Total Flavonoids Of Hawthorn Leaves Combined With Astrocytes Promote Neuronal Repair Of Oxidative Damage

Background and purpose:Spinal cord injury often causes irreversible damage to the structure and function of neurons,leading to the destruction of nerve function,partial or complete loss of sensory and motor abilities,and even paraplegia or quadriplegia^[1].According to the pathological process,spinal cord injury is generally divided into primary spinal cord injury and secondary spinal cord injury.Primary spinal cord injury is often a mechanical injury caused by external or internal factors that directly act on the spinal cord,and once it appears,it cannot be recovered.After acute spinal cord injury,the cascade of self-destructive cascades of waterfall-mediated tissue mediated by inflammatory response,free radical-induced oxidative damage,etc.is called secondary injury,which can lead to the expansion of the injury area and the paralysis to a higher segment.The degree of damage is much greater than the primary spinal cord injury.At present,the main therapeutic drugs for spinal cord injury include methylprednisolone and ganglioside,but these clinical drugs have the disadvantages of long drug time,ineffective long-term efficacy,lack of safety,and poor patient compliance.Efficient drugs have been particularly urgent^[2].The previous study of the research group found that the main component of the hawthorn leaves in Guangxi’s local medicinal material,hawthorn leaves,can significantly reduce the expression of inflammatory factors in the injured spinal cord of rats with spinal cord injury,reduce the infiltration of inflammatory cells in the spinal cord tissue of rats,and reduce The swelling of the endoplasmic reticulum of nerve cells reduces the rate of syringomyelia^[3-4].From the in vitro level,there is little research on the repair of damaged spinal neurons by hawthorn leaves flavonoids.After spinal cord injury,astrocytes are activated in large quantities and regulate the microenvironment of spinal neurons,which plays an important role in regulating the repair and regeneration of spinal nerves^[5].Therefore,this experiment explored the molecular mechanism of hawthorn leaf total flavonoids combined with astrocytes to regulate the microenvironment of injured spinal neurons and promote the repair of spinal neurons through in vitro experiments.Methods:1.Dissect neonatal rats to isolate spinal neurons and culture them in serum-free Neurosouls medium,and useβ-Tubulin3 immunofluorescence staining to identify spinal neurons.2.Take spinal neurons cultured for 7 days in vitro,intervene with different concentrations of hydrogen peroxide for different times,MTT detects the survival rate of spinal neurons,so as to construct a suitable spinal neuron oxidative injury model.3.To evaluate the toxicity of hawthorn leaves flavonoids to spinal neurons and astrocytes by MTT method.4.Build a co-culture model of spinal neurons and astrocytes through the Transwell chamber,divide the spinal neurons into 5 groups,Con group（spinal neurons without any treatment）;Hurt group（200μM H₂O₂ treatment spinal cord neurons24h）;Astrocyte group（spinal neurons were treated with H₂O₂ for 24h,then co-cultured with AS via transwell chamber）;Direct drug group（spinal neurons treated with H₂O₂ for 24h,125μg/m L TFHL for 24h）;Co-Culture group（After spinal neurons were treated with H₂O₂ for 24h,co-culture was established with AS through transwell chamber,and 125μg/m L TFHL was added for 24h）.5.MTT detects the activity of spinal neurons under different treatment methods.6.Invert the phase contrast microscope to observe the morphological changes of spinal cord neurons under different treatment methods.7.Immunofluorescence staining to observe the changes of spinal neurons under different treatment methods.8.Electron microscopy to detect the changes of autophagic vesicles of spinal neurons under different treatment methods.9.ROS kit detects the changes of reactive oxygen species in spinal neurons under different treatment methods.10.MDC kit detects the formation of autophagosomes of spinal cord neurons under different treatment methods.11.Immunoblotting and PCR detection of astrocyte inflammatory factors and signal molecules on inflammatory pathway NF-k B under different treatment methods.Results:1.Immunofluorescence staining found that the positive rate of spinal neuron cells was>90%,and the isolation and cultivation of neurons in vitro was successful.2.MTT test found that when the concentration of H₂O₂ is less than or equal to 100μM,there is no obvious toxic effect on the cells,but as the concentration increases（>400μM）,the cell activity decreases significantly.The effect of oxidative stress injury caused by 200μM H₂O₂ for 24h is obviously better than 12h,so in this experiment,the treatment of spinal neurons with200μM H₂O₂ for 24h is the ideal model condition.3.The MTT cytotoxicity experiment found that as long as the dose concentration of total flavonoids in hawthorn leaves is less than 125μg/ml,this dose will not produce cytotoxic effects on spinal neurons.4.Compared with Hurt group,the cell survival rate of Co-culture group was 68.21%,which was significantly higher than that of Hurt group.5.Morphological observation revealed that the neurons of spinal cord in the Hurt group shrank,the protrusions disappeared,and a large number of cells died.Compared with the Hurt group,the number of spinal neurons in the Co-culture group was significantly increased,the morphology was complete,and there were more protrusions.6.Immunofluorescence showed that compared with Hurt group,the cytoskeleton of Co-culture group was complete,and the content of tubulin Map-2 was significantly increased.7.MDC staining found that compared with Hurt group,the number of autophagic vesicles in Co-culture group increased significantly.8.ROS staining found that compared with Hurt group,the content of reactive oxygen in Co-culture group was significantly reduced.9.Electron microscope observation showed that compared with Hurt group,the number of autophagosomes in Co-culture group increased significantly.10.PCR found that compared with the Hurt group,the m RNA expression of pro-inflammatory cytokines in the Drug group was significantly reduced,and the m RNA expression of anti-inflammatory cytokines was significantly increased.11.WB results also confirmed that compared with the Hurt group,the protein expression of pro-inflammatory cytokines in the Drug group was significantly reduced,and the protein expression of the anti-inflammatory cytokines was significantly increased.12.Compared with the Hurt group,the expression level of signaling pathway proteins in the Drug group was significantly reduced.Conclusion:1.Hawthorn leaf total flavonoids can be combined with astrocytes to promote the repair of spinal neurons injured by oxidative damage.2.Hawthorn leaf total flavonoids can inhibit the expression level of NF-κB signaling pathway protein in astrocytes,inhibit the expression of inflammatory cytokines,improve the microenvironment of spinal neurons,and thus inhibit the oxidative damage of spinal neurons.Promote autophagy and repair of spinal neurons.3.Hawthorn leaf total flavonoids may become a potential new drug for the treatment of secondary spinal cord injury,and may be used for nutritional intervention after spinal cord injury.