Andrographolide在血管平滑肌細胞中誘導細胞凋亡與抗發炎作用之分子機轉探討

Molecular mechanisms of andrographolide-induced apoptosis and anti-inflammatory effect in vascular smooth muscle cells

陳右穎(Yu-Ying Chen)

指導教授 ： 許準榕

臺北醫學大學/醫學院/醫學科學研究所/博士(2014年)

摘要

在心血管發炎疾病中，不正常的血管平滑肌細胞增生(VSMCs)常被視為一個重要的病理因素。而在本實驗室之前的研究中亦發現，一個新穎的NF-κB抑制劑andrographolide可以藉由調控蛋白質磷酸酶2A (PP2A)使得NF-κB去磷酸化，進而達到抗發炎的作用。在本篇研究中，將分為兩部分：(I) 探討andrographolide誘導大鼠血管平滑肌細胞凋亡的詳細機轉；(II) 研究andrographolide應用於腫瘤壞死因子(TNF-α)活化血管平滑肌細胞的發炎反應與分子機轉。
(I) 轉染pp2a小片段干擾RNA (siRNA)後，發現可以抑制andrographolide所誘導的血管平滑肌細胞p38MAPK、p53磷酸化與後續的caspase-3活化程度；加入Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1)抑制劑SSG或shp-1 siRNA亦可以觀察到降低andrographolide刺激血管平滑肌細胞SHP-1磷酸化與後續所誘使的PP2A去磷酸化。此外，當給予NAC (ROS清除劑)或diphenylene iodonium (Nox抑制劑)可使andrographolide誘導血管平滑肌細胞的p53、Bax、caspase-3活化反轉；並且發現3-OMS (中性神經鞘磷脂酶抑制劑)可有效的減弱andrographolide所誘導的血管平滑肌細胞p47phox (Nox次單元蛋白)磷酸化與後續的Bax、caspase-3活化程度。(II) 給予andrographolide可濃度相關性抑制TNF-α刺激血管平滑肌的iNOS蛋白表現，同時也觀察到可降低JNK、Akt、p65磷酸化。而當給予LY294002 (PI3K/Akt抑制劑)或SP600125 (JNK抑制劑)可顯著的反轉andrographolide抑制TNF-α刺激血管平滑肌的p65磷酸化。此外，兩種抑制劑皆可降低Akt磷酸化，LY294002卻並不能影響JNK磷酸化進程。
Andrographolide經由活化SHP-1-PP2A-p38MAPK-p53-Bax與ceramide-p47phox-ROS這兩條訊息傳遞路徑去誘使血管平滑肌細胞粒線體功能失常導致細胞凋亡；另一方面，藉由JNK-Akt-p65訊息機轉去調控血管平滑肌內的NF-κB活化發炎反應。綜合以上結果發現andrographoldie具有潛力開發作為心血管疾病之治療藥物。

關鍵字

血管平滑肌細胞 ； 細胞凋亡 ； 發炎反應 ； 蛋白磷酸酶2A ； SHP-1 ； ROS ； 腫瘤壞死因子

並列摘要

The abnormal growth of vascular smooth muscle cells (VSMCs) is considered a critical pathogenic process in inflammatory vascular diseases. We have previously demonstrated that protein phosphatase 2 A (PP2A)-mediated NF-κB dephosphorylation contributes to the anti-inflammatory properties of andrographolide, a novel NF-κB inhibitor. In this study, (I) we investigated whether andrographolide causes apoptosis, and characterized its apoptotic mechanisms in rat VSMCs. On the other hand, (II) we found the mechanisms of the inhibitory effects of andrographolide in VSMCs exposed to a pro-inflammatory stimulus, tumor necrosis factor-α (TNF-α).
(I) Transfection with pp2a small interfering RNA (siRNA) suppressed andrographolide-induced p38MAPK activation, p53 phosphorylation, and caspase 3 activation. Andrographolide also activated the Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1), and induced PP2A dephosphorylation, both of which were inhibited by the SHP-1 inhibitor sodium stibogluconate (SSG) or shp-1 siRNA. Furthermore, andrographolide induced reactive oxygen species (ROS) formation, p53 activation, Bax and active caspase-3 expression, and these phenomena were suppressed by pretreating the cells with NAC, a ROS scavenger, or diphenylene iodonium, a NADPH oxidase (Nox) inhibitor. However, pretreatment with 3-O-methyl-sphingomyeline, a neutral sphingomyelinase inhibitor, significantly inhibited andrographolide-induced p47phox (Nox subunit protein) phosphorylation as well as Bax and active caspase-3 expression. (II) Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced JNK, Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation.
Andrographolide activates the SHP-1-PP2A-p38MAPK-p53-Bax and ceramide-p47phox-ROS signaling cascades, causing mitochondrial dysfunction and VSMC death. Furthermore, andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases.

並列關鍵字

VSMC ； apoptosis ； inflammation ； PP2A ； SHP-1 ； ROS ； TNF-α

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