Effects Of Diosmetin And Reduced Graphene Oxide-Supported Palladium Nanoparticles On Neural Stem Cells

With the increase of aging population,the incidence of neurodegenerative diseases increased rapidly in these years.Thus,now there is a research hotspot focused on how to promote nerve regeneration in brain to repair the desrupted neural network,reduce oxidative stress or chronic inflammation-induced injury,and then rescure cognitive function,especially for the treatment of intractable neurodegenerative diseases such as Alzheimer's disease?AD?.Neural stem cells?NSCs?have a potential to repair the nerve tissues,that can be propagated,differentiated and then integrated into the existing neural network.However,changes in the internal and external environment can affect cell's function,the redox imbalance in the growth microenvironment and changes of extracellular matrix will affect its survival,proliferation and differentiation.The recent studies have shown that graphene?G?and graphene oxide?GO?have the characteristics of non-cell toxicity,transferability and accessibility,which can be used as a stem cell culture platform,to promote the proliferation and differentiation of stem cells.Reduced Graphene Oxide@Palladium?rGO@Pd?can not only keep the advantages on graphene biological safety,excellent electrical,optical,mechanical properties and large specific surface area,but also have good catalytic hydrogenation properties.Combined with G or GO,rGO@Pd can enhance the electrochemical capacitance of the materials.In the first part of this study,we used rGO@Pd as a substrate in culture medium for the growth of NSCs to explore whether rGO@Pd would affect the development of NSCs and observe the effect on the proliferation of NSCs.NSCs is sensitive to changes in the level of reactive oxygen species?ROS?which accumulation reduces the neurogenesis in the brain.Thus,it is important to study the antioxidation mechanism to improve the survival of NSCs under stress conditions.In recent years,many studies have found that traditional Chinese medicine monomer can protect NSCs from damage,promote the proliferation,migration and differentiation of them.Diosmetin,one of the natural flavonoids,has multiple biological activities,such as anti-inflammation,bacteriostasis,anti-oxidation,anti-tumor,and so on.With the development of nanotechnology and traditional Chinese medicine?TCM?research,there are more and more researches on nerve regeneration by combination of traditional Chinese medicine monomers and nano-materials.Thus,in the second part of this study,we used H₂O₂-induced NSCs as an oxidative damage model to investigate the effects of diosmetin on NSCs in vitro.This work is mainly exploring the experimental basis for the treatment of neurodegenerative diseases with nanomaterials in combination with diosmetin,evaluating the effects of traditional Chinese medicine monomer on pathogenesis of neurodegenerative diseases,and providing novel insights into developing new treatment stragegies against neurodegenerative diseases.Part ? Effects of rGO@Pd on the proliferation of NSCsWe used the reduced graphene oxide-supported palladium nanoparticles?rGO@Pd?as a substrate for the growth of mouse NSCs to investigate the effects of rGO@Pd on the proliferation of NSCs in vitro.The rGO@Pd were obtained by ultrasonic treatment of rGO@Pd.We used scanning electron microscope?SEM?,fourier transform infrared spectroscopy?FTIR?and dynamic light scattering?DLS?to characterize rGO@Pd.The effect of rGO@Pd on NSCs activity was detected by Calcein-AM staining and the optimal concentration was selected.The effect of rGO@Pd on the proliferation of NSCs was evaluated using BrdU labeling and immunocytochemistry staining.We found that the characterization of rGO@Pd in the experiment is uniform and is a kind of microporous structure.In the rGO@Pd,each functional group has almost disappeared and the oxygen-containing group is completely reduced,indicating that the material is rGO@Pd,and has well stability and dispersibility in water.The results of Calcein-AM staining showed that cell activity of NSCs was increased after treatment with 0.005 mg/mL of rGO@Pd.As the concentration of rGO@Pd increased to 0.01 mg/mL,it was significantly increased?P<0.05?.The result of BrdU immunocytochemistry staining showed that the appropriate concentration of rGO@Pd could induce the proliferation of NSCs,the effect of rGO@Pd on cell proliferation was the greatest at a concentration of 0.01 mg/mL?P<0.05?,and the percentage of BrdU positive cells in 0.01 mg/mL rGO@Pd group?23.7±1.2%?was significantly higher than that in control group?19.3±0.6%??P<0.05?.Thus,using rGO@Pd as a substrate for culture of mouse NSCs in vitro,the above results indicate that rGO@Pd could enhance the activity of NSCs and promote the proliferation of NSCs under the appropriate concentration.The above work has certain scientific significance and theoretical support for the development of new materials and the use of new methods to regulate the function of neural stem cells and further apply to the process of repairing brain aging injury.Part ? Effects of diosmetin on H₂O₂-induced oxidative stress of NSCsWe used H₂O₂-induced mouse NSCs as an oxidative damage model to investigate the effect of diosmetin on oxidative damage of NSCs.We screened the concentration of H₂O₂ and the optimal drug concentrations,using CCK8 assay.The effects of diosmetin on the survival rate of NSCs after injury were detected by CCK8 assay and Calcein-AM staining,and the mortality of NSCs was detected by PI/Hoechst staining.We found that the optimum concentration of H₂O₂ was200?M,and when the concentration of diosmetin increased to 2400 nM,it had toxic effect on NSCs.After pretreatment NSCs with different concentrations of diosmetin for 4 hours,we added 200?M H₂O₂ into the culture medium for 12hours,the results showed that the death of NSCs induced by H₂O₂ could be blocked by diosmetin,and cell viability was enhanced as shown in CCK8 assay and Calcein-AM staining.The result of PI/Hoechst staining showed that the number of PI positive cells significantly decreased in the treatment group.When the concentration of diosmetin was 800 nM,the drug had the strongest inhibitory effect on cell death induced by H₂O₂-induced oxidative stress of NSCs.Thus,our results showed that the oxidative damage of H₂O₂-induced mouse NSCs was protected by diosmetin.The specific underlying mechanism has yet to be further explored.