是否透過其他作用抑制TNFα誘發發炎反應，則有待進一步釐清。

結論，穿心蓮內酯誘發GSTP、HO-1和GCLM基因表現和活化PI3K/Akt/AP-1和PI3K/Akt/Nrf2路徑有關；穿心蓮內酯也會透過減弱TNFα誘發Src磷酸化及p47phox和p67phox膜轉移，抑制ICAM-1表現及HL-60黏附作用。綜合以上研究，穿心蓮內酯透過上調初代肝細胞GSTP表現，抑制內皮細胞NADPH oxidase活化及上調HO-1和GCLM表現來達到解毒、抗氧化及抗發炎作用。

關鍵字

穿心蓮內酯 ； pi屬麩胱甘肽硫轉移酶解毒 ； 抗發炎 ； 抗氧化

並列摘要

Department of Health, Executive Yuan, R.O.C. (TAIWAN) reported that cancer, cardiovascular disease, chronic liver disease and liver cirrhosis are among the top 10 death causes in 2012. In recent years, various phytochemicals from foods and herbs exhibited different biological properties via regulation of gene expression. Andrographis paniculata (A. paniculata) is a traditional medicinal herb which is widely used in many Asia countries. Andrographolide, a diterpenoid, is the major bioactive component of A. paniculata, which has been demonstrated to have various biological actions including anti-diabetes, anti-inflammation, antioxidation, and anti-hepatotoxicity. In this study, we investigated the effect of andrographolide on detoxification and anti-inflammation.

The pi class of glutathione S-transferase (GSTP) is one of the phase II biotransformation enzymes. Our previous study showed that andrographolide upregulates the expression of GSTP. The aim of the first study was to investigate the mechanism by which andrographolide induces GSTP gene expression in rat primary hepatocytes. Andrographolide was dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO in medium is 0.1%. Hepatocytes treated with 40 μM andrographolide had increased Akt phosphorylation at 0.5 h and c-jun phosphorylation at 3 h. However, pretreatment with 20 μM PI3K inhibitors, wortmannin and LY294002 for 1 h, or siPI3K abolished the andrographolide-induced phosphorylation of c-jun at 3 h and GSTP protein expression at 48 h. EMSA showed that pretreatment with wortmannin, LY294002, or siPI3K attenuated andrographolide-induced AP-1-DNA-binding activity. Results of immunoprecipitation indicated that andrographolide increased nuclear c-fos/c-jun heterodimer accumulation. Addition of antibodies against c-jun and c-fos to nuclear extract decreased nuclear protein binding to the AP-1 consensus of DNA sequence. In summary, andrographolide induction of GSTP expression in rat primary hepatocytes is through activation of the PI3K/Akt, phosphorylation of c-jun, nuclear accumulation of AP-1, and subsequent binding to the response element in the target gene.

Oxidative stress is considered to be a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is one of the major sources of endogenous reactive oxygen species (ROS). In the second study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. EA.hy926 cells were pretreated with 7.5 μM andrographolide [1:1000 (vol/vol) DMSO/DMEM] for 16 h and then challenged with 1 ng/ml TNFα for an additional 5 min to 6 h. Andrographolide attenuated TNFα-induced ROS generation at 20 min, Src phosphorylation at 5 min, and NADPH oxidase subunits, p47phox and p67phox, membrane translocation at 15 min as well as ICAM-1 gene expression at 6 h. In the small hairpin RNA interference assay, shp47phox abolished TNFα-induced p65 nuclear translocation at 1 h, ICAM-1 gene expression and adhesion of HL-60 cells at 6 h. Moreover, pretreatment with the NADPH oxidase inhibitors, 2 mM apocynin (APO) and 5 μM diphenyleneiodonium (DPI), for 1 h attenuated TNFα-induced p47phox and p67phox membrane translocation and ICAM-1 gene expression. Andrographolide induced heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) gene expression in a time-dependent manner. Cellular GSH content was increased by andrographolide after 24 h treatment. shGCLM attenuated andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed the similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. Pretreatment with the 20 μM PI3K inhibitor, LY294002, for 1 h attenuated andrographolide-induced HO-1 and GCLM gene expression and cellular GSH content, but MEK inhibitor, PD98059, had no effect on andrographolide-induced HO-1 and GCLM gene expression. Moreover, pretreatment with LY294002 attenuated andrographolide-induced Nrf2 nuclear translocation and c-jun phosphorylation at 2 h. shNrf2 and shJUN decreased andrographolide-induced HO-1 and GCLM gene expression. The mechanism underlying up-regulation of HO-1 and GCLM by andrographolide was dependent on PI3K/Akt pathway, and both Nrf2 and AP-1 transcriptional factors were involved. Taken together, our results suggest that andrographolide attenuates TNFα-induced inflammation at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM gene expression, which is PI3K/Akt pathway-dependent.

In summary, we suggested that andrographolide has detoxification, antioxidant and anti-inflammatory activities not only through the induction of GSTP in rat primary hepatocytes and HO-1 and GCLM in EA.hy926 cells but also through the suppression of TNFα-induced NADPH oxidase activation. Moreover, the induction of GSTP, HO-1 and GCLM is dependent on PI3K/Akt pathway and Nrf2 as well as AP-1 were demonstrated to be the transcriptional factors involved.

並列關鍵字

Andrographolide ； the pi class of glutathione S-transferase ； detoxification ； antiinflammation ； antioxidation

參考文獻

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