脆葉馬蘭水萃液抑制子宮肌瘤之分子機轉研究

子宮肌瘤是一種良性的腫瘤，也是女性在生育年齡中最好發的婦科疾病。根據研究，美國有70-80%的女性曾經罹患子宮肌瘤，而衛福部的資料顯示2018年在台灣有超過36萬人被診斷為此疾病。子宮肌瘤的症狀包括經期異常出血、子宮壓力升高、下背痛、甚至是胚胎著床不易。 脆葉馬蘭(Strobilanthes crispus)是一種原生於馬達加斯加的植物，也被稱為黑面將軍，在新馬地區的傳統醫學上使用於癌症、糖尿病及子宮肌瘤的治療。某些研究指出脆葉馬蘭的萃取物可以抑制癌細胞增生、抗氧化及抑制血管增生，但目前沒有研究分析脆葉馬蘭萃取物對於子宮肌瘤的分子機轉。首先，本研究發現脆葉馬蘭水萃液可以抑制大鼠子宮肌瘤細胞株ELT3-Luc的生長，並且可以造成細胞自噬的標誌蛋白LC3-II以及ATG5-ATG12複合體的增加；反之，以氯喹(chloroquine)抑制細胞自噬作用則可以部份回復脆葉馬蘭水萃液的抑制效果。我們進一步發現脆葉馬蘭水萃液會增加細胞內活性氧化物質的產生，而彗星試驗則證明細胞DNA損傷的發生；此外，以N-acetyl-L-cysteine抑制細胞內活性氧物質產生則可完全消除脆葉馬蘭水萃液對細胞的生長抑制以及DNA損傷的產生，顯示脆葉馬蘭水萃液可誘導活性氧物質進而造成子宮肌瘤細胞產生DNA損傷。接著我們使用流式細胞儀分析細胞週期，發現脆葉馬蘭水萃液會造成細胞濃度依賴性地累積於G1期，同時也確認了細胞週期G1檢查點蛋白cyclin D1/CDK6的表現量會在處理後下降。為了確認脆葉馬蘭水萃液藉由何種機制導致G1期停滯，我們使用了西方點墨法分析了細胞經過治療後的蛋白表現，發現ATM、IGF1R、以及Akt的磷酸化形式蛋白表現量都會下降。而IGF1R以及ATM的抑制劑也分別能夠抑制p-Akt/cyclin D1/CDK6 的表現；我們更進一步發現ATM的抑制劑ku-55933也同樣會導致細胞的G1期停滯。總結本研究結果，我們確認了脆葉馬蘭水萃液會藉由抑制p-ATM/p-IGF1R/p-Akt的路徑導致cyclin D1及CDK6的表現下降，進而誘導大鼠子宮肌瘤細胞株ELT3-Luc產生G1期停滯；脆葉馬蘭水萃液也會誘導細胞內活性氧化物質的產生導致細胞DNA損傷。未來本研究將以氣相層析質譜儀鑑定脆葉馬蘭水萃液的有效成分，以期能進而開發以純化之脆葉馬蘭植化素作為子宮肌瘤之單方治療藥物。

Uterine fibroids (UF), the benign tumors of the uterus, are one of the most common diseases among women in the 40s and early 50s. UF may lead to heavy bleeding, pelvic pressure, pain, or even reproductive dysfunction. The percentage of women who suffered from UF is up to 70-80% in the USA. Moreover, there are over 360,000 women diagnosed as uterine fibroids in Taiwan in 2018. Strobilanthes crispus (also called black face general, BFG) is a traditional medication in Singapore and Malaysia. It's used for the treatment of tumor, diabetic, and UFs. Several studies have confirmed that the extraction of BFG displays anti-cancer, anti-oxidant, and anti-angiogenic activities. However, how BFG extraction influences UFs is yet to be elucidated. We firstly found that the BFG water extract inhibited the proliferation of a rat UF cell line, ELT3-Luc. The autophagy marker, LC3-II, and the autophagosome elongation factor, ATG5-ATG12 complex, were increased. Treatment of chloroquine, the autophagy inhibitor, partially recovered the inhibitory effect of BFG extract. Furthermore, the intracellular production of ROS was increased in BFG treated ELT3 cells, as well as the enhanced DNA damage was observed by comet assay. The treatment of N-acetyl-L-cysteine abolished the inhibitory effect of BFG in proliferation or the induction of DNA damage indicating that BFG extract could cause ROS mediated DNA damage. Cell cycle analysis by flow cytometry revealed that the treatment of BFG in ELT3 cells induced G1 arrest, as well as the reduction of cyclin D1 and CDK6 expression, in a dose-dependent manner. The downregulation of p-ATM, p-IGF1R, and p-Akt proteins in ELT3 cells was also observed in BFG-treated ELT3 cells. In addition, the treatment of ku-55933, an ATM inhibitor, caused G1 arrest at the similar level of BFG treatment. In conclusion, we demonstrate that BFG water extracts display a growth inhibition effect to a rat UF cell line, which is associated with the induction of G1 arrest through the downregulation of p-ATM and the ROS mediated DNA damage. In the future, we will further identify the functional component of BFG extract in the UF inhibition by Gas chromatography–mass spectrometry, which may lead to the development of phytochemicals for UF treatment.