Study On The Protective Effect And Mechanism Of Morroniside On Oxidative Stress Injury Of Oln-93 Cells

IntroductionBackground and purpose Neurodegenerative disease have always been a worldwide problem,and nowadays have not an appropriate way to treat it.It is an important factor to lead to neurodegenerative disease that neuron axonal demyelination cause signal black.Oligodendrocyte precursor cells(OPC)proliferate,migrate and differentiate into oligodrocytes and eventual LY it forms a myelin sheath in the central nervous system.In the physiological LY,in order to ensure the integrality of neuronal axon myelin and smooth signal transmission,OPC consume a large number of ATP for continuous proliferation and differentiation to assure myelin sheath shed were replaced during physiology or pathology in time.Because the high metabolism and high oxygen consumption of OPC,so OPCs are vulnerable to oxidative attack.If OPC were injuried,the shed myelin sheath can't be timely replaced and signal transmission is blocked,which is likely to induce neurodegenerative disease.Hence,preventing oxidative damage of OPC in central nervous system is a necessary means to prevent and treat neurodegenerative disease.Morroniside is a naturally active small molecule extracted from cornus officinalis.Studies have shown that it has anti-oxidative and anti-apoptotic protective effects on neurons,but its effect on glial cells has been little reported.Therefore,in this study,Oln-93 cells(oligodendrocyte precursor cells in rats)were used research object to explore the protective of Morroniside on oxidative stress injury of Oln-93 cells and its mechanism.Hoping to provide new experimental basis and ideas for preventing and treatment neurodegenerative disease.Method Oln-93 cells' model of oxidative stress was established: 1.Oln-93 cells were adjusted to logarithmic growth phase,H2O2(?M)of different concentrations were incubated to determine the optimal damage concentrations and time by MTT.Cells were pretreated by different concentrations of Morroniside(?M)for 24 h and the appropriate concentrationof H2O2 were used damage cells to determine the optimal dosing concentration.2.Using inverted microscope to observed the morphological changes of cells in each group.3.LDH kit was used to detect cytotoxicity in each group.4.MDA kit was used to detect cells lipid oxidation degree.5.ROS kit was used to detect each intracellular ROS release quantity.6.The changes of mitochondrial membrane potential(MMP)in each group were detected by JC-1 fluorescence probe and fluorescence microscope.7.TUNEL kit was used to detect TUNEL expression in cells of each group.8.Using Hoechst33342 staining to observe the nuclear morphology changes of cells in each group.9.Extracting the protein of each group cells,and expression of Oxidation-related protein i NOS,SOD2 and apoptosis-related proteins Bcl-2,Bax,AKT,P-AKT and Caspase3 were detected by Western Blot.Result 1.Cell activity was detected by MTT,result showed that Morroniside significantly improved the decrease of cell activity induced by H2O2.2 Observing the morphology of each cell,the results showed Morroniside can obviously improve H2O2 damage caused by broken,shrinkage of cells.3.The detection results of LDH cytotoxicity test showed that the LDH release of cells increased significantly after H2O2 injury,but Morroniside could significantly reduce the release of LDH in cells.4.Lipid oxidation test results showed that Morroniside could significantly reduce the production of MDA.5.ROS test results showed that Moroniside could significantly reduce the ROS release of Oln-93 cells induced by H2O2.6.Mitochondrial membrane potential test results showed that Morroniside could significantly inhibit the H2O2-induced reduction of mitochondrial membrane potential in cells.7.TUNEL test results showed that Morroniside could significantly reduce the expression of TUNEL induced by H2O2.8.Hoechst33342 staining results showed that after H2O2 injury,the nucleus showed a shrivelled,fragmented and irregular morphology,but Morroniside could significantly decrease the number of apoptotic.9.Western-blot results showed that Morroniside could significantly reduce the expression of i NOS,increase the expression of antioxidant proteins SOD2,decrease the expression of Pro-apoptotic proteins Bax and Caspase3,and increase the expression of anti-apoptotic proteins Bcl-2and P-AKT.10.Oln-93 cells were preincubated by LY294002 and detected,and the results showed(1)Morphology showed Morroniside's effect was inhibited,after LY294002 advance incubation.(2)ROS test showed LY294002 inhibit Morroniside's effect that Morroniside reduce the ROS release of Oln-93 cells induced by H2O2.(3)TUNEL test showed the improvement of TUNEL expression by Morroniside was inhibited after incubation with LY294002.(4)The result of Hoechst33342 showed the effect that Morroniside reduce the cells apoptosis was inhibited after LY294002 incubation.(5)WB result showed after cells were preincubated by LY294002,effect of Morroniside to regulate apoptotic proteins(Bcl-2?P-AKT?Bax?caspase-3)and i NOS was significantly reduced.Conclusion Monoside significant LY inhibited H2O2-induced oxidative damage in Oln-93 cells.The mechanism may be based on the PI3K/AKT pathway,which is closely related to inhibiting the expression of oxidative stress and Pro-apoptotic molecule(Caspase-3)in cells,promoting the expression of Bcl-2,and thereby blocking the mitochondrial apoptosis pathway.