The Protective Effect Of Morroniside Against H₂O₂-induced Oxidative Damage In Sk-N-SH Cells And Its Mechanism

Objective:To explore the protective effect of morroniside against oxidative damage of SK-N-SH cells using H2O2-induced oxidative damage model.Methods:Human neuroblastoma SK-N-SH cells were pretreated with different dose of morroniside(1, 10, and 100 μM) for 24 h followed by incubated with H2O2(200-400μM) for 24 h to induce neurotoxicity. The growth and morphological changes in each group were observed under an inverted phase contrast microscope. MTT assay was conducted to detect the changes in cell viability. The cell membrane integrity was monitored using a permeability assay to determine the release of LDH into the medium.Lipid oxidation level was detected using lipid peroxidation(MDA) test kit. Intracellular Superoxide dismutase was determined by the SOD kit. Intracellular reactive oxygen species(ROS) level was quantified using a 2′,7′-dichlorofluorescein(DCF) fluorescence assay. The level of intracellular superoxide anion was detected using superoxide anion fluorescent probe(Dihydroethidium). Changes in mitochondrial membrane potential(△Ψm) and Caspase-3 activity were detected by detection kit(JC-1) and spectrophotometric detection, respectively. FCM and Hoechst staining were used to observe the cell apoptosis. Western blot assay was used to detect the expression of apoptosis-related proteins.Results:1. MTT assay showed that cell viability was significantly reduced after treated withH2O2 compared with the control group. However, morroniside obviously attenuatedH2O2-induced loss of cell viability.2. After 24 h treatment with 200 μM H2O2, SK-N-SH cells displayed significantmorphological changes, such as cell projections disappear, body swelling round andshrink, aggregation and broken phenomenon. These morphological changes werereverted by the pretreatment of morroniside.3. LDH cytotoxicity assay showed that morroniside could reduce intracellular enzymeLDH release.4. Flow cytometry and Hoechst staining showed that morroniside significantlyinhibited H2O2-induced apoptosis of SK-N-SH cells.5. DCFH-DA and Dihydroethidium fluorescent probe assay showed that morronisidecould significantly inhibit intracellular production of ROS in SK-N-SH cells.6. Lipid oxidation detection showed that morroniside effectively inhibited intracellularlipid peroxidation. SOD testing suggested that morroniside significantly inhibitedH2O2-induced decrease of SOD.7. JC-1 fluorescent probe and spectrophotometry showed that morronisidesignificantly inhibited H2O2-induced mitochondrial membrane potential decreaseand caspase-3 activation in SK-N-SH cells.8. Western blot and RT-PCR indicated that morroniside effectively inhibited H2O2-induced BAX up-regulation and stimulated the expression of Bcl-2.Conclusion:Morroniside could inhibit H2O2-induced oxidative damage by inhibiting the ROS-initiated oxidative stress, decreasing the expression of BAX and stimulating the expression of Bcl-2, and blocking mitochondrial apoptotic pathway.