以順鉑誘導的小鼠急性腎損傷模式探討高良薑素的保護機制

順鉑(cisplatin)為常用於治療腫瘤的化學藥劑，臨床上主要的限制原因，為使用cisplatin後有30%的病人有腎臟損傷的副作用出現，近年來許多研究都在尋找可以減緩此副作用的化合物，提供病人更多選擇性。在本實驗室發現高良薑素(Galangin)於誘導細胞發炎實驗中發現有抗發炎的功效；動物實驗中發現，以propacetamol誘導肝臟腎臟損傷，有增加抗氧化能力進而減少肝腎損傷的功效，所以我們將探討高良薑素處理後是否可以減緩cisplatin所造成的腎臟損傷。實驗設計主要分成三部分，第一部分我們先單純以高良薑素處理，探討三種劑量(50、 75、 100 mg/kg)對小鼠腎臟是否有毒性，經實驗結果我們得知不同劑量的高良薑素對小鼠的腎臟並無明顯毒性；第二部分我們將探討高良薑素處理後是否能減緩cisplatin造成的腎臟損傷，發現不同濃度的高良薑素處理後會減緩cisplatin所造成的體重變輕、腎臟腫脹、腎臟生化指標(Blood urea nitrogen, BUN、Creatinine, CREA)升高和腎臟組織的病變；第三部分我們以75 mg/kg高良薑素處理，探討高良薑素是透過那些機制來減緩cisplatin所造成的腎損傷；在抗氧化能力實驗中，我們發現以高良薑素處理(75 mg/kg)後會透過提升nuclear factor erythroid 2–related factor 2 (Nrf-2)表現，進而增加抗氧化物質穀胱甘肽(Glutathione, GSH)和抗氧化酵素穀胱甘肽過氧化物酶(Glutathione peroxidase, GPx)、過氧化氫酶(Catalase)的表現，中和cisplatin所產生的氧化壓力；在發炎反應實驗中，我們發現以高良薑素處理後會減少腫瘤壞死因子(Tumor Necrosis Factor-α, TNF-α)、白細胞介素1β(Interleukin 1 β, IL-1 β)和白細胞介素6(Interleukin 6, IL-6)的表現，進而減少發炎反應的產生；在細胞凋亡實驗中，高良薑素處理後會減緩ERK的磷酸化，進而降低促凋亡蛋白腫瘤蛋白p53 (Tumor protein p53, p53)表現，回升抑凋亡蛋白B細胞淋巴瘤基因(B cell lymphoma 2, Bcl-2)表現，透過降低Bax/Bcl-2的比值，降低腎臟細胞凋亡的比例。綜合抗氧化能力、發炎反應和細胞凋亡的實驗，我們發現高良薑素會透過Nrf2 路徑活化抗氧化酵素，中和氧化壓力，經由降低ERK磷酸化及P53表現，減少細胞的凋亡，降低主要的發炎因子TNF-α及其它發炎因子IL-1 β及IL-6的表現，來降低發炎反應，所以我們發現高良薑素有開發成保護腎臟藥物或保健食品的潛力。

Cisplatin (C) is an effective clinical used anti-neoplastic drug, but its major side effect, nephrotoxicity, limits its usage. A new compound which can ameliorate this side effect but do not affect its anti-neoplastic activity will show a great clinical benefit. In previous tests, galangin (G) was found to have anti-oxidative and anti-inflammatory effects in cellular and mouse experiments, and could ameliorate propacetamol-induced liver and kidney injuries. The aim of this study is to investigate the protective effects and mechanisms of G against C-induced kidney injury in mice. The experiments are divided into three parts. Part one: to test whether different dosages of G show nephrotoxicity in mice. Administrations of different concentrations (50、75、100 mg/kg) of G did not affect body weight and induce kidney injury in mice. Part two: the administration of different concentrations of G (50、75、100 mg/kg) ameliorate C-induced nephrotoxicity or not. Our finding suggest that G decrease C-induced nephrotoxicity. Furthermore, we chose the middle dose of 75 mg/kg for subsequent analysis. Part three: the possible mechanisms of G against a mouse model of cisplatin-induced renal injury. Pretreatment of G decreased the oxidative stress induced by C by increasing nuclear translocation of Nrf-2, restoring the major antioxidant, glutathione, and increasing glutathione peroxidase and catalase activities. Pretreatment of G also inhibited the cisplatin-induced expressions of inflammatory cytokines TNF-α, IL-1β and IL-6. Moreover, pretreatment of G inhibited cisplatin-induced apoptosis by suppressing cisplatin-induced p53 and p-ERK expressions and also Bax/Bcl-2 imbalance. Our findings suggest that pretreatment of G alleviates the C-induced nephrotoxicity through up-regulation of antioxidative mechanisms and down-regulations of proteins involved in the inflammation and apoptosis. Thus G might be clinically used with C to ameliorate the nephrotoxicity of C.