| Background and Aims: Spinal cord injury(SCI)refers to the spinal cord nervous system injury caused by the high energy external force acting on the spine in different ways,causing the spinal column to shift,burst,and then involve the spinal cord in the spinal canal.It brings great harm and heavy burden to the patients and their families.At present,the clinical use of drugs to treat spinal cord injury,although the effect is significant,the toxic side effects are obvious and uncertain long-term efficacy,some high prices of drugs cause low compliance of patients.So it is a trend to find a natural,effective,safe,and inexpensive drug.Chinese herbal medicine has the advantages of small toxicity,safety,low price and so on,which has become the focus of our research.Our research group has selected the plant chemicals with anti-inflammatory effect and found that the main pharmacological component of hawthorn leaves in Guangxi.It is the main pharmacological components of hawthorn leaves,which is mainly produced in Guangxi Hezhou,Guilin,Liuzhou and Baise.It can improve the tissue ischemia and hypoxia,decrease the expression of inflammatory factors and alleviate theinflammatory reaction.These effects can effectively improve secondary spinal cord injury,so we speculate that hawthorn leaf total flavonoids may have potential therapeutic effect on spinal cord injury.In order to verify the hypotheses,we carried out preliminary pre-experiments and confirmed that the total flavonoids of hawthorn leaves could significantly improve the motor function of rats,improve the apoptosis of injured spinal cord tissues,protect the Nissl bodies of neurons and down-reguLate the expression of inflammatory factors.Increase the expression of protein related to spinal cord injury repair.These studies suggest that total flavonoids of hawthorn leaves can improve the local microenvironment of injured spinal cord and improve the recovery of motor function in rats with spinal cord injury.Total flavonoids from hawthorn leaves can be a potential new drug for secondary spinal cord injury.In this experiment,we will explore the effect of total flavonoids of hawthorn leaves on secondary spinal cord of rats in vivo.To explore the relationship between the total flavonoids of hawthorn leaves and the activation of inflammatory reaction of microglia and the possible molecular mechanism,which provides a theoretical basis for the clinical application of total flavonoids of hawthorn leaves?Methods: 1.(1)The model was established.The SPF 12-week-old SD rats were randomly divided into three groups: sham operation group(Sham group),spinal cord-striking injury group(SCI group),and total flavonoids of hawthorn leaves treatment group(SCI+TFHL group).(2)SCI group and SCI+TFHL group established the model of spinal cord injury in SD rats according to the ALLEN's method.In the SCI+TFHL group,the total flavonoids of hawthorn leaves were injected intraperitoneally 100mg/kg at 0,6,and 12 hours respectively.The same amount of saline was injected into the SCI group and Sham group.(3)Thefunction of spinal motion of the rat was assessed by the BBB score;(4)Fluorescence staining was used to observe the regeneration of the spinal cord and the formation of glial scar;(5)The neurite neurite growth was observed by the reverse-tracing technique;(6)The expression of the inflammatory reaction factor and the pathway protein was detected by Western-blot and Real-time PCR.2.Cell model was established.(1)BV2 microglia was cultured in cell bottle containing 10% fetal bovine serum and 1% streptomycin at 37? and 5% CO2 was cultured in the incubator.(2)The experiment was divided into three groups:Sham group: normal culture;LPS group: BV2 microglia activated by LPS;LPS+TFHL group: TFHL was pretreated for 2 h and co-cultured with LPS.After24 hours of culture,the cells were collected for follow-up experiments.(3)MTT was used to evaluate the toxicity of the drug to cells;(4)In order to compare the difference between inactive microglia and activated microglia under confocal microscope.(5)In order to detect the expression level of inflammatory response factor by ELISA,and to detect the expression of inflammatory pathway protein by Western-blot.Results:(1)The results of BBB showed that compared with the Sham group,the BBB score in the SCI group was significantly lower than that in the SCI+TFHL group,while the BBB score in the SCI+TFHL group was increased.(2)After HE staining,the spinal cord tissue structure in the SCI group was confused and a large number of inflammatory cells infiltrated.There was no obvious confusion in the spinal cord tissue structure in SCI+TFHL group,the infiltration of inflammatory cells was decreased obviously,and the path morphology of spinal cord tissue was improved obviously.(3)Compared with SCI group,the levels of inflammatory factor protein and mRNA in SCI+TFHL group were comparedwith those in SCI+TFHL group detected by Western Blot and PCR.(4)The fluorescent expression of inflammatory pathway protein in SCI +TFHL group was lower than that in SCI group under fluorescence microscope.(5)Nissl staining showed that the number of Nissl bodies in SCI group decreased and the morphology of Nissl bodies was difficult to recognize.The number and the morphology of Nissl bodies in SCI+TFHL group were higher than that in SCI group.(6)TUNEL staining showed that the number of apoptotic cells in SCI group was significantly higher than that in SCI group.(7)The levels of neurofilament protein 200(NF200)and NeuN in SCI group were lower than those in SCI group.(8)Immunofluorescence assay showed that the levels of neurofilament protein(NF200)and NeuN were lower in SCI+TFHL group.The content of NF200 in the SCI+TFHL group without axonal regeneration was significantly higher than that in the SCI group.(9)The MTT test showed that2.5?g/mL TFHL had no cytotoxic effect on microglia.The expression of mRNA in SCI+TFHL group was significantly lower than that in SCI group.By ELISA assay,we found that the protein expression of inflammatory factors in SCI+TFHL group was significantly lower than that in SCI group.(10)Compared with SCI group,the activation of SCI+TFHL cells was significantly inhibited and the fluorescence intensity of TLR4 and Iba-1 was decreased.(11)The level of pathway protein in SCI+TFHL group was significantly lower than that in SCI group.Conclusion:(1)In vivo,total flavonoids of hawthorn leaves can decrease the expression of inflammatory factors,gradually improve the local microenvironment of injured spinal cord,decrease the apoptosis of spinal cord tissue,and enhance the repair degree of spinal cord function.(2)Total flavonoids from hawthorn leaves could inhibit the activation of microglia and decrease theexpression of inflammatory factors in TLR4/NF-?B pathway.(3)Total flavonoids of hawthorn leaf could inhibit the activation of microglial cells and decrease the expression of inflammatory factors in NF-?B pathway in vitro.It is suggested that total flavonoids of hawthorn leaves may have anti-inflammatory effects by activating microglia cells and inhibiting TLR4/NF-?B signaling pathway after spinal cord injury.(4)Total flavonoids of hawthorn leaves may become a potential new drug for secondary spinal cord injury. |
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