A Dual-fluorescent Assay For Screening Active Compounds From Traditional Chinese Medicine Against Doxorubicin Toxicity

With the development of modern biotechnology, cell-based high-throughput and high-content screening are widely used in biomedical area. It also provides the new thoughts and methods for the discovery of active components of traditional Chinese medicine (TCM).Doxorubicin (DOX) has been widely used for the chemotherapy of solid tumors, leukemia, lymphoma, breast and other cancers. Unfortunately, the usage of DOX is hampered due to its severe cardiotoxicity (including acute myocardial injury in the form of acute tachyarrhythmia and acute heart failure) and chronic cardiotoxicity, which may result in cardiomyopathy even eventually severe heart failure and death. Multiple mechanisms of DOX-induced cardiotoxicity have been proposed, including the increase of cardiac oxidative stress, calcium overload, abnormalities in mitochondria, caspase activation induced apoptosis in myocardial cells, energy metabolism change and so on.Based on an automated cellular fluorescent imaging platform, the present study established a dual-fluorescent assay for screening active compounds against doxorubicin toxicity from traditional Chinese medicine. The major contents and contributions of this thesis includes the following aspects:1. According to the potential mechanisms of DOX-induced cardiotoxicity, three fluoresent dyes were investigated, in which FDA can indicate the whole activity of cells, Hoechst can stain the nucle of cells, and MTR can exhibit the alteration of mitochondrial membrane potential. Cultured H9c2cardiac muscle cells were used to perform cell-based assay. The linear relationship between cell density and fluorescent intensity of three dyes in present or absent of varied concentrations of DOX was investigated. The results demonstrated that FDA and Hoechst were appropriate for staining H9c2cells for determing Dox-induced cardiotoxicity.2. After compared the performance of mono dye or dual dying of cells to study DOX-induced cardiotoxicity, we propose an assay for screening cardioprotective compounds by dual-fluorescent dyes FDA ang Hoechst. The IC50of DOX was0.82and0.17μM calculated by fluorescent intensity of FDA and Hoechst respectively. The proposed approach was evaluated and validated by a known cardioprotective compound rutin. At the concentration of100μM, rutin has the protective percent of53.6%(FDA staining) and38.9%(Hoechst staining).3. The proposed method has also applied in identifying potential cardioprotective constituent of a clinical used Chinese medicine Zhenqifuzheng, a complementary drug for chemotherapy. At last, we found five components with strong cardioprotective effect by screening52components from Zhenqifuzheng. LC-MS was used to putatively identify the chemical structures of active components. Two active compounds, hydroxytyrosol and neonuezhenide showed significant cardioprotective effect in the dose-dependent manner. The cardioprotective percent of hydroxytyrosol and neonuezhenide were19.1%and29.6%(FDA staining) while30.0%and24.2%(Hoechst staining) at the concentration of80μM.