Supplementary Materialsijms-20-05832-s001

Supplementary Materialsijms-20-05832-s001. glycoside, fraxin, isolated from Column chromatographic separation of the EtOAc-soluble fraction of the extract led to the isolation of 15 compounds, followed by high-performance liquid chromatography (HPLC) purification. In this study, cell-based protection screening was first performed to evaluate the neuroprotective effects of the isolated compounds 1C15 using human dopaminergic neuroblastoma SH-SY5Y cells. To elucidate the underlying neuroprotection mechanism, we studied the mechanism of action of the most active compound 5, hyperoside (quercetin 3-bark was extracted with water at 90 C and then filtered. The filtrate was concentrated under vacuum to obtain a crude aqueous extract and then solvent-partitioned with hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and bark. The isolated compounds were identified as (7< 0.01 and < 0.001, respectively). Furthermore, cells pretreated with 0.25, 0.5, 1 and 2 M hyperoside showed significantly reduced 6-OHDA-induced LDH release, compared to control cells treated with 6-OHDA alone (Determine 2D, < 0.05 and < 0.001, respectively). To observe the neuroprotective effect of hyperoside on 6-OHDA-induced cell death and DNA fragmentation, we performed a TUNEL staining assay. In representative images, this TUNEL staining revealed significant DNA fragmentation after exposure to 6-OHDA, whereas pretreatment with hyperoside (5) significantly prevented DNA fragmentation (Physique 2E). Open in another window Body 2 Ramifications of hyperoside on SH-SY5Y cells (A and C). Cells had been Ellagic acid pretreated using the indicated concentrations of hyperoside or = 6). Hyperoside prevents 6-OHDA-induced DNA fragmentation (E). DNA fragmentation was assayed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Cells had been pretreated using the hyperoside (0.5C2 M) for 4 h and subjected to 200 M 6-OHDA for 24 h. Pictures proven are consultant of three tests and visualized by fluorescence microscopy (20). *** 0.001 vs. the control group. # < 0.05, ## < 0.01 and ### < 0.001 vs. the 6-OHDA-treated group. n.s.: not really significant. Scale club: 200 m. 2.4. Hyperoside Prevents 6-OHDA-Induced Intracellular ROS Mitochondrial and Deposition Membrane Potential Dysfunction in SH-SY5Y Cells Following, we looked into intracellular ROS deposition and mitochondrial membrane potential (MMP) dysfunction, that are well-known initiators from the oxidative stress that induces cell injury. Treatment with 200 M 6-OHDA significantly increased the intracellular ROS, compared to the control (Physique 3A, < 0.001 and Figure 3C, upper); however, the 6-OHDA-mediated increase in intracellular ROS was significantly prevented by pretreatment with hyperoside at Rabbit Polyclonal to CXCR4 0.5, 1 and 2 M (< Ellagic acid 0.01 and < 0.001, respectively). Conversely, treatment with 200 M 6-OHDA significantly decreased this MMP, compared to the control (Physique 3B, < 0.001 and Figure 3C, bottom); however, the 6-OHDA-mediated decrease in intracellular MMP was significantly prevented by pretreatment with hyperoside (5) at 0.5, 1, and 2 M (< 0.001, respectively). Open in a separate window Physique 3 Hyperoside prevents 6-OHDA-induced intracellular Reactive Oxygen Species (ROS) accumulation (A,C, upper) and mitochondrial membrane potential (MMP) dysfunction (B,C, bottom) in SH-SY5Y cells. Representative images were observed under fluorescence microscopy (20). Cells were pretreated with the hyperoside (0.25C2 M) for 4 h and then exposed to 200 M 6-OHDA for 1 or 24 h. Error bars show the mean SEM (= 6). Images shown are representative of three experiments and visualized by fluorescence microscopy (40). *** 0.001 vs. control group. ## < 0.01 and ### < 0.001 Ellagic acid vs. the 6-OHDA-treated group. Level bar: 200 m. 2.5. Hyperoside-Mediated Activation of Nrf2 Occurred in a Time- and Concentration-Dependent Manner in SH-SY5Y Cells To examine whether hyperoside (5) induces the HO-1 transcriptional signaling pathway, which is usually directly linked to Nrf2-dependent activation, we used both western blot analysis and immunostaining. Treatment with hyperoside induced significant nuclear translocation of Nrf2 in both a concentration-dependent (Physique 4A, < 0.05 and < 0.001) and time-dependent (Physique 4B, < 0.01 and < 0.001) Ellagic acid manner. Similarly, after treatment with 2 M hyperoside, the nuclear protein levels of Nrf2 were significantly increased for 1 h, peaked at 4 h, and then decreased at 6 h. Based on these results, treatment with 2 M hyperoside for 4 h was used to induce nuclear translocation of Nrf2 in subsequent experiments. In an attempt to determine whether induction of HO-1 by hyperoside (5) was indeed responsible via the activation of the ARE-binding ability of Nrf2, the cells were transfected with luciferase reporters under the control of the ARE promoter. As shown in Physique 4C, the transcriptional activity of ARE was significantly increased by treatment with hyperoside in a concentration-dependent manner, compared to the control (< 0.001, respectively). Representative images reveal the nuclear inclusion of Nrf2 in cells treated with hyperoside (Physique 4D). As expected, pretreatment with hyperoside (5) activated the nuclear translocation of Nrf2 in the cells. Open up in another window Amount 4 Hyperoside activates nuclear translocation.