活體組織經歷缺血-再灌流傷害時，組織常會產生大量的自由基和發炎等反應，讓損害比灌流前更加嚴重，因此發展有效降低血液再灌流傷害的方法，是臨床醫學上重要的工作。過去研究顯示銀杏萃取物EGb761具有抗氧化、抗發炎及清除自由基的能力，本研究將測試EGb761是否有成為缺血-再灌流傷害保護藥物之潛力。體外實驗上利用ABTS和DPPH先行測試EGb761清除自由基的能力，以及利用Mouse Macrophage細胞株RAW 264.7進行細胞毒性試驗，並取得MDA和MPO之IC50。加LPS誘導後收集細胞培養液測定NO (16hr)、IL-6 (6hr)、TNF-α (6hr) 的濃度，來觀察自由機方面反應的機制。在動物實驗上，30分鐘前於靜脈注射，給予大鼠不同劑量的EGb761，造成缺血2小時候，鬆開止血帶形成再灌流傷害。在缺血前與再灌流後24小時分別測量血漿NO濃度；並取左右腿的Skeletal muscle切片分別做H&E染色，和MPO與Macrophage的IHC染色；測定GSH level、MDA lelve和MPO活性。結果顯示於體外試驗中，事前給予EGb761可以降低LPS所誘導的發炎反應。在體內中，事前給予EGb761依濃度由低至高，對於缺血-再灌流引發大鼠後肢Skeletal muscle的傷害可能藉由EGb761抗氧化、抗發炎和清除自由基的能力，有減輕Skeletal muscle組織的受傷的效果。 Ischemia-reperfusion injury could causes free radical attacks and localized inflammatory reactions in tissue environment. The EGb761, extracted from the Ginkgo biloba tree, is effective in anti-inflammation, anti-oxidation and free radical scavenger. We would like to clarify whether EGb761 have the potential to help ischemia-reperfusion injury. The free radical cleaning ability was test by ABTS test and DPPH test. Cytotoxicity and the IC50 of ABTS, DPPH, MDA and MPO in mouse macrophage cell line RAW 264.7 are measured. NO, IL-6, and TNF-α in culture medium that induced by LPS was also evaluated. In vivo, the EGb761 was injected by i.v. before tourniquet properly tied limbs of rat for 30min, after 2-hours ischemia, loosen the tourniquet to reperfusion 24-hours. Plasma NO concentration before ischemia and after reperfusion was measured. H&E stain, IHC stain (Antibody: MPO, Macrophage), GSH level, MDA level, and MPO activity was also evaluated in skeletal muscles. Our results suggest that EGB761 could reduce inflammation effect induced by LPS in vitro. Treatment of EGB761 before ischemia could reduce macrophage accumulation and tissue damage. In conclusion, EGb761 shows great potential for anti-inflammatory, and anti-oxidation in ischemia-reperfusion injury skeletal muscles.
