| Based on the bioactivity screening results of the compounds isolated in our lab, homoeriodictyol was found to be a good protector against cellular oxidative damage, and alisol B-23-acetate can inhibit the proliferation of lung cancer cells. The bioactive mechanisms of the two compounds were further studied in this project.Oxidative stress leads to vascular endothelial cell damage which plays a key role in several kinds of vascular disease, such as atherosclerosis and chronic kidney diseases. Oxidative stress is caused by an imbalance in the anti-oxidative stress defense systems and reactive species, such as reactive oxygen species (ROS) in cells. One of the effective methods to reduce oxidative damage induced by oxidative stress is activating the cellular antioxidant system. The nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway is one of the main defense mechanisms against oxidative or electrophilic stress, and activation this pathway can rebuild cellular antioxidant system and maintain the stability of cellular redox homeostasis. Nrf2, principal transcription factor that translocate to the nucleus where it binds with ARE (antioxidant response element) in the promoter region of its target genes thereby inducing phase II detoxifying enzymes and anti-oxidative enzymes, and boost the cellular anti-oxidative defense system to protect cells against oxidative stress for long time. Many studies have shown that activating the Nrf2/ARE pathway can effectively prevent the vascular disease induced by oxidative stress. Hence, one of the most effective methods to cure cardiovascular and cerebrovascular diseases is to finding Nrf2 inducer and activating the Nrf2/ARE pathway to protect vascular endothelial cells against damage caused by oxidative stress.Homoeriodictyol, a flavanone isolated from Viscum coloratum (Kom.) Nakai, has been established as an antioxidant and free radical scavenger. The present study was designed to expfore the protective effects of homoeriodictyol against hydrogen peroxide (H2O2) induced toxicity on human umbilical vein endothelial cells (EA.hy926 cells) and the effects on Nrf2 signaling pathway induced by this compound was investigated also.MTT assay and the xCELLigence live cell analysis (RTCAassay) results showed that homoeriodictyol protected EA.hy926 cells against H2O2-induced oxidative damage in a dose-dependent manner. DAPI staining and Annexin V/PI staining indicated that homoeriodictyol prevented H2O2-induced apoptosis in EA.hy926 cells. By immunofluorescence analysis, homoeriodictyol was found to induce translocation of Nrf2 into nucleus. Western blot, real time RT-PCR and cycloheximide chase analysis results indicated that homoeriodictyol upregulated the expression of Nrf2, NQO1 and yGCS, delayed the degradation of Nrf2 protein, and also upregulated the mRNA level of Nrf2, NQ01 and GCLM. Further studies showed that homoeriodictyol increased intracellular level of reduced glutathione, inhibited H2O2-induced ROS production, and regulated the expression of apoptosis-associated proteins induced by H2O2. Finally, we transfected EA.hy926 cells with Nrf2 small interfering RNA (Nrf2-siRNA) to knockdown the expression of Nrf2 and tested the protective effects of homoeriodictyol by MTT assay, RTCA assay and Western blot analysis. Results showed that the protective effects of homoeriodictyol on cells were attenuated, thus suggested the protective effects exerted by homoeriodictyol was mediated byNrf2 signaling pathway. In conclusion, homoeriodictyol is a natural Nrf2 inducer and protects EA.hy926 cells against H2O2-induced apoptosis through the activation of Nrf2/ARE pathway.Cancer is one of the leading causes of human health. The lung cancer with high incidence and mortality was so much focused on. The development of new effective drugs is an important concern with the increasing of lung cancer.Alisol B-23-acetate, a tetracyclic triterpenoid, was isolated from Alisma orientale (Samuels) Juzep. Ailsol A (A1), alisol A-23-acetate (A2), alisol O (A3) and alisol B-23-acetate (A4) were tested the cytotoxic activities on lung cancers by MTT assay. We found that compounds of A1 and A4 showed strong cytotoxic activity against the A549 and NCI-H292 cells. Subsequently A4 was chosen for further evaluation, which has the best activities of the four compounds and the quantity is enough. A4 induced cellular apoptosis suggested by DAPI staining and Annexin V/PI staining. The intracellular ROS level increased after treatment with A4, and the mitochondrial member potential was decreased. A4 induced S phase arrest in NC1-H292 cells. The expression of cytochrome c, AIF, Bax, Be 1-2, Bcl-xL, caspase-9, caspase-3 and PARP were tested by Western blot and immunofluorescence analysis. In conclusion, A4 triggers apoptosis of A549 and NCI-H292 cells through mitochondrial-dependent pathway, and it will be a promising candidate as a therapeutic agent for lung carcinoma. |
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